论文部分内容阅读
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 m RNA水平较空白对照组和空病毒对照组明显升高(P<0.01),c-met m RNA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。
OBJECTIVE: To construct a recombinant lentiviral vector containing the NK4 gene (LV-NK4) and to investigate the effect of NK4 on the proliferation and apoptosis of lung cancer A549 cells. Methods: NK4 gene was cloned into lentiviral vector GV358 with enhanced green fluorescent protein (EGFP) by DNA recombination technique. 293T cells were co-transfected with lentivirus packaging system and packaged as lentivirus , Infected A549 cells and observed the infection efficiency. The expression of NK4 gene and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. A549 / NK4 (experimental group) and A549 / LV (empty virus control group) were established. Cell c-met gene levels were determined by RT-PCR. MTT colorimetric assay The proliferation of cells in each group was measured from day 1 to day 7, and the growth curve was drawn. The apoptosis rate of each group was detected by flow cytometry. Results: The successful construction of LV-NK4 was confirmed by gene sequencing. The expression of NK4 protein was obvious in A549 cells after infection, and Western blot showed 50 000 protein bands. RT-PCR results showed that the level of NK4 m RNA in experimental group was significantly higher than that in control group (P <0.01). The level of c-met m RNA in the experimental group was significantly lower than that in the other two groups (P <0.05). The MTT results showed that the cells in the experimental group grew more on day 4 than the control group and empty virus The control group was slow (P <0.05). The results of flow cytometry showed that the apoptosis rate of the experimental group was higher than that of the normal control group and empty virus control group (P <0.05). CONCLUSION: NK4 lentiviral vector is successfully constructed and transfected into A549 cells. NK4 can inhibit the proliferation and promote the apoptosis of A549 cells, which may be related to the down-regulation of c-met gene level.