大鼠脂肪干细胞的分离培养和鉴定  

Isolation,culture and identification of rat adipose derived stem cells

作　　者：刘娜[1] 温秀杰[1] 刘宁[1] 李向杰[1] 刘鲁川[1] 邓蔓菁[1] 

机构地区：[1]第三军医大学第三附属医院野战外科研究所口腔科,重庆400042

出　　处：《牙体牙髓牙周病学杂志》2010年第12期675-678,683,共5页Chinese Journal of Conservative Dentistry

基　　金：重庆市自然科学基金(2007BB5075)

摘　　要：目的:体外分离培养大鼠脂肪干细胞(Adipose derived stem cell,ADSCs),并进行鉴定。方法:取出生后4 d SD大鼠的腹股沟皮下脂肪组织,经胶原酶消化获得脂肪细胞,通过有限稀释法克隆化培养、分离得到大鼠ADSCs,用含100 mL/L FBS的DMEM培养液培养并传代。测定克隆形成率;流式细胞仪进行干细胞分子表型(Stro-1,CD90,CD105,CD34)检测;并进行成骨、成脂、成神经诱导。结果:有限稀释法所得细胞,有较强的克隆形成能力,STRO-1、CD90、CD105阳性表达,CD34阴性表达。成骨、成脂、成神经诱导实验证明所得细胞有多向分化能力。结论:有限稀释法克隆化培养可成功分离得到高纯度的ADSCs,为ADSCs在组织再生中的研究提供了可靠依据。AIM： To obtain and identify a large number of adipose derived stem cells（ADSCs） by monoclonal culture,and to investigate the characteristics and differentiation potential of ADSCs derived from the monoclone in vitro.METHODS： Adipose tissue was isolated from 4-day-old rat.After sufficient mixing and filtering,the rat adipose cells were seeded onto culture plates at a low density.Monoclonal cells were indentified through microscope and were picked out with micromanipulative technique when these monoclones were obvious.The ADSCs were identified by colony-forming assay and membrane antigens expression.Expression of Stro-1,CD90,CD105,CD34 were detected with flowcytometry（FCM）.Multipotential differentiations of ADSCs into adipocyte,bone and neuron were identified by oil red O staining,alizarin red staining and morphological observation.RESULTS： The obtained cells had high colony-forming efficiency.FCM results indicated that the positive rate of Stro-1,CD90,CD105 and CD34 were 8.1%,78.2%,96.9% and 1.2%,respectively.Under specific conditions,they could differentiate into adipocytes,osteoblasts and nerve cells in vitro.CONCLUSION： Limiting dilution assay is an effective method to isolate ADSCs and the single-cell-derived clonal cell populations demonstrate the properties of stem cells in vitro.

关 键 词：脂肪干细胞 分离 鉴定 细胞克隆 

分 类 号：R780.2[医药卫生—口腔医学]