本文選題：結(jié)核分枝桿菌 + H37Ra　； 參考：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：目的：探討小鼠腹腔接種結(jié)核分枝桿菌活菌H37Ra、滅活菌H37Rv后的免疫物質(zhì)的表達差異,了解它們的免疫物質(zhì)的表達情況和相關(guān)的信號通路，探討活菌H37Ra及滅活菌H37Rv作為新的結(jié)核候選疫苗的可行性。 方法：1.分別培養(yǎng)BALB/c小鼠腹腔巨噬細胞，用活菌H37Ra，滅活菌H37Rv感染10h后用抗酸染色方法，在顯微鏡下觀察各組巨噬細胞吞噬情況并計算兩種結(jié)核分枝桿菌的吞噬率。2.將活菌H37Ra，滅活菌H37Rv，生理鹽水分別接種BALB/c小鼠腹腔，免疫30d后取小鼠腹腔巨噬細胞，，RT-PCR法檢測小鼠腹腔巨噬細胞IL-12p40，TNF-a，IFN-γ的基因轉(zhuǎn)錄水平；ELISA法檢測細胞因子IL-12p40，TNF-a，IFN-γ的表達；Griess法，化學法檢測小鼠腹腔巨噬細胞分泌NO、H_2O_2水平；流式細胞儀檢測IFN-γ誘導(dǎo)的CD40L的變化情況。3.將活菌H37Ra，滅活菌H37Rv，生理鹽水分別接種BALB/c小鼠腹腔，免疫30d后取小鼠腹腔巨噬細胞，用IFN-γ刺激1天，用流式細胞儀，共聚焦及Western檢測巨噬細胞表面CD40L的變化情況。 結(jié)果：1.巨噬細胞對活菌H37Ra和滅活菌H37Rv的吞噬率分別為（55.71±8.42）%,（14.82±2.12）%，與滅活菌相比，活菌H37Ra有更強的被吞噬能力（P0.01）。2.小鼠腹腔巨噬細胞被活菌H37Ra免疫后能顯著誘導(dǎo)巨噬細胞的IL-12P40、TNF及IFN-γ基因的轉(zhuǎn)錄，細胞因子IL-12P40、TNF-a、IFN-γ、NO及H_2O_2高水平分泌;3.外源性細胞因子IFN-γ更能刺激活菌H37Ra誘導(dǎo)巨噬細胞表面CD40L的高表達。 結(jié)論：活的結(jié)核分枝桿菌減毒株H37Ra能顯著誘導(dǎo)小鼠腹腔巨噬細胞產(chǎn)生更多的保護性免疫物質(zhì)，有利于免疫應(yīng)答和免疫保護，有可能作為新的結(jié)核候選疫苗，同時，IFN-γ是誘導(dǎo)刺激小鼠腹腔巨噬細胞產(chǎn)生免疫應(yīng)答的重要因子，而CD40L信號途徑是介導(dǎo)刺激巨噬細胞產(chǎn)生免疫應(yīng)答的信號通路之一；IFN-γ更能刺激活菌CD40L信號通道產(chǎn)生免疫應(yīng)答反應(yīng)。而滅活菌H37Rv不能顯著激活小鼠腹腔巨噬細胞分泌保護性免疫物質(zhì)，不宜作為新的結(jié)核候選疫苗。
[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- 緯 and the expression of cytokine IL-12p40, TNF-aIFN- 緯 was detected by Griess method, and the expression of IL-12p40, TNF-aIFN- 緯 was detected by Elisa, and the expression of IL-12p40 and TNF-aIFN- 緯 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- 緯 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- 緯 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 鹵8.42 and 14.82 鹵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- 緯 genes in macrophages, and the cytokines IL-12P40, TNF-a, IFN- 緯, and H_2O_2 secreted high levels of TNF- 緯 and H_2O_2. Exogenous cytokines IFN- 緯 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- 緯 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- 緯 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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.11

【參考文獻】

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1 拾莉;丁元生;楊華;劉耀婷;劉忠華;胡忠義;黃瑞;;結(jié)核分枝桿菌19kD-ESAT6融合蛋白的克隆表達及純化[J];微生物與感染;2009年01期

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