【作者】 魏攀； 陈峰； 华红；

【Author】 Pan Wei;Feng Chen;Hong Hua;Peking University School and Hospital of Stomatology;

【机构】 北京大学口腔医院；

【摘要】 目的:通过液相色谱-串联质谱(LC-MS/MS)法筛选p SS唾液生物标记物,选取其中可能与pSS发病相关的α烯醇化酶(ENO1)进行验证,并初步探讨ENO1与pSS可能的发病机制。方法:在唾液蛋白标记物筛选阶段纳入10例pSS患者和10例非SS受试者。使用乙醇沉淀法提取唾液全蛋白,对提取的样本进行一维SDS-PAGE蛋白分离、质谱前样本处理,进行LC-MS/MS分析。通过生物信息数据库检索,发现两组间差异表达蛋白。验证阶段选择差异表达的ENO1作为研究对象,分别使用ELISA和Western Blot法验证其在pSS和对照组唾液中的表达水平,使用RealTime PCR法验证ENO1 mRNA在唾液腺中表达水平,使用免疫组化研究ENO1蛋白在唾液腺中的表达和分布。ENO1基因功能探索阶段,通过对比突变型ENO1质粒转染的大鼠颌下腺上皮细胞SMG-16(SMG-16-ENO1)与空载质粒转染的SMG-16细胞(SMG-16-Ctr)的LC-MS/MS检测结果,发现两组间差异表达蛋白。经蛋白分类、功能和参与的生物过程分析,初步探索ENO1基因在SMG-16细胞中的作用,探索ENO1与pSS发病的关系。结果在唾液蛋白生物标记物筛选阶段,发现在pSS组和对照组出现表达差异的唾液蛋白共21个,其中包括ENO1在内的17个蛋白在pSS组高表达,另外4个蛋白在pSS组低表达。在验证阶段,ELISA和Western Blot结果发现ENO1在pSS患者唾液中表达量显著高于对照组(p<0.05);RealTime PCR结果表明pSS患者唇腺组织中ENO1基因表达量显著高于对照组(p<0.05);免疫组化结果显示pSS组唇腺内ENO1表达高于对照组,且高表达的ENO1蛋白主要分布于腺泡和导管上皮细胞胞质中,淋巴细胞浸润灶中未发现ENO1表达。细胞实验发现SMG-16-ENO1和SMG-16-Ctr两组细胞间共有204个差异表达蛋白,其中SMG-16-ENO1组ENO1蛋白表达水平为对照组的1.5倍。通过基于进化关系的蛋白质分析发现,差异表达蛋白中Na+K+ATP酶涉及唾液的跨细胞分泌,紧密连接ZO-1、ZO-2蛋白与旁细胞分泌途径相关,STAT3和STAT6与唾液腺上皮细胞凋亡及免疫应答有关。结论 1.本研究通过LC-MS/MS的方法发现了一组pSS唾液蛋白生物标记物,共包括21个唾液蛋白。2.使用ELISA和Western Blot的方法,在新受试者中证实pSS唾液ENO1表达水平显著高于对照组,并首次在pSS患者唾液腺组织中发现ENO1基因高表达。通过免疫组化方法验证pSS患者唾液腺高表达的ENO1蛋白分布于腺泡和导管上皮细胞。根据以上结果推测pSS患者唾液ENO1可能直接来源于唾液腺,并提示ENO1可能与pSS发病机制相关。更多还原

【Abstract】 Objective: To identify new potential protein biomarkers in human whole saliva(WS) that may be used in the early detection of primary Sjogren’s syndrome. To validate the expression of α-enolase in saliva and salivary gland of pSS, and to explore its role in the pathogenesis of pSS. Methods: Proteins in saliva samples were purified by Protein G-Sepharose CL-4B and ethanol respectively. The purified protein samples were separated by SDS-PAGE. Proteomic analysis and candidate protein biomarkers of primary SS were identified by LC-MS/MS. The level of salivary α-enolase(ENO1) was validated by ELISA and Western blot. The gene expression of ENO1 in labial glands of pSS was verified by Real-time quantitative polymerase chain reaction. The expression and distribution of ENO1 in libial salivary glands were observed by use of Immunohistochemical staining. To explore the function of ENO1 gene in salivary epithelial cells, plasmid containing the ENO1 gene and empty plasmid were transfected into SMG-16 cells respectively. LC-MS/MS analysis was performed to detect the differentially expressed proteins. The molecular function and biological processes the proteins involved in were analyzed. Results: Four salivary proteins were found to be down-regulated and 17 proteins, including ENO1, were found to be up-regulated in primary SS patients compared with control subjects. The ELISA and Western blot results showed that ENO1 was highly expressed in the saliva of pSS patients. Real-time PCR results showed that the expression of ENO1 mRNA in libial salivary glands of pSS patients were significantly higher than that of in healthy controls. Immunohistochemical staining of libial salivary glands indicated high expression of ENO1 protein in the acinar and duct cells of pSS patients, and low expression in healthy controls. The ENO1 expression level was 1.5 times higher in the plasmid containing the ENO1 gene transfected SMG-16 cells than the empty plasmid transfected cells.Totally, 204 proteins were found to be expressed differently between the two cell groups, including tight junction ZO-1 and ZO-2 proteins and Na+K+ATPase which are related to the serection function of salivary epithelial cells, and the STAT3/6 proteins, which is related to the apoptosis of salivary epithelial cells and immune response. Conclusion: 1. Our study suggests that ENO1 is a potential biomarker of pSS, which could be used in the detection of pSS. 2. The highly expression of ENO1 were found in saliva and labial gland of pSS.更多还原