髓样分化蛋白-2模拟肽抑制巨噬细胞吞噬革兰阴性菌

发布时间：2018-04-08 23:07

本文选题：髓样分化蛋白-模拟肽　切入点：革兰阴性菌　出处：《第三军医大学学报》2016年08期

【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(P0.01),且细胞培养上清TNF-α和IL-6的分泌水平显著降低(P0.01)。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。
[Abstract]:Objective to investigate the effect of myeloid differentiation protein 2 mimic peptide MD-2 mimetic peptidem on phagocytosis of gram-negative bacteria by macrophages and its inhibitory effect on the secretion of inflammatory factors.Methods the gene fragment expressing EGFP was amplified by PCR from p EGFP-C3 plasmid. The prokaryotic expression vector pET-32a (pET-32a) was constructed by gene recombination. After sequencing, the recombinant strain pET-32a (-EGFP-BL21DE3DE3PLys), which was stably expressed in Escherichia coli BL21(DE3)PLys S, was screened out.After the expression of recombinant plasmid was induced by IPTG, the phagocytic ability of RAW264.7 cells to EGFP-BL21 was detected by immunofluorescence and flow cytometry to verify the construction of EGFP-BL21.The phagocytic effect of RAW264.7 cells on EGFP-BL21 pretreated with different concentrations of MDMP1. 0 渭 g / mL and the levels of TNF- 伪 and IL-6 in the supernatant of cell culture were detected by ELISA.Results Recombinant Escherichia coli BL21(DE3)PLys Ss with stable expression of EGFP was obtained. After phagocytosis of recombinant Escherichia coli BL21(DE3)PLys Sby macrophage RAW264.7 and laser confocal detection, obvious green fluorescence was observed in the cells.The results of flow cytometry showed that the phagocytic ability of RAW264.7 cells to EGFP-BL21 was significantly inhibited, and the phagocytic coefficient of 10 100 渭 g / mL in pretreatment group was significantly lower than that of positive control group treated with serum (P 0.01), and the levels of TNF- 伪 and IL-6 in the supernatant of cell culture decreased significantly (P 0.01).Conclusion MDMP pretreatment can significantly inhibit the phagocytosis of RAW264.7 cells against Gram-negative bacteria and decrease the secretion of inflammatory cytokines TNF- 伪 and IL-6 during this process.
【作者单位】：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;第三军医大学西南医院全军临床病理学研究所;
【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)~~
【分类号】：R392

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