Effect of gap junctions on RAW264.7 macrophages infected with H37Rv.
Effect of gap junctions on RAW264.7 macrophages infected with H37Rv.
复制标题
间隙连接对感染H37RV的RAW264.7巨噬细胞的影响。
DOI：
10.1097/md.0000000000012125
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发表时间：
2018-08
期刊：
影响因子：
1.6
通讯作者：
Zhang L
中科院分区：
文献类型：
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作者：
Lu Y;Wang XM;Yang P;Han L;Wang YZ;Zheng ZH;Wu F;Zhang WJ;Zhang L
细胞凋亡和炎症反应在结核分枝杆菌（Mycobacterium tuberculosis，MTB）感染的发病机制中起重要作用。当巨噬细胞发生凋亡和极化时，可能需要间隙连接（GJ）为其功能提供条件。连接蛋白43（Cx43）和连接蛋白37（Cx 37）是巨噬细胞中参与GJ通道形成的主要连接蛋白。建立H37 Rv感染RAW 264. 7巨噬细胞模型，研究结核分枝杆菌感染后连接蛋白与宿主巨噬细胞免疫防御反应的关系。首先，采用实时荧光定量聚合酶链反应（RT-PCR）检测Cx43和Cx 37 mRNA的表达。免疫印迹法和免疫荧光法检测Cx43蛋白的表达和定位。共聚焦显微镜观察细胞间差距连接通讯（gapjunctionalintercellularcommunication，GJIC）。电镜观察巨噬细胞形态。流式细胞术检测RAW 264. 7巨噬细胞凋亡和线粒体膜电位，Real-time PCR和ELISA法检测炎症因子CD 86、CD 206、IL-6、IL-10、TNF-α和TGF-β的表达。H37 Rv感染显著促进宿主巨噬细胞Cx43 mRNA和蛋白表达（分别增加1.6倍和0.3倍），并增强宿主巨噬细胞GJIC。H37 Rv感染诱导宿主巨噬细胞细胞间通讯时，细胞凋亡率和炎症因子表达也随之增加。结果表明，H37 Rv感染可明显诱导宿主巨噬细胞Cx43表达，增强GJIC，可能参与宿主巨噬细胞的炎症反应，调节炎症因子的释放和/或启动细胞凋亡，激活宿主免疫防御反应。
Apoptosis and inflammation have been shown to play an important role in the mechanisms involved in the pathogenesis of Mycobacterium tuberculosis (MTB) infection. When macrophages undergo apoptosis and polarization, gap junctions (GJs) may be needed to provide conditions for their functions. Connexin 43 (Cx43) and connexin 37 (Cx37) are the main connexins in macrophages that participate in the formation of GJ channels. An H37Rv infection RAW264.7 macrophage model was established to investigate the associate between connexins and host macrophage immune defense response after MTB infection. First, Real-time Polymerase Chian Reaction (RT-PCR) was used to detect the mRNA expression of Cx43 and Cx37. Cx43 protein expression and location was detected by western blotting and immunofluorescence. Confocal microscope was used to assay the gap junctional intercellular communication (GJIC). Then, electron microscope used to observe the morphology of macrophages. Finally, RAW264.7 macrophage apoptosis and mitochondrial membrane potential was detected by flow cytometry, and the expression of inflammation factors such as CD86, CD206, and IL-6, IL-10, TNF-α, and TGF-β were detected by Real-time PCR and enzyme-linked-immunosorbent serologic assay (ELISA). H37Rv infection significantly promoted host macrophage Cx43 mRNA and protein expression (increased 1.6-fold and 0.3-fold respectively), and enhanced host macrophage GJIC. When host macrophage cell-to-cell communication induced by H37Rv infection, the apoptosis rate and inflammatory factors expression also increased. The results confirm that H37Rv infection can obviously induce host macrophage Cx43 expression and enhance GJIC, which may implicated in host macrophage inflammatory reaction, to regulate the release of inflammatory factors and/or initiate apoptosis to activate host immune defense response.