中华临床感染病杂志
中華臨床感染病雜誌
중화림상감염병잡지
CHINESE JOURNAL OF CLINICAL INFECTIOUS DISEASES
2015年
4期
301-305
,共5页
周舟%陈小梅%李富强%唐翠兰
週舟%陳小梅%李富彊%唐翠蘭
주주%진소매%리부강%당취란
巨噬细胞%内毒素类%受体,胆碱能%烟碱
巨噬細胞%內毒素類%受體,膽堿能%煙堿
거서세포%내독소류%수체,담감능%연감
Macrophages%Endotoxins%Receptors,cholinergic%Nicotine
目的:探讨大鼠巨噬细胞α7-乙酰胆碱受体(α7-AChR )介导的胆碱能抗炎通路在内毒素所致炎症反应中的作用及机制。方法采用密度梯度离心的方法原代分离培养大鼠巨噬细胞,经流式细胞仪对细胞纯度进行鉴定。采用荧光共聚焦显微镜和蛋白质免疫印迹试验( Western blot )鉴定大鼠巨噬细胞α7-AChR的表达特征。烟碱预处理原代分离培养的大鼠巨噬细胞后给予内毒素脂多糖(LPS)刺激,采用酶联免疫吸附试验(ELISA)测定上清液中肿瘤坏死因子(TNF)-α的浓度变化。 Western blot检测原代培养巨噬细胞中p-NF-κB蛋白表达。采用方差分析比较不同浓度烟碱下TNF-α的水平差异。结果荧光共聚焦显微镜下观察到原代分离培养大鼠巨噬细胞膜上存在α7-AChR。给予烟碱预处理能显著降低LPS刺激后巨噬细胞培养上清中TNF-α浓度,在烟碱浓度为0、1、10、100μmol/L 时, TNF-α的浓度分别为(2123±86)、(1486±80)、(1316±83)和(1090±77) pg/mL,随着烟碱浓度的增加,TNF-α释放量减少(t=16.33、20.18和26.83,P<0.05)。同时,不同浓度的烟碱预处理后,巨噬细胞内的p-NF-κB水平明显下降。结论激活大鼠巨噬细胞α7-AChR可以抑制内毒素诱导的炎症因子TNF-α的释放,其作用机制为通过NF-κB信号通路抑制炎症反应。
目的:探討大鼠巨噬細胞α7-乙酰膽堿受體(α7-AChR )介導的膽堿能抗炎通路在內毒素所緻炎癥反應中的作用及機製。方法採用密度梯度離心的方法原代分離培養大鼠巨噬細胞,經流式細胞儀對細胞純度進行鑒定。採用熒光共聚焦顯微鏡和蛋白質免疫印跡試驗( Western blot )鑒定大鼠巨噬細胞α7-AChR的錶達特徵。煙堿預處理原代分離培養的大鼠巨噬細胞後給予內毒素脂多糖(LPS)刺激,採用酶聯免疫吸附試驗(ELISA)測定上清液中腫瘤壞死因子(TNF)-α的濃度變化。 Western blot檢測原代培養巨噬細胞中p-NF-κB蛋白錶達。採用方差分析比較不同濃度煙堿下TNF-α的水平差異。結果熒光共聚焦顯微鏡下觀察到原代分離培養大鼠巨噬細胞膜上存在α7-AChR。給予煙堿預處理能顯著降低LPS刺激後巨噬細胞培養上清中TNF-α濃度,在煙堿濃度為0、1、10、100μmol/L 時, TNF-α的濃度分彆為(2123±86)、(1486±80)、(1316±83)和(1090±77) pg/mL,隨著煙堿濃度的增加,TNF-α釋放量減少(t=16.33、20.18和26.83,P<0.05)。同時,不同濃度的煙堿預處理後,巨噬細胞內的p-NF-κB水平明顯下降。結論激活大鼠巨噬細胞α7-AChR可以抑製內毒素誘導的炎癥因子TNF-α的釋放,其作用機製為通過NF-κB信號通路抑製炎癥反應。
목적:탐토대서거서세포α7-을선담감수체(α7-AChR )개도적담감능항염통로재내독소소치염증반응중적작용급궤제。방법채용밀도제도리심적방법원대분리배양대서거서세포,경류식세포의대세포순도진행감정。채용형광공취초현미경화단백질면역인적시험( Western blot )감정대서거서세포α7-AChR적표체특정。연감예처리원대분리배양적대서거서세포후급여내독소지다당(LPS)자격,채용매련면역흡부시험(ELISA)측정상청액중종류배사인자(TNF)-α적농도변화。 Western blot검측원대배양거서세포중p-NF-κB단백표체。채용방차분석비교불동농도연감하TNF-α적수평차이。결과형광공취초현미경하관찰도원대분리배양대서거서세포막상존재α7-AChR。급여연감예처리능현저강저LPS자격후거서세포배양상청중TNF-α농도,재연감농도위0、1、10、100μmol/L 시, TNF-α적농도분별위(2123±86)、(1486±80)、(1316±83)화(1090±77) pg/mL,수착연감농도적증가,TNF-α석방량감소(t=16.33、20.18화26.83,P<0.05)。동시,불동농도적연감예처리후,거서세포내적p-NF-κB수평명현하강。결론격활대서거서세포α7-AChR가이억제내독소유도적염증인자TNF-α적석방,기작용궤제위통과NF-κB신호통로억제염증반응。
Objective To investigate the effect of rat macrophage α7-acetylcholine receptor (α7-AChR )-mediated cholinergic anti-inflammatory pathway on endotoxin-induced inflammation reaction . Methods Density gradient centrifugation method was used to isolate rat primary macrophages and flow cytometry was used to identify the cell purity .α7-AChR in macrophages was detected by fluorescence confocal microscopy and Western blot .After 1ipopolysaccharides ( LPS) was added to the culture media of primary culture of macrophages , the concentration of tumor necrosis factor ( TNF)-αin the supernatant was determined by enzyme linked immunosorbent assay (ELISA).The expression of p-NF-κB protein in primary cultured macrophages was detected by Western blot .ANOVA was used to analyze TNF-αlevels after adding different concentrations of nicotine .Results α7-AChR was observed by fluorescence confocal microscope in primary macrophages .Nicotine could significantly reduce the concentration of TNF-αin culture supernatants of macrophages after LPS stimulation .When the concentrations of nicotine were 0, 1, 10, 100μmol/L, the concentrations of TNF-αwere (2 123 ±86), (1 486 ±80), (1 316 ±83) and (1 090 ±77)pg/mL, respectively (t=16.33, 20.18 and 26.83, P<0.05).The level of p-NF-κB in macrophages was also reduced when nicotine added .Conclusion Activation of macrophage α7-AChR can inhibit the endotoxin-induced release of inflammatory factor TNF-α, which may be through NF-κB signal pathway .