Toll样受体基因与葡萄膜炎遗传易感性关系的研究

发布时间：2018-05-04 18:25

本文选题：TLRs + TLR2　；参考：《重庆医科大学》2014年博士论文

【摘要】：研究背景：葡萄膜炎是一类常见又重要的致盲性眼病，在我国约有300-500万患者，其中有相当一部分为不可治盲。临床上多发于青壮年，多反复发作、治疗棘手，常伴有全身自身免疫性疾病或者炎症性疾病，被认为是眼科的顽疾，但该类疾病发病机制复杂，尚未得到充分阐明。TLR家族属于能识别病原体引发的大分子的模式识别受体，在固有免疫中起关键性的作用。不同种类的淋巴细胞及其它一些细胞可以表达TLR，这些受体位于细胞表面或细胞壁内。目前的研究发现人类有10种不同类型的TLR（TLR1-TLR10）,这些受体能分别被它们对应的配体识别并激活。研究发现TLR2,4,7,8,9通过不同的通路参与了一些自身免疫性疾病发生发展的过程。也有文献报道了发现TLR基因多态或拷贝数变异和一些自身免疫性疾病的易感性之间存在联系。基于以上研究发现的TLR在多种自身免疫性疾病发生过程中所起的重要作用这一背景，我们进行了以下两个方面的研究：1.探讨Toll样受体的基因多态与中国汉族的葡萄膜炎疾病（包括Beh et病、VKH综合征、急性前葡萄膜炎伴或不伴强直性脊柱炎）是否存在联系。2.探讨Toll样受体的拷贝数变异与中国汉族的葡萄膜炎疾病（包括Beh et病、VKH综合征、急性前葡萄膜炎伴或不伴强直性脊柱炎）是否存在联系。通过这些研究，以期对这些葡萄膜炎的发病基因易感性有更深入的认识，并为防治提供新研究靶点和策略。第一部分Toll样受体（TLR）基因多态与葡萄膜炎遗传易感性研究目的：TLR2，TLR4，TLR8和TLR9被报道和好几个自身免疫性疾病有关系。本实验探讨了这些TLR基因多态和中国汉族人群Beh et病、VKH综合征及急性前葡萄膜炎伴或不伴强直性脊柱炎（AAU+AS±）遗传易感性是否有相关性。方法：基因分型采用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）方法。第一阶段包括眼部Beh et病患者400例、VKH综合征400例、AAU+AS±400例和健康对照600例。第二阶段包括438例眼部Beh et病患者和1000例正常人对照。使用卡方检验比较患者和对照组的等位基因和基因型的频率。利用实时荧光定量PCR从正常人外周血单核细胞（PBMC）中检测mRNA的表达。并利用ELISA检测正常人外周血单核细胞培养上清中TNF-α,IL-6,IL-10,和IL-1β的水平。结果：在第一阶段，仅有rs2289318/TLR2的CC基因型和C等位基因及rs3804099/TLR2的CT基因型频率，在眼部Beh et病患者和对照组之间存在统计学差异（分别为Pc=0.048,Pc=0.008和Pc=0.005）。在第二阶段和两阶段总计阶段确认了这一联系（分别为Pc=0.001，Pc=6.89E-06，，Pc=2.426E-06）。携带rs2289318的CC型及rs3804099的 TT型的正常人，其PBMC经肽聚糖（PGN）刺激后，TLR2的mRNA表达分别高于该单核苷酸多态性位点（SNP）其它的基因型（分别为Pc=0.028，Pc=0.004）。TLR2的这两个SNP的不同基因型在培养上清中TNF-α,IL-6,IL-10,和IL-1β的表达水平方面没有发现有统计学差异。结论：本研究提供了证据证明TLR2基因多态与眼部Beh et病的发病风险显著相关。第二部分Toll样受体基因拷贝数变异与葡萄膜炎遗传易感性研究目的：TLR被报道参与了多个自身免疫性疾病的发生和发展。本研究探讨了在患Beh et病、VKH综合征及急性前葡萄膜炎伴或不伴强直性脊柱炎（AAU+AS±）中国汉族人群中，TLR1-10的基因拷贝数变异是否与遗传易感性有关系。方法：利用实时荧光定量PCR检测TLR的基因拷贝数。实验分成2个阶段，第一阶段包括眼部Beh et病患者400例、VKH综合征400例、AAU+AS±400例和健康对照600例。第二阶段包括眼部Beh et病578例和正常人对照1000例。使用卡方检验比较患者和对照组的拷贝数频率。利用实时荧光定量PCR，检测TLR7不同拷贝数的正常人外周血单核细胞（PBMC）中mRNA的表达。利用ELISA检测TLR7不同拷贝数的正常人外周血单核细胞培养上清中TNF-α, IL-6,和IL-1β的水平。结果：在第一阶段，发现TLR7的基因拷贝数频率，在拷贝数大于1的眼部Beh et病男性患者和男性对照组之间存在统计学差异,拷贝数大于2的眼部Beh et病女性患者和女性对照组之间也存在统计学差异（分别为Pc=0.021, OR=2.452,95%CI=1.440to4.176; Pc=0.048,OR=2.620,95%CI=1.406to4.882）。在第二阶段和两阶段混合后均确认了这一联系（分别为pc=1.14E-06, OR=2.324,95%CI1.680to3.215;Pc=9.12E-05, OR=2.492,95%CI=1.603to3.873）。携带拷贝数大于1的正常男性或大于2的正常女性，其PBMC经TLR7特异性配体R848刺激后，TLR7的mRNA表达分别高于其它拷贝数的正常男性或女性（分别为Pc=0.021，Pc=0.006）。无论男性或女性TLR7的不同拷贝数在培养上清中TNF-α,IL-6,和IL-1β的水平方面没有发现有统计学差异。结论：本研究提供了证据证明TLR7基因拷贝数变异与眼部Beh et病的发病风险显著相关。
[Abstract]:TLR2 , 4 , 7 , 8 and 9 have been implicated in the pathogenesis of autoimmune diseases .

Study on the Genetic Susceptibility of Toll - like Receptor ( TLR ) Gene in the First Part

Objective : TLR2 , TLR8 and TLR9 were reported to be related to several autoimmune diseases . These TLR polymorphisms and Beh et ' s disease , VKH syndrome ( VKH ) syndrome and acute preveitis with or without ankylosing spondylitis ( AAU + AS 卤 ) were investigated .

Methods : Polymerase chain reaction - restriction fragment length polymorphism ( PCR - RFLP ) method was used in the first stage , including 400 patients with Beh et ' s disease , 400 cases of VKH syndrome , AAU + AS 卤 400 cases and healthy control 600 cases . The second stage consisted of 438 cases of ocular Beh et patients and 1000 healthy controls .

Results : In the first stage , only rs2289318 / TLR2 CC genotype and genotype C allele and rs3804099 / TLR2 were significantly different ( Pc = 0.048 , Pc = 0.008 and Pc = 0.005 , respectively ) . This contact ( Pc = 0.001 , Pc = 6.89E - 06 , Pc = 2.426E - 06 , respectively ) was confirmed during the second and two - stage totals . CC type with rs2289318 and rs3804099

After stimulated with peptidoglycan ( PGN ) , the mRNA expression of TLR2 was higher than that of the single nucleotide polymorphism site ( SNP ) ( Pc = 0.028 , Pc = 0.004 ) . There was no statistically significant difference in the levels of TNF - . alpha . , IL - 6 , IL - 10 , and IL - 1尾 in the culture supernatant of the two SNPs of TLR2 .

Conclusion : This study provides evidence that TLR2 gene polymorphism is significantly associated with the onset of ocular Beh et disease .

Study on the copy number variation of Toll - like receptor gene in the second part and the genetic susceptibility of diveitis

Objective : TLR was reported to be involved in the development and development of multiple autoimmune diseases . The genetic copy number variation of TLR1 - 10 in Chinese Han population with Beh et ' s disease , VKH syndrome and acute preveitis with or without ankylosing spondylitis ( AAU + AS 卤 ) was investigated .

Methods : The gene copy number of TLR was detected by real - time fluorescence quantitative PCR . The experiment was divided into 2 stages . The first stage consisted of 400 cases of Beh et ' s disease in eyes , 400 cases of VKH syndrome , AAU + AS 卤 400 cases and healthy control 600 cases . The second stage consisted of 578 cases of Beh et ' s disease and 1000 healthy controls . The expression of mRNA in peripheral blood mononuclear cells ( PBMC ) was detected by means of real - time fluorescence quantitative PCR . The levels of TNF - 伪 , IL - 6 , and IL - 1尾 in peripheral blood mononuclear cells ( PBMC ) were detected by ELISA .

Results : In the first stage , the gene copy number frequency of TLR7 was found , there was statistical difference between the male patients with Beh et disease and the male control group with the copy number greater than 1 . There was also statistical difference between the female patients with Beh et ' s disease and the female control group ( Pc = 0.021 , OR = 2.452 , 95 % CI = 1.4404.176 ; Pc = 0.048 , OR = 2.620 , 95 % CI = 1.406374.882 ) . This contact was confirmed after the second and two stages ( pc = 1.14e - 06 , OR = 2.324 , 95 % CI 1.680 to3.215 ; Pc = 9.12 - 05 , OR = 2.492 , 95 % CI = 1.603to3.873 ) . After stimulation with TLR7 specific ligand R848 , the mRNA expression of TLR7 was higher than that of normal male or female ( Pc = 0.021 , Pc = 0.006 ) . No statistical difference was found in the levels of TNF - . alpha . , IL - 6 , and IL - 1尾 in the culture supernatant , regardless of the number of copies of the male or female TLR7 .

Conclusion : This study provides evidence that the variation of TLR7 gene copy number is associated with the risk of ocular Beh et disease .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.9

【共引文献】