devR基因调控的结核分枝杆菌细胞毒性的研究

Research on devR regulated cytotoxicity of Mycobacterium tuberculosis

目的阐明devR基因表达对分枝杆菌生长及感染能力的影响。方法确认野生型牛分支杆菌卡介苗（M.bovis BCG,简称BCG）、devR基因敲除的BCG菌株（简称ΔdevR）和devR基因回补的BCG菌株（简称devR.com）的devR序列正确;在不同时间点测定600 nm下的吸光度（A）值,绘制3种细菌的生长曲线;以感染复数（MOI）=10感染人肺泡Ⅱ型上皮细胞A549（简称A549细胞）和小鼠巨噬细胞Raw264.7（简称Raw264.7细胞）,分别采用流式细胞仪和酶联免疫吸附试验于0、24、48、72 h测定细胞凋亡率和乳酸脱氢酶（LDH）释放水平;采用实时定量PCR（RT-PCR）测定凋亡相关基因bcl-2和bad的表达水平。结果ΔdevR菌株在早期（培养2-10 d）生长较BCG菌株和devR.com菌株明显加快（P〈0.01）。ΔdevR菌株感染A549细胞和Raw264.7细胞后,细胞的凋亡和坏死效应明显增加,与BCG菌株和devR.com菌株比较差异有统计学意义（P〈0.05）。3种菌株感染A549细胞均导致凋亡相关基因bcl-2表达上调,且ΔdevR菌株感染组bcl-2基因表达明显高于BCG和devR.com菌株感染组;3种菌株感染Raw264.7细胞均导致凋亡相关基因bad表达上调,且ΔdevR菌株感染组bad基因表达明显高于BCG和devR.com菌株感染组。结论 devR基因敲除的BCG菌株毒力明显增强。

Objective To identify the influence of devR expression on the growth and cytotoxicity of tuberculosis.Methods Culture wild Mycobacterium bovis Bacillus Calmette-Guérin（ BCG）,devR knockout BCG（ ΔdevR） and devR compensated BCG（ devR. com） were identified for devR sequence,and the growth curves at different times were drawn under absorbance（ A） 600 nm. A549 cells and Raw264. 7 cells were infected with the strains at multiplicity of infection（MOI） = 10. The apoptosis ratio was detected by flow cytometry,and lactate dehydrogenase（LDH） released by host cells was measured by enzyme-linked immunosorbent assay（ ELISA） at 0,24,48 and 72 h. The expressions of apoptosis related genes,bcl-2 and bad,were determined by real-time quantitation polymerase chain reaction（ PCR）.Results Comparing to BCG and devR. com,the growth of ΔdevR was significantly accelerated at early phase（2-10 d,P 〈0. 01）. Apoptosis and necrosis ratio of A549 cells and Raw264. 7 cells infected by ΔdevR were significantly higher than those infected by BCG and devR. com（ P〈0. 05）. All 3 strains could up-regulate the expressions of bcl-2 of A549 cells,and ΔdevR stimulated higher level of bcl-2 expression than BCG and devR. com. They also could increase the bad expression of Raw264. 7 cells. ΔdevR induced more bad gene expression than BCG and devR. com. Conclusions The knockout of devR could enhance the cytotoxicity of BCG.