釉原蛋白羧基末端肽加速细胞周期促进成釉细胞系ALC细胞增殖  

The C-terminus of the amelogenin peptide promotes the proliferation of ALC ameloblasts through accelerating cell cycle

作　　者：刘敏[1] 王睿捷 宋丹阳 杨随 谭陶[2] 王磊[1] 王衣祥[3] Liu Min;Wang Ruijie;Song Danyang;Yang Sui;Tan Tao;Wang Lei;Wang Yixiang(Department of Prosthodontics,Peking University Hospital of Stomatology,Beijing 100081,China;Department of Stomatology,Peking University Shougang Hospital,Beijing 100144,China;Clinical Laboratory,Peking University Hospital of Stomatology,Beijing 100081,China)

机构地区：[1]北京大学口腔医院修复科,北京市100081 [2]北京大学首钢医院口腔科,北京市100144 [3]北京大学口腔医院中心实验室,北京市100081

出　　处：《中国组织工程研究》2020年第1期99-105,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81470770),项目负责人:王磊;北京市自然科学基金(7172240),项目负责人:王衣祥;北京市自然科学基金(7182181),项目参与人:王衣祥~~

摘　　要：背景:釉原蛋白羧基末端肽(C-terminus of the amelogenin peptide,AMG-CP)作为一种小分子内源性短肽,在物种间的序列高度保守,提示其在牙齿发育过程中参与重要的生理过程。有研究表明AMG-CP对成牙骨质细胞、骨髓间充质干细胞、牙周膜成纤维细胞的增殖分化有调控作用,但是AMG-CP对成釉细胞生物学功能的影响尚未见报道。目的:探究不同质量浓度AMG-CP对成釉细胞系ALC细胞增殖的影响及相关机制。方法:人工合成并通过液相色谱和质谱检测AMG-CP合成情况。使用xCELLigence RTCA实时细胞分析系统观察0,0.5,1,2 mg/L AMG-CP对ALC细胞增殖的影响。流式细胞仪检测0,1,2 mg/L AMG-CP对ALC细胞周期的影响。Real-time PCR检测0,1,2 mg/L AMG-CP对ALC细胞的细胞周期蛋白Cyclin D1、CDK4、MCM2、MCM5 mRNA表达水平的影响。Western blot检测0,1 mg/L AMG-CP对ALC细胞表达细胞增殖相关通路中p-ERK1/2、total-ERK1/2表达水平的影响。利用MAPK-ERK1/2通路抑制实验,在ERK阻断剂U0126作用下阻断ERK1/2磷酸化,检测AMG-CP对ALC细胞增殖能力的影响。结果与结论:①与对照组比较,AMG-CP促进ALC细胞增殖,群体倍增时间降低,并存在浓度梯度依赖性;②与对照组比较,AMG-CP具有加速细胞周期的作用;③与对照组比较,AMG-CP可以上调细胞周期相关基因Cyclin D1、CDK4、MCM2、MCM5的表达;④与对照组比较,AMG-CP可以上调p-ERK1/2表达,激活MAPK-ERK1/2信号通路;⑤U0126抑制MAPK-ERK1/2通路激活后,AMG-CP失去对ALC细胞促增殖作用;⑥以上结果证实AMG-CP可以激活MAPK-ERK1/2通路,加速细胞周期,进而促进ALC细胞增殖,提示AMG-CP具有促进成釉细胞增殖的潜能。BACKGROUND:C-terminus of the amelogenin peptide(AMG-CP)is a small molecular endogenous peptide that is highly conserved among species.It is involved in important physiological processes during tooth development.Some studies have shown that AMG-CP can regulate the proliferation and differentiation of cementoblasts,bone marrow mesenchymal stem cells and periodontal ligament fibroblasts,but the biological function of AMG-CP on ameloblasts has not been elucidated.OBJECTIVE:To investigate the effects of different concentrations of AMG-CP on the proliferation of ALC ameloblasts and its underlying mechanisms.METHODS:AMG-CP was successfully synthesized and determinated by liquid chromatography and mass spectrometry.The effects of AMG-CP at 0,0.5,1,2 mg/L on the proliferation of ALC ameloblasts were observed by xCELLigence RTCA cell analysis system in real time.The effect of AMG-CP at 0,1,2 mg/L on cell cycle of ALC was detected by flow cytometry.Real-time PCR was used to detect the expression of cyclin D1,CDK4,MCM2,MCM5 mRNA in ALC cells treated with AMG-CP at 0,1,2 mg/L.Western blot was carried out to evaluate the effect of AGM-CP at 0,1 mg/L on MAPK-ERK1/2 pathway by detecting the expression of phosphorylated ERK1/2 and total ERK1/2 in ALC cells.Pathway blockade assay was performed by using ERK1/2 blocker U0126 to pretreat ALC cells.Then cell proliferation ability as well as phosphorylated ERK1/2 expression was analyzed by xCELLigence RTCA cell analysis system and western blot.RESULTS AND CONCLUSION:Compared with the control group,AMG-CP promoted the proliferation of ALC cells,and decreased the population doubling time in a dose-depending manner.Flow cytometry detected the acceleration of cell cycle after treatment with AMG-CP.The results of Real-time PCR showed that AMG-CP upregulated cell cycle-related genes(cyclin D1,CDK4,MCM2,MCM5)expression.Western blot results showed that AMG-CP could upregulate the expression of phosphorylated ERK1/2 and activate MAPK-ERK1/2 signaling pathway in ALC cells.After U0126 was used to

关 键 词：釉原蛋白羧基末端肽 釉原蛋白 成釉细胞 细胞增殖 细胞周期 MAPK-ERK信号通路 牙釉质发育 

分 类 号：R459.5[医药卫生—治疗学] R394.2[医药卫生—临床医学]