大鼠脂肪干细胞的提取与鉴定

Extraction and identification of rat adipose-derived stem cells

目的:提取大鼠脂肪干细胞并进行成脂、成骨、成内皮诱导分化及鉴定。方法:取大鼠腹股沟周围脂肪组织,使用Ⅰ型胶原酶消化后获得原代脂肪干细胞进行培养;流式细胞术检测脂肪干细胞表面分子标志物;使用成骨、成脂、成内皮诱导液诱导脂肪干细胞向骨细胞、脂肪细胞、血管内皮细胞定向分化,并用相应的染色剂进行鉴定。结果 :脂肪干细胞呈长梭型生长,传代之后形态趋于稳定。流式细胞术检测显示CD29、CD44、CD90表达阳性,CD45表达阴性。成脂诱导分化后油红O染色显示细胞内出现大小不等的橘红色脂滴。成骨诱导后茜素红染色显示橘红色的钙化结节。成内皮诱导后细胞形态呈现"铺路石"样,细胞能吞噬Dil标记的乙酰化低密度脂蛋白并具有分泌一氧化氮能力。结论:大鼠脂肪干细胞易分离培养且具有多向分化的潜能。

Objective : To extract rat adipose-derived stem cells and identify the cells by induction into adipocyte, osteocyte and vascular endothelial cells. Methods : Get the adipose tissue around the groin of rats, primary adipose-derived stem cells were obtained after digestion with type Ⅰ collagenase and then it was cultured in the flask;identify the adipose-derived stem cell surface molecular markers by flow cytometry. After differentiations into adipocyte, osteocyte and vascular endothelial cells, the cells were identified by the corresponding stain. Results : Adipose-derived stem cells grew in a long shuttle type and their morphology tended to be stable after passage. The surface marker CD29, CD44, CD90 were positive, and CD45 was negative. After adipogenic and osteogenic induction, respectively, oil red O staining showed orange lipid droplets of varying sizes in the cells, ailzarin red staning showed the orange calcified nodules. After differentiations into the endothelial cells, the cells looked like paving stones. The cells can swallow Dil-labeled acetylated low-density lipoprotein and have the abilitiy of nitric oxide secretion. Conclusion : Rat adipose-derived stem cells are easily isolated and cultured and they have the potential to differentiate into multiple directions.