小鼠巨噬细胞转染Rv0901基因后活性的改变

目的 将重组结核分枝杆菌Rv0901基因的重组质粒pcDNA3.1-Rv0901转染小鼠腹腔巨噬细胞,研究结核分枝杆菌Rv0901基因在结核分枝杆菌感染巨噬细胞的过程中所起的作用.方法 制备小鼠腹腔巨噬细胞,分别将pcDNA3.1、pcDNA3.1-Rv0901质粒DNA与绿色荧光蛋白(GFP)的质粒DNA混合,以脂质体转染的方式共转染小鼠腹腔巨噬细胞.转染后72 h,分别收集细胞用流式细胞仪检测GFP的表达和细胞凋亡的发生情况,用反转录聚合酶链式反应(RT-PCR)检测质粒的转染情况.收集转染72 h后的细胞培养液上清,检测细胞NO、IFN-γ的释放水平.结果 pcDNA3.1和pcDNA3.1-Rv0901转染的巨噬细胞中均检测到了GFP的表达,转染后的巨噬细胞RT-PCR能扩增出Rv0901的目的 片段,pcDNA3.1-Rv0901转染巨噬细胞后细胞的凋亡率高于空载体对照组(56.4±2.0)% vs (19.9±1.5)%,P＜0.05], pcDNA3.1-Rv0901转染巨噬细胞后细胞培养液上清中NO和IFN-γ的释放量高于空载体对照组NO:(40.4±3.0) μmol/L vs (27.5±3.2) μmol/L, IFN-γ:(2.11±0.031) ng/mL vs (0.62±0.025) ng/mL,P均＜0.05].结论 pcDNA3.1-Rv0901转染小鼠巨噬细胞后使巨噬细胞的凋亡增加,NO和IFN-γ的释放量增加,提示结核分枝杆菌Rv0901基因可能导致巨噬细胞的活性增强,与结核分枝杆菌感染巨噬细胞的机理有关.

Effect of Transfected Rv0901 Gene on the Activity of Mice Macrophages

OBJECTIVE: To test the effect of Rv0901 gene of Mycobacterium tuberculosis on the activity of mice macrophages. METHODS: Peritoneal macrophages of mice were isolated and transfected with pcDNA3. 1 or pcDNA3. 1-Rv0901 plasmid DNA, along with the GFP DNA. The effectiveness of the transfection was detected by RT-PCR. The expression of the GFP and the apoptosis ratio of the macrophages were detected by FCM 72 hours after the transfection. The level of nitric oxide and IFN-gamma in cultural supernatant were also measured 72 hours after transfection. RESULTS: All of the macrophages being transfected had the expression of GFP. After being transfected with pcDNA3. 1-Rv0901, the 528 bp gene of Rv0901 was amplified by RT-PCR. The macrophages transfected with pcDNA3. 1-Rv0901 had higher apoptosis ratio (56.4 +/- 2.0)% vs (19.9 +/- 1.5)%] and released more nitric oxide (40.4 +/- 3.0) micromol/L vs (27.5 +/- 3.2) micromol/L] and IFN-gamma (2.11 +/- 0.031) ng/mL vs (0.62 +/- 0.025) ng/mL] in the cultural supernatants than those transfected with pcDNA3. 1 (P < 0.05). CONCLUSION: Transient transfection of pcDNA3. 1-Rv0901 increases the apoptosis ratio of the macrophages, which could increase the release of nitric oxide and IFN-gamma.