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目的研究海分枝杆菌感染PC-12神经元细胞的感染潜能及其诱导促炎性细胞因子表达的免疫特性。方法用海分枝杆菌感染神经营养因子(nerve growth factor,NGF)诱导分化的PC-12神经元,采用激光共聚焦显微镜观察细菌在PC-12神经元细胞内的定位分布,并计数细菌的菌落形成单位(CFU)以观察其在PC-12神经元细胞内的增殖情况;采用实时荧光定量PCR(qRT-PCR)、酶联免疫吸附试验(ELISA)及免疫印迹法(Western blot)检测肿瘤坏死因子(tumor necrosis factor,TNF)-α、白介素(Interleukin)-1β、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和环氧合酶(cyclooxygenase-2,COX-2)mRNA及蛋白的表达情况。结果海分枝杆菌感染PC-12细胞后,激光共聚焦显微镜观察细菌侵入PC-12神经元胞浆内并进行增殖。当细菌以MOI=20感染神经元细胞时,感染后24h与感染后6h相比,细菌在细胞内增殖了约25倍。同时海分枝杆菌感染后6h即可诱导促炎性分子TNF-α、IL-1β、iNOS和COX-2的mRNA表达明显增加,感染24h后TNF-α、IL-1β、iNOS和COX-2的蛋白表达也明显增加。结论 PC-12能够作为海分枝杆菌感染的宿主细胞,并引起促炎相关因子的分泌,从而促进炎症反应。
Objective To study the infectious potential of Mycobacterium tuberculosis infection in PC-12 neurons and the immunological characteristics of the cells induced by proinflammatory cytokines. Methods PC12 cells differentiated by nerve growth factor (NGF) were infected with Mycobacterium maritima. The localization of bacteria in PC-12 neurons was observed by laser scanning confocal microscopy and the colonies of bacteria were counted (CFU) in order to observe its proliferation in PC-12 neurons. Real-time quantitative PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blot were used to detect tumor necrosis Tumor necrosis factor (TNF) -α, interleukin-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) The expression of the situation. Results After infection of Mycobacterium maritima with PC-12 cells, the bacteria invaded the cytoplasm of PC-12 neurons by laser confocal microscopy and proliferated. When bacteria infected neuronal cells at a MOI = 20, the bacteria proliferated about 25-fold in the cells 24 h after infection compared to 6 h after infection. At the same time, the mRNA expression of TNF-α, IL-1β, iNOS and COX-2 were significantly increased 6h after infection with Mycobacterium maritime. The mRNA expression of TNF-α, IL-1β, iNOS and COX- The protein expression also increased significantly. Conclusion PC-12 can be used as a host cell for Mycobacterium maritime infection and induce the secretion of proinflammatory factors, thereby promoting the inflammatory response.