人脐带间充质干细胞分离培养建立及其生物学特性观察  

Experimental study of humam umbilical cord mesenchymal stem cell isolation,culture and biological characteristics

作　　者：谢扬虎[1] 李玉云[2] 

机构地区：[1]蚌埠医学院临床检验诊断学实验中心,安徽蚌埠233030 [2]蚌埠医学院临床检验诊断学教研室,安徽蚌埠233030

出　　处：《蚌埠医学院学报》2010年第10期982-985,共4页Journal of Bengbu Medical College

基　　金：安徽省教育厅自然科学研究资助项目(2006KJ392B)

摘　　要：目的:建立从人的脐带组织中分离和培养脐带间充质干细胞(UC-MSCs)的方法,并探讨UC-MSCs的生物学特性。方法:分别采用原代贴壁培养法和酶消化法(胶原酶Ⅱ和胰酶)分离培养UC-MSCs,瑞氏染色观察细胞形态,流式细胞术检测第3代以后的UC-MSCs表面特异标志物表达,MTT法检测P7细胞增殖情况。UC-MSCs同外周血单个核细胞共培养后,用MTT法测定细胞增殖率,Hoechst与PI双染色,观察UC-MSCs对健康人淋巴细胞增殖的影响。结果:原代贴壁培养法1周左右可见成纤维样细胞从组织块边缘爬出,成簇生长;酶消化法3～5天可见成纤维样细胞均匀生长。传代培养后,UC-MSCs均呈长梭形、旋涡状生长;细胞核大,核仁清晰;免疫表型:CD29阳性率为(95.71±2.23)%,CD31和CD34阳性率分别为(2.47±0.54)%和(3.24±0.34)%;第7代UC-MSCs仍具有较强的分裂增殖能力。UC-MSCs对淋巴细胞的增殖反应具有抑制作用,呈细胞数量剂量依赖性,而淋巴细胞对UC-MSCs的生长未见影响。结论:从人脐带中成功分离培养的细胞具有UC-MSCs生物学特性。Objective：To establish a method for isolation and culture of umbilical cord mesenchymal stem cells（UC-MSCs）from human umbilical cord in vitro,and explore its biological characterstics.Methods：Human UC-MSCs were isolated and expanded in culture with explantation technique and enzyme digestion（collagenase Ⅱand trypsin）,respectively;the cell morphology was observed by Wright-staining;after the third passage,the surface markers of UC-MSCs were detected and idntified by flow cytometry,and the seventh passage,the cell proliferation was analyzed by MTT assay.After UC-MSCs were co-cultured with peripheral blood mononuclear cells,the impact on the T cells were observed by Hoechst and PI double staining.Results：About one week after explantation,the fibroblast-like cells sprawled out from the edge of the tissue,growing in cluster;3-5 days after enzyme digestion,cells were watched growing fiber-shaped.After passaged,UC-MSCs showed the status of long,spindle-shaped and circinate growth;they had large nucleus and clear nucleolus.Immunophenotype analysis showed that they had strong positive expression for CD29（95.71±2.23）%,but week expression for CD31,and CD34 were（2.47±0.54）% and（3.24±0.34）%,respectively;the seventh passage UC-MSCs still had strong proliferation ability;UC-MSCs suppressed T cells proliferation in a dose-dependent manner;lymphocytes did not affect the growth of UC-MSCs.Conclusions：The cells isolated successfully from the umbilical cord have biological characteristics of the mesenchymal stem cell.

关 键 词：干细胞 间充质 脐带 分离 培养 淋巴细胞 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学]