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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。
OBJECTIVE: To establish a lung cancer A549 cell model stably overexpressing integrin LK (ILK) and investigate the effect of ILK overexpression on the biological activity of lung cancer A549 cells. Methods: Human ILK gene was amplified by RT-PCR and constructed recombinant plasmid pEGFP-ILK. After identification by restriction enzyme and sequencing, the plasmid pEGFP-ILK was transfected into lung cancer A549 cells by lipofectamine. The stable transfected cells were screened by G418 and expanded (A549 / pEGFP-ILK group) A549 cells (A549 / pEGFP-C1 group) and A549 control group were transfected. The expression and intracellular localization of EGFP-ILK fusion protein were observed by fluorescence microscopy. The transcription level of ILK mRNA in each group was detected by RT-PCR. The expression of ILK protein in each group was detected by Western blot. Cell proliferation was measured by flow cytometry. The apoptosis of cells in each group was detected by flow cytometry. The morphological changes of cells were observed by HE staining. Results: Restriction endonuclease digestion and sequencing confirmed that pEGFP-ILK recombinant plasmid was successfully constructed. The plasmid was transfected into A549 cells and screened by G418 to obtain a stable transfectant expressing green fluorescent protein. The ILK gene mainly localized in the cytoplasm. Compared with the control group, the mRNA and protein expressions of ILK in A549 / pEGFP-ILK cells were significantly increased, the overexpression rates of ILK were 218.18% and 245.45% (P <0.05), respectively. (P <0.05). The level of apoptosis was significantly inhibited (P <0.05). The mitotic phase of A549 cells over-expressing ILK increased. Conclusion: The model of overexpressing ILK lung cancer cells was established successfully. Overexpression of ILK promoted the proliferation, anti-apoptotic and mitotic phases of cancer cells.