1、蒙，汉族TIM1基因多态性和IL13表达与PNS的相关性研究 2、蒙，汉族TIM3基因多态性和IFN-γ表达与PNS

发布时间：2018-05-17 11:12

本文选题：T细胞免疫球蛋白域黏蛋白域蛋白-1水相逢 + 肾病综合征　；参考：《南方医科大学》2016年博士论文

【摘要】：第一部分背景和目的：儿童原发性肾病综合征发病率占儿童泌尿系统第二位,发病率逐年提高,但发病机制目前仍不清楚,从最初发现白细胞介素-4等在肾病综合征患者表达异常到发现TH1/TH2免疫平衡紊乱可能参与该发病机制,再到TIM基因家族在调控人类效应T细胞活化及分化为TH1/TH2可能的作用,实现了从宏观到微观,从分子水平到基因水平研究的重大进展,这与前人所做的研究努力及科学技术水平的提升密不可分。研究表明,遗传因素参与原发性肾病综合征的发生、发展过程,且具有遗传易感性,不同地区、不同种族,其基因多态性变异也具有差异。越来越多的研究表明,T细胞免疫球蛋白域黏蛋白域蛋白-1(T-cell immunoglobulin domain and mucin domain-1, TIM-1)基因存在多态性,并且基因多态性可能与某些疾病如自身免疫性疾病、糖尿病、支气管哮喘、类风湿性关节炎的发病存在关联,甚至与某些疾病的遗传易感性相关。研究指出PNS可能的发病机制与T淋巴细胞免疫失衡有关,往往涉及Thl与Th2数量及功能紊乱。TIM基因家族被发现其在调控T淋巴细胞活化及细胞因子分泌等方面具有重要作用,而这些均与PNS的尿蛋白形成有关。目前国内外对TIM基因家族的研究也越来越多。由于单核苷酸多态性的存在,使得疾病的发病机制非单一因素所决定,遗传因素也参与疾病的发生、发展过程,不同地区、不同种族,其基因多态性变异也具有差异。因此,TIM-1基因多态性研究为探讨PNS的易感性,疾病的进展、预后,甚至通过干预TH1/TH2之间的平衡来治疗该疾病成为一种可能具有重大意义。本实验目的在探讨内蒙地区蒙汉两族TIM-1基因多态性差异及其与儿童原发性肾病综合征的关系；探索IL-13与PNS之间的关联,为PNS的治疗提供新思路。方法1.样本：病例组的选择：2014-10至2015-12期间,在内蒙古医科大学附属医院儿内科和国际蒙医院住院的确诊为初发PNS的患儿21例；对照组：于内蒙古医科大学附属医院及国际蒙医院同一时期同地区的健康体检汉族儿童20例、蒙古族儿童20例作为汉族及蒙古族正常对照。2.纳入标准：病例组依照我国儿童PNS诊断标准,确诊为PNS；对照组除外变应性疾病、肾脏病史及肾脏病家族史、近期免疫抑制剂应用史。3.采集受试对象外周静脉血液标本2m1各2管,均取自于清晨空腹,按要求妥善存放,将所提取的血液标本按步骤提取DNA及检测IL-13浓度。4.实验方法：TIM-1-1454G/A位点多态性检测用到的方法包括聚合酶链反应和限制性内切酶片段长度多态性(PCR-RFLP)、琼脂糖凝胶电泳和基因测序等方法。IL-13检测采用酶联免疫吸附试验完成。5.数据分析：将所得数据运用正确的统计学方法,采用统计学相关软件,得出各组的基因型以及频率等结果,将数据结果进行统计学比较,检验水准为α=0.05,当P0.05,差异有统计学意义,反之则为无意义。结果1、PNS患儿启动子-1454G/A基因型GG、AG、AA的频率为0.905,0.095,0.0；汉族对照组基因型GG、AA、AG频率为0.55、0.4、0.05；蒙古族对照组基因型GG、AA、AG频率为0.6,0.35,0.05。2.汉族PNS组与汉族正常组的基因频率比较差异有统计学意义(χ2=5.64,P0.025)；蒙古族与汉族对照组基因型频率相比较,差异无统计学意义(χ2=0.11,P0.05)。3.PNS组与健康对照组基因型频率分布比较,差异有统计学意义(χ2=7.09 P=0.030.05)。4.PNS患儿中TIM-1-1454G等位基因频率较正常组频率明显增高(OR=6.23, 95%CI=1.38-28.22,P=0.01)。5.PNS患儿急性期的IL-13含量较健康对照组相比差异有统计学意义。6.蒙古族与汉族正常对照组中IL-13的水平相比较,两者无明显差异。结论1.TIM-1-1454G/A位点的基因多态性可能与内蒙地区汉族儿童PNS的发病相关；-1454G等位基因可能是内蒙地区汉族儿童PNS发病的危险因素,或者说TIM-1--1454G等位基因与PNS的易感性相关。2.虽然本实验不能说明汉族与蒙古族正常儿童的TIM-1基因多态性存在差异,但在未扩大样本及对TIM-1其它基因位点进行进一步研究的前提下,并不能认为TIM-1在蒙古族与汉族对照组间不存在基因多态性。3.IL-13的水平异常与PNS的发病密切相关,IL-13参与PNS的发病。4.TIM-1基因多态性和IL-13水平参与了儿童PNS的发病,两者之间具体的调控途径仍然有待进一步研究。第二部分背景和目的：TIM(T细胞免疫球蛋白黏蛋白分子)是一组由TIM基因编码的T细胞表面分子。TIM蛋白家族各成员在T细胞亚群上的选择性表达使之成为区分Thl和Th2细胞亚群的重要标志,同时它们在T细胞分化、T细胞效应功能、巨噬细胞活化等方面的作用被认为参与了多种自身免疫性疾病的发生、发展,从而引起了人们的关注。TIM家族是新近研究的与临床疾病密切相关的基因家族,其中TIM-3是备受关注的重要成员,其具有多重生物学效应,同时是区分Th1/Th2细胞特异性的表面标志分子,有研究其同时表达于Th17分子表面,参与机体的各项免疫应答调节,如调节IFN-r的表达,与临床疾病的发病有密切联系。Th1和Th2是效应性T细胞,但同时也具有免疫调节作用,Th1主要介导细胞免疫和炎症反应,抗病毒和抗胞内寄生菌感染,参与移植物排斥；Th2主要涉及B细胞增殖,抗体产生、超敏反应和抗寄生虫免疫。两群细胞分泌的细胞因子不同,其中关键性细胞因子是Th1分泌IFN-γ,Th2分泌IL-4.然而,Th1/Th2又显示免疫调节作用。Th1产生的IFN-γ可激活胞内一种称为T-bet的亚群专一性转录因子,T-bet可促进IFNG基因转录而抑制IL4基因转录；相反,Th2产生的IL-4可激活Th2亚群专一性转录因子Gata-3,后者促进IL4基因而抑制IFNG基因的转录。其结果,Thl和Th2成为一类各自以对方及相关疾病为调节对象的细胞亚群。IFN-γ能诱导Mφ及APC上调MHCⅡ类分子,增强抗原处理机提呈作用。IFN-γ自身及其诱导的Mφ产生的IL-12可诱导Thl细胞功能,增强迟发型超敏反应及效应CTL产生。儿童原发性肾病综合征据目前研究分析,是一种以免疫介导为主的,以部分免疫沉着为主,损害肾小球组织的疾病。很多研究提示,该疾病的发生发展存在免疫紊乱的表现,主要是以Th1/Th2平衡紊乱为主[2,15,28]；在该疾病的研究中,我们考虑加强对TIM家族的有关基因进行试验对比研究。TIM-3有多重的生物学效应,以调节免疫反应为主,其与特异性受体相结合,产生特定的细胞信号,在某些特殊细胞因子的刺激作用下,出现Th1类细胞的活化或增殖受到抑制,程度较为显著。从而导致出现T细胞为主的免疫朝向现负方调节的现象。增强或抑制TIM-3与其特异性配体的结合,对有关TH1/Th2失衡相关的疾病可起到改变其导向的作用.在各类临床疾病中均有其独特的治疗思路研究意义。因此在临床疾病的研究过程中对TIM-3分子的深入研究,对相关疾病的治疗及临床症状的缓解会起到不可估量的作用。且IFN-γ在PNS患儿体内表达降低,提示Th1细胞的失活有可能参与PNS的发病过程。TIM-3基因的转录发生在机体受抗原刺激的初始阶段,该阶段对T细胞分化及定型为THI或TH2有至关重要的作用。对TIM-3基因关于肾脏疾病的研究才刚刚开始,其分子调节功能的许多机制仍然需要大量研究。TIM-3可能成为Th细胞上免疫调节治疗的靶向分子。本实验目的是研究TIM-3基因多态性及IFN-γ水平与儿童原发性肾病综合征(PNS)发病机制的相关性。方法：采用病例对照研究,选取居住于内蒙古自治区汉族PNS患儿21例,正常蒙古族(祖籍三代以上均为蒙古族,居住于锡盟地区的蒙古族)、汉族(祖籍三代以上均为汉族,家族中无蒙汉通婚史)对照组各为20例,采用PCR-限制性片段多态性分析、PCR-聚丙烯酰胺凝胶电泳分析和毛细管电泳分析,基因测序技术检测PNS患儿和40例健康儿童(健康对照组)的TIM-3基因外显子-574A/C的SNP的多态性,计算基因型及等位基因频率的统计。采用酶联免疫吸附实验检测血IFN-γ水平,分析其与原发性肾病综合征发生发展中的作用及检测IFN-γ的临床诊断意义。结果：(1)健康汉族对照组TIM-3外显子区-574位A/A,A/C, C/C基因型频率分别为：30%,45%,25%,蒙古族该位点的出现频率为：20%,55%,25%；蒙古族与汉族的正常对照组比较差异无统计学的意义(χ2=0.6,P0.05,)而PNS组频率分别为9.5%,28.6%,61.9%；该数据统计的基因型频率与汉族健康对照组比较出现的差异统计学有意义。(χ2=7.08,P=0.05)；将总体正常对照组与病例组比较差异具有显著性,有统计学意义(χ2=7.16, P=0.05); PNS患儿携带-574C/C等位基因的频率增高(OR=4.875, ORL=1.58, ORu=14.1;P=0.05);(2)由于TIM-3对IFN-γ的转录翻译有一定的调控作用,我们在进一步对其细胞因子水平进行研究。21例PNS患儿及40例正常对照组血清IFN-γ的水平进行检测,分析其与PNS临床指标的关系。PNS组与正常对照组相比,血清IFN-γ的水平无明显差异性。各组间差异无统计学意义(P0.05)。结论：TIM-3基因外显子-574A/C的单核苷酸多态性可能与儿童肾病综合征的发病机制相关。但在细胞因子水平,IFN-γ无明显的差异性变化。
[Abstract]:Background and objective: the incidence of primary nephrotic syndrome in children is second of the urinary system in children. The incidence of the disease is increasing year by year, but the pathogenesis is still unclear. From the initial discovery of the abnormal expression of interleukin -4 in patients with nephrotic syndrome to the discovery of TH1/TH2 immune balance disorder may be involved in the pathogenesis, and then to TIM The gene family plays an important role in regulating the activation and differentiation of human effect T cells into TH1/TH2. It has achieved great progress from macro to microcosmic, from molecular level to gene level. This is inseparable from the previous research efforts and the advancement of science and technology. The genetic polymorphisms of the T cell immunoglobulin domain protein -1 (T-cell immunoglobulin domain and mucin domain-1, TIM-1) gene are polymorphic, and the genetic polymorphism may be associated with some diseases. Diseases such as autoimmune diseases, diabetes, bronchial asthma, and rheumatoid arthritis are associated, and even related to genetic susceptibility to certain diseases. The study indicates that the possible pathogenesis of PNS is related to the imbalance of T lymphocyte immunity, which often involves the number and dysfunction of the.TIM gene family of Thl and Th2 and is found to be in the control of T drenching. The activation of the cells and the secretion of cytokine are important, and these are related to the formation of PNS's urinary protein. There are more and more studies on the TIM gene family at home and abroad. The existence of single nucleotide polymorphisms makes the pathogenesis of the disease determined by no single factor, and the genetic factors are also involved in the development of the disease. The genetic polymorphisms of different regions and different races are also different. Therefore, the study of TIM-1 gene polymorphism is of great significance to explore the susceptibility of PNS, the progress of the disease, the prognosis, and even the balance between the TH1/TH2 and the treatment of the disease. The purpose of this experiment is to explore the two ethnic TI of Mongolian and Han nationality in Inner Mongolia region. M-1 gene polymorphism and its relationship with children's primary nephrotic syndrome; explore the association between IL-13 and PNS to provide new ideas for the treatment of PNS. Method 1.: the selection of the case group: 2014-10 to 2015-12, in the hospital of the Affiliated Hospital of Inner Mongolia Medical University and the international Mongolian Hospital of the Inner Mongolia Medical University, the primary PNS patients were diagnosed. 21 children in the control group: 20 cases of Han children in the same area of Inner Mongolia Medical University Affiliated to Inner Mongolia Medical University and international Mongolian hospital, and 20 cases of Mongolian children as the normal control of Han and Mongolian. The case group was diagnosed as PNS according to the diagnostic standard of Chinese children's PNS; the control group except the allergic disease, kidney The history of the disease and family history of kidney disease and the history of the application of immunosuppressive agents in the recent history of immunosuppressive agents.3. collected 2 tubes of 2M1 from the peripheral venous blood specimens of the subjects, which were taken from the early morning empty stomach and properly stored in accordance with the requirements. The extracted blood specimens were extracted according to the steps of DNA and the IL-13 concentration.4. test method: the method package used for the TIM-1-1454G/A locus polymorphism detection. .IL-13 detection, including polymerase chain reaction and restriction endonuclease fragment length polymorphism (PCR-RFLP), agarose gel electrophoresis and gene sequencing, used enzyme linked immunosorbent assay to complete the analysis of.5. data: using the correct statistical method and using statistical software to obtain the genotype and frequency of each group. The results were compared statistically, the test level was alpha =0.05, when P0.05, the difference was statistically significant, and on the contrary, it was meaningless. Results 1, the frequency of -1454G/A genotype GG, AG, AA in PNS children was 0.905,0.095,0.0, and the genotype GG, AA, AG frequency of the Han control group was 0.55,0.4,0.05; Mongolian control group genotype, frequency, frequency There was a significant difference in the frequency of gene frequency between the PNS group of 0.6,0.35,0.05.2. Han and the normal Han group (x 2=5.64, P0.025), and there was no statistical difference between the Mongolian and the Han control group (x 2=0.11, P0.05), and the difference was statistically significant (x 2=7.09 P) between the.3.PNS group and the healthy control group (x 2=7.09 P). =0.030.05) the frequency of TIM-1-1454G allele in children with.4.PNS was significantly higher than that of the normal group (OR=6.23, 95%CI=1.38-28.22, P=0.01), the IL-13 content in the acute phase of children with.5.PNS was significantly higher than that of the healthy control group. There was no significant difference in the level of IL-13 in the normal control group between the Mongolian and the Han people. The genetic polymorphism of the -1454G/A loci may be associated with the incidence of PNS in Han children in Inner Mongolia. The -1454G allele may be a risk factor for the incidence of PNS in Han children in Inner Mongolia, or the TIM-1--1454G allele is associated with the susceptibility of PNS to.2. although this experiment does not explain the TIM-1 gene polymorphism of the Han and Mongolian normal children. There is a difference in sex, but on the premise of no enlargement of the sample and the further study of other TIM-1 gene loci, it is not considered that the abnormal level of genetic polymorphism of TIM-1 between the Mongolian and the Han control groups is closely related to the incidence of PNS, and IL-13 participates in the polymorphism of the.4.TIM-1 gene and IL-13 level in the pathogenesis of PNS. The specific regulatory pathway between PNS and children remains to be further studied. Second background and purpose: TIM (T cell immunoglobulin mucin molecule) is a group of T cell surface molecules encoded by TIM gene.TIM protein family members in the T cell subgroup selectively expressed to differentiate Thl and Th2 cell subgroups The important markers of group, and their role in T cell differentiation, T cell effect function, macrophage activation and so on, are considered to be involved in the occurrence and development of a variety of autoimmune diseases, which have aroused the concern that the.TIM family is a recently studied gene family closely related to clinical diseases, of which TIM-3 is the most important concern. As a member, it has multiple biological effects and is a surface marker to distinguish the specific Th1/Th2 cells. It is expressed on the surface of the Th17 molecule and participates in the regulation of the immune response of the body, such as regulating the expression of IFN-r, and closely related to the pathogenesis of clinical diseases:.Th1 and Th2 are effector T cells, but they also have immune response. Pestilence, Th1 mainly mediates cell immunity and inflammatory reaction, antiviral and anti cytosolic infection, and participates in graft rejection; Th2 mainly involves B cell proliferation, antibody production, hypersensitivity and anti parasitic immunity. The cytokines secreted by two groups are different, and the key cytokines are Th1 secreting IFN- gamma, Th2 secretes IL-4. ran. In addition, Th1/Th2 also shows that IFN- gamma produced by immunoregulatory action.Th1 activates a subgroup specific transcription factor called T-bet. T-bet can promote the transcription of IFNG gene and inhibit the transcription of IL4 gene. On the contrary, IL-4 activates Th2 subgroup specific transcription factor Gata-3, which promotes the IL4 gene to inhibit the transcription of the gene. As a result, Thl and Th2 become a class of cell subgroups, each of which are regulated by each other and related diseases,.IFN- gamma can induce M phi and APC up regulation of MHC II molecules, enhance the function of.IFN- y and its induced M Phi induced IL-12 inducible Thl cells, enhance late hypersensitivity and effect CTL production. Nephrotic syndrome, according to current research and analysis, is an immune mediated disease which is mainly mediated by immunization and damages glomerular tissue. Many studies suggest that the development of the disease is manifested in the presence of immune disorders, mainly based on Th1/Th2 balance disorder [2,15,28]; in the study of the disease, we consider strengthening TIM A comparative study of the related genes in the family,.TIM-3 has multiple biological effects, which regulates the immune response mainly. It combines with the specific receptor to produce specific cell signals. Under the stimulation of certain specific cytokines, the activation or proliferation of Th1 like cells is inhibited, which leads to the emergence of T. Cell based immunization toward negative side regulation. Enhancing or inhibiting the binding of TIM-3 to its specific ligands can change its guiding role for diseases related to TH1/Th2 imbalance. It has its unique therapeutic significance in all kinds of clinical diseases. Therefore, the TIM-3 molecule in the study of the disease of the bed. In depth study, it can play an inestimable role in the treatment of related diseases and the relief of clinical symptoms. And the expression of IFN- gamma is reduced in PNS children, suggesting that the inactivation of Th1 cells may participate in the pathogenesis of PNS, and the transcription of.TIM-3 gene occurs at the initial stage of the body being stimulated by antigen, and the differentiation and stereotype of T cells is THI or TH2 has a crucial role to play. The study of the TIM-3 gene about kidney disease has just begun. Many mechanisms of its molecular modulating function still require a lot of study of.TIM-3 as a target for immunomodulatory therapy on Th cells. The purpose of this experiment is to study the TIM-3 gene polymorphism and the level of IFN- gamma in children with primary nephrotic synthesis. The correlation of the pathogenesis (PNS). Methods: a case control study was used to select 21 cases of PNS children living in the Han nationality in the Inner Mongolia Autonomous Region, the normal Mongolian nationality (all over three generations of the ancestors are Mongolian, the Mongolian people living in Xilong area), the Han nationality (all of the three generations of the ancestors were Han, and the family without the marriage history of the Mongolian Han Tong) in the control group, each of the 20 cases, used in the control group. PCR- restriction fragment polymorphism analysis, PCR- polyacrylamide gel electrophoresis analysis and capillary electrophoresis analysis, gene sequencing technique to detect the SNP polymorphism of TIM-3 exon -574A/C in children with PNS and 40 healthy children (healthy control group), to calculate the genotype and allele frequency statistics. The enzyme linked immunosorbent assay (ELISA) test was used. The serum IFN- gamma level was used to analyze its role in the development of primary nephrotic syndrome and the clinical diagnostic significance of IFN- gamma. Results: (1) the frequencies of A/A, A/C, C/C genotype of -574 in exon TIM-3 of the healthy Han control group were 30%, 45%, 25% respectively, and the frequencies of the Mongolian loci were 20%, 55%, 25%, and the Mongolian and Han nationality were positive. There was no statistical significance in the control group (x 2=0.6, P0.05), and the frequency of the PNS group was 9.5%, 28.6%, 61.9%, respectively. The difference between the genotype frequency and the Han healthy control group was significant. (x 2=7.08, P=0.05), the difference between the general control group and the case group was significant, and the statistical difference was statistically significant. Significance (x 2=7.16, P=0.05); the frequency of -574C/C allele in children with PNS increased (OR=4.875, ORL=1.58, ORu=14.1; P=0.05); (2) because TIM-3 had a certain regulatory effect on the transcription of IFN- gamma, we were further investigating the level of cytokines in.21 cases of PNS children and 40 normal controls. There was no significant difference in serum IFN- gamma level between group.PNS and normal control group. There was no significant difference between each group (P0.05). Conclusion: the single nucleotide polymorphisms of TIM-3 gene exon -574A/C may be associated with the pathogenesis of children with nephrotic syndrome, but at the level of cytokines, there is no IFN- gamma. Distinctly different changes.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R726.9

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