摘要
目的:研究BMP9是否能够激活i SCAP细胞中的Smad信号通路,以及Smad信号通路在BMP9诱导i SCAP细胞成骨/成牙本质向分化过程中的作用。方法:首先,采用Western印迹实验检测Ad-BMP9转染i SCAP后Smad1/5/8蛋白的磷酸化水平。随后,利用dn ALK1重组腺病毒和BMP9条件培养基作用于i SCAP,Western印迹实验检测Smad1/5/8蛋白磷酸化水平;采用碱性磷酸酶(ALP)活性检测和染色方法分析早期成骨/成牙本质指标变化,茜素红染色法检测钙盐沉积程度;RT-PCR成骨/成牙本质相关基因Runx2、OCN、OPN和DMP1表达的影响。结果:BMP9可上调i SCAP中Smad1/5/8的磷酸化水平;dn ALK1抑制BMP9条件培养基作用后,可抑制Smad1/5/8的磷酸化,i SCAP细胞中早期成骨/成牙本质标志物ALP活性和晚期成骨/成牙本质标志钙盐结节减少,重要成骨转录因子Runx2基因表达减少,成骨/成牙本质相关基因OCN、OPN、DMP1的表达也受到了抑制。结论:Smad信号通路在BMP9诱导i SCAP成骨/成牙本质过程中存在并起着重要作用。
Objective ： differentiation of stem cells Although BMP9 has a prominent effect on m the apical papilla （iSCAP） in the previous clear. Smad signaling pathway is one of the most canonical pathway in BMP promoting osteogenic/odontogenic experiment, the mechanism is not signaling pathways. Thus, the aims are to investigate the effects of Smad signaling pathway on BMP9-induced osteogenic/odontogenic differentiation of iSCAP. Methods ： First of all, BMP9 was introduced into the iSCAP by using recombinant adenoviruses method and phosphorylated forms of Smadl/5/8 protein was assayed by Western blot after 36 hours. Subsequently, taking the dominant negative mutant receptor ALK1 （ dnALK1 ） recombinant adenoviruses and BMP9 conditioned medium together into the iSCAP. Then, activity of Smadl/5/8 was detected by Western blot. Chemoluminescence quantitative and staining assay were applied to measure the activity of alkaline phosphatase （ALP）, Alizarin red S staining was used to examine calcium deposition. At last, expression of transcription factor RUNX2 and osteogenic/odontogenic target genes （such as OCN,OPN,DMP1 ） were measured by RT-PCR. Results：It was found that BMP9 stimulated the activation of Smadl/5/8 in iSCAP, and this phenomenon was decreased by dnALK1 in iSCAP. BMP9-induced osteogenic/odontogenic differentiation （early marker ALP, expression of OCN and OPN, late osteogenic/odontogenic marker calcium deposition） were significantly inhibited by dnALK1, which is a competitive inhibitor of BMP9. Furthermore, expression of transcription factor Runx2 and osteogenic/odontogenic target genes were reduced by dnALK1. Conclusion：Taken together, these results reveal that Smadl/5/8 was activated in BMP9-induced osteogenic differentiation of iSCAP. And inhibition of Smadl/5/ 8 activity results in reduction of BMP9-induced osteogenic/odontogenic differentiation of iSCAP, which implies that BMP9 can induce osteogenic/odontogenic differentiation of iSCAP through Smad signaling pathway. A1K1 is an importa
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2016年第8期16-22,共7页
China Biotechnology
基金
重庆市科委自然科学基金资助项目(cstc2013jcyjA10025)
关键词
永生化根尖牙乳头干细胞
BMP9
显性负性突变ALK1受体成骨/成牙本质分化
Immortalized dental apical papilla stem cellsBone morphogenetic proteins9 Dominantnegative mutant receptor ALK1 Osteogenic/odontogenic differentiation
