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目的拟利用转化生长因子β1(TGF-β1)诱导人肝癌细胞发生上皮-间质转换(epithelial-mesenchymal transition,EMT),建立EMT模型。方法在低血清培养条件下,用TGF-β1处理肝癌细胞2~6 d,观察细胞形态变化,采用Western印迹检测EMT典型标志物的表达变化,流式细胞术检测肝癌肿瘤干细胞标志物CD13和Ep CAM的表达变化,并利用微小RNA芯片技术(miRNA chip)筛选和分析TGF-β1处理前后差异表达的miRNA。结果经10 ng/ml TGF-β1诱导2~6 d后,多数细胞形态由上皮样转变为成纤维样,细胞间隙明显增大;Western印迹定量分析结果显示,10 ng/ml TGF-β1处理后,肝癌细胞E-钙黏蛋白(cadherin)表达水平显著性下调;流式细胞术结果显示,CD13和Ep CAM显著性上调;对miRNA芯片结果分析得到35种差异表达比较显著的miRNA,经过生物信息学分析,这些差异表达的miRNA在转录调控中发挥了重要作用。结论在低血清培养条件下,用10 ng/ml TGF-β1处理肝癌细胞2~6 d,细胞发生明显的EMT过程,利用该法可成功建立肝细胞癌的体外EMT细胞模型,在此模型中,可对差异表达的miRNA进行筛选分析和进一步的功能研究。
OBJECTIVE: To induce the epithelial-mesenchymal transition (EMT) of human hepatocarcinoma cells by transforming growth factor-β1 (TGF-β1) and establish EMT model. Methods The hepatocellular carcinoma cells were treated with TGF-β1 for 2 ~ 6 days under low serum culture conditions. The morphological changes of the cells were observed. Western blotting was used to detect the expression of EMT markers. Flow cytometry was used to detect CD13 and Ep CAM expression changes, and the use of microRNA microarray (miRNA chip) screening and analysis of TGF-β1 differentially expressed miRNA before and after treatment. Results After 2 ~ 6 days of induction with 10 ng / ml TGF-β1, most of the cells changed from epithelial to fibroblast and the interstitial space increased significantly. Western blot analysis showed that after 10 ng / ml TGF-β1 treatment , And the expression levels of E-cadherin in hepatocellular carcinoma cells were significantly down-regulated. The results of flow cytometry showed that CD13 and Ep CAM were significantly up-regulated. Among the 35 differentially expressed miRNAs, miRNAs were significantly differentially expressed, According to the analysis, these differentially expressed miRNAs play an important role in transcriptional regulation. Conclusions Under low serum culture conditions, the hepatoma cells treated with 10 ng / ml TGF-β1 for 2-6 d have obvious EMT process. The EMT cell model of hepatocellular carcinoma can be successfully established by this method. In this model , Can differentially expressed miRNA screening analysis and further functional studies.