兔骨髓间充质干细胞体外培养向成骨和成脂方向的诱导分化

Differentiation of mesenchymal stem cells from rabbit bone marrow into osteoblasts and lipoblasts in vitro

目的：骨髓间充质干细胞是存在于骨髓组织中多种干细胞的混和体，因其具有取材方便、扩增迅速、可自体移植并且具有多分化潜能等特点，是近年研究的热点。实验建立了体外分离、培养及鉴定兔骨髓间充质干细胞的方法，并通过此进一步验证其诱导成骨及成脂多向分化的潜能。方法：实验于2006-08/2007—06在吉林大学药学院生物工程实验室完成，实验室属吉林大学重点实验室。①实验材料：四五月龄新西兰大白兔2只，由吉林大学基础医学院动物培养中心提供，体质量1．0～1．5kg，雌雄不限，实验动物级别为二级，实验过程中对动物处置符合动物伦理学标准。②实验方法：实验应用了密度梯度离心法与贴壁培养法相结合的方法从兔股骨、胫骨中分离、纯化骨髓间充质干细胞并在体外进行培养，以形态学及细胞表面标志的方法鉴定间充质干细胞，在倒置显微镜下观察细胞的形态特征，并利甩成骨诱导剂包括0．1μmol／L的地塞米松、50mg/L的维生素C、10mmol／L β-磷酸甘油钠和成脂诱导剂含10％FBS的L．DMEM、0．25μmol／L地塞米松、50μmol／L吲哚美辛、0．5mmol／LIBMX、10mg／L牛胰岛素诱导其向软骨细胞及脂肪细胞分化。结果：①经原代及传代培养的骨髓间充质干细胞呈梭形，类似于成纤维细胞，骨髓间充质干细胞均一地表达CD29、CD44，而CD34、CD45均阴性表达。②成骨诱导14d后细胞不明显，未形成钙结节，21d后碱性磷酸酶钙钴法染色后大多数细胞的胞质呈棕黑色。③成脂诱导48h后，细胞内有小脂滴出现，2周后脂滴数量增加并相互融合，细胞（有）由长梭形变为圆形或多边形，油红O染色显示有大量脂质沉积。结论密度梯度离心法与贴壁培养法相结合的方法是比较理想的骨髓间充质干细胞培养法，骨髓间充质干细胞经诱导培养后具有多向分化潜能。

AIM： Bone marrow mesenchymal stem cells （BMSCs） are admixture of many kinds of stem cells in bone marrow, which have many characteristics including convenient use, prompt amplification, autotransplantation and multi-directional differentiation potency. It has been the hotspot in recent study. This study establishes the method of isolation and culture of rabbit BMSCs in vitro and confirms the potency of multi-directional differentiation by osteoblast and lipoblast formation induction. METHODS： Experiments were performed at the Bioengineering Laboratory in Pharmacy School of Jilin University from August 2006 to June 2007. ①Two New Zealand rabbits （1.0-1.5 kg） aged 4 5 months after birth, of either sex, were bought from Animal Cultivation Center in Preclinical Medicine School of Jilin University. The hierarchy of animals was second grade. The disposition of the rabbits met the ethics standards. ② BMSCs were isolated from the femurs and tibias bones and purified and cultured in vitro by density gradient centrifugation and adherent culture. BMSCs were evaluated by morphology and surface marker. BMSCs culture was observed with an inverted microscope. BMSCs were induced to osteoblasts and lipoblasts by osteogenic inductor including 0.1 μmol/L dexamethasone, 50 mg/L vitamin C, 10 mmol/L β -phosphoglycerol and adipogenic inductor including 10% FBS-L-DMEM, 0.25 μ mol/L dexamethasone, 50 μmol/L antifani, 0.5 mmol/L IBMX, 10 mg/L bovine insulin. RESULTS： ①The BMCSs was fusiform, fibroblast-shape after cultivation of primary and subcultured. CD29 and CD44 were expressed, but CD34 and CD45 were not expressed. ②The cells did not form calcium nodus in the 14th day after osteogenic induction and the cytoplasm of the most cells presented brownish-black in the 21^st day after alkaline phosphatase calcium-cobalt staining. ③Intra-cellular lipid droplet appeared after 48-hours adipogenic induction. The quantity of lipid droplet increased and were fused each other 2 weeks later. The morphous of fusiform shape became round or polygon, and a great quantity lipid deposited after oil red O staining. CONCLUSION： Combination of density gradient centrifugation and adherent culture is an ideal method to culture BMSCs. BMSCs have the potency of multi-directional differentiation after induction.