超短程化疗对脊柱结核患者外周血基因表达谱变化的研究

发布时间：2018-03-17 00:27

  本文选题：超短程化疗　切入点：脊柱结核　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：目的探讨超短程化疗前后脊柱结核患者临床疗效与外周血m RNA基因表达谱差异基因变化的关系;研究Krüppel样因子4(KLF4)调控的TLR-2/p38MAPK/NF-κB信号通路在脊柱结核发病机制中的作用。方法1.收集宁夏医科大学总医院脊柱骨科收治的27例采用超短程化疗及彻底病灶清除术治疗的脊柱结核患者为实验组。所有脊柱结核患者均经病理及手术证实,男13例,女14例,年龄17-72岁,平均38.8±15.6岁。将27例脊柱结核患者分为三组:未治疗组(A组)8例、超短程化疗结束组(B组)9例、随访一年组(C组)10例。采用2SHRZ/2-4HRZ方案且小于6个月的超短程化疗方案,手术方案均采用彻底病灶清除、病椎间植骨融合及器械内固定术式。对照组为5例非结核住院患者,男3例,女2例,年龄25-50岁,平均35.2±12.7岁。2.采集所有患者外周静脉血10 mL,提取总RNA,利用昂飞Affymetrix U133Plus 2.0 Array芯片进行外周血表达谱芯片的检测。通过芯片显著性分析软件(SAM)分析得到各组之间的差异表达基因,对差异基因进行聚类分析,并利用分子功能注释系统(MAS)系统获得pathway通路,然后进行差异基因功能分析,对获得的差异表达基因采用反转录PCR(RT-PCR)法验证。3.收集并对比分析脊柱结核患者的临床资料,观察超短程化疗治疗脊柱结核的临床疗效,同时对不同治疗阶段脊柱结核患者外周血中的肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)、白介素12(IL-12)、白介素8(IL-8)及核转录因子κB(NF-κB)进行酶联免疫吸附(ELISA)法检测并应用Western Blot法检测外周血中的NF-κB蛋白。4.挑选脊柱结核患者组即A组和正常对照组两组的外周血白细胞差异表达的mRNA基因进行研究,对比分析A组和正常对照组两组患者的临床表现、血常规、血沉(ESR)、C-反应蛋白(CRP)及影像学检查等。应用RT-PCR检测A组和正常对照组两组外周血中KLF4、Toll样受体2(TLR-2)、p38丝裂原活化蛋白激酶(mapk)、nf-κb基因在mrna水平的表达;应用elisa法对ifn-γ、tnf-α、白介素1(il-1)进行检测;应用westernblot法检测外周血白细胞中klf4、tlr-2、磷酸化p38(p-p38)mapk、p38mapk、nf-κb蛋白的表达。利用佛波酯(pma)诱导人单核细胞(thp-1)为巨噬细胞,脂多糖(lps)作用于巨噬细胞,在mrna和蛋白水平检测巨噬细胞中tlr-2、p-p38、p38、nf-κb、klf4的表达,同时检测ifn-γ、tnf-α、il-1的表达。结果1.临床结果显示a组结核病灶活动;b、c两组结核病灶治愈且差异无统计学意义。a、b、c三组表达谱芯片sam分析结果显示,a、b组之间显著差异表达基因共942条,上调基因936条、下调基因6条;a、c组之间显著差异表达基因276条,上调基因256条、下调基因20条;b、c组之间无显著差异表达基因。mas分析显示差异基因主要参与炎症反应及宿主的免疫调节机制的过程。rt-pcr验证结果与芯片结果一致。a组较b、c两组tnf-α、ifn-γ、il-12、il-8表达水平明显升高,差异具有统计学意义,这与nf-κb的表达水平相一致。2.脊柱结核组即a组和正常对照组两组的临床结果显示,相对于正常对照组,脊柱结核组esr、crp、中性粒细胞相对值升高,淋巴细胞绝对值、淋巴细胞相对值及血红蛋白值降低,差异具有统计学意义。芯片结果提示脊柱结核患者与正常对照组之间显著差异表达基因共68条,上调基因16条,下调基因52条,其中klf4基因在脊柱结核患者外周血中表达最为显著,升高2.89倍。rt-pcr结果显示脊柱结核患者较正常对照组外周血klf4、tlr-2、p38、nf-κb基因在mrna水平均高表达,ifn-γ、tnf-α、il-1表达亦升高,同时klf4、tlr-2、p-p38、nf-κb蛋白表达水平增加,差异具有统计学意义,但p38蛋白水平却无明显变化,差异无统计学意义。3.在100ng/mllps作用后的巨噬细胞中tlr-2、p-p38、nf-κb、klf4在mrna水平和蛋白表达水平明显增加,ifn-γ、tnf-α及il-1的表达也明显升高,差异具有统计学意义。结论1.超短程化疗治疗脊柱结核的临床疗效与外周血mrna差异基因、炎性细胞因子及核转录因子κB蛋白表达水平一致。2.外周血mRNA表达谱芯片可用于评估在彻底病灶清除术基础上采用超短程化疗治疗的脊柱结核患者的临床疗效。3.外周血Krüppel样因子4可能通过诱导单核细胞向巨噬细胞分化而参与脊柱结核的发病,其可能通过TLR-2/p38MAPK/NF-κB途径参与脊柱结核炎症反应的发生。
[Abstract]:Objective to explore the relationship between gene expression profiles in patients with spinal tuberculosis clinical curative effect and peripheral blood m RNA gene before and after ultra short course chemotherapy; study of Kr ppel like factor 4 (KLF4) TLR-2/p38MAPK/NF- kappa B signal transduction pathway in the pathogenesis of spinal tuberculosis. Methods 1. collect spinal admitted to the Department of orthopedics, Ningxia Medical College Hospital 27 cases with ultra short course chemotherapy and spinal tuberculosis patients with radical debridement as the therapy group. All patients with spinal tuberculosis were confirmed by surgery and pathology, 13 cases were male, 14 were female, age 17-72 years, average 38.8 + 15.6 years. 27 cases of spinal tuberculosis were divided into three groups: the treatment group (A group) 8 cases, ultra short end of chemotherapy group (B group) 9 cases, one year follow-up group (C group) 10 cases. Using ultra short term chemotherapy scheme of 2SHRZ/2-4HRZ and less than 6 months, the operation scheme adopts thorough debridement, interbody fusion and vertebral disease The internal fixation operation. The control group was 5 cases of non tuberculosis patients, male 3 cases, female 2 cases, age 25-50 years, average 35.2 + 12.7.2. collected peripheral venous blood in patients with 10 mL, total RNA was extracted using Affymetrix Affymetrix U133Plus 2 Array chip peripheral blood expression microarray detection through the significance analysis of microarrays software (SAM) analysis of gene expression differences between the groups, clustering analysis of differential gene expression, and the use of molecular function annotation system (MAS) system to obtain the pathway pathway, and then analyze the difference of gene function, gene expression by reverse transcription PCR (RT-PCR) to obtain the difference method.3. collect and analyze the clinical data of patients with spinal tuberculosis, clinical efficacy of ultra short course chemotherapy for the treatment of spinal tuberculosis, and the different stages of treatment of spinal tuberculosis in peripheral blood of patients with tumor necrosis factor alpha (TNF- alpha, interferon gamma (IF) N-纬),鐧戒粙绱，

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