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背景:骨髓间充质干细胞取材方便,易于体外扩增,其体外培养方法及诱导分化仍需要改良。目的:对大鼠骨髓间充质干细胞体外培养方法及诱导剂配比进行改良,观察骨髓间充质干细胞向成骨细胞分化的潜能。设计:观察实验。单位:解放军广州军区广州总医院医学实验科。材料:实验于2004-07/2006-06在解放军广州军区总医院医学实验科干细胞组织工程实验室完成,选用20只成年SD大鼠,清洁级,雌雄不拘,体质量140~180 g,由解放军广州军区广州总医院动物实验中心提供。实验过程中对动物的处置符合动物伦理学标准。β-甘油磷酸纳,地塞米松,维生素C均为美国Sigma公司产品,羊抗大鼠骨钙素抗体购自美国DSL公司,S-P免疫组化试剂盒为福建迈新生物技术开发有限公司产品。方法:采用改良法进行细胞的培养及诱导细胞成骨。①骨髓间充质干细胞分离及培养:大鼠麻醉后处死分离双侧股骨、胫骨骨髓制备单细胞悬液接种于培养瓶中,培养后48及96 h半量更换培养液,以去除未贴壁的造血细胞,以后每3 d换液1次,进一步去除未贴壁的细胞,待细胞汇合约80%时,用0.25%胰蛋白酶消化,按1∶2传代培养。将第2代骨髓间充质干细胞接种于6孔培养板和玻璃平皿中,48 h后吸去基础培养液。②骨髓间充质干细胞向成骨细胞诱导分化:应用含终浓度分别为10 mmol/L、10-7mol/L、50 mg/L的地塞米松、β-甘油磷酸纳和维生素C的诱导分化培养液定向诱导传代细胞向成骨细胞分化。主要观察指标:①应用改良的培养方法及自行配比的培养液诱导分化后10 d采用钙钴法测定碱性磷酸酶活性。②培养后12 d采用免疫组织化学法检测骨钙素分泌情况。③诱导培养后2周采用Von kossa染色检测细胞矿化作用。结果:①碱性磷酸酶活性:经诱导后细胞碱性磷酸酶染色明显,胞质中阳性反应呈现灰黑色颗粒或块状沉淀,碱性磷酸酶活性部位呈棕黑色。②骨钙素分泌情况:细胞经诱导后骨钙素阳性较明显,胞核呈蓝色,胞浆呈棕色。③细胞矿化作用:细胞经诱导培养后呈复层生长,并出现不透明结节,Von kossa染色可见黑色的矿化结节颗粒,颗粒大小不均一,提示有矿化基质沉积。结论:改良法可成功培养及诱导大鼠骨髓间充质干细胞成骨。
BACKGROUND: Bone marrow-derived mesenchymal stem cells are easy to be drawn and easy to expand in vitro. The methods of in vitro culture and differentiation need to be improved. OBJECTIVE: To improve the method of rat bone marrow mesenchymal stem cells culture in vitro and the ratio of inducer, and to observe the potential of bone marrow mesenchymal stem cells to differentiate into osteoblasts. Design: observation experiment. Unit: PLA General Hospital of Guangzhou Military Command, Guangzhou. MATERIALS: The experiment was performed in the Stem Cell and Tissue Engineering Laboratory of Medical Laboratory of PLA General Hospital of PLA from July 2004 to June 2006. Twenty adult SD rats were selected to be clean, male and female. The weight ranged from 140 to 180 g. Guangzhou Military Region Guangzhou General Hospital Animal Experimental Center. The handling of animals during the experiment is in line with animal ethics standards. β-glycerophosphate sodium, dexamethasone, vitamin C are the United States Sigma products, goat anti-rat osteocalcin antibody purchased from the United States DSL company, S-P immunohistochemistry kit Fujian step new biotechnology Development Co., Ltd. products. Methods: The modified method was used to culture the cells and induce cell osteogenesis. ① Bone marrow-derived mesenchymal stem cells were isolated and cultured: Rats were sacrificed after anesthesia, bilateral femur and tibia bone marrow were isolated and prepared single cell suspension was inoculated in culture flasks, half and 48 h after culture medium replacement medium to remove unattached Hematopoietic cells, after every 3 d fluid change 1, further removal of unattached cells, until the cells confluent about 80%, with 0.25% trypsin digestion, 1: 2 subcultures. The second generation of bone marrow mesenchymal stem cells were seeded on 6-well plates and glass dishes and basal medium was aspirated after 48 h. (2) Bone marrow mesenchymal stem cells differentiate into osteoblasts: The induced differentiation of osteoblasts was induced by dexamethasone, β-glycerophosphate and vitamin C at final concentration of 10 mmol / L, 10-7 mol / L and 50 mg / Culture medium directed differentiation of the passaged cells to osteoblasts. MAIN OUTCOME MEASURES: ① Alkaline phosphatase activity was assayed by calcium and cobalt method on the 10th day after induction of differentiation using a modified culture method and self-adapted culture medium. ②12 days after culture, immunohistochemistry was used to detect the secretion of osteocalcin. ③ Von kossa staining was used to detect cell mineralization 2 weeks after induction. Results: ① Alkaline phosphatase activity: Alkaline phosphatase staining of cells was obvious after induction. The positive reaction in cytoplasm showed gray-black granules or massive sediment, and the active site of alkaline phosphatase was brown-black. Osteocalcin secretion: osteoblast-positive cells were significantly positive after induction, the nucleus was blue, brown cytoplasm. ③ Cell mineralization: The cells grew in multiple layers after induction and cultured, and opaque nodules appeared. Von kossa staining showed black mineralized nodules with uneven particle size, indicating the deposition of mineralized matrix. Conclusion: The modified method can successfully culture and induce rat bone marrow mesenchymal stem cells osteogenesis.