本文選題：樹突狀細胞 + 結節(jié)硬化復合物1　； 參考：《北京協(xié)和醫(yī)學院》2017年碩士論文

【摘要】：背景:樹突狀細胞(dendritic cells,DCs)是一種誘導T細胞活化和增殖反應的重要細胞。結節(jié)性硬化物1(tuberous sclerosis complex 1,Tsc1)是哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)上游重要的負調節(jié)因子。最近的研究突出Tsc1在樹突狀細胞的發(fā)育方面的作用,但是在體內Tsc1是否直接調節(jié)或者怎么調節(jié)成熟樹突狀細胞對T細胞活化增殖作用的機制還不是很明晰。目的:本實驗主要研究穩(wěn)態(tài)下成熟樹突狀細胞表達的Tsc1對T細胞活化增殖作用的機制。方法:(1)Tsc1f/f小鼠和CD11cCre轉基因小鼠雜交來建立特異性敲除樹突狀細胞中Tsc1的小鼠(CD11cCreTsc1f/f)系統(tǒng),普通PCR和westernblot分別在基因水平和蛋白水平檢測Tsc1基因是否敲除。(2)流式細胞術檢測T細胞的活化、Ki67表達和細胞因子的表達。(3)應用體外樹突狀細胞-初始T細胞共培養(yǎng)系統(tǒng),流式細胞術檢測阻斷或不阻斷Nrp1時T細胞增殖;流式細胞術檢測樹突狀細胞中Nrp1的表達。(4)流式細胞儀檢測樹突狀細胞主要信號通路的激活和變化;流式細胞術檢測樹突狀細胞胞內S6K磷酸化水平、Nrp1表達水平和T細胞的活化比例。結果:(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基因敲除后與對照組相比,脾臟CD4+T細胞和CD8+T細胞活化增加,脾臟中初始CD4+T細胞和CD8+T細胞的細胞數(shù)隨年齡增加而減少,T細胞細胞增殖增加和細胞因子表達升高。(3)Tc1基因敲除后與對照組相比,樹突狀細胞誘導的初始T細胞增殖是增加的,樹突狀細胞中表達大量的Nrp1,阻斷Nrp1,可以部分抑制由Tsc1敲除的樹突狀細胞啟動的初始CD4+T細胞的活化。(4)Tc1基因敲除后與對照組相比,S6K和PPAR-y的活化明顯增加;在CD11cCreTsc1f/f Raptorf/+小鼠樹突狀細胞中Nrp1表達及T細胞活化的表型被極大地挽救,mTORC1的過度表達被降低;抑制PPAR-γ,可以使Tsc1敲除的樹突狀細胞中Nrp1的表達降低。結論:(1)成功建立了Tsc 基因敲除小鼠CD11cCreTsc1f/f;(2)樹突狀細胞的Tsc1基因可以防止體內自發(fā)的T細胞活化;(3)在樹突狀細胞中Tsc1通過抑制Nrp1表達來阻止初始T細胞的增殖;(4)Tsc1通過阻止mTORC1-PPAR-γ信號通路來抑制Nrp1的表達。目的探索轉化生長因子β(transforming growth factor-β,TGF-β)通過調節(jié)樹突狀細胞(dendriticcells,DCs)的功能從而抑制CD4+T細胞增殖活化的可能作用機制。方法應用CD11c+免疫磁珠分選小鼠脾臟DCs,并檢測其純度。TGF-β處理DCs后,進行基因芯片檢測。實時定量PCR驗證基因芯片結果。DCs經(jīng)TGF-β處理后和CD4+T細胞共培養(yǎng),流式細胞術檢測CD4+T細胞的活化和增殖。結果CD11c磁珠分選DCs純度可以達到95%。TGF-β處理DCs后,抑制DCs表面CD53、CD69、CD33、CD74、CD93 分子的表達;抑制趨化因子 Cc13、Ccl5、Ccl9、Cc16、Cc117、Cxcl10、Cc122、Cc14、Ccr7、Cc12、Cxc19、Ccr7 的表達;抑制 IL-2ra、IL12-rb2、IL-15ra、IL-1b、IL-15炎性細胞因子及其受體的表達;抑制CD4+T細胞的增殖和活化。結論TGF-β可以抑制DCs部分重要的表面CD分子、趨化因子及其受體、細胞因子及其受體的表達,最終抑制CD4+T細胞的促活化和增殖。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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