【作者】 李红；

【导师】 吴德生；

【作者基本信息】 四川大学 ， 劳动卫生与环境卫生， 2006， 博士

【摘要】 目的 1、建立和完善小鼠骨髓间充质干细胞(bone mesenchymal stem cells，BMSCs)的体外培养体系，最大限度地获得BMSCs。2、建立BMSCs的体外诱导分化模型。3、探讨氟化钠和酒精对间充质干细胞的毒性作用的机制，从而为防治氟骨病和酒精引起的股骨头坏死、骨质疏松等损伤机制研究提供科学依据。 方法 1、无菌取C57／BL6小鼠骨髓，用全骨髓培养方法体外扩增BMSCs，免疫组织化学染色鉴定BMSCs的阳性表面标志物CD90和阴性表面标志物的CD34，绘制细胞的生长曲线。2、以含地塞米松、β-磷酸甘油钠和维生素C的a-MEM培养液诱导BMSCs向成骨细胞方向分化，分别于不同时间收获细胞进行成骨细胞的早、中、晚期指标鉴定。以含地塞米松和胰岛素的a-MEM培养液诱导BMSCs向脂肪细胞方向分化，油红O染色鉴定脂肪细胞。3、分别用含不同浓度的NaF、Et和Ach的基础培养液染毒细胞，3天后收获细胞行MTT试验、c-fos免疫组织化学染色检测毒物对细胞的毒性；分别用含NaF、Et或Ach的成骨诱导培养液染毒细胞，于3，8，15天后分别行ALP含量测定，VEGF、TAZ、OPG、Collagen type Ⅰ、PPAR γ更多还原

【Abstract】 Objectives to establish and optimize in-vitro culture system of murine mesenchymal stem cells (BMSCs) in order to obtain more BMSCs; to establish the model of BMSCs differentiation in vitro; to investigate the mechanism of adverse effects on BMSCs by sodium fluoride and alcohol and therefore to provide theoretical basis for the prevention and cure of osteofluorosis, osteoporosis and osteonecrosis by alcohol abuse.Methods 1 the marrow was harvested from C57/BL6 male mice. BMSCs were amplificated in vitro by the method of whole marrow culture and identified surface marker CD90 and CD34 by immunohistochemistry. Finally the growth curve of BMSCs was described. 2 BMSCs were cultured in a-MEM supplemented by dexamethasone, β-sodium phosphoglycerol and vitamin C to differentiate toward ostoblasts. Cells were harvested at different time to be identified the early, middle and late function markers of osteoblasts. Furthermore BMSCs were induced in a-MEM supplemented by dexamethasone and insulin to differentiate toward adipocytes. Cells were obtained to conduct oil更多还原