RNA干扰沉默Ghrelin基因对仔猪胃酸分泌的调节作用  

Regulating effect of RNAi-mediated Ghrelin gene silencing on gastric acid secretion in piglets

作　　者：杜改梅[1] 胡志华[1] 罗碧平[1] 吴结革[1] 张淼[1] 晏文梅[1] 刘茂军[2] 

机构地区：[1]金陵科技学院动物科学与技术学院,江苏金陵210038 [2]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,南京210014

出　　处：《南方农业学报》2016年第8期1396-1400,共5页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31302054);江苏省自然科学基金项目(BK20131086)

摘　　要：【目的】研究生长素(Ghrelin)基因沉默对仔猪胃酸分泌的调节作用,为防治仔猪断奶后某些消化系统功能障碍或分泌异常研究提供新靶点,进而阐明Ghrelin对胃酸分泌的调节作用机制。【方法】采用脂质体将RNA干扰沉默的shGhrelin转染体外培养的猪胃黏膜上皮细胞,优化确立最佳转染条件,并通过荧光定量RT-PCR检测shGrelin转染对胃黏膜上皮细胞中Ghrelin基因mRNA表达及H+-K+-ATPase(质子泵)活性的影响。【结果】质粒(sh Ghrelin)与脂质体(Lipofectamine 2000)的最佳转染比例为1∶3,最佳转染时间24 h。与正常对照组相比,以shGhrelin转染胃黏膜上皮细胞后,其Ghrelin基因m RNA的表达显著降低(P<0.05,下同),H+-K+-ATPase活性也显著下降。【结论】采用RNA干扰技术能沉默胃黏膜上皮细胞Ghrelin基因表达和抑制H+-K+-ATPase活性,表明Ghrelin对胃酸分泌具有重要的调节作用。[Objective]The regulating effect of Ghrelin gene silencing on gastric acid secretion were investigated, in order to provide new target for digestive system dysfunctions or diacrisis of weaning piglet, and then clarify regulation mechanism of ghrelin in the gastric aeid secretion. [Method]shGhrelin was transfected into in vitro poreine gastric mucosal cells using liposome, and the transfection conditions were further optimized. The effects of shGhrelin transfection on Ghre- lin gene mRNA expression and H＋-K＋-ATPase （proton pump） activity were detected by fluorescent quantitative RT-PCR. [Result]The results showed that, the optimal transfection ratio of plasmid （shGhrelin） to lipofectamine 2000 was 1：3, and the optimal transfection time was 24 h. Furthermore, after shGhrelin was transfected into in vitro porcine gastric mucosal cells, Ghrelin gene mRNA expression and H＋-K＋-ATPase activity were reduced significantly（P〈O.05）, compared with control group. [Conclusion ] Ghrelin gene expression is silenced successfully by using RNA interference （RNAi） technology, and H＋-K＋-ATPase activity is inhibited. It indicated that ghrelin plays an important role in regulating gastric acid secretion.

关 键 词：生长素(Ghrelin) 胃黏膜上皮细胞 胃酸分泌 RNA干扰 

分 类 号：S828[农业科学—畜牧学]