细胞色素P4501A1对巨噬细胞吞噬功能的调控作用及机制研究  

Effect and mechanism of cytochrome P4501A1 on regulating phagocytosis of macrophage

作　　者：田李星 万凌辉 朱俊宇 梁华平 Tian Lixing;Wan Linghui;Zhu Junyu;Liang Huaping(Department of Military Preventive Medicine,Frontier Medical Service Training Brigade,Army Medical University,Hutubi 831200,Xinjiang Uygur Autonomous Region,China;Department of Basic Medicine,Frontier Medical Service Training Brigade,Army Medical University,Hutubi 831200,Xinjiang Uygur Autonomous Region,China;Department of Wound Infection and Drug,Army Medical Center,Army Medical University,Chongqing 400042,China)

机构地区：[1]陆军军医大学边防卫勤训练大队军事预防医学教研室,新疆维吾尔自治区呼图壁831200 [2]陆军军医大学边防卫勤训练大队基础医学教研室,新疆维吾尔自治区呼图壁831200 [3]陆军军医大学陆军特色医学中心战伤感染与特需药品研究室,重庆400042

出　　处：《中华危重病急救医学》2023年第2期158-163,共6页Chinese Critical Care Medicine

基　　金：陆军军医大学科技创新能力提升专项项目(2021XQN11);重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0234)。

摘　　要：目的探讨细胞色素P4501A1(CYP1A1)对巨噬细胞吞噬大肠杆菌(E.coli)能力的调控作用及机制。方法①体外培养小鼠单核/巨噬细胞白血病细胞株RAW264.7(RAW),用感染复数(MOI)为30的E.coli刺激细胞40 min,并设甘油对照组,观察感染时CYP1A1的表达变化。②体外培养CYP1A1过表达RAW细胞(CYP1A1/RAW)、CYP1A1敲除RAW细胞(CYP1A1 KO/RAW),用MOI为30的E.coli刺激细胞40 min,并以阴性对照RAW细胞(NC/RAW)为对照,观察细胞吞噬功能与CYP1A1表达的关系,以及CYP1A1对吞噬受体〔清道夫受体A(SR-A)〕及其信号通路〔丝裂素活化蛋白激酶(MAPK)通路〕的调控作用。③体外培养NC/RAW、CYP1A1 KO/RAW细胞,用1μmol/L细胞外信号调节激酶(ERK)抑制剂U0126预处理2 h后,再用MOI为30的E.coli刺激细胞40 min,并设磷酸盐缓冲液(PBS)对照组,观察CYP1A1是否通过调节MAPK通路调控巨噬细胞的吞噬功能。④体外培养RAW细胞,用100 nmol/L的CYP1A1羟化酶活性产物12(S)-羟基二十碳四烯酸〔12(S)-HETE〕预处理2 h后,再用MOI为30的E.coli刺激细胞40 min,并设PBS对照组,观察巨噬细胞的吞噬功能是否与CYP1A1羟化酶代谢产物有关。⑤体外培养CYP1A1过表达且羟化酶活性位点突变的RAW细胞(CYP1A1m/RAW),用MOI为30的E.coli刺激细胞40 min,并设CYP1A1/RAW对照组,观察巨噬细胞的吞噬功能是否与CYP1A1羟化酶活性有关。结果①与甘油对照组相比,E.coli刺激后RAW细胞中CYP1A1 mRNA表达显著增加(2^(-ΔΔCt):7.79±0.71比1.00±0.00,P<0.05),提示CYP1A1可能参与调控感染进程。②与NC/RAW细胞相比,E.coli刺激40 min后CYP1A1/RAW细胞吞噬E.coli菌落数明显降低(×10^(3) CFU/mL:4.67±3.06比15.67±5.03,P<0.05),而CYP1A1 KO/RAW细胞吞噬E.coli菌落数则显著增加(×10^(3) CFU/mL:46.00±5.29比15.67±5.03,P<0.05),说明CYP1A1可负性调控巨噬细胞吞噬功能。同时,与NC/RAW细胞相比,CYP1A1/RAW细胞中SR-A mRNA表达明显下调(2^(-ΔΔCt):0.31±0.03比1.00±0.00,P<0.05),且ERK的Objective To explore the effect and mechanism of cytochrome P4501A1(CYP1A1)on regulating phagocytosis of macrophage treated with Escherichia coli(E.coli).Methods①The mouse leukemia cells lines of monocyte macrophage RAW264.7(RAW)were cultured in vitro and treated with 30 multiplicity of infection(MOI)dosages of E.coli for 40 minutes,glycerin control group was set up to observe the change of CYP1A1 during infection.②The RAW cells with CYP1A1 overexpression(CYP1A1/RAW)and knock out(CYP1A1 KO/RAW)were cultured in vitro and treated with 30 MOI E.coli for 40 minutes,while the negative controlled RAW cells(NC/RAW)were established as control to observe the relationship between cell phagocytosis and CYP1A1 expression,and the effect of CYP1A1 on phagocytic receptor[scavenger receptor-A(SR-A)]and its signal pathway[mitogen-activated protein kinase(MAPK)pathway].③NC/RAW and CYP1A1 KO/RAW cells were cultured in vitro and pretreated with 1μmol/L extracellular signal-regulated kinase(ERK)inhibitor(U0126)for 2 hours,and then treated with 30 MOI E.coli for 40 minutes,phosphate buffered solution(PBS)control group was set up to observe whether the effect of CYP1A1 on phagocytosis through controlled the MAPK pathway.④The RAW cells were cultured in vitro and pretreated with 100 nmol/L CYP1A1 hydroxylase active product 12(S)-hydroxyeicosatetraenoic acid[12(S)-HETE]for 2 hours,and then treated with 30 MOI E.coli for 40 minutes,and PBS control group was set up to observe whether the effect of CYP1A1 on phagocytosis was related to CYP1A1 hydroxylating metabolite.⑤The RAW cells with overexpression CYP1A1 hydroxylase-activity mutation(CYP1A1m/RAW)were cultured in vitro and treated with 30 MOI E.coli for 40 minutes,the CYP1A1/RAW cells were set up as control group to observe whether the effect of CYP1A1 on phagocytosis was related to CYP1A1 hydroxylase-activity.Results①Compared with glycerin control group,CYP1A1 mRNA expression was significantly increased by E.coli stimulation(2^(-ΔΔCt):7.79±0.71 vs.1.00±0.00,P<0.05),indic

关 键 词：细胞色素P4501A1 大肠杆菌 巨噬细胞 吞噬 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]