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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。
OBJECTIVE: To study the isolation and culture of rat bone marrow stromal stem cells (BMSCs), and to explore the osteogenic differentiation of rat BMSCs by differentiating into osteoblasts in vitro. Methods The experiment was carried out in the Central Laboratory of Stomatology, China Medical University in 2010. The whole bone marrow culture was used to isolate and culture BMSCs in rats. The morphology of the cells was observed under light microscope. After the cells were passaged to the required number, In the induction group, the osteogenic induction was induced by the drug method (0.1μmol / L dexamethasone, 10mmol / L sodium β-phosphoglycerate, 0.2mmol / L vitamin C). The non-induced group was not induced by any method Bone induction. On the 1st, 3rd and 5th day of induction, the two groups of cells were sampled, and the cell viability and alkaline phosphatase activity were detected. Scanning electron microscopy was performed on the fifth day after induction. Compare the osteogenic ability of two groups of cells. Results Under the light microscope, the cultured cells were round, polygonal, fusiform and other fibroblast-like appearance with a larger proportion of nuclear cytoplasm and higher transmittance. After continuous culture, the cells showed a bundle-like and swirling growth, Morphological characteristics of stem cells. On the first day after osteogenic induction, there was a significant difference between the two groups (P <0.05) and no significant difference in alkaline phosphatase expression (P> 0.05). On the 3rd and 5th day The activity of alkaline phosphatase and alkaline phosphatase in both groups were significantly different (P <0.05). On the fifth day of induction culture, the morphological changes of the two groups were observed under the scanning electron microscope. CONCLUSION: BMSCs can be effectively isolated and cultured in rat bone marrow by using whole bone marrow culture method. The isolated cells are in line with the characteristics of stromal stem cells. Dexamethasone, β-phosphoglycerate and vitamin C can effectively induce osteoblast formation.