小分子化合物体外诱导肝上皮样干细胞-WB细胞表达PDX1

Small Molecules Induced Hepatic Epithelial-Like Stem Cells WB Cells Expression of PDX1 in Vitro

目的:近年来干细胞治疗糖尿病一直是国内外研究人员关注的焦点,而肝细胞向胰岛样细胞转变也是热点之一。本实验应用小分子化合物在体外诱导WB-F344大鼠来源肝上皮样干细胞(简写WB细胞)表达胰腺内分泌前体细胞基因PDX1,建立一种体外诱导WB细胞分化为胰腺内分泌前体细胞的实验方法。方法:选用5-AZA TSA,RA,ITS等小分子化合物,分两步法直接诱导WB细胞分化为表达PDX1的胰腺内分泌前体细胞,用含有不同浓度5-AZA分化培养基诱导WB分化,摸索诱导分化的最佳条件。观察细胞形态变化,RT-PCR及实时定量PCR检测部分基因表达情况,免疫荧光检测PDX1的表达。结果:5AZA 5 uM处理2 d,TSA 1 d,RA联合ITS诱导7天,诱导的WB细胞表达PDX1较1-4 uM 5-AZA诱导强,并表达胰腺内分泌前体细胞的相关基因,NGN3,Neurod,NKX2.2,WB表达的Nestin仍持续表达,Insulin1有少量表达。WB表达的肝干细胞基因如ALB,AFP大量下调,标志分化的基因C/EBP下调。结论:5-AZA,TSA,RA,ITS等小分子化合物能够诱导肝上皮样细胞WB表达PDX1,并且这种诱导分化的细胞具有胰腺内分泌前体细胞特征。本实验进一步证明在体外微环境中,肝干细胞能向胰腺内分泌细胞转化,而肝细胞增极强。

Objective： Recent years,stem cells in the treatment of diabetes has been the focus of attention of the researchers at home and abroad,and liver cells to islet-like cells shift is also one of the researches hot.To apply hepatic epithelial-like stem cells mall molecule compounds to induce WB-F344 rat Source（WB cells） expression of PDX1 in pancreatic endocrine precursor cells in vitro,and establish an experimental methods that WB cells induced to differentiate into pancreatic endocrine precursor cells in vitro.Methods： 5-AZA,TSA,RA,ITS small molecules compound,a two-step method induced WB cells directly differentiation expression of PDX1 in pancreatic endocrine precursor cells,differentiation medium containing different concentrations of 5-AZA,and explore the best conditions.WB cells morphological changes were observed,RT-PCR and quantitative PCR detect part of the gene expression,immunofluorescence detection of PDX1 expression.Results： 5AZA 5 uM induced 2 d,then TSA treatmented 1 d,RA,ITS treatmented 7 d,WB cells induced expression PDX1 upregulated compared with 5-AZA 1-4 uM and 6-10 uM Related gene expression in pancreatic endocrine precursor cells,NGN3,Neurod,NKX2.2,a small amount Insulin1 expression.Nestin persistent expression.WB expression ALB,AFP large number of downregulated,hepatocytes differentiation gene C/EBP is downregulation.Conclusion： 5-AZA,TSA,RA,ITS compounds capable of inducing WB expression of PDX1,and the induction of WB cells differentiation has pancreatic endocrine precursor cells characterized.The experiments further demonstrated in vitro micro-environment,the liver stem cells can be transformed into pancreatic endocrine cells,hepatocyte growth strong to provide sufficient cell-derived stem cells for future treatment of diabetes.