摘要
目的研究人脐带Wharton胶中间充质干细胞（mesenchymal stem cells，MSCs）向软骨诱导分化的特性。方法去除新鲜人脐带动、静脉和外膜组织，获得Wharton胶并剪碎，胶原酶消化法进行原代培养。经传代培养、流式细胞检测表面标志、克隆形成率测定后，用软骨诱导培养液使MSCs向软骨细胞分化，并以组织化学和免疫组织化学染色进行鉴定。逆转录聚合酶链反应（reverse transcription—polymerase chain reaction，RT-PCR）检测软骨特征性标志。结果人脐带Wharton胶富含干细胞。采用酶消化法可快速分离培养原代细胞。流式细胞检测表明这些细胞不表达造血干细胞标志，表达CD44、CDl05、CD27l等间质干细胞表面标志；HLA-ABC阳性表达，HLA—DPDQDR阴性表达。不同代MSCs的克隆形成率差异无统计学意义（P〉O．05）。未进行软骨诱导的细胞弱表达软骨细胞标志，经软骨诱导分化后，组织化学和免疫组织化学检测表明其能够较好地表达软骨细胞标志。RT—PCR结果显示人脐带Wharton胶中MSCs诱导前后均表达Sox-9、Col-2Al。结论人脐带Wharton胶中MSCs来源广泛，无伦理学限制；且具有前软骨细胞的特性，有望成为软骨组织工程新型的种子细胞。
Objective To investigate the characterics of mesenchymal stem cells （MSCs） derived from Wharton jelly of human umbilical cord and possibility of ehondrogenie induction. Methods After removal of umbilical arteries, vein and tuniea extema of umbilical cord, the remaining tissue was Wharton jelly. To obtain adherent cells, Wharton jelly of umbilical cord was cut into pieces and digested with collagenase of type I with concentration of 0.075%. The stem cells of umbilical cord were passaged. Phenotypes were detected with flow cytometry and colony-forming unit-fibroblast （CFU-F） frequency of different passages of the MSCs were calculated to evaluate their reproductive activity. The MSCs were cultured in chondrogenic media and detected with histochemistry and immunohistochemistry assay. Cartilage markers were detected by reverse transcription-polymerase chain reaction （RT-PCR） analysis. Results Wharton jel- ly of human umbilical cord had abundant stem cells. The isolation of these cells had been proved easily when collagenase was used to detach the cells from Wharton jelly. Flow cytometric analyses revealed that the stem cells from Wharton jelly represented a nonhematopoietic cell population. However they expressed amounts of MSCs markers, such as CD44, CD105 and CD271. Meanwhile positive expression of HLA-ABC was found in the stem cells, but not HLA-DPDQDR. The CFU-F frequency of different passages of the MSCs derived from Wharton jelly showed no statistical significance （P 〉0.05）. The histochemistry and im- munohistochemistry showed that the MSCs which weakly expressed chondrocyte markers strongly expressed these markers after chondr, genic induction. Both Sox-9 and Col-2AI gene detected by RT-PCR were found in the MSCs derived from Wharton jelly whatever undergone chondrogenic induction or not. Conclusion The MSCs resource derived from Wharton jelly of human umbilical cord is abundant without limitation of ethics problems. The MSCs with the characteristic of pre-chondrocyte maybe the newly stem cells for tissue engineering of cartilage.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2007年第12期I0050-I0055,共6页
Chinese Journal of Orthopaedics
基金
国家自然科学基金资助项目（30672134）
北京市科学技术委员会科技计划重大项目（H060920050630）
关键词
脐带
间质干细胞
软骨
Umbilical cord
Mesenchymal stem cell
Cartilage
