电针“委中”大鼠血清对多裂肌卫星细胞成肌分化因子和周期蛋白表达的影响
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摘要
目的观察电针"委中"大鼠血清对大鼠腰多裂肌卫星细胞成肌分化因子MyoD、周期蛋白CDK4、P57和CyclinD表达的影响,从细胞周期角度分析电针"委中"血清促进损伤多裂肌再生修复的起效机制。方法SD大鼠随机分为空白组、模型组和电针"委中"组,每组8只。通过肌肉注射0.5%布比卡因制备多裂肌损伤模型,电针组选取"委中"穴进行电针治疗,每日1次,每次20 min,第4日收集血清。培养原代大鼠腰多裂肌卫星细胞,随机分为空白血清组、模型血清组、电针"委中"血清组、抑制剂血清组(电针"委中"血清中加PI3K抑制剂LY294002),以对应血清分别培养1 d,免疫印迹法检测肌卫星细胞MyoD、CDK4、P57和CyclinD蛋白表达。结果造模后,各血清组MyoD、CDK4和P57表达水平均显著升高(P <0.01);与模型血清组对比,电针"委中"血清组MyoD、CDK4升高(P <0.05或P <0.01)而P57显著降低(P <0.01);与电针"委中"血清组对比,加入LY294002抑制剂后MyoD、CDK4降低(P <0.05或P <0.01)而P57显著升高(P <0.01)。与空白血清组对比,电针"委中"血清组CyclinD升高(P <0.05),其他各组对比差异无统计学意义(P> 0.05)。结论电针"委中"血清可通过上调MyoD、CDK4的表达,并下调P57的表达,加速肌卫星细胞增殖的周期性进程,促进损伤多裂肌的良性修复。
Objective: To observe the effect of electro-acupuncture "Weizhong" serum on the expression of MyoD,CDK4,P57 and Cyclin D in rat lumbar multifidus muscle satellite cells,and to analyze the mechanism of electro-acupuncture "Weizhong" serum promoting the regeneration and repair of injured multifidus muscle from the perspective of cell cycle. Methods: SD rats were randomly divided into the blank group,the model group and electro-acupuncture "Weizhong" group,with 8 rats in each group. The model of polyfid muscle injury was established by intramuscular injection of 0.5% bupivacaine. The electro-acupuncture group selected "Weizhong" point for electro-acupuncture treatment,once a day,20 minutes a time,and collected serum on the fourth day. The primary rat lumbar multifidus muscle satellite cells were cultured and randomly divided into blank serum group,model serum group,electro-acupuncture "Weizhong" serum group and electro-acupuncture "Weizhong" plus LY294002 inhibitor serum group. The corresponding serum was cultured for 1 day. The expression of MyoD,CDK4,CyclinD and P57 protein in muscle satellite cells was detected by Western blot. Results: The expression levels of MyoD,CDK4 and P57 in serum groups were significantly increased after modeling(P < 0.01);compared with model serum group,MyoD and CDK4 in electroacupuncture "Weizhong" serum group increased(P < 0.05 or P < 0.05) and P57 decreased significantly(P < 0.01);compared with electroacupuncture "Weizhong" serum group,MyoD and CDK4 decreased(P < 0.05 or P < 0.01) and P57 increased significantly after adding LY294002 inhibitor(P < 0.01). Compared with the blank serum group,CyclinD in the serum group of electro-acupuncture "Weizhong" increased(P < 0.05),but there was no significant difference among the other groups(P > 0.05). Conclusion: Electroacupuncture "Weizhong" serum can accelerate the cyclical process of muscle satellite cell proliferation by up-regulating the expression of MyoD and CDK4 and down-regulating the expression of P57,and promote the benign repair of damaged polyfidus muscle.
Objective: To observe the effect of electro-acupuncture "Weizhong" serum on the expression of MyoD,CDK4,P57 and Cyclin D in rat lumbar multifidus muscle satellite cells,and to analyze the mechanism of electro-acupuncture "Weizhong" serum promoting the regeneration and repair of injured multifidus muscle from the perspective of cell cycle. Methods: SD rats were randomly divided into the blank group,the model group and electro-acupuncture "Weizhong" group,with 8 rats in each group. The model of polyfid muscle injury was established by intramuscular injection of 0.5% bupivacaine. The electro-acupuncture group selected "Weizhong" point for electro-acupuncture treatment,once a day,20 minutes a time,and collected serum on the fourth day. The primary rat lumbar multifidus muscle satellite cells were cultured and randomly divided into blank serum group,model serum group,electro-acupuncture "Weizhong" serum group and electro-acupuncture "Weizhong" plus LY294002 inhibitor serum group. The corresponding serum was cultured for 1 day. The expression of MyoD,CDK4,CyclinD and P57 protein in muscle satellite cells was detected by Western blot. Results: The expression levels of MyoD,CDK4 and P57 in serum groups were significantly increased after modeling(P < 0.01);compared with model serum group,MyoD and CDK4 in electroacupuncture "Weizhong" serum group increased(P < 0.05 or P < 0.05) and P57 decreased significantly(P < 0.01);compared with electroacupuncture "Weizhong" serum group,MyoD and CDK4 decreased(P < 0.05 or P < 0.01) and P57 increased significantly after adding LY294002 inhibitor(P < 0.01). Compared with the blank serum group,CyclinD in the serum group of electro-acupuncture "Weizhong" increased(P < 0.05),but there was no significant difference among the other groups(P > 0.05). Conclusion: Electroacupuncture "Weizhong" serum can accelerate the cyclical process of muscle satellite cell proliferation by up-regulating the expression of MyoD and CDK4 and down-regulating the expression of P57,and promote the benign repair of damaged polyfidus muscle.
引文
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[21]Li X,Liu H,Wang H,et al.Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling[J].Bioscience reports,2014,34(5):609-620.
[2]Carosio S,Berardinelli MG,Aucello M,et al.Impact of ageing on muscle cell regeneration[J].Ageing Res Rev,2011,10:35-42.
[3]贺静,于建春.针灸血清作用的研究进展[J].中国针灸,2014,34(10):1042-1046.
[4]刘通,于佳妮,邹德辉,等.电针血清对多裂肌卫星细胞增殖及Pax-7、成肌分化抗原、磷酸化蛋白激酶B表达的影响[J].针刺研究,2016,41(5):402-409.
[5]邹德辉,陈玉佩,刘通,等.电针“委中”对布比卡因致大鼠腰多裂肌损伤后形态学及CK、IL-17表达的影响[J].中国针灸,2017,37(9):971-976.
[6]陈玉佩,刘通,邹德辉,等.局部注射布比卡因建立大鼠骨骼肌损伤模型的组织形态学评价[J].中国组织工程研究,2016,20(18):2615-2621.
[7]李忠仁.实验针灸学[M].2版.北京:中国中医药出版社,2007:255-257.
[8]陈玉佩,王淑艳,刘通,等.大鼠腰多裂肌卫星细胞的分离培养与鉴定[J].细胞与分子免疫学杂志,2018(8):714-717.
[9]周同,陆爱云.多裂肌与慢性腰痛康复的研究[J].中国康复医学杂志2012,27(4):387-389.
[10]Yu ZG,Wang RG,Xiao C,et al.Effects of zusanli and ashi acupoint electroacupuncture on repair of skeletal muscle and neuromuscular junction in a rabbit gastrocnemius contusion model[J].Evidence-based complementary and alternative medicine:eCAM,2016,2016(6):7074563.
[11]Su Z,Robinson A,Hu L,et al.Acupuncture plus Low-Frequency electrical stimulation(Acu-LFES)attenuates diabetic myopathy by enhancing muscle regeneration[J].Plos One,2015,10(7):jap.00175.2015.
[12]梁文娜,李西海,李灿东,等.基于血清药理学理论从含证血清探讨中医证的形成机制[J].中华中医药杂志,2014,29(7):2104-2106.
[13]赵丹丹,唐成林,黄思琴,等.电针干预对大鼠腓肠肌适应性肥大及肌卫星细胞增殖分化的影响[J].针刺研究,2017,42(6):489-495.
[14]金晶,卢健.肌卫星细胞代谢重编程研究进展及其展望[J].中国体育科技,2017,53(6):65-70.
[15]Farina NH,Hausburg M,Betta ND,et al.A role for RNApost-transcriptional regulation in satellite cell activation[J].Skelet Muscle,2012,2(1):21.
[16]Macaluso F,Myburgh KH.Current evidence that exercise can increase the number of adult stem cells[J].J Muscle Res Cell Motil,2012,33(3-4):187-198.
[17]Blain SW.Switching cyclin D-Cdk4 kinase activity on and off[J].Cell Cycle,2008,7(7):892.
[18]张也频,章宗籍.Cip/Kip家族的独特成员-p57Kip2的研究进展[J].中国肿瘤生物治疗杂志,2011,18(5):574-580.
[19]王前,邓晶,蒋永新.cyclinD1的研究进展[J].现代肿瘤医学,2009,17(2):350-353.
[20]李俊华,王正珍,王军力,等.针刺对急性骨骼肌钝挫伤大鼠肌卫星细胞增殖的影响[J].中国体育科技,2012,48(4):137-140.
[21]Li X,Liu H,Wang H,et al.Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling[J].Bioscience reports,2014,34(5):609-620.