过表达整合素连接激酶肺癌A549细胞的建立及生物学活性

Establishment of a lung cancer A549 cell line stable over-expressing integrin linked kinase protein and its biological activity

目的:建立稳定过表达整合素连接激酶(ILK)的肺癌A549细胞模型,探讨ILK过表达对肺癌A549细胞生物学活性的影响。方法:以RT-PCR法扩增人ILK基因,构建pEGFP-ILK重组质粒。经酶切及测序鉴定后,将pEGFP-ILK质粒用脂质体转染肺癌A549细胞,G418压力筛选出稳定转染细胞克隆并扩大培养(A549/pEGFP-ILK组),设pEGFP-C1空质粒转染A549细胞(A549/pEGFP-C1组)及空白A549细胞对照组。用荧光显微镜观察EGFP-ILK融合蛋白的表达及细胞内定位,RT-PCR法检测各组细胞中ILK mRNA的转录水平,Western blot法检测各组细胞中ILK蛋白表达水平,MTT比色法检测各组细胞的增殖活力,流式细胞术检测各组细胞的凋亡情况,HE染色观察细胞形态变化。结果:酶切及测序证实,pEGFP-ILK重组质粒构建成功。该质粒转染A549细胞、并经G418压力筛选后,获得了表达绿色荧光蛋白的稳定转染株,ILK基因表达产物主要定位于细胞质内。与对照组相比,A549/pEGFP-ILK细胞ILK的mRNA及蛋白表达水平明显升高,其过表达率分别为218.18%和245.45%(P<0.05);过表达ILK的A549细胞增殖活力显著增强(P<0.05);凋亡水平被显著抑制(P<0.05);过表达ILK的A549细胞的核分裂相增多。结论:成功建立过表达ILK肺癌细胞模型,ILK过表达可促进癌细胞增殖、抗凋亡及核分裂相增多。

AIM： To establish a lung cancer A549 cell line stable over-expressing human integrin linked kinase （ILK） and study the effect of over-expression of ILK on biological activity of A549 cells. METHODS： Human ILK gene was amplified by RT-PCR, then cloned into pEGFP-C1 vector to construct pEGFP-ILK. After confirmed by restriction analysis and sequencing, the recombinant plasmid was transtected into A549 cells mediated with liposome, then G418-resistant clones of A549 cells （A549/pEGFP-ILK） as experimental group were obtained, and paralleled with the vector control （A549/pEGFP-C1） and A549 cell control. The expression and localization of EGFP-ILK fusion protein in A549 cells was observed by fluorescence microscopy. RT-PCR and Western blot were performed to detect the level of ILK mRNA and ILK protein of each group cells respectively. The cell proliferation was tested by methylthia- zolyl tetrazolium （MTT） assay, and the cell apoptosis was measured by flow cytometry, and the morphologic changes of cells were observed by HE staining. RESULTS： Both restriction analysis and sequencing proved that the pEGFP-ILK plasmid was constructed correctly. The distribution of fluorescence of stable transfected A549 cells indicated that the product of ILK gene was mainly located in cytoplasm. Compared with A549/pEGFP-C1 group and A549 group, the level of ILK mRNA and ILK protein of A549/pEGFP-ILK cells were significantly increased, which over-expression ratio was 218.18% and 24.5.4.5% respectively （ P 〈 0.0.5 ）. The proliferation ability of the A549 cells over-expressing ILK was increased significantly （ P 〈 0.05 ）. However, the apoptosis of A549/pEGFP-ILK cell was inhibited significantly by over-expression of ILK （P〈0.05）. After HE staining, the increased mitosis were observed only in A549/pEGFP-ILK group cells. CONCLUSION： The lung cancer cell line stable over-expressing ILK protein was constructed successfully, and ILK over-expression could promote cell proliferation, inhibit cell apoptosis and increase mitosis.