地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素、吲哚美辛定向诱导兔骨髓间充质干细胞分化为脂肪细胞的研究(英文)

Differentiation of rabbit bone marrow mesenchymal stem cells into adipocytes in media containing dexamethasone,3-isobutyl-1-methylxanthine,insulin and indomethacin

背景：研究证明，脂肪细胞来源于脂肪前体细胞，而脂肪前体细胞又是由间充质干细胞分化而来。探讨间充质干细胞分化为脂肪细胞的机制，可能为肥胖防治提供新的思路。目的：探讨兔骨髓间充质干细胞体外分离培养、诱导分化为脂肪细胞的方法。设计：观察对比，体外实验。单位：滨州医学院组织学与胚胎学教研室及云南省天然药物药理重点实验室。材料：选用健康6-8周龄日本大耳兔12只，体质量200～300g，雌雄不拘，由昆明医学院实验动物中心提供。定向诱导细胞用地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素为Sigma公司产品，吲哚美辛为ALEXIS公司产品。方法：实验于2004-07／2005-01在云南省天然药物药理重点实验室完成。由兔股骨、胫骨及肱骨中分离骨髓间充质干细胞，对其进行纯化、培养。用1μmol几地塞米松、0．5mmol／L3-异丁基-1-甲基黄嘌呤、10mg／L胰岛素、0．2mmol／L吲哚美辛定向诱导骨髓间充质干细胞分化为脂肪细胞。实验过程中对动物的处置符合动物伦理学标准。主要观察指标：采用改良MTT法测定其生长曲线，油红-O染色：光镜下观察橙红色脂滴沉着的脂肪细胞比例。结果：骨髓间充质干细胞经定向诱导72h后，细胞内有脂滴出现，随着诱导时间的延长，脂滴逐渐增加并融合为脂泡，细胞由梭形转变为类圆形或多角形：第3周油红-O染色显示80％以上的细胞转变为脂肪细胞。结论：兔骨髓间充质干细胞在体外分离、培养后，经1μmol／L地塞米松、0．5mmol／L3-异丁基-1-甲基黄嘌呤、10mg／L胰岛素、0．2mmol／L吲哚美辛定向诱导，可以分化为脂肪细胞。

BACKGROUND： Adipocytes are derived from preadipocytes, which are induced by mesenchymal stem cell differentiation. This study explored the mechanism of mesenchymal stem cell differentiation into adipocytes and provided a new thinking for preventing and treating obesity. OBJECTIVE： To investigate the isolation and culture of BMSCs from rabbits in vitro, and the method of differentiating into adipocytes. DESIGN： An observational controlled in vitro experiment. SETTING： Department of Histology and Embryology, Binzhou Medical College, and Yunnan Pharmacological Laboratories of Natural Products. MATERIALS： Totally 12 healthy Japanese rabbits aged 6-8 weeks of 200 to 300 g of either sex were provided by Experimental Animal Center of Kunming Medical College. Dexamethasone, 3-isobutyl-l-methylxanthine and insulin （Sigma, USA） and indomethacin （ALEXIS, USA） were used in this study. METHODS： Experiments were performed at the Yunnan Pharmacological Laboratories of Natural Products between July 2004 and January 2005. BMSCs were harvested from the femurs, tibias and humerus bones of the rabbits, and then purified and proliferated. After primary culture, the subcultured cells were cultured in media including 1 μ mol/L, dexamethasone, 0,5 mmol/L 3-isobutyl-1-ethylxanthine, 10 mg/L insulin and 0.2 mmol/L indomethacin. The disposal was accorded with the animal ethical standards. MAIN OUTCOME MEASURES： Growth curve of the third passage of cells was evaluated by modified MTT method. Salmon pink lipid drop appeared after Oil Red O staining under a light microscope. RESULTS： After 72 hours of adipocyte induction, lipid droplet appeared in BMSCs. With the prolongation Of induction time, lipid vacuoles were found and cells changed from fusiform to round or polygon. At week 3, approximately 80% BMSCs were induced into adipocytes as detected by Oil Red O. CONCLUSION： Rabbit BMSCs isolated and cultured in vitro can be induced into adipocytes in the media containing 1 μ mol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 10 mg/L insulin and 0.2 mmol/L indomethacin.