水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究

Function Analysis of Buffalo BMP1 Gene on Proliferation and Apoptosis of Granulosa Cells

本研究旨在探讨BMP1基因对体外培养水牛颗粒细胞增殖与凋亡的影响,采用qRT-PCR检测体外培养0、24、48、72和96h水牛颗粒细胞中BMP1基因的表达规律,通过在水牛颗粒细胞培养液中添加BMP1基因重组蛋白和抗BMP1抗体,观察其对体外培养水牛颗粒细胞增殖变化的影响,并应用qRT-PCR和Western blotting方法检测与细胞增殖和细胞凋亡相关因子表达的变化规律。结果显示,随着颗粒细胞体外培养时间的延长,BMP1基因的相对表达量显著上调(P<0.05),96h培养组BMP1表达最高。添加BMP1重组蛋白可显著促进水牛颗粒细胞的体外增殖(P<0.05),并能够显著上调细胞周期关键蛋白Cyclin D1、Cyclin D2和PCNA mRNA表达水平(P<0.05),同时显著抑制了细胞凋亡信号通路中促凋亡相关因子(Fas、FasL、TNFR1、TNF-α、Cytochrome C、Apaf1、Chop、Caspase-3、Caspase-8和Caspase-9)mRNA表达(P<0.05)。Western blotting检测结果表明,BMP1重组蛋白能够抑制Fas、Bax和Caspase-9蛋白表达,蛋白与mRNA表达一致。而添加抗BMP1抗体的试验则得出相反的结果。综上表明,BMP1基因能通过调控相关基因的表达促进体外培养水牛颗粒细胞的增殖并抑制其凋亡,在水牛卵泡发生过程中具有重要的调控功能,为阐明BMP1基因参与家畜卵泡发生的分子机制奠定基础。

This study focus on the affection of BMP1 gene on proliferation and apoptosis of in vitro cultured buffalo granulosa cells.Expression pattern of BMP1 in different in vitro cultured time of buffalo granulosa cells including 0,24,48,72 and 96hwere determined by qRT-PCR separately.Different concentrations of BMP1 recombinant protein or BMP1 antibody were added to the culture medium,and the effect to proliferation of granulose cells were detected,further effect on expression pattern of cell proliferation and apoptosis associated genes were also analyzed by qRT-PCR and Western blotting.BMP1 gene was significantly increased in the in vitro cultured granulosa cells with the extended recording time(P<0.05),and reached a peak at 96 h.Adding BMP1 recombinant protein significantly promoted the proliferation of granulosa cells(P<0.05),as well as up-regulated the expression of Cyclin D1,Cyclin D2 and PCNAgenes in mRNA level.In addition,mRNA expression level of pro-apoptosis factors(Fas,FasL,TNFR1,TNF-α,Cytochrome C,Apaf1,Chop,Caspase-3,Caspase-8and Caspase-9)were down regulated.Further-more,Western blotting results showed that the protein expression level of Fas,Baxand Caspase-9was also down-regulated.The contrast results were observed by adding BMP1 antibody investigation.BMP1 promotes proliferation and inhibits apoptosis of in vitro cultured buffalo granulosa cells by regulation the expression pattern of associated genes,which indicated that it took an important role in the follicular development of buffalo,and laid a theoretical foundation to clarify the livestock follicular genesis.