树突状细胞的TSC1对抗原非特异性初始T细胞活化和增殖的作用机制

发布时间：2018-05-31 06:01

  本文选题：树突状细胞 + 结节硬化复合物1　； 参考：《北京协和医学院》2017年硕士论文

【摘要】：背景:树突状细胞(dendritic cells,DCs)是一种诱导T细胞活化和增殖反应的重要细胞。结节性硬化物1(tuberous sclerosis complex 1,Tsc1)是哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)上游重要的负调节因子。最近的研究突出Tsc1在树突状细胞的发育方面的作用,但是在体内Tsc1是否直接调节或者怎么调节成熟树突状细胞对T细胞活化增殖作用的机制还不是很明晰。目的:本实验主要研究稳态下成熟树突状细胞表达的Tsc1对T细胞活化增殖作用的机制。方法:(1)Tsc1f/f小鼠和CD11cCre转基因小鼠杂交来建立特异性敲除树突状细胞中Tsc1的小鼠(CD11cCreTsc1f/f)系统,普通PCR和westernblot分别在基因水平和蛋白水平检测Tsc1基因是否敲除。(2)流式细胞术检测T细胞的活化、Ki67表达和细胞因子的表达。(3)应用体外树突状细胞-初始T细胞共培养系统,流式细胞术检测阻断或不阻断Nrp1时T细胞增殖;流式细胞术检测树突状细胞中Nrp1的表达。(4)流式细胞仪检测树突状细胞主要信号通路的激活和变化;流式细胞术检测树突状细胞胞内S6K磷酸化水平、Nrp1表达水平和T细胞的活化比例。结果:(1)成功建立了Tsc 基因敲除小鼠,Tsc1在基因水平和蛋白水平均被敲除。(2)Tc1基因敲除后与对照组相比,脾脏CD4+T细胞和CD8+T细胞活化增加,脾脏中初始CD4+T细胞和CD8+T细胞的细胞数随年龄增加而减少,T细胞细胞增殖增加和细胞因子表达升高。(3)Tc1基因敲除后与对照组相比,树突状细胞诱导的初始T细胞增殖是增加的,树突状细胞中表达大量的Nrp1,阻断Nrp1,可以部分抑制由Tsc1敲除的树突状细胞启动的初始CD4+T细胞的活化。(4)Tc1基因敲除后与对照组相比,S6K和PPAR-y的活化明显增加;在CD11cCreTsc1f/f Raptorf/+小鼠树突状细胞中Nrp1表达及T细胞活化的表型被极大地挽救,mTORC1的过度表达被降低;抑制PPAR-γ,可以使Tsc1敲除的树突状细胞中Nrp1的表达降低。结论:(1)成功建立了Tsc 基因敲除小鼠CD11cCreTsc1f/f;(2)树突状细胞的Tsc1基因可以防止体内自发的T细胞活化;(3)在树突状细胞中Tsc1通过抑制Nrp1表达来阻止初始T细胞的增殖;(4)Tsc1通过阻止mTORC1-PPAR-γ信号通路来抑制Nrp1的表达。目的探索转化生长因子β(transforming growth factor-β,TGF-β)通过调节树突状细胞(dendriticcells,DCs)的功能从而抑制CD4+T细胞增殖活化的可能作用机制。方法应用CD11c+免疫磁珠分选小鼠脾脏DCs,并检测其纯度。TGF-β处理DCs后,进行基因芯片检测。实时定量PCR验证基因芯片结果。DCs经TGF-β处理后和CD4+T细胞共培养,流式细胞术检测CD4+T细胞的活化和增殖。结果CD11c磁珠分选DCs纯度可以达到95%。TGF-β处理DCs后,抑制DCs表面CD53、CD69、CD33、CD74、CD93 分子的表达;抑制趋化因子 Cc13、Ccl5、Ccl9、Cc16、Cc117、Cxcl10、Cc122、Cc14、Ccr7、Cc12、Cxc19、Ccr7 的表达;抑制 IL-2ra、IL12-rb2、IL-15ra、IL-1b、IL-15炎性细胞因子及其受体的表达;抑制CD4+T细胞的增殖和活化。结论TGF-β可以抑制DCs部分重要的表面CD分子、趋化因子及其受体、细胞因子及其受体的表达,最终抑制CD4+T细胞的促活化和增殖。
[Abstract]:Background: dendritic cells (DCs) is an important cell to induce the activation and proliferation of T cells. Nodular sclerosis 1 (tuberous sclerosis complex 1, Tsc1) is an important negative regulator in the upstream of the mammalian target protein of rapamycin (mammalian target of rapamycin). The role of cell development, but the mechanism of direct regulation of Tsc1 in vivo or how to regulate the activation and proliferation of T cells by mature dendritic cells is not clear. Objective: this experiment mainly studied the mechanism of Tsc1 on the activation and proliferation of T cells expressed by mature dendritic cells in the steady state. Methods: (1) Tsc1f/f mice and CD11 CCre transgenic mice were hybridized to establish a specific knockout Tsc1 mouse (CD11cCreTsc1f/f) system. Normal PCR and Westernblot detected the Tsc1 gene at the gene level and protein level. (2) flow cytometry was used to detect the activation of T cells, the expression of Ki67 and the expression of cytokine. (3) the application of dendritic cells in vitro - Initial T cell co culture system, flow cytometry was used to detect the proliferation of T cells when blocking or blocking Nrp1; flow cytometry was used to detect the expression of Nrp1 in dendritic cells. (4) flow cytometry was used to detect the activation and change of the main signal pathways in dendritic cells; flow cytometry was used to detect the level of S6K phosphorylation in dendritic cells, Nrp1 expression level and T Results: (1) Tsc gene knockout mice were successfully established, and Tsc1 was knocked out at both the gene level and the protein level. (2) after Tc1 knockout, the activation of the spleen CD4+T and CD8+T cells increased, and the number of the initial CD4+T cells and CD8+T cells in the spleen decreased with age, and the proliferation of T cell cells. Increase and increase the expression of cytokine. (3) after Tc1 knockout, the proliferation of initial T cells induced by dendritic cells is increased compared with the control group. The expression of a large number of Nrp1 in dendritic cells, blocking the Nrp1, can partially inhibit the activation of the initial CD4+T cells initiated by Tsc1 knockout dendritic cells. (4) Tc1 gene knockout and the control group In contrast, the activation of S6K and PPAR-y increased significantly; the expression of Nrp1 and the activation of T cells in the CD11cCreTsc1f/f Raptorf/+ mouse dendritic cells were greatly saved, the overexpression of mTORC1 was reduced, and the inhibition of PPAR- gamma could reduce the expression of Nrp1 in the Tsc1 knockout dendritic cells. Conclusion: (1) the Tsc gene knockout mice were successfully established. (2) (2) the Tsc1 gene of dendritic cells can prevent spontaneous T cell activation in the body; (3) in dendritic cells, Tsc1 inhibits the proliferation of initial T cells by inhibiting the expression of Nrp1; (4) Tsc1 inhibits the expression of Nrp1 by blocking the mTORC1-PPAR- gamma signaling pathway. Objective to explore transforming growth factor beta (transforming growth factor-). The possible mechanism of inhibiting the proliferation and activation of CD4+T cells by regulating the function of dendritic cells (DendriticCells, DCs) to inhibit the proliferation and activation of CD4+T cells. Methods the splenic DCs of mice was sorted with CD11c+ immunomagnetic beads, and the purity of.TGF- beta treated DCs was detected by gene chip detection. The real time quantitative PCR verification gene chip result was processed by TGF- beta treatment. CD4+T cell co culture, flow cytometry to detect the activation and proliferation of CD4+T cells. Results the purity of CD11c magnetic bead separation DCs can reach 95%.TGF- beta DCs, inhibit the DCs surface CD53, CD69, CD33, CD74, the expression of CD93 molecules; inhibit the expression of chemoattractant The expression of IL-2ra, IL12-rb2, IL-15ra, IL-1b, IL-15 inflammatory cytokines and their receptors, and inhibit the proliferation and activation of CD4+T cells. Conclusion TGF- beta inhibits the important surface CD molecules, chemokines and their receptors, the expression of cytokines and their receptors, and ultimately inhibits the activation and proliferation of CD4+T cells.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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