髓样分化蛋白-2模拟肽抑制巨噬细胞吞噬革兰阴性菌  

Inhibitory effect of myeloid differentiation protein-2 mimetic peptide on phagocytosis of macrophage to Gram-negative bacteria

作　　者：王虎[1] 杨俊霞[1] 黄家君[1] 周运江[1] 随何欢 朱江[2] 吴友莉[2] 段光杰[2] 

机构地区：[1]重庆医科大学药学院药理学教研室,重庆市生物化学与分子药理学重点实验室,重庆400016 [2]第三军医大学西南医院全军临床病理学研究所,重庆400038

出　　处：《第三军医大学学报》2016年第8期805-810,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81171848);第三军医大学青年人才创新基金(SWH2011023)~~

摘　　要：目的探讨髓样分化蛋白-2模拟肽(MD-2 mimetic peptide,MDMP)对巨噬细胞吞噬革兰阴性菌的影响及所致炎症因子分泌的抑制效应。方法采用PCR从p EGFP-C3质粒中扩增出表达EGFP的基因片段,通过基因重组构建原核表达载体p ET-32a(+)-EGFP,经测序鉴定后,转化至大肠杆菌BL21(DE3)PLys S中筛选出稳定表达的重组菌p ET-32a(+)-EGFP-BL21(DE3)PLys S(简称为EGFPBL21)。以IPTG诱导重组质粒表达后,通过免疫荧光和流式细胞术检测RAW264.7细胞对EGFP-BL21的吞噬能力以验证EGFP-BL21的构建情况。进一步检测RAW264.7细胞对经不同浓度MDMP(1.0、10、100μg/m L)预处理后的EGFP-BL21的吞噬效应;采用ELISA检测细胞培养上清液中TNF-α和IL-6的分泌水平。结果筛选获得稳定表达EGFP的重组大肠杆菌BL21(DE3)PLys S,重组菌经巨噬细胞RAW264.7吞噬后流式和激光共聚焦检测结果显示细胞内出现明显的绿色荧光。MDMP预处理后,流式检测结果显示RAW264.7细胞对EGFP-BL21的吞噬能力受到明显抑制,预处理组(10、100μg/m L)的吞噬系数(PI)显著低于经血清调理的阳性对照组(P<0.01),且细胞培养上清TNF-α和IL-6的分泌水平显著降低(P<0.01)。结论 MDMP预处理可明显抑制RAW264.7细胞对革兰阴性菌的吞噬作用,并降低该过程中炎症因子TNF-α和IL-6的分泌。Objective To investigate the inhibitory effect of myeloid differentiation protein-2 mimetic peptide（ MDMP） on the phagocytosis of macrophage to Gram-negative bacteria and on TNF-α and IL-6secretion in this process. Methods Enhanced green fluorescent protein（ EGFP） gene fragment was amplified from p EGFP-C3 plasmid by PCR. The prokaryotic expression vector p ET-32a（ ＋）-EGFP was constructed by gene recombination. After sequencing,Escherichia coli BL21（ DE3） PLys S was transformed with the vector to screen recombinant bacteria p ET-32a（ ＋）-EGFP-BL21（ DE3） PLys S（ EGFP-BL21） with stable expression of EGFP. The expression of recombinant plasmid was induced with IPTG,and the phagocytic ability of RAW264. 7 cells to EGFP-BL21 was detected by immunofluorescence assay and flow cytometry. And then,the effect of MDMP（ 1,10 and 100 μg/m L） on the phagocytosis of RAW264. 7 cells to EGFP-BL21 was analyzed by flow cytometry,and the secretion of IL-6 and TNF-α was measured by ELISA. Results Recombinant Escherichia coli EGFP-BL21 with stable expression of EGFP was obtained. After pretreatment with MDMP,the phagocytosis of RAW264. 7 cells to EGFP-BL21 was significantly inhibited, and the phagocytic index（ PI） of the pretreatment group（ 10 and 100 μg/m L） was significantly lower than that of the positive control group（ P〈0. 01）. Furthermore,MDMP（ 10 and 100 μg/m L） significantly decreased the secretion of TNF-α and IL-6 in EGFP-BL21-stimulated RAW264. 7 cells. Conclusion MDMP pretreatment can inhibit the phagocytosis of RAW264. 7 cells to Gram-negative bacteria,and reduce the secretion of inflammatory factors such as TNF-α and IL-6.

关 键 词：髓样分化蛋白-2模拟肽 革兰阴性菌 吞噬 

分 类 号：R378[医药卫生—病原生物学] R392-33[医药卫生—基础医学]