布鲁氏菌外膜蛋白L16、L19对THP-1细胞免疫调控因子表达的影响  

The effect of Brucella outer membrane protein L16,L19 on the expression of immune regulatory factors in THP-1 cells

作　　者：杨恒 谢红珍 易嘉敏 胡飞环 任慧 王文敬 Yang Heng;Xie Hongzhen;Yi Jiamin;Hu Feihuan;Ren Hui;Wang Wenjing(Department of Blood Transfusion,Zhujiang Hospital of Southern Medical University,Guangzhou 510280,China;Department of Transfusion Medicine,School of Laboratory and Biotechnology,Southern Medical University,Guangzhou 510515,China;First Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou 510400,China;Department of Blood Transfusion,Guangdong General Hospital,Guangzhou 510080,China;Blood Transfusion Research Institute of Guangzhou Blood Center,Guangzhou 510095,China)

机构地区：[1]南方医科大学珠江医院输血科,广州510280 [2]南方医科大学检验与生物技术学院输血医学系,广州510515 [3]广州中医药大学第一临床医学院,广州510400 [4]广东省人民医院输血科,广州510080 [5]广州血液中心输血研究所,广州510095

出　　处：《中华地方病学杂志》2023年第5期345-350,共6页Chinese Journal of Endemiology

基　　金：广东省基础与应用基础研究基金(2021A1515011885);南方医科大学珠江医院院长基金(yzjj2020qn16)。

摘　　要：目的初步探究布鲁氏菌外膜蛋白16、19脂化型重组蛋白(L16、L19)对人单核细胞白血病细胞系(THP-1细胞)免疫调控因子表达的影响。方法以佛波酯(PMA)活化的THP-1细胞为体外实验细胞模型,采用成组设计,分别将L16、L19与THP-1细胞共培养(L16刺激组、L19刺激组),并以PMA活化的THP-1细胞为对照组。共培养4 h时,分别采用免疫荧光染色(IFS)法和蛋白免疫印迹(Western blot)法检测L16、L19是否进入细胞。共培养12、24 h时,实时荧光定量PCR法测定干扰素调节因子1(IRF-1)、Ⅱ类主要组织相容性复合物反式激活蛋白(CⅡTA)mRNA表达水平;Western blot法检测T细胞免疫球蛋白黏蛋白-3(Tim-3)和γ干扰素受体1(IFNGR1)蛋白表达水平。结果共培养4 h时,可见L16、L19分别进入L16、L19刺激组THP-1细胞内。共培养12 h时,L16刺激组IRF-1 mRNA表达水平(0.16±0.15)明显低于对照组(1.00±0.00,P<0.05);共培养24 h时,L16刺激组CⅡTA mRNA表达水平(0.17±0.10)明显低于对照组(1.00±0.00,P<0.05);共培养12、24 h时,L19刺激组IRF-1、CⅡTA mRNA表达水平与对照组比较差异均无统计学意义(均P>0.05)。Western blot结果显示,共培养12、24 h时,对照组、L16刺激组、L19刺激组INFGR1、Tim-3蛋白表达水平比较,差异均有统计学意义(F=50.92、6.80、148.73、156.57,均P<0.05)。其中,共培养12 h时,L16、L19刺激组INFGR1蛋白表达水平均明显低于对照组,且L19刺激组高于L16刺激组(均P<0.05);而L19刺激组Tim-3蛋白表达水平高于对照组(P<0.05)。共培养24 h时,L16、L19刺激组INFGR1蛋白表达水平均低于对照组,且L19刺激组高于L16刺激组(均P<0.05);而L16刺激组Tim-3蛋白表达水平高于对照组和L19刺激组(均P<0.05)。结论布鲁氏菌L16可以下调THP-1细胞IRF-1、CⅡTA mRNA表达水平;L16、L19均能够下调IFNGR1、上调Tim-3蛋白表达水平。Objective To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16,19(L16 and L19)on the expression of immune regulatory factors in human monocytic leukemia cell line(THP-1 cells).Methods THP-1 cells activated with phorbol ester(PMA)were used as an in vitro experimental cell model,and a group design was used to co-culture L16,L19 and THP-1 cells(L16 stimulated group,L19 stimulated group),respectively.THP-1 cells activated with PMA were used as the control group.When co-cultured for 4 hours,immunofluorescence staining(IFS)and Western blotting were used to detect whether L16 and L19 entered the cells,respectively;when co-cultured for 12,24 hours,real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1(IRF-1)and trans activator protein of major histocompatibility complex class Ⅱ(CⅡTA);Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3(Tim-3)and γ interferon receptor 1(IFNGR1).Results When co-cultured for 4 hours,L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group,respectively.When co-cultured for 12 hours,the expression level of IRF-1 mRNA in the L16 stimulated group(0.16±0.15)was significantly lower than that in the control group(1.00±0.00,P<0.05).When co-cultured for 24 hours,the expression level of CⅡTA mRNA in the L16 stimulated group(0.17±0.10)was significantly lower than that in the control group(1.00±0.00,P<0.05).When co-cultured for 12 and 24 hours,there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group(P>0.05).Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group,L16 stimulated group,and L19 stimulated group after co-cultured for 12 and 24 hours(F=50.92,6.80,148.73,156.57,P<0.05).Among them,when co-c

关 键 词：布鲁杆菌属 外膜蛋白 免疫调控因子 

分 类 号：R392[医药卫生—免疫学]