新金分枝杆菌感染对小鼠脾脏Tregs亚群表型及功能的影响

发布时间：2018-06-02 11:53

本文选题：新金分枝杆菌 + 调节性T细胞　；参考：《吉林农业大学》2017年硕士论文

【摘要】：近年来,临床上感染非结核分枝杆菌的病例不断增加,非结核分枝杆菌病已经成为危害人类生命和健康的重要问题。目前尚无治疗非结核分枝杆菌病的特效药。调节性T细胞是负向调节机体免疫反应的一类T细胞亚群,它既能够抑制机体不恰当的免疫反应,还能限定免疫应答的范围和程度。因此本试验通过对感染新金分枝杆菌的C57BL/6小鼠进行研究,分析其感染期间对Tregs亚群表型及功能的影响,使我们对非结核分枝杆菌感染期间机体Tregs亚群免疫机制有一个新的认识。本试验选取雌性C57BL/6小鼠,经腹腔注射感染新金分枝杆菌。分别于感染后1、2、3、4、5、6、7、9、11、13、16、18、23周采用流式细胞术检测脾脏CD_4~+CD_(25)~+Fox P3~+Treg与CD8~+CD_(25)~+Fox P3~+Treg比例变化;并用CD152检测表型变化明显的时间点检测其抑制性功能;利用免疫磁珠分选CD_4~+CD_(25)~+Treg与CD_4~+CD_(25)-T细胞进行MTT增殖抑制试验;通过荧光定量PCR技术检测CD_4~+CD_(25)~+Treg中Fox P3、TGF-β、IL-10和IFN-γ4种细胞因子m RNA水平表达量的变化。结果显示:1、新金分枝杆菌感染的小鼠脾脏中,Tregs亚群主要以CD_4~+CD_(25)~+FoxP3~+Treg表型存在,并在第6周其所占细胞比例达到最高,约占2%。CD_4~+CD_(25)~+CD152~+细胞与CD_4~+CD_(25)~+Fox P3~+细胞比例呈正相关。2、利用免疫磁珠分选的CD_4~+CD_(25)-T细胞及CD_4~+CD_(25)~+Treg细胞纯度较高,CD_4~+CD_(25)-T细胞为82.5%,CD_4~+CD_(25)~+Treg细胞可以达到79.1%。CD_4~+CD_(25)~+Treg对CD_4~+CD_(25)-T细胞产生抑制性作用,CD_4~+CD_(25)-T细胞的抑制率为89.8%。3、CD_4~+CD_(25)~+Treg分泌的FoxP3、IL-10与TGF-β的mRNA相对表达量均与对照组差异显著,IFN-γm RNA表达量与对照组无统计学差异。综上,新金分枝杆菌感染小鼠脾脏Tregs亚群主要以CD_4~+CD_(25)~+Fox P3存在,发挥其免疫抑制作用。体外增殖抑制试验中,CD_4~+CD_(25)~+Treg能够抑制CD_4~+CD_(25)-T细胞的增殖作用,验证了CD_4~+CD_(25)~+Treg能够通过直接接触发挥抑制性功能。CD_4~+CD_(25)~+Treg细胞主要通过分泌细胞因子TGF-β与IL-10来发挥其免疫抑制作用。
[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- 纬 in CD4 ~ CDGF-尾 -IL-10 and IFN- 纬 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- 纬 m RNA and TGF- 尾 secreted by 25 ~ Treg of CD4 ~ CDD. There was no significant difference in the expression of IFN- 纬 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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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