CTLA4在T调节细胞诱导异种抗原免疫耐受中的机制研究  

The Mechanism Study on CTLA4 involved in Regulatory T cell Mediating Immune Tolerance for Xenogeneic Response

作　　者：马小倩[1] 王佳[1] 王维[1] MA Xiaoqian WANG Jia WANG Wei(Cell Transplantation and Gene Therapy Institute, the 3rd Xiangya Hospital of Central South University, Changsha 410013, Hunan, China)

机构地区：[1]细胞移植与基因治疗研究中心,中南大学湘雅三医院,湖南长沙410013

出　　处：《激光生物学报》2016年第4期342-349,共8页Acta Laser Biology Sinica

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)

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(P<0.05);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。Objective：To evaluate whether CTLA4 plays an important role in regulatory T （Treg）cells mediating im-mune tolerance for xeno-antigen and to explore the mechanism.Methods：Regulatory T cells which were separated by CD4 ＋CD25 ＋CD127dim regulatory T cell kit were cultured with IL2,rapamycin and CD3 /CD28 microbeads in vitro for 4 weeks.The viability of expanded Treg was examined by AO /PI staining and the growth curve of Treg was calculated through cell counting.Fluorescence Activating Cell Sorter （FACS）was used to analyze the phenotype of expanded Treg and the siRNA transfection efficiency by labeled with Alex488.The down-regulation efficiency of CTLA4 siRNA was ex-amined by Real-time PCR and flow cytometry.Mixed lymphocyte reaction was used to explore the function of Treg cells incubated with or without DCs.Results：After expanded in vitro for 4 weeks,the number of Treg cells which possessed with high activity and purity was up to 1200 times compared to the freshly isolated cells.The transfection efficiency of siRNA was 61.8% ＆#177;4.5%.The expression of CTLA4 was down-regulated either in mRNA level or in protein level, which had significant difference compared to control group （P ＆lt;0.05）.And the CTLA4 targeted siRNA did not affect the expression of other function related genes of Treg.Mixed lymphocyte reaction （MLR）showed that Treg cells down-regulation of CTLA4 could not inhibit the effector T cells＆#39;proliferation which was induced by xeno-antigen.Moreover,in MLR with DCs the suppression ability of CTLA4 siTreg was also reduced compared to the control Treg at all ratios.Con-clusion：Taken together,CTLA4 plays an important role in human Treg mediating suppression for xenogeneic response and CTLA4 involves the suppression through both direct way and indirect way.Down-regulation of CTLA4 could abrogate the suppressive ability of Treg for xeno-antigen.

关 键 词：细胞毒性T淋巴细胞相关抗原4 小分子RNA T调节细胞 异种抗原 免疫应答 

分 类 号：R392.4[医药卫生—免疫学]