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目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。
Objective To observe the expression and maintenance of osteogenic activity of osteogenic induced rabbit bone marrow stromal stem cells (BMSCs) in vitro and in vivo. Methods The osteogenic and differentiation characteristics of BMSCs were observed under osteogenic induction in vitro. The co-culture model of rabbit BMSCs and live bones was established to simulate the “osteogenic environment” in vivo and the osteogenic MSCs were placed under co-culture and normal subculturing conditions The subculture was carried out to observe the phenotype maintenance of osteoblast-induced BMSCs under in vitro and in vivo simulated culture conditions. Results The ALP activity and osteocalcin in BMSCs induced by drug-induced osteoblast were significantly higher than those in normal group (P <0.05). The type Ⅰ collagen and osteocalcin in BMSCs induced by drug-induced osteosarcoma were all immunohistochemical positive. Semi-quantitative RT-PCR assay showed that the expression of type I collagen mRNA was significantly higher than that of the normal subculture group. After osteogenic induction, the cells were maintained in a high level for 5 days, and their alkaline phosphatase (ALP) activity, osteocalcin level and type Ⅰ collagen Type; in the co-culture conditions, ALP activity, osteocalcin level of type I collagen expression remained high, and ALP activity, osteocalcin levels at most time points were higher than normal subculture. CONCLUSION: The osteoblast-induced culture shows the characteristics of promoting the transformation of BMSCs into osteoblasts. The expression of ALP, osteocalcin and type I collagen can reach a high level within a short period of time. The osteogenic induced BMSCs in vitro or in vivo were subcultured Under, can maintain the osteogenic phenotype, maintain osteal vitality.