Foxo3a基因 z-bo 2020-11-12 395

　　Objective: To study the regulation of Foxo3a gene on the development of rat ovarian granulosa cells in vitro and its prevention of cisplatin ovarian toxicity.

　　Methods: Rat primary granule cells were cultured in vitro, and primary granule cells were identified by HE staining and immunofluorescence; recombinant adenovirus AD-rFoxo3a carrying Foxo3a gene was constructed, and Foxo3a expression was detected by RT-PCR and Western-blot methods The experiment is divided into 3 groups: experimental group (AD-rFoxo3a group), negative control group (rAD group) and blank control group (Control group). The cell growth curves of each group are drawn respectively, and the Western-blot method detects cyclin D1 in each group. The expression of p27, Bax and Bim proteins were detected by flow cytometry and Hoechst33342/PI method to detect cell cycle and apoptosis. Flow cytometry was used to detect the apoptosis of each group of cells after adding the optimal concentration of cisplatin.

　　Results: (1) The survival rate of rat primary ovarian granulosa cells isolated and cultured in vitro was >90%, and the cell purity was >95%; (2) The Foxo3a gene was highly expressed at the mRNA level after the recombinant adenovirus AD-rFoxo3a infected granulosa cells for 36 h. Highly expressed at protein level after 48 hours of infection; (3) The cell proliferation of the experimental group was inhibited, and the proportion of cells in G1 phase and the apoptosis rate were increased compared with the blank control group and the experimental control group (P<0.01). There was no statistical difference between the latter two groups Scientific significance; (3) The Bim, p27, and cyclin D1 proteins of the experimental group were higher than those of the control group, but there was no significant difference in the expression of Bax protein in each group; (4) The apoptosis rate of the experimental group was higher than that of the blank control after adding cisplatin for 24 hours Group and experimental control group (P<0.01), there was no significant difference in apoptosis rate between the latter two groups.

　　Conclusion: Overexpression of Foxo3a gene in vitro may induce rat ovarian granulosa cells to quiescence in G1 phase by promoting the expression of cyclin D1 and p27 protein, and increase Bim protein expression to induce apoptosis of granulosa cells; at the same time, overexpression of Foxo3a gene cannot reverse cisplatin in vitro The resulting granulosa cell apoptosis, its regulation of follicular development and the prevention of cisplatin’s toxicity to the ovaries need to be further studied in vivo.