补肾法、疏肝法对排卵障碍模型大鼠HPOA及人卵巢颗粒细胞分泌功能影响的比较研究
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摘要
第一部分:补肾法、疏肝法对排卵障碍模型大鼠HPOA的影响
目的:通过研究比较补肾法与疏肝法对排卵障碍模型大鼠HPOA影响的异同,探讨补肾法、疏肝法治疗排卵障碍性疾病的作用机制,以丰富中医生殖理论,促进中医妇科学的发展。
方法:模型一:选用同批出生9日龄健康雌性SD大鼠204只,体重约15g。随机取170只大鼠,于颈背部皮下一次性注射丙酸睾丸酮1.25mg/只(0.05ml/只)造模。第22日龄断奶,自由进食,同条件饲养。第70日龄阴道开口,造模组连续阴道上皮细胞涂片两个性周期(一个性周期4-5天),阴道上皮细胞持续角化,提示雄激素致排卵障碍大鼠模型造模成功。余34只,于颈背部皮下一次性注射中性茶油0.05ml作为正常对照组。
模型二:选用3月龄健康雌性Wistar大鼠204只,体重180~250g。随机取170只大鼠改进郑家润,梁文波等方法。雷公藤多甙片35mg/kg/d(给药剂量为临床成人用量的20倍),灌胃法给药,连续57天。造模组连续阴道上皮细胞涂片两个性周期,动情周期转为不规则,主要为周期不完整及周期延长,提示雷公藤致排卵障碍大鼠模型造模成功。其余34只灌胃给蒸馏水作为正常对照组。
将两种排卵障碍成功模型大鼠分别随机分为模型组和治疗组,治疗组包括:补肾调经方高剂量组、补肾调经方低剂量组、逍遥丸高剂量组、逍遥丸低剂量组,每组分别各34只;治疗组分别灌服补肾调经方与逍遥丸,模型组和正常组灌服蒸馏水,每日1次。灌胃给药5周,并阴道细胞涂片两个性周期。最后一次给药后禁食8小时,选择相同动情周期(动情前期、动情后期)断头取血,制备血浆、血清,放免法检测血浆卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)、孕酮(P)、睾酮(T)、催乳素(PRL)。同时立即无菌操作,在冰盘上取下部分腺垂体、卵巢、子宫,称重后快速、轻柔地投入预冷的光镜固定液内固定。
结果:1造模结果:(1)雷公藤致排卵障碍模型大鼠血清生殖激素与正常组比较,动情前、后E_2降低(均P<0.05)、动情前、后PRL降低(P<0.05,P<0.01),动情前、后LH升高(均P<0.01)动情后期T升高(P<0.05)、动情前期P升高(P<0.01)。卵巢子宫重量指数与正常组比较无变化(均P>0.05)。对垂体形态、卵巢形态、子宫形态均有轻度异常。(2)雄激素致无排卵模型大鼠血清生殖激素与正常组比较,动情前、后FSH降低(均P<0.01)、动情前期LH降低(P<0.05),动情前、后PRL升高(均P<0.01)、动情前、后T升高(均P<0.01)、动情前、后E_2升高(均P<0.01)。动情前、后卵巢重量指数、动情前、后子宫重量指数与正常组比较降低(均P<0.01)对垂体形态、卵巢形态、子宫形态均有异常。
2补肾法对两种模型大鼠HPOA的影响:(1)雷公藤致排卵障碍模型组补肾法治疗后,与模型组比较,补肾调经低组可使造模大鼠动情后期E_2增加(P<0.05),恢复至正常水平(P>0.05)。动情前期LH明显降低(P<0.01),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫恢复正常。补肾调经高组可使造模大鼠动情前期E_2明显增加(P<0.01),恢复至正常水平(P>0.05)。动情后期E_2明显增加(P<0.01),高于正常(P<0.05)。动情后期PRL增加(P<0.05),恢复至正常水平(P>0.05)。动情前期P明显降低(P<0.01),恢复至正常水平(P>0.05)。动情前、后LH降低(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情后期T降低(P<0.05),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。(2)雄激素致无排卵模型组补肾法治疗后,与模型组比较,补肾调经低组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情后期卵巢重量指数明显增加(P<0.01),恢复至正常水平(P>0.05)。动情后期子宫重量指数明显增加(P<0.01),恢复至正常水平(P>0.05)。动情前、后E_2降低(均P<0.05),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低,(均P<0.01),恢复至正常水平(P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体。补肾调经高组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情前期LH明显增加(P<0.01),恢复至正常水平(P>0.05)。动情前、后卵巢重量指数明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后子宫重量指数明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后E_2明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可使受损的卵巢、子宫、垂体基本恢复正常。
3疏肝法对两种模型大鼠HPOA的影响:(1)雷公藤致排卵障碍模型组疏肝法法治疗后,与模型组比较,逍遥丸低组可使造模大鼠动情前、后PRL明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前期LH降低(P<0.01),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。逍遥丸高组可使造模大鼠动情后期E_2增加(P<0.05),恢复至正常水平(P>0.05)。动情前、后PRL增加(均P<0.01),恢复至正常水平(均P>0.05)。降低造模大鼠动情前期P至正常水平(P>0.05)。动情前、后LH明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。(2)雄激素补肾法治疗后,与模型组比较,逍遥丸低组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。增加造模大鼠动情前期LH至正常(P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体形态。逍遥丸高组可使造模大鼠动情前期FSH明显增加(P<0.01),高于正常(P<0.05)。动情后期FSH增加(P<0.05),恢复至正常水平(P>0.05)。增加造模大鼠动情前期LH高于正常(P<0.05)。增加造模大鼠动情前、后子宫重量指数至正常水平(均P>0.05)。动情后期E_2降低(P<0.05),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体形态。
4补肾法、疏肝法对两种模型大鼠HPOA影响的比较:(1)雷公藤致排卵障碍模型组,补肾调经高组可使造模大鼠动情后期E_2高于逍遥丸高组(P<0.05),动情前期PRL低于逍遥丸高组(P<0.05),补肾调经低组动情后期E_2明显高于逍遥丸低组(P<0.01),动情后期LH高于逍遥丸低组(P<0.05),动情前期PRL低于逍遥丸低组(P<0.05),动情后期PRL低于逍遥丸低组(P<0.05)在改善卵巢形态方面,补肾调经高、低组卵泡数目,成熟卵泡直径,GC的层数,个数和排列致密程度要优于逍遥丸高、低组。(2)雄激素致无排卵模型组,补肾调经高组动情前、后卵巢重量指数明显高于逍遥丸高组(均P<0.01),动情后期子宫重量指数高于逍遥丸高组(P<0.05),补肾调经低组动情前期LH低于逍遥丸低组(P<0.05)。在改善垂体形态方面,逍遥丸高、低组腺垂体腺细胞数量、血窦数量要多于补肾调经高、低剂量组。在改善卵巢形态方面,补肾调经高组外形要粗于逍遥丸高组,卵巢卵泡膜细胞层薄于逍遥丸高组,卵泡数量、卵泡直径、黄体数量要高于逍遥丸高组,GC的层数、数量及排列致密度要高于逍遥丸高组。在改善子宫形态方面,补肾高、低组子宫肌层厚度、腺体的数量、内膜层柱状细胞的连续性和完整性要优于逍遥丸高、低组。
第二部分:补肾法、疏肝法对人卵巢颗粒细胞分泌功能的影响
目的:通过卵巢颗粒细胞培养,观察两法对卵巢颗粒细胞分泌功能的影响,探讨两法对卵巢自分泌系统的影响,研究排卵障碍的发生与卵巢颗粒细胞分泌功能的关系,比较补肾调经方与逍遥丸对人卵巢颗粒细胞分泌功能影响的异同,为补肾调经方与逍遥丸治疗排卵障碍性疾病的临床应用提供实验依据。
方法:取21只6周龄健康雌性大鼠按体重随机分为7组,空白对照组每日灌服生理盐水2ml/100g;补肾调经方组低、中、高剂量组和逍遥丸高、中、低剂量组分别给予补肾调经方和逍遥丸。各组均于末次给药后1小时大鼠股动脉采血。室温静置2h,3000r/min离心10min,取上清,56℃水浴30 min灭活补体,0.22μm无菌滤器过滤除菌、分装,-20℃保存备用。
选取年龄在20-30周岁,身体健康,因排卵障碍不孕就诊于河北医科大学中医学院门诊部的女性志愿者4名。B超监测未见成熟卵泡,并且排除卵巢,子宫器质性疾病,双侧输卵管造影结果显示为通畅,近三月未服用激素。分别口服补肾调经方和逍遥丸。连续服药3天,第4天清晨空腹服药(服药前禁食12h),一次服用全天剂量,于末次给药1小时后静脉取血5ml,室温静置2h,3000r/min离心10min,取上清,56℃水浴30 min灭活补体,0.22μm无菌滤器过滤除菌、分装。空白血清的制备:年龄在20-30周岁,身体健康,月经规律,B超监测有成熟卵泡,并且无卵巢,子宫器质性疾病的女性志愿者1名,清晨空腹静脉采血5ml,,其余同人含药血清制备。-20℃保存备用。
将从河北省第二附属医院辅助生殖科取来的含有颗粒细胞的卵泡液经2000r/min离心,弃上清液,沉淀颗粒细胞,用DMEM培养基洗涤后,经密度梯度为0.5Percoll悬液2500 r/min离心30min分离除去红细胞。加入0.25%的胰蛋白酶,37℃消化5-10min左右,加入含有10%胎牛血清和1640培养液终止消化。PBS冲洗,离心。经台盼兰染色细胞存活率85%-90%,用含有10%胎牛血清和1640培养液将颗粒细胞浓度调整为2×105的单细胞悬液,将单细胞悬液放入在24孔的的培养板,每板1ml,在5%的CO2,37℃孵箱中培养。24h后细胞贴壁。分别加入补肾调经方和逍遥丸人含药血清和大鼠含药血清制成高剂量(18%)、中剂量(12%)、低剂量(6%)处理细胞48h。设加正常人和大鼠血清者为空白对照。
结果:1补肾调经方含药血清对人黄素化颗粒细胞分泌功能的影响:(1)人含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,补肾调经高、中、低组E_2高于空白组(P<0.05, P<0.01, P<0.01)。补肾调经中组P明显低于空白组(P<0.01)。补肾调经中、高组cAMP明显高于空白组(均P<0.01)。两两比较,补肾调经高组E_2高于补肾调经中、低组(P<0.05, P<0.01),补肾调经中组E_2高于补肾调经低组(P<0.01)。补肾调经高、低组P高于补肾调经中组(P<0.05,P<0.01)。补肾调经中、高组cAMP明显高于补肾调经低组(均P<0.01)。②对FSHR表达的影响:蛋白印迹法:补肾调经高、中、低组FSHR表达均高于空白组,补肾调经高、中组FSHR表达要高于补肾调经低组。RT-PCR法:与空白组比较,补肾调经中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,补肾调经中FSHR值明显高于补肾调经高、低组(均P<0.01),补肾调经高组FSHR明显高于补肾调经低组(P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,补肾调经中、高组P450表达高于空白组(P<0.01,P<0.05)。补肾调经高、中、低组PPARγ表达低于空白组(均P<0.01)。两两比较,补肾调经中组P450表达明显高于补肾调经高、低组(均P<0.01)。补肾调经低、中组PPARγ表达明显低于补肾调经高组(均P<0.01)。(2)大鼠含药血清组,①对E_2、P、cAMP水平的影响:与空白组比较,补肾调经高、中组E_2高于空白组(均P<0.01)。补肾调经中组P明显低于空白组(P<0.01)。补肾调经中、高组cAMP明显高于空白组(P<0.05,P<0.01)。两两比较,补肾调经高、中组E_2高于补肾调经低组(均P<0.01)。补肾调经中组P低于补肾调经高、低组(P<0.05,P<0.01)。补肾调经高组cAMP高于补肾调经低组(P<0.05)。②对FSHR表达的影响:蛋白印迹法:补肾调经高、中、低组FSHR表达均高于空白组,补肾调经高、中组要高于补肾调经低组。RT-PCR法:与空白组比较,补肾调经中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,补肾调经中、高组FSHR表达明显高于补肾调经低组(均P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,补肾调经中、高组P450表达高于空白组(P<0.05,P<0.01)。补肾调经中组PPARγ表达明显低于空白组(P<0.01)。两两比较,补肾调经高组P450表达高于补肾调经低、中组(P<0.01,P<0.05)。补肾调经中组PPARγ表达明显低于补肾调经低、高组(P<0.05,P<0.01)。
2逍遥丸含药血清对人黄素化颗粒细胞分泌功能的影响(1)人含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,逍遥丸高、中、低组E_2高于空白组(均P<0.01)。逍遥丸高组P明显低于空白组(P<0.05)。逍遥丸低、中、高组cAMP明显高于空白组(均P<0.01)。两两比较,逍遥丸高组E_2高于逍遥丸低组(P<0.01)。逍遥丸高组cAMP明显高于逍遥丸中、低组(均P<0.01)。②对FSHR表达的影响:蛋白印迹法:逍遥丸高、中、低组表达均高于空白组,逍遥丸高组高于逍遥丸低、中组,逍遥丸中组高于逍遥丸低组。RT-PCR法:与空白组比较,逍遥丸中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,逍遥丸高组FSHR高于逍遥丸中、低组(均P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,逍遥丸中、高组P450表达明显高于空白组(P<0.01,P<0.05)。逍遥丸高、中、低组PPARγ表达低于空白组(P<0.01,P<0.01,P<0.05)。两两比较,逍遥丸高、中组P450表达明显高于逍遥丸低组(均P<0.01)。逍遥丸中、低组PPARγ表达低于逍遥丸高组(P<0.01,P<0.05)。(2)大鼠含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,逍遥丸高、中组E_2高于空白组( P<0.01, P<0.01,P<0.05)。逍遥丸高组P明显高于空白组(P<0.05)。逍遥丸中、高组cAMP明显高于空白组(均P<0.01)。两两比较,逍遥丸高组E_2高于逍遥丸低、中组(均P<0.01),逍遥丸中组E_2明显高于逍遥丸低组(P<0.01)。逍遥丸中、低组P明显低于逍遥丸高组(均P<0.01)。逍遥丸高组cAMP高于逍遥丸中、低组(P<0.05,P<0.01)。②对FSHR表达的影响。蛋白印迹法:逍遥丸高、中、低组表达均高于空白组,逍遥丸高组高于逍遥丸低、中组,逍遥丸中组高于逍遥丸低组。RT-PCR法:与空白组比较,逍遥丸高组FSHR表达明显高于空白组(P<0.01)。两两比较,逍遥丸高组高于逍遥丸中、低组(P<0.05,P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,逍遥丸高组P450表达高于空白组(P<0.05)。逍遥丸中组PPARγ表达明显低于空白组(P<0.01)。
3两种含药血清对人黄素化颗粒细胞分泌功能影响的比较:(1)人含药血清组:①对E_2、P、cAMP水平的影响:补肾调经低组E_2低于逍遥丸高组(P<0.01)。补肾调经中组E_2高于逍遥丸低组(P<0.05)。补肾调经高组E_2高于逍遥丸高、中、低组(均P<0.01)。补肾调经中组P低于逍遥丸中、低组(均P<0.01)。补肾调经低组cAMP低于逍遥丸高、中组(均P<0.01)。补肾调经中组cAMP高于逍遥丸低组(P<0.05),低于逍遥丸高组(P<0.01)。补肾调经高组cAMP低于逍遥丸高组(P<0.01)。②对FSHR表达的影响。蛋白印迹法:补肾调经高、中、低组FSHR的表达均高于相应的逍遥丸高、中、低组。且补肾调经低组FSHR的表达高于逍遥丸高组FSHR的表达。RT-PCR法:补肾调经低组FSHR的表达低于逍遥丸高、中组(P<0.05,P<0.01)。补肾调经中组FSHR的表达高于逍遥丸高、中、低组(均P<0.01)。补肾调经高组FSHR的表达高于逍遥丸低、中组(均P<0.01)。③对P450、PPARγ表达的影响:补肾调经低组P450低于逍遥丸中、高组(P<0.01,P<0.05)。补肾调经中组P450高于逍遥丸低组(P<0.01)。补肾调经高组P450高于逍遥丸中、低组(均P<0.05)。补肾调经低组PPARγ低于逍遥丸高组(P<0.05)。补肾调经中组PPARγ高于逍遥丸低组(P<0.05),低于逍遥丸高组(P<0.01)。补肾调经高组PPARγ高于逍遥丸中、低组(均P<0.01)。(2)大鼠含药血清组:①对E_2、P、cAMP水平的影响:补肾调经低组E_2低于逍遥丸高、中组(均P<0.01)。补肾调经中组E_2高于逍遥丸中、低组(P<0.05,P<0.01)。补肾调经高组E_2高于逍遥丸中、低组(均P<0.01)。补肾调经低组P低于逍遥丸高组(P<0.01)。补肾调经中组P低于逍遥丸中、高组(P<0.05,P<0.01)。补肾调经高组P低于逍遥丸高组(P<0.01)。补肾调经低组cAMP低于逍遥丸高、中组(均P<0.01)。补肾调经中组cAMP低于逍遥丸高组(P<0.01)。补肾调经高组cAMP低于逍遥丸高组(P<0.01)。②对FSHR表达的影响。蛋白印迹法:补肾调经中、低组人黄素化颗粒细胞FSHR的表达均高与相应的逍遥丸中、低组。且补肾调经中组人黄素化颗粒细胞FSHR的表达高于逍遥丸高、中、低组FSHR的表达。RT-PCR法:补肾调经低组FSHR的表达低于逍遥丸高组(P<0.05)。补肾调经中组FSHR的表达高于逍遥丸中、低组(P<0.05,P<0.01)。补肾调经高组FSHR的表达高于逍遥丸中、低组(均P<0.01)。③对P450、PPARγ表达的影响:补肾调经低组P450低于逍遥丸中、高组(P<0.01,P<0.05)。补肾调经中组P450高于逍遥丸低组(P<0.01)。补肾调经高组P450高于逍遥丸低、中、高组(均P<0.05)。补肾调经中组PPARγ低于逍遥丸低、高组(均P<0.05)。补肾调经高组PPARγ低于逍遥丸中组(P<0.05)。
结论:(1)两种排卵障碍模型血清生殖激素均紊乱,垂体、卵巢、子宫形态均有异常;雄激素致无排卵模型卵巢、子宫重量指数降低。雄激素致无排卵模型对卵巢、子宫形态及重量指数降低要重于雷公藤致排卵障碍模型。(2)补肾法、疏肝法通过改善垂体、卵巢、子宫形态,调节生殖内分泌激素水平,增加卵巢、子宫重量指数,发挥促进卵泡发育和成熟,治疗排卵障碍性疾病的作用。补肾法在改善卵巢、子宫形态,增加卵巢、子宫重量指数,调节卵巢分泌血清生殖激素方面优于疏肝法,疏肝法在改善垂体形态,调节垂体分泌血清生殖激素方面优于疏肝法。(3)补肾法、疏肝法可以改善人卵巢黄素化颗粒细胞分泌功能,调节卵巢的自分泌系统,达到改善卵巢功能的作用。补肾法在提高人卵巢黄素化颗粒细胞E_2、FSHR,降低PPARγ水平及表达方面优于疏肝法,疏肝法在提高人卵巢黄素化颗粒细胞cAMP水平方面法优于补肾法
Part one: Comparative study of the effect about Bushen treatment and Shugan treatment on the anovulatory model rats HPOA
Objective: Through the study of comparative Bushen method and Shugan method anovulation similarities and differences between the impact of rats HPOA to explore the Bushen method and Shugan method Treatment of ovulation disorders mechanism of action in order to enrich the theory of reproductive medicine and promote the development of traditional Chinese medicine gynecology .
Methods: 204 healthy female Wistar rats of 3 months old, weighing 180-250g were fed as well, randomly 170 of these rats were improved as the methods of Zheng Jia Yun, Liang Wenbo Inc. TWP-chip 35mg/kg/d (a dose was about an adult dose for clinical use 20 times, weighing every week, adjusting the delivery quantity), Gastric lavage method for medicine once a day continued 57 days. Making module two consecutive smears of vaginal epithelial cells in cycles estrous cycles became irregular, mainly the cycle was extended and not complete, suggesting that triptolide-induced rat models of dysfunction ovulation were made successfully. The remaining 34 rats were treated with the distilled water as a normal control group.
204 healthy female SD rats of 9-day age from the same batch, whose weight was about 15g, were adopted in our research. 170 of those rats were made as models by subcutaneous injection of testosterone 1.25mg/only (0.05ml/only) at nucha. All these 204 rats were weaned when they were 22-day old, which were raised by Special manager under free feeding and others same conditions. When they were 70-day old, their vaginals debouched. We began to make the smear of the vaginal epithelial cells for 2 successive sexual cycle for the model group, the vaginal epithelial cells showed continuously cutinization, which suggested us that models of anovulatory had been set up successfully. The remaining 170 rats were chosen randomly as normal group, each of them was injected 0.05ml neutral tea oil subcutaneously at nucha. Following the methods of Yu Jin etc.
The two batchs of model rats were randomly divided into five groups: the model group, the Bushentiaojing Recipe high-dose group, the Bushentiaojing Recipe low-dose group, the Xiaoyao Pill high-dose group, the Xiaoyao Pill low-dose group, each group was made up of 34 rats; the treatment groups were drenched with Bushentiaojing Recipe and Xiaoyao Pill, model groups and normal groups were drenched with distilled water once a day. Treatments continued for 5 weeks, and made the smear of vaginal epithelial cells for 2 successive sexual cycle. Prohibited feeding them within 8 hours after administration, each group were decapitated at proestrus and estrus periods. We take blood to determination E_2, P, FSH, LH, T, PRL, take out the part of the pituitary, ovarian and uterus tissues from those rats by Aseptic caesarean, soaking up blood, after weighing, fixed them in LM liquid.
Results: 1 Two models results: (1) Triptolide-induced ovulation disorder model reproductive hormones in serum compared with normal group, estrogen before and after E_2 decreased (all P<0.05), androgen cause anovulation model before and after PRL decrease (P<0.05, P<0.01), estrogen before and after LH increased (all P<0.01) metaestrus T increased (P<0.05), proestrus P increased (P<0.01). Ovarian and Uterus mass index did not change compared with normal group (all P>0.05). Pituitary morphology, ovarian morphology, uterine shape are mild injury. (2) Triptolide-induced ovulation disorder model in serum compared with normal group, estrogen before and after FSH decreased (all P<0.01), proestrus LH lower (P <0.05), estrogen before and after the PRL increase (all P<0.01), estrogen before and after T increased (both P<0.01), estrogen before and after E_2 increased (all P<0.01). Estrogen before and after ovarian index, estrogen before and after uterine index lower compared with the normal group (all P <0.01) on the pituitary morphology, ovarian morphology, uterine injury patterns.
2 The effect of Bushenfa Recipe on ovulation disorder HPOA of rats: (1) Triptolide-induced ovulation disorder model group, compared the model group with Bushentiaojing Recipe low group would enable model rats metaestrus increased E_2 metaestrus (P<0.05), restored to the normal level (P> 0.05). Proestrus LH decreased significantly (P<0.01), restored to the normal level (P>0.05). The pituitary, ovary, uterus damage can back to normal. Bushentiaojing Recipe high group would enable proestrus E_2 model rats increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus E_2 significantly increased (P <0.01), higher than normal (P<0.05). Metaestrus PRL increased (P<0.05), restored to the normal level (P>0.05). Proestrus P significantly decreased (P <0.01), restored to the normal level (P> 0.05). Estrus before and after the LH lower (P<0.01, P<0.05), restored to the normal level (all P>0.05). Metaestrus T reduced (P<0.05), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Bushentiaojing Recipe, compared with model group, Bushentiaojing Recipe low group would enable to regulate menstruation model rats with esturs before and after the FSH increased (P<0.01, P<0.05), restored to the the normal level (all P>0.05). Metaestrus ovarian index increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus uterine index increased significantly (P <0.01), restored to the normal level (P>0.05). Estrus before and after E_2 decreased (all P<0.05), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus and pituitary. Bushengtiaojing Recipe high group would enable model rats estrus before and after the FSH increased (P<0.01, P<0.05), restored to the normal level (all P>0.05). Proestrus LH significantly increased (P<0.01), restored to the normal level (P>0.05). Estrus before and after ovarian index was significantly increased (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after uterine index was significantly increased (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after E_2 decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged ovaries, uterus, pituitary basically can back to normal.
3 The effect of Shuganfa on HPOA of ovulation disorder rats: (1) Triptolide-induced ovulation disorder model group after shuganfa treatment, compared with model group, the Xiaoyao pill low group would enable model rats’PRL of estrus before and after increased significantly (all P<0.01), restored to the normal level (all P>0.05). Proestrus LH lower (P<0.01), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. Xiaoyao pill high group would enable model rats metaestrus E_2 increased (P<0.05), restored to the normal level (P> 0.05). Estrus before and after PRL increased (both P<0.01), restored to the normal level (all P>0.05). Reduced model rats proestrus P to a normal level (P>0.05). Estrus before and after the LH decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Shuganfa, compared with model group, the Xiaoyao pill low group would enable model rats estrus before and after FSH increased (P <0.01, P <0.05), restored to the normal level (all P>0.05). Increase in model rats proestrus LH to normal (P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape. Xiaoyao pill high group would enable model rats proestrus FSH increased significantly (P<0.01), higher than normal (P<0.05). Metaestrus FSH increased (P<0.05), restored to the normal level (P>0.05). Increase in model rats proestrus LH higher than normal (P<0.05). Increase in model rats with estrus before and after the uterine index to a normal level (all P>0.05). Metaestrus E_2 reduced (P<0.05), restored to the normal level (all P> 0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape.
4 Comparison of the effects of Bushenfa and Shuganfa on gonad axis in rats with ovulation disorder: (1) Triptolide-induced ovulation disorder model group, the Bushentiaojing Recipe high group to regulate model rats metaestrus E_2 higher than Xiaoyao pill group (P<0.05), proestrus PRL lower than Xiaoyao pill high group (P<0.05), the Bushentiaojing Recipe low group to regulate metaestrus E_2 was significantly higher than the Xiaoyao pill low group (P<0.01), higher than the Xiaoyao pill metaestrus LH the low group (P <0.05), proestrus PRL lower than Xiaoyao pill low group (P<0.05), metaestrus PRL lower than Xiaoyao pill low group (P<0.05).Improving in the ovarian morphology, the Bushentiaojing Recipe high and low group the number of follicles, mature follicle diameter, the number of GC storeys and level and dense arrangement better than Xiaoyao pill high and low groups. (2) Androgen-induced anovulation model group, the Bushentiaojing Recipe high group to regulate the ovarian index of estrus before and after was significantly higher than Xiaoyao pill high group (P<0.01), the metaestrus uterus index higher than Xiaoyao pill high group (P<0.05 ), the Bushentiaojing Recipe low group to regulate proestrus LH lower than Xiaoyao pill low group (P<0.05). Patterns to improve the pituitary, Xiaoyao pill high and low group of anterior pituitary gland cells, the number is greater than the Bushentiaojing Recipe high and low group. Improving in the ovarian morphology, the the Bushentiaojing Recipe high group have a higher rough profile than Xiaoyao pill low group, ovarian follicle cells thiner than the Xiaoyao pill high group, the number of follicles, follicle diameter and corpus luteum volume to be higher than Xiaoyao pill high group, the number of storeys, number and arrangement density of GC higher than Xiaoyao pill high group. Patterns in improving the uterus, the myometrial thickness, the number of glands, lining layer of columnar cells of continuity and integrity of the Bushentiaojing Recipe high and low group is superior to Xiaoyao pill high and low groups.
Part two: Comparative study of the effect about Bushen treatment and Shugan treatment people luteinized granulose cells secretory function
Objective: To explore the Bushen and Shugan treatment of ovulation disorders in the mechanism, through the ovarian granulosa cell cultures to observe the two method of ovarian granulosa cell function, and to explore two methods of ovarian secretion system of self-study of ovulation barriers to the occurrence of ovarian granulosa cell function and the relationship between the Bushentiaojing Recipeand and Xiaoyao pill compare pairs of ovarian granulosa cell secretion function of the similarities and differences between, for treatment of Bushentiaojing Recipeand and Xiaoyao pill ovulation disorders provide experimental basis for clinical application.
Methods: 21 6-week-old healthy female rats were randomly divided into 7 groups, and control group were fed daily with normal saline 2ml/100g; Bushentiaojing Recipeand group of low, middle and high-dose group and Xiaoyao pill high, middle and low-dose group were given Bushentiaojing Recipeand and Xiaoyao pill. In each group were taken at 1 hour after the last administration of rat femoral artery blood. Put it aside at room temperature 2h, 3000r/min centrifugal 10min, take the supernatant, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-loaded, -20℃to save backup.
Select the age of 20-30 years of age, physical health and treatment of infertility due to ovulatory dysfunction, Hebei Medical University School of Medicine clinics in the female volunteers 4. B- super-monitoring no mature follicle, and the exclusion of ovarian, uterine organic diseases, bilateral tubal patency imaging results showed that as the last in March did not take hormones. Each side oral Bushentiaojing Recipeand and Xiaoyao pill. Continuous medication 3 days, the first 4 days early morning empty stomach medication (medication before the fasting 12h), one taking a day dose, at 1 hour after the last administration venous blood taken 5ml, put it aside at room temperature 2h, 3000r/min centrifugal 10min, taking on the Ching, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-installed. Preparation of blank serum: the age of 20-30 years of age, physical health, menstrual laws, B super-monitoring of mature follicles, and no ovaries, uterus organic diseases of female volunteers, an early morning fasting venous blood 5ml, the rest of my colleagues Preparation of drug-containing serum. -20℃to save backup.
From the Second Affiliated Hospital of Hebei Province, assisted reproductive branches fetch the follicular fluid containing granulosa cells by 2000r/min centrifuge, discard supernatant, precipitation granulosa cells, after washing with DMEM medium by density gradient 0.5Percoll suspension 2500 r / min centrifugation to remove red blood cells separated 30min. Adding 0.25% trypsin, 37℃to digest 5-10min or so, to join with 10% fetal bovine serum and the 1640 termination of digestion. PBS washing, centrifugation. After trypan blue staining cells in 85% -90% survival, with 1640 containing 10% fetal bovine serum and the concentration of granule cell culture medium will be adjusted to 2×105 single-cell suspension, single-cell suspension into a 24-hole of the culture plate, each plate 1ml, at 5% CO2, 37℃incubation box training. 24h of cell adhesion. Were added to Bushentiaojing recipeand and Xiaoyao pill containing human serum and rat serum is made with 6%, 12%, 18%, high, medium and low-dose-treated cells 48h. Canadian-based normal and control rat serum were blank.
Results: 1 Bushentiaojing Recipeand containing serum on human luteinized granulosa cell function: (1) Human containing serum groups:①E_2, P, cAMP levels of impact: compared with the blank group, Bushentiaojing high, medium, Low group E_2 are higher than control group (P<0.05, P<0.01, P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group is significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group E_2 is higher than Bushentiaojing medium, low group (P <0.05, P<0.01), Bushentiaojing medium group E_2 is higher than the low group (P<0.01). Bushentiaojing high and low group P is higher than medium group (P<0.05, P<0.01). Bushentiaojing middle and high group cAMP are significantly higher than low group (P<0.01).②FSHR expression: Protein blotting: Bushentiaojing high, medium and low groups FSHR expression are higher than control group, Bushentiaojing high, medium group of FSHR expression are higher than low group. RT-PCR method: comparison with the control group, Bushentiaojing middle and high group FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium group FSHR is significantly higher than high, low group (all P<0.01), Bushentiaojing high group FSHR is significantly higher than low group (P<0.01).③P450, PPARγexpression: compared with the control group, kidney regulate menstruation, the high expression group of P450 is higher than the control group (P<0.01, P<0.05). Bushentiaojing high, medium and low groups PPARγexpression are lower than control group (all P <0.01). Pairwise comparison, Bushentiaojing high group of P450 expression is significantly higher than that low group (all P<0.01). Bushentiaojing low, medium group of PPARγexpression are significantly lower than the Bushentiaojing high group (all P<0.01). (2) rats containing serum group①E_2, P, cAMP levels of impact: compared with the control group, Bushentiaojing high, medium group E_2 are higher than control group (all P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group are significantly higher than control group (P<0.05, P<0.01). Pairwise comparison, Bushentiaojing high, medium group E_2 are higher than Bushentiaojing low group (all P <0.01). Bushentiaojing medium group P is lower than low group (P<0.05, P <0.01). Bushentiaojing high group cAMP is higher than low group (P<0.05).②FSHR expression: Protein blotting: Bushentiaojing high, medium and low group FSHR expression are higher than control group. Bushentiaojing high, middle group are higher than low group. RT-PCR method: comparison with control group, Bushentiaojing middle and high groups FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium, high group FSHR are sign ificantly higher than low group (all P<0.01).③P450, PPARγexpression: compared with control group, Bushentiaojing medium, high group P450 expression are higher than the control group (P<0.05, P<0.01). Bushentiaojing medium group PPARγexpression are significantly lower than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group P450 expression is higher than low group (P<0.01, P<0.05). Bushentiaojing medium group PPARγexpression is lower than low and high group (P <0.05, P <0.01).
2 Xiaoyao pill containing serum on human luteinized granulosa cell function .(1) Human containing serum groups:①E_2, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, medium and low groups E_2 are higher than control group (all P<0.01). Xiaoyao pill high group P is significantly lower than control group (P<0.05). Xiaoyao pill low, medium and high group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E_2 is higher than low group (P<0.01). Xiaoyao pill medium group cAMP significantly higher than high group and low group (all P<0.01).②FSHR expression: Protein blotting: Xiaoyao pill high, medium and low expression groups are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill medium group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill FSHR is higher than the high group and low group (all P<0.01).③P450, PPARγexpression: comparison with control group, Xiaoyao pill medium high group expression P450 expression is significantly higher than control group (P <0.01, P <0.05). Xiaoyao pill high, medium and low group PPARγexpression are lower than control group (P <0.01, P<0.01, P<0.05). Pairwise comparison, Xiaoyao pill high, medium group of P450 expression are significantly higher than the low group (all P<0.01). Xiaoyao pill low group PPARγexpression is lower than the medium,high group (P <0.01, P<0.05). (2) rats containing serum groups:①pairs of E_2, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, E_2 are higher than the control group (P<0.01, P<0.01, P<0.05) . Xiaoyao pill high group P is higher than control group (P<0.05). Xiaoyao pill high, medium group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E_2 is higher than low, medium group (all P<0.01), Xiaoyao pill medium group E_2 is significantly higher than low group (P<0.01). Xiaoyao pill medium, low group P are significantly lower than high group (all P<0.01). Xiaoyao pill high group cAMP is higher than medium, low group (P<0.05, P<0.01).②FSHR expression. Western blotting: Xiaoyao pill high, medium and low groups expression are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill high group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (P<0.01). Pairwise comparison, Xiaoyao pill high group is higher than medium , low group (P<0.05, P<0.01).③P450, PPARγexpression: compared with control group, Xiaoyao pill high group P450 expression is higher than control group (P<0.05). Xiaoyao pill medium group PPARγexpression is significantly lower than that control group (P <0.01).
3 Two kinds of drug-containing serum on human luteinized granulosa cell function affect the comparison of: (1) Human containing serum groups:①E_2, P, cAMP levels of impact: Bushentiaojing low group E_2 is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group E_2 is higher than Xiaoyao pill low group (P<0.05). Bushentiaojing high group E_2 is higher than Xiaoyao pill high, medium and low group (all P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill low group (P<0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium groups cAMP is higher than Xiaoyao pill low group (P<0.05), lower than Xiaoyao pill high group (P <0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P<0.01).②FSHR. Western blotting: Bushentiaojing high, medium and low groups expression of FSHR are higher than Xiaoyao pill high, medium and low groups, and Bushentiaojing low group FSHR expression is higher Xiaoyao pill high group. RT-PCR method: Bushentiaojing low group FSHR is lower than Xiaoyao pill high, medium group (P<0.05,P<0.01). Bushentiaojing medium group FSHR is higher than Xiaoyao pill high, medium and low group (all P <0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low, medium group (all P<0.01).③P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P <0.01, P <0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing medium group P450 expression is higher than Xiaoyao pill high, low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05), lower than the Xiaoyao pill high group (P<0.01). Bushentiaojing high group PPARγis higher than Xiaoyao pill low group (P<0.01). (2) rats containing serum groups:①E_2, P, cAMP levels of impact: Bushentiaojing low group E_2 is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium group E_2 is higher than Xiaoyao pill medium, low group (P<0.05, P<0.01). Bushentiaojing high group E_2 is higher than Xiaoyao pill high ,low group (all P<0.01). Bushentiaojing low group P is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill medium, high group (P<0.05,P<0.01). Bushentiaojing high group P ia lower than Xiaoyao pill high group (P <0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, (all P<0.01). Bushentiaojing medium group cAMP is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P <0.01).②FSHR expression. Western blotting: Bushentiaojing middle, low groups FSHR are higher than Xiaoyao pill high, low group. Bushentiaojing medium group is higher than Xiaoyao pill high, medium and low groups. RT-PCR method: Bushentiaojing FSHR low group is higher than Xiaoyao pill high group (P<0.05). Bushentiaojing medium group FSHR is higher than Xiaoyao pill medium, low group (P<0.05,P<0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low group (P<0.01).③P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P<0.01,P<0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing high group P450 is higher than Xiaoyao pill low, medium and high group(all P<0.05). Bushentiaojing medium group PPARγis ower than Xiaoyao pill low, high group (all P<0.05). Bushentiaojing high group PPARγis lower than Xiaoyao pill medium group (P<0.05).
Conclusions: 1 two kinds of ovulation disorder models are serum reproductive hormone disorders, pituitary, ovarian, uterine morphology are abnormal; Triptolide-induced ovulation disorder model rats ovaries, uterus weight index are lower, Androgen-induced ovulation model rats ovarian, uterine shape and weight index is more severe than Triptolide-induced ovulation disorder model. 2 Bushen and Shugan method both by improve the pituitary gland, ovarian, uterine shape, regulating reproductive endocrine hormone levels, increase ovary, uterus weight index, to play the promotion of follicular development and maturation, ovulation disorders treatment effect. Bushen method in improving the ovarian, uterine morphology, increased ovary, uterus weight index, regulation of ovarian secretion of reproductive hormones is better than Shugan, Shugan method improve pituitary morphology, regulation of pituitary secretion of reproductive hormones are better than Bushen. 3 Bushen, Shugan method can improve human luteinized granulosa cell function, self-regulation of ovarian secretion system, improve ovarian function. Bushen method in improve E_2, FSHR, reduce PPARγis better than Shugan, Sugan method improve cAMP is better than Bushen.
目的:通过研究比较补肾法与疏肝法对排卵障碍模型大鼠HPOA影响的异同,探讨补肾法、疏肝法治疗排卵障碍性疾病的作用机制,以丰富中医生殖理论,促进中医妇科学的发展。
方法:模型一:选用同批出生9日龄健康雌性SD大鼠204只,体重约15g。随机取170只大鼠,于颈背部皮下一次性注射丙酸睾丸酮1.25mg/只(0.05ml/只)造模。第22日龄断奶,自由进食,同条件饲养。第70日龄阴道开口,造模组连续阴道上皮细胞涂片两个性周期(一个性周期4-5天),阴道上皮细胞持续角化,提示雄激素致排卵障碍大鼠模型造模成功。余34只,于颈背部皮下一次性注射中性茶油0.05ml作为正常对照组。
模型二:选用3月龄健康雌性Wistar大鼠204只,体重180~250g。随机取170只大鼠改进郑家润,梁文波等方法。雷公藤多甙片35mg/kg/d(给药剂量为临床成人用量的20倍),灌胃法给药,连续57天。造模组连续阴道上皮细胞涂片两个性周期,动情周期转为不规则,主要为周期不完整及周期延长,提示雷公藤致排卵障碍大鼠模型造模成功。其余34只灌胃给蒸馏水作为正常对照组。
将两种排卵障碍成功模型大鼠分别随机分为模型组和治疗组,治疗组包括:补肾调经方高剂量组、补肾调经方低剂量组、逍遥丸高剂量组、逍遥丸低剂量组,每组分别各34只;治疗组分别灌服补肾调经方与逍遥丸,模型组和正常组灌服蒸馏水,每日1次。灌胃给药5周,并阴道细胞涂片两个性周期。最后一次给药后禁食8小时,选择相同动情周期(动情前期、动情后期)断头取血,制备血浆、血清,放免法检测血浆卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)、孕酮(P)、睾酮(T)、催乳素(PRL)。同时立即无菌操作,在冰盘上取下部分腺垂体、卵巢、子宫,称重后快速、轻柔地投入预冷的光镜固定液内固定。
结果:1造模结果:(1)雷公藤致排卵障碍模型大鼠血清生殖激素与正常组比较,动情前、后E_2降低(均P<0.05)、动情前、后PRL降低(P<0.05,P<0.01),动情前、后LH升高(均P<0.01)动情后期T升高(P<0.05)、动情前期P升高(P<0.01)。卵巢子宫重量指数与正常组比较无变化(均P>0.05)。对垂体形态、卵巢形态、子宫形态均有轻度异常。(2)雄激素致无排卵模型大鼠血清生殖激素与正常组比较,动情前、后FSH降低(均P<0.01)、动情前期LH降低(P<0.05),动情前、后PRL升高(均P<0.01)、动情前、后T升高(均P<0.01)、动情前、后E_2升高(均P<0.01)。动情前、后卵巢重量指数、动情前、后子宫重量指数与正常组比较降低(均P<0.01)对垂体形态、卵巢形态、子宫形态均有异常。
2补肾法对两种模型大鼠HPOA的影响:(1)雷公藤致排卵障碍模型组补肾法治疗后,与模型组比较,补肾调经低组可使造模大鼠动情后期E_2增加(P<0.05),恢复至正常水平(P>0.05)。动情前期LH明显降低(P<0.01),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫恢复正常。补肾调经高组可使造模大鼠动情前期E_2明显增加(P<0.01),恢复至正常水平(P>0.05)。动情后期E_2明显增加(P<0.01),高于正常(P<0.05)。动情后期PRL增加(P<0.05),恢复至正常水平(P>0.05)。动情前期P明显降低(P<0.01),恢复至正常水平(P>0.05)。动情前、后LH降低(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情后期T降低(P<0.05),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。(2)雄激素致无排卵模型组补肾法治疗后,与模型组比较,补肾调经低组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情后期卵巢重量指数明显增加(P<0.01),恢复至正常水平(P>0.05)。动情后期子宫重量指数明显增加(P<0.01),恢复至正常水平(P>0.05)。动情前、后E_2降低(均P<0.05),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低,(均P<0.01),恢复至正常水平(P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体。补肾调经高组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。动情前期LH明显增加(P<0.01),恢复至正常水平(P>0.05)。动情前、后卵巢重量指数明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后子宫重量指数明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后E_2明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可使受损的卵巢、子宫、垂体基本恢复正常。
3疏肝法对两种模型大鼠HPOA的影响:(1)雷公藤致排卵障碍模型组疏肝法法治疗后,与模型组比较,逍遥丸低组可使造模大鼠动情前、后PRL明显增加(均P<0.01),恢复至正常水平(均P>0.05)。动情前期LH降低(P<0.01),恢复至正常水平(P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。逍遥丸高组可使造模大鼠动情后期E_2增加(P<0.05),恢复至正常水平(P>0.05)。动情前、后PRL增加(均P<0.01),恢复至正常水平(均P>0.05)。降低造模大鼠动情前期P至正常水平(P>0.05)。动情前、后LH明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可使受损的垂体、卵巢、子宫形态恢复正常。(2)雄激素补肾法治疗后,与模型组比较,逍遥丸低组可使造模大鼠动情前、后FSH增加(P<0.01,P<0.05),恢复至正常水平(均P>0.05)。增加造模大鼠动情前期LH至正常(P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体形态。逍遥丸高组可使造模大鼠动情前期FSH明显增加(P<0.01),高于正常(P<0.05)。动情后期FSH增加(P<0.05),恢复至正常水平(P>0.05)。增加造模大鼠动情前期LH高于正常(P<0.05)。增加造模大鼠动情前、后子宫重量指数至正常水平(均P>0.05)。动情后期E_2降低(P<0.05),恢复至正常水平(均P>0.05)。动情前、后PRL明显降低(均P<0.01),恢复至正常水平(均P>0.05)。动情前、后T明显降低(均P<0.01),恢复至正常水平(均P>0.05)。可改善受损的卵巢、子宫、垂体形态。
4补肾法、疏肝法对两种模型大鼠HPOA影响的比较:(1)雷公藤致排卵障碍模型组,补肾调经高组可使造模大鼠动情后期E_2高于逍遥丸高组(P<0.05),动情前期PRL低于逍遥丸高组(P<0.05),补肾调经低组动情后期E_2明显高于逍遥丸低组(P<0.01),动情后期LH高于逍遥丸低组(P<0.05),动情前期PRL低于逍遥丸低组(P<0.05),动情后期PRL低于逍遥丸低组(P<0.05)在改善卵巢形态方面,补肾调经高、低组卵泡数目,成熟卵泡直径,GC的层数,个数和排列致密程度要优于逍遥丸高、低组。(2)雄激素致无排卵模型组,补肾调经高组动情前、后卵巢重量指数明显高于逍遥丸高组(均P<0.01),动情后期子宫重量指数高于逍遥丸高组(P<0.05),补肾调经低组动情前期LH低于逍遥丸低组(P<0.05)。在改善垂体形态方面,逍遥丸高、低组腺垂体腺细胞数量、血窦数量要多于补肾调经高、低剂量组。在改善卵巢形态方面,补肾调经高组外形要粗于逍遥丸高组,卵巢卵泡膜细胞层薄于逍遥丸高组,卵泡数量、卵泡直径、黄体数量要高于逍遥丸高组,GC的层数、数量及排列致密度要高于逍遥丸高组。在改善子宫形态方面,补肾高、低组子宫肌层厚度、腺体的数量、内膜层柱状细胞的连续性和完整性要优于逍遥丸高、低组。
第二部分:补肾法、疏肝法对人卵巢颗粒细胞分泌功能的影响
目的:通过卵巢颗粒细胞培养,观察两法对卵巢颗粒细胞分泌功能的影响,探讨两法对卵巢自分泌系统的影响,研究排卵障碍的发生与卵巢颗粒细胞分泌功能的关系,比较补肾调经方与逍遥丸对人卵巢颗粒细胞分泌功能影响的异同,为补肾调经方与逍遥丸治疗排卵障碍性疾病的临床应用提供实验依据。
方法:取21只6周龄健康雌性大鼠按体重随机分为7组,空白对照组每日灌服生理盐水2ml/100g;补肾调经方组低、中、高剂量组和逍遥丸高、中、低剂量组分别给予补肾调经方和逍遥丸。各组均于末次给药后1小时大鼠股动脉采血。室温静置2h,3000r/min离心10min,取上清,56℃水浴30 min灭活补体,0.22μm无菌滤器过滤除菌、分装,-20℃保存备用。
选取年龄在20-30周岁,身体健康,因排卵障碍不孕就诊于河北医科大学中医学院门诊部的女性志愿者4名。B超监测未见成熟卵泡,并且排除卵巢,子宫器质性疾病,双侧输卵管造影结果显示为通畅,近三月未服用激素。分别口服补肾调经方和逍遥丸。连续服药3天,第4天清晨空腹服药(服药前禁食12h),一次服用全天剂量,于末次给药1小时后静脉取血5ml,室温静置2h,3000r/min离心10min,取上清,56℃水浴30 min灭活补体,0.22μm无菌滤器过滤除菌、分装。空白血清的制备:年龄在20-30周岁,身体健康,月经规律,B超监测有成熟卵泡,并且无卵巢,子宫器质性疾病的女性志愿者1名,清晨空腹静脉采血5ml,,其余同人含药血清制备。-20℃保存备用。
将从河北省第二附属医院辅助生殖科取来的含有颗粒细胞的卵泡液经2000r/min离心,弃上清液,沉淀颗粒细胞,用DMEM培养基洗涤后,经密度梯度为0.5Percoll悬液2500 r/min离心30min分离除去红细胞。加入0.25%的胰蛋白酶,37℃消化5-10min左右,加入含有10%胎牛血清和1640培养液终止消化。PBS冲洗,离心。经台盼兰染色细胞存活率85%-90%,用含有10%胎牛血清和1640培养液将颗粒细胞浓度调整为2×105的单细胞悬液,将单细胞悬液放入在24孔的的培养板,每板1ml,在5%的CO2,37℃孵箱中培养。24h后细胞贴壁。分别加入补肾调经方和逍遥丸人含药血清和大鼠含药血清制成高剂量(18%)、中剂量(12%)、低剂量(6%)处理细胞48h。设加正常人和大鼠血清者为空白对照。
结果:1补肾调经方含药血清对人黄素化颗粒细胞分泌功能的影响:(1)人含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,补肾调经高、中、低组E_2高于空白组(P<0.05, P<0.01, P<0.01)。补肾调经中组P明显低于空白组(P<0.01)。补肾调经中、高组cAMP明显高于空白组(均P<0.01)。两两比较,补肾调经高组E_2高于补肾调经中、低组(P<0.05, P<0.01),补肾调经中组E_2高于补肾调经低组(P<0.01)。补肾调经高、低组P高于补肾调经中组(P<0.05,P<0.01)。补肾调经中、高组cAMP明显高于补肾调经低组(均P<0.01)。②对FSHR表达的影响:蛋白印迹法:补肾调经高、中、低组FSHR表达均高于空白组,补肾调经高、中组FSHR表达要高于补肾调经低组。RT-PCR法:与空白组比较,补肾调经中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,补肾调经中FSHR值明显高于补肾调经高、低组(均P<0.01),补肾调经高组FSHR明显高于补肾调经低组(P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,补肾调经中、高组P450表达高于空白组(P<0.01,P<0.05)。补肾调经高、中、低组PPARγ表达低于空白组(均P<0.01)。两两比较,补肾调经中组P450表达明显高于补肾调经高、低组(均P<0.01)。补肾调经低、中组PPARγ表达明显低于补肾调经高组(均P<0.01)。(2)大鼠含药血清组,①对E_2、P、cAMP水平的影响:与空白组比较,补肾调经高、中组E_2高于空白组(均P<0.01)。补肾调经中组P明显低于空白组(P<0.01)。补肾调经中、高组cAMP明显高于空白组(P<0.05,P<0.01)。两两比较,补肾调经高、中组E_2高于补肾调经低组(均P<0.01)。补肾调经中组P低于补肾调经高、低组(P<0.05,P<0.01)。补肾调经高组cAMP高于补肾调经低组(P<0.05)。②对FSHR表达的影响:蛋白印迹法:补肾调经高、中、低组FSHR表达均高于空白组,补肾调经高、中组要高于补肾调经低组。RT-PCR法:与空白组比较,补肾调经中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,补肾调经中、高组FSHR表达明显高于补肾调经低组(均P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,补肾调经中、高组P450表达高于空白组(P<0.05,P<0.01)。补肾调经中组PPARγ表达明显低于空白组(P<0.01)。两两比较,补肾调经高组P450表达高于补肾调经低、中组(P<0.01,P<0.05)。补肾调经中组PPARγ表达明显低于补肾调经低、高组(P<0.05,P<0.01)。
2逍遥丸含药血清对人黄素化颗粒细胞分泌功能的影响(1)人含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,逍遥丸高、中、低组E_2高于空白组(均P<0.01)。逍遥丸高组P明显低于空白组(P<0.05)。逍遥丸低、中、高组cAMP明显高于空白组(均P<0.01)。两两比较,逍遥丸高组E_2高于逍遥丸低组(P<0.01)。逍遥丸高组cAMP明显高于逍遥丸中、低组(均P<0.01)。②对FSHR表达的影响:蛋白印迹法:逍遥丸高、中、低组表达均高于空白组,逍遥丸高组高于逍遥丸低、中组,逍遥丸中组高于逍遥丸低组。RT-PCR法:与空白组比较,逍遥丸中、高组FSHR表达明显高于空白组(均P<0.01)。两两比较,逍遥丸高组FSHR高于逍遥丸中、低组(均P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,逍遥丸中、高组P450表达明显高于空白组(P<0.01,P<0.05)。逍遥丸高、中、低组PPARγ表达低于空白组(P<0.01,P<0.01,P<0.05)。两两比较,逍遥丸高、中组P450表达明显高于逍遥丸低组(均P<0.01)。逍遥丸中、低组PPARγ表达低于逍遥丸高组(P<0.01,P<0.05)。(2)大鼠含药血清组:①对E_2、P、cAMP水平的影响:与空白组比较,逍遥丸高、中组E_2高于空白组( P<0.01, P<0.01,P<0.05)。逍遥丸高组P明显高于空白组(P<0.05)。逍遥丸中、高组cAMP明显高于空白组(均P<0.01)。两两比较,逍遥丸高组E_2高于逍遥丸低、中组(均P<0.01),逍遥丸中组E_2明显高于逍遥丸低组(P<0.01)。逍遥丸中、低组P明显低于逍遥丸高组(均P<0.01)。逍遥丸高组cAMP高于逍遥丸中、低组(P<0.05,P<0.01)。②对FSHR表达的影响。蛋白印迹法:逍遥丸高、中、低组表达均高于空白组,逍遥丸高组高于逍遥丸低、中组,逍遥丸中组高于逍遥丸低组。RT-PCR法:与空白组比较,逍遥丸高组FSHR表达明显高于空白组(P<0.01)。两两比较,逍遥丸高组高于逍遥丸中、低组(P<0.05,P<0.01)。③对P450、PPARγ表达的影响:与空白组比较,逍遥丸高组P450表达高于空白组(P<0.05)。逍遥丸中组PPARγ表达明显低于空白组(P<0.01)。
3两种含药血清对人黄素化颗粒细胞分泌功能影响的比较:(1)人含药血清组:①对E_2、P、cAMP水平的影响:补肾调经低组E_2低于逍遥丸高组(P<0.01)。补肾调经中组E_2高于逍遥丸低组(P<0.05)。补肾调经高组E_2高于逍遥丸高、中、低组(均P<0.01)。补肾调经中组P低于逍遥丸中、低组(均P<0.01)。补肾调经低组cAMP低于逍遥丸高、中组(均P<0.01)。补肾调经中组cAMP高于逍遥丸低组(P<0.05),低于逍遥丸高组(P<0.01)。补肾调经高组cAMP低于逍遥丸高组(P<0.01)。②对FSHR表达的影响。蛋白印迹法:补肾调经高、中、低组FSHR的表达均高于相应的逍遥丸高、中、低组。且补肾调经低组FSHR的表达高于逍遥丸高组FSHR的表达。RT-PCR法:补肾调经低组FSHR的表达低于逍遥丸高、中组(P<0.05,P<0.01)。补肾调经中组FSHR的表达高于逍遥丸高、中、低组(均P<0.01)。补肾调经高组FSHR的表达高于逍遥丸低、中组(均P<0.01)。③对P450、PPARγ表达的影响:补肾调经低组P450低于逍遥丸中、高组(P<0.01,P<0.05)。补肾调经中组P450高于逍遥丸低组(P<0.01)。补肾调经高组P450高于逍遥丸中、低组(均P<0.05)。补肾调经低组PPARγ低于逍遥丸高组(P<0.05)。补肾调经中组PPARγ高于逍遥丸低组(P<0.05),低于逍遥丸高组(P<0.01)。补肾调经高组PPARγ高于逍遥丸中、低组(均P<0.01)。(2)大鼠含药血清组:①对E_2、P、cAMP水平的影响:补肾调经低组E_2低于逍遥丸高、中组(均P<0.01)。补肾调经中组E_2高于逍遥丸中、低组(P<0.05,P<0.01)。补肾调经高组E_2高于逍遥丸中、低组(均P<0.01)。补肾调经低组P低于逍遥丸高组(P<0.01)。补肾调经中组P低于逍遥丸中、高组(P<0.05,P<0.01)。补肾调经高组P低于逍遥丸高组(P<0.01)。补肾调经低组cAMP低于逍遥丸高、中组(均P<0.01)。补肾调经中组cAMP低于逍遥丸高组(P<0.01)。补肾调经高组cAMP低于逍遥丸高组(P<0.01)。②对FSHR表达的影响。蛋白印迹法:补肾调经中、低组人黄素化颗粒细胞FSHR的表达均高与相应的逍遥丸中、低组。且补肾调经中组人黄素化颗粒细胞FSHR的表达高于逍遥丸高、中、低组FSHR的表达。RT-PCR法:补肾调经低组FSHR的表达低于逍遥丸高组(P<0.05)。补肾调经中组FSHR的表达高于逍遥丸中、低组(P<0.05,P<0.01)。补肾调经高组FSHR的表达高于逍遥丸中、低组(均P<0.01)。③对P450、PPARγ表达的影响:补肾调经低组P450低于逍遥丸中、高组(P<0.01,P<0.05)。补肾调经中组P450高于逍遥丸低组(P<0.01)。补肾调经高组P450高于逍遥丸低、中、高组(均P<0.05)。补肾调经中组PPARγ低于逍遥丸低、高组(均P<0.05)。补肾调经高组PPARγ低于逍遥丸中组(P<0.05)。
结论:(1)两种排卵障碍模型血清生殖激素均紊乱,垂体、卵巢、子宫形态均有异常;雄激素致无排卵模型卵巢、子宫重量指数降低。雄激素致无排卵模型对卵巢、子宫形态及重量指数降低要重于雷公藤致排卵障碍模型。(2)补肾法、疏肝法通过改善垂体、卵巢、子宫形态,调节生殖内分泌激素水平,增加卵巢、子宫重量指数,发挥促进卵泡发育和成熟,治疗排卵障碍性疾病的作用。补肾法在改善卵巢、子宫形态,增加卵巢、子宫重量指数,调节卵巢分泌血清生殖激素方面优于疏肝法,疏肝法在改善垂体形态,调节垂体分泌血清生殖激素方面优于疏肝法。(3)补肾法、疏肝法可以改善人卵巢黄素化颗粒细胞分泌功能,调节卵巢的自分泌系统,达到改善卵巢功能的作用。补肾法在提高人卵巢黄素化颗粒细胞E_2、FSHR,降低PPARγ水平及表达方面优于疏肝法,疏肝法在提高人卵巢黄素化颗粒细胞cAMP水平方面法优于补肾法
Part one: Comparative study of the effect about Bushen treatment and Shugan treatment on the anovulatory model rats HPOA
Objective: Through the study of comparative Bushen method and Shugan method anovulation similarities and differences between the impact of rats HPOA to explore the Bushen method and Shugan method Treatment of ovulation disorders mechanism of action in order to enrich the theory of reproductive medicine and promote the development of traditional Chinese medicine gynecology .
Methods: 204 healthy female Wistar rats of 3 months old, weighing 180-250g were fed as well, randomly 170 of these rats were improved as the methods of Zheng Jia Yun, Liang Wenbo Inc. TWP-chip 35mg/kg/d (a dose was about an adult dose for clinical use 20 times, weighing every week, adjusting the delivery quantity), Gastric lavage method for medicine once a day continued 57 days. Making module two consecutive smears of vaginal epithelial cells in cycles estrous cycles became irregular, mainly the cycle was extended and not complete, suggesting that triptolide-induced rat models of dysfunction ovulation were made successfully. The remaining 34 rats were treated with the distilled water as a normal control group.
204 healthy female SD rats of 9-day age from the same batch, whose weight was about 15g, were adopted in our research. 170 of those rats were made as models by subcutaneous injection of testosterone 1.25mg/only (0.05ml/only) at nucha. All these 204 rats were weaned when they were 22-day old, which were raised by Special manager under free feeding and others same conditions. When they were 70-day old, their vaginals debouched. We began to make the smear of the vaginal epithelial cells for 2 successive sexual cycle for the model group, the vaginal epithelial cells showed continuously cutinization, which suggested us that models of anovulatory had been set up successfully. The remaining 170 rats were chosen randomly as normal group, each of them was injected 0.05ml neutral tea oil subcutaneously at nucha. Following the methods of Yu Jin etc.
The two batchs of model rats were randomly divided into five groups: the model group, the Bushentiaojing Recipe high-dose group, the Bushentiaojing Recipe low-dose group, the Xiaoyao Pill high-dose group, the Xiaoyao Pill low-dose group, each group was made up of 34 rats; the treatment groups were drenched with Bushentiaojing Recipe and Xiaoyao Pill, model groups and normal groups were drenched with distilled water once a day. Treatments continued for 5 weeks, and made the smear of vaginal epithelial cells for 2 successive sexual cycle. Prohibited feeding them within 8 hours after administration, each group were decapitated at proestrus and estrus periods. We take blood to determination E_2, P, FSH, LH, T, PRL, take out the part of the pituitary, ovarian and uterus tissues from those rats by Aseptic caesarean, soaking up blood, after weighing, fixed them in LM liquid.
Results: 1 Two models results: (1) Triptolide-induced ovulation disorder model reproductive hormones in serum compared with normal group, estrogen before and after E_2 decreased (all P<0.05), androgen cause anovulation model before and after PRL decrease (P<0.05, P<0.01), estrogen before and after LH increased (all P<0.01) metaestrus T increased (P<0.05), proestrus P increased (P<0.01). Ovarian and Uterus mass index did not change compared with normal group (all P>0.05). Pituitary morphology, ovarian morphology, uterine shape are mild injury. (2) Triptolide-induced ovulation disorder model in serum compared with normal group, estrogen before and after FSH decreased (all P<0.01), proestrus LH lower (P <0.05), estrogen before and after the PRL increase (all P<0.01), estrogen before and after T increased (both P<0.01), estrogen before and after E_2 increased (all P<0.01). Estrogen before and after ovarian index, estrogen before and after uterine index lower compared with the normal group (all P <0.01) on the pituitary morphology, ovarian morphology, uterine injury patterns.
2 The effect of Bushenfa Recipe on ovulation disorder HPOA of rats: (1) Triptolide-induced ovulation disorder model group, compared the model group with Bushentiaojing Recipe low group would enable model rats metaestrus increased E_2 metaestrus (P<0.05), restored to the normal level (P> 0.05). Proestrus LH decreased significantly (P<0.01), restored to the normal level (P>0.05). The pituitary, ovary, uterus damage can back to normal. Bushentiaojing Recipe high group would enable proestrus E_2 model rats increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus E_2 significantly increased (P <0.01), higher than normal (P<0.05). Metaestrus PRL increased (P<0.05), restored to the normal level (P>0.05). Proestrus P significantly decreased (P <0.01), restored to the normal level (P> 0.05). Estrus before and after the LH lower (P<0.01, P<0.05), restored to the normal level (all P>0.05). Metaestrus T reduced (P<0.05), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Bushentiaojing Recipe, compared with model group, Bushentiaojing Recipe low group would enable to regulate menstruation model rats with esturs before and after the FSH increased (P<0.01, P<0.05), restored to the the normal level (all P>0.05). Metaestrus ovarian index increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus uterine index increased significantly (P <0.01), restored to the normal level (P>0.05). Estrus before and after E_2 decreased (all P<0.05), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus and pituitary. Bushengtiaojing Recipe high group would enable model rats estrus before and after the FSH increased (P<0.01, P<0.05), restored to the normal level (all P>0.05). Proestrus LH significantly increased (P<0.01), restored to the normal level (P>0.05). Estrus before and after ovarian index was significantly increased (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after uterine index was significantly increased (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after E_2 decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged ovaries, uterus, pituitary basically can back to normal.
3 The effect of Shuganfa on HPOA of ovulation disorder rats: (1) Triptolide-induced ovulation disorder model group after shuganfa treatment, compared with model group, the Xiaoyao pill low group would enable model rats’PRL of estrus before and after increased significantly (all P<0.01), restored to the normal level (all P>0.05). Proestrus LH lower (P<0.01), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. Xiaoyao pill high group would enable model rats metaestrus E_2 increased (P<0.05), restored to the normal level (P> 0.05). Estrus before and after PRL increased (both P<0.01), restored to the normal level (all P>0.05). Reduced model rats proestrus P to a normal level (P>0.05). Estrus before and after the LH decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Shuganfa, compared with model group, the Xiaoyao pill low group would enable model rats estrus before and after FSH increased (P <0.01, P <0.05), restored to the normal level (all P>0.05). Increase in model rats proestrus LH to normal (P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape. Xiaoyao pill high group would enable model rats proestrus FSH increased significantly (P<0.01), higher than normal (P<0.05). Metaestrus FSH increased (P<0.05), restored to the normal level (P>0.05). Increase in model rats proestrus LH higher than normal (P<0.05). Increase in model rats with estrus before and after the uterine index to a normal level (all P>0.05). Metaestrus E_2 reduced (P<0.05), restored to the normal level (all P> 0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape.
4 Comparison of the effects of Bushenfa and Shuganfa on gonad axis in rats with ovulation disorder: (1) Triptolide-induced ovulation disorder model group, the Bushentiaojing Recipe high group to regulate model rats metaestrus E_2 higher than Xiaoyao pill group (P<0.05), proestrus PRL lower than Xiaoyao pill high group (P<0.05), the Bushentiaojing Recipe low group to regulate metaestrus E_2 was significantly higher than the Xiaoyao pill low group (P<0.01), higher than the Xiaoyao pill metaestrus LH the low group (P <0.05), proestrus PRL lower than Xiaoyao pill low group (P<0.05), metaestrus PRL lower than Xiaoyao pill low group (P<0.05).Improving in the ovarian morphology, the Bushentiaojing Recipe high and low group the number of follicles, mature follicle diameter, the number of GC storeys and level and dense arrangement better than Xiaoyao pill high and low groups. (2) Androgen-induced anovulation model group, the Bushentiaojing Recipe high group to regulate the ovarian index of estrus before and after was significantly higher than Xiaoyao pill high group (P<0.01), the metaestrus uterus index higher than Xiaoyao pill high group (P<0.05 ), the Bushentiaojing Recipe low group to regulate proestrus LH lower than Xiaoyao pill low group (P<0.05). Patterns to improve the pituitary, Xiaoyao pill high and low group of anterior pituitary gland cells, the number is greater than the Bushentiaojing Recipe high and low group. Improving in the ovarian morphology, the the Bushentiaojing Recipe high group have a higher rough profile than Xiaoyao pill low group, ovarian follicle cells thiner than the Xiaoyao pill high group, the number of follicles, follicle diameter and corpus luteum volume to be higher than Xiaoyao pill high group, the number of storeys, number and arrangement density of GC higher than Xiaoyao pill high group. Patterns in improving the uterus, the myometrial thickness, the number of glands, lining layer of columnar cells of continuity and integrity of the Bushentiaojing Recipe high and low group is superior to Xiaoyao pill high and low groups.
Part two: Comparative study of the effect about Bushen treatment and Shugan treatment people luteinized granulose cells secretory function
Objective: To explore the Bushen and Shugan treatment of ovulation disorders in the mechanism, through the ovarian granulosa cell cultures to observe the two method of ovarian granulosa cell function, and to explore two methods of ovarian secretion system of self-study of ovulation barriers to the occurrence of ovarian granulosa cell function and the relationship between the Bushentiaojing Recipeand and Xiaoyao pill compare pairs of ovarian granulosa cell secretion function of the similarities and differences between, for treatment of Bushentiaojing Recipeand and Xiaoyao pill ovulation disorders provide experimental basis for clinical application.
Methods: 21 6-week-old healthy female rats were randomly divided into 7 groups, and control group were fed daily with normal saline 2ml/100g; Bushentiaojing Recipeand group of low, middle and high-dose group and Xiaoyao pill high, middle and low-dose group were given Bushentiaojing Recipeand and Xiaoyao pill. In each group were taken at 1 hour after the last administration of rat femoral artery blood. Put it aside at room temperature 2h, 3000r/min centrifugal 10min, take the supernatant, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-loaded, -20℃to save backup.
Select the age of 20-30 years of age, physical health and treatment of infertility due to ovulatory dysfunction, Hebei Medical University School of Medicine clinics in the female volunteers 4. B- super-monitoring no mature follicle, and the exclusion of ovarian, uterine organic diseases, bilateral tubal patency imaging results showed that as the last in March did not take hormones. Each side oral Bushentiaojing Recipeand and Xiaoyao pill. Continuous medication 3 days, the first 4 days early morning empty stomach medication (medication before the fasting 12h), one taking a day dose, at 1 hour after the last administration venous blood taken 5ml, put it aside at room temperature 2h, 3000r/min centrifugal 10min, taking on the Ching, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-installed. Preparation of blank serum: the age of 20-30 years of age, physical health, menstrual laws, B super-monitoring of mature follicles, and no ovaries, uterus organic diseases of female volunteers, an early morning fasting venous blood 5ml, the rest of my colleagues Preparation of drug-containing serum. -20℃to save backup.
From the Second Affiliated Hospital of Hebei Province, assisted reproductive branches fetch the follicular fluid containing granulosa cells by 2000r/min centrifuge, discard supernatant, precipitation granulosa cells, after washing with DMEM medium by density gradient 0.5Percoll suspension 2500 r / min centrifugation to remove red blood cells separated 30min. Adding 0.25% trypsin, 37℃to digest 5-10min or so, to join with 10% fetal bovine serum and the 1640 termination of digestion. PBS washing, centrifugation. After trypan blue staining cells in 85% -90% survival, with 1640 containing 10% fetal bovine serum and the concentration of granule cell culture medium will be adjusted to 2×105 single-cell suspension, single-cell suspension into a 24-hole of the culture plate, each plate 1ml, at 5% CO2, 37℃incubation box training. 24h of cell adhesion. Were added to Bushentiaojing recipeand and Xiaoyao pill containing human serum and rat serum is made with 6%, 12%, 18%, high, medium and low-dose-treated cells 48h. Canadian-based normal and control rat serum were blank.
Results: 1 Bushentiaojing Recipeand containing serum on human luteinized granulosa cell function: (1) Human containing serum groups:①E_2, P, cAMP levels of impact: compared with the blank group, Bushentiaojing high, medium, Low group E_2 are higher than control group (P<0.05, P<0.01, P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group is significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group E_2 is higher than Bushentiaojing medium, low group (P <0.05, P<0.01), Bushentiaojing medium group E_2 is higher than the low group (P<0.01). Bushentiaojing high and low group P is higher than medium group (P<0.05, P<0.01). Bushentiaojing middle and high group cAMP are significantly higher than low group (P<0.01).②FSHR expression: Protein blotting: Bushentiaojing high, medium and low groups FSHR expression are higher than control group, Bushentiaojing high, medium group of FSHR expression are higher than low group. RT-PCR method: comparison with the control group, Bushentiaojing middle and high group FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium group FSHR is significantly higher than high, low group (all P<0.01), Bushentiaojing high group FSHR is significantly higher than low group (P<0.01).③P450, PPARγexpression: compared with the control group, kidney regulate menstruation, the high expression group of P450 is higher than the control group (P<0.01, P<0.05). Bushentiaojing high, medium and low groups PPARγexpression are lower than control group (all P <0.01). Pairwise comparison, Bushentiaojing high group of P450 expression is significantly higher than that low group (all P<0.01). Bushentiaojing low, medium group of PPARγexpression are significantly lower than the Bushentiaojing high group (all P<0.01). (2) rats containing serum group①E_2, P, cAMP levels of impact: compared with the control group, Bushentiaojing high, medium group E_2 are higher than control group (all P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group are significantly higher than control group (P<0.05, P<0.01). Pairwise comparison, Bushentiaojing high, medium group E_2 are higher than Bushentiaojing low group (all P <0.01). Bushentiaojing medium group P is lower than low group (P<0.05, P <0.01). Bushentiaojing high group cAMP is higher than low group (P<0.05).②FSHR expression: Protein blotting: Bushentiaojing high, medium and low group FSHR expression are higher than control group. Bushentiaojing high, middle group are higher than low group. RT-PCR method: comparison with control group, Bushentiaojing middle and high groups FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium, high group FSHR are sign ificantly higher than low group (all P<0.01).③P450, PPARγexpression: compared with control group, Bushentiaojing medium, high group P450 expression are higher than the control group (P<0.05, P<0.01). Bushentiaojing medium group PPARγexpression are significantly lower than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group P450 expression is higher than low group (P<0.01, P<0.05). Bushentiaojing medium group PPARγexpression is lower than low and high group (P <0.05, P <0.01).
2 Xiaoyao pill containing serum on human luteinized granulosa cell function .(1) Human containing serum groups:①E_2, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, medium and low groups E_2 are higher than control group (all P<0.01). Xiaoyao pill high group P is significantly lower than control group (P<0.05). Xiaoyao pill low, medium and high group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E_2 is higher than low group (P<0.01). Xiaoyao pill medium group cAMP significantly higher than high group and low group (all P<0.01).②FSHR expression: Protein blotting: Xiaoyao pill high, medium and low expression groups are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill medium group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill FSHR is higher than the high group and low group (all P<0.01).③P450, PPARγexpression: comparison with control group, Xiaoyao pill medium high group expression P450 expression is significantly higher than control group (P <0.01, P <0.05). Xiaoyao pill high, medium and low group PPARγexpression are lower than control group (P <0.01, P<0.01, P<0.05). Pairwise comparison, Xiaoyao pill high, medium group of P450 expression are significantly higher than the low group (all P<0.01). Xiaoyao pill low group PPARγexpression is lower than the medium,high group (P <0.01, P<0.05). (2) rats containing serum groups:①pairs of E_2, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, E_2 are higher than the control group (P<0.01, P<0.01, P<0.05) . Xiaoyao pill high group P is higher than control group (P<0.05). Xiaoyao pill high, medium group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E_2 is higher than low, medium group (all P<0.01), Xiaoyao pill medium group E_2 is significantly higher than low group (P<0.01). Xiaoyao pill medium, low group P are significantly lower than high group (all P<0.01). Xiaoyao pill high group cAMP is higher than medium, low group (P<0.05, P<0.01).②FSHR expression. Western blotting: Xiaoyao pill high, medium and low groups expression are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill high group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (P<0.01). Pairwise comparison, Xiaoyao pill high group is higher than medium , low group (P<0.05, P<0.01).③P450, PPARγexpression: compared with control group, Xiaoyao pill high group P450 expression is higher than control group (P<0.05). Xiaoyao pill medium group PPARγexpression is significantly lower than that control group (P <0.01).
3 Two kinds of drug-containing serum on human luteinized granulosa cell function affect the comparison of: (1) Human containing serum groups:①E_2, P, cAMP levels of impact: Bushentiaojing low group E_2 is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group E_2 is higher than Xiaoyao pill low group (P<0.05). Bushentiaojing high group E_2 is higher than Xiaoyao pill high, medium and low group (all P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill low group (P<0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium groups cAMP is higher than Xiaoyao pill low group (P<0.05), lower than Xiaoyao pill high group (P <0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P<0.01).②FSHR. Western blotting: Bushentiaojing high, medium and low groups expression of FSHR are higher than Xiaoyao pill high, medium and low groups, and Bushentiaojing low group FSHR expression is higher Xiaoyao pill high group. RT-PCR method: Bushentiaojing low group FSHR is lower than Xiaoyao pill high, medium group (P<0.05,P<0.01). Bushentiaojing medium group FSHR is higher than Xiaoyao pill high, medium and low group (all P <0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low, medium group (all P<0.01).③P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P <0.01, P <0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing medium group P450 expression is higher than Xiaoyao pill high, low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05), lower than the Xiaoyao pill high group (P<0.01). Bushentiaojing high group PPARγis higher than Xiaoyao pill low group (P<0.01). (2) rats containing serum groups:①E_2, P, cAMP levels of impact: Bushentiaojing low group E_2 is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium group E_2 is higher than Xiaoyao pill medium, low group (P<0.05, P<0.01). Bushentiaojing high group E_2 is higher than Xiaoyao pill high ,low group (all P<0.01). Bushentiaojing low group P is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill medium, high group (P<0.05,P<0.01). Bushentiaojing high group P ia lower than Xiaoyao pill high group (P <0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, (all P<0.01). Bushentiaojing medium group cAMP is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P <0.01).②FSHR expression. Western blotting: Bushentiaojing middle, low groups FSHR are higher than Xiaoyao pill high, low group. Bushentiaojing medium group is higher than Xiaoyao pill high, medium and low groups. RT-PCR method: Bushentiaojing FSHR low group is higher than Xiaoyao pill high group (P<0.05). Bushentiaojing medium group FSHR is higher than Xiaoyao pill medium, low group (P<0.05,P<0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low group (P<0.01).③P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P<0.01,P<0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing high group P450 is higher than Xiaoyao pill low, medium and high group(all P<0.05). Bushentiaojing medium group PPARγis ower than Xiaoyao pill low, high group (all P<0.05). Bushentiaojing high group PPARγis lower than Xiaoyao pill medium group (P<0.05).
Conclusions: 1 two kinds of ovulation disorder models are serum reproductive hormone disorders, pituitary, ovarian, uterine morphology are abnormal; Triptolide-induced ovulation disorder model rats ovaries, uterus weight index are lower, Androgen-induced ovulation model rats ovarian, uterine shape and weight index is more severe than Triptolide-induced ovulation disorder model. 2 Bushen and Shugan method both by improve the pituitary gland, ovarian, uterine shape, regulating reproductive endocrine hormone levels, increase ovary, uterus weight index, to play the promotion of follicular development and maturation, ovulation disorders treatment effect. Bushen method in improving the ovarian, uterine morphology, increased ovary, uterus weight index, regulation of ovarian secretion of reproductive hormones is better than Shugan, Shugan method improve pituitary morphology, regulation of pituitary secretion of reproductive hormones are better than Bushen. 3 Bushen, Shugan method can improve human luteinized granulosa cell function, self-regulation of ovarian secretion system, improve ovarian function. Bushen method in improve E_2, FSHR, reduce PPARγis better than Shugan, Sugan method improve cAMP is better than Bushen.
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15刘平,王宁生,雷燕等.关于血清药理学的若干思考.中国中西医结合杂志,1999,10(5):263-266
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17 Abel MH,Wooton AN,Wilkins V, et al.The efect of a null mutation in the follicle stimulating hormone receptor gene on mouse reproduction. Endocrinology,2003,141(5):1795-1803
18 Guan Q,Ma HG,Wang YY,et al.Effects of co-administration of growth hormone(GH) and aspirin to women during in vitro fertilization and embryo transfer (IVF-ET) cycles.Zhong hua Nan Ke Xue, 2007,13(9):798-800
19 Abdennebi L,Monget G,Pisselet C,et al.Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian folicles from high and low prolific sheep breeds.Biol Reprod,1999;60(4):845-854
20 Araki, Miller WR. Aromatase activity in breast tissue. J Steroid Biochem Mol Biol, 1991,39:783-790
21 Carolyn M K,OlivierB,WalterW,et a1. Expression and Localization of PP-ARs in the Rat Ovary During Follicu-lar Devdopment and the Periovulatory Period. Endo-crinology, 2001, 142(11): 4831.
22 Komar CM,Curry Jr. Inverse relationship between the expression of messenger ribonucleic acid for peroxisome proliferator-activated receptor and P450 side chain cleavage in the rat ovary. Biol Reprod, 2003, 69:549-555
23李明,母义明,汪保安,等.PPARγ和RXR激动剂抑制人卵巢颗粒细胞芳香化酶活性和其mRNA的水平.中华内分泌代谢杂志,2003 19(6):471-473
24吕时铭,吕红等.转化生长因子参与调节颗粒细胞雌孕激素的分泌.中华检验医学杂志,2001,24(5):278一281
1李桂玲,归绥琪.补肾中药对雄激素致不孕大鼠胰岛素样生长因子-l及胰岛素样生长因子-1受体的影响.中国中西医结合杂志,2000,20(9):677-678
2余运初.关于针刺和中药促排卵机制的研究.中西医结合杂志,1985,(5):201
3俞瑾,孙月丽,邵公权,等.补肾化痰治疗多囊卵巢综合征中对下丘脑-垂体-卵巢功能的调节.中西医结合杂志, 1986,6(4): 218.
4罗元恺.实用中医妇科学.上海:上海科学技术出版社,1996: 290
5范春茹,车胜男,崔兆琴.补肾法治疗排卵障碍103例.陕西中医,2002,23(5):389-390
6李新荣.中西医结合治疗排卵障碍性不孕症168例.河南中医,2004,24(4):53-54
7刘淑珍,王秀珍,李春荣.中西医结合治疗排卵障碍性不孕症168例.生殖医学杂志,2005,14(2):106-107
8邓卫红.疏肝补肾周期疗法治疗无排卵性不孕症160例.山东中医杂志2009 ,28(2):101-102
9赵瑞华,补肾调经汤促排卵的临床研究.中国中西医结合杂志,1996 18(5):264
10李研芬,周素玲,杨雅梅.黄素化未破裂卵泡综合症病因及治疗探讨,生殖医学杂志,2005 14(3):169
11罗凌等.补肾调冲方治疗对排卵障碍性不孕症临床观察.新中医,2008 40(4):31-32
12徐莲薇,倪晓容,叶玉妹等.补肾活血调周法治疗多囊卵巢综合征疗效观察.上海中医杂志,2008,42(12):37-38
13周璐,章巧萍,徐孝平,等.生精汤对雄激素致无排卵模型大鼠卵巢功能及形态学的影响.浙江中医药大学学报, 2006, 30(6): 621-622
14邓阿黎,周忠明,张亚兵.补肾化痰祛瘀类中药对PCOS模型大鼠生殖内分泌系统影响的实验研究.河南中医,2009 29(6):551-553
15邓阿黎,周忠明,张亚兵.补肾化痰祛疲中药对PCOS模型大鼠卵巢组织形态学作用的实验研究.2009 12(2):143-145
16李翠萍,黄霞,牛丽丽等.补肾调周法对PCO大鼠排卵障碍干预的实验研究.山西中医,2008,24(5):46-47
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14冯伟.MTT法分析中药含药血清对体外软骨细胞增殖影响的研究.上海中医药大学学报,2000,14(1):43-45
15刘平,王宁生,雷燕等.关于血清药理学的若干思考.中国中西医结合杂志,1999,10(5):263-266
16 Huhtaniemi IT.LH and FSH receptor mutations and their effects on puberty.Horm Res,2002,57,2:35-38
17 Abel MH,Wooton AN,Wilkins V, et al.The efect of a null mutation in the follicle stimulating hormone receptor gene on mouse reproduction. Endocrinology,2003,141(5):1795-1803
18 Guan Q,Ma HG,Wang YY,et al.Effects of co-administration of growth hormone(GH) and aspirin to women during in vitro fertilization and embryo transfer (IVF-ET) cycles.Zhong hua Nan Ke Xue, 2007,13(9):798-800
19 Abdennebi L,Monget G,Pisselet C,et al.Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian folicles from high and low prolific sheep breeds.Biol Reprod,1999;60(4):845-854
20 Araki, Miller WR. Aromatase activity in breast tissue. J Steroid Biochem Mol Biol, 1991,39:783-790
21 Carolyn M K,OlivierB,WalterW,et a1. Expression and Localization of PP-ARs in the Rat Ovary During Follicu-lar Devdopment and the Periovulatory Period. Endo-crinology, 2001, 142(11): 4831.
22 Komar CM,Curry Jr. Inverse relationship between the expression of messenger ribonucleic acid for peroxisome proliferator-activated receptor and P450 side chain cleavage in the rat ovary. Biol Reprod, 2003, 69:549-555
23李明,母义明,汪保安,等.PPARγ和RXR激动剂抑制人卵巢颗粒细胞芳香化酶活性和其mRNA的水平.中华内分泌代谢杂志,2003 19(6):471-473
24吕时铭,吕红等.转化生长因子参与调节颗粒细胞雌孕激素的分泌.中华检验医学杂志,2001,24(5):278一281
1李桂玲,归绥琪.补肾中药对雄激素致不孕大鼠胰岛素样生长因子-l及胰岛素样生长因子-1受体的影响.中国中西医结合杂志,2000,20(9):677-678
2余运初.关于针刺和中药促排卵机制的研究.中西医结合杂志,1985,(5):201
3俞瑾,孙月丽,邵公权,等.补肾化痰治疗多囊卵巢综合征中对下丘脑-垂体-卵巢功能的调节.中西医结合杂志, 1986,6(4): 218.
4罗元恺.实用中医妇科学.上海:上海科学技术出版社,1996: 290
5范春茹,车胜男,崔兆琴.补肾法治疗排卵障碍103例.陕西中医,2002,23(5):389-390
6李新荣.中西医结合治疗排卵障碍性不孕症168例.河南中医,2004,24(4):53-54
7刘淑珍,王秀珍,李春荣.中西医结合治疗排卵障碍性不孕症168例.生殖医学杂志,2005,14(2):106-107
8邓卫红.疏肝补肾周期疗法治疗无排卵性不孕症160例.山东中医杂志2009 ,28(2):101-102
9赵瑞华,补肾调经汤促排卵的临床研究.中国中西医结合杂志,1996 18(5):264
10李研芬,周素玲,杨雅梅.黄素化未破裂卵泡综合症病因及治疗探讨,生殖医学杂志,2005 14(3):169
11罗凌等.补肾调冲方治疗对排卵障碍性不孕症临床观察.新中医,2008 40(4):31-32
12徐莲薇,倪晓容,叶玉妹等.补肾活血调周法治疗多囊卵巢综合征疗效观察.上海中医杂志,2008,42(12):37-38
13周璐,章巧萍,徐孝平,等.生精汤对雄激素致无排卵模型大鼠卵巢功能及形态学的影响.浙江中医药大学学报, 2006, 30(6): 621-622
14邓阿黎,周忠明,张亚兵.补肾化痰祛瘀类中药对PCOS模型大鼠生殖内分泌系统影响的实验研究.河南中医,2009 29(6):551-553
15邓阿黎,周忠明,张亚兵.补肾化痰祛疲中药对PCOS模型大鼠卵巢组织形态学作用的实验研究.2009 12(2):143-145
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