过表达SLC7A5基因对大鼠卵巢颗粒细胞凋亡的影响及其机制OA北大核心CSTPCD

Objective:To discuss the effect of over-expression of solute carrier family 7 member 5(SLC7A5)gene on the apoptosis of ovarian granulosa cells of the rats,and to clarify its related mechanism.Methods:Four 3-week-old SPF grade SD female rats were used to extract the primary ovarian granulosa cells of the rats.These cells were divided into negative control group(NC group)and follicle-stimulating hormone receptor(FSHR)staining group(FSHR group).Immunofluorescence staining was used to detect the expressions of FSHR protein in the ovarian granulosa cells of the rats to identify the successful isolation of the primary ovarian granulosa cells of the rats.The ovarian granulosa cells were divided into control group(transfected with empty vector plasmid)and OE-SLC7A5 group(transfected with SLC7A5 over-expression plasmid).Real-time fluoresscence quantitative PCR(RT-qPCR)and Western blotting methods were used to verify the transfection efficiency of the cells;flow cytometry was used to detect the apoptotic rates and cell cycle percentages of the ovarian granulosa cells in two groups;RT-qPCR method was used to detect the expression levels of SLC7A5,cysteinyl aspartate specific proteinase(Caspase)-3,Caspase-8,and tumor necrosis factor-α(TNF-α)mRNA in the ovarian granulosa cells in two groups;Western blotting method was used to detect the expression levels of SLC7A5,Caspase-3,cleaved Caspase-3,Caspase-8,cleaved Caspase-8,and TNF-α proteins in the ovarian granulosa cells in two groups.Results:The fluorescence microscope observation results showed that the ovarian granulosa cells appeared spindle-shaped or irregular and specifically expressed FSHR.No FSHR green fluorescence was observed in NC group,while FSHR green fluorescence expression was observed in FSHR group,indicating successful isolation of primary ovarian granulosa cells of the rats.Compared with control group,the expression levels of SLC7A5 mRNA and protein in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05),indicating successful transfection of SLC7A5 over-expression plasmid into the ovarian granulosa cells.The flow cytometry results showed that compared with control group,the apoptotic rate of the cells in OE-SLC7A5 group was significantly increased(P<0.05).Compared with control group,the percentage of the ovarian granulosa cells at S phase in OE-SLC7A5 group was significantly decreased(P<0.05).The RT-qPCR results showed that compared with control group,the expression levels of TNF-α,Caspase-3,and Caspase-8 mRNA in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of TNF-α,Caspase-8,cleaved Caspase-8,Caspase-3,cleaved Caspase-3,and SLC7A5 proteins in the ovarian granulosa cells in OE-SLC7A5 group were significantly increased(P<0.05).Conclusion:The increased expression of SLC7A5 protein promotes the apoptosis of the granulosa cells by upregulating the expressions of TNF-α,Caspase-8,and Caspase-3 apoptotic pathways.