本文選題：miRNA + 調(diào)節(jié)T細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：研究背景和目的:近年來,大量研究表明Treg細(xì)胞在器官移植免疫排斥反應(yīng)中起著重要的調(diào)控作用,通過對Treg細(xì)胞的調(diào)控能夠控制免疫排斥反應(yīng)。如果在移植前或者在排斥反應(yīng)的早期階段采取措施在受者體內(nèi)誘導(dǎo)產(chǎn)生足夠數(shù)量的Treg細(xì)胞或增強(qiáng)其抑制功能,將可能會減輕排斥反應(yīng),實現(xiàn)對移植器官的免疫耐受。盡管如此,Treg細(xì)胞的誘導(dǎo)產(chǎn)生、分化發(fā)育以及發(fā)揮抑制作用的確切機(jī)制仍不明確,有關(guān)Treg細(xì)胞在器官移植中的合理誘導(dǎo)方式、如何保持其最佳作用也有待于進(jìn)一步的深入研究。近年來隨著對microRNA(miRNA)在免疫學(xué)領(lǐng)域的研究深入,人們發(fā)現(xiàn)miRNA在Treg細(xì)胞的穩(wěn)態(tài)和功能方面起著重要的調(diào)控作用,很多基因在Treg細(xì)胞內(nèi)持續(xù)性地上調(diào)或者下調(diào)也是依賴于miRNA的。研究人員還進(jìn)一步篩選出Treg細(xì)胞和效應(yīng)T細(xì)胞中差異表達(dá)的miRNA,其中包括與機(jī)體免疫應(yīng)答關(guān)系十分密切的miR-146a。這種miRNA是在細(xì)胞膜表面由參與固有免疫應(yīng)答中的TLRs信號通路所調(diào)控的,而TLRs配體、TNF-α以及IL-1β等多種因素都能夠誘導(dǎo)miR-146a的表達(dá)。在后續(xù)的研究中證實miR-146a能夠在轉(zhuǎn)錄后水平調(diào)控TRAF6、IRAK1、STAT1三者的表達(dá),進(jìn)而對免疫信號轉(zhuǎn)導(dǎo)通路起到反饋調(diào)節(jié)作用,從而影響機(jī)體的免疫應(yīng)答過程。但是在器官移植免疫應(yīng)答過程中,有關(guān)miR-146a和Treg的關(guān)系卻未見報道。本研究主要目的是探討miR-146a調(diào)控Treg細(xì)胞的分子機(jī)制,并在體內(nèi)通過調(diào)控miR-146a表達(dá)水平來增強(qiáng)Treg細(xì)胞的免疫抑制功能,從而建立一種抑制器官移植免疫排斥的新方法。研究方法:本研究分為三個部分:體外轉(zhuǎn)染實驗、體內(nèi)轉(zhuǎn)染實驗和心臟移植模型實驗。在第一部分,通過體外轉(zhuǎn)染調(diào)控Treg細(xì)胞中miR-146a的表達(dá)水平來研究其對Treg細(xì)胞的影響,并探討其機(jī)制。在第二部分,通過陰莖背靜脈注射miR-146a agomir和antagomir進(jìn)行體內(nèi)轉(zhuǎn)染,評價轉(zhuǎn)染效果并篩選出調(diào)控Treg細(xì)胞mi R-146a表達(dá)的最佳轉(zhuǎn)染方案,并進(jìn)一步探討體內(nèi)轉(zhuǎn)染調(diào)控miR-146a的表達(dá)對小鼠Treg細(xì)胞的影響。在第三部分,以第二部分的轉(zhuǎn)染小鼠作為受體,建立腹腔異位心臟移植模型,探討調(diào)控treg細(xì)胞mir-146a的表達(dá)對同種異系小鼠心臟移植急性免疫排斥的影響,并探討其機(jī)制。研究結(jié)果:1.在體外實驗中,mir-146aagomir和antagomir可以分別顯著上調(diào)和下調(diào)treg細(xì)胞mir-146a的表達(dá)水平(p0.01)。與對照組相比,agomir組traf6、irak1、stat1mrna轉(zhuǎn)錄水平及蛋白的表達(dá)水平均顯著下調(diào)(p0.01);antagomir組traf6、irak1、stat1mrna轉(zhuǎn)錄水平及蛋白的表達(dá)水平顯著上調(diào)(p0.01)。antagomir組treg細(xì)胞的cd62l和ki67的表達(dá)水平較對照組和agomir組顯著升高(p0.01)。另外,treg細(xì)胞的體外抑制功能實驗中,antagomir組cd4+t細(xì)胞的增殖指數(shù)較其他兩組也明顯降低(p0.05)。2.在體內(nèi)實驗中,與對照組相比,mir-146aagomir和antagomir轉(zhuǎn)染劑量分別為10nmol和150nmol時,脾臟中treg細(xì)胞的mir-146a表達(dá)水平顯著改變(p0.01),而相應(yīng)劑量下的cd4+cd25-t細(xì)胞的mir-146a表達(dá)卻不受影響(p0.05)。agomir組脾臟中treg細(xì)胞traf6、irak1、stat1mrna及蛋白的表達(dá)水平顯著下調(diào)(p0.01),antagomir組三者mrna及蛋白的表達(dá)水平顯著上調(diào)(p0.01)。各組外周血、胸腺及脾臟中t細(xì)胞亞群(cd3+、cd4+、cd8+)的比例無顯著統(tǒng)計學(xué)差異(p0.05)。antagomir組外周血和脾臟中treg細(xì)胞比例較對照組和agomir組明顯升高(p0.01),而三組胸腺中treg細(xì)胞比例卻無顯著性差異(p0.05)。單向混合淋巴細(xì)胞培養(yǎng)實驗中,與對照組和agomir組相比,antagomir組cd4+t細(xì)胞比例顯著降低(p0.05),treg細(xì)胞比例顯著升高(p0.01),ifn-γ陽性表達(dá)的細(xì)胞(th1細(xì)胞)比例顯著升高(p0.01)。antagomir組混合培養(yǎng)體系上清液ifn-γ的表達(dá)也顯著升高(p0.01),而三組il-4、il-17的表達(dá)卻無顯著性差異(p0.05)。3.在心臟移植模型實驗中,與生理鹽水對照組、antagomir組和αifn-γ組相比,antagomir+αifn-γ組心臟移植物的存活時間明顯延長(p0.05),急性排斥反應(yīng)病理分級亦顯著降低(p0.05)。另外,antagomir組和antagomir+αifn-γ組的cd4+t細(xì)胞比例較生理鹽水對照組明顯降低(p0.05),而treg細(xì)胞比例以及foxp3在心臟移植物的表達(dá)水平均較其余兩組顯著增高(p0.05)。與生理鹽水對照組比較,antagomir組術(shù)后第5天心臟移植物ifn-γ的表達(dá)顯著增高(p0.05),stat1和pstat1的蛋白表達(dá)水平顯著上調(diào)(p0.05)。antagomir協(xié)同ifn-γ中和抗體干預(yù)后,pSTAT1的表達(dá)水平顯著下調(diào)(P0.05),而各組TRAF6和IRAK1的表達(dá)水平卻無顯著差異(P0.05)。研究結(jié)論:經(jīng)小鼠陰莖背靜脈注射miR-146a agomir和antagomir轉(zhuǎn)染試劑能夠分別有效的上調(diào)和下調(diào)Treg細(xì)胞內(nèi)miR-146a的表達(dá)。正常生理狀態(tài)下,mi R-146a在Treg細(xì)胞中能夠調(diào)控TRAF6、IRAK1、STAT1三個靶基因的表達(dá)。下調(diào)Treg細(xì)胞mi R-146a的表達(dá)能夠增強(qiáng)Treg細(xì)胞的增殖能力和對CD4+T細(xì)胞的抑制功能。在小鼠心臟移植免疫排斥過程中,miR-146a主要參與調(diào)控Treg細(xì)胞的IFN-γ/STAT1信號通路,其靶基因是STAT1。雖然miR-146a下調(diào)仍能夠有效的誘導(dǎo)受體Treg細(xì)胞的增殖,但是Treg細(xì)胞抑制Th1分泌IFN-γ的功能受損,若協(xié)同應(yīng)用IFN-γ中和抗體則能夠有效抑制免疫排斥,從而改善移植心臟存活狀況。
[Abstract]:Research background and purpose: in recent years, a large number of studies have shown that Treg cells play an important regulatory role in the immune rejection of organ transplantation, and can control immune rejection through the regulation of Treg cells. A sufficient number of Treg fines are induced in the recipients before or in the early stages of rejection. In spite of this, the induction of Treg cells, differentiation and development, and the exact mechanism to play a inhibitory role are still unclear. The rational inducement of Treg cells in organ transplantation and how to keep their best effects remain to be made. In recent years, with the in-depth study of microRNA (miRNA) in the field of immunology, people have found that miRNA plays an important role in the regulation of the homeostasis and function of Treg cells. Many genes are continuously up-regulated or downregulated in Treg cells and are also dependent on miRNA. Researchers have further screened the Treg cells and the Treg cells. The differential expression of miRNA in effect T cells, including miR-146a., which is closely related to the immune response of the body, is regulated by the TLRs signaling pathway involved in the intrinsic immune response on the surface of the cell membrane, while a variety of factors such as TLRs ligands, TNF- a, and IL-1 beta can induce the expression of miR-146a. Real miR-146a can regulate the expression of TRAF6, IRAK1, and STAT1 three in the post transcriptional level, and then regulate the immune signal transduction pathway, thus affecting the immune response process. However, the relationship between miR-146a and Treg has not been reported in the process of organ transplantation immune response. The main purpose of this study is to explore miR-146a. The molecular mechanism of Treg cells is regulated and the immunosuppressive function of Treg cells is enhanced by regulating the expression level of miR-146a in the body. A new method of inhibiting immune rejection in organ transplantation is established. The research method is divided into three parts: in vitro transfection experiment, in vivo transfection experiment and heart transplantation model experiment. The expression level of miR-146a in Treg cells was regulated by transfection in vitro, and its effect on Treg cells was studied and its mechanism was discussed. In the second part, miR-146a agomir and antagomir were injected into the dorsal vein of the penis to be transfected in vivo. The transfection effect was evaluated and the optimal transfection scheme to regulate the R-146a expression of Treg MI was selected and further improved. To investigate the effect of the expression of miR-146a on mouse Treg cells in vivo transfection. In the third part, a second part of the transfected mice was used as a receptor to establish a model of heterotopic heart transplantation in the abdominal cavity. The effect of the expression of Treg cell miR-146a on the acute immunization rejection of the allogeneic mice was investigated and its mechanism was discussed. The results were as follows: 1 In vitro, mir-146aagomir and antagomir could significantly up-regulate and downregulate the expression level of miR-146a in Treg cells (P0.01). Compared with the control group, the level of TRAF6, irak1, stat1mrna transcriptional level and protein expression in the agomir group were significantly down (P0.01), antagomir group TRAF6, transcription and protein expression level. The expression level of CD62L and Ki67 in Treg cells in group.Antagomir (P0.01) was significantly higher than that in the control group and agomir group (P0.01). In addition, the proliferation index of antagomir group cd4+t cells was significantly lower than that of the other two groups in the inhibition function experiment of Treg cells (P0.05).2. in vivo, compared with the control group. When the transfection dose of tagomir was 10nmol and 150nmol, the miR-146a expression level of the Treg cells in the spleen was significantly changed (P0.01), while the miR-146a expression of the cd4+cd25-t cells at the corresponding dose was not affected (P0.05).Agomir group Treg cell TRAF6 in the spleen. The expression level of RNA and protein was significantly up-regulated (P0.01). The proportion of T cell subgroups (cd3+, cd4+, cd8+) in peripheral blood, thymus and spleen of each group had no significant difference (P0.05), the proportion of Treg cells in peripheral blood and spleen of.Antagomir group was significantly higher than that of control group and agomir group (P0.01), but there was no significant difference in the proportion of Treg cells in the three groups of thymus. (P0.05) in the unidirectional mixed lymphocyte culture experiment, compared with the control group and the agomir group, the proportion of cd4+t cells in the antagomir group decreased significantly (P0.05), the proportion of Treg cells increased significantly (P0.01), and the proportion of ifn- y positive cells (Th1 cells) increased significantly (P0.01).Antagomir group and the expression of ifn- gamma in the mixed culture system was also significantly increased. 0.01) and there was no significant difference in the expression of IL-4 and IL-17 in the three groups (P0.05).3. in the model experiment of heart transplantation, compared with the saline control group. Compared with the antagomir group and the group of alpha ifn- gamma, the survival time of the cardiac allograft in the antagomir+ alpha ifn- group was significantly prolonged (P0.05), and the pathological grading of the acute rejection was also significantly decreased (P0.05). In addition, antagomir group The proportion of cd4+t cells in the group of antagomir+ alpha ifn- gamma was significantly lower than that in the normal saline control group (P0.05), while the proportion of Treg cells and the expression level of Foxp3 in the heart grafts were significantly higher than those in the other two groups (P0.05). Compared with the saline control group, the expression of ifn- gamma in the fifth days after antagomir group was significantly higher (P0.05), stat. The protein expression level of 1 and pstat1 was significantly up-regulated (P0.05) and the expression level of pSTAT1 was significantly down (P0.05), but there was no significant difference in the expression level of TRAF6 and IRAK1 in each group (P0.05). The conclusion was that the miR-146a agomir and the antagomir transfection reagents could be effective by injecting the dorsal vein of the penis in mice. The expression of miR-146a in Treg cells is up and down. Under normal physiological state, MI R-146a can regulate the expression of three target genes of TRAF6, IRAK1, STAT1 in Treg cells. Down regulation of Treg cell mi R-146a can enhance the proliferation of Treg cells and inhibit the inhibitory function of the cells. R-146a mainly participates in the IFN- gamma /STAT1 signaling pathway regulating Treg cells, the target gene is STAT1. although the miR-146a downregulation can effectively induce the proliferation of receptor Treg cells, but Treg cells inhibit the function of Th1 secreting IFN- gamma, and the synergistic application of IFN- gamma neutralization antibody can effectively inhibit immune rejection, thus improving the transplanted heart. Survival.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R654.2

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3 史軍;剝開黏合的細(xì)胞之書[N];健康報;2012年

4 張佳星;分化之路 殊途同歸[N];科技日報;2008年

5 記者 施嘉奇　通訊員 王姿英;探索中藥對干細(xì)胞分化的作用[N];文匯報;2009年

6 常麗君;美生物學(xué)家打造出“細(xì)胞黑客”[N];科技日報;2010年

7 記者陳超;日開發(fā)細(xì)胞間隔離培養(yǎng)技術(shù)[N];科技日報;2003年

8 陳超;科學(xué)家發(fā)現(xiàn)引發(fā)細(xì)胞分化的分子通道[N];科技日報;2010年

9 記者 白毅;我國科學(xué)家用iPS細(xì)胞首獲克隆豬[N];中國醫(yī)藥報;2013年

10 記者 錢錚;日本用人類iPS細(xì)胞首次制成血小板[N];新華每日電訊;2009年

相關(guān)碩士學(xué)位論文 前10條

1 申芳芳;B1-a細(xì)胞和B2細(xì)胞二者在發(fā)育和分化方面的相關(guān)性的研究[D];中國醫(yī)科大學(xué);2010年

2 彭勇;低強(qiáng)度瞬態(tài)電磁場導(dǎo)致細(xì)胞膜穿孔的研究[D];四川大學(xué);2001年

3 劉麗萍;mTOR/MAPK信號轉(zhuǎn)導(dǎo)途徑對細(xì)胞倍體化的作用及修飾機(jī)制[D];大連醫(yī)科大學(xué);2006年

4 鄭琛;大鼠骨髓間充質(zhì)干細(xì)胞生物反應(yīng)器灌注培養(yǎng)細(xì)胞數(shù)量即時監(jiān)測與分化的研究[D];上海交通大學(xué);2009年

5 李珍;胰島素樣生長因子-1聯(lián)合外源性轉(zhuǎn)錄因子誘導(dǎo)小鼠iPS細(xì)胞的研究[D];華中科技大學(xué);2012年

6 鄭宇;支架蛋白palladin在細(xì)胞分化和侵襲中的作用[D];吉林大學(xué);2013年

7 劉，

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