结核分枝杆菌RelE毒素蛋白对肺癌A-549细胞的生长抑制作用

RelE Toxin Protein of Mycobacterium Tuberculosis Induces Growth Inhibition of Lung Cancer A-549 Cell

目的探索结核分枝杆菌RelE毒素蛋白是否对肺癌A-549细胞有生长抑制作用。方法采用PCR技术克隆结核分枝杆菌RelE、RelB、RelBE基因,分别构建真核质粒PcDNA3.1-RelE、PcDNA3.1-RelB和PcDNA3.1-RelBE。经酶切分析和DNA测序证实重组质粒构建成功后,采用脂质体转染方法分别转入肺癌A-549细胞,经RT-PCR鉴定后,建立稳定表达相关蛋白的细胞株。通过细胞生长曲线、活细胞蛋白含量测定、MTT细胞增殖实验、HE染色观察细胞凋亡、流式细胞术检测瞬时转染细胞的凋亡率等,观察结核分枝杆菌RelE毒素蛋白是否对肺癌A-549细胞具有生长抑制和促凋亡的作用。结果结核分枝杆菌RelE毒素蛋白组的肺癌A-549细胞增殖速度低于其他各组,对营养缺乏更为敏感,相同范围视野下凋亡细胞多于其他各组,瞬时转染细胞的凋亡率也高于其他组(P<0.05)。结论结核分枝杆菌RelE毒素蛋白对肺癌A-549细胞具有生长抑制和促凋亡的作用。

Objective To investigate whether the RelE toxin protein of mycobacterium tuberculosis has a growth inhibition effect on lung cancer A-549 cell. Methods The complete open-reading frame sequences of RelE, RelB and RelBE genes were amplified by PCR with using M. tuberculosis H37Rv genomic DNA as the template. The RelE, RelB and RelBE genes were subcloned into PcDNA3. 1 （＋）. After being verified with restriction endonuclease digestion and DNA sequence determination, the recombinant vectors were applied to transfect lung cancer A-549 cells by liposome transfection method. By determining the growth curve and protein level of living cells, MTT cell proliferation assay, the apoptosis of cells with HE staining and the apoptosis rate of transient transfection cells detected by Flow Microfluorimetry, it was observed and analyzed whether the RelE toxin protein of mycobacterium tuberculosis had a growth inhibition and apoptosis effects on lung cancer A-549 cell. Results The cell proliferation rate of lung cancer A-549 cells effected by the RelE toxin protein of mycobacterium tuberculosis was lower than that of the other groups, and this group cells with RelE protein effect showed more sensitive to nutrition starving. HE staining revealed that this group cells which included the transient transfection with RelE expression plasmid appeared to have more apoptosis cells and higher apoptotic rate than other groups did （P〈 0.05）. Conclusion The RelE toxin protein of mycobacterium tuberculosis has growth inhibition and apoptotic effect on lung cancer A-549 cell.