血浆Exosomes小体对Treg细胞功能的影响及B细胞体外扩增Treg细胞的初步研究

【作者】 马越娥；

【导师】 范华骅；

【作者基本信息】 复旦大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 1.通过将人血浆Exosomes小体、重组Wnt分子分别与人外周血CD4+CD25+CD1271ow调节性T细胞(Regulatory T Cells, Treg)作用,观察Treg细胞在体外的存活情况及其Wnt信号通路的变化。2.通过将同种异基因人外周血B淋巴细胞体外扩增Treg细胞,观察Treg细胞扩增前后在数量上的变化及表型和功能是否改变,建立体外扩增Treg细胞的有效方法,并与树突细胞(dendritic cell, DC)扩增Treg细胞的方法相比较。1.应用免疫磁珠分离方法分离纯化人外周血CD4+CD25+CD1271owTreg细胞,将Wnt分子和从多个健康供者血浆中分离得到的Exosomes小体分别加入到Treg细胞中,加入人白介素2(IL-2,50U/ml)。利用RT-PCR检测共培养12h后Wnt信号通路涉及的凋亡相关基因的改变,同时采用流式技术检测Wnt信号通路中磷酸化β-catenin水平的改变,并在共培养14天后采用流式技术检测各组细胞的凋亡情况。设立未经处理的Treg细胞组为对照组。2.将异体健康供者外周血分离得到的B淋巴细胞与磁珠分选得到的Treg细胞共孵育,用IL-2(IOOU/ml)和CD28单抗(500ng/ml)共同刺激,在培养14天的过程中通过流式检测Treg细胞表型的变化,同时与树突状细胞扩增Treg细胞的方法相比较(培养条件相同)。14天培养结束后,将细胞与CFSE处理过的经强刺激(加入CD3单抗500ng/ml,IL-2300U/ml)的CD4+效应性T细胞共培养,在第五天通过流式检测Treg细胞对CD4+T细胞的增殖抑制,设立只经强刺激的CFSE处理过的CD4+T细胞为对照组。1.通过RT-PCR分析纯化的Treg细胞上的Frizzled(FRZ)受体及低密度脂蛋白相关蛋白5(LRP5)和LRP6的基因表达,结果显示Treg细胞表达FRZ2、3、4,LRP6基因,通过Western Blotting技术检测血浆中Exosomes小体上Wnt3a和Wnt5a的蛋白表达,均有条带出现。共培养12h后,RT-PCR检测凋亡相关基因Bcl-xL、Bcl-2、c-Myc的表达,结果显示Treg细胞与血浆exosomes样小体或Wnt分子共培养时,抗凋亡基因Bcl-2相对于对照组均有明显的上调,另一抗凋亡基因Bcl-xL和诱导凋亡的基因c-Myc无明显变化。培养14天后,通过流式检测凋亡,加入Exosomes样小体或Wnt分子的Treg细胞生存率均比对照组高。2.经B细胞联合IL-2和CD28单抗刺激的Treg细胞在培养14天后流式检测Foxp3和CD4/CD25,阳性率均很高,此时计数Treg细胞为最初数量的20倍。利用CFSE检测技术,将扩增14天的Treg细胞与CD3单抗和IL-2强刺激的CD4+T细胞共培养,5天后流式检测,扩增后的Treg细胞能够有效地抑制CD4+T细胞的增殖功能。1.初步判断Wnt信号通路能够影响Treg细胞的生存状态,血浆Exosomes小体能够延长Treg细胞的生存时间,其中Exosomes小体携带Wnt分子与Treg细胞上的Frizzled受体作用可以活化Treg细胞的Wnt信号通路,在提高Treg细胞的生存率方面起了一定的作用,即血浆Exosomes小体可能通过Wnt信号通路途径影响Treg细胞的存活。2.同种异基因B淋巴细胞能够在体外有效的扩增人外周血Treg细胞,扩增效率可达20倍以上,并且能显著抑制CD4+T细胞的增殖。更多还原

【Abstract】 1. Through the effect of human plasma exosomes-like vesicles or recombinant Wnt molecules on CD4+ CD25+ CD127low regulatory T cells (Treg), observed the survival of Treg cells in vitro and the changes of the Wnt signaling pathway.2. Through the expansion of Treg cells by allogeneic human peripheral blood B lymphocytes in vitro, observed the changes of Treg cells in the number, phenotype and function after the expansion, established an effective way of the expansion of Treg cells in vitro, and compared with the expansion method by regulatory dendritic cells (rDC).1.CD4+CD25+CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) using Magnetic cell sorting, Wnt molecules and human plasma exosomes-like vesicles isolated from several healthy donors were cocultured with the Treg cells respectively, with interleukin 2 (IL-2,50U/ml). Analyzed the changes of apoptosis related genes what Wnt signaling pathway involved in after 12h by RT-PCR, detected the changes of phosphorylatedp-catenin level in Wnt signaling pathway and the apoptosis of co-culture cells in every group after 14 days by flow cytometry. The untreated Treg cells were the control group.2. The purified Treg cells handled by B-lymphocyte isolated from allogeneic peripheral blood with IL-2 (100U/ml) and CD28 monoclonal antibody (500ng/ml), detected the changes of phenotype of Treg cells in the 14 days co-culture. On the 14th day, the expanded Treg cells were cultured with CD4+ effector T cells which were stimulated strongly (addition of 500ng/ml CD3 monoclonal antibody and 300U/ml IL-2), detected the inhibition of the proliferation of CD4+ effector T cells by flow cytometry on the 5th day. The CD4+ effector T cells stimulated strongly only were established as the control group. 1. Analyzed the Frizzled receptors (FRZ) and LDL receptor related protein 5 (LRP5) and LRP6 gene expression on Treg cells by RT-PCR, the results showed that the Treg cells expressed FRZ2,3,4, LRP6 genes. Detected the protein expression of Wnt3a and Wnt5a in Plasma Exosomes by Western Blotting, both appeared the bands. Detection of apoptosis-related genes Bcl-xL, Bcl-2, c-Myc expression after 12h co-culture by RT-PCR showed that anti-apoptosis gene Bel-2 increased significantly in Treg cells cultured with plasma exosomes-like vesicles or the Wnt molecules, the expression of Bcl-xL and c-Myc had no significant change. On the 14th day, the survival rate of Treg cells which added exosomes-like vesicles or the Wnt molecules were higher than the control group by detecting the apoptosis with flow cytometry.2. After 14 days of coculturation with B cells combined with IL-2 and CD28 monoclonal antibody, the positive rate of Foxp3 and CD4/CD25 of the Treg cells were both high, and the absolute number of Treg cells were amplified 20 times. The expanded Treg cells cultured with the CD4+ T cells which were stimulated by CD3 monoclonal antibody and IL-2, the results detected by flow cytometry using CFSE technology after 5 days showed that the expanded Treg cells can effectively inhibit the proliferation of CD4+ effector T cells.1. We can initially judge that the Wnt signaling pathway can influence the survival status of Treg cells, plasma Exosomes-like vesicles were able to prolong the survival time of the Treg cells, in which the interaction between Wnt molecules carried by Exosomes-like vesicles and the Frizzled receptors on Treg cells can activate the Wnt signaling pathway of the Treg cells, which played a certain role in improving the survival rate of the Treg cells. The plasma Exosomes-like vesicles can affect the survival of Treg cells in vitro through the Wnt singaling pathway.2. Allogeneic B lymphocytes can effectively amplify human peripheral blood Treg cells in vitro, the expansion efficiency can be up to 20 times compared to the initial, and the expanded Treg cells can inhibit the proliferation of CD4+ effector T cell significantly.更多还原