人脂肪来源干细胞的分离、培养及成骨分化的研究

Study on Isolation,Cultivation and Osteogenic differentiation of Human Adipose-Derived Stem Cells

目的体外分离、培养、扩增及鉴定人脂肪来源干细胞(human adipose-derived stem cells,hASCs),并向成骨诱导分化,为临床骨组织重建提供种子细胞。方法应用胶原酶消化法和贴壁筛选法相结合从人手术切除的脂肪组织中提取hASCs。MTS法绘制hASCs生长曲线;利用流式细胞术检测hASCs表面标志;以低糖DMEM含10%FBS及1%青/链霉素为基础培养基,含1nM地塞米松,10mM磷酸甘油,0.05mM维生素C的条件培养基连续培养4w进行体外成骨诱导,并利用硫酸茜红素S(Alizarin Red S)染色检测钙沉积情况。结果成功从人脂肪组织中分离、提取了hASCs并在体外扩增。hASCs呈成纤维细胞样生长。生长曲线显示hASCs增生分裂能力活跃。hASCs表面标志表达CD29,CD44,CD73,CD105和CD166,不表达CD31,CD34,CD45和HLA-DR。成骨诱导4w后,经硫酸茜红素S染色可见:细胞呈复层生长,细胞外富集细胞外基质,基质经染色呈现橘红色,数量多,成结节状或成片状存在,并出现钙盐结节,证明有钙盐沉积。结论成功建立了一种简单有效的体外分离和培养hASCs的方法。获得的hASCs生长增殖旺盛、保存方便,具有多向分化能力。hASCs可以作为细胞移植治疗和组织工程理想的种子细胞。

Objective Isolation,cultivation,amplification,charateristics and osteogenetic differentiation of human adipose-derived stem cells(hASCs),and to evaluate the possibility of hASCs as seed cells for clinical bone reconstruction.Methods hASCs were isolated from exairesis fat tissue by collagen digestion and adherent screening.Cell proliferative ability was assayed by cell growth curve using MTS.Cell surface markers of hASCs were examined by flow cytometry.Osteogenic differentiaton in vitro:DMEM(low-glucose) medium with 10% FBS,1% P/S,100 nM dexamethasone,10 mM β-glycerophosphate and 0.05mM ascorbic acid-2-phosphate was used as induced medium.hASCs were seeded at a density of 4,000 cells/cm2 and cultured for 28 days with media changed every third day.Calcium deposits were stained orange-red by the Alizarin Red Solution.Results hASCs were successfully isolated and culured from human adipose tissue.They appeared fibroblast-like morphology.Growth curve revealed that hASCs could proliferate rapidly in vitro.hASCs showed positive of CD29,CD44,CD73,CD105 and CD166,but negative of CD31,CD34,CD45 and HLA-DR.After 4 weeks induction of osteogenic differentiation,the cells cultured multilayer.There were extracellular matrix enriched and stained orange red by Alizarin Red staining.Conclusion A simple and effective method of ioslation and cultivation of hASCs was successfully established in vitro.hASCs could grow and proliferate rapidly in vitro,readily available,easy to preserve,and capable of multilineage differentiation.hASCs can be an ideal seed cells for cell therapy and tissue engineering.