摘要
目的研究Foxo3a基因对大鼠卵巢颗粒细胞体外发育的调控及其预防顺铂卵巢毒性作用。方法大鼠原代颗粒细胞体外培养,利用HE染色法及免疫荧光法进行原代颗粒细胞鉴定;构建携带Foxo3a基因的重组腺病毒AD-r Foxo3a,利用RT-PCR及Western-blot法检测Foxo3a表达;实验分为3组:实验组(AD-r Foxo3a组)、阴性对照组(r AD组)及空白对照组(Control组),分别绘制各组细胞生长曲线,Western-blot法检测各组cyclin D1、p27、Bax、Bim蛋白表达,利用流式细胞术、Hoechst33342/PI法分别检测细胞周期及细胞凋亡情况,流式细胞术检测各组细胞加入最适浓度顺铂后细胞凋亡情况。结果(1)体外分离培养大鼠原代卵巢颗粒细胞存活率>90%,细胞纯度>95%;(2)重组腺病毒AD-r Foxo3a感染颗粒细胞36 h后Foxo3a基因在m RNA水平高表达,感染48 h后在蛋白水平高表达;(3)实验组细胞增殖受到抑制,G1期细胞比例及细胞凋亡率较空白对照组及实验对照组增加(P<0.01),后两组差异无统计学意义;(3)实验组Bim、p27、cyclin D1蛋白较对照组高表达,而各组Bax蛋白表达无明显差异;(4)加入顺铂24 h后实验组细胞凋亡率高于空白对照组及实验对照组(P<0.01),后两组细胞凋亡率差异无统计学意义。结论过表达Foxo3a基因在体外可能通过促进cyclin D1和p27蛋白表达诱导大鼠卵巢颗粒细胞静止在G1期,通过增加Bim蛋白表达诱导颗粒细胞凋亡;同时过表达Foxo3a基因在体外不能逆转顺铂导致的颗粒细胞凋亡,其对于卵泡发育的调控及预防顺铂对卵巢的毒性作用还有待体内实验进一步研究。
Objective To evaluate the effect of Foxo3 a gene over- expression on the development of rat ovarian granulosa cells and in prevention of cisplatin- induced ovarian damage in rats. Methods Rat ovarian granulose cells released mechanically from the ovaries were cultured in vitro and identified with HE staining and immunohistochemical staining for FSHR. A recombinant adenovirus carrying Foxo3 a gene was constructed for infecting the granulose cells, and the cell growth and expressions of cyclin D1, p27, Bax, and Bim were detected; the cell apoptosis and cell cycle changes were detected using Hoechst/PI 33342 staining and flow cytometry, respectively. The transfected cells were challenged with cisplatin and the cell apoptosis was detected with flow cytometry. Results Over 90% of the cultured cells survived and contained more than 95%ovarian granulose cells. Infection of the cells with the recombinant adenovirus resulted in over- expressions of Foxo3 a at the m RNA and protein levels at 36 h and 48 h after the infection, respectively. The infected cells showed suppressed proliferation,increased apoptotic rate and cell cycle arrest in G1 phase with increased expressions of Bim, p27, and cyclin D1 but without significant changes in Bax expression. Cisplatin exposure caused a significantly higher apoptosis rate in the infected cells than in the control cells. Conclusion Over- expression of Foxo3 a gene can promote granulose cell apoptosis by increasing Bim expression and cause cell cycle arrest in G1 phase by increasing cyclin D1 and p27 expressions, but can not prevent the toxic effects of cisplatin on ovarian granulosa cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2016年第6期796-801,813,共7页
Journal of Southern Medical University
基金
国家自然科学基金(81041101)
广东省科技计划(2013B021800145)~~
