Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨

发布时间：2018-09-03 18:34

【摘要】：目的:探讨Notch信号对骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导间充质干细胞成骨分化的影响以及作用机制。方法:(1)DAPT或Ad-dominant-negative mutants of Notch1(Addn Notch1)和BMP4-CM处理小鼠胚胎成纤维细胞,检测早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP);(2)茜素红S染色实验检测晚期成骨钙盐沉积情况;(3)半定量反转录聚合酶链反应(RT-PCR)检测成骨分化相关基因ALP,Runx2,Col1a1的表达;(4)免疫细胞化学检测p-Smad1/5/8的表达;(5)结晶紫染色和流式细胞术检测细胞的增殖及周期改变。结果:(1)DAPT抑制BMP4诱导的早期成骨分化,且呈浓度依赖性;(2)Delta-like 1(DLL1)促进BMP4诱导的成骨分化,DAPT和dn Notch1抑制BMP4诱导的成骨分化;(3)DLL1促进BMP4诱导的成骨相关基因ALP,Runx2,Col1a1的表达,DAPT抑制这些基因的表达;(4)DLL1促进BMP4诱导的细胞核内p-Smad1/5/8的表达,而DAPT抑制其表达;(5)DLL1促进BMP4诱导的细胞增殖,而DAPT抑制BMP4诱导的细胞增殖。结论:Notch信号通过BMP/Smads信号通路促进BMP4诱导的MSCs成骨分化,在此过程中也有促细胞增殖的作用。
[Abstract]:Aim: to investigate the effect of Notch signal on osteogenic differentiation of mesenchymal stem cells induced by bone morphogenetic protein 4 (BMP4) and its mechanism. Methods: (1) Mouse embryonic fibroblasts were treated with DAPT or Ad-dominant-negative mutants of Notch1 (Addn Notch1) and BMP4-CM. Detection of early osteogenesis index (alkaline phosphatase,ALP); (2) alizarin red S staining for late osteogenic calcium deposition; (3) semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of osteoblast differentiation related gene ALP,Runx2,Col1a1; (4) immunocytochemistry (5) Crystal violet staining and flow cytometry were used to detect cell proliferation and cell cycle change. Results: (1) DAPT inhibited early osteogenic differentiation induced by BMP4. (2) Delta-like 1 (DLL1) promoted BMP4 induced osteogenic differentiation and dn Notch1 inhibited BMP4 induced osteogenic differentiation; (3) DLL1 enhanced BMP4 induced osteoblast related gene ALP,Runx2,Col1a1 expression; (4) DLL1 enhanced BMP4 induced nuclear p-Smad1/5/8 expression. DAPT inhibited its expression, (5) DLL1 promoted BMP4 induced cell proliferation, while DAPT inhibited BMP4 induced cell proliferation. Conclusion the BMP/Smads signaling pathway promotes the osteogenic differentiation of MSCs induced by BMP4, and it also promotes the proliferation of MSCs cells.
【作者单位】：重庆医科大学检验医学院临床检验诊断学教育部重点实验室;
【基金】：重庆市自然科学基金(CSTC2012jj A10004;KJ130305)资助项目
【分类号】：R329.2

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