人脐带Wharton胶中间充质干细胞的分离、培养与鉴定  

Isolation,culture and characterization of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord

作　　者：侯克东[1] 卢世璧[1] 张莉[1] 袁玫[1] 孙明学[1] 彭江[1] 赵斌[1] 眭翔[1] 许文静[1] 

机构地区：[1]解放军总医院骨科研究所,北京100853

出　　处：《解放军医学杂志》2008年第4期375-378,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助课题(30672134);北京市科学技术委员会科技计划重大项目(H060920050630)

摘　　要：目的研究人脐带Wharton胶中间充质干细胞(MSCs)的生物学特性。方法去除新鲜人脐带动静脉和脐带外膜组织,获得Wharton胶,分别采用酶消化法和组织块培养法获得脐带Wharton胶中的干细胞,通过细胞传代培养、生长曲线测定、活细胞鉴定进行细胞生长动力学研究,流式细胞仪分析细胞表面分子特点,组织化学和免疫组化染色鉴定软骨特征性标志物表达情况。RT-PCR检测Sox-9及Col-2A1mRNA表达情况。结果采用酶消化法可以快速分离Wharton胶中MSCs,且细胞迅速贴壁生长;组织块法培养获得种子细胞时间较长。流式细胞仪检测显示所获得的细胞表达CD44、CD105等MSCs标志物,HLA-ABC表达阳性,HLA-DPDQDR表达阴性。经过冻存复苏后免疫表型无显著变化。组织化学和免疫组化染色显示脐带Wharton胶MSCs弱表达软骨特征性标志。RT-PCR显示Wharton胶MSCs表达Sox-9和Col-2A1。结论人脐带Wharton胶MSCs不向造血系分化,且具有前软骨细胞的特性,有望作为软骨组织工程新型的种子细胞。Objective To investigate the bionomics of mesenchymal stem cells （MSCs） derived from Wharton＇s jelly of human umbilical cord. Methods Umbilical arteries, vein and umbilical cord tunica externa were removed, and the remaining tissue （Wharton＇s jelly） was harvested. To gather MSCs, the umbilical cord was cut into small fragments and digested with 0.075% type Ⅰ collagenase, or the small fragments were cultured with DMEM. The cells derived from Wharton＇s jelly of umbilical cord were serially subcultivated, the growth curve was drawn, and the identification of living cells was made for studying the cell growth kinetics. The surface antigens were detected with flow cytometry, the cartilage markers were detected by histochemistry and immunohistochemistry, and the expressions of Sox-9 and Col-2A1 mRNA were analyzed by RT-PCR. Results The confluence time of primary culture cells was 3-5d in cells harvested with enzyme digestion, and 10-15d in cells harvested with micro mass. The results of flow cytometry showed that the MSCs derived from umbilical cord expressed CD44 and CD105; the expression of HLA-ABC was positive, while HLA-DPDQDR was negative. There was no significant change on the immunophenotype of umbilical cord MSCs after cryopreservation and thawing. The findings of histochemistry and immunohistochemistry showed that the expression of chondrocyte markers in Wharton＇s jelly MSCs was weakly positive. The results of RT-PCR showed the chondrocyte markers Sox-9 and Col-2A1 were positively expressed in Wharton＇s jelly MSCs. Conclusion The MSCs derived from Wharton jelly of human umbilical cord do not differentiate into hematopoietic cells, may express the chondrocyte markers （they are suggested to have characteristics of pre-chondrocytes）, and is expected to be a new type of stem cells of tissue engineering cartilage.

关 键 词：脐带 间质干细胞 细胞培养 

分 类 号：R394.26[医药卫生—医学遗传学]