成骨诱导或干扰素γ预处理对胎儿骨髓源Flk-1^+间充质干细胞免疫活性的调节  

Osteogenic differentiation or interferon-gamma pretreatment regulates immunological activities of human fetal bone marrow-derived Flk-1^+ mesenchymal stem cell

作　　者：张斌[1] 刘瑞[1] 朱希山[1] 豆晓伟[1] 刘星霞[1] 陈媛[1] 赵春华[1] 

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院组织工程研究中心,北京市100005

出　　处：《中国组织工程研究与临床康复》2008年第43期8529-8534,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：中国科技部"八六三"计划专项基金(2006AA02A109;2006AA02A115);国家自然科学基金(30570771)~~

摘　　要：背景:骨髓间充质干细胞在修复异基因组织最终分化为特定细胞后,理论上会因MHC及共刺激分子上调而引起组织排斥,但实际上并没有发生排斥反应。推测骨髓间充质干细胞在异基因组织分化诱导及炎性因子微环境作用下,有可能分化成一种仍具有免疫调节活性的终末细胞,不被机体的免疫识别系统清除掉。目的:在体外运用成骨诱导及干扰素γ预处理来模拟体内组织分化及炎性因子微环境,观察在此作用下胎儿骨髓源Flk-1+间充质干细胞是否能够保持其在自然条件下所呈现的免疫学活性。设计、时间及地点:细胞学体外对照观察,于2007-09/2008-05在北京协和医学院基础学院完成。材料:2例骨髓样品取自流产胎儿,由北京回龙冠妇产医院提供。外周血样品取自15~35岁健康志愿者,由北京三○七医院提供。地塞米松等诱导因子和干扰素γ为美国Sigma公司产品。方法:Ficoll法分离胎儿骨髓单个核细胞,取白环层以上细胞,以1×106/cm2密度接种,贴壁法纯化,采用阴性分选法去除CD45+、GlyA+和CD34+细胞,分离扩增得到Flk-1+骨髓间充质干细胞。以淋巴细胞分离液梯度离心分离外周血单个核细胞,用于混合淋巴细胞培养实验及有丝分裂原刺激的淋巴细胞增殖实验。主要观察指标:流式细胞仪鉴定细胞表型,用成骨、成脂、成软骨和成内皮诱导液检测其分化潜能,成骨诱导或干扰素γ预处理后细胞表面抗原表达、对淋巴细胞增殖及活化抗原CD69、CD25表达的影响,对白细胞介素10和转化生长因子β分泌水平的影响。结果:光镜下骨髓来源的间充质干细胞为成纤维细胞样贴壁生长,均高表达Flk-1,同时高表达黏附分子CD29、CD44、CD105,造血细胞标志CD34、CD45呈阴性;可向成骨、成脂肪、成软骨和成内皮方向分化;低表达MHC-Ⅰ类分子,不表达MHC-Ⅱ类分子,成骨诱导能上调MHC-Ⅰ表达,干扰素γ预处理可上调MHBACKGROUND： Mesenchymal stem cells （MSCs） may differentiate into committed ceils in the process of repairing allogenic tissue damage, and no rejection happens although the upregulation of major histocompatibility complex （MHC） and costimulatory molecules may induce the rejection in a theoretical manner. It is presumed that MSCs under allogenic tissue differentiation and inflammatory factor microenvironment are potential to differentiate into terminal cells with immunoregulatory activity and escape immune surveillance. OBJECTIVE： To utilize in vitro osteogenic differentiation and interferon- γ pretreatment to simulate the microenvironment of differentiation and inflammation in vivo, and to explore whether human fetal bone marrow-derived Flk-1^＋ MSCs still remain their immunological activities. DESIGN, TIME AND SETTING： Controlled cytological observation in vitro was performed in School of Basic Medicine, Peking Union Medical College from September 2007 to May 2008. MATERIALS： Two bone marrow samples were taken from aborted fetus provided by Beijing Huilongguan Maternity Hospital, and peripheral blood samples from healthy volunteers aged 15 35 years recruited from Beijing 307 Hospital. Interferon-γ and such osteogenic inductor as dexamethasone, β -glycerophosphate and ascorbic acid were purchased from Sigma-Aldrich. METHODS： Fetal bone marrow mononuclear cells were isolated by means of Ficoll method, and cells above white circular layer were harvested and inoculated at a density of 1×10^6/cm^2, followed by removing CD45^＋, GlyA^＋ and CD34^＋ cells using negative separation and purifying using adherence method. Flk-1^＋ MSCs were obtained following isolated and expanded from bone marrow of aborted fetus. Peripheral blood mononuclear cells were isolated with gradient centrifugation using lymphocyte isolation solution, and they were used for mixed lymphocyte culture and mitogen-stimulated lymphocyte proliferation experiments. MAIN OUTCOME MEASURES： Flow cytometry was used to identify th

关 键 词：骨髓间充质干细胞 成骨 干扰素Γ 免疫调节活性 

分 类 号：R394.2[医药卫生—医学遗传学]