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

发布时间：2018-01-28 04:09

本文关键词： CD4+CD25+CD127low调节性T细胞血浆Exosomes小体 Wnt信号通路 B淋巴细胞体外扩增增殖抑制　出处：《复旦大学》2010年硕士论文　论文类型：学位论文

【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。
[Abstract]:1., we observed the survival of Treg cells in vitro and the changes of Wnt signaling pathway through the action of human plasma Exosomes bodies and recombinant Wnt molecules on CD4+CD25+CD1271ow regulatory T cells (Regulatory T Cells, Treg), respectively.
2. by allogeneic peripheral blood B lymphocytes in vitro amplification of Treg cells, Treg cells were observed before and after expansion in the number of changes and phenotypic and functional changes in the effective method to establish the amplification of Treg cells in vitro, and dendritic cells (dendritic cell, DC) compared with amplification of Treg cells.
Separation and purification of CD4+CD25+CD1271owTreg cells from peripheral blood of 1. application of immunomagnetic separation method, the isolated plasma Wnt molecules and from multiple healthy donors in Exosomes bodies were added to Treg cells, adding human interleukin 2 (IL-2,50U/ml). RT-PCR is used to detect the co culture of 12h Wnt pathway involved in apoptosis related genes. At the same time, using flow cytometry to detect phosphorylation of Wnt signaling pathway in the beta -catenin level changes, and after 14 days of CO cultivation with apoptosis was detected by flow cytometry. The cells were set up without Treg cell group treatment as control group.
2. healthy allogeneic donor peripheral blood B lymphocytes were isolated and multisort obtained Treg cells incubated with IL-2 monoclonal antibody (IOOU/ml) and CD28 (500ng/ml) Co stimulation, by changing the flow cytometry phenotype of Treg cells during the 14 day culture, compared the simultaneous amplification of Treg cells and dendritic cells (cultured in the same conditions).14 days after culture, the cells treated with CFSE by strong stimulation (adding CD3 mAb 500ng/ml, IL-2300U/ml) co cultured CD4+ effector T cells, Treg cells were detected by flow type CD4 on +T cells proliferation in fifth days, set up by only CFSE strong stimulation of CD4+T cells as the control group.
1. through RT-PCR analysis of purified Treg cell Frizzled (FRZ) and low density lipoprotein receptor related protein 5 (LRP5) and expression of LRP6 gene, the results showed that Treg FRZ2,3,4 expression of LRP6 gene was detected by Exosomes, plasma Western Blotting technology on Wnt3a and Wnt5a were both protein expression bands. Co culture 12h, RT-PCR detection of apoptosis related gene Bcl-xL, Bcl-2, c-Myc expression showed that Treg cells were co cultured with plasma exosomes like bodies or Wnt molecules, the anti apoptotic gene Bcl-2 was significantly up-regulated compared with the control group, another anti apoptotic gene Bcl-xL and apoptosis gene c-Myc had no obvious change. Culture 14 days later, apoptosis by flow cytometry, adding Exosomes like bodies or Wnt molecules Treg cell survival rate were higher than the control group.
2. after B cells combined with IL-2 and CD28 monoclonal antibody stimulated Treg cells cultured for 14 days in flow cytometry detection of Foxp3 and CD4/CD25, the positive rate is very high, the Treg cell count was 20 times the original number. The use of CFSE detection technology, the amplification of 14 days of Treg cells with CD3 monoclonal antibody and IL-2 strong stimulation of CD4 +T cells after 5 days of co culture, flow cytometry, expanded Treg cells can effectively inhibit the proliferation of CD4+T cells.
1. preliminary judgment can Wnt pathway affects the survival of Treg cells, plasma Exosomes bodies can prolong the survival time of Treg cells, Wnt signaling pathway in which Exosomes bodies carry Frizzled receptors and Wnt molecule on Treg cells can activate Treg cells, play a role in improving the survival rate of Treg cells, namely plasma Exosomes bodies may through Wnt signal pathway. The influence of Treg cell survival.
2. allogeneic B lymphocytes can effectively amplify human peripheral blood Treg cells in vitro, the amplification efficiency can be more than 20 times, and can significantly inhibit the proliferation of CD4+T cells.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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