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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (8): 16-22    DOI: 10.13523/j.cb.20160803
研究报告     
BMP9经Smad信号通路调控iSCAP成骨/成牙本质分化
陈冰, 孔令姣, 雷金霞, 沈露, 张彩, 王金华
重庆医科大学附属口腔医院 口腔疾病与生物医学重庆市重点实验室 重庆 401147
BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway
CHEN Bing, KONG Ling-jiao, LEI Jin-xia, SHEN Lu, ZHANG Cai, WANG Jin-hua
Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Stomatological Hospital of Chongqing Medical University, Chongqing 401147, China
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摘要：

目的：研究BMP9是否能够激活 iSCAP细胞中的Smad信号通路，以及Smad信号通路在BMP9诱导iSCAP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iSCAP后Smad1/5/8蛋白的磷酸化水平。随后，利用dnALK1重组腺病毒和BMP9条件培养基作用于iSCAP，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iSCAP中Smad1/5/8的磷酸化水平；dnALK1抑制BMP9条件培养基作用后，可抑制Smad1/5/8的磷酸化，iSCAP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iSCAP成骨/成牙本质过程中存在并起着重要作用。
关键词： 显性负性突变ALK1受体;  成骨/成牙本质分化;  BMP9;  永生化根尖牙乳头干细胞    
Abstract:

Objective:Although BMP9 has a prominent effect on promoting osteogenic/odontogenic differentiation of stem cells from the apical papilla (iSCAP) in the previous experiment, the mechanism is not clear. Smad signaling pathway is one of the most canonical pathway in BMP 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 Smad1/5/8 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 Smad1/5/8 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 Smad1/5/8 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 Smad1/5/8 was activated in BMP9-induced osteogenic differentiation of iSCAP.And inhibition of Smad1/5/8 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. AlK1 is an important receptor of BMP9-Smad signaling pathway, dnALK1 can inhibit BMP9 effectively, however, whether can it inhibit the other signal pathways are remained to be studied. In addition, animal experiments should be performed to verify the inhibition of dnALK1 in BMP9-induced osteogenic/odontogenic differentiation of iSCAP.
Key words: Immortalized dental apical papilla stem cells    Dominant negative mutant receptor ALK1    Osteogenic/odontogenic differentiation    Bone morphogenetic proteins9
收稿日期: 2016-04-26 出版日期: 2016-08-25
ZTFLH:  R78  
基金资助:

重庆市科委自然科学基金资助项目（cstc2013jcyjA10025）
通讯作者: 王金华     E-mail: dentistwangjh@163.com
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引用本文:

陈冰, 孔令姣, 雷金霞, 沈露, 张彩, 王金华. BMP9经Smad信号通路调控iSCAP成骨/成牙本质分化[J]. 中国生物工程杂志, 2016, 36(8): 16-22.

CHEN Bing, KONG Ling-jiao, LEI Jin-xia, SHEN Lu, ZHANG Cai, WANG Jin-hua. BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway. China Biotechnology, 2016, 36(8): 16-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160803    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I8/16

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