本文選題：新金分枝桿菌 + 調(diào)節(jié)性T細胞�。� 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：近年來,臨床上感染非結(jié)核分枝桿菌的病例不斷增加,非結(jié)核分枝桿菌病已經(jīng)成為危害人類生命和健康的重要問題。目前尚無治療非結(jié)核分枝桿菌病的特效藥。調(diào)節(jié)性T細胞是負向調(diào)節(jié)機體免疫反應(yīng)的一類T細胞亞群,它既能夠抑制機體不恰當?shù)拿庖叻磻?yīng),還能限定免疫應(yīng)答的范圍和程度。因此本試驗通過對感染新金分枝桿菌的C57BL/6小鼠進行研究,分析其感染期間對Tregs亞群表型及功能的影響,使我們對非結(jié)核分枝桿菌感染期間機體Tregs亞群免疫機制有一個新的認識。本試驗選取雌性C57BL/6小鼠,經(jīng)腹腔注射感染新金分枝桿菌。分別于感染后1、2、3、4、5、6、7、9、11、13、16、18、23周采用流式細胞術(shù)檢測脾臟CD_4~+CD_(25)~+Fox P3~+Treg與CD8~+CD_(25)~+Fox P3~+Treg比例變化;并用CD152檢測表型變化明顯的時間點檢測其抑制性功能;利用免疫磁珠分選CD_4~+CD_(25)~+Treg與CD_4~+CD_(25)-T細胞進行MTT增殖抑制試驗;通過熒光定量PCR技術(shù)檢測CD_4~+CD_(25)~+Treg中Fox P3、TGF-β、IL-10和IFN-γ4種細胞因子m RNA水平表達量的變化。結(jié)果顯示:1、新金分枝桿菌感染的小鼠脾臟中,Tregs亞群主要以CD_4~+CD_(25)~+FoxP3~+Treg表型存在,并在第6周其所占細胞比例達到最高,約占2%。CD_4~+CD_(25)~+CD152~+細胞與CD_4~+CD_(25)~+Fox P3~+細胞比例呈正相關(guān)。2、利用免疫磁珠分選的CD_4~+CD_(25)-T細胞及CD_4~+CD_(25)~+Treg細胞純度較高,CD_4~+CD_(25)-T細胞為82.5%,CD_4~+CD_(25)~+Treg細胞可以達到79.1%。CD_4~+CD_(25)~+Treg對CD_4~+CD_(25)-T細胞產(chǎn)生抑制性作用,CD_4~+CD_(25)-T細胞的抑制率為89.8%。3、CD_4~+CD_(25)~+Treg分泌的FoxP3、IL-10與TGF-β的mRNA相對表達量均與對照組差異顯著,IFN-γm RNA表達量與對照組無統(tǒng)計學(xué)差異。綜上,新金分枝桿菌感染小鼠脾臟Tregs亞群主要以CD_4~+CD_(25)~+Fox P3存在,發(fā)揮其免疫抑制作用。體外增殖抑制試驗中,CD_4~+CD_(25)~+Treg能夠抑制CD_4~+CD_(25)-T細胞的增殖作用,驗證了CD_4~+CD_(25)~+Treg能夠通過直接接觸發(fā)揮抑制性功能。CD_4~+CD_(25)~+Treg細胞主要通過分泌細胞因子TGF-β與IL-10來發(fā)揮其免疫抑制作用。
[Abstract]:In recent years, the number of patients infected with non-tuberculous mycobacterium has been increasing, and non-tuberculosis mycoidosis has become an important problem that endangers human life and health. At present, there is no specific drug for the treatment of non-tuberculosis mycobacterium disease. Regulatory T cells are a subgroup of T cells that negatively regulate the immune response of the body. It can not only inhibit the inappropriate immune response, but also limit the scope and degree of immune response. Therefore, through the study of C57BL/6 mice infected with Mycobacterium neoformans, the effects of the infection on the phenotype and function of Tregs subgroup were analyzed, so that we could have a new understanding of the immune mechanism of Tregs subgroup during the period of non-tuberculosis mycobacterium infection. In this study, female C57BL/6 mice were injected intraperitoneally with Mycobacterium neoformans. The changes of CD4 ~ Fox P3 ~ Treg in spleen and CD8 ~ CDT 25 ~ Fox P3 ~ Treg in spleen were detected by flow cytometry at week 1, 2, 2, 2, 3, 5, 5, 5, 9, 11, 13, 16, 18, 23 weeks after infection, and the inhibitory function was detected by CD152 at a time point when phenotypic changes were obvious. The expression of Fox P3TGF- 尾 IL-10 and IFN- 緯 in CD4 ~ CDGF-尾 -IL-10 and IFN- 緯 were detected by fluorescence quantitative PCR technique. The results showed that Tregs subsets in the spleen of mice infected with Mycobacterium neoformans mainly existed in the phenotype of CD4 ~ CDSP-25 ~ FoxP3 ~ Treg, and reached the highest percentage of cells in the 6th week. There was a positive correlation between the proportion of CD4- Fox P3~ cells and the proportion of CD4- Fox P3~ cells. The purity of CD4- CD_(25)-T cells and CD4- Treg cells was 82.5 CD4CD4- CD152- CD_(25)-T cells. The CD4- CD_(25)-T cells could reach 79.1. CD4CD4- Fox P3~ + cells. The CD4- CD_(25)-T cells could inhibit the CD4- CD_(25)-T cells in the cells of 79.1. CD4CD4CDT / Treg. The CD4- CD_(25)-T cells of the population could reach 79.1.CD4CD4CDT / Treg could inhibit the proliferation of CD4- CD_(25)-T cells by using immunomagnetic beads to separate the CD4- CD_(25)-T cells and the CD4- Treg cells with the purity of CD4- CD_(25)-T cells. The inhibitory rate of CD4 ~ CD_(25)-T cells was 89.8.3. there was no significant difference in the relative expression of mRNA between the two groups in the expression of FoxP3- 緯 m RNA and TGF- 尾 secreted by 25 ~ Treg of CD4 ~ CDD. There was no significant difference in the expression of IFN- 緯 m RNA between the two groups. In conclusion, the spleen Tregs subsets of mice infected with Mycobacterium neoformans were mainly CD4 ~ CDSP-25 ~ Fox P3, which played an immunosuppressive effect. In vitro proliferation inhibition test, CD4 ~ CDS ~ Treg can inhibit the proliferation of CD4- CD_(25)-T cells, which proves that CD4 ~ CDS-1 ~ Treg can exert inhibitory function by direct contact. CD4- 尾 and IL-10 can play its immunosuppressive effect mainly by secreting cytokines TGF- 尾 and IL-10.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R52

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