活菌H37Ra与灭活菌H37Rv感染小鼠腹腔巨噬细胞实验研究

发布时间：2018-05-10 18:26

  本文选题：结核分枝杆菌 + H37Ra　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：目的：探讨小鼠腹腔接种结核分枝杆菌活菌H37Ra、灭活菌H37Rv后的免疫物质的表达差异,了解它们的免疫物质的表达情况和相关的信号通路，探讨活菌H37Ra及灭活菌H37Rv作为新的结核候选疫苗的可行性。 方法：1.分别培养BALB/c小鼠腹腔巨噬细胞，用活菌H37Ra，灭活菌H37Rv感染10h后用抗酸染色方法，在显微镜下观察各组巨噬细胞吞噬情况并计算两种结核分枝杆菌的吞噬率。2.将活菌H37Ra，灭活菌H37Rv，生理盐水分别接种BALB/c小鼠腹腔，免疫30d后取小鼠腹腔巨噬细胞，，RT-PCR法检测小鼠腹腔巨噬细胞IL-12p40，TNF-a，IFN-γ的基因转录水平；ELISA法检测细胞因子IL-12p40，TNF-a，IFN-γ的表达；Griess法，化学法检测小鼠腹腔巨噬细胞分泌NO、H_2O_2水平；流式细胞仪检测IFN-γ诱导的CD40L的变化情况。3.将活菌H37Ra，灭活菌H37Rv，生理盐水分别接种BALB/c小鼠腹腔，免疫30d后取小鼠腹腔巨噬细胞，用IFN-γ刺激1天，用流式细胞仪，共聚焦及Western检测巨噬细胞表面CD40L的变化情况。 结果：1.巨噬细胞对活菌H37Ra和灭活菌H37Rv的吞噬率分别为（55.71±8.42）%,（14.82±2.12）%，与灭活菌相比，活菌H37Ra有更强的被吞噬能力（P0.01）。2.小鼠腹腔巨噬细胞被活菌H37Ra免疫后能显著诱导巨噬细胞的IL-12P40、TNF及IFN-γ基因的转录，细胞因子IL-12P40、TNF-a、IFN-γ、NO及H_2O_2高水平分泌;3.外源性细胞因子IFN-γ更能刺激活菌H37Ra诱导巨噬细胞表面CD40L的高表达。 结论：活的结核分枝杆菌减毒株H37Ra能显著诱导小鼠腹腔巨噬细胞产生更多的保护性免疫物质，有利于免疫应答和免疫保护，有可能作为新的结核候选疫苗，同时，IFN-γ是诱导刺激小鼠腹腔巨噬细胞产生免疫应答的重要因子，而CD40L信号途径是介导刺激巨噬细胞产生免疫应答的信号通路之一；IFN-γ更能刺激活菌CD40L信号通道产生免疫应答反应。而灭活菌H37Rv不能显著激活小鼠腹腔巨噬细胞分泌保护性免疫物质，不宜作为新的结核候选疫苗。
[Abstract]:Objective: to investigate the expression of immunoreactive substances after inoculating live Mycobacterium tuberculosis strain H37Raand with inactivated H37Rv in mice, and to understand the expression of immune substances and related signal pathways. To explore the feasibility of live bacteria H37Ra and inactivated H37Rv as new vaccine candidates for tuberculosis. Method 1: 1. The peritoneal macrophages of BALB/c mice were cultured separately. The phagocytosis of macrophages in each group was observed under microscope and the phagocytic rate of two mycobacterium tuberculosis was calculated. Live bacteria H37Ra, inactivated bacteria H37Rvand saline were inoculated intraperitoneally into BALB/c mice respectively. After 30 days of immunization, macrophages of mice were immunized with RT-PCR to detect the gene transcription level of IL-12p40, TNF-atIFN- 纬 and the expression of cytokine IL-12p40, TNF-aIFN- 纬 was detected by Griess method, and the expression of IL-12p40, TNF-aIFN- 纬 was detected by Elisa, and the expression of IL-12p40 and TNF-aIFN- 纬 was detected by Elisa. The level of No-H _ 2O _ 2 secreted by mouse peritoneal macrophages was detected by chemical method, and the change of CD40L induced by IFN- 纬 was detected by flow cytometry. Live bacteria H37Ra, inactivated bacteria H37Rv and normal saline were inoculated intraperitoneally into BALB/c mice respectively. After 30 days of immunization, peritoneal macrophages were taken from mice. The macrophages were stimulated with IFN- 纬 for one day. The changes of CD40L on macrophages were detected by flow cytometry, confocal focusing and Western. The result is 1: 1. The phagocytosis rates of macrophages to H37Ra and H37Rv were 55.71 卤8.42 and 14.82 卤2.12, respectively. Compared with inactivated bacteria, H37Ra had a stronger phagocytosis ability. Murine peritoneal macrophages immunized with live bacteria H37Ra could significantly induce the transcription of IL-12P40TNF- and IFN- 纬 genes in macrophages, and the cytokines IL-12P40, TNF-a, IFN- 纬, and H_2O_2 secreted high levels of TNF- 纬 and H_2O_2. Exogenous cytokines IFN- 纬 could stimulate the overexpression of CD40L on the surface of macrophages induced by living bacteria H37Ra. Conclusion: live Mycobacterium tuberculosis attenuated strain H37Ra can significantly induce more protective immune substances in mouse peritoneal macrophages, which is beneficial to immune response and protection, and may be used as a new vaccine candidate for tuberculosis. IFN- 纬 is an important factor in inducing the immune response of murine peritoneal macrophages, and the CD40L signaling pathway is one of the signaling pathways that mediate the immune response of macrophages. IFN- 纬 can stimulate the immune response of live bacteria CD40L signal channels. But inactivated H37Rv could not activate the secretion of protective immune substances by peritoneal macrophages of mice, so it was not suitable to be used as a new vaccine candidate for tuberculosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.11

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相关期刊论文 前1条

1 拾莉;丁元生;杨华;刘耀婷;刘忠华;胡忠义;黄瑞;;结核分枝杆菌19kD-ESAT6融合蛋白的克隆表达及纯化[J];微生物与感染;2009年01期

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