【作者】 黄健；

【导师】 郑月慧；

【作者基本信息】 南昌大学 ， 生理学， 2008， 硕士

【摘要】 目的完善原始卵泡的体外培养技术;探索原癌基因c-erbB2在大鼠原始卵泡启动生长过程中的表达变化及可能的作用。方法(1)选择2日龄SD大鼠,建立Waymouth和DMEM两种卵巢体外培养体系,用组织切片和HE染色法观察原始卵泡启动生长情况,用免疫组化和Western blot方法检测卵泡活化生长的重要标志物——增殖细胞核抗原(PCNA)的表达情况,以判断原始卵泡在两种培养体系中的生长状态,挑选出适合原始卵泡生长发育的体外培养体系。(2)用原位杂交、RT-PCR和免疫组化方法检测c-erbB2 mRNA和蛋白在原始卵泡启动生长中及在表皮生长因子(EGF)作用下的表达情况。(3)用Western blot方法同步测定p-ERK1/2蛋白在原始卵泡启动生长中及在EGF作用下的表达情况,并分析与c-erbB2 mRNA表达变化的相关关系。结果(1)经组织形态学的比较、各级正常卵泡数量的计算及PCNA表达情况的分析显示,原始卵泡在Waymouth培养体系中存活和生长情况优于DMEM培养体系;并观察到在Waymouth培养体系中加入50ng/ml EGF能进一步促进原始卵泡启动生长。(2)原位杂交结果显示,c-erbB2 mRNA在大鼠原始卵泡生长过程中均有表达,主要位于卵母细胞胞浆内,有随卵泡生长逐渐增加趋势;半定量RT-PCR结果显示,c-erbB2 mRNA表达在2日龄大鼠卵巢培养8d后与培养0d(对照组)相比显著增加(相对光密度值分别为0.297±0.018和0.178±0.011,P<0.05),并在50ng/ml EGF作用下进一步增强;免疫组化结果显示,ErbB2蛋白在大鼠原始卵泡生长过程中均有表达,主要表达于卵母细胞胞膜上,也有随卵泡生长逐渐表达增强的趋势。(3)Western blot结果显示,磷酸化细胞外信号调节激酶1/2 (p-ERK1/2)也随卵泡发育其含量不断增加,在50ng/ml EGF作用下进一步增强。经Spearman相关分析表明,在原始卵泡启动生长过程中,p-ERK1/2含量的变化与c-erbB2 mRNA的表达变化呈显著的正相关关系(rs=0.900,P<0.05)。结论(1)建立了新生大鼠卵巢体外培养体系。(2)原始卵泡中有c-erbB2 mRNA及蛋白的表达,且随原始卵泡的启动生长过程表达增强,EGF在促进原始卵泡启动生长的同时,也使c-erbB2 mRNA及蛋白的表达增强,表明原癌基因c-erbB2在原始卵泡启动生长中起重要作用。(3)在原始卵泡启动生长中,p-ERK1/2含量的变化与c-erbB2 mRNA的表达变化非常一致,两者呈显著的正相关关系,ERK-MAPK信号通路可能在介导原癌基因c-erbB2调控原始卵泡生长中起作用。更多还原

【Abstract】 ObjectiveTo establish the culture system of rat primordial follicles in vitro and study the possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.Methods(1) Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth and the DMEM culture systems; the morphous and the quantity of every stage follicles of ovaries cultured for 0d、4d and 8d were observed by histological sections stained with hematoxylin; the expressions of PCNA in the ovaries were examined by immunohistochemistry and western blot.(2) In-situ hybridization, RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF.(3) Western blot was used to observe the p-ERK1/2 contents during the initiation growth of primordial follicles and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth.Results(1) Compared with the DMEM culture system according to the results of the morphous and the quantity of every stage follicles of ovaries, the expressions of PCNA in the ovaries,the Waymouth culture system is a more ideal model to study the initiation growth of primordial follicles. 50ng/ml EGF could further promote the initiation growth of primordial follicles in the Waymouth culture system.(2) In-situ hybridization showed c-erbB2 mRNA existed in the oocytes endochylema, the expressions of c-erbB2 mRNA appeared to be more intense when primordial follicles were cultured for 8d than cultured for 0d in the Waymouth culture system and were further increased with 50ng/ml EGF for 4d and 8d. The same results were observed by RT-PCR,too. Immunohistochemistry showed ErbB2 existed in the oocytes membrane, the expressions of ErbB2 appeared to be more intense when primordial follicles were cultured for 8d than cultured for 0d in the Waymouth culture system and were further increased with 50ng/ml EGF for 4d and 8d.(3) Western blot analysis showed that p-ERK1/2 protein levels were increased when primordial follicles were cultured for 4d and 8d or supplyment with 50ng/ml EGF. These changes were consistent with the changes of c-erbB2 mRNA and protein. Furthermore, Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF(rs=0.900,P<0.05).Conclusion(1) The culture system of primordial follicles in vitro was successfully established in this study.(2) C-erbB2 mRNA and protein existed in the primordial follicles, the expressions increased as the growth of the primordial follicles and after the effect of EGF, it was suggested proto-oncogene c-erbB2 maybe play an important role during the initiation growth of primordial follicles.(3) There was a significant positive correlation relationship between the contents of p-ERK1/2 and the expressions of c-erbB2 mRNA during the primordial follicles growth, and it was indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.更多还原