大鼠脂肪间充质干细胞体外分离培养及CM-DiI标记后的传代示踪

摘要

Objective To establish a method for isolation, culture and cell labeling of rat adipose-derived stem cells( ADSCs) and investigate the passage tracing feasibility of CM-DiI labeled ADSCs in vitro. Methods Adipose tissue was harvested from the inguinal fat pad of Sprague-Dawley rats under sterile conditions. By means of combi-ning zymogen digestion with differential velocity adherence, the primary cells were isolated. They were used on ser-ial subcultivation. The morphology of cells was observed by inverted phase contrast microscope. The molecular phe-notype and differentiation potentiality of three passaged ADSCs were identified. Three passaged ADSCs were labeled by CM-DiI and they were used on serial subcultivation. Fluorescence labeling of cells were observed under the fluo-rescence inverted microscope and laser scanning confocal microscope. The labeling rate was detected by flow cytom-etry. Results ADSCs showed long spindle shape and vortex like growth. After passages, the growth and prolifera-tion rate of cells were accelerated. The cells were identified positive for CD29 and CD90 makers, and they showed the lack of CD34,CD45 and CD31 expressions. Alizarin red and oil red O staining showed ADSCs could be differ-entiated to osteoblasts and adipocytes. The red fluorescence of CM-DiI labeling existed in the cell membrane and cytoplasm, which did not exist in the cell nucleus. After passages, the fluorescence was attenuated, and the fluores-cence labeling rates of the third, sixth and ninth passage were 97. 09%, 66. 21% and 37. 86%, respectively. Con-clusion Rat ADSCs can grow and proliferate rapidly, and they have multiple differentiation potential. CM-DiI can label ADSCs simply and effectively. These can provide the basis for the following experiment in vivo.%目的：建立大鼠脂肪间充质干细胞( ADSCs )体外分离培养和细胞标记的方法，探讨CM-DiI标记ADSCs在体外传代示踪的可行性。方法无菌切取SD大鼠腹股沟脂肪组织，运用酶原消化并差速贴壁的方法获取原代细胞并进行传代培养。观察细胞形态，对第3代ADSCs进行细胞表型鉴定和成骨成脂分化能力鉴定。采用 CM-DiI标记第3代ADSCs并进行传代培养，荧光显微镜和激光扫描共聚焦显微镜观察荧光标记情况，流式细胞仪检测标记率。结果 AD-SCs呈长梭形、漩涡样生长，传代后细胞生长增殖速度加快。流式细胞仪检测结果显示 CD29、CD90阳性表达， CD34、CD45、CD31阴性表达。茜素红和油红O染色显示ADSCs具有成骨成脂分化潜能。 CM-DiI标记的红色荧光存在于细胞膜和细胞质，不存在于细胞核，传代培养后荧光有所衰减，第3、6、9代的荧光标记率分别为97．09%、66．21%和37．86%。结论大鼠ADSCs的生长增殖能力强，具有多向分化潜能，采用CM-DiI标记ADSCs简单易行且示踪效果良好，可为后期体内实验提供依据。