去乙酰化酶SIRT1过表达对猪卵巢颗粒细胞中AMPK活性的影响

Effect of the overexpression from deacetylase gene SIRT1 on AMPK activity in porcine granulosa cells

构建猪SIRT1基因全长编码区(coding region sequence,CDS)的真核表达载体,转染猪原代卵巢颗粒细胞,探讨SIRT1基因过表达对猪卵巢颗粒细胞中AMPK基因的转录及其蛋白活性的影响。测序结果表明,猪SIRT1基因的CDS区全长大小为2 229 bp,与NCBI发布的猪SIRT1基因m RNA序列(EU030283.2)一致;转染p EGFP–C1–SIRT1载体的颗粒细胞中SIRT1的m RNA表达水平和蛋白表达水平极显著高于空载体对照组(P<0.01);p EGFP–C1–SIRT1转染组细胞中,AMPK–α1和AMPK–α2基因的m RNA表达量显著高于空载体对照组,且细胞中AMPKαThr172位点的磷酸化水平显著升高(P<0.05)。结果表明,体外培养的原代猪卵巢颗粒细胞中的SIRT1基因过表达使AMPK的表达显著增加,影响AMPK的活性,推测SIRT1可能通过AMPK在猪卵巢颗粒细胞凋亡过程中发挥重要的调控作用。

A eukaryotic expression vector of full-length gene SIRT1 was constructed and transfected into porcine primary ovary granulosa cells for investigating the effects of overexpression from gene SIRT1 on AMPK m RNA expression and its activity. The results showed that the full-length coding sequence of porcine gene SIRT1 was identical to NCBI reference sequence（EU030283.2）, it had 2 229 bp. Both m RNA and protein expression level of gene SIRT1 were significantly increased in the group transfected with p EGFP–C1–SIRT1（P〈0.01）. The m RNA expression of AMPK–α1 and AMPK–α2 were both significantly increased, so did the phosphorylation level at Thr172 sites in AMPKα（P〈0.05）. The overexpression of gene SIRT1 was significantly increased the AMPK expression and its activity in ovarian granulosa cells via in vitro cultured from primary porcine. These results suggested that SIRT1 might play an important role in regulating the apoptosis of porcine granulosa cell through activating AMPK.