论文部分内容阅读
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 for the isolation and culture of adipose-derived stem cells (ASCs) from human lipoaspirate and to identify the morphology, growth kinetics, surface markers and differentiation ability. Methods Adipose tissue was obtained from 4 healthy adult female patients undergoing abdominal liposuction. Adipose-derived stem cells were isolated and cultured by enzymatic digestion and subcultured for 20 passages to observe cell morphology. MTT, The activity of cell growth at the 3rd, 9th, 15th and 20th generation was assayed and the cell growth curve was drawn. Cell cycle and surface antigen expression were measured by flow cytometry. Oil red O and alizarin red staining Qualitative. RESULTS: Human adipose-derived stem cells were fibroblast-like in shape and swirling in shape. The results of MTT assay showed that ASCs still had strong proliferative activity until 15 passages and then gradually slowed down, The results of flow cytometry showed that MSCs labeled with CD90 and CD44 were positive (P <0.05). The cell cycle analysis showed that ASCs had the characteristics of stem cells. , Hematopoietic stem cell marker CD34, hematopoietic marker CD45 and endothelial marker CD31 were negative. In addition, CD49d expression was low and CD 106 expression was negative. After adipogenic induction, a large number of lipid droplets were found in the cells and positive with oil red O staining. After the induction of calcification under visible nodules, alizarin red staining. Conclusions Human lipoaspirate contains a large number of stem cells, which can maintain stable proliferation and express stem cell surface markers. Under the action of inducer, it can differentiate into adipogenic and osteogenic.