过表达NK4对人肺腺癌A549细胞增殖和凋亡的影响

目的:构建含有NK4基因的重组慢病毒载体(LV-NK4),感染人肺腺癌A549细胞后观察NK4基因的表达,并研究NK4对肺癌A549细胞增殖和凋亡的影响?方法:采用DNA重组技术,将NK4基因克隆至带增强型绿色荧光蛋白(EGFP)的慢病毒载体GV358,脂质体介导法将其与慢病毒包装系统共转染293T细胞,包装为慢病毒,感染A549细胞,观察感染效率?采用逆转录聚合酶链反应(RT-PCR)及免疫印迹法(Western blot)检测NK4基因和蛋白的表达?建立A549(空白对照组)?A549/NK4(实验组)?A549/LV(空病毒对照组)3组细胞;RT-PCR 法测定各组细胞c-met基因水平;噻唑蓝(MTT)比色法测定各组细胞第1~7天增殖情况,绘制生长曲线;流式细胞术检测各组细胞凋亡率?结果:经基因测序证实LV-NK4构建成功;感染后A549细胞中可见明显的NK4蛋白表达,Western blot显示50 000处有蛋白条带;RT-PCR结果显示实验组NK4 mRNA水平较空白对照组和空病毒对照组明显升高(P < 0.01),c-met mRNA水平较其他两组下降(P < 0.05);MTT结果显示实验组细胞从第4天起生长比正常对照组和空病毒对照组均缓慢(P < 0.05);流式细胞术结果显示实验组细胞凋亡率高于正常对照组和空病毒对照组(P < 0.05)?结论:成功构建NK4慢病毒载体且有效转染A549细胞;NK4具有抑制A549细胞增殖并促进其凋亡的作用,可能与下调c-met基因水平有关?

Effects of overexpression of NK4 on cell proliferation and apoptosis of human adenocarcinoma A549 cells

Objective:To study the transfection and expression level of human NK4 gene in A549 cells by constructing NK4 of recombinant lentiviral vector, and observe the effect of NK4 on cell proliferation and apoptosis. Methods: The NK4 gene was cloned into lentiviral expression vector by recombining DNA technology. The recombinant plasmid was cotransfected with lentiviral packaged systems in 293T cells by lipofectin reagent to produce lentiviral particles. A549 cells were infected with the lentivirus, and the infection efficiency was observed under fluorescence microscope. Expression of NK4 gene and protein was identified by RT-PCR and Western blot, respectively. Three groups of cells were established, including the A549/NK4 group, the negative control group (A549/LV), and the blank control group (A549). Expression of c-met mRNA in the three groups of cells was examined by RT-PCR analysis. Through MTT colorimetric, we assayed the growth of the three groups of cells from the first day to the seventh day, and drew a growth curve to compare the proliferations of cells. The rates of apoptosis of the cells were detected by flow cytometry. Results: LV-NK4 transfected A549 cells expressed NK4, and the cells expressed less c-met than the blank and negative group. In MTT test, the A549/NK4 group cells grew slower than the other group from the fourth day (P < 0.05). With flow cytometry, the apoptosis rate of the A549/NK4 group was higher than the blank and negative control group. Conclusion: The NK4 recombinant lentiviral vector had been successfully constructed and effectively transfected A549 cells. NK4 can inhibit the proliferation and promote the apoptosis of A549 cells, the mechanism may be related to down-regulated c-met.