人类白细胞抗原-G阳性的脐带间充质干细胞体外诱导调节性T细胞的实验研究  

Experimental study on the in vitro induction of regulatory T cells by umbilical cord mesenchymal stem cells with positive human leukocyte antigen-G

作　　者：白剑[1,2] 肖漓[1,2] 苗兰英[2] 林大勇 刘鸿[2] 高钰[1] 陈文[1] 毕丽丽[1] 孔祥瑞[1] 黄海燕[1] 石炳毅[1] Bai Jian;Xiao Li;Miao Lanying;Lin Dayong;Liu Hong;Gao Yu;Chen Wen;Bi Lili;Kong Xiangrui;Huang Haoyan;Shi Bingyi(Basic Research Laboratory of Organ Transplant Institute, the 309th Hospital of Chinese People＇s Liberation Army, Beijing Key Laboratory of Organ Transplantation and Immunology Regulatory , Beijing 100091, China)

机构地区：[1]解放军第309医院全军器官移植研究所移植研究室北京市器官移植与免疫调节重点实验室,北京100091 [2]辽宁中医药大学

出　　处：《器官移植》2018年第2期97-102,共6页Organ Transplantation

基　　金：国家自然基金面上项目(81571555);辽宁省自然科学基金(2015020714)

摘　　要：目的探讨人类白细胞抗原(HLA)-G阳性的脐带间充质干细胞在体外诱导调节性T细胞(Treg)产生的效果。方法从新生儿脐带中分离脐带间充质干细胞,采用脂质体转染的方式将PEGFP-N1-HLA-G质粒转染到脐带间充质干细胞中,设为PEGFP-N1-HLA-G组;转染空载体PEGFP-N1质粒的脐带间充质干细胞设为PEGFP-N1组;相同条件下,未加入空载体的脐带间充质干细胞设为空白对照组。采用流式细胞仪检测脐带间充质干细胞标志物;采用蛋白质免疫印迹法鉴定各组细胞HLA-G蛋白的表达;各组细胞与健康人外周血中CD4+T细胞混合培养24 h和48 h后,采用流式细胞仪检测CD4+CD25+Foxp3+Treg占全部T细胞的比例。结果脐带间充质干细胞CD45、CD34和HLA-DR呈阴性表达,CD29、CD44和CD105呈阳性表达;PEGFP-N1-HLA-G组可以表达HLA-G蛋白,与空白对照组和PEGFP-N1组比较差异均有统计学意义(均为P<0.01)。PEGFP-N1-HLA-G组细胞在与CD4+T细胞混合培养24 h后,CD4+CD25+Foxp3+Treg占全部T细胞的(15.3±1.9)%,在培养48 h后,CD4+CD25+Foxp3+Treg占全部T细胞的(14.3±2.1)%,与空白对照组和PEGFP-N1组比较,差异均有统计学意义(均为P<0.05)。结论 HLA-G基因修饰后脐带间充质干细胞能够有效地在体外诱导CD4+CD25+Foxp3+Treg的产生。Objective To explore the effect of umbilical cord mesenchymal stem cells with positive human leukocyte antigen（HLA）-G on inducing the production of regulatory T cells（Treg） in vitro. Methods Umbilical cord mesenchymal stem cells were isolated from umbilical cord of neonates. PEGFP-N1-HLA-G plasmid was transfected into the human umbilical cord mesenchymal stem cells by liposome transfection, as PEGFP-N1-HLA-G group. PEGFP-N1 empty vector plasmid was transfected into the human umbilical cord mesenchymal stem cells, as PEGFP-N1 group. The human umbilical cord mesenchymal stem cells without empty vector under the same conditions were set as blank control group. Markers of the umbilical cord mesenchymal stem cells were detected using flow cytometry. The expression of HLA-G protein in each group of cells was identified by Western Blot. After mixed-culturing with CD4＋T cells in peripheral blood of healthy subjects for 24 h and 48 h, the proportion of CD4＋ CD25＋ Foxp3＋Treg in total T cells of each group was detected by flow cytometry. Results CD45, CD34 and HLA-DR presented negative expression on umbilical cord mesenchymal stem cells, while CD29, CD44 and CD105 presented positive expression. HLA-G protein could be expressed in the PEGFP-N1-HLA-G group, which had statistically significant difference compared with the blank control group and PEGFP-N1 group（both P〈0.01）. After PEGFP-N1-HLA-G group and CD4＋T cells were mixed-cultured for 24 h and 48 h, CD4＋ CD25＋ Foxp3＋Treg accounted for（15.3±1.9）% and（14.3±2.1）% of the total T cells respectively, both of which presented statistically significant difference compared with the blank control group and PEGFP-N1 group（all P〈0.05）. Conclusions Umbilical cord mesenchymal stem cells with HLA-G gene modified can effectively induce the production of CD4＋ CD25＋ Foxp3＋Treg in vitro.

关 键 词：人类白细胞抗原(HLA)-G 脐带间充质干细胞 T细胞 调节性T细胞(Treg) 转染 修饰 免疫排斥 免疫耐受 CD4+T细胞 

分 类 号：R392.4[医药卫生—免疫学]