摘要
目的初步探讨Notch信号通路对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导多潜能干细胞成骨分化的作用和机制。方法腺病毒表达载体(AdBM P9/AdGFP、AdHey1/AdRFP)和BMP9/GFP条件培养基处理C2C12细胞,细胞化学染色和活性定量检测早期成骨标志物碱性磷酸酶(ALP),定量RT-PCR检测Notch信号通路靶基因Hey1,以了解Hey1在AdBMP9介导的成骨分化作用中的表达和作用;采用细胞密度实验和Notch信号通路抑制剂(DAPT)检测Notch信号通路对成骨的影响;在AdBMP9/AdGFP处理下,检测细胞上Notch信号通路配体、受体表达变化。结果 AdBMP9作用下,1、3、5、7dALP活性持续增加(P<0.05);Hey1一过性升高,其与AdBMP9呈剂量依赖性;Hey1可协助BMP9成骨(P<0.05),不能单独起效。80%和40%细胞密度组ALP量和Hey1表达均高于20%组(P<0.05);DAPT组中ALP活性明显降低(P<0.05);在AdBMP9作用下,Notch信号通路配受体有不同程度的上调,其中受体Notch4和配体Jag-1上调最为明显(分别为11.3倍和5.1倍)。结论 Notch信号通路参与BMP9介导的多潜能干细胞成骨分化,其可能的机制是BMP9通过诱导Notch通路配体、受体表达上调使Hey1升高,后者协同成骨。
Objective To study the role of Notch signaling pathway in bone morphogenetic protein 9 （BMP9） induced osteogenic differentiation of multipotent stem cells and its mechanism. Methods Adenovirus vectors （AdBMP9/AdGFP, AdHey1/AdRFP） and C2C12 cells treated with BMP9/GFP were stained with cell chemistry. Alkaline phosphatase （ALP）, an osteogenesis marker, was detected with a quantitative method. Target gene Hey1 of Notch signaling pathway was detected by quantitative RT-PCR in order to study its role in BMP9-induced osteogenic differentiation of multipotent stem cells. Effect of Notch signaling pathway on osteogenesis was detected by cell-density assay and Notch signaling pathway inhibitor （DAPT）. After treatment with AdBMP9/AdGFP, ligands of Notch receptor and its expression were detected. Results AdBMP9 increased the activity of ALP on days 1, 3, 5 and 7, while transiently increased the Hey1 expression level in a dose-dependent manner. Although Hey1 itself could not increase ALP, it could help BMP9 to enhance ALP （P0.05）. The expression level of ALP and Hey1 was higher in 40% and 80% cell density groups than in 20% cell density group （P0.05）. The ALP activity was lower in DAPT group than in control group （P0.05）. The number of Notch receptors and ligands was greater in BMP9 group than in GFP group, in which the receptor （Notch 4） and the ligand （Jag-1） increased by 11.3 and 5.1 times, respectively. Conclusion Notch signaling pathway can enhance BMP9-induced osteogenic differentiation of multipotent stem cells possibly by up-regulating the expression of its ligands and receptors through BMP9,thus leading to osteogenesis.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第23期2492-2496,共5页
Journal of Third Military Medical University
基金
重庆市自然科学基金(CSTC2008BB5396)~~
