兔BMSCs体外培养及定向诱导为脂肪细胞、成骨细胞的研究

发布时间：2018-03-16 15:54

本文选题：骨髓间充质干细胞　切入点：细胞培养　出处：《昆明医学院》2005年硕士论文　论文类型：学位论文

【摘要】：目的:探讨兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)体外分离培养、诱导分化为脂肪细胞及成骨细胞的方法,并研究其生物学特性,为组织工程及干细胞药理学研究奠定实验基础。方法:采用体外细胞培养技术自兔股骨、胫骨及肱骨中分离BMSCs进行纯化、培养。用地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素、吲哚美辛定向诱导BMSCs分化为脂肪细胞,油红-O染色鉴定。用地塞米松、β-甘油磷酸钠、维生素C定向诱导BMSCs分化为成骨细胞,NBT/BCIP染色法、P-对硝基苯基质法及茜素红染色法检测BMSCs的成骨能力。结果:原代及传代培养显示,兔BMSCs具有活跃的增殖能力。成脂诱导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,细胞形态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BMSCs转变为脂肪细胞。成骨诱导第1～5天,培养的细胞增殖旺盛,3～5天即可融合成单层,随着诱导时间的延长,细胞增殖速度减慢,出现细胞聚集的现象,培养细胞逐渐汇合呈铺路石状,细胞形态变为胞体较小的立方形,继续诱导,可出现多角形成骨样细胞,胞外基质分泌逐渐增多,胶原堆积、钙盐沉积,10～12天形成不透光矿化结节,ALP活性增高,茜素红染色阳性,表示BMSCs向成骨细胞分化。结论:应用本实验方法培养的兔BMSCs,体外生长稳定、增殖速度快、贴壁率高、可连续传代;经体外诱导可以向脂肪细胞、成骨细胞分化。
[Abstract]:Objective: to investigate the method of inducing adipocytes and osteoblasts from rabbit bone marrow mesenchymal stem cells bone marrow mesenchymal stem cells in vitro, and to study their biological characteristics. Methods: BMSCs was purified from rabbit femur, tibia and humerus by cell culture in vitro. Indomethacin induced the differentiation of BMSCs into adipocytes, which was identified by oil red-O staining. The differentiation of BMSCs into osteoblasts was induced by vitamin C. The osteogenic ability of BMSCs was detected by P- p-nitrobenzene matrix method and alizarin red staining method by NBT / BCIP staining. Results: the primary culture and passage culture showed that rabbit BMSCs had active proliferative ability. After 72 hours of lipogenesis, lipid droplets appeared in the cells. With the prolongation of the induction time, the lipid droplets gradually increased and fused into lipid bubbles. The morphology of the cells changed from fusiform to round or polygonal, and the oil red O staining at the 3rd week showed that more than 80% BMSCs were transformed into adipocytes. After 1 to 5 days of osteogenic induction, the cultured cells could be fused into monolayers. With the prolongation of induction time, cell proliferation slowed down, cell aggregation appeared, the culture cells gradually converged into a paving stone, the shape of the cells became square with smaller cell body, and the polygonal osteoblasts appeared after the induction. The secretion of extracellular matrix increased gradually, collagen accumulation, calcium salt deposition for 10 ~ 12 days to form impermeable mineralized nodules, ALP activity increased, alizarin red staining positive. Conclusion: the rabbit BMSCs cultured by this method has the advantages of stable growth in vitro, rapid proliferation, high adhesion rate, and can be subcultured continuously, and can differentiate into adipocytes and osteoblasts after induction in vitro.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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