摘要
目的体外分离培养人皮下脂肪来源的细胞群,依据国际标准规定通过形态观察、免疫表型检测及多种分化潜能的鉴定证实体外获得的细胞群为脂肪来源的间充质干细胞。方法采用0.1%胶原酶I及0.25%胰酶双消化法进行细胞的体外分离,LG-DMEM培养基加入体积分数为10%胎牛血清,于37℃、5%CO2的饱和湿度条件下培养,待细胞生长至70%～80%融合时消化传代扩增。通过倒置显微镜观察细胞形态;流式细胞仪检测细胞免疫表型表达;取第3代细胞,于培养基中分别加入成骨及成脂诱导剂进行定向诱导分化,并对诱导后的细胞染色鉴定。结果分离培养的细胞群呈长梭状,漩涡样贴壁生长;流式细胞仪检测P2代细胞,其中CD73,CD105,C90,CD44,CD29及CD49d呈阳性表达,而CD14,CD34,CD45,HLA-DR及CD106呈阴性表达;成骨诱导3周后,茜素红染色、VON-KOSSA染色及细胞碱性磷酸酶染色都呈阳性;成脂诱导2周后,细胞内有小脂滴出现,油红O染色呈阳性。结论胶原酶胰酶双消化法从脂肪组织中分离获得的细胞可贴壁生长;流式细胞仪检测证实细胞表达与间充质干细胞相符的免疫表型;定向诱导后具有成骨及成脂分化能力;符合间充质干细胞的特性。
Objective Subcutaneous adipose-derived cell populations would be isolated and cultured in vitro. In accordance with international standard, we will confirm that cell populations obtained in vitro are adipose-derived mesenchymal stem cells by morphology,immunophenotyping and identifying of multiple differentiation potential. Methods Using dualdigestion method for separation of cells in vitro with 0. 1% collagenase I and 0. 25% trypsin to get it, adding LG-DMEM medium containing the volume fraction of 10% fetal bovine serum,culturing at 37℃ and 5% of CO2 saturated humidity conditions, then amplifying of digestion and passage until the cell growth to 70% to 80%. Cell morphology would be observed through inverted microscope. Flow cytometry detect cell phenotype expression. The third passage cells would be directed induction of differentiation by added osteogenic and adipogenic induction agents into the culture medium, and then induced cells would be staining. Results Isloated cell populations are long fusiform and whirlpool-like adherent growth. P2 cells detected by Flow cytometry,which of CD73 and CD105, C90, CD44, CD49d and CD29 are positive expression, but of CD14 and CD34, CD45, CD106and HLA-DR are negative expression. Alizarin red staining, alkaline phosphatase staining and VON-KOSSA stain tested positive 3 weeks after osteogenic induction. 2 weeks after the adipogenic induction,there are small lipid droplets appearing,and oil red O staining is positive. Conclusion The Cells that using coltagenase and trypsin digestion method isolated from adipose tissue can be attached to the wall growth. Flow cytometry confirme that isolated cells can express the mesenchyme stem ceils consistent phenotype and have osteogenic and adipogenic differentiation ability after directed induced, the cultured cells are consistent with the international standard about the characteristics of mesenchyme stem cells.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2013年第8期708-712,共5页
Chinese Journal of Blood Transfusion
