| Cathepsin B(CTSB)is a member of the lysosomal proteinase family,which participates in the maturation of mammalian oocytes and affects subsequent embryonic development.Granulosa cells are an important part of follicles,which play a regulatory role in follicular development and oocyte maturation.The purpose of this study was to explore the effect of CTSB on the function of sheep granulosa cells and its mechanism.CTSB overexpression vector plasmid pcDNA3.1-CTSB and siRNA inhibiting CTSB expression were constructed,And transfected sheep glume cells cultured in vitro as experimental groups(CTSB overexpression group and CTSB expression inhibition group),A blank group and a negative control group were set up,the proliferation of granulosa cells was detected by CCK8 method,Annexiny-FIICPI method was used to detect apoptosis and cell cycle of granulosa cells,and the expression level of CTSB signaling pathway related genes was detected by qRT-PCR.The effects of CTSB on the proliferation,apoptosis,cell cycle and related genes of sheep ovarian glume were systematically analyzed.The results were as follows:Amplifying the CDS coding region of CTSB gene,the CTSB overexpression vector(pcDNA3.1-CTSB)containing EcoR I+Nhe I double restriction site was constructed,and the RNA(siCTSB)interfering with CTSB gene was synthesized.Fluorescence quantitative PCR and Western Blot were used to detect the overexpression and interference efficiency of CTSB.The results showed that pcDNA3.1-CTSB significantly increased the expression of CTSB,while siCTSB significantly decreased the expression of CTSB,Indicating that the CTSB overexpression vector plasmid pcDNA3.1-CTSB and the siRNA that inhibit CTSB expression were successfully constructed.CCK8 method detected cell proliferation after pcDNA3.1-CTSB and siRNA transfection particle cells 24h,and the results showed that the CTSB over-expression group significantly inhibited particle cell proliferation(P<0.01),while the CTSB expression inhibitor group significantly promoted particle cell proliferation(P<0.01).Annexinv-FITC/PI double-dyed method to detect the apoptosis of granulocyte transfection 24h,the results showed that the apoptosis rate of CTSB over-expression group particle cells increased significantly(P<0.01),and the apoptosis-related gene mRNA was detected RNA expression levels were found to have significantly increased the expression of Bax and Caspase3 apoptosis genes(P<0.01)and the apoptosis gene p53 expression(P<0.05).The difference in Bcl-2 gene expression was not significant(P>0.05),interference inhibition of CTSB gene expression significantly reduced the apoptosis of particulate cells(P<0.01),and significantly increased the Bcl-2 gene Expression(P<0.01),significantly reduced bax,p53 expression(P<0.05),Caspase3 apoptosis gene expression difference was not significant(P>0.05).Results of cell cycle after transfection 24h were detected by flow cytometer:CTSB over-expression resulted in a significant increase in the proportion of granulated cells in G0/G1 cells(P<0.01),and a significant decrease in the proportion of cells in stage S and G2/M(P<0.01)and significantly increased the mRNA expression of cell cycle proteins Cyclin B1,Cyclin D1,p21(P<0.01);There was a significant decrease in the cell ratio in Phase 1(P<0.01),a significant increase in the proportion of cells in Stage S(P<0.01),and no significant difference between the G2/M cell ratio and the control group(P>0.05),while The mRNA expression of cell cycle proteins Cyclin B1 and Cyclin D1 was significantly reduced(P<0.01)and the mRNA expression of cell cycle inhibitor p21 was significantly increased(P<0.05).In summry,CTSB negatively regulates granulosa cell proliferation and cell cycle progression by affecting the expression of Cyclin B1,Cyclin D1 and p21 genes,and positively regulates granulosa cell apoptosis by affecting the expression of Bax and p53 genes.The results can provide a theoretical basis for an in-depth study of CTSB gene regulation of sheep follicle development.. |
