CTLA4在T调节细胞诱导异种抗原免疫耐受中的机制研究

发布时间：2018-01-01 21:06

本文关键词：CTLA4在T调节细胞诱导异种抗原免疫耐受中的机制研究　出处：《激光生物学报》2016年04期 　论文类型：期刊论文

【摘要】：目的:通过靶向CTLA4 siRNA探讨CTLA4在T调节细胞诱导异种抗原免疫耐受中是否发挥功能及其作用机制。方法:体外扩增培养利用磁珠分选出的T调节细胞,AO/PI染色计算活率并通过细胞计数作出生长曲线,流式细胞仪检测扩增后细胞表型;采用Alex488染料标记siRNA,通过流式细胞仪检测siRNA的转染效率;实时定量PCR检测靶向CTLA4 siRNA的沉默效率;流式细胞术检测CTLA4在蛋白水平的变化;混合淋巴实验检测CTLA4表达下调后,T调节细胞的功能变化。结果:经过4周体外扩增,T调节细胞能够增长约1 200倍,并具有高活率和高纯度;siRNA的转染效率为61.8%±4.5%;Realtime PCR和流式细胞术检测CTLA4在mRNA水平和蛋白水平均有不同程度下降,其结果与对照组相比差异显著(P0.05);混合淋巴实验结果显示T效应细胞在受到异种抗原刺激时会发生增殖,Treg细胞能够抑制这种增殖,但是CTLA4表达下调后明显减弱了T调节细胞的抑制能力。进一步,在DC细胞参与的混合淋巴实验中发现Treg能够通过DC抑制T效应细胞对异种抗原的应答,而si CTLA4-Treg不能通过DC抑制T效应细胞增殖。结论:CTLA4在Treg介导的异种免疫应答中发挥着重要作用,这种作用方式可能是通过直接作用于T效应细胞或者间接通过DC细胞作用于T效应细胞发挥作用。靶向CTLA4 siRNA能够下调Treg细胞CTLA4的表达,影响Treg细胞抑制异种抗原引起T效应细胞应答的能力。
[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% 卤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.
【作者单位】：细胞移植与基因治疗研究中心中南大学湘雅三医院;
【基金】：国家自然科学基金青年科学基金(81201171) 湖南省自然科学基金重点项目(13JJ2003) 湖南省卫计委科研计划课题项目(20160125)
【分类号】：R392
【正文快照】： T调节细胞是一组具有抑制自身免疫反应、阻止损伤性免疫病理发生和维持机体免疫平衡作用的T细胞亚群,可用于预防和治疗免疫排斥、移植物抗宿主病(GVHD)以及自身免疫性疾病[1,2]。CD4+CD25+T细胞是T调节细胞家族中最主要的一群,由Sakaguchi等在1995年首先发现,CD25是IL-2受体的

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