CGRP影响人牙髓干细胞增殖分化的研究

摘要

Objective To study the effects of calcitonin gene-related peptide (CGRP) on proliferation and dif-ferentiation of human dental pulp stem cells, and to probe the role of CGRP signaling pathway. Methods Firstly, the healthy pulp was collected, and the pulp stem cells were cultured and selected in vitro. The tetrazolium-based colorimet-ric assay (MTT test) was used to detect the proliferation of dental pulp stem cells with different concentrations of CGRP, and the appropriate concentration was obtained. The effect of CGRP on the formation of mineralized nodules in dental pulp stem cells was observed by alizarin red staining. The expression levels of the Col-1 and Runx2 mRNA and protein acted by CGRP in dental pulp stem cells were detected by real-time RT-PCR and Western blot. At the last, we used re-al-time RT-PCR to observe the expression levels of Col-1 and Runx2 mRNA effect by mitogen-activated protein kinase (MAPK) signal pathway inhibitors. Results The dental pulp stem cells grew stably by cultivating and filtering the cells. MTT method showed that the proliferation of dental pulp stem cells was enhanced by CGRP, and the effect was most significant when CGRP concentration was 10-7 mol/L. The mineralize nodules were observed by alizarin red stain-ing when the dental pulp stem cells enchanced by CGRP for 28 d. Real-time RT-PCR and Western blot showed that the expression levels of mRNA and protein of Col-1 and Runx2 were enhanced when CGRP acted on the dental pulp stem cells. In MAPK signal pathways, real-time RT-PCR showed that the mRNA expression level of Runx2 enhanced by CGRP was regulated by ERK kinase pathway, while the mRNA expression level of Col-1 enhanced by CGRP was regu-lated by ERK kinase pathway and P38 signal pathway. Conclusion CGRP can enhance the differentiation and prolifera-tion of human dental pulp stem cells, which is regulated by the MAPK signal pathway.%目的 研究降钙素基因相关肽(CGRP)对人牙髓干细胞增殖分化的调控作用,并初步探讨CGRP的调控信号通路.方法 首先获取健康的牙髓,体外培养并筛选牙髓干细胞;采用MTT法检测不同浓度的CGRP对牙髓干细胞的增殖作用,获取适宜的作用浓度;利用茜素红染色法观察CGRP作用下牙髓干细胞形成矿化结节的效果;在实时定量RT-PCR法及免疫印迹法检测CGRP作用下牙髓干细胞中Ⅰ型胶原(Col-1)、转录因子Runx2的mRNA和蛋白表达水平;最后利用实时定量RT-PCR法检测MAPK信号通路抑制剂作用下CGRP对牙髓干细胞中Ⅰ型胶原、Runx2 mRNA表达水平的影响.结果 经过筛选培养获得生长稳定的牙髓干细胞;MTT法检测可知CGRP可促进牙髓干细胞的增殖,在CGRP的浓度达到10-7 mol/L时效果最显著;在CGRP作用牙髓干细胞28 d后,经茜素红染色可见矿化结节;经实时定量RT-PCR法和免疫印迹法检测可知CGRP作用下牙髓干细胞中Col-1、Runx2的mRNA和蛋白表达水平得到显著提高;MAPK信号通路中ERK激酶通路能够调控CGRP作用下牙髓干细胞中Runx2表达的水平,ERK激酶和p38通路能够调控CGRP作用下牙髓干细胞中Col-1表达的水平.结论 CGRP能够促进牙髓干细胞的增殖分化,这一作用受到MAPK信号通路的调控.