本文關(guān)鍵詞：CTLA4在T調(diào)節(jié)細(xì)胞誘導(dǎo)異種抗原免疫耐受中的機(jī)制研究　出處：《激光生物學(xué)報(bào)》2016年04期 　論文類型：期刊論文

  更多相關(guān)文章： 細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原 小分子RNA T調(diào)節(jié)細(xì)胞 異種抗原 免疫應(yīng)答

【摘要】：目的:通過(guò)靶向CTLA4 siRNA探討CTLA4在T調(diào)節(jié)細(xì)胞誘導(dǎo)異種抗原免疫耐受中是否發(fā)揮功能及其作用機(jī)制。方法:體外擴(kuò)增培養(yǎng)利用磁珠分選出的T調(diào)節(jié)細(xì)胞,AO/PI染色計(jì)算活率并通過(guò)細(xì)胞計(jì)數(shù)作出生長(zhǎng)曲線,流式細(xì)胞儀檢測(cè)擴(kuò)增后細(xì)胞表型;采用Alex488染料標(biāo)記siRNA,通過(guò)流式細(xì)胞儀檢測(cè)siRNA的轉(zhuǎn)染效率;實(shí)時(shí)定量PCR檢測(cè)靶向CTLA4 siRNA的沉默效率;流式細(xì)胞術(shù)檢測(cè)CTLA4在蛋白水平的變化;混合淋巴實(shí)驗(yàn)檢測(cè)CTLA4表達(dá)下調(diào)后,T調(diào)節(jié)細(xì)胞的功能變化。結(jié)果:經(jīng)過(guò)4周體外擴(kuò)增,T調(diào)節(jié)細(xì)胞能夠增長(zhǎng)約1 200倍,并具有高活率和高純度;siRNA的轉(zhuǎn)染效率為61.8%±4.5%;Realtime PCR和流式細(xì)胞術(shù)檢測(cè)CTLA4在mRNA水平和蛋白水平均有不同程度下降,其結(jié)果與對(duì)照組相比差異顯著(P0.05);混合淋巴實(shí)驗(yàn)結(jié)果顯示T效應(yīng)細(xì)胞在受到異種抗原刺激時(shí)會(huì)發(fā)生增殖,Treg細(xì)胞能夠抑制這種增殖,但是CTLA4表達(dá)下調(diào)后明顯減弱了T調(diào)節(jié)細(xì)胞的抑制能力。進(jìn)一步,在DC細(xì)胞參與的混合淋巴實(shí)驗(yàn)中發(fā)現(xiàn)Treg能夠通過(guò)DC抑制T效應(yīng)細(xì)胞對(duì)異種抗原的應(yīng)答,而si CTLA4-Treg不能通過(guò)DC抑制T效應(yīng)細(xì)胞增殖。結(jié)論:CTLA4在Treg介導(dǎo)的異種免疫應(yīng)答中發(fā)揮著重要作用,這種作用方式可能是通過(guò)直接作用于T效應(yīng)細(xì)胞或者間接通過(guò)DC細(xì)胞作用于T效應(yīng)細(xì)胞發(fā)揮作用。靶向CTLA4 siRNA能夠下調(diào)Treg細(xì)胞CTLA4的表達(dá),影響Treg細(xì)胞抑制異種抗原引起T效應(yīng)細(xì)胞應(yīng)答的能力。
[Abstract]:Aim: to investigate the function and mechanism of CTLA4 in T cell mediated xenoantigen tolerance by targeting CTLA4 siRNA. T-regulated cells isolated from magnetic beads were amplified and cultured in vitro. The viability was calculated by AO/PI staining and the growth curve was made by cell count. The phenotypes of expanded cells were detected by flow cytometry. Alex488 dye labeled siRNAs were used to detect the transfection efficiency of siRNA by flow cytometry. The silencing efficiency of target CTLA4 siRNA was detected by real-time quantitative PCR. The changes of CTLA4 in protein level were detected by flow cytometry. Mixed lymphoid assay was used to detect the functional changes of T regulatory cells after down-regulation of CTLA4 expression. Results: after 4 weeks of expansion in vitro, the T regulated cells could increase about 1 200 times, with high viability and high purity. The transfection efficiency of siRNA was 61.8% 鹵4.5. Realtime PCR and flow cytometry showed that the mRNA and protein levels of CTLA4 were decreased in different degrees, and the results were significantly different from those of the control group (P 0.05). The results of mixed lymphoid assay showed that T effector cells could proliferate when stimulated by xenoantigen and Treg cells could inhibit the proliferation. However, the down-regulation of CTLA4 expression significantly weakened the inhibitory ability of T regulatory cells. In the mixed lymphoid experiment in which DC cells participated, Treg could inhibit the response of T effector cells to xenoantigen through DC. Si CTLA4-Treg could not inhibit the proliferation of T effector cells through DC. Conclusion: CTLA4 plays an important role in Treg mediated xenoimmune response. This action may be done by acting directly on T effector cells or indirectly by DC cells on T effector cells. SiRNA can down-regulate the expression of CTLA4 in Treg cells. The ability of Treg cells to inhibit the response of T effector cells induced by xenoantigen was affected.
【作者單位】： 細(xì)胞移植與基因治療研究中心中南大學(xué)湘雅三醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金青年科學(xué)基金(81201171) 湖南省自然科學(xué)基金重點(diǎn)項(xiàng)目(13JJ2003) 湖南省衛(wèi)計(jì)委科研計(jì)劃課題項(xiàng)目(20160125)
【分類號(hào)】：R392
【正文快照】： T調(diào)節(jié)細(xì)胞是一組具有抑制自身免疫反應(yīng)、阻止損傷性免疫病理發(fā)生和維持機(jī)體免疫平衡作用的T細(xì)胞亞群,可用于預(yù)防和治療免疫排斥、移植物抗宿主病(GVHD)以及自身免疫性疾病[1,2]。CD4+CD25+T細(xì)胞是T調(diào)節(jié)細(xì)胞家族中最主要的一群,由Sakaguchi等在1995年首先發(fā)現(xiàn),CD25是IL-2受體的

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