【摘要】 目的:观察维甲酸和地塞米松对兔骨髓基质细胞的成骨诱导影响,探讨二者联合诱导兔骨髓基质细胞向成骨细胞分化的可能性和适当条件,使兔骨髓基质细胞向成骨诱导得以优化,从而为临床治疗骨缺损提供实验基础。方法:实验于2005-12/2006-3在锦州医学院附属第一医院骨科实验室完成。①体外培养兔骨髓基质干细胞。②用不同的诱导剂将其向类成骨细胞诱导。实验分非诱导组(用DMEM培养基培养),地塞米松诱导组(培养基中加入地塞米松、β-甘油磷酸钠和维生素C),维甲酸诱导组(培养基中加入维甲酸),地塞米松与维甲酸联合诱导组(培养基中加入地塞米松、β-甘油磷酸钠、维生素C和维甲酸)。③通过倒置相差显微镜对细胞的形态进行观察;于第4,6,8,10,12天定量检测其分泌的碱性磷酸酶;应用原位杂交技术检测Ⅰ型胶原的表达情况。结果:①原代和传代培养时不同组别兔骨髓基质细胞的生长情况:地塞米松诱导组细胞形态向类成骨细胞转化,维甲酸诱导组细胞与神经干细胞类似,地塞米松与维甲酸联合诱导组细胞与地塞米松诱导组细胞形态变化大体一致,但细胞更饱满,多角形细胞比例更高,部分区域细胞呈现漩涡状分布。②各组碱性磷酸酶活性检测结果:非诱导组碱性磷酸酶表达量低,随时间略有增加;地塞米松诱导组碱性磷酸酶表达呈曲线升高[6,8,10,12d依次为(265.9±47.3),(445.4±69.4),(650.3±52.0),(703.6±62.5)nkat/L];维甲酸诱导组基本无碱性磷酸酶表达;地塞米松与维甲酸联合诱导组碱性磷酸酶表达趋势与地塞米松诱导组相似,但绝对值有所增高[6,8,10,12d依次为(355.4±62.2),(631.3±84.4),(925.0±69.0),(1039.5±85.2)nkat/L]。③各组原位杂交技术检测Ⅰ型胶原结果:地塞米松诱导组和地塞米松、维甲酸联合诱导组检测Ⅰ型胶原结果发现胞浆内可见大量棕黄色颗粒,非诱导组与维甲酸诱导组为阴性。结论:适当浓度地塞米松可成功的将兔骨髓基质细胞向成骨细胞诱导,维甲酸与地塞米松联合诱导可使成骨能力进一步加强,表现为碱性磷酸酶表达明显增高,并表达Ⅰ型胶原,具体浓度条件有待进一步研究。更多还原

【Abstract】 AIM: To observe the possibility that dexamethasone (DXM) and retinoic acid (RA) are associated to induce rabbit bone marrow stromal cells (BMSCs) into osteoblasts and the suitable content, and further investigate an optimization for bone induction, so as to provide the experimental base for the treatment of bone defect in clinic. METHODS: The experiment was carried out in the Laboratory of Orthopaedic Department of the First Affiliated Hospital of Jinzhou Medical College from December 2005 to March 2006. Rabbit BMSCs were cultured in vitro, and then induced to osteoblasts by different inductors. The experiment was divided into four groups. Group A: no induction in DMEM. Group B: induced by DXM, β-sodium glycerophosphate and vitamin C. Group C: induced by RA. Group D: induced by the combination of DXM, β-sodium glycerophosphate, vitamin C and RA. The forms of the cells were observed under inverted phase contrast microscope, and the type I collagen and alkaline phosphatase (ALP) secreted by osteoblasts were detected by In Situ Hybridization and quantitative analysis on days 4, 6, 8, 10 and 12. RESULTS: ①The growth of rabbit BMSCs in different groups of primary culture and subculture: The cells in group B was converted to like-osteoblasts; The cells induced by RA became like-nerve stem cells; The cells in group D were similar to that of group B in forms, but they were more satiate, the proportion of polygon cells was higher than that of group B, and some cells were distributed as whirlpool.②The expression of ALP was much higher in group B than in group A, which was lower but increased a little with time extended Days 6, 8, 10 and 12 in group B: (265.9±47.3), (445.4±69.4), (650.3±52.0), (703.6±62.5) nkat/L. The expression in group D was similar to that of group B, but absolute value was more evident Days 6, 8, 10 and 12 in group D: (355.4±62.2), (631.3±84.4), (925.0±69.0), (1039.5±85.2) nkat/L. There was no ALP expressing in group C.③In group B and group D, detection of type I collagen with In Situ Hybridization blotting showed that there were lots of yellow-brown materials in cytoplasm, but the results in group A and C were negative. CONCLUSION: Rabbit BMSCs can be induced into marrow stromal osteoblasts by suitable contents of DXM. And the combination of DXM and RA can enhance this effect that the expression of ALP is increased obviously, and the expression of type I collagen is observed. The suitable density of DXM and RA need future researches.更多还原