| Granulosa cells can promote the activation of primordial follicles in the ovary,regulate the development of follicles during the anagen phase,regulate estrogen production in response to follicle stimulating hormones,and also induce apoptosis of follicles.The in vitro culture of granulosa cells can be used to study some genes and m RNA interaction mechanisms related to reproductive traits,but its inability to be cultured in vitro for a long period of time has limited some of the related scientific studies.In this study,we conducted an in vitro culture test on the ovarian granulosa cells of Shaanbei White Cashmere Goats to optimize the isolation method,culture conditions,and to investigate the freezing and recovery of the cells after passaging,in order to establish a good primary and passaging culture system and to provide more options and basis for the isolation and culture method of granulosa cells.The results of this study are as follows:1.The cell viability obtained by syringe aspiration method for isolating ovarian granulosa cells is higher than that of the dissection method,but the dissection method can obtain a larger number of cells;cell viability was detected by CCK-8 assay and cell proliferation by Ed U doping method,and the cell viability of MEM group was extremely significantly higher than that of DMEM and DMEM/F12 group(P<0.05),and the proliferation ability was significantly higher than that of DMEM(P<0.05);RT-PCR results showed that The expression of PCNA Cyclin D1 in the MEM group was significantly higher than the other two groups(P<0.05),the expression of CDK2 was highly significantly higher than the other two groups(P<0.01),the expression of BAX/BCL was highly significant lower than the other two groups,and the expression of caspase3 was lower than the other two groups(P<0.01).When the volume ratio of serum,culture medium and DMSO in the lyophilization solution was 5:4:1,the cell survival rate of GCs after resuscitation was40.77%±1.32,and the 24 h apposition rate was significantly higher than that of serum with40% and 20% of the lyophilization solution(P<0.05).2.The most suitable concentration of EGF for GCs cultured in vitro was 10 ng/m L.RT-PCR results showed that the addition of EGF up-regulated the expression of Cyclin D1 and down-regulated the expression of BAX/BCL and P21;the most suitable concentration of b FGF was 10ng/m L,the addition of b FGF up-regulated the expression of PCNA and Cyclin D1 expression level,otherwise,BAX/BCL expression were down-regulated by the addition of b FGF;the secretion of estrogen and progesterone in the test group and the control group after the addition of growth factors was not significantly different by ELISA(P>0.05),the expression of granulosa cell differentiation and steroid hormone production-related genes such as St AR,3βHSD and CYP11A1 was extremely significantly lower than that of the blank group(P<0.01).3.After transduction of GCs cultured in vitro with the lentiviral plasmid Plox-Ttag-ires TK,the cells maintained normal growth morphology,and the fluorescence detection results showed successful viral transduction,and the whole genome of ovarian granulosa cells was extracted and successful expression of SV40 T gene was detected.In summary,ovarian granulosa cells isolated by aspiration method and supplemented with MEM medium culture can achieve effective in vitro culture of ovarian granulosa cells from Shaanbei White Cashmere Goat;after the addition of growth factors,in vitro cultured ovarian granulosa cells can be better passaged and cultured with no significant changes in steroid hormone secretion and no luteinization,transferred into lentiviral plasmid Plox-Ttag-ires TK,successful expression of SV40 T gene was detected. |
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