本文選題：樹突狀細(xì)胞 + 結(jié)節(jié)硬化復(fù)合物1�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文

【摘要】：背景:樹突狀細(xì)胞(dendritic cells,DCs)是一種誘導(dǎo)T細(xì)胞活化和增殖反應(yīng)的重要細(xì)胞。結(jié)節(jié)性硬化物1(tuberous sclerosis complex 1,Tsc1)是哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)上游重要的負(fù)調(diào)節(jié)因子。最近的研究突出Tsc1在樹突狀細(xì)胞的發(fā)育方面的作用,但是在體內(nèi)Tsc1是否直接調(diào)節(jié)或者怎么調(diào)節(jié)成熟樹突狀細(xì)胞對(duì)T細(xì)胞活化增殖作用的機(jī)制還不是很明晰。目的:本實(shí)驗(yàn)主要研究穩(wěn)態(tài)下成熟樹突狀細(xì)胞表達(dá)的Tsc1對(duì)T細(xì)胞活化增殖作用的機(jī)制。方法:(1)Tsc1f/f小鼠和CD11cCre轉(zhuǎn)基因小鼠雜交來建立特異性敲除樹突狀細(xì)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(cè)Tsc1基因是否敲除。(2)流式細(xì)胞術(shù)檢測(cè)T細(xì)胞的活化、Ki67表達(dá)和細(xì)胞因子的表達(dá)。(3)應(yīng)用體外樹突狀細(xì)胞-初始T細(xì)胞共培養(yǎng)系統(tǒng),流式細(xì)胞術(shù)檢測(cè)阻斷或不阻斷Nrp1時(shí)T細(xì)胞增殖;流式細(xì)胞術(shù)檢測(cè)樹突狀細(xì)胞中Nrp1的表達(dá)。(4)流式細(xì)胞儀檢測(cè)樹突狀細(xì)胞主要信號(hào)通路的激活和變化;流式細(xì)胞術(shù)檢測(cè)樹突狀細(xì)胞胞內(nèi)S6K磷酸化水平、Nrp1表達(dá)水平和T細(xì)胞的活化比例。結(jié)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(duì)照組相比,脾臟CD4+T細(xì)胞和CD8+T細(xì)胞活化增加,脾臟中初始CD4+T細(xì)胞和CD8+T細(xì)胞的細(xì)胞數(shù)隨年齡增加而減少,T細(xì)胞細(xì)胞增殖增加和細(xì)胞因子表達(dá)升高。(3)Tc1基因敲除后與對(duì)照組相比,樹突狀細(xì)胞誘導(dǎo)的初始T細(xì)胞增殖是增加的,樹突狀細(xì)胞中表達(dá)大量的Nrp1,阻斷Nrp1,可以部分抑制由Tsc1敲除的樹突狀細(xì)胞啟動(dòng)的初始CD4+T細(xì)胞的活化。(4)Tc1基因敲除后與對(duì)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(xì)胞中Nrp1表達(dá)及T細(xì)胞活化的表型被極大地挽救,mTORC1的過度表達(dá)被降低;抑制PPAR-γ,可以使Tsc1敲除的樹突狀細(xì)胞中Nrp1的表達(dá)降低。結(jié)論:(1)成功建立了Tsc 基因敲除小鼠CD11cCreTsc1f/f;(2)樹突狀細(xì)胞的Tsc1基因可以防止體內(nèi)自發(fā)的T細(xì)胞活化;(3)在樹突狀細(xì)胞中Tsc1通過抑制Nrp1表達(dá)來阻止初始T細(xì)胞的增殖;(4)Tsc1通過阻止mTORC1-PPAR-γ信號(hào)通路來抑制Nrp1的表達(dá)。目的探索轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor-β,TGF-β)通過調(diào)節(jié)樹突狀細(xì)胞(dendriticcells,DCs)的功能從而抑制CD4+T細(xì)胞增殖活化的可能作用機(jī)制。方法應(yīng)用CD11c+免疫磁珠分選小鼠脾臟DCs,并檢測(cè)其純度。TGF-β處理DCs后,進(jìn)行基因芯片檢測(cè)。實(shí)時(shí)定量PCR驗(yàn)證基因芯片結(jié)果。DCs經(jīng)TGF-β處理后和CD4+T細(xì)胞共培養(yǎng),流式細(xì)胞術(shù)檢測(cè)CD4+T細(xì)胞的活化和增殖。結(jié)果CD11c磁珠分選DCs純度可以達(dá)到95%。TGF-β處理DCs后,抑制DCs表面CD53、CD69、CD33、CD74、CD93 分子的表達(dá);抑制趨化因子 Cc13、Ccl5、Ccl9、Cc16、Cc117、Cxcl10、Cc122、Cc14、Ccr7、Cc12、Cxc19、Ccr7 的表達(dá);抑制 IL-2ra、IL12-rb2、IL-15ra、IL-1b、IL-15炎性細(xì)胞因子及其受體的表達(dá);抑制CD4+T細(xì)胞的增殖和活化。結(jié)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(xì)胞因子及其受體的表達(dá),最終抑制CD4+T細(xì)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(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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