骨形态发生蛋白4/Smad信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用  

Regulation of bone morphologenetic protein 4 /Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes

作　　者：张海玉[1] 张晓丽[1] 纪淑芳[1] 邴鲁军[1] 郝晶[1] 

机构地区：[1]山东大学医学院组织学与胚胎学教研室,教育部实验畸形学重点实验室,济南250012

出　　处：《解剖学报》2014年第3期375-382,共8页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31071269)

摘　　要：目的探讨骨形态发生蛋白(BMP)4/Smad信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用。方法取出生3d的雌性昆明小鼠卵巢,采用两步酶消化法,分离纯化卵母细胞,进行原代培养。将卵母细胞分为3组:正常对照组(Con组)、添加重组人BMP4组(BMP4组)、添加BMP4和BMP4抑制剂6-{4-[2-(1-呱啶基)乙氧基]苯基}-3-(4-吡啶基)吡唑并(1,5-A)嘧啶(dorsomorphin)组(BMP4+抑制剂组)。采用TUNEL法,检测BMP4对卵母细胞凋亡的影响;采用免疫组织化学染色与实时定量PCR,检测BMP4对卵母细胞中p-Smad1/5/8、sohlh2、ckit及foxo3a表达的影响。采用RNA干扰技术、转染sohlh2质粒和LY294002处理,探讨BMP4/Smad信号通路在小鼠原始卵泡卵母细胞凋亡中的调控机制。结果 TUNEL结果显示,BMP4组凋亡卵母细胞比例明显低于Con组(P<0.05)和BMP4+抑制剂组(P<0.05);加入BMP4可明显促进细胞Smad入核,上调sohlh2和c-kit表达,抑制foxo3a入核;转染sohlh2 siRNA后,可明显阻断BMP4抑制卵母细胞凋亡的作用;转染sohlh2质粒后,p-foxo3a表达量显著增加,LY294002可明显抑制sohlh2促进foxo3a磷酸化的作用。结论激活BMP4/Smad信号通路可抑制小鼠原始卵泡卵母细胞的凋亡,其作用机制可能是通过上调sohlh2和c-kit基因表达,进而调控foxo3a入核而实现的。Objective To explore the effect and mechanism of the bone morphologenetic protein 4 （BMP4）/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes .Methods Three-day-old Kunming mouse ovarine tissues were digested by the two-step enzymatic method to extract and purify oocytes .The cultured oocytes were divided into three groups： the normal culture medium （Con group）, the medium with BMP4 （BMP4 group）, and the medium with BMP4 and BMP4 inhibitor （ BMP4＋inhibitor group ） .TUNEL was used to examine the effects of BMP 4 on the survival of the primordial follicle oocyte;Immunohistochemical staining and Real-time quantitative PCR were performed to investigate the expressions of p-Smad1/5/8, sohlh2, c-kit and foxo3a;siRNA interference, sohlh2 plasmid transfection and LY294002 treatments were performed to explore the mechanism of the BMP 4/Smad signaling pathway on the apoptosis of oocytes . Results TUNEL results demonstrated that the ratio of apoptotic oocytes in BMP 4 group was significantly lower than that in the Con group （ P 〈0.05 ） and the BMP4 ＋inhibitor group （ P 〈0.05 ）; BMP4 significantly promoted the nuclear translocation of Smad and inhibited the nuclear translocation of foxo 3a, the mRNA and protein levels of sohlh2 and c-kit remarkably increased in BMP4 group.The effect of BMP4 on the oocyte survival was significantly repressed after sohlh 2 siRNA transfection.Sohlh2 overexpression up-regulated the expression of p-foxo3a, and this activity was abolished by LY 294002.Conclusion BMP4/Smad signaling pathway may inhibit primordial follicle oocyte apoptosis , via up-regulation of the expression of sohlh2 and c-kit, and then down-regulation of the nuclear translocation of foxo 3a.

关 键 词：骨形态发生蛋白4 SMAD信号通路 卵母细胞 Sohlh2 原位缺口末端标记 免疫组织化学 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]