本文選題：血栓閉塞性脈管炎 + 血管內(nèi)皮細(xì)胞　； 參考：《新疆醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:1)研究新疆阿克蘇地區(qū)阿瓦提縣維吾爾族人群血栓閉塞性脈管炎(TAO)的流行病學(xué)狀況分析導(dǎo)致疾病的危險(xiǎn)因素;2)篩選維吾爾族血栓閉塞性脈管炎的易感SNP及易感基因,分析可能參與疾病的生物學(xué)過程及可能的通路;3)結(jié)合危險(xiǎn)因素及全基因組關(guān)聯(lián)分析結(jié)果進(jìn)行體外驗(yàn)證,研究可替寧處理下的HUVEC,TLR-4/MyD88介導(dǎo)的免疫炎癥通路與血栓閉塞性脈管炎發(fā)生調(diào)控的機(jī)制。方法:第一部分:對(duì)新疆阿克蘇地區(qū)阿瓦提縣常住維吾爾族人口進(jìn)行抽樣調(diào)查,結(jié)合流行病學(xué)調(diào)查問卷、體格檢查、血液檢查及超聲造影檢查結(jié)果,分析人群的環(huán)境因素個(gè)人習(xí)慣與血清可替寧、白介素-6、17(IL-6,IL-17)、腫瘤壞死因子(TNF-α)濃度進(jìn)行測(cè)定。所獲資料根據(jù)資料的類型計(jì)算單因素危險(xiǎn)因素并計(jì)算患病率,研究該病的影響因素;將結(jié)果納入多因素條件logistic回歸模型進(jìn)行多因素分析,綜合分析環(huán)境危險(xiǎn)因素。第二部分:選擇維吾爾族患者80名為病例組,年齡在20~47歲之間;對(duì)照組選取同樣遺傳背景的維吾爾族正常人群按照1∶1對(duì)照配比。樣本基因型檢測(cè)采用全基因組Affymetrix Genome-Wide Human SNP Array 6.0芯片。通過質(zhì)量控制及分析,確定維吾爾族人群發(fā)病的易感SNP。對(duì)易感SNP所在基因及上下游相關(guān)基因進(jìn)行富集分析,找到富集差異基因的Pathway條目,尋找差異基因可能的功能和細(xì)胞通路。第三部分:體外培養(yǎng)人臍靜脈血管內(nèi)皮細(xì)胞(HUVEC),復(fù)蘇,傳代,進(jìn)行細(xì)胞鑒定,檢測(cè),達(dá)到穩(wěn)態(tài)。進(jìn)行MTT實(shí)驗(yàn),使用可替寧處理細(xì)胞,摸索可替寧的處理濃度。將細(xì)胞分為正常對(duì)照可替寧1ug和100ug/ml處理?xiàng)l件;使用QPCR技術(shù)檢測(cè)可替寧處理HUVEC的TLR-4總蛋白及細(xì)胞表面蛋白表達(dá);使用ELISA方法檢測(cè)可替寧處理下的細(xì)胞培養(yǎng)液中TNF-α和IL-6濃度變化;使用western-blot方法檢測(cè)NF-κB和IκB-α蛋白水平的變化。綜合分析可替寧對(duì)HUVEC TLR-4的表達(dá)及TLR-4/MyD88/NF-κB炎癥信號(hào)通路的活化。結(jié)果:第一部分:1)成功對(duì)新疆阿克蘇地區(qū)阿瓦提縣進(jìn)行了血栓閉塞性脈管炎相關(guān)的流行病學(xué)調(diào)查;阿瓦提縣血栓閉塞性脈管炎的患病率為8.7%,全部為維吾爾族男性;2)收入(P0.05),吸煙量(X2=13.48,P0.05),疾病史(P0.05),對(duì)寒冷的耐受程度(X2=941.30,P0.05),四肢缺血病史(X2=769.67,P0.05),游走性靜脈炎(X2=523.11,P0.05),間歇性跛行史(X2=489.89,P0.05),四肢末端疼痛及壞疽(X2=331.48,P0.05),足背動(dòng)脈搏動(dòng)(P0.05),血清可替寧濃度(Z=16.17,P0.05),血清IL-6濃度(Z=15.60,P0.05),血清IL-17濃度(Z=12.76,P0.05),血清TNF-α濃度(Z=11.57,P0.05)與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義,是疾病的影響因素;3)經(jīng)過篩選后的多因素logistic回歸分析后發(fā)現(xiàn)血清可替寧水平(X2=14.45,P0.05,OR=20.25,95%CI:3.96-145.38)與疾病的關(guān)聯(lián)性很明顯,是疾病的危險(xiǎn)因素。第二部分:1)經(jīng)過嚴(yán)格的質(zhì)量控制后,本節(jié)共研究包含70例維吾爾族患者,64例正常維吾爾族對(duì)照的484730個(gè)常染色體SNP的TAO全基因組關(guān)聯(lián)分析數(shù)據(jù)庫。該數(shù)據(jù)庫病例-對(duì)照樣本匹配良好。發(fā)現(xiàn)26個(gè)SNP為TAO的易感位點(diǎn),同時(shí)篩選出這26個(gè)易感SNPs所在的基因及附近的基因共84個(gè)基因;2)TAO易感SNP主要富集于:細(xì)胞粘附,生物粘附,細(xì)胞與細(xì)胞間粘附,細(xì)胞真菌起源的分子應(yīng)答,調(diào)節(jié)細(xì)胞組件大小,細(xì)胞內(nèi)的信號(hào)級(jí)聯(lián),腫瘤壞死因子生成的正向調(diào)控,模式識(shí)別受體信號(hào)通路,激活先天性免疫,先天性免疫激活反應(yīng)等生物學(xué)過程。第三部分:1)可替寧對(duì)HUVEC細(xì)胞增殖沒有顯著的影響,而1000 ug/ml對(duì)細(xì)胞的抑制影響可能由于藥物濃度過大(P0.05);2)可替寧處理對(duì)HUVEC遷移無顯著影響(P0.05);3)可替寧處理下可顯著抑制TLR-4 mRNA表達(dá)(P0.05);4)可替寧處理下對(duì)TLR-4蛋白表達(dá)沒有顯著的影響(P0.05);5)可替寧處理下,HUVEC細(xì)胞TLR-4上膜的比例在3h和24h顯著增加�？商鎸幙赡芡ㄟ^增加TLR-4上膜比例,進(jìn)一步激活TLR-4介導(dǎo)的炎癥相關(guān)信號(hào)通路(P0.05);6)經(jīng)過可替寧處理可顯著減少IκB-α表達(dá),進(jìn)而使得NF-κB表達(dá),從而激活NF-κB信號(hào)通路(P0.05);7)可替寧可顯著增加胞外TNF-α和IL-6的分泌(P0.05)。結(jié)論:第一部分:1)不同民族和性別之間的TAO患病率存在差異,維吾爾族TAO的患病率高于漢族,男性高于女性;2)TAO患病率與血清可替寧濃度,血清IL-6濃度,血清IL-17濃度,血清TNF-α濃度關(guān)系密切。收入,吸煙量,疾病史,對(duì)寒冷的耐受程度,四肢缺血病史,游走性靜脈炎,間歇性跛行史,四肢末端疼痛及壞疽,足背動(dòng)脈搏動(dòng),與TAO的聯(lián)系精密。TAO與可替寧代謝,內(nèi)皮細(xì)胞損傷,炎癥因子釋放,雌激素水平有關(guān),是自身免疫性炎癥疾病的組成成分。第二部分:1)本研究在維吾爾族人群中進(jìn)行了一次全基因關(guān)聯(lián)分析,報(bào)道了26個(gè)血栓閉塞性脈管炎的易感SNP,討論了血栓閉塞性脈管炎與免疫性炎癥的關(guān)系,強(qiáng)調(diào)了粘附因子和炎癥因子在發(fā)病中的重要作用,為進(jìn)一步結(jié)石發(fā)病機(jī)制提供了理論基礎(chǔ),但是需要后期的重復(fù)驗(yàn)證;2)用生物信息學(xué)分析工具對(duì)血栓閉塞性脈管炎的易感SNP進(jìn)行功能富集分析發(fā)現(xiàn)三條與疾病相關(guān)的重要信息(一條通路,粘附因子,IL-17炎癥因子)。為該病的病因?qū)W研究提供了重要線索。第三部分:1)可替寧增加HUVEC膜表面TLR-4 RNA表達(dá);2)可替寧活化HUVEC炎癥相關(guān)信號(hào)通路;3)可替寧激活HUVEC的TLR-4/MyD88/NF-κB炎癥信號(hào)通路。
[Abstract]:Objective: 1) to study the epidemiological situation of thrombovasculitis obliterans (TAO) in Uygur population in Awati County, Akesu, Xinjiang, to analyze the risk factors of the disease; 2) to screen the susceptibility and susceptibility genes of Uygur thrombotic obliterans, and to analyze the possible biological processes and possible pathways involved in the disease; 3) combine the risk of the disease.) In vitro validation of factors and total genome association analysis to study the mechanism of HUVEC, TLR-4/MyD88 mediated immuno inflammatory pathways and thrombotic vasculitis induced by cotinine. Methods: Part 1: a sample survey on the population of the permanent vouuer ethnic group in Awati, Akesu, Xinjiang, combined with epidemiological studies Questionnaire, physical examination, blood examination and ultrasound examination results were used to determine the concentration of serum cotinine, interleukin -6,17 (IL-6, IL-17), and tumor necrosis factor (TNF- alpha). The data obtained by the data were calculated and the risk factors were calculated and the influencing factors were calculated. The results were included in multi factor conditional logistic regression model for multi factor analysis and comprehensive analysis of environmental risk factors. The second part: 80 cases of Uygur patients were selected as case group, the age was between the age of 20~47, and the control group selected the normal Uygur population of the same genetic background according to the ratio of 1 to 1. Genomic Affymetrix Genome-Wide Human SNP Array 6 chip. Through quality control and analysis, determine the susceptibility SNP. of the Uygur population to enrich and analyze the genes of the susceptible SNP and the related genes in the upper and lower reaches, find the Pathway entries to enrich the differential genes, find the possible functions and cell pathways of the differential genes. The third part: In vitro culture of human umbilical vein endothelial cells (HUVEC), resuscitation, generation, cell identification, detection, and steady state. MTT experiments were conducted, cells were treated with cotinine to handle the concentration of cotinine. The cells were divided into normal control cotinine 1ug and 100ug/ml, and QPCR technique was used to detect the TLR-4 total egg of cotinine to treat HUVEC. The expression of the surface protein of Rhizoma Bletillae cells; the changes in the concentration of TNF- alpha and IL-6 in the cell culture medium under the treatment of cotinine using the ELISA method; the changes in the level of NF- kappa B and I kappa B- alpha by Western-blot method. The expression of HUVEC TLR-4 and the activation of TLR-4/MyD88/NF- kappa B inflammatory signaling pathway were analyzed comprehensively. Results: the first part 1) the epidemiological investigation of thrombosis obliterans in Awati County, Akesu, Xinjiang, was carried out. The prevalence rate of thrombosis obliterans in Awati county was 8.7%, all were Uygur men; 2) income (P0.05), smoking volume (X2=13.48, P0.05), disease history (P0.05), cold tolerance (X2=941.30, P0.05), limbs X2=769.67 (P0.05), X2=523.11 (P0.05), intermittent claudication (X2=489.89, P0.05), extremities end pain and gangrene (X2=331.48, P0.05), dorsal artery pulsation of the foot (P0.05), serum cotinine concentration (Z=16.17, P0.05), serum IL-6 concentration, serum concentrations of alpha concentration, serum alpha concentration 57, P0.05) and the control group had statistical significance, it was the influence factor of the disease; 3) after the screening of multiple factor Logistic regression analysis, the serum cotinine level (X2=14.45, P0.05, OR=20.25,95%CI:3.96-145.38) was associated with the disease obviously, it was the risk factor of the disease. The second part: 1) after strict quality control, this The joint study consisted of 70 Uygur patients and 64 normal Uygur controls of 484730 autosomal SNP TAO whole genome database. The database case - control samples were well matched. 26 SNP were found to be the susceptible loci of TAO. At the same time, these 26 susceptible SNPs genes and a total of 84 genes in the nearby genes were screened. 2) TAO susceptibility SNP is mainly enriched in cell adhesion, bioadhesion, cell adhesion to cell and cell, molecular response of cell fungal origin, modulating cell assembly size, intracellular signal cascade, positive regulation of tumor necrosis factor generation, pattern recognition receptor signaling pathway, activation of congenital immunity, congenital immune activation reaction and so on. Process. Third part: 1) cotinine had no significant effect on the proliferation of HUVEC cells, and the inhibitory effect of 1000 ug/ml on the cell might be due to the excessive concentration of drug (P0.05); 2) cotinine treatment had no significant effect on HUVEC migration (P0.05); 3) cotinine treatment could significantly inhibit TLR-4 mRNA expression (P0.05); 4) cotinine treatment of the TLR-4 protein table There was no significant impact (P0.05); 5) the proportion of TLR-4 on the HUVEC cells increased significantly in the cotinine treatment. Cotinine might further activate the TLR-4 mediated inflammation related signaling pathway (P0.05) by increasing the proportion of the upper membrane of the TLR-4; 6) the expression of I kappa B- alpha could be significantly reduced through the cotinine, and thus the NF- kappa B was expressed. Activation of NF- kappa B signaling pathway (P0.05); 7) cotinine significantly increased extracellular TNF- alpha and IL-6 secretion (P0.05). Conclusion: the first part: 1) there is a difference in the prevalence rate of TAO between different nationalities and sexes. The prevalence rate of Uygur TAO is higher than that of the Han, male and female; 2) the TAO prevalence rate and serum cotinine concentration, serum IL-6 concentration, serum IL-17. Concentration, serum TNF- alpha concentration is closely related. Income, smoking, disease history, tolerance to cold, history of limb ischemia, walking phlebitis, intermittent claudication, extremities pain and gangrene, dorsal artery pulsation in the extremities, and the association of TAO with.TAO and cotinine, endothelial cell injury, release of inflammatory factors, estrogen levels. It is a component of autoimmune inflammatory diseases. Second part: 1) a total gene association analysis was carried out in the Uygur population. The susceptibility SNP of 26 thrombovasculitis obliterans was reported. The relationship between thrombus occlusive vasculitis and immune inflammation was discussed, and the importance of adhesion and inflammatory factors in the disease was emphasized. It provides a theoretical basis for further pathogenesis of stones, but requires repeated verification in the later period; 2) the functional enrichment analysis of the susceptible SNP of thrombotic thrombovasculitis by bioinformatics analysis tool found three important information related to the disease (a path, adhesion factor, IL-17 inflammatory factor). The study provides important clues. Third parts: 1) cotinine increases the expression of TLR-4 RNA on the surface of HUVEC membrane; 2) cotinine activates HUVEC inflammation related signaling pathway; 3) cotinine activates the TLR-4/MyD88/NF- kappa B inflammatory signaling pathway of HUVEC.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R543
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本文編號(hào)：2117243