CAJ | 학술논문

目的评价乙酰胆碱受体对胞壁酰二肽(MDP)激活小鼠巨噬细胞Nod样受体2/受体相互作用蛋白2(NLR2/RIP2)通路的调控作用.方法 RAW264.7细胞长至对数生长期时,接种于12孔培养板(细胞密度1×106个/ml,2 ml/孔),108个培养孔.采用随机数字表法,将其分为3组(n=36),正常对照组(C组)常规培养;M组加入MDP,终浓度为10 μg/ml;G组同时加入MDP和α7烟碱型乙酰胆碱受体特异性激动剂GTS-21,终浓度分别为10、50 μg/ml.分别于MDP孵育1、6、24h时取12个培养孔,取细胞悬液,采用实时荧光定量PCR法检测NLR2 mRNA表达,采用Western blot印迹法检测RIP2表达,采用ELISA法检测培养液TNF-α和高迁移率族蛋白-1(HMGB1)的浓度.结果与C组比较,M组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平升高(P＜0.05);与M组比较,G组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平降低(P＜0.05).结论乙酰胆碱受体可抑制MDP激活小鼠巨噬细胞NLR2/RIP2通路转导.
목적 평개을선담감수체대포벽선이태(MDP)격활소서거서세포Nod양수체2/수체상호작용단백2(NLR2/RIP2)통로적조공작용.방법 RAW264.7세포장지대수생장기시,접충우12공배양판(세포밀도1×106개/ml,2 ml/공),108개배양공.채용수궤수자표법,장기분위3조(n=36),정상대조조(C조)상규배양;M조가입MDP,종농도위10 μg/ml;G조동시가입MDP화α7연감형을선담감수체특이성격동제GTS-21,종농도분별위10、50 μg/ml.분별우MDP부육1、6、24h시취12개배양공,취세포현액,채용실시형광정량PCR법검측NLR2 mRNA표체,채용Western blot인적법검측RIP2표체,채용ELISA법검측배양액TNF-α화고천이솔족단백-1(HMGB1)적농도.결과 여C조비교,M조불동시점NLR2 mRNA、RIP2、TNF-α화HMGB1적수평승고(P＜0.05);여M조비교,G조불동시점NLR2 mRNA、RIP2、TNF-α화HMGB1적수평강저(P＜0.05).결론 을선담감수체가억제MDP격활소서거서세포NLR2/RIP2통로전도.
Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.