牙龈卟啉单胞菌脂多糖通过髓样细胞触发受体-1调控巨噬细胞极化状态的研究  

Porphyromonas gingivalis lipopolysaccharide regulates macrophage polarization via triggering receptors expressed on myeloid cells-1

作　　者：刘琳 杨芸 周婕妤 吴亚菲[1] 赵蕾[1] Liu Lin;Yang Yun;Zhou Jieyu;Wu Yafei;Zhao Lei(Department of Periodontology,West China Hospital of Stomatology,Sichuan University&State Key Laboratory of Oral Diseases&National Clinical Research Center for Oral Diseases,Chengdu 610041;3E Dental,Chengdu 610041,China)

机构地区：[1]四川大学华西口腔医院牙周科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,成都610041 [2]成都武侯三益匠心口腔门诊部,610041

出　　处：《中华口腔医学杂志》2021年第2期175-181,共7页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81991500,81991502);四川省重点研发项目(2018SZ0161)。

摘　　要：目的探究牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)刺激巨噬细胞后,髓样细胞触发受体-1(triggering receptors expressed on myeloid cells-1,TREM-1)表达与M1、M2型极化的关系,进一步探讨巨噬细胞TREM-1在牙周炎发生发展中的作用机制。方法使用豆蔻酰佛波醇乙酯诱导人单核细胞系THP-1分化为巨噬细胞,予以0(空白对照组)和1μg/ml的Pg-LPS(LPS组)刺激,同期加入终质量浓度为0.1μg/ml的TREM-1抑制剂LP17(LPS+LP17组)或对照肽(LPS+对照肽组),培养24 h后用实时荧光定量PCR(real-time quantitative-PCR,RT-qPCR)检测TREM-1和巨噬细胞M1、M2型极化标志物(分别为CD86、CD206)及其极化相关细胞因子[分别为肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-10]mRNA表达变化,蛋白质印迹法检测TREM-1、CD86、CD206蛋白含量,酶联免疫吸附测定法检测巨噬细胞培养上清液中TNF-α、IL-1β及IL-10的表达。结果细胞培养24 h后,LPS组巨噬细胞中TREM-1相对mRNA表达量(1.40±0.14)及蛋白表达量(3.85±0.24)均较空白对照组(分别为1.01±0.18、1.00±0.05)显著升高(P<0.05),同时M1型极化标志物CD86 mRNA及蛋白表达(分别为1.42±0.01、1.55±0.07)均较空白对照组(分别为1.00±0.09、1.00±0.10)显著上调(P<0.01),且M1型极化相关细胞因子TNF-α、IL-1βmRNA及蛋白表达量均显著增加(P<0.05);加入TREM-1阻断剂LP17后,与LPS组相比,TREM-1 mRNA及蛋白表达水平差异均无统计学意义(P>0.05),CD86 mRNA(0.96±0.00)和蛋白(1.36±0.02)表达水平均显著下降(P<0.05),TNF-α、IL-1βmRNA及蛋白表达水平均显著降低(P<0.05)。对于M2型极化标志物CD206及极化相关细胞因子IL-10,细胞培养24 h后,CD206 mRNA(0.56±0.05)及蛋白表达(0.25±0.04)均较空白对照组(分别为1.02±0.25、1.00±0.10)显著下调(P<0.01),IL-10 mRNA较空白对照组表达显著上调(P<0.05),其蛋白表达水平与空白对照组相比差异无统�Objective To investigate the relationship between pattern recognition receptor triggering receptors expressed on myeloid cells-1(TREM-1)and M1/M2 polarization in macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS),so as to explore the mechanism of TREM-1 in periodontitis.Methods Human monocytic cell line THP-1 were induced to differentiate into macrophages by phorbol-12-myristate-13-acetate and stimulated by 0(blank control group)and 1μg/ml Pg-LPS(LPS group),respectively.LP17,the TREM-1 inhibitor(LPS+LP17 group)and its control peptide(LPS+control peptide group)with final concentration of 0.1μg/ml were added at the same time.After 24 hours stimulation,the expression of TREM-1,M1 markers and related cytokines[CD86,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β],M2 markers and related cytokines(CD206,IL-10)mRNA were detected by real-time quantitative-PCR(RT-qPCR),the level of TREM-1,CD86 and CD206 proteins were detected by Western blotting,and TNF-α,IL-1βand IL-10 in the macrophage culture supernatant were detected by enzyme linked immunosorbent assay.Results After 24 h of cell culture,the relative expressions of TREM-1 mRNA(1.40±0.14)and protein(3.85±0.24)in macrophages in the LPS group increased compared with the blank control group(1.01±0.18 and 1.00±0.05,respectively)(P<0.05).Meanwhile,the expression levels of M1 markers CD86 mRNA and protein[LPS group vs blank control group were(1.42±0.01 vs 1.00±0.09)and(1.55±0.07 vs 1.00±0.10),respectively]were up-regulated(P<0.01),and the expressions of mRNA and protein of M1 related cytokines TNF-αand IL-1 increased(P<0.05).After the addition of TREM-1 blocker LP17,the levels of mRNA and protein of TREM-1 showed no significant changes(P>0.05),while the levels of CD86 mRNA(0.96±0.00)and protein(1.36±0.02)decreased(P<0.05),and the levels of TNF-αand IL-1 further decreased(P<0.05).For M2 marker CD206 and related cytokine IL-10,CD206 mRNA(0.56±0.05)and protein(0.25±0.04)were significantly down-regulated(P<0.01)compared with the

关 键 词：牙周炎 紫单胞菌 龈 髓样细胞触发受体‑1 巨噬细胞 极化 

分 类 号：R781.42[医药卫生—口腔医学]