血浆外泌体样小体免疫调节作用的初步研究  

A preliminary study on the regulatory function of human plasma exosomes-like vesicles

作　　者：马越娥[1] 任亚娜[2] 杨洁[2] 李丹[1] 郭陈智[3] 郭娟[4] 倪兵[5] 范华骅[2] 

机构地区：[1]复旦大学附属华山医院临床检验科,上海200040 [2]上海市血液中心血液工程研究室 [3]华东师范大学生命科学学院 [4]上海市交通大学附属第六人民医院血液科 [5]华东师范大学电镜中心

出　　处：《中华血液学杂志》2010年第6期376-380,共5页Chinese Journal of Hematology

摘　　要：目的 鉴定血浆中外泌体（exosomes）样小体,研究其生物学特性及免疫调节作用.方法 利用多次超速离心结合膜超滤的方法,从健康供者血浆中分离纯化外泌体样小囊泡.采用透射电镜从形态学方面鉴定此囊泡为外泌体,用流式细胞术检测其生物学特性.通过磁珠分选从健康供者外周血单个核细胞中分选CD4^＋T细胞及CD4^＋CD25^＋CD127^low调节性T细胞（Treg）,外泌体样小体与CD4^＋T细胞或Treg细胞共孵育,通过增殖和凋亡实验分析其免疫调节作用.采用流式细胞术检测外泌体样小体与Treg细胞共孵育后,Treg细胞中Wnt经典信号转导通路中磷酸化β-catenin的变化.结果 人血浆中的外泌体样小体与外泌体在形状和大小方面均相似,表达外泌体的标志性蛋白CD63和CD81,还表达MHC-Ⅱ类分子和共刺激分子CD86等.血浆外泌体样小体与CD4^＋T细胞孵育后能抑制CD4^＋T细胞的增殖,且呈剂量依赖性.血浆外泌体样小体与Treg细胞孵育后,Treg细胞的生存时间延长,培养14 d后与血浆外泌体样小体共孵育的Treg细胞存活率为57.07%,而对照Treg细胞存活率为30.91%.RT-PCR检测出Treg细胞有Frizzled受体FRZ2、3、4及LRP6 mRNA表达（相对灰度值分别为48.50、34.84、23.85、49.73）,而血浆中外泌体样小体携带Wnt分子.与血浆外泌体孵育后,Treg细胞磷酸化β-catenin平均荧光强度降低（由20.06±2.99降低到12.41±2.08）,抗凋亡基因bcl-2明显上调（相对灰度值由0.45上调到84.97）.结论 血浆中存在外泌体样小体,表达免疫调节分子,并且体外能够抑制活化的CD4^＋T细胞增殖,通过Wnt信号转导途径延长Treg细胞生存时间.Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CD4^＋T ceils and CD4^＋ CD25^＋CD127 ^low Treg cells were purified from peripheral blood mononuclear cells（PBMCs）by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4^＋ T cells or CD4^＋ CD25^＋CD127^low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4^＋ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed（the relative gray value was 48.50、34.84、23.85、49.73）in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased（from 20.06±2.99 to 12.41±2.08）,and the expression of Bcl-2 mRNA was upregulated significantly（the relative gray value from 0.45 to 84.97）.Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4^＋ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.

关 键 词：CD4^+T细胞 TREG细胞 WNT信号转导途径 血浆外泌体样小体 

分 类 号：R392[医药卫生—免疫学]