脐血间充质干细胞的分离培养鉴定及诱导分化研究

目的：分离培养人脐血间充质干细胞（human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSC）,体外观察其生长特性,并在特定条件下诱导分化,探讨其成脂成骨分化能力.方法：采用沉降法和密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,倒置显微镜下观察其形态及生长情况；流式细胞仪分析细胞周期并检测细胞表面标志物；用茜素红染色和油红0染色分别鉴定其成骨成脂分化能力.结果：纯化的hUCB-MSC贴壁生长,呈均一梭形,具有较强的增值能力,流式细胞仪分析P3代hUCB-MSC稳定表达间充质干细胞表面抗原标志CD73,CD105和CD90等,不表达造血标志CD34和CD45;成骨诱导后3周后细胞茜素红染色阳性；成脂诱导3周后细胞油红0染色阳性.结论：本实验分离的hUCB-MSC具有较强的增殖能力,表达间充质干细胞的表面标记,具有成骨成脂分化潜能.

Isolation,characterization and differentiation of human umbilical cord blood-derived mesenchymal stem cells

Objective To isolate and culture human umbilical cord blood-derived mesenchymal stem cells （hUCB-MSC）, and to observe the biological characteristics of hUCB-MSC and to investigate the osteogenic and adipogenic differentiation potential with proper condition in vitro. Methods Umbilical cord blood mononuclear cells were separated by the method of density gradient centrifugation and MSCs were obtained by adhere culture. The cell morphology and their growth characteristics in vitro were observed under an inverted phase contrast microscope,and the cell cycles were tested by flowing cytometry. Mesenchymal like stem cells were identified by surface marker with flow cytometry. The osteogenic potential was determined by alizarin red staining and the adipogenic potential was detected by oil-red O staining. Results UCB-MSCs were adherent with a fibroblast like morphology and whirlpool like growth alinement. Flow cytometry showed that The third passage cells were positive for CD105, CD73 and CD90 expression, but they failed to express hematopoictic cell surface markers, such as CD34 and CD45. When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks, the formation of a mineral extracellular matrix and neutral lipid vacuoles detected by alizarin red staining and oil-red O staining respectively. Conclusions The MSCs from umbilical cord blood in this study show great capability of proliferation and regeneration and can be induced into osteoblasts and adipobiasts in a suitable condition in vitro.