Islet-1(Isl1),a LIM homeodomain protein,is expressed in the embryonic pancreatic epithelium.As a key transcription factor,Isl1 can not only regulate insulin gene expression in normal glucose condition but also maintai...Islet-1(Isl1),a LIM homeodomain protein,is expressed in the embryonic pancreatic epithelium.As a key transcription factor,Isl1 can not only regulate insulin gene expression in normal glucose condition but also maintainβ-cell function and impact pancreaticβ-cell target genes.Some experiments have suggested that Micro RNA(miRNA)can play a critical role during the induction of insulinproducing cells(IPCs).However,it is unclear whether miRNA may regulate Isl1 expression during differentiation of human umbilical cord mesenchymal stem cells(HUMSCs)into IPCs.In this investigation,we induced HUMSCs into IPCs with a modified two-step protocol,activin A,retinoic acid(step1)and conophylline,nicotinamide(step2).To find the miRNA regulating Isl1 expression,we respectively used Target Scan,miRDB and RNAhybrid to predict and got the result,miR-128 and miR-216a.The miRNAs can inhibit Isl1 expression by dual luciferase assay.The results of real-time Polymerase Chain Reaction(PCR)showed that Isl1 expression level was almost reciprocal to that of miR-128 and miR-216a during differentiation of HUMSCs into IPCs.Furthermore,over-expression of miR-128 or miR-216a downregulated expression levels of Isl1 and Maf A.Therefore,miR-128 or miR-216a may regulate expression of islet-specific transcription factors to control differentiation of HUMSCs into IPCs.展开更多
文摘目的探讨过表达胰岛素基因增强子结合蛋白1(insulin gene enhancer binding protein 1,ISL-1)的慢病毒在体外转染脂肪干细胞(adipose-derived stem cells,ADSCs),能否诱导ADSCs向起搏样细胞分化。方法取第3～5代ADSCs,随机分成Bank、m Cherry和胰岛素基因增强子结合蛋白1(insulin gene enhancer binding protein 1,ISL-1) 3组,按分组分别转染病毒,经荧光强度和流式分析确定最适感染复数,与乳鼠心室肌细胞(neonatal rat cardiomyocytes,NRVMs)共培养7天后进行实时荧光定量聚合酶式反应(quantitative real time polymerase chain reaction,qRT-PCR)、蛋白质印迹、免疫荧光检测分析,期间观察细胞形态和搏动频率变化,并用膜片钳技术记录细胞内电流活动。结果分离贴壁后的ADSCs呈长梭形,慢病毒转染ADSCs的最适感染复数为50。ISL-1组ADSCs形态呈多样化,窦房结特异性基因HCN4、Cx45和Tbx3的mRNA表达水平上调,而工作心肌特异性基因Nkx2. 5下调,组间比较差异有统计学意义(P <0. 05)。ISL-1组大多数细胞可检测到HCN4表达并且可记录到超极化内向电流。结论经ISL-1基因修饰的ADSCs通过体外心肌微环境诱导,产生了一定的高表达窦房结标志性基因并具有细胞内典型超极化电活动的起搏样细胞。
基金Supported by Liaoning Province Education Administration Funded Program of China(LJKZ1374)。
文摘Islet-1(Isl1),a LIM homeodomain protein,is expressed in the embryonic pancreatic epithelium.As a key transcription factor,Isl1 can not only regulate insulin gene expression in normal glucose condition but also maintainβ-cell function and impact pancreaticβ-cell target genes.Some experiments have suggested that Micro RNA(miRNA)can play a critical role during the induction of insulinproducing cells(IPCs).However,it is unclear whether miRNA may regulate Isl1 expression during differentiation of human umbilical cord mesenchymal stem cells(HUMSCs)into IPCs.In this investigation,we induced HUMSCs into IPCs with a modified two-step protocol,activin A,retinoic acid(step1)and conophylline,nicotinamide(step2).To find the miRNA regulating Isl1 expression,we respectively used Target Scan,miRDB and RNAhybrid to predict and got the result,miR-128 and miR-216a.The miRNAs can inhibit Isl1 expression by dual luciferase assay.The results of real-time Polymerase Chain Reaction(PCR)showed that Isl1 expression level was almost reciprocal to that of miR-128 and miR-216a during differentiation of HUMSCs into IPCs.Furthermore,over-expression of miR-128 or miR-216a downregulated expression levels of Isl1 and Maf A.Therefore,miR-128 or miR-216a may regulate expression of islet-specific transcription factors to control differentiation of HUMSCs into IPCs.
