人脐血间充质干细胞培养及对异体外周血T淋巴细胞增殖的影响  

Culture of umbilical cord blood-derived mesenchymal stem cells and its effect on the proliferation of allogeneic peripheral blood T lymphocytes

作　　者：白海[1] 王茜[1] 吴涛[1] 杨小亮[1] 司志刚[1] 薛志文[1] 

机构地区：[1]解放军兰州军区兰州总医院血液科,全军血液病中心,甘肃省兰州市730050

出　　处：《中国组织工程研究与临床康复》2008年第3期438-441,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：军队“十一五”杰出人才基金(06J005)~~

摘　　要：目的:研究发现间充质干细胞具有独特的免疫调节特性,体外能够抑制混合淋巴细胞培养或有丝分裂原刺激中的T淋巴细胞增殖。实验观察体外分离培养的脐血间充质干细胞对异体外周血T淋巴细胞增殖的抑制效果。方法:实验于2004-10/2006-06在解放军兰州军区兰州总医院完成。①对象:足月正常分娩产妇脐血及健康志愿者外周血分别由解放军兰州军区兰州总医院妇产科、血液科提供,产妇与志愿者对实验均签署知情同意书,实验经医院医学伦理委员会批准。②实验方法:Percoll法分离脐血单个核细胞,贴壁纯化,达80%～90%融合时胰蛋白酶消化,按2.0×108L-1密度传代,取第4～5代细胞用于实验。密度梯度法分离外周血单个核细胞,以含体积分数为0.2胎牛血清的RPMI 1640培养基调整细胞浓度至1×109L-1时加入尼龙棉柱内,用培养基冲出非黏附细胞即为T淋巴细胞。脐血间充质干细胞分为2×108,1×108,0.5×108L-1组,各100μL接种于96孔培养板,加入2×108L-1T淋巴细胞悬液100μL,再给予植物血凝素刺激T淋巴细胞转化。以T淋巴细胞+植物血凝素为阳性对照。③实验评估:观察体外培养的脐血间充质干细胞形态,应用流式细胞仪检测细胞表面抗原的表达。MTT法测定脐血间充质干细胞对异体外周血T淋巴细胞增殖的影响。结果:①形态特征与表面抗原表达:原代培养10～18d形成平行排列、辐射状或旋涡状的成纤维样细胞,即为脐血源性的间充质干细胞,传代7d左右细胞达90%融合。流式细胞仪检测90%细胞稳定表达间充质干细胞相关抗原CD13,CD29,CD44,CD166,不表达造血细胞系的表面抗原CD34,CD45。②脐血间充质干细胞对异体T淋巴细胞增殖的抑制:与阳性对照比较,经脐血间充质干细胞共培养后植物血凝素刺激T淋巴细胞的增殖作用受到抑制,增殖指数明显降低(P<0.01)。间充质干细胞与T淋巴细胞比值为1:1,1:2和1:4�AIM： Mesenchymal stem cells （MSCs） have special immunoregulations, in vitro MSCs can inhibit mixed lymphocyte reaction or Iymphocyte proliferation stimulated by mitogen. This study cultured human umbilical cord blood （UCB） MSCs and observe its immune regulation on allogeneic peripheral blood T lymphocytes in vitro. METHODS： The experiment was conducted at Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from October 2004 to June 2006. （1）UCB from full-term normal delivery was provided by Department of Gynaecology and Obstetrics of the Hospital, peripheral blood from healthy donors was obtained from Department of Hematology of the Hospital. Mothers of neonates and the donors signed the informed consent, and the experiment was permitted by Medical Ethics Committee of the Hospital. （2）Mononuclear cells （MNCs） of UCB were separated at 80% - 90% confluence then passaged at a density of 2.0 ×10^8 L^-1. The MSCs of fourth or fifth passage were used. Peripheral blood MNCs were separated by density gradient method. When the density of cells was adjusted to 1.0×10^9 L^-1 by RPMI1640 medium containing 0.2 volume fraction fetal bovine serum, cells were added to nylon column, and the cells washed by medium were T lymphocytes. MSCs were cultured at a density of 2×10^8 , 1×10^8 and 0.5×10^8 L^-1 in 96-well plate, 100 μ L in each well, and added 100μ L T lymphocytes （2×10^8 L^-1） and phytohemagglutinin （PHA）. T lymphocytes and PHA served as positive control group, （3）MSC morphologies were observed, and the surface marks were examined with flow cytometer. The effect of MSCs on T cells proliferation was measured by MTT method. RESULTS： （1）MSCs morphologies and surface marks： Umbilical cord blood MSCs showed parallelly arranged, radiat or whirlpool-shaped fibroblast-like cells after 10 - 18 days culture. After 7 days passage, the cell colony reached 90% confluence Flow cytometer showed that more than 90% of these cells were positive for CD13, CD29, CD44, a

关 键 词：脐血 间充质干细胞 异基因外周血 T淋巴细胞 免疫抑制作用 

分 类 号：R394.2[医药卫生—医学遗传学]