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  本文選題：骨髓間充質干細胞　切入點：細胞培養(yǎng)　出處：《昆明醫(yī)學院》2005年碩士論文　論文類型：學位論文

【摘要】：目的:探討兔骨髓間充質干細胞(bone marrow mesenchymal stem cells,BMSCs)體外分離培養(yǎng)、誘導分化為脂肪細胞及成骨細胞的方法,并研究其生物學特性,為組織工程及干細胞藥理學研究奠定實驗基礎。方法:采用體外細胞培養(yǎng)技術自兔股骨、脛骨及肱骨中分離BMSCs進行純化、培養(yǎng)。用地塞米松、3-異丁基-1-甲基黃嘌呤、胰島素、吲哚美辛定向誘導BMSCs分化為脂肪細胞,油紅-O染色鑒定。用地塞米松、β-甘油磷酸鈉、維生素C定向誘導BMSCs分化為成骨細胞,NBT/BCIP染色法、P-對硝基苯基質法及茜素紅染色法檢測BMSCs的成骨能力。 結果:原代及傳代培養(yǎng)顯示,兔BMSCs具有活躍的增殖能力。成脂誘導72h后,細胞內(nèi)有脂滴出現(xiàn),隨著誘導時間的延長,脂滴逐漸增加并融合為脂泡,細胞形態(tài)由梭形轉變?yōu)轭悎A形或多角形,第3周油紅-O染色顯示80%以上的BMSCs轉變?yōu)橹炯毎�。成骨誘導第1～5天,培養(yǎng)的細胞增殖旺盛,3～5天即可融合成單層,隨著誘導時間的延長,細胞增殖速度減慢,出現(xiàn)細胞聚集的現(xiàn)象,培養(yǎng)細胞逐漸匯合呈鋪路石狀,細胞形態(tài)變?yōu)榘w較小的立方形,繼續(xù)誘導,可出現(xiàn)多角形成骨樣細胞,胞外基質分泌逐漸增多,膠原堆積、鈣鹽沉積,10～12天形成不透光礦化結節(jié),ALP活性增高,茜素紅染色陽性,表示BMSCs向成骨細胞分化。結論:應用本實驗方法培養(yǎng)的兔BMSCs,體外生長穩(wěn)定、增殖速度快、貼壁率高、可連續(xù)傳代;經(jīng)體外誘導可以向脂肪細胞、成骨細胞分化。
[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.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R329.2

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