基于TALE技术的哈萨克族食管上皮DNA甲基转移酶1高表达细胞株模型的构建

Construction of esophageal epithelial cell with high DNA methyltransferase 1 expression

目的:构建哈萨克族食管上皮细胞DNA甲基转移酶1(DNMT1)高表达细胞株模型。方法:将构建的WV0133、WV0132和WV072质粒转染至哈萨克族食管上皮细胞,利用嘌呤霉素对DNMT1高表达细胞进行筛选,并通过荧光显微镜、实时荧光定量PCR(q PCR)和Western blot法检测细胞转染情况和目的基因DNMT1的表达情况。结果:WV0133和WV0132质粒转染组细胞DNMT1m RNA表达水平是正常细胞组的1.786倍和2.721倍,DNMT1蛋白表达水平是正常细胞组的2.734倍和3.100倍,均显著高于正常细胞组(P<0.01)。其中,WV0132质粒转染组细胞DNMT1 m RNA和蛋白表达水平较高。结论:成功构建了哈萨克族食管上皮DNMT1高表达细胞株模型,为深入研究食管癌的发生机制提供了研究工具。

OBJECTIVE：To construct esophageal epithelial cells which express high levels of DNA methyltransferase 1（DNMT1）.METHODS：Plasmids WV0133,WV0132 and WV0132 were constructed and transfee ted into esophageal epithelial cells.Transfection of the cells was observed under the inverted fluorescence microscope.Cells that expressed high levels of DNMT1 were selected by puromycin.Expressions of DNMT1 mRNA and protein were determined by using quantitative real-time PCR and Western blot.RESULTS：Compared with the normal cell group,the mRNA expression levels were 1.786 times and 2.721 times in the WV0133- and WV0132-transfected cells,respectively,and their protein expression levels were 2.734 times and 3.100 times,respectively.They were significantly higher than those in the normal cell group（P〈0.01）.CONCLUSION：An esophageal epithelial cell line which expressed high levels of DNMT1 was successfully constructed.The cell line can be a valuable tool to study mechanisms for esophageal cancer development.