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目的研究大鼠骨髓基质干细胞(BMSCs)分离培养的方法,并采用药物对其进行成骨诱导,探讨大鼠BMSCs在体外向成骨细胞分化的能力。方法本实验于2010年在中国医科大学口腔医学院中心实验室进行,采用全骨髓培养法进行大鼠BMSCs的分离与培养,光镜下观察细胞形态,之后细胞传代到所需数量后,随机分为2组,诱导组采用药物法(0.1μmol/L的地塞米松、10mmol/L的β-磷酸甘油钠、0.2mmol/L维生素C)进行成骨诱导,非诱导组未采用任何方法进行成骨诱导。在诱导的第1、3、5天,分别对两组细胞取样,进行细胞活性检测和碱性磷酸酶检测;在诱导的第5天进行扫描电镜观察。比较两组细胞成骨能力。结果光镜下,分离培养的细胞呈圆形、多角形、梭形等成纤维细胞样外观,细胞核浆比例较大,透光度较高,连续培养后呈现束状和漩涡状生长,符合基质干细胞的形态学特征。在成骨诱导的第1天,两组细胞的活性检测差异有统计学意义(P<0.05),碱性磷酸酶表达差异无统计学意义(P>0.05);在诱导的第3、5天,两组细胞的活性检测、碱性磷酸酶表达差异均有统计学意义(P<0.05)。在诱导培养的第5天,扫描电镜下观察可见两组细胞形态不同。结论采用全骨髓培养法可对大鼠BMSCs进行有效的分离培养,分离出的细胞符合基质干细胞特点;通过地塞米松、β-磷酸甘油钠、维生素C可对其进行有效的成骨诱导。

Culture and osteogenic potential of bone marrow stromal cells in rats

Objective To study the rat bone marrow stromal cells （BMSCs） isolation and culture methods, and the potential of rat BMSCs differentiating into osteoblast through drug induction. Methods In this study, in 2010, using rat whole bone marrow culture method for isolation and culture of BMSCs, cell morphology was observed under light microscope, after the passage to the desired number of cells were randomly divided 2 groups, one group with drug laws （0.1 μtool / L dexamethasone, 10 mmol / L of sodium β-glycerophosphate, 0.2 mmol / L vitamin C） for bone induction, the other group did not use other methods osteogenic induction. 1,3,5 in the first day of induction, cells were two groups of samples, the alkaline phosphatase, MTT test; in the induction of the first five days scanning electron microscopy. Osteogenic cells were compared the difference in ability. Results The results of light microscopy, isolation and culture of cells were round, polygonal, spindle-shaped fibroblast-like appearance, etc., a larger proportion of nuclear plasma, high transmittance, after continuous culture showed growth of bundle and swirling, in line with mesenchymal stem cells morphological features. Bone induction in the first day, two groups of cells, activity detection was significantly （P 〈 0.05）, alkaline phosphatase expression was no significant difference （P 〉 0.05） ; in the induction of the first 3,5 days, two groups of cells, activity detection, alkaline phosphatase expression differences were statistically significant （P 〈 0.05）. Induction training in the first 5 days, making two sets of cells SEM specimens, scanning electron microscopy shows that two different cell morphology. Conclusion The whole bone marrow culture method can be effective in rat BMSCs culture and cells isolated from cell line with characteristics of mesenchymal stem; dexamethasone, β-glycerophosphate sodium, vitamin C can induce rat BMSCs differentiating into osteoblast.