devR基因调控的结核分枝杆菌细胞毒性的研究

doi:10.3969/j.issn.1673-8640.2015.06.016

摘要/Abstract

摘要： 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。

关键词: devR基因, 卡介苗, 感染, 凋亡, 乳酸脱氢酶

Abstract: 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.

Key words: devR, Bacillus Calmette-Guérin, Infection, Apoptosis, Lactate dehydrogenase

中图分类号:

R446.7

旦正尖措, 马越云, 周磊, 付晓蕊, 苏明权, 郝晓柯. devR基因调控的结核分枝杆菌细胞毒性的研究[J]. 检验医学, 2015, 30(6): 607-612.

DANZHENG Jiancuo, MA Yueyun, ZHOU Lei, FU Xiaorui, SU Mingquan, HAO Xiaoke. Research on devR regulated cytotoxicity of Mycobacterium tuberculosis[J]. Laboratory Medicine, 2015, 30(6): 607-612.

图/表 5

图1 BCG、ΔdevR和devR.com菌株目的基因的PCR鉴定注:(a)慢生长分支杆菌特异性基因Rv0577PCR(786 bp)鉴定,1为BCG菌株,2为ΔdevR菌株,3为devR.com菌株;(b)目的基因devR(654 bp)PCR鉴定,1为ΔdevR菌株,2为BCG菌株,3为devR.com菌株;M为DNA Maker;+为阳性对照(H37Rv),-为阴性对照(DH5α)

图2 BCG菌株、ΔdevR菌株和devR.com菌株的生长特性鉴定注:(a)生长曲线,▲BCG;◆devR.com;■ΔdevR;(b)抗酸染色鉴定

图3 BCG菌株、ΔdevR菌株和devR.com菌株分别感染A549细胞和Raw264.7细胞后2种细胞的凋亡率比较注:(a)A549细胞凋亡率比较;(b)RAW264.7细胞凋亡率比较;与ΔdevR菌株比较,*P<0.01

图4 BCG菌株、ΔdevR菌株和devR.com菌株分别感染A549细胞和Raw264.7细胞后的细胞毒性比较注:(a)A549细胞毒性;(b)Raw264.7细胞毒性;与未处理组比较,*P<0.05;与ΔdevR菌株感染组比较,#P<0.01

图5 BCG菌株、ΔdevR菌株和devR.com菌株感染A549和Raw264.7细胞后对bad和bcl-2基因表达的影响注:(a)bad基因表达;(b)bad表达情况(与β-actin的比值);(c)bcl-2基因表达;(d)bcl-2表达情况(与β-actin的比值);与ΔdevR菌株比较,*P<0.05

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