TLR7在结核分枝杆菌感染中的作用研究

Inhibitory effects of TLR7 on Mycobacterium tuberculosis

目的将TLR7激活基序ssRNA加入结核分枝杆菌感染的小鼠巨噬细胞,研究TLR7被激活后在结核分枝杆菌感染中的作用。方法结核分枝杆菌感染RAW264.7细胞,感染后加入化学合成的TLR7激活基序ssRNA,RT-PCR法检测感染不同时间之后细胞对细菌的吞噬率;无菌TritonX-100裂解细胞,罗氏培养基培养计数活细菌;电镜观察细胞内自噬小体;ELISA检测细胞上清中IL-12、TNF-α与IL-4的含量。结果感染3h后RAW264.7细胞吞噬率最高(P>0.05);处理3h后电镜观察,ssRNA组细胞状态明显好于对照组;36h后ssRNA组IL-12(P<0.05)、IL-4(P<0.05)水平高于对照组,细胞内活菌数量ssRNA组低于对照组(P<0.05),48h后IL-4(P<0.05)水平下降,TNF-α的含量上升(P<0.05)。结论TLR7能通过诱导细胞自噬、调节细胞因子产生等机制增强细胞杀结核分枝杆菌的能力,TLR7激活基序ssRNA可以用于治疗结核分枝杆菌感染。

The aim of the present study was to investigate the inhibitory effects of TLR7 on Mycobacterium tuberculosis. TLR7 on infected RAW264.7 cells was activated by chemical synthesis of TLR7 activation motif ssRNA. Activated RAW264.7 cells were inoculated with Mycobacterium tuberculosis, quantitative PCR method was applied to detect the phagocytosis rate of cell to bacteria at different time after infection. Cytokine production was measured by ELISA from cell supernatant. Cells were cultured on Roche medium and counted after sterile cracked with TritonX-100 and diluted with PBS. Scanning electronic micro- scope （ SEM ） was applied to detect the morphological changes of ceils treated with TLR7 activation motif ssRNA. The highest phagocytosis rate of bacteria of RAW264.7 cells was at 3 hours post infection （P〈0.05）. Compared with that of the control group, treatment after 36 hours intracellular bacterial quantity in ssRNA treated group was lower （P〈0.05）, levels of IL-12 （P〈0.05） and IL-4 （P〈0.05） were increased. For treatment after 48 hours, level of IL-4 （P〈0.05） was decreased, and TNF-α （P〈0.05） was increased. For treatment after 3 hours, cell morphology of the ssRNA group was obviously better than the control group and appeared lots of phagosomes. Results suggested that TLR7 could enhance macrophages in killing Myco-bacterium tuberculosis by forming phagosomes and regulating cytokines production, and TLR7 activation motif ssRNA could be used in the treatment of tuberculosis.