脐带源性和脐血源性间充质干细胞分离培养方法的比较  

Comparison of Isolation and Culture Methods between Umbilical Cord Mesenchymal Stem Cells and Umbilical Cord Blood Mesenchymal Stem Cells

作　　者：管英华[1] 谢扬虎[2] 魏晓巍[2] 李玉云[3] 

机构地区：[1]巢湖职业技术学院生物技术系临检教研室,安徽省巢湖市238000 [2]蚌埠医学院临床检验诊断学实验中心,安徽省蚌埠市233030 [3]蚌埠医学院医学检验系

出　　处：《中华全科医学》2011年第9期1331-1333,F0003,共4页Chinese Journal of General Practice

基　　金：安徽省教育厅自然科学研究资助项目(2006KJ392B)

摘　　要：目的建立分离培养人脐带间充质干细胞(UC MSCs)和脐带血间充质干细胞(UCB MSCs)的方法,并比较其效果。方法无菌条件下采集健康足月新生儿脐带及脐血各30份,分别通过原代贴壁培养法、酶消化法培养UCMSCs,及淋巴细胞分离液法、羟乙基淀粉沉降与淋巴细胞分离两步分离法获得脐血中单个核细胞培养UCB MSCs。应用含10%胎牛血清的DMEM/F12培养基,比较UC MSCs和UCB MSCs分离培养的效果与生长形态。流式细胞术检测其表面标志及诱导分化鉴定其分化能力。结果 UC MSCs原代贴壁培养法8 d左右可见成纤维样细胞从组织块边缘爬出且成簇生长,酶消化法培养UC MSCs 5 d左右均匀生长;UCB MSCs分离培养用羟乙基淀粉沉降与淋巴细胞分离两步法得到的细胞数量较淋巴细胞分离法明显增多。两种来源MSCs培养方法相比较,UC MSCs原代培养的时间短,培养成功率明显增高。流式细胞仪检测两种来源的MSCs具有MSCs表面标志特征。定向诱导分化结果表明MSCs具有被诱导为成骨细胞、脂肪细胞的分化能力。结论人脐带来源间充质干细胞原代培养周期短,培养效率更高;脐血间充质干细胞的分离方法中羟乙基淀粉沉降与淋巴细胞分离两步法效率更高。Objective To establish methods for isolation and culture of umbilical cord mesenchymal stem cells（UC MSCs） and umbilical cord blood mesenchymal stem cells （ UCB MSCs） in vitro, and compare the effects. Methods Umbilical cord blood （n = 30） and Umbilical cord（ n = 30） were collected from healthy full-term deliveries. Human UC MSCs were isolated and cultured using adherence technique and enzyme digestion, and mononuclear cells were isolated from umbilical cord blood with the lymphocyte isolation method and hydroxyethyl starch sedimentation ＋ lymphocyte isolation method to culture UCB MSCs. Cells were cultured in DMEM/F12 containing 10% fetal bovine serum,and compared cell morphologic change and isolation outcomes. The surface markers of MSCs were detected by flow cytometry, and the ability of MSCs differentiation was analyzed by inducing differentiated in vitro. Results About 8 days after adherence,the fibroblast-like cells were observed sprawled out from the edge of the tissue, growing in cluster;and 5 days ＇after enzyme digestion, cells growing fiber shaped. In UCB MSCs isolation and culture, compared with the lymphocyte isolation method, the number of mononuclear cells significantly increased using the hydroxyethyl starch sedimentation ＋ lymphocyte isolation method. Two sources of MSCs surface markers analyzed by flow cytometry showed that they expressed the characteristics of MSCs. Inducing differentiated MSCs in vitro showed they could be induced to osteoblast and adipocyte. Conclusion The period of UC MSCs primary culture was shorter, and with a better culture efficiency. The hydroxyethyl starch sedimentation ＋ lymphocyte isolation method isolated and cultured UCB MSCs was more effective.

关 键 词：脐带 脐带血 间充质干细胞 分离 培养 

分 类 号：R446.11[医药卫生—诊断学]