本文選題：負壓　切入點：結(jié)核　出處：《山西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:通過成功構(gòu)建負壓調(diào)控MTB感染巨噬細胞的體外模型,篩選并檢測信號軸相關(guān)基因及巨噬細胞極化相關(guān)指標,初步探索負壓調(diào)控對結(jié)核性創(chuàng)面免疫相關(guān)信號軸及巨噬細胞功能的影響,為結(jié)核性創(chuàng)面的治療提供新的思路和方法。方法:1、成功構(gòu)建負壓調(diào)控MTB感染巨噬細胞的體外模型。2、利用高通量測序及生物信息學分析初步篩選結(jié)核創(chuàng)面免疫相關(guān)目的基因及負壓調(diào)控后免疫相關(guān)信號軸,并利用聚類分析篩選差異性表達的巨噬細胞極化相關(guān)基因,初步探索負壓調(diào)控對MTB感染巨噬細胞功能的影響。3、選擇PPD試驗陰性、家族中無結(jié)核病史的健康體檢者為對照組;選擇脊柱結(jié)核伴創(chuàng)面患者為感染組,經(jīng)VSD負壓治療30d后再次復(fù)查,并將其納入治療組。分別抽取上述人群的外周血,用Ficoll法分離外周血單個核細胞(PBMCs),提取RNA,利用RT-PCR技術(shù)驗證人體中目的基因A20及其相關(guān)信號軸基因的差異性表達。4、體外培養(yǎng)RAW264.7巨噬細胞,并將其分為3組:對照組、MTB感染組、負壓治療組。至細胞生長對數(shù)期時,分別給予上述不同條件的干預(yù),24h后消化、收集上述細胞,再次利用RT-PCR技術(shù)對目的基因A20及其相關(guān)信號軸基因的差異性表達進行驗證。同時利用流式細胞術(shù)(FCM)檢測不同干預(yù)條件下巨噬細胞表面標志CD80、CD206的表達,以此來研究不同干預(yù)條件對巨噬細胞功能的影響。結(jié)果:1、高通量測序篩選及生物信息學分析發(fā)現(xiàn)基因A20相關(guān)信號通路基因呈顯著差異性表達,且巨噬細胞極化相關(guān)基因IL-1、IL-6、IL-23、TNF-a較對照組呈顯著升高。2、人外周血(PBMCs)及體外細胞實驗RT-PCR發(fā)現(xiàn),A20-NF-k B呈負調(diào)控:2.1在人外周血(PBMCs)中A20的表達量在感染組(4.05±0.17)較對照組(1.85±0.13)明顯偏高(P0.05);而NF-k B在感染組為(0.61±0.17)較對照組(1.13±0.37)卻明顯偏低(P0.05)。經(jīng)VSD負壓治療后,A20的表達量(2.36±0.21)較感染組(4.05±0.17)有所降低(P0.05),相應(yīng)的NF-k B的表達量(0.81±0.21)較前(0.61±0.17)有所升高(P0.05)。2.2在MTB感染RAW264.7細胞模型中A20的表達量感染組(6.92±0.21)低于對照組(9.75±0.33);而NF-k B的表達量感染組(24.13±0.31)卻高于對照組(8.91±0.13),差異有統(tǒng)計學意義(P0.05)。在負壓干預(yù)條件下,A20的表達量(5.57±0.17)比感染組更低(6.92±0.21),相應(yīng)的NF-k B的表達量(33.41±0.17)比感染組(24.13±0.31)卻更高(P0.05)。3、MAl T1基因與A20呈同向變化:在人外周血(PBMCs)中MALT1的表達量在感染組(5.01±0.09)較對照組(3.08±0.25)偏高(P0.05),經(jīng)VSD負壓治療后,其表達量(2.05±0.08)較感染組(5.01±0.09)有所降低(P0.05)。在MTB感染RAW264.7細胞模型中MALT1的表達量感染組(6.53±0.28)低于對照組(8.41±0.37);經(jīng)負壓干預(yù)后,MALT1的表達量(3.69±0.44)比感染組更低(6.53±0.28)(P0.05)。4、用FCM檢測巨噬細胞表面標志發(fā)現(xiàn):MTB感染組CD80呈顯著高表達(P0.05),而經(jīng)負壓干預(yù)后CD80的表達量更高(P0.05);而CD206的表達量差異無統(tǒng)計學意義(P0.05)。結(jié)論:1、利用高通量測序及生物信息學初步預(yù)測了負壓對A20相關(guān)信號軸及巨噬細胞極化功能的影響。2、利用RT-PCR驗證了在人外周血(PBMCs)及體外細胞模型中A20-NF-k B呈負調(diào)控,與生物信息學篩查結(jié)果一致,且在人(PBMCs)及體外細胞模型中負壓調(diào)控均能降低A20、升高NF-k B。3、而MALT1與A20呈同向變化趨勢,說明MALT1并非是A20的唯一上游基因,可能還受其他異常基因的調(diào)控,這有待進一步的研究。4、利用FCM發(fā)現(xiàn)經(jīng)MTB感染后的巨噬細胞,呈現(xiàn)M1狀態(tài);經(jīng)負壓干預(yù)后巨噬細胞更趨于M1極化。
[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 嫻嬪簭絳涢，

本文編號：1620324