【摘要】 目的探讨卡巴胆碱对脂多糖(LPS)刺激后人外周血单核细胞释放炎症介质的影响及其受体机制。方法取正常献血者外周血,密度梯度离心法分离单个核细胞,再利用单核细胞的贴壁性,分离获得单核细胞。用含10%胎牛血清的RPMI 1640培养液重悬,调整细胞浓度为2×105/ml后,加入包被了特异性捕获抗体(TNF-α、IL-6、IL-10)的96孔板,观察不同浓度卡巴胆碱(0.01～100μmol/L)对LPS刺激下单核细胞产生TNF-α、IL-6、IL-10的影响,并取最佳浓度进行受体机制探讨研究。实验分6组:空白对照(C)组仅加RPMI 1640培养液;LPS单独刺激(L)组仅加100ng/ml LPS刺激;烟碱预处理(N)组及卡巴胆碱预处理(K)组先用卡巴胆碱或烟碱(100μmol/L)预处理单核细胞5min,再加入100ng/ml LPS刺激;阿托品+卡巴胆碱预处理(A+K)组和α-银环蛇毒素(α-Bgt)+卡巴胆碱预处理(α-Bgt+K)组先在单核细胞悬液中加入胆碱能M受体拮抗剂阿托品或胆碱能N受体α7亚基特异性拮抗剂α-Bgt,5min后给予卡巴胆碱,5min后再给予LPS刺激。孵育12h(TNF-α、IL-6)或24h(IL-10)后,洗去细胞,加入生物素标记的检测抗体,经链霉亲和素偶联的辣根过氧化物酶催化底物显色后,全自动免疫斑点图像分析仪检测TNF-α、IL-6、IL-10含量。结果LPS单独刺激时,TNF-α、IL-6、IL-10含量较对照组明显升高(P<0.01)。用卡巴胆碱或烟碱预处理细胞后,TNF-α、IL-6含量明显下降,与LPS单独刺激组比较差异显著(P<0.01),但IL-10含量则无明显变化。在0.01～100μmol/L浓度范围内,随着卡巴胆碱浓度增加,其对LPS刺激下单核细胞产生TNF-α、IL-6的抑制作用也更加明显。阿托品预处理细胞后,卡巴胆碱对LPS刺激下单核细胞释放TNF-α、IL-6的抑制作用无明显变化,而α-Bgt预处理细胞后,卡巴胆碱对TNF-α、IL-6释放的抑制作用明显减弱。结论卡巴胆碱同烟碱一样具有强大的抗炎作用,该作用在单核细胞是通过N样胆碱能受体α7亚基实现的。更多还原

【Abstract】 Objective To investigate the effects of carbachol on production of cytokines from human monocytes stimulated by lipopolysaccharide and its receptor mechanism.Methods Pripheral blood monocytes of healthy donors were isolated by density-gradient centrifugation with Ficol/Hypaque,suspended in RPMI 1640 medium with 10% heat inactivated fetal calf serum,and seeded in 6-well plates.After incubation for 2h at 37℃,adherent cells were detached with 0.25% Trypsin-EDTA,resuspended(2×105 cells/ml) in RPMI 1640 medium with 10% heat inactivated fetal calf serum,and seeded in 96-well plate coated by capture antibody(TNF-α,IL-6 and IL-10).Effect of carbachol in different concentrations(0.01-100μmol/L) on production of TNF-α,IL-6 and IL-10 was tested.The optimal concentration was adopted for mechanism study.The cells for test were divided into 6 groups: control,LPS stimulation alone,LPS stimulation + carbachol,LPS stimulation + nicotine,LPS stimulation + carbachol + atropine(an antagon of cholinergic M receptor),LPS stimulation + carbachol + α-Bungarotoxin(an antagon of α7 subunit of cholinergic N receptor) groups.After the manipulation,cells were incubated at 37℃ for an appropriate length of time.Remove cells,and add biotinylated detection antibody,streptavidin-alkaline phosphatase conjugated,and substrate BCIP/NBT.Contents of TNF-α,IL-6 and IL-10 were determined by Automated ELISA-Spot Assay Video Analysis instrument.Results When monocytes were stimulated by LPS alone,concentrations of TNF-α,IL-6 and IL-10 were obviously higher than those of control group(P<0.01).After pretreated by carbachol or nicotine,concentrations of TNF-α and IL-6 obviously decreased compared with those of LPS stimulation alone group(P<0.01),but there was no significant change in IL-10 concentration.Decrease of TNF-α and IL-6 showed concentration-dependent manner when stimulated with carbachol fluctuated within 0.01-100μmol/L.When pretreated by atropine before addition of carbachol,there was no significant changes in concentrations of TNF-α and IL-6.When pretreated with α-Bungarotoxin before carbachol administration,the inhibitive effect of carbachol on production of TNF-α and IL-6 was blocked.Conclusion Carbachol exerts strong anti-inflammatory effect on monocytes by activating α7 subunit of cholinergic N receptor.更多还原