目的：建立人脐带间充质干细胞（MSCs）分离培养方法，并进行生物学鉴定及定向诱导分化研究。方法采用酶消化 Wharton 胶法体外分离培养人脐带干细胞，通过流式细胞仪分析其表面标记分子，并向成骨、成脂方向诱导分化，钙钴法染色检测 ALP，茜素红染色检测矿化结节，油红“O”染色检测脂肪滴，进行干细胞的成骨成脂活性鉴定。结果分离培养的脐带间充质干细胞 CD73，CD90和 CD105均表达阳性，阳性率为92.45％，95.45％和96.45％，CD34表达阴性，阳性率仅为1.07％。成骨诱导3周后 ALP 染色胞质内可见黑色颗粒，4周后可见大量矿化结节。成脂诱导2周后细胞内出现大量脂肪小滴，多数细胞形态变圆，油红“O”染色阳性。结论脐带来源的间充质干细胞具有成骨、成脂多向分化潜能，为临床干细胞治疗研究的种子细胞来源奠定了基础。%Objective To establish the method of isolation,culture and identify biological characterization of mesenchymal stem cells from human umbilical cord (hUCMSCs);and study their multiple differentiation potency.Methods Stem cells from human umbilical cord were cultured by enzyme Wharton jelly method in vitro.The surface markers were identified by flow cytometry.Multi-differentiation capacity was identified by osteogenic and adipogenic differentiation.ALP was detected with Calcium cobalt staining.The mineralized ability in vitro was measured with Alizarin red staining.Theadipocyte differen-tiation ability was measured with oil red-O staining.Results Flow cytometry analysis revealed that CD73 (92.45%),CD90 (95.45%)and CD105 (96.45%)were highly expressed on these cells’surface,while CD34 (1.07%)were negative ex-pressed.Cells were cultured with induced-osteogenic medium after 3 weeks,ALP staining in the cytoplasm of black parti-cles,and a large amount of mineralized nodules within cells was observed after 4 weeks.Cells were cultured with induced-adi-pogenic medium after 2 weeks,the majority of these cells were round,oil red O staining of lipid droplets generated within cells was observed.Conclusion Mesenchymal stem cells from human umbilical cord have the potential of multi-directional differentiation.These cells could be induced to differentiate into adipocytes and osteoblasts,which laid the foundation for clinical stem cell therapy research source of seed cells.
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