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目的探讨Toll样受体2(TLR2)、TLR4介导的免疫调节信号通路在结核分枝杆菌感染诱导的单核细胞趋化蛋白-1(monocyte chemo attractant protein-1,MCP-1)产生中所起的作用。方法建立人型结核分枝杆菌H37Rv感染小鼠模型,获取模型组及对照组小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)及肺泡巨噬细胞(alveolar macro-phage,AM),对BALF中AM进行计数,采用酶联免疫吸附试验(ELISA)法测定BALF中MCP-1水平,Western免疫蛋白印迹(Western blot)方法检测肺组织中MCP-1蛋白含量,逆转录聚合酶链反应(RT-PCR)测定模型组及对照组AM中TLR2、TLR4mRNA的表达;抗体干预组的AM用TLR2、TLR4抗体预处理并与H37Rv菌株共培养,并设未予以抗体预处理的PBS组及未作任何处理的对照组,分别用ELISA、Western blot法检测AM培养液中及AM中的MCP-1水平,以观察TLR2、TLR4在H37Rv诱导MCP-1生成所起的作用。结果模型组BALF中AM计数及MCP-1蛋白含量显著高于对照组(P<0.05);模型组小鼠肺组织中MCP-1蛋白含量显著高于对照组(P<0.05);模型组AM内TLR2、TLR4mRNA表达显著高于对照组(P<0.05);模型组BALF中MCP-1水平与AM数目呈正相关(r=0.92,P<0.05),并分别与TLR2、TLR4mRNA表达呈正相关(r=0.88,P<0.05;r=0.82,P<0.05)。PBS组AM及其培养上清液中MCP-1蛋白水平显著高于对照组(P<0.05);TLR2、TLR4抗体干预组AM及其培养上清液中MCP-1蛋白水平均显著低于PBS组(P<0.05)。结论在结核宿主免疫中,MCP-1参与诱导单核细胞聚集到感染部位这一过程,AM膜上的TLR2、TLR4受体通道介导了人型结核分枝杆菌H37Rv所诱导的MCP-1表达。
Objective To investigate the role of TLR2 and TLR4-mediated immunoregulatory signaling pathway in the production of monocyte chemo attractant protein-1 (MCP-1) induced by Mycobacterium tuberculosis Play a role. Methods The mouse model of Mycobacterium tuberculosis H37Rv infection was established and the bronchoalveolar lavage fluid (BALF) and alveolar macrophages (AM) in the model group and the control group were obtained. The BALF The levels of MCP-1 in BALF were detected by enzyme linked immunosorbent assay (ELISA). The levels of MCP-1 protein in lung tissue were detected by Western blot. The mRNA and protein expression of MCP-1 in lung tissue were measured by reverse transcriptase-polymerase chain reaction -PCR) were used to detect the expression of TLR2 and TLR4 mRNA in AM and AM groups. AM was pretreated with TLR2 and TLR4 antibody and co-cultured with H37Rv strain in antibody-treated group. PBS without antibody pretreatment and without any The levels of MCP-1 in AM and AM were detected by ELISA and Western blot respectively to observe the role of TLR2 and TLR4 in H37Rv-induced MCP-1 production. Results The level of MCP-1 and MCP-1 protein in BALF of model group were significantly higher than those of control group (P <0.05). The content of MCP-1 protein in lung tissue of model group was significantly higher than that of control group (P <0.05) The expression of TLR2 and TLR4 mRNA in BALF of model group was positively correlated with the number of AM (r = 0.92, P <0.05), and was positively correlated with the expression of TLR2 and TLR4 mRNA (r = 0.88, P <0.05; r = 0.82, P <0.05). The protein level of MCP-1 in AM and its culture supernatant of PBS group was significantly higher than that of control group (P <0.05). The levels of MCP-1 protein in AM and its supernatant of TLR2 and TLR4 antibody intervention group were significantly lower than those of PBS Group (P <0.05). Conclusion MCP-1 is involved in the induction of monocyte aggregation to the site of infection in tuberculosis host immunity. The TLR2 and TLR4 receptor channels on AM membrane mediate the expression of MCP-1 induced by M. tuberculosis H37Rv .