嗅素蛋白4对卡介苗诱导巨噬细胞凋亡和细菌胞内生存率的影响

doi:10.3969/j.issn.1673-8640.2020.08.017

摘要/Abstract

摘要：

目的探讨嗅素蛋白4（OLFM4）对卡介苗（BCG）感染的巨噬细胞RAW264.7凋亡及BCG存活率的影响及机制。方法采用逆转录聚合酶链反应（RT-PCR）检测OLFM4 mRNA表达水平。采用免疫印迹法检测OLFM4及凋亡相关蛋白Bcl-2和Bax的表达。采用平板菌落计数法分析BCG存活率,通过酶联免疫吸附试验评估巨噬细胞凋亡率。结果 BCG感染的巨噬细胞RAW264.7中OLFM4 mRNA和蛋白表达水平均显著升高,并呈时间依赖性。此外,敲除OLFM4能够抑制BCG诱导的细胞凋亡,同时增加抑凋亡蛋白Bcl-2表达,降低促凋亡蛋白Bax的表达。OLFM4沉默使RAW264.7细胞中BCG生存率明显升高。机制研究结果显示,下调OLFM4可抑制p-p65和TLR2的表达,进而阻断TLR2/NF-κB信号通路。结论沉默OLFM4可能通过阻断TLR2/NF-κB信号通路抑制BCG诱导的巨噬细胞RAW264.7凋亡,从而增加BCG存活率,初步阐明了OLFM4在巨噬细胞抗BCG感染中的作用机制,为结核病（TB）的诊断和治疗奠定了理论基础。

关键词: 嗅素蛋白4, 巨噬细胞, 凋亡, 结核分枝杆菌

Abstract:

Objective To investigate the role and mechanism of olfactomedin（OLFM4） in Bacillus Calmette-Guerin（BCG）-induced macrophage RAW264.7 apoptosis and BCG survival rate. Methods The mRNA expression of OLFM4 was determined by reverse transcription-polymerase chain reaction（PCR）. Western blotting was conducted to determine the expression level of OLFM4 and apoptosis-associated protein Bcl-2 and Bax. The survival rate of BCG was determined by colony-forming unit. Enzyme-linked immunosorbent assay was performed to analyze cell apoptosis rate. Results The expression of OLFM4 at both mRNA and protein levels was dramatically elevated in a time dependent manner in macrophage RAW264.7 cells stimulated with BCG. The depletion of OLFM4 apparently repressed BCG-induced macrophage apoptosis. Meanwhile,the knockdown of OLFM4 enhanced strongly the expression of anti-apoptotic Bcl-2 concomitant with reducing the expression of pro-apoptotic protein Bax in RAW264.7 cells followed by BCG infection. Moreover,the silencing of OLFM4 enhanced obviously the intracellular survival of BCG during the infection of macrophages with BCG. Mechanically,the deletion of OLFM4 blocked TLR2/NF-κB signaling pathway through inhibiting the expression of p-p65 and TLR2. Conclusions The silencing of OLFM4 represses BCG-induced macrophage apoptosis and increases BCG survival rate in macrophage RAW264.7 through blocking TLR2/NF-κB signaling pathway. The underlying mechanism of OLFM4 in host cells on BCG infection warrants further study for the development of novel agents for clinical treatment of tuberculosis （TB）.

Key words: Olfactomedin, Macrophage, Apoptosis, Mycobacterium tuberculosis

中图分类号:

R446.1

王启源, 姬文兰, 虞秀锋. 嗅素蛋白4对卡介苗诱导巨噬细胞凋亡和细菌胞内生存率的影响[J]. 检验医学, 2020, 35(8): 818-822.

WANG Qiyuan, JI Wenlan, YU Xiufeng. Influence of OLFM4 on BCG-induced apoptosis and intracellular survival rate in macrophage[J]. Laboratory Medicine, 2020, 35(8): 818-822.

图/表 4

图1 OLFM4在BCG诱导的巨噬细胞RAW264.7中的表达注：（a）、（b）BCG感染巨噬细胞OLFM4 mRNA及蛋白水平;（c）、（d）OLFM4沉默转染效率;与对照比较,*P<0.05;与BCG+si-NC比较,#P<0.05

图2 OLFM4沉默抑制BCG诱导的巨噬细胞凋亡注：（a）酶联免疫吸附试验检测细胞凋亡结果;（b）免疫印迹法检测凋亡相关蛋白Bcl-2和Bax表达结果;与对照比较,*P<0.05;与BCG+si-NC比较,#P<0.05;Bax;Bci-2

图3 OLFM4沉默促进胞内BCG存活注：si-NC;si-OLFM4;与si-NC比较,*P<0.05

图4 沉默OLFM4抑制BCG诱导的TLR2/NF-κB通路注：（a）免疫印迹法检测TLR2、p65、p-p65表达;（b）TLR2、p65、p-p65相对表达量;与对照比较,*P<0.05;与BCG+si-NC比较,#P<0.05

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