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

【摘要】：目的探討超短程化療前后脊柱結(jié)核患者臨床療效與外周血m RNA基因表達(dá)譜差異基因變化的關(guān)系;研究Krüppel樣因子4(KLF4)調(diào)控的TLR-2/p38MAPK/NF-κB信號通路在脊柱結(jié)核發(fā)病機(jī)制中的作用。方法1.收集寧夏醫(yī)科大學(xué)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(shù)治療的脊柱結(jié)核患者為實驗組。所有脊柱結(jié)核患者均經(jīng)病理及手術(shù)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(jié)核患者分為三組:未治療組(A組)8例、超短程化療結(jié)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(shù)方案均采用徹底病灶清除、病椎間植骨融合及器械內(nèi)固定術(shù)式。對照組為5例非結(jié)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(jìn)行外周血表達(dá)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(dá)基因,對差異基因進(jìn)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(jìn)行差異基因功能分析,對獲得的差異表達(dá)基因采用反轉(zhuǎn)錄PCR(RT-PCR)法驗證。3.收集并對比分析脊柱結(jié)核患者的臨床資料,觀察超短程化療治療脊柱結(jié)核的臨床療效,同時對不同治療階段脊柱結(jié)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(zhuǎn)錄因子κB(NF-κB)進(jìn)行酶聯(lián)免疫吸附(ELISA)法檢測并應(yīng)用Western Blot法檢測外周血中的NF-κB蛋白。4.挑選脊柱結(jié)核患者組即A組和正常對照組兩組的外周血白細(xì)胞差異表達(dá)的mRNA基因進(jìn)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(yīng)蛋白(CRP)及影像學(xué)檢查等。應(yīng)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(dá);應(yīng)用elisa法對ifn-γ、tnf-α、白介素1(il-1)進(jìn)行檢測;應(yīng)用westernblot法檢測外周血白細(xì)胞中klf4、tlr-2、磷酸化p38(p-p38)mapk、p38mapk、nf-κb蛋白的表達(dá)。利用佛波酯(pma)誘導(dǎo)人單核細(xì)胞(thp-1)為巨噬細(xì)胞,脂多糖(lps)作用于巨噬細(xì)胞,在mrna和蛋白水平檢測巨噬細(xì)胞中tlr-2、p-p38、p38、nf-κb、klf4的表達(dá),同時檢測ifn-γ、tnf-α、il-1的表達(dá)。結(jié)果1.臨床結(jié)果顯示a組結(jié)核病灶活動;b、c兩組結(jié)核病灶治愈且差異無統(tǒng)計學(xué)意義。a、b、c三組表達(dá)譜芯片sam分析結(jié)果顯示,a、b組之間顯著差異表達(dá)基因共942條,上調(diào)基因936條、下調(diào)基因6條;a、c組之間顯著差異表達(dá)基因276條,上調(diào)基因256條、下調(diào)基因20條;b、c組之間無顯著差異表達(dá)基因。mas分析顯示差異基因主要參與炎癥反應(yīng)及宿主的免疫調(diào)節(jié)機(jī)制的過程。rt-pcr驗證結(jié)果與芯片結(jié)果一致。a組較b、c兩組tnf-α、ifn-γ、il-12、il-8表達(dá)水平明顯升高,差異具有統(tǒng)計學(xué)意義,這與nf-κb的表達(dá)水平相一致。2.脊柱結(jié)核組即a組和正常對照組兩組的臨床結(jié)果顯示,相對于正常對照組,脊柱結(jié)核組esr、crp、中性粒細(xì)胞相對值升高,淋巴細(xì)胞絕對值、淋巴細(xì)胞相對值及血紅蛋白值降低,差異具有統(tǒng)計學(xué)意義。芯片結(jié)果提示脊柱結(jié)核患者與正常對照組之間顯著差異表達(dá)基因共68條,上調(diào)基因16條,下調(diào)基因52條,其中klf4基因在脊柱結(jié)核患者外周血中表達(dá)最為顯著,升高2.89倍。rt-pcr結(jié)果顯示脊柱結(jié)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(dá),ifn-γ、tnf-α、il-1表達(dá)亦升高,同時klf4、tlr-2、p-p38、nf-κb蛋白表達(dá)水平增加,差異具有統(tǒng)計學(xué)意義,但p38蛋白水平卻無明顯變化,差異無統(tǒng)計學(xué)意義。3.在100ng/mllps作用后的巨噬細(xì)胞中tlr-2、p-p38、nf-κb、klf4在mrna水平和蛋白表達(dá)水平明顯增加,ifn-γ、tnf-α及il-1的表達(dá)也明顯升高,差異具有統(tǒng)計學(xué)意義。結(jié)論1.超短程化療治療脊柱結(jié)核的臨床療效與外周血mrna差異基因、炎性細(xì)胞因子及核轉(zhuǎn)錄因子κB蛋白表達(dá)水平一致。2.外周血mRNA表達(dá)譜芯片可用于評估在徹底病灶清除術(shù)基礎(chǔ)上采用超短程化療治療的脊柱結(jié)核患者的臨床療效。3.外周血Krüppel樣因子4可能通過誘導(dǎo)單核細(xì)胞向巨噬細(xì)胞分化而參與脊柱結(jié)核的發(fā)病,其可能通過TLR-2/p38MAPK/NF-κB途徑參與脊柱結(jié)核炎癥反應(yīng)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，

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