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信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。
Signal transducer and activator of transcription 5 (STAT5) play an important role in the development of mammary gland, immune response, cell metabolism, hematopoiesis and tumorigenesis, but little is known about the function of islet cells. This study aimed to investigate the effect of STAT5 on insulin secretion in mouse pancreatic islets MIN6 cells and its mechanism. After transfection of small interfering RNA (siRNA) by electroporation, STAT5 gene expression was knocked down by real-time quantitative PCR and Western blotting. Stat5 siRNA transfected cells showed similar mRNA and protein expression levels compared with those transfected with control siRNA 70% and 67% respectively (P <0.01), suggesting that Stat5 gene silencing model was successfully constructed. Enzyme-linked immunosorbent assay combined with Western blotting showed that Stat5 siRNA-transfected cells enhanced insulin secretion by about 1-fold (P <0.05) at different concentrations of glucose stimulation compared with control cells; meanwhile, calmodulin-dependent protein The level of phosphorylation of calmodulin-dependent protein kinase Ⅱ (Ca MKⅡ) also increases. The addition of CaMPⅡ inhibitor AIPⅡ significantly inhibited the increase of cell insulin secretion induced by Stat5 siRNA transfection (P <0.01). The above results show that silencing Stat5 gene can promote the insulin secretion of MIN6 cells by enhancing the phosphorylation of CaMKII.