【作者】 林恒； 许莉莉； 刘欢； 孙琴； 陈卓； 袁国华； 陈智；

【Author】 Heng Lin,Lili Xu,Huan Liu,Qin Sun,Zhuo Chen,Guohua Yuan,Zhi Chen~*(State Key Laboratory Breeding Base of Basic Science of Stomatology(Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education(KLOBM),School and Hospital of Stomatology,Wuhan University,Wuhan,China)

【机构】 口腔医学教育部重点实验室(武汉大学)； 武汉大学口腔医院/口腔医学院；

【摘要】 目的:KLF4在细胞分化和增殖中发挥着重要作用。过去的研究发现KLF4在极化期和分泌期的成牙本质细胞中有特异性表达,但是KLF4在成牙本质细胞分化过程中发挥何种作用尚未可知。本研究是为了明确KLF4在人牙髓细胞向成牙本质细胞样细胞分化过程中的作用。研究方法:用矿化诱导液诱导人牙髓细胞向成牙本质细胞样细胞分化,使用real-time RT-PCR和Weste rn blot方法检测此过程中成牙本质细胞分化相关基因(DSPP、DMP1、ALP)的表达、ALP活性,并通过EdU掺入法来检测细胞的增殖能力,同时检测KLF4的mRNA和蛋白质表达水平。构建KLF4过表达质粒转染人牙髓细胞,检测KLF4过表达对成牙本质细胞分化相关基因表达、ALP活性和细胞增殖能力的影响。结果:在人牙髓细胞向成牙本质细胞样细胞的分化过程中,ALP活性明显增强,成牙本质细胞分化的相关指标显著增加,同时细胞的增殖能力下降。KLF4的mRNA和蛋白质表达水平分别在矿化诱导后第5天和第7天显著增加,并持续增加至第14天。免疫荧光结果同样显示KLF4在矿化诱导后第7天表达显著增加。当KLF4过表达时,ALP活性显著增强;成牙本质细胞分化相关基因表达均上调;而细胞增殖能力显著下降。结论:KLF4可促进人牙髓细胞向成牙本质细胞样细胞分化并抑制人牙髓细胞的增殖。更多还原

【Abstract】 Objective:KLF4 plays an important role in cyto-differentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts.However,the role of KLF4 in odontoblast differentiation was still unknown.The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells(hDPCs).Methods:hDPCs were treated with odontoblastic induction medium.Odontoblastic differentiation was determined by detection of alkaline phosphatase(ALPase) activity,expression of mineralization-related genes including ALP,dentin sialophosphoprotein(DSPP) and dentin matrix protein-1 (DMP-1).Also,cell proliferation ability was examined by 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay.Simultaneously,mRNA and protein levels of KLF4 were detected.pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4,and biologic effects of KLF4 on hDPCs were investigated by evaluation of ALPase activity,detection of ALP,DSPP and DMP-1 expression and analysis of cell proliferation ability.Results:ALPase activity and expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile,the proliferation ability decreased in this procedure.mRNA and protein levels of KLF4 increased significantly on day 5 after odontoblastic induction of hDPCs and kept increasing until day 14.hDPCs showed upregulated activity of ALPase and expression of mineralization-related genes,including ALP,DMP-1 and DSP,after KLF4 overexpression. Besides,proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.Conclusion:Our findings suggest that KLF4 is able to promote odontoblastic differentiation of hDPCs and inhibit proliferation of hDPCs.更多还原