大鼠脂肪源性干细胞的分离培养及鉴定

背景：脂肪源性干细胞在体外易于培养,增殖快,具有多向分化潜能。目的：构建一种体外分离培养SD大鼠脂肪源性干细胞的方法,并对其部分生物学特性与表型进行分析。方法：切取SD大鼠腹股沟脂肪垫,应用胶原酶Ⅰ消化,分离大鼠脂肪源性干细胞,进行体外培养、传代,倒置显微镜观察细胞的生长增殖及形态变化,诱导成骨、成脂,分别行碱性磷酸酶、茜素红、von Kossa染色及油红O染色,绘制生长曲线及用流式细胞仪检测细胞表面标记。结果与结论：体外培养的脂肪源性干细胞呈梭形,增殖活跃,传代后形态均一,多次传代后细胞仍保持较强增殖能力,生长曲线呈＂S＂型。成骨诱导实验组碱性磷酸酶、茜素红、von Kossa染色阳性;成脂诱导实验组油红O染色阳性;对照组均为阴性。细胞CD29,CD44,CD105表达阳性,CD31,CD45表达阴性。提示SD大鼠腹股沟脂肪垫分离的脂肪源性干细胞在体外易于分离培养和传代扩增,特定条件下可诱导分化为成骨细胞和脂肪细胞,并表达间充质干细胞相关的表型。

Isolation,culture and identification of rat adipose-derived stem cells

BACKGROUND：Adipose tissue contains abundant adipose-derived stem cells（ADSCs） ,these cells can be cultured in vitro easily and proliferate rapidly. With the potential of multi-directional differentiation,ADSCs are promising to be seed cells of tissue engineering and have attracted much attention. OBJECTIVE：To establish a method for the isolation and culture of SD rat ADSCs in vitro,and to investigate some biological characteristics and phenotypes. METHODS：Collagenase Ⅰwas used to digest and isolate ADSCs from SD rat inguinal fat pads. ADSCs were cultured and passaged in vitro,viewed under inverted microscope and were induced toward osteoblasts and adipocytes. Osteogenic differentiation was confirmed by alkaline phosphatase,alizarin red and von Kossa staining,adipogenic differentiation was confirmed by oil red O staining. Growth curve was drawn and cell surface antigens were identified by flow cytometer. RESULTS AND CONCLUSION：In vitro cultured ADSCs were spindle-shaped and easily proliferated,well-proportioned after passage. Following many times of passages,ADSCs could still maintain strong proliferative ability. The growth curve was ＂S＂ shaped. After osteogenic induction,ADSCs showed a positive reaction for alkaline phosphatase,alizarin red and von Kossa staining,while the adipogenetic induction group showed a positive reaction for oil red O staining. The control group showed negative reaction for every staining. ADSCs were positive for CD29,CD44 and CD105,but negative for CD31 and CD45. ADSCs isolated from SD rat inguinal fat pads can be isolated,cultured,passaged and proliferated easily in vitro,and can be induced into osteoblasts and adipocytes under certain conditions. Furthermore,ADSCs expressed related phenotypes of mesenchymal stem cells.