本文選題：超短程化療　切入點：脊柱結核　出處：《天津醫(yī)科大學》2016年博士論文　論文類型：學位論文

【摘要】：目的探討超短程化療前后脊柱結核患者臨床療效與外周血m RNA基因表達譜差異基因變化的關系;研究Krüppel樣因子4(KLF4)調控的TLR-2/p38MAPK/NF-κB信號通路在脊柱結核發(fā)病機制中的作用。方法1.收集寧夏醫(yī)科大學總醫(yī)院脊柱骨科收治的27例采用超短程化療及徹底病灶清除術治療的脊柱結核患者為實驗組。所有脊柱結核患者均經(jīng)病理及手術證實,男13例,女14例,年齡17-72歲,平均38.8±15.6歲。將27例脊柱結核患者分為三組:未治療組(A組)8例、超短程化療結束組(B組)9例、隨訪一年組(C組)10例。采用2SHRZ/2-4HRZ方案且小于6個月的超短程化療方案,手術方案均采用徹底病灶清除、病椎間植骨融合及器械內固定術式。對照組為5例非結核住院患者,男3例,女2例,年齡25-50歲,平均35.2±12.7歲。2.采集所有患者外周靜脈血10 mL,提取總RNA,利用昂飛Affymetrix U133Plus 2.0 Array芯片進行外周血表達譜芯片的檢測。通過芯片顯著性分析軟件(SAM)分析得到各組之間的差異表達基因,對差異基因進行聚類分析,并利用分子功能注釋系統(tǒng)(MAS)系統(tǒng)獲得pathway通路,然后進行差異基因功能分析,對獲得的差異表達基因采用反轉錄PCR(RT-PCR)法驗證。3.收集并對比分析脊柱結核患者的臨床資料,觀察超短程化療治療脊柱結核的臨床療效,同時對不同治療階段脊柱結核患者外周血中的腫瘤壞死因子α(TNF-α)、干擾素γ(IFN-γ)、白介素12(IL-12)、白介素8(IL-8)及核轉錄因子κB(NF-κB)進行酶聯(lián)免疫吸附(ELISA)法檢測并應用Western Blot法檢測外周血中的NF-κB蛋白。4.挑選脊柱結核患者組即A組和正常對照組兩組的外周血白細胞差異表達的mRNA基因進行研究,對比分析A組和正常對照組兩組患者的臨床表現(xiàn)、血常規(guī)、血沉(ESR)、C-反應蛋白(CRP)及影像學檢查等。應用RT-PCR檢測A組和正常對照組兩組外周血中KLF4、Toll樣受體2(TLR-2)、p38絲裂原活化蛋白激酶(mapk)、nf-κb基因在mrna水平的表達;應用elisa法對ifn-γ、tnf-α、白介素1(il-1)進行檢測;應用westernblot法檢測外周血白細胞中klf4、tlr-2、磷酸化p38(p-p38)mapk、p38mapk、nf-κb蛋白的表達。利用佛波酯(pma)誘導人單核細胞(thp-1)為巨噬細胞,脂多糖(lps)作用于巨噬細胞,在mrna和蛋白水平檢測巨噬細胞中tlr-2、p-p38、p38、nf-κb、klf4的表達,同時檢測ifn-γ、tnf-α、il-1的表達。結果1.臨床結果顯示a組結核病灶活動;b、c兩組結核病灶治愈且差異無統(tǒng)計學意義。a、b、c三組表達譜芯片sam分析結果顯示,a、b組之間顯著差異表達基因共942條,上調基因936條、下調基因6條;a、c組之間顯著差異表達基因276條,上調基因256條、下調基因20條;b、c組之間無顯著差異表達基因。mas分析顯示差異基因主要參與炎癥反應及宿主的免疫調節(jié)機制的過程。rt-pcr驗證結果與芯片結果一致。a組較b、c兩組tnf-α、ifn-γ、il-12、il-8表達水平明顯升高,差異具有統(tǒng)計學意義,這與nf-κb的表達水平相一致。2.脊柱結核組即a組和正常對照組兩組的臨床結果顯示,相對于正常對照組,脊柱結核組esr、crp、中性粒細胞相對值升高,淋巴細胞絕對值、淋巴細胞相對值及血紅蛋白值降低,差異具有統(tǒng)計學意義。芯片結果提示脊柱結核患者與正常對照組之間顯著差異表達基因共68條,上調基因16條,下調基因52條,其中klf4基因在脊柱結核患者外周血中表達最為顯著,升高2.89倍。rt-pcr結果顯示脊柱結核患者較正常對照組外周血klf4、tlr-2、p38、nf-κb基因在mrna水平均高表達,ifn-γ、tnf-α、il-1表達亦升高,同時klf4、tlr-2、p-p38、nf-κb蛋白表達水平增加,差異具有統(tǒng)計學意義,但p38蛋白水平卻無明顯變化,差異無統(tǒng)計學意義。3.在100ng/mllps作用后的巨噬細胞中tlr-2、p-p38、nf-κb、klf4在mrna水平和蛋白表達水平明顯增加,ifn-γ、tnf-α及il-1的表達也明顯升高,差異具有統(tǒng)計學意義。結論1.超短程化療治療脊柱結核的臨床療效與外周血mrna差異基因、炎性細胞因子及核轉錄因子κB蛋白表達水平一致。2.外周血mRNA表達譜芯片可用于評估在徹底病灶清除術基礎上采用超短程化療治療的脊柱結核患者的臨床療效。3.外周血Krüppel樣因子4可能通過誘導單核細胞向巨噬細胞分化而參與脊柱結核的發(fā)病,其可能通過TLR-2/p38MAPK/NF-κB途徑參與脊柱結核炎癥反應的發(fā)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-緯),鐧戒粙绱，

本文編號：1622364