动物造模 z-bio 2022-02-25 374

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

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

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

　　Conclusion: Overexpression of Foxo3a gene may induce rat ovarian granulosa cells to quiescent in G1 phase by promoting the expression of cyclin D1 and p27 proteins in vitro, and induce granulosa cell apoptosis by increasing the expression of Bim protein. At the same time, overexpression of Foxo3a gene cannot reverse cisplatin in vitro. The resulting apoptosis of granulosa cells, the regulation of follicle development and the prevention of the toxic effect of cisplatin on the ovary need to be further studied in vivo experiments.