摘要
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1.0~1.5 kg, 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，并利用成骨诱导剂包括0.1 μmol/L的地塞米松、50 mg/L的维生素C、10 mmol/Lβ-磷酸甘油钠和成脂诱导剂含10%FBS的L-DMEM、0.25 μmol/L地塞米松、50 μmol/L吲哚美辛、0.5 mmol/L IBMX、10 mg/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14 d后细胞不明显，未形成钙结节，21 d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48 h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。
关键词：骨髓；间充质干细胞；诱导；兔；多向分化

于音，赵刚，许侃，房学迅，邵佳甲.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[J].中国组织工程研究与临床康复，2008，12(8):1449-1452 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1449(ps).pdf]

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

Abstract

AIM：To study the proliferation, migration and differentiation of neural stem cells to give a useful proof of appearance and development of nervous system, and to observe the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the proliferation and the ability of differentiation into neurons of neural stem cells-derived from basal forebrain in vitro.

METHODS：Experiments were performed at the Department of Anatomy, Guangzhou Medical College from December 2005 to July 2006. ① Thirty SPF class new-born SD rats (less than 24 h) were selected. All the manipulation was according to animal ethnic standards. ②The brains of new-born rats were taken out under aseptic condition. The single cell suspension from the brain tissues of the basal forebrain by gentle mechanical dissociation with the use of trypsin was cultured in DMEM/F12 medium containing B27, EGF and bFGF (10 μg/L), (4-6)×105 cells/flask. Neurospheres were generated and floated in the culture. Neurospheres were collected and labeled with BrdU (6 mg/L) in the culture medium. The experiments were divided into three groups: adding 0.1 (V/V) bovine serum, EGF (10 μg/L) or bFGF (10 μg/L) into the culture. ③BrdU labeling was used to confirm the proliferation potential. The expression of Nestin antigen, BrdU labeling and the ability of differentiation into neurons of neural stem cells were detected by immunofluorescence techques.

RESULTS：①Morphology observation: Neural stem cells-derived from basal forebrain of new-born rats were bright ball shape in primary culture. The number of cells decreased after primary culture and some cells began to divide. One week after culture, the neurospheres were made of decades and hundreds of cells. The shape and verge of neural stem cells was regular and clear, and refraction ability was strong. The color of cytoplasm was deep and ratio of nucleus/cytoplasm was big. These results showed neural stem cells-derived from basal forebrain had the ability of self-renewing and could be obtained by subculture. ②Nestin expression: All the generated cells showed Nestive positive. ③BrdU labeling detection: All the neurospheres were BrdU positive, showing neurospheres were made of sustained self-renewing cells. ④Differentiation results: Under the conditions of 0.1 (v/v) bovine serum, EGF (10 μg/L) and bFGF (10 μg/L) into the culture, the ratios of neural stem cells differentiating into neurons were 20%, 22% and 40%, respectively.

CONCLUSION：① EGF and bFGF can activate neural stem cells-derived from basal forebrain of new-born rats to proliferate and make them maintain embryo characteristics. ② Serum serving as a control group, the ability of bFGF differentiating neural stem cells into neurons is stronger than that of EGF.

Gu HG, Long DH, Li XB, Zhang GP, Luo M, Li JM, Leng SL. Proliferation and differentiation of neural stem cells from neonatal rat basal forebrain of newborn rats into neurons in different culture conditions.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1445-1448(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1445(ps).pdf]