湖羊PGR基因对卵泡颗粒细胞体外增殖与凋亡的影响

旨在探究孕酮受体(progesterone receptor,PGR)基因对湖羊卵泡颗粒细胞体外增殖与凋亡的影响。利用RT-PCR技术扩增和克隆获得PGR基因编码序列(CDS),通过生物信息学软件对其氨基酸序列及同源性进行比对;用所获得序列构建过表达载体和干扰siRNA,分别转染湖羊颗粒细胞,并用CCK8技术检测颗粒细胞的细胞活力;采用RT-qPCR和Western blot技术,检测细胞周期和凋亡的相关基因或蛋白表达水平。结果显示,羊PGR基因的CDS区全长2736 bp,编码911个氨基酸;与其他物种氨基酸序列的同源性为39.66%~95.44%。干扰PGR基因通过下调CDK4、Bcl-2和上调Caspas3、Caspase8、BAX基因mRNA的表达(P<0.05),进而抑制颗粒细胞的增殖,但对CyclinD1未产生影响(P>0.05);过表达PGR基因通过上调CDK4、CyclinD1、Bcl-2和下调Caspase3、Caspase8、BAX基因mRNA的表达(P<0.05),进而促进颗粒细胞的增殖;BAX蛋白表达变化与对应mRNA的表达趋势一致(P<0.05);过表达PGR基因显著上调PCNA基因表达,而干扰PGR基因则下调PCNA蛋白表达(P<0.05)。研究表明,PGR基因通过调控细胞周期和凋亡关键基因的表达,影响湖羊颗粒细胞的增殖与凋亡,进而调节湖羊卵泡发育。

Effects of PGR on proliferation and apoptosis in vitro of Hu sheep graunlosa cells

This experiment was to investigate the effects of the progesterone receptor(PGR)gene on in vitro proliferation and apoptosis of Hu sheep granulosa cells(GCs)by overexpressing and silencing.The coding sequence(CDS)of PGR gene was amplified and cloned using RT-PCR,and its amino acid sequence and homology analyzed using a bioinformatic software.According to the sequence of the in vitro proliferation obtained,PGR overexpression vector and siRNA were constructed,and they were transfected into Hu sheep GCs.Next,CCK8 was used to detect the viability of the cell proliferation.The mRNA and protein expression levels of the cell cycle-and apoptosis-related genes were detected using RT-qPCR and Western blot.The results showed that the CDS of PGR was 2736 bp,and it encoded 911 amino acids.The PGR amino acid sequence showed a 39.66%-95.44% homology with the sequences of proteins from the other species.Knockdown PGR significantly inhibited the proliferation of GCs by down-regulating the mRNA expression of CDK4,Bcl-2 and up-regulating Caspas3,Caspase8 and BAX(P<0.05);these had no effect on CyclinD1(P>0.05).Overexpressed PGR significantly promoted GC proliferation by up-regulating the mRNA expression of CDK4,CyclinD1,Bcl-2 and down-regulating Caspase3,Caspase8 and BAX(P<0.05).The protein expression of BAX was consistent with its mRNA expression(P<0.05).Overexpressed PGR significantly up-regulated the expression of PCNA,while the interfering PGR significantly down-regulated the expression of PCNA(P<0.05).To sum up,PGR affected the proliferation and apoptosis of GCs by regulating the cell cycle-and apoptosis-related genes,and further influenced follicular development in Hu sheep.