骨髓间充质干细胞成骨分化过程中碱性磷酸酶基因表达含量的变化

Changes of alkaline phosphatase gene expression level in osteogenic differentiation of bone marrow mesenchymal stem cells

目的:提取鼠骨髓间充质干细胞(BMSCs)进行成骨诱导并对BMSCs成骨分化过程中碱性磷酸酶(ALP)的表达量进行定量监测,揭示BMSCs在成骨分化过程中活性的变化。方法:采用全骨髓贴壁法提取鼠BMSCs,培育、扩增至第3代时,以地塞米松、β-甘油磷酸钠、维生素C为主要成分配制诱导培养基进行成骨诱导3周。在成骨诱导过程中,通过实时荧光定量(qRT-PCR)探针法对目标细胞的ALP表达量进行实时监测。结果:成骨诱导BMSCs,经显微镜观察、ALP染色、矿化结节染色等检测方法证实诱导成功。qRT-PCR实时检测的ALP表达量在1周后快速上升并维持1周的高值,2周后快速下降至初始值。结论:采用全骨髓贴壁法提取、培养的BMSCs,利用经典的成骨诱导方法可分化为成骨细胞。成骨分化过程中,目标细胞的成骨活性1周后开始快速增强,并可在高水平维持1周,2周后活性开始下降。

Objective To investigate the changes of bone marrow mesenchymal stem cells（BMSCs） activity during osteogenic differentiation by performing osteogenic induction for the extracted rat BMSCs and quantitatively monitoring the alkaline phosphatase（ALP） expression level during osteogenic differentiation of BMSCs. Methods BMSCs extracted with full-adherent method were cultivated until cells spread to the third generation. The prepared induction medium with the main components of dexamethasone, beta glycerophosphate, vitamin C was used for 3 weeks of osteogenic induction. During osteogenic induction,the ALP expression level of target cells was monitored in real-time with quantitative real-time polymerase chain reaction（qRTPCR） probe method. Results The osteogenic induction of BMSCs was confirmed by microscopy, ALP staining, staining of mineralized nodules and so on. The ALP expression level detected with qRT-PCR in real-time increased rapidly after 1 week and maintained high level for 1 week, but decreased rapidly to initial value after 2 weeks. Conclusion BMSCs extracted and cultured with full-adherent method were differentiated into osteoblasts by classical osteogenic induction. During osteogenic differentiation,the osteogenic activity of target cells increases rapidly after 1 week, and maintains high level for 1 week, but declines after 2 weeks.