人脂肪源性干细胞的分离及生物学性状的鉴定  

Isolation of human adipose-derived stem cells and the identification of biological characteristics

作　　者：王洁晴[1] 柏树令[1] 侯伟健[1] 佟浩[1] 田晓红[1] 徐赫[1] 

机构地区：[1]中国医科大学人体解剖学教研室,沈阳110001

出　　处：《解剖学报》2011年第2期205-209,共5页Acta Anatomica Sinica

摘　　要：目的建立从人脂肪抽吸物中分离培养脂肪来源干细胞(ASCs)的方法,并从形态学、生长动力学、表面标记物和分化能力等方面进行鉴定。方法从4例行腹部脂肪抽吸术的健康成年女性患者获得脂肪组织,通过酶消化法分离和培养脂肪干细胞,并传代培养至20代,观察细胞形态;四甲基偶氮唑盐(MTT)法测定和比较第3、9、15和20代细胞生长活性并绘制细胞生长曲线;流式细胞仪测定细胞周期和表面抗原表达;成脂成骨诱导后分别行油红O和茜素红染色定性。结果人脂肪源性干细胞形态类似成纤维细胞,呈漩涡状生长;MTT结果显示,ASCs传至15代仍有较强的增殖活性,之后逐渐减慢,至20代时细胞增殖速度明显降低,统计学分析显示,与第3、9、15代细胞有差异(P<0.05);细胞周期分析发现,ASCs具有干细胞的特性;流式细胞仪检测结果显示,间充质干细胞标记CD90、CD44表达阳性,造血干细胞标记CD34、血细胞标记CD45、内皮细胞标记CD31表达均为阴性,另外,CD49d低表达,CD 106表达阴性;成脂诱导后细胞内可见大量脂滴形成,油红O染色阳性;成骨诱导后镜下可见钙结节形成,茜素红染色阳性。结论人脂肪抽吸物中含有大量干细胞,能保持稳定增殖和表达干细胞表面标志,在诱导剂的作用下能向成脂和成骨分化。Objective To establish a method to isolate and culture adipose-derived stem ceils （ASCs） from the human liposuction aspirates, and conduct observations of the cell morphology, growth kinetics, surface markers and differentiating capacity. Method Adipose tissues were obtained from 4 healthy adult women who were experienced abdominal liposuction. ASCs, from liposuction aspirates, were isolated by enzymatic digestion, and were cultured to passage 20, the morphology of the cultured cells was observed. The cell viability was evaluated with MTT, and compared among passage 3,9,15 and 20. Cell growth curve was generated. The cell cycle and the surface marker profiles were detected by flow cytometry. Adipogenic differentiation and osteogenic differentiation of ASCs was assessed by oil red O and Alizarin Red staining respectively. Results The ASCs present a vortex pattern growth with a fibroblast-like appearance, and as shown by MTT, proliferation activity was strong when they subcultured to passage 15, then gradually slowed down, significantly reduced when they passed to passage 20. Statistical analysis showed that passage 20 and passage 3,9,15 were significantly different （P 〈 0. 05 ）. ASCs also showed characteristics of stem cell cycle. The positive expression of mesenchymal stem cell markers CDg0, CD44 and negative expression of hematopoietic stem cell marker CD34, the blood cell marker CD45,the endothelial cell marker CD31 were observed in ASCs by flow cytometry. In addition, the expression of CD49d was low and of CD106 was negative. Oil red O staining of ASCs after adipogenic induction demonstrated numerous intracellular lipid droplets. Calcium nodules could seen after osteogenic induction and Alizarin red staining was positive. Conclusion ASCs can be isolated from human liposuction aspirates and expressing cell surface markers of stem cells with strong proliferative ability. ASCs also can be induced to differentiate into adipose tissue and osseous tissue under the action of the induction agent.

关 键 词：脂肪来源干细胞 组织工程 分化 四甲基偶氮唑盐检测法 人 

分 类 号：Q813.11[生物学—生物工程]