牙龈卟啉单胞菌脂多糖刺激巨噬细胞表达髓样细胞触发受体-1的研究  

Expression of triggering receptors expressed by myeloid cells-1 in macrophages stimulated by Porphyromonas gingivalis-lipopolysaccharide

作　　者：杨芸 陈珊珊 许春梅 吴亚菲[1] 赵蕾[1] Tang Yun;Chen Shanshan：;Xu Chunmei;Wu Yafei;Zhao Lei(State Key Laboratory of Oral Diseases ＆ National Clinical Research Center for Oral Diseases ＆ Dept.of Periodontology,West China Hospital of Sto-matology,Sichuan University,Chengdu 610041,China;Dept.of Stomatology,Beijing Anzhen Hospital,Capital Medical University,Beijing 100029,China)

机构地区：[1]口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院牙周科,成都610041 [2]首都医科大学附属北京安贞医院口腔科,北京100029

出　　处：《华西口腔医学杂志》2018年第5期475-481,共7页West China Journal of Stomatology

基　　金：国家自然科学基金(81371150;81771077);教育部留学回国人员科研启动基金(2013-693-11-11)~~

摘　　要：目的探索牙龈卟啉单胞菌脂多糖(Pg-LPS)刺激的巨噬细胞中髓样细胞触发受体-1(TREM-1)、可溶性TREM-1(sTREM-1)及肿瘤坏死因子-α(TNF-α)的表达,探讨TREM-1与牙周炎发病机制的可能关联。方法使用豆蔻酰佛波醇乙酯诱导人单核细胞系细胞THP-1分化为巨噬细胞,分别予以0(空白对照)、0.5、1.0μg·mL^(-1)的Pg-LPS刺激,并根据不同时间分组:孵育4、6、12、24 h组。实时定量聚合酶链反应检测巨噬细胞中TREM-1 mRNA的表达,Western blot分析TREM-1蛋白表达的差异,细胞免疫荧光染色检测TREM-1在巨噬细胞中的表达部位,并通过酶联免疫吸附试验对细胞培养液中的sTREM-1及TNF-α进行检测。结果与空白对照组相比,Pg-LPS刺激巨噬细胞后TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的s TREM-1表达均显著增强(P<0.05);1.0μg·mL^(-1) Pg-LPS组TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的sTREM-1表达量高于0.5μg·mL^(-1)组,且分别从6、4、4 h时间点开始差异具有统计学意义(P<0.05);细胞免疫荧光染色结果显示,Pg-LPS(1.0μg·mL^(-1))刺激巨噬细胞24 h后,可见TREM-1蛋白呈阳性染色,且TREM-1的染色部位主要位于巨噬细胞的细胞膜区域;Pg-LPS刺激巨噬细胞后TNF-α的分泌水平升高(P<0.05),其中1.0μg·mL^(-1) Pg-LPS组表达量高于0.5μg·mL^(-1)组,且从12 h开始差异具有统计学意义(P<0.05);0.5μg·mL^(-1) Pg-LPS刺激巨噬细胞后TREM-1 mRNA、TREM-1蛋白、sTREM-1间表达呈正相关(r=1,P<0.05),但与TNF-α表达不存在相关性;在1.0μg·mL^(-1) Pg-LPS刺激巨噬细胞后检测到了具有统计学意义的sTREM-1与TNF-α表达的正相关性(r=1,P<0.05)。结论巨噬细胞受到Pg-LPS刺激后可出现Pg-LPS浓度依赖性的TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的sTREM-1表达上调,且能观测到三者之间表达的正相关性;同时细胞培养上清液中的TNF-α也出现Pg-LPS浓度依赖性的表达上调,在高浓度Pg-LPS(1.0μg·mL^(-1))刺激�Objective Soluble triggering receptors expressed by myeloid cells-1（sTREM-1） and inflammatory cytokine tumor necrosis factor-α（TNF-α） in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide（Pg-LPS） to investigate the expression of triggering receptors expressed by myeloid cells-1（TREM-1） and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. Methods THP-1 cells（a human monocytic cell line derived from an acute monocytic leukemia patient） were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0（blank control）, 0.5, or 1.0 μg·mL^-1 Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. Results Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated（P〈0.05）. The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL^-1 Pg-LPS group than in the 0.5 μg·mL^-1 group; this expression was statistically significant since the 6, 4, and 4 h time point（P〈0.05）. Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS（1.0 μg·mL^-1） for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages（P〈0.05）. The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TN

关 键 词：髓样细胞触发受体-1 牙周炎 牙龈卟啉单胞菌 巨噬细胞 脂多糖 肿瘤坏死因子-α 

分 类 号：R781.4[医药卫生—口腔医学]