TNF-α调控HODs样细胞瞬时感受器电位TRVP2通道蛋白表达的初步研究

Preliminary study on TNF-α regulating the expression of TRVP2 channel protein in human odontoblast-like cells

目的分析肿瘤坏死因子α(TNF-α)调控人成牙本质细胞(HODs)样细胞中瞬时感受器电位香草酸受体2(TRPV2)离子通道的表达特征和相关受体,进一步丰富对人牙髓TRPV2离子通道生理功能研究的认识。方法收集2016年9月—2017年1月在天津医科大学口腔医院口腔颌面外科门诊因正畸拔除的健康完整第三磨牙20颗,患者年龄18~25岁。获取牙髓组织,在含10%胎牛血清的培养基中孵育、诱导和传代,选取第4~6代细胞进行后续实验。免疫荧光染色检测诱导HODs样细胞标志性蛋白特异性抗体牙本质涎磷蛋白(DSPP)和nestin表达特征,实时荧光定量聚合酶链反应(RT-qPCR)、蛋白质印迹法(WB)和流式细胞术(FCM)等方法分别检测0、1、10μg/L TNF-α对TRPV2离子通道表达水平的影响。使用肿瘤坏死因子受体(TNFR)1拮抗剂R-7050处理细胞,分析TNFR1在TNF-α调控TRPV2离子通道表达中的作用。结果免疫荧光染色鉴定HODs样细胞DSPP和nestin表达阳性,RT-qPCR和WB证实,与无TNF-α处理的对照组相比,10μg/L TNF-α能够明显上调TRPV2离子通道mRNA和蛋白表达水平(P<0.05),FCM也发现TNF-α能够提高TRPV2蛋白表达水平。研究进一步证实,与未用R-7050处理的阴性对照组相比,使用R-7050后TNF-α诱导的HODs样细胞TRPV2离子通道表达水平明显下调(P<0.05)。结论TNF-α主要通过TNFR1调控HODs样细胞TRPV2离子通道表达水平上升。

Objective To analyze the expression characteristics and related receptors of tumor necrosis factor-alpha(TNF-α)on the regulation of transient receptor potential vanilloid 2(TRPV2)in human odontoblasts(HODs)-like cells,which would further enrich the study on the physiological functions of TRPV2 ion channels in human dental pulp.Methods Twenty intact and healthy third molars extracted for orthodontic purpose were collected from adults aged between 18 and 25 years in the department of maxillofacial surgery,stomatological hospital of Tianjin Medical University from September 2016 to January 2017.The pulp tissues were obtained,cultured and induced and passaged in the minimum essential medium(MEM).The 4-6 passages of cells were utilized in the subsequent experiments.Immunofluorescence staining was used to detect the expression characteristics of HDDs-like protein dentin sialophospho protern(DSPP)and nestin.The influences of 1μg/L and 10μg/L TNF-αon TRPV2 ion channel expression in HODs-like cells were investigated by RT-qPCR,Western blot assay and flow cytometry.Tumor necrosis factor receptor(TNFR)1 antagonist R-7050 was used to treat cells,and analyze its role in the regulation of TRPV2 ion channel by TNF-α.Results Immunofluorescence staining showed positive expressions of DSPP and nestin in HODs-like cells.Results of RT-qPCR and Western blot assay confirmed that 10μg/L of TNF-αsignificantly up-regulated the mRNA and protein expression of TRPV2 ion channel compared with that of control group without TNF-αtreatment(P<0.05).Flow cytometry also showed that TNF-αcould obviously up-regulate the expression level of TRPV2 protein.Further study confirmed that the increasing expression level of TRPV2 ion channels in human HODs-like cells treated with TNF-αwas obviously downregulated after R-7050 treatment compared with that of control group without R-7050 treatment(P<0.05).Conclusion TNF-αup-regulates TRPV2 ion channel expression level by TNFR1 in HODs-like cells.