摘要
目的意义：从细胞和分子水平上研究胎盘在发生、发展及成熟过程中，组织结构变化的规律和凋亡的机理，探讨牦牛妊娠期胎盘细胞凋亡及其与早期妊娠中断的关系，对揭示胎盘组织生理功能的发挥和流产发病机制提供一条新的思路。在牦牛的生殖调控和疾病防制工作中具有重要意义，也可为人和其他动物生殖机理的研究提供参考资料。
试验方法：（1）采集了总计47头4～8岁分别处于正常妊娠期、分娩前后及地塞米松（DEX）诱导流产病例的牦牛胎盘组织。采用常规石蜡组织切片制作和HE染色技术，观察了牦牛胎盘组织的基本结构、凋亡细胞的形态特征及其在不同组别之间的动态变化；采用体视学方法对胎盘组织胎儿绒毛、肉阜隐窝和双核细胞体密度及绒毛面密度等主要参数的动态变化进行了分析；采用原位荧光标记（DAPI）技术和DNA末端标记（TUNEL）技术，对胎盘组织中细胞凋亡状况做了进一步观察和分析；采用免疫组织化学（SP法）技术，对牦牛胎盘组织中凋亡相关蛋白Fas、FasL、Bcl-2和Bax的表达进行了检测。
（2）采集妊娠5月龄左右的牦牛胎盘绒毛组织，体外分离培养绒毛滋养细胞。加入不同浓度的DEX（10-10～10-5mol/L），培养24小时后，采用原位荧光标记（DAPI）技术检测了DEX不同浓度对培养细胞凋亡状况的影响；采用FITC标记的荧光二抗免疫细胞化学法(FITC)，对DEX（10-9～10-7mol/L）干预后的胎盘滋养细胞爬片进行Fas、FasL、Bcl-2和Bax蛋白表达的检测。
试验结果：（1）妊娠期牦牛胎盘组织经历了从形成、生长到成熟的生理过程。在不同的生长发育阶段，胎盘组织具有不同的形态学特征。到妊娠45天左右，胎盘基本具备了上皮绒毛膜型胎盘的典型结构，胎儿绒毛上皮主要由柱状滋养细胞和滋养巨细胞细胞组成，隐窝上皮为一层完整的立方上皮。随着妊娠的进行，胎儿绒毛逐渐分支，并深入到母体肉阜隐窝中，胎盘组织中双核滋养细胞体密度也逐渐增高。妊娠6个月，子宫隐窝上皮开始出现明显的细胞空泡化等退行性变化。到分娩前，整个隐窝上皮基本缺失。妊娠中期药物流产的胎盘组织中胎儿绒毛有明显的退形性变化。
（2）妊娠期牦牛胎盘组织中具有明显的细胞凋亡现象。尽管整个妊娠期凋亡细胞数量一直很少，但凋亡细胞也表现出了明显的时空特异性分布规律。整个妊娠期TUNEL阳性细胞主要分布在胎儿绒毛上皮、母体隐窝上皮中，在妊娠早期的子宫肉阜基质中也偶有分布。随着妊娠的进行，TUNEL阳性细胞的数量在两层上皮组织中总体呈上升趋势，且胎儿绒毛上皮中明显高于母体隐窝上皮，产前胎儿绒毛上皮中显著升高；产后绒毛上皮中最高；药物流产组胎儿绒毛上皮中也明显高于同期正常妊娠组。
（3）Fas在妊娠早、中期阶段（5个月以内）的胎儿绒毛中表达一直较强，在妊娠3个月时阳性指数达到了最大值。之后Fas蛋白在胎儿绒毛中的表达迅速降低。在隐窝基质中，妊娠的绝大部分时间都可以清晰地看到单个分布的被染成棕色的Fas阳性细胞。FasL蛋白在胎儿绒毛组织中始终以一个相对较为稳定的水平表达，主要是一些单核、和多核滋养巨细胞呈现阳性反应；在子宫肉阜隐窝中，FasL的表达普遍较弱，且阳性指数随着妊娠的进行逐渐降低。Bcl-2蛋白主要在绒毛滋养上皮和肉阜隐窝上皮中表达，而且随着妊娠阶段的进行，表达量逐渐降低。分娩后的胎儿绒毛组织中，Bcl-2阳性指数显著高于分娩前绒毛组织。Bax蛋白主要在胎儿绒毛上皮中表达，随妊娠的进展表达量明显增强，另外在妊娠早期的胎盘绒毛上皮中也有很强的表达。药物流产胎盘组织的胎儿绒毛中，Bcl-2和Bax蛋白阳性程度明显高于同期正常妊娠组，并且Bax蛋白表达量的升高比Bcl-2更为明显。
（4）当DEX浓度增加到10-9mol/L时，能显著促进胎盘滋养细胞的凋亡。在10-9～10-7mol/L浓度范围内，体外培养的牦牛胎盘滋养细胞Fas、FasL蛋白的表达受DEX的影响不明显。而Bcl-2和Bax蛋白的表达随着药物浓度的增加呈明显的上升趋势，且Bax蛋白的上升更加明显。
结论：（1）妊娠期牦牛胎盘组织经历了从形成、生长到成熟的生理过程。胎盘组织在形态结构上也发生一系列变化，以适应胎儿营养供应及分娩前胎盘成熟的生理需要。
（2）牦牛胎盘组织中少量的细胞凋亡维持了胎盘组织细胞的正常更新和动态平衡；分娩前胎盘成熟过程伴随着凋亡细胞数量的明显上升；药物流产使胎儿绒毛上皮细胞凋亡增加。胎盘组织凋亡相关蛋白的表达特征提示，Fas、FasL信号系统可能与胎儿绒毛的生长以及妊娠免疫豁免机制有一定的关系；胎盘滋养细胞的凋亡可能主要通过Bcl-2和Bax蛋白调控。早期牦牛胎盘组织中Bcl-2和Bax蛋白的高表达可能反映了在胎盘形成阶段胎盘组织具有很高的细胞更新率；药物流产胎盘组织中细胞凋亡的增加可能是由Bcl-2和Bax蛋白表达量的改变引起的。
（3）体外实验表明，DEX能诱导体外培养的牦牛胎盘滋养细胞发生凋亡，并且DEX对体外培养的牦牛胎盘滋养细胞凋亡的诱导作用可能与Bcl-2和Bax蛋白的表达有关。
Objective: It would offer a new insight for us to understanding the physiological function andmechanism of abortion by discussing the relationship between apoptosis and the interruptionof pregnency during ealier gestation and investigating the morphological changes ofplacentome and apoptosis at cellula and molecular level. These works would play animportant role in yaks reproduction and disease prevention. On the orther hand, it also providesome usefull data concern to the reproduction mechanism of human being and other animals.
Methords:(1)47yaks of4～8years old during pregnancy, pre and post parturation, afterdexamethasone(DEX) intramuscular injection were used to collect the placentomes. Aferfixed with4%（W/V）paraformaldehyde, paraffins embedding tissue sections and HE dyeingtechnology was used to investigate the basic structure and the apoptotic morphologicalcharacteristics of placentome cells. Stereoscopy methord was used to analyse the changes ofsome stereological parameters of yak placentomes in each stage of gestation. In situfluorescence labeling(DAPI) and DNA terminal labaling(TUNEL) techniques were also usedto confirm the apoptotic cells in yak placentomes. The expression of Fas、FasL、Bcl-2and Baxproteins was detected by immunohistochemistry methord.(2) Placental villi were collected from5months pregnant yak plcentomes. After enzymicdigested, villous trophoblast cells were separated and cultured in vitro. The influence ofdifferent concentrated DEX on apoptosis of trophoblast cells was inspected using in situfluorescence labeling(DAPI) techniques. The expression of Fas、FasL、Bcl-2and Bax proteinswas detected by FITC labeled fluorescence scecond antibody using immunocytochemistrytechniques.
Results:(1) yaks placentomes experienced different physiological process as generation,development and maturation during pregnancy. Different morphological characteristics wereobserved during different stage of gestation. About in45days of gestation, yaks placentomes demonstrated typical epitheliochorial placental structure. Fetal villous epithelium was consistof columnar trophoblast cells and gaint trophoblast cells. Meternal crypt was covered with alayer of intact cubic epithelium. With the progress of gestation, fetal villi branched and deeplypenetrated into the meternal caruncula crypts. The volume density of binucleate cells wasincreased in placentomes. In6months of gestation, obviously degenerated changes such ascell vacuolization can be observed in caruncula crypt epithelium. At pre-parturition, most ofthe crypt epithelium were disappear. obviously degenerated changes was also found in yakplacentomes of DEX induced abortion case.
(2) Apoptotic cells were found in yak placentomes. These cells displayed obvious changesafter HE, DAPI and TUNEL dyeing. Though the number was little, the apoptotic cellsdistributed in the placentomes by a spatiotemporal specific manner. The positive cells ofTUNEL reaction mainly located in the fetal villious trophoblast, meternal caruncula cryptepithelium during whole gestation and in the caruncula stroma during early stage of gestation.With the progress of gestation, the number of positive cells was increased in these two layersof epithelium, especially in the fetal villious epithelium of pre and post-parturation and DEXinduced abortion case.
(3) the expression of Fas protein was strong in the fetal villi during early and middle stage ofgestation. The positive index of Fas reached the maximum in the third month of gestation.Then Fas protein was decreased in fetal villi. In caruncula stroma, brown stained and singlelydistributed Fas positive cells were clearly observed in most time of gestation. The expressionof FasL protein was more stable in fetal villi, most positive reation was presented in somemononuclear, binuclear and multinuclear trophoblast cells. In caruncula crypts, FasLexpression was more weak and the positive index was declined with the progress of gestation.Bcl-2protein was expressed mainly in villious and caruncula crypt epithelium,the quantity ofBcl-2protein was declined with the progress of gestation.but the positive index in fetal villi ofpost-parturation was higher than pre-parturation. Bax protein was expressed mainly in fetalvillious epithelium and the quantity was increased during whole gestation. In DEX inducedabortion cases, both Bcl-2and Bax proteins were all strong expressed, especailly Bax protein.
(4)10-9mol/L DEX significantly promoted the apoptosis of in vitro cultured trophoblast cells.The effection of DEX on expression of Fas and FasL was unconspicuous at the concentration of10～10mol/L. but Bcl-2and Bax protein expression was obviously increased after DEXtreatment,especially Bax protein.
Conclusion:(1) Yaks placentomes experienced different physiological process as generation,development and maturation during pregnancy. The changes of morphological characteristicswere taken placed to accommodate the physiological requirements of cyotrophy supplementand placental maturation during pre-parturation.
(2) A small number of apoptotic cells maintained the normal replacement and dynamicbalance of placental tissue. The process of placental maturation during pre-parturation wasaccompanied with the obviously increase of apoptotic cells. In DEX induced abortion cases,the number of apoptotic cells was increased. The status of apoptosis related proteinsexpression indicated that Fas and FasL probably involved in the process of fetal villi growingand the mechanism of immune privilege during pregnancy. The apoptosis of the trophblastcells was related to the Bcl-2and Bax protein. The strong expression of Bcl-2and Bax proteinin early stage of gestation perhaps reflected a higher cell replacement with the placentalgeneration. The increase of the number of apoptotic cells in DEX induced abortion cases wasresulted by the change of Bcl-2and Bax expression.
(3) in vitro experiment indicate that DEX can induced the apoptosis of cultured yak placentaltrophoblast cells and the mechanism of the apoptosis probably related to the expression ofBcl-2and Bax protein.
引文
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