人骨髓基质干细胞的单细胞克隆培养与鉴定***☆

背景：骨髓基质干细胞缺乏特异的表面识别分子，其鉴定一直是研究中的难题。目的：探索人骨髓基质干细胞培养条件，获得体外克隆化培养的成人骨髓基质干细胞，并对其进行表型分析及分化潜能鉴定。方法：外科手术取髂骨术中抽取骨髓，采用密度梯度离心法初步分离骨髓基质干细胞，用极限稀释法进行克隆化培养；流式细胞仪对克隆化培养的骨髓基质干细胞进行细胞表面标志检测，并进行体外成软骨、成骨及心肌细胞诱导，免疫组织化学及RT-PCR检测成软骨、成骨及心肌细胞表达，确定其表型及分化潜能。结果与结论：单个细胞来源骨髓基质干细胞在体外1∶3传代一般可以传28代左右，24代以前生长状态良好；经流式细胞仪检测，骨髓基质干细胞表达CD29，CD44，CD106，不表达CD14，CD34，CD45，HLA-DR；骨髓基质干细胞体外可向成骨、成软骨及心肌细胞分化，提示体外克隆化培养的骨髓基质干细胞能维持良好的成体干细胞生物学特性。关键词：单克隆培养；骨髓基质干细胞；多能成体干细胞；鉴定；分化doi:10.3969/j.issn.1673-8225.2012.10.008

Single-cell clonal culture and identification of human bone marrow stromal stem cells

BACKGROUND: Bone marrow stromal stem cells lack of specific surface marker and their identification challenges scholars all the time. OBJECTIVE: To explore the culture condition for clonal isolation of human bone marrow stromal stem cells (BMSCs) and identify their surface markers and differentiation potentials. METHODS: Human bone marrow was taken and BMSCs were isolated using density gradient centrifugation. Clone-like cells were selected by single cell limiting dilution. The surface antigens of the cloned BMSCs were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by inducing BMSCs into osteoblasts, chondrocytes and cadiocytes. RT-PCR and immunocytochemistry were applied to detect the three mutli-differentiations. RESULTS AND CONCLUSION: Single cell-derived BMSCs could be expanded to passage 28 in vitro which still maintaued active proliferation ability.The expanded BMSCs expressed CD29, CD44, CD106, but not CD14, CD34, CD45, HLA-DR. They could be induced to differentiate into osteoblasts, chondrocytes and cadiocytes. These findings suggest that cloned BMSCs can maintain the phenotypes of stem cells during in vitro culture.