FSH对体外培养仔猪睾丸支持细胞skp2表达的影响

The Effect of FSH on the Expression of skp2 in Cultured Immature Boar Sertoli Cells

本研究旨在确定促卵泡素是否可通过cAMP、Ca2+内流和细胞外调节的蛋白激酶(ERK1/2)调节培养条件下未成熟仔猪睾丸支持细胞中S期激酶相关蛋白2(skp2)的表达。以培养的仔猪睾丸支持细胞为实验材料,通过添加各种信号通路的抑制剂,运用Western blot检测skp2、p27kip1蛋白的表达,运用实时荧光定量PCR检测skp2mRNA的表达。结果发现,促卵泡素(50ng.mL-1)以时间依赖的方式促进了skp2蛋白和mRNA的表达(P<0.05),这一作用在30min时达到高峰。FSH(50ng.mL-1)和Forskolin(10μmol.L-1)均促进了skp2蛋白和mRNA的表达(P<0.05),但降低了p27kip1蛋白的水平,而Rp-cAMP、Verapamil和U0126都抑制了FSH的作用,使skp2蛋白和mRNA的表达有所下降,但提高了p27kip1蛋白的表达,但3种抑制剂单独作用时对skp2蛋白、mR-NA以及p27kip1蛋白的表达没有显著影响(P>0.05)。这表明FSH可能主要通过影响cAMP的产生和Ca2+内流,并激活ERK1/2通路调节skp2蛋白的表达,而skp2蛋白的水平与p27kip1蛋白成负相关。

The objective of the study was to identify whether FSH regulated the expression of skp2 mRNA and protein via the Ca2＋ influx,cAMP and extracellular signal-regulated kinase（ERK1/2） pathway in cultured immature boar Sertoli cells.In the study,cultured immature boar Sertoli cells were used as material,and Western blot and Real-time RT-PCR were used to detect the expression of skp2 protein,p27kip1 protein and skp2 mRNA,respectively.FSH（50 ng·mL-1） increased the protein concentration and the mRNA abundance of skp2 from 15 to 90 min（P0.05）.FSH activity was peaking at 30 min,compared to control（P0.05）.Both FSH（50 ng·mL-1） and Forskolin（10 μmol·L-1） increased the level of skp2 protein and mRNA（P0.05）,and reduced the expression of p27kip1 protein.Rp-cAMP,Verapamil and U0126 reduced FSH-induced the expression of skp2 protein and mRNA,but increased the expression of p27kip1 protein（P0.05）,however Rp-cAMP,Verapamil and U0126 alone had no significant effect on the expression of skp2 protein,skp2 mRNA and p27kip1 protein.The result indicate that FSH regulates the expression of skp2 via affecting the production of cAMP,Ca2＋ influx and ERK1/2 activation,and the expression levels of skp2 and p27kip1 protein is negatively correlated.