可替宁与TLR-4交互作用对血栓闭塞性脉管炎影响的基因组学研究

发布时间：2018-07-12 13:02

  本文选题：血栓闭塞性脉管炎 + 血管内皮细胞　； 参考：《新疆医科大学》2016年博士论文

【摘要】：目的:1)研究新疆阿克苏地区阿瓦提县维吾尔族人群血栓闭塞性脉管炎(TAO)的流行病学状况分析导致疾病的危险因素;2)筛选维吾尔族血栓闭塞性脉管炎的易感SNP及易感基因,分析可能参与疾病的生物学过程及可能的通路;3)结合危险因素及全基因组关联分析结果进行体外验证,研究可替宁处理下的HUVEC,TLR-4/MyD88介导的免疫炎症通路与血栓闭塞性脉管炎发生调控的机制。方法:第一部分:对新疆阿克苏地区阿瓦提县常住维吾尔族人口进行抽样调查,结合流行病学调查问卷、体格检查、血液检查及超声造影检查结果,分析人群的环境因素个人习惯与血清可替宁、白介素-6、17(IL-6,IL-17)、肿瘤坏死因子(TNF-α)浓度进行测定。所获资料根据资料的类型计算单因素危险因素并计算患病率,研究该病的影响因素;将结果纳入多因素条件logistic回归模型进行多因素分析,综合分析环境危险因素。第二部分:选择维吾尔族患者80名为病例组,年龄在20~47岁之间;对照组选取同样遗传背景的维吾尔族正常人群按照1∶1对照配比。样本基因型检测采用全基因组Affymetrix Genome-Wide Human SNP Array 6.0芯片。通过质量控制及分析,确定维吾尔族人群发病的易感SNP。对易感SNP所在基因及上下游相关基因进行富集分析,找到富集差异基因的Pathway条目,寻找差异基因可能的功能和细胞通路。第三部分:体外培养人脐静脉血管内皮细胞(HUVEC),复苏,传代,进行细胞鉴定,检测,达到稳态。进行MTT实验,使用可替宁处理细胞,摸索可替宁的处理浓度。将细胞分为正常对照可替宁1ug和100ug/ml处理条件;使用QPCR技术检测可替宁处理HUVEC的TLR-4总蛋白及细胞表面蛋白表达;使用ELISA方法检测可替宁处理下的细胞培养液中TNF-α和IL-6浓度变化;使用western-blot方法检测NF-κB和IκB-α蛋白水平的变化。综合分析可替宁对HUVEC TLR-4的表达及TLR-4/MyD88/NF-κB炎症信号通路的活化。结果:第一部分:1)成功对新疆阿克苏地区阿瓦提县进行了血栓闭塞性脉管炎相关的流行病学调查;阿瓦提县血栓闭塞性脉管炎的患病率为8.7%,全部为维吾尔族男性;2)收入(P0.05),吸烟量(X2=13.48,P0.05),疾病史(P0.05),对寒冷的耐受程度(X2=941.30,P0.05),四肢缺血病史(X2=769.67,P0.05),游走性静脉炎(X2=523.11,P0.05),间歇性跛行史(X2=489.89,P0.05),四肢末端疼痛及坏疽(X2=331.48,P0.05),足背动脉搏动(P0.05),血清可替宁浓度(Z=16.17,P0.05),血清IL-6浓度(Z=15.60,P0.05),血清IL-17浓度(Z=12.76,P0.05),血清TNF-α浓度(Z=11.57,P0.05)与对照组差异有统计学意义,是疾病的影响因素;3)经过筛选后的多因素logistic回归分析后发现血清可替宁水平(X2=14.45,P0.05,OR=20.25,95%CI:3.96-145.38)与疾病的关联性很明显,是疾病的危险因素。第二部分:1)经过严格的质量控制后,本节共研究包含70例维吾尔族患者,64例正常维吾尔族对照的484730个常染色体SNP的TAO全基因组关联分析数据库。该数据库病例-对照样本匹配良好。发现26个SNP为TAO的易感位点,同时筛选出这26个易感SNPs所在的基因及附近的基因共84个基因;2)TAO易感SNP主要富集于:细胞粘附,生物粘附,细胞与细胞间粘附,细胞真菌起源的分子应答,调节细胞组件大小,细胞内的信号级联,肿瘤坏死因子生成的正向调控,模式识别受体信号通路,激活先天性免疫,先天性免疫激活反应等生物学过程。第三部分:1)可替宁对HUVEC细胞增殖没有显著的影响,而1000 ug/ml对细胞的抑制影响可能由于药物浓度过大(P0.05);2)可替宁处理对HUVEC迁移无显著影响(P0.05);3)可替宁处理下可显著抑制TLR-4 mRNA表达(P0.05);4)可替宁处理下对TLR-4蛋白表达没有显著的影响(P0.05);5)可替宁处理下,HUVEC细胞TLR-4上膜的比例在3h和24h显著增加。可替宁可能通过增加TLR-4上膜比例,进一步激活TLR-4介导的炎症相关信号通路(P0.05);6)经过可替宁处理可显著减少IκB-α表达,进而使得NF-κB表达,从而激活NF-κB信号通路(P0.05);7)可替宁可显著增加胞外TNF-α和IL-6的分泌(P0.05)。结论:第一部分:1)不同民族和性别之间的TAO患病率存在差异,维吾尔族TAO的患病率高于汉族,男性高于女性;2)TAO患病率与血清可替宁浓度,血清IL-6浓度,血清IL-17浓度,血清TNF-α浓度关系密切。收入,吸烟量,疾病史,对寒冷的耐受程度,四肢缺血病史,游走性静脉炎,间歇性跛行史,四肢末端疼痛及坏疽,足背动脉搏动,与TAO的联系精密。TAO与可替宁代谢,内皮细胞损伤,炎症因子释放,雌激素水平有关,是自身免疫性炎症疾病的组成成分。第二部分:1)本研究在维吾尔族人群中进行了一次全基因关联分析,报道了26个血栓闭塞性脉管炎的易感SNP,讨论了血栓闭塞性脉管炎与免疫性炎症的关系,强调了粘附因子和炎症因子在发病中的重要作用,为进一步结石发病机制提供了理论基础,但是需要后期的重复验证;2)用生物信息学分析工具对血栓闭塞性脉管炎的易感SNP进行功能富集分析发现三条与疾病相关的重要信息(一条通路,粘附因子,IL-17炎症因子)。为该病的病因学研究提供了重要线索。第三部分:1)可替宁增加HUVEC膜表面TLR-4 RNA表达;2)可替宁活化HUVEC炎症相关信号通路;3)可替宁激活HUVEC的TLR-4/MyD88/NF-κB炎症信号通路。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543
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