稳定转染Dicer基因对HELF细胞功能的影响

Effect of Stable Transfection of Dicer Gene on the Function of Human Embryonic Luag Fibroblasts

目的观察正常人胚肺成纤维细胞(HELF)在稳定转染Dicer基因后增殖能力及迁移能力的变化。方法应用脂质体介导方法转染Dicer基因表达载体于HELF细胞,G418筛选,PCR检测整合情况,RT-PCR检测表达情况,Western-blot检测蛋白表达水平,确定稳定转染细胞株,MTT方法检测细胞增值能力,Transwell方法检测细胞迁移能力。结果Dicer基因稳定转染HELF细胞系建立,用RT-PCR和Western-blot方法检测到Dicer基因mRNA和蛋白表达水平都明显增强。与转染空载体的HELF细胞相比,转染Dicer基因的HELF细胞48、72、96h的MTT光吸收值明显升高(P<0.05),并且穿过人工重构基底膜的细胞数明显增多。结论Dicer基因稳定转染HELF细胞后,HELF细胞增殖能力和迁移能力都明显增强。

Objective To observe the changes of cell proliferation and migration abilities of the normal human embryonic lung fibroblasts（HELF） after stable transfection of Dicer gene.Methods The transfection of Dicer gene into HELF cells was done by liposome-mediated method.G418 was used to screen HELF cells stably expressing Dicer gene.PCR was used to detect the integration of Dicer gene to HELF cells.RT-PCR was used to detect the expression of mRNA,and Western blot was used to determine the Dicer protein expression level.MTT was used to detect the proliferation ability of HELF cells stably expressing Dicer gene,and the cell migration ability was detected by transwell chamber assay.Results The HELF cells stably expressing Dicer gene was established.A significant increase of the mRNA and protein expression levels of gene Dicer in HELF cells stably expressing Dicer was shown by RT-PCR and Western-blot.Compared with the empty vector-transfected cells,MTT absorption of HELF cells stably expressing Dicer was significantly higher at 48,72 and 96 hours（P 0.05）,and the cells through Matrigel were prominently increased.Conclusions The cell proliferation and migration ability of Dicer gene stably transfected HELF cells are markedly increased.