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Notch1在BMP9诱导iMEF细胞成骨分化中的作用

, PP. 39-45

Keywords: Notch,骨髓间充质干细胞,永生化小鼠胚胎成纤维细胞,骨形态发生蛋白

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Abstract:

目的研究Notch信号中Notch1受体在骨形态发生蛋白9(bonemorphogeneticproteins9,BMP9)诱导的永生化小鼠胚胎成纤维细胞(immortalizedmouseembryonicfibroblasts,iMEF)成骨分化中的作用。方法利用Notch1受体显性负性突变体(dominantnegativemutant,dnNotch1)处理iMEF细胞,首先分别采用细胞化学染色、活性测定和RT-PCR了解Notch1对早期成骨指标碱性磷酸酶(alkalinephosphatase,ALP)和晚期成骨指标骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)的影响;其次用Westernblot、荧光素酶和RT-PCR检测Notch1对BMP9经典信号通路中Smad1/5/8磷酸化和Smad结合元件(smad-bindingelement,SBE)活性影响;最后用结晶紫染色和流式细胞仪分析其对细胞增殖、周期及凋亡的影响。结果与对照组相比,dnNotch1处理iMEF第3、5、7天后,ALP活性均降低(P<0.05),并呈剂量依赖性,11d时OPN和OCN表达量也下调(P<0.05);BMP9经典通路中Smad1/5/8蛋白量没有明显变化,而p-Smad1/5/8蛋白及SBE活性受到了抑制(P<0.05);结晶紫染色及流式细胞仪分析结果显示dnNotch1能够抑制BMP9诱导的iMEF早期细胞增殖,但对细胞凋亡无影响。结论dnNotch1竞争抑制Notch1信号,使BMP9诱导iMEF的成骨分化能力显著下降,其可能通过抑制BMP信号通路的活化和iMEF早期增殖两方面来实现。

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