家蝇幼虫血淋巴MAC-1对LPS诱导小鼠腹腔巨噬细胞分泌炎性因子的影响  

Effect of MAC-1 in the hemolymph of housefly larvae on the secretion of inflammatory factors by peritoneal macrophages induced with LPS in mice

作　　者：王芳胜 庞芸 迟茜文 张霞 朱家洪 魏洪[2] WANG Fang-sheng;PANG Yun;CHI Qian-wen;ZHANG Xia;ZHU Jia-hong;WEI Hong(Department of Pathogenic Biology of Guizhou Medical University,Key Laboratory of Pathogen Biology of Guizhou Province,Guiyang 550025,China;Laboratory of Infection and Immunology of Guizhou Medical Univer-sity)

机构地区：[1]贵州医科大学微生物学教研室,贵州省高校病原生物学特色重点实验室,贵州贵阳550025 [2]贵州医科大学感染与免疫学实验室

出　　处：《中国病原生物学杂志》2018年第12期1375-1379,1408,共6页Journal of Pathogen Biology

基　　金：贵州省教育厅博士基金项目S2012-11(103015);贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2012)45号];教育部2016年高校大学生创新创业训练计划项目(No.201610660310).

摘　　要：目的探讨家蝇幼虫血淋巴多肽MAC-1对脂多糖(LPS)诱导小鼠腹腔巨噬细胞分泌肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)水平的影响及其基于NF-κB转录因子可能的作用机制。方法用5%淀粉经小鼠腹腔注射诱导巨噬细胞产生,采用差速贴壁法分离纯化。96孔板中细胞随机分成6组:5mg/L LPS分别与50、200、500、1 000μg/ml浓度MAC-1共同作用组,阴性对照组(10%MEM),阳性对照组(5mg/L LPS);12孔板细胞随机分6组:MEM阴性对照组,50μg/ml MAC-1组,5mg/L LPS组,50μg/ml MAC-1+5mg/L LPS组,5mg/L LPS+10μmol/L PDTC组,5mg/L LPS+10μmol/L PDTC+50μg/ml MAC-1。两种分组均给药培养12h,收集细胞上清液并提取细胞mRNA,采用酶联免疫吸附试验(ELISA)和逆转录实时荧光定量PCR(RT-qPCR)法检测细胞炎性因子及其基因表达和Toll样受体(TLR4)、NF-κBp50基因相对表达量。结果阴性对照组和LPS组巨噬细胞IL-1β和IL-6分别为220.9±20.3pg/ml和269.06±13.54pg/ml以及212.21±13.54pg/ml和269.43±21.12pg/ml,差异有统计学意义(F=11.02,15.60,P<0.01);TNF-α为185.00±7.38和204.77±34.30pg/ml,差异无统计学意义(F=0.64,P>0.05)。50μg/ml组与其他MAC-1浓度组相比IL-1β、IL-6、TNF-α均显著降低(F=10.46,68.13,16.45,P<0.05);IL-1β、IL-6、TNF-α、TLR4、NF-κB基因相对表达量LPS组较其他组显著增高(F=13.39,10.45,21.25,30.81,17.53,P<0.05或P<0.01),LPS组与50μg/ml MAC-1+LPS组比较差异有统计学意义(F=14.62,15.11,18.78,128.46,12.81,P<0.01),PDTC+LPS组与PDTC+LPS+MAC-1组比较差异均无统计学意义(F=0.25,2.40,0.27,1.55,1.22,P>0.05)。结论 MAC-1能抑制LPS诱导小鼠腹腔巨噬细胞分泌相关炎性因子,其机制可能与NF-κB转录因子有关,尚待进一步研究。Objective To investigate the effect that MAC-1from Musca domestica larvae has of secretion of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)induced with lipopolysaccharide(LPS)in peritoneal macrophages and the mechanism of action of NF-κB transcription factors. Methods Macrophage production was induced by intraperitoneal injection of 5%starch,and macrophages were separated and purified using differential adherence.Cells in 96-well plates were randomly divided into 6groups:groups treated with 5mg/L LPS and 50,200,500,or 1,000μg/ml of MAC-1,a negative control group(10% MEM),and a positive control group(5mg/L LPS).Cells in12-well plates were randomly divided into 6groups:a MEM negative control group,a 50μg/ml MAC-1group,a 5mg/L LPS group,a 50μg/ml MAC-1+5mg/L LPS group,a 5mg/L LPS+10μmol/L PDTC group,and a 5mg/L LPS+10μmol/L PDTC+50μg/ml MAC-1.Cells in both groups were cultured with the respective agent for 12 h,the cell supernatant was collected,and mRNA was extracted.The expression of inflammatory factors,gene expression,and therelative expression of Toll-like receptor(TLR4)and NF-κBp50 genes were detected using an enzyme-linked immunosorbent assay(ELISA)and reverse transcription real-time fluorescent quantitative PCR(RT-qPCR). Results Levels of IL-1βand IL-6 expressed in macrophages in the negative control groups were 220.9±20.3 pg/ml and 269.06±13.54 pg/ml,which differed significantly from levels of 212.21±13.54 pg/ml and 269.43±21.12 pg/ml in the LPS groups(F=11.02,15.60,P<0.01).The level of TNF-αwas 185.00±7.38 pg/ml in the negative control group and 204.77±34.30 pg/ml in the LPS group,so the levels did not differ significantly(F=0.64,P >0.05).Compared to other groups treated with different MAC-1 concentrations,levels of IL-1β,IL-6,and TNF-αdecreased significantly in the 50μg/ml group(184.06±8.83 pg/ml,78.88±3.17 pg/ml,88.04±2.77 pg/ml,F=10.46,68.13,16.45,P<0.05).The relative levels of expression of the IL-1β,IL-6,TNF-α,TLR4,and NF-κB genes in the LPS g

关 键 词：家蝇幼虫血淋巴MAC-1 脂多糖 巨噬细胞 细胞因子 信号分子 

分 类 号：R384.2[医药卫生—医学寄生虫学]