脂磷壁酸诱导人成牙本质样细胞炎症微环境的机制研究  

The mechanism of LTA-induced inflammatory microenvironment in human odontoblast-like cells

作　　者：孟润莎 莫泽欢 徐琼[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2017年第5期257-265,共9页Chinese Journal of Stomatological Research(Electronic Edition)

摘　　要：目的研究脂磷壁酸(LTA)诱导的体外培养人成牙本质样细胞(hOB)炎症反应及其上游信号通路的作用机制。方法从人牙髓组织中分离并培养hOB,实时荧光定量聚合酶链反应(PCR)和蛋白印迹法(Western blot)检测成牙本质细胞标志物,对hOB进行鉴定,结果采用独立样本t检验;采用Western blot和免疫荧光法检测hOB中TLR2的表达,结果采用t检验;以10μg/ml LTA刺激hOB,炎性因子蛋白芯片检测42种炎性因子蛋白的表达,实时荧光定量PCR及酶联免疫吸附法(ELISA)对白细胞介素6(IL-6)和IL-8的表达进行验证,Western blot检测不同时间点LTA刺激下TLR2的表达情况,结果采用单因素方差分析;Western blot检测LTA对核转录因子(NF-κB)和活化的丝裂酶原活化蛋白激酶(MAPK)信号通路关键蛋白IKKα/β、IκBα、p65、ERK、JNK及p38磷酸化程度的影响,结果采用方差分析。结果分离培养获得的hOB中牙本质基质蛋白1(DMP1;t_(mRNA)=6.206,P_(mRNA)=0.0024;t_(蛋白)=11.48,P_(蛋白)=0.001)、牙本质涎磷蛋白(DSPP;t_(mRNA)=4.284,P_(mRNA)=0.0155;t_(蛋白)=34.93,P_(蛋白)<0.0001)和巢蛋白(Nestin;t_(mRNA)=6.397,P_(mRNA)=0.0021;t_(蛋白)=19.04,P_(蛋白)=0.0001)的表达均较人牙髓细胞(hDPC)高,提示已成功获得hOB。炎性因子蛋白芯片结果显示,LTA刺激后14种炎性因子均升高(P<0.05),其中IL-6和IL-8升高最为明显且经实时荧光定量PCR(FIL-6=40.62,PIL-6<0.0001;FIL-8=1768,PIL-8<0.0001)和ELISA(FIL-6=380.9,PIL-6<0.0001;FIL-8=252.5,PIL-8<0.0001)验证。10μg/ml LTA刺激16 h后,hOB中TLR2蛋白表达显著性升高,差异具有统计学意义(F=3.175,P=0.0469)。LTA刺激可使NF-κB和MAPK信号通路关键蛋白IKKα/β(F=28.7,P<0.0001)、IκBα(F=106.3,P<0.0001)、p65(F=44.58,P<0.0001)、ERK(F=45.49,P<0.0001)、JNK(F=12.43,P=0.0007)及p38(F=28.28,P<0.0001)磷酸化程度升高。结论 LTA可能通过TLR2/NF-κB/MAPK信号通路促进hOB释放炎性因子。Objective To investigate the mechanism of lipoteichoic acid （LTA） -induced human odontoblast-like cells （hOBs） and its upstream signaling pathways in vitro. Methods HOBs were cultured and differentiated from human healthy tooth pulp. Real-time quantitative polymerase chain reaction （qRT- PCR） and western blot were used to identify hOBs by detecting odontoblast markers like DSPP, DMP1, NESTIN. The expression of TLR2 in hOBs was detected by western blot and immunofluorescence and analyzed by independent-samples t test. Antibody arrays were used to examine the levels of 42 cytokines related to immunity and inflammation when hOBs exposing to 10 μg/ml LTA. The results of antibody arrays were verified by qRT-PCR and Enzyme-linked immunosorbent assay （ELISA）. After stimulating with LTA for the indicated times, TLR2 expression in hOBs were detected by western blot. To investigate the role of nuclear factor-KB （NF-KB） and mitogen-activated protein kinase （MAPK） signaling pathways in LTA-induced inflammation, western blot was used to analyzed the phosphorylation of IKKct / 13, IKBα, p65, ERK, JNK and p38. The results mentioned above except the detection of phenotypic characterization in hOBs was evaluated by One- Way ANOVA. Results The expression of DMP1 （tmRNA = 6.206, PmRN~. = 0.0024 ; t,,,,,~, = 11.48, P = 0.001 ）, DSPP （t,,,RNA = 4.284, PmRNA = 0.0155 ; t ~ 34.93, Pprotein〈 0.0001 ） and NESTIN （tmj^NA = 6.397, PmRNA = 0.0021 ; tprotein = 19.04, Pp = 0.0001 ） in hOBs obtained and differentiated from dental pulp tissue were significantly higher than human dental pulp cells （hDPCs）, suggesting that hOBs were successfully obtained. Antibody arrays showed that 14 cytokines significantly increase with LTA stimulation in hOBs （P 〈 0.05）, meanwhile, IL-6 and IL-8 were the most obviously among them. The results of qRT-PCR （FIL-6 = 40.62, PIE-6 〈 0.0001 ; F1L-S = 1768, P,.8 〈 0.0001 ） and ELISA （FIL-6=380.9, PtL-6 〈 0.0001 ; FIL-8 = 252.5, PIL-8 〈 0.0001 ） confirm

关 键 词：成牙本质样细胞 脂磷壁酸 炎症微环境 机制 

分 类 号：R781.1[医药卫生—口腔医学]