过敏毒素C3a在健康人气道上皮间质转化中的作用研究

摘要

目的 观察过敏毒素C3a在正常人气道上皮间质转化(EMT)中的作用及其相关分子机制.方法 培养正常人气道上皮细胞BEAS-2B,分为对照组,重组人(rhC3a)刺激组,rhC3a+C3a受体拮抗剂(C3aRA)的拮抗组,通过显微镜观察细胞形态学变化情况;四甲基偶氮唑蓝(MTT)法检测细胞增殖能力;ELISA检测细胞上清液中转化生长因子-β1(TGF-β1)的蛋白表达情况;RT-PCR检测C3a受体(C3aR)和EMT相关指标mRNA变化情况;Western blot检测C3aR、Smad2/3、p38-MAPK蛋白表达情况.结果 30、50 nmol/L rhC3a刺激组细胞形态由正常鹅卵石样变为梭形,加入1μmol/L C3aRA拮抗组细胞形态较对照组比较无明显改变.50 nmol/L rhC3a刺激组的细胞增殖较对照组降低(P=0.047);30 nmol/L rhC3a刺激组中TGF-β1蛋白水平较对照组升高(P＜0.05),C3aR mRNA及蛋白水平较对照组均升高(P＜0.05),p-Smad2/3、p-p38-MAPK蛋白水平较对照组升高(P＜0.05),加入1 μmol/L C3aRA可以降低以上指标表达(P＜0.05).30 nmol/L rhC3a刺激组中α-平滑肌肌动蛋白、N-钙黏蛋白mRNA表达上调,E钙黏蛋白mRNA表达下降,加入1μmol/L C3aRA可以拮抗这一过程(P＜0.05).结论 过敏毒素C3a通过结合C3aR,诱导正常人气道上皮细胞发生EMT,其机制可能是通过激活Smad2/3、p38-MAPK通路来参与.%Objective To investigate the role of anaphylatoxin C3a on epithelial mesenchymal transition(MTT) in normal human bronchial epithelium cells and its molecular mechanism.Methods Normal human bronchial epithelium cells BEAS-2B were cultured,and divided into control group,rhC3a stimulation group,rhC3a+ C3a receptor(C3aRA)antagonist group,the morphological changes of cells were observed by microscope;cell proliferation was detected by MTT;the expression of TGF-β1 protein level in cell supernatant was evaluated by ELISA;the expression of C3aR mRNA and EMT related indicators mRNA changes were detected by RT-PCR;the expression of C3aR and Smad2/3,p38 MAPK pathway proteins were detected by Western blot.Results Cell morphology in 30,50 nmol/L rhC3a stimulation group was changed from normal cobblestone like to spindle shape,cell morphology in C3aRA antagonist group had no significant change when compared with the control group.The cell proliferation was reduced in 50 nmol/L rhC3a stimulation group(P=0.047);the levels of TGF-β1,p-Smad2/3,p-p38-MAPK protein were increased(P＜0.05),C3aR mRNA and protein levels were also significantly increased (P＜0.05) in 30 nmol/L rhC3a stimulation group when compared with control group,but the addition of 1 μmol/L C3aRA could reduce their expressions(P＜0.05).30 nmol/L rhC3a could induce the up regulation of α-SMA and N-cadherin mRNA,and decrease the expression of E-cadherin mRNA,adding 1 μmol/L C3aRA can antagonize this process (P＜0.05).Conclusion Anaphylatoxin C3a can induce EMT in normal human bronchial epithelium cells by combining C3aR,its mechanism may be involved in activating Smad2/3 and p38-MAPK pathway.