Rho/ROCK抑制剂Y27632对小鼠腔前卵泡体外培养的影响  

Effect of Rho/ROCK Inhibitor Y27632 on the Culture of Mouse Preantral Follicles in Vitro

作　　者：俞晓丽[1,2] 蒲静 罗晓强[1,2] 刘心蕊 邱意开[1,2] 张樱馨 裴秀英 YU Xiaoli;PU Jing;LUO Xiaoqiang;LIU Xinrui;QIU Yikai;ZHANG Yingxin;PEI Xiuying(Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Key Laboratory of Reproduction and Genetics in Ningxia,Yinchuan 750004,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室,宁夏生殖与遗传重点实验室,银川750004 [2]宁夏医科大学基础医学院,银川750004

出　　处：《宁夏医科大学学报》2019年第8期767-772,共6页Journal of Ningxia Medical University

基　　金：宁夏回族自治区重点研发计划(2019BFG02007);国家自然科学基金(31560329);宁夏自然科学基金(NZ16062);宁夏医科大学基础医学西部一流学科资助项目(NXYLXK2017B07);宁夏生殖与遗传基础与临床科技创新团队(NXRGT201503)

摘　　要：目的探讨Rho/ROCK信号通路抑制剂Y27632对小鼠卵泡体外发育的影响。方法取出生后12.5d的小鼠卵巢,采用机械法收集单个腔前卵泡,用微滴培养法对其进行培养。设置对照组和含有Y27632的处理组,培养期间观察卵泡和卵母细胞生长状态,同时,测量对照组和处理组的卵泡直径和卵母细胞直径,用荧光定量PCR技术检测颗粒细胞、卵母细胞以及凋亡相关基因的表达情况,同时用细胞荧光染色技术监测卵泡中颗粒细胞的凋亡情况。结果通过形态学观察,培养基中添加Y27632后,整个卵泡生长过程中,卵泡的增长直径为(25.90±3.69)μm和卵母细胞增长直径为(4.37±1.89)μm,与对照组相比差异均无统计学意义(P均>0.05)。经荧光定量PCR技术检测发现,随着卵泡的不断发育,颗粒细胞表达相关基因Amh、Lhr、Fshr和Foxl2,与对照组相比有差异,而卵母细胞特异性基因Bmp15则无变化。卵母细胞成熟后经微管染色发现,Y27632的添加并不影响卵母细胞完成第一次减数分裂。通过卵泡活力检测的结果得知,处理组的颗粒细胞凋亡现象少于对照组,凋亡基因Bad低于对照组,且能引起凋亡抑制基因Xiap的上调。结论小鼠卵泡经微滴法培养后,添加Y27632能提高颗粒细胞的基因Amh、Lhr、Fshr和Foxl2的表达。同时可降低颗粒细胞的凋亡,从而维持卵泡的正常发育。Objective To investigate the effect of Rho/Rock signaling pathway inhibitor Y27632 on the development of mouse follicles in vitro.Methods Ovaries of 12.5-day-old ICR mice were collected and preantral follicles were isolated by mechanical method.The follicle was cultured by microdroplet culture,and a blank control group and a treatment group containing Y27632 were set in the experiment.Results The average growth diameter of the follicles (25.90±3.69)μm and oocytes (4.37±1.89)μm treated by Y27632 were not significantly different from the control group.qRT-PCR result showed that with the development of follicles,granulosa cells expressed related genes Amh,Lhr,Fshr and Foxl2,which showed significant differences compared with the control group,while the oocyte-specific gene Bmp15 showed no significant changes.Moreover,after oocyte maturation,microtubule staining revealed that the addition of Y27632 did not affect the oocyte to complete the first meiosis.According to the results of follicle viability assay,the granulosa cell apoptosis in the treatment group was less than that in the control group,and the apoptosis gene Bad was significantly lower than the control group,and it could cause significant up-regulation of the apoptosis inhibitory gene Xiap.Conclusion After the mice follicles were cultured by microdroplet method,Y27632 was added to increase the expression of the granulosa cells genes Amh,Lhr,Fshr and Foxl2.At the same time,it can reduce the apoptosis of granulosa cells,thus maintaining the normal development of follicles.

关 键 词：小鼠 腔前卵泡 体外培养 卵母细胞成熟 Y27632 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]