NALP3炎性体在牙龈卟啉单胞菌脂多糖刺激RAW264.7细胞中的作用  

Study on the role of NALP3 inflammasome in Porphyromonas gingivalis lipopolysaccharide induced RAW264.7

作　　者：杨芳[1] 陈明月[2] 胡英英[1] 汪昌宁[3] 

机构地区：[1] 武汉大学口腔医学院口腔基础医学省部共建国家重点实验室培育基地和口腔生物医学教育部重点实验室,430079 [2]太和医院口腔科,湖北十堰442000 [3] 武汉大学口腔医学院牙周科,430079

出　　处：《中华口腔医学杂志》2017年第5期289-293,共5页Chinese Journal of Stomatology

基　　金：国家自然科学基金（30973314）

摘　　要：目的 研究NALP3（NACHT-LRR-PYD结构域蛋白3）炎性体在牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）脂多糖刺激小鼠巨噬细胞系RAW264.7细胞中调控炎症因子的作用.方法 对RAW264.7细胞常规培养并平均分为3组,每组4×105个细胞：A组为对照组（细胞未处理）;B组为1 mg/L Pg脂多糖刺激组;C组为胱天蛋白酶1抑制剂（AC-YVAD-CMK）预处理＋1 mg/L Pg脂多糖刺激组.C组RAW264.7细胞采用连续稀释（5、10、25、50、75、100、200μmol/L）的AC-YVAD-CMK预处理2 h,随后Pg脂多糖（1 mg/L）孵育24 h,通过细胞计数试剂盒检测细胞生存率.采用实时荧光定量PCR检测3组RAW264.7细胞6 h后NALP3和白细胞介素1β（interleukin-1β,IL-1β）基因转录水平;蛋白质印迹法和酶联免疫吸附测定法检测3组细胞24 h后上述两种蛋白的表达.结果 不同浓度（0、5、10、25、50、75、100、200μmol/L）AC-YVAD-CMK处理RAW264.7细胞对细胞活力无明显影响;实时荧光定量PCR结果显示,A、B、C组IL-1βmRNA表达分别为1.03±0.08、5.48±0.22和4.31±0.20;NALP3 mRNA分别为0.96±0.05、2.62±0.44、1.73±0.09;蛋白质印迹法结果显示,A、B、C组NALP3蛋白的表达量分别为1.00±0.10、2.34±0.04、1.64±0.04;酶联免疫吸附测定结果显示,A、B、C组IL-1β的分泌值分别为（40.20±0.25）、（61.50±1.81）、（52.40±1.91）ng/L.在Pg脂多糖刺激下,IL-1β基因和蛋白表达显著增加（P〈0.01,P〈0.01）,同时NALP3基因和蛋白表达显著升高（P〈0.01,P〈0.01）;运用AC-YVAD-CMK预处理后IL-1β基因表达和蛋白质合成显著减少（P=0.002,P=0.027）,NALP3基因表达和蛋白质合成显著降低（P〈0.01,P〈0.01）.结论 Pg脂多糖可激活RAW264.7细胞中NALP3炎性体信号通路,阻断该通路可以抑制炎症因子分泌.Objective To illuminate the effect of NALP3 inflammasome on regulating the expression of cytokines of macrophages in periodontitis. Methods RAW264.7 cells were cultured and divided into three groups. The first group stayed normal as control, the second group was stimulated by 1 mg/L Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS）, the third group was pretreated with AC-YVAD-CMK （caspase-1 inhibitor） before stimulated with 1 mg/L Pg LPS. RAW264.7 cells pretreated with various concentrations （0, 5, 10, 25, 50, 75, 100, 200 μmol/L） of AC-YVAD-CMK for 2 h, and stimulated by 1 mg/L Pg LPS for 24 h in the third group. After that, cell survival rate were detected by cell＆amp;nbsp;counting kit-8. Every group cells gene transcription of NALP3 and interleukin-1β（IL-1β） were detected by quantitative real-time PCR （qPCR） after 6 h, protein expression of NALP3 and IL-1β were separately detected by Western blotting and enzyme linked immunosorbent assay （ELISA） after 24 h, respectively. Results It is observed that treatment with 5, 10, 25, 50, 75, 100, 200 μmol/L AC-YVAD-CMK did not significantly affect the viability of RAW264.7 cells. qPCR showed that mRNA expression of IL-1β level （1.03±0.08, 5.48±0.22, 4.31±0.20） and NALP3 level （0.96±0.05,2.62±0.44,1.73±0.09）. Western blotting showed that protein expression of NALP3 level （1.00 ± 0.10, 2.34 ± 0.04, 1.64 ± 0.04）, ELISA showed protein secretion of IL-1βlevel （[40.20±0.25], [61.50±1.81], [52.40±1.91] ng/L）. After stimulated by Pg LPS, mRNA and protein expression of IL-1β（P〈0.01, P〈0.01） and NALP3 （P〈0.01, P〈0.01） significantly increased;but the expression of IL-1β（P=0.002, P=0.027） and NALP3 （P〈0.01, P〈0.01） were decreased when pretreated with AC-YVAD-CMK. Conclusions NALP3 inflammasome signal pathway can be activated by Pg LPS in RAW264.7. Block of the pathway can inhibit Pg LPS-induced secretion of cytokines.

关 键 词：牙周炎 紫单胞菌 龈 脂多糖类 

分 类 号：R781[医药卫生—口腔医学]