负压对结核创面免疫相关信号轴及细胞功能的影响

发布时间：2018-03-16 14:29

  本文选题：负压　切入点：结核　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过成功构建负压调控MTB感染巨噬细胞的体外模型,筛选并检测信号轴相关基因及巨噬细胞极化相关指标,初步探索负压调控对结核性创面免疫相关信号轴及巨噬细胞功能的影响,为结核性创面的治疗提供新的思路和方法。方法:1、成功构建负压调控MTB感染巨噬细胞的体外模型。2、利用高通量测序及生物信息学分析初步筛选结核创面免疫相关目的基因及负压调控后免疫相关信号轴,并利用聚类分析筛选差异性表达的巨噬细胞极化相关基因,初步探索负压调控对MTB感染巨噬细胞功能的影响。3、选择PPD试验阴性、家族中无结核病史的健康体检者为对照组;选择脊柱结核伴创面患者为感染组,经VSD负压治疗30d后再次复查,并将其纳入治疗组。分别抽取上述人群的外周血,用Ficoll法分离外周血单个核细胞(PBMCs),提取RNA,利用RT-PCR技术验证人体中目的基因A20及其相关信号轴基因的差异性表达。4、体外培养RAW264.7巨噬细胞,并将其分为3组:对照组、MTB感染组、负压治疗组。至细胞生长对数期时,分别给予上述不同条件的干预,24h后消化、收集上述细胞,再次利用RT-PCR技术对目的基因A20及其相关信号轴基因的差异性表达进行验证。同时利用流式细胞术(FCM)检测不同干预条件下巨噬细胞表面标志CD80、CD206的表达,以此来研究不同干预条件对巨噬细胞功能的影响。结果:1、高通量测序筛选及生物信息学分析发现基因A20相关信号通路基因呈显著差异性表达,且巨噬细胞极化相关基因IL-1、IL-6、IL-23、TNF-a较对照组呈显著升高。2、人外周血(PBMCs)及体外细胞实验RT-PCR发现,A20-NF-k B呈负调控:2.1在人外周血(PBMCs)中A20的表达量在感染组(4.05±0.17)较对照组(1.85±0.13)明显偏高(P0.05);而NF-k B在感染组为(0.61±0.17)较对照组(1.13±0.37)却明显偏低(P0.05)。经VSD负压治疗后,A20的表达量(2.36±0.21)较感染组(4.05±0.17)有所降低(P0.05),相应的NF-k B的表达量(0.81±0.21)较前(0.61±0.17)有所升高(P0.05)。2.2在MTB感染RAW264.7细胞模型中A20的表达量感染组(6.92±0.21)低于对照组(9.75±0.33);而NF-k B的表达量感染组(24.13±0.31)却高于对照组(8.91±0.13),差异有统计学意义(P0.05)。在负压干预条件下,A20的表达量(5.57±0.17)比感染组更低(6.92±0.21),相应的NF-k B的表达量(33.41±0.17)比感染组(24.13±0.31)却更高(P0.05)。3、MAl T1基因与A20呈同向变化:在人外周血(PBMCs)中MALT1的表达量在感染组(5.01±0.09)较对照组(3.08±0.25)偏高(P0.05),经VSD负压治疗后,其表达量(2.05±0.08)较感染组(5.01±0.09)有所降低(P0.05)。在MTB感染RAW264.7细胞模型中MALT1的表达量感染组(6.53±0.28)低于对照组(8.41±0.37);经负压干预后,MALT1的表达量(3.69±0.44)比感染组更低(6.53±0.28)(P0.05)。4、用FCM检测巨噬细胞表面标志发现:MTB感染组CD80呈显著高表达(P0.05),而经负压干预后CD80的表达量更高(P0.05);而CD206的表达量差异无统计学意义(P0.05)。结论:1、利用高通量测序及生物信息学初步预测了负压对A20相关信号轴及巨噬细胞极化功能的影响。2、利用RT-PCR验证了在人外周血(PBMCs)及体外细胞模型中A20-NF-k B呈负调控,与生物信息学筛查结果一致,且在人(PBMCs)及体外细胞模型中负压调控均能降低A20、升高NF-k B。3、而MALT1与A20呈同向变化趋势,说明MALT1并非是A20的唯一上游基因,可能还受其他异常基因的调控,这有待进一步的研究。4、利用FCM发现经MTB感染后的巨噬细胞,呈现M1状态;经负压干预后巨噬细胞更趋于M1极化。
[Abstract]:Objective: to construct an in vitro model of MTB infection of macrophages in negative pressure control, screening and detection of the index correlated signal axis gene and macrophage polarization, explore the effect of negative pressure on the function of regulation of related signal axis and macrophage of tuberculous wound, provide new ideas and methods for the treatment of tuberculous wounds. Methods: 1, construct in vitro model of.2 negative regulation of MTB infection of macrophages, using high-throughput sequencing and bioinformatics analysis of preliminary screening of tuberculosis wound immune related target gene and negative regulation of immune related signal, and using cluster analysis of macrophage polarization related genes differentially expressed, preliminary exploration of negative pressure effects on the function of macrophage MTB infected.3, select the PPD test was negative, no family history of tuberculosis in healthy control group; spinal tuberculosis with wound patients As the infection group, the VSD 30d negative pressure treatment again after the review, and the treatment group. Peripheral blood samples were extracted from the crowd, the separation of peripheral blood mononuclear cells by Ficoll method (PBMCs), RNA extraction, using RT-PCR technology to verify the differences in the human A20 gene and related genes of signal axis the expression of.4 and RAW264.7 macrophages cultured in vitro and divided into 3 groups: control group, MTB infection group, negative pressure treatment group. Cell growth to logarithmic phase, were given the different conditions of the intervention, after 24h digestion, collecting the cells, again using RT-PCR technology to verify the expression of A20 gene and its related differences the signal of shaft gene. At the same time using flow cytometry (FCM) detection of different interference conditions of macrophage surface markers CD80, CD206 expression, in order to study different intervention conditions on macrophages. Results: 1. High throughput 娴嬪簭绛涢，

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