【作者】 李静；

【导师】 叶冬青；

【作者基本信息】 安徽医科大学 ， 流行病与卫生统计学， 2012， 博士

【摘要】 研究背景系统性红斑狼疮（systemic lupus erythematosus，SLE）是一种全身性的自身免疫性疾病，其特征主要表现为多种自身抗体形成，免疫复合物沉积和补体活化，造成多系统和多器官损害。SLE的发病机制至今尚不明确，一般认为其受遗传易感性、环境因素、性激素紊乱和免疫异常等多个方面影响。天然免疫和获得性免疫系统紊乱在SLE的免疫发病机制中起着重要作用。SLE的免疫异常与多条免疫信号通路（NF-κB信号通路、Toll-like受体信号通路、JAK∕STAT信号通路、MAPK信号通路、PI3-AKT信号通路及Th17信号通路等）紊乱密切相关，各条通路之间可能相互作用、异常调节从而导致机体免疫调节紊乱。本课题组前期采用病例对照研究的方法，利用Real-time PCR芯片技术筛选Toll-like受体信号通路上SLE的差异表达基因，结果发现受体相互作用蛋白2（receptor interacting protein2，RIP2）在SLE患者外周血白细胞中的mRNA表达上调，提示RIP2可能参与了SLE的发病。RIP2，也称为RIPK2、RICK或CARDAK，是信号转导途径中关键的丝氨酸∕苏氨酸激酶，能参与Toll-like受体和NOD样受体等介导的天然免疫和获得性免疫应答，诱导产生炎症细胞因子。已有研究表明RIP2可能参与了炎症性肠病、多发性硬化症等自身免疫性疾病的发病，但是迄今为止国内外尚无关于RIP2与SLE关联性研究的相关报道。基于上述内容，本课题拟采用病例对照研究，利用TaqMan探针技术、ELISA方法，检测SLE患者及正常对照者RIP2基因单核苷酸多态性及血清水平，探讨其与SLE发病之间的关联性。本课题的完成将有助于阐明RIP2在SLE发病中的作用，进一步揭示SLE的发病机制，并为寻找新的SLE治疗靶点提供科学依据。目的比较SLE患者和正常对照者RIP2基因的2个SNP位点（rs16900617、rs16900627）的等位基因和基因型频率的差异，探讨RIP2基因单核苷酸多态性与系统性红斑狼疮的关联性。方法病例来自安徽省立医院和安徽医科大学第一附属医院风湿免疫科的SLE患者（均符合1997年美国风湿病学会修订的SLE分类诊断标准），共590例。对照来自健康志愿者，共660例。采用自行设计的问卷，调查并记录研究对象的一般情况和临床资料，在获得知情同意后，采集SLE患者及正常对照者的外周静脉血。应用TaqMan探针及ABI7300PCR仪检测SLE患者和正常对照者RIP2基因的等位基因和基因型频率。采用SPSS10.01软件进行统计分析，χ~2检验或确切概率法用于比较两组间等位基因频率、基因型频率的分布差异，Stata7.0软件用于分析哈迪-温伯格平衡，应用SHEsis软件进行单倍型分析。检验水准α=0.05。结果（1）rs16900617位点多态性与SLE的关联性研究SLE患者组AA、AG、GG的基因型频率分别为86.4%、13.4%和0.2%，对照组为77.9%、20.3%和1.8%；SLE患者组A和G等位基因频率为93.1%和6.9%，对照组则为88.0%和12.0%。两组基因型频率的分布差异有统计学意义（AG vs.AA：OR=0.59，95%CI0.44-0.81，P=0.001；GG vs. AA：OR=0.08，95%CI0.01-0.65，P=0.006；AG+GG vs. AA：OR=0.55，95%CI0.41-0.75，P <0.001），等位基因频率的分布差异也具有统计学意义（G vs.A：OR=0.54，95%CI0.41-0.72，P <0.001）。狼疮肾炎（lupus nephritis，LN）患者的基因型分布状况为：A和G等位基因频率为94.0%和6.0%；AA、AG、GG的基因型频率分别为88.0%、12.0%和0%。非LN患者组中：A和G等位基因频率为93.2%和6.8%；AA、AG、GG的基因型频率分别为86.6%、13.2%和0.2%。两组的基因型频率的分布差异无统计学意义（AG vs.AA：OR=0.90，95%CI0.52-1.55，P=0.698；GG vs.AA：OR=1.003，95%CI0.997-1.008，P=1.000；AG+GG vs. AA：OR=0.88，95%CI0.51-1.52，P=0.651），等位基因频率的分布差异也没有统计学意义（G vs. A：OR=0.87，95%CI0.52-1.48，P=0.612）。（2）rs16900627位点多态性与SLE的关联性研究SLE患者的基因型分布状况为：A和G等位基因频率为80.7%和19.3%；AA、AG、GG的基因型频率分别为64.6%、32.2%和3.2%。对照组中：A和G等位基因频率为84.2%和15.8%；AA、AG、GG的基因型频率分别为72.3%、23.9%和3.8%。两组的基因型频率分布差异有统计学意义（AG vs.AA：OR=1.51，95%CI1.17-1.93，P=0.001；AG+GG vs.AA：OR=1.43，95%CI1.13-1.82，P=0.003），等位基因频率的分布差异也有统计学意义（G vs.A：OR=1.28，95%CI1.04-1.58，P=0.019）。LN患者的基因型分布状况为：A和G等位基因频率为77.1%和22.9%；AA、AG、GG的基因型频率分别为58.4%、37.3%和4.2%。非LN患者组中：A和G等位基因频率为82.2%和17.8%；AA、AG、GG的基因型频率分别为67.5%、29.5%和3.1%。两组等位基因的频率分布差异有统计学意义（G vs.A：OR=1.37，95%CI1.00-1.87，P=0.046），基因型频率分布差异也具有统计学意义（AG+GG vs. AA：OR=1.47，95%CI1.02-2.13，P=0.039）。（3）RIP2基因rs16900617和rs16900627位点的单倍型分析将RIP2基因的2个SNP位点rs16900617和rs16900627构建单倍型，单倍型AG和GA在SLE患者组和正常对照组中的分布差异具有统计学意义（OR=1.37，95%CI1.11-1.70，P=0.003；OR=0.60，95%CI0.45-0.79，P=0.0003）。（4）RIP2基因SNP位点与SLE临床特征的关系RIP2基因的2个SNP位点rs16900617、rs16900627的等位基因、基因型与SLE患者主要临床特征，如关节炎、颧部红斑、口腔溃疡等之间不存在统计学关联（P均>0.05）。结论RIP2基因的rs16900617和rs16900627位点及其单倍型在SLE患者和正常对照者中的分布差异有统计学意义，提示RIP2基因多态性可能与中国汉族人群SLE遗传易感性有关。目的比较SLE患者和正常对照者，狼疮肾炎患者和非狼疮肾炎患者，疾病活动期患者和非疾病活动期患者之间血清RIP2表达水平差异，分析血清RIP2水平与SLE患者实验室检测指标、临床特征之间的关联性，探讨其在SLE发生发展中的作用。方法病例来自安徽省立医院和安徽医科大学第一附属医院风湿免疫科的门诊及住院患者，共80例，SLE的诊断依据1997年美国风湿病学会修订的SLE分类标准。对照来自健康志愿者，共76例，与病例按照年龄、性别匹配。问卷调查收集研究对象的一般情况和临床资料，并进行SLE疾病活动指数（systemic lupuserythematosus disease activity index，SLEDAI）评分。获得研究对象知情同意后，抽取外周静脉血。采用ELISA试剂盒检测血清RIP2水平，比较SLE患者和正常对照者，狼疮肾炎患者和非狼疮肾炎患者，疾病活动期患者和非疾病活动期患者之间血清RIP2表达水平差异。采用SPSS10.01软件进行统计分析，检验水准α=0.05。结果（1）不同组别间血清RIP2水平的比较SLE患者组与正常对照组的血清RIP2浓度分别为：2.725（1.901，3.876）ng/mL、2.627（1.906，3.309）ng/mL，差异无统计学意义（Z=-1.009，P=0.313）；SLE合并肾炎组与未合并肾炎组的血清RIP2浓度分别为：2.684（1.906，3.529）ng/mL、2.970（1.897，4.258）ng/mL，差异无统计学意义（Z=-1.353，P=0.176）；活动期与非活动期SLE患者血清RIP2浓度分别为：2.813（2.038，3.876）ng/mL、2.022（1.491，3.825）ng/mL，差异无统计学意义（Z=-1.550，P=0.121）。（2）血清RIP2水平与SLE疾病活动指数的关系SLE患者血清RIP2浓度与SLE疾病活动指数无相关性（r=0.094，P=0.409）。（3）血清RIP2水平与临床特征和实验室检测指标的关联SLE患者血清RIP2水平与患者的视神经受损存在显著关联，视神经受损的患者血清RIP2水平降低（Z=-2.096，P=0.023）；SLE患者血清RIP2水平与抗RNP抗体、血沉、C反应蛋白存在显著关联，在抗RNP抗体阳性、血沉升高、C反应蛋白升高的SLE患者中血清RIP2水平显著升高（Z=-2.469，P=0.014；Z=-2.218，P=0.027；Z=-1.967，P=0.049）。结论本研究未发现SLE患者组与正常对照组、狼疮肾炎组与非狼疮肾炎组、疾病活动组与非疾病活动组之间的血清RIP2水平表达差异，但是血清RIP2水平与SLE患者的部分临床特征、实验室检测指标之间存在关联。更多还原

【Abstract】 Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease,it is characterized by a diverse array of autoantibody production, immune complexesdeposition and complement activation, resulting in multiple system and organinvolvement. The etiology of SLE remains unclear, genetic susceptibility, environmentalfactors, sexual hormone disorder and immune dysfunction are thought to contribute todisease development.Dysfunction of innate immunity and adaptive immunity plays an importment rolein the immune pathogenesis. Multiple immune signaling pathways have been reportedto participate in the immune pathogenesis of SLE, including NF-κB signaling pathway,Toll-like receptor signaling pathway, JAK/STAT signaling pathway, MAPK signalingpathway, PI3-AKT signaling pathway, Th17signaling pathway and so on. Thesesignaling pathways interaction might regulate abnormally, resulting in the immunedysregulation.Our previous study has found that the level of RIP2mRNA in SLE patients wassignificantly elevated compared to the healthy controls using case-control design andReal-time PCR assay to detect differentially expressed genes in Toll-like receptorsignaling pathway. This result indicated that RIP2might take part in the progress ofSLE.RIP2, also known as RIPK2, RICK, CARDAK, is a key serine/threonine kinase insignal transduction pathway. RIP2mediates innate and adaptive immune responsesinduced through multiple receptors, such as Toll-like receptor and NOD-like receptorand so on, resulting in the production of inflammatory cytokines. Several studies have identified that RIP2might be associated with autoimmune diseases, such as Crohn’sdisease, multiple sclerosis. However, so far no study has reported the association ofRIP2and SLE at home and abroad.Thus, on the basis of the content above, our study adopted case-control design todetect association of RIP2gene polymorphisms, serum RIP2level and SLE by theTaqMan SNP Genotyping Assay kit and ELISA. The accomplishment of this studywould help to further reveal the role of RIP2in the pathogenesis of SLE and developnew potential therapeutic targets. Objective To compare the differences of allele and genotype frequencies about twoSNPs (rs16900617, rs16900627) of RIP2between SLE patients and normal controls,and to explore the association of RIP2polymorphisms with susceptibility to SLE.Methods590SLE patients were recruited from Department of Rheumatology andImmunology of Anhui Provincial Hospital and the First Affiliated Hospital of AnhuiMedical University. All SLE patients fulfilled the1997revised American College ofRheumatology classification criteria.660normal controls were recruited from healthyvolunteers. The demographic and clinical data were collected by self-designedquestionnaire. After obtaining the informed consent, blood samples from all studiedsubjects were collected. The genotypes of the rs16900617and rs16900627SNPs weredetermined by the TaqMan SNP Genotyping Assay kit using an ABI7300Real-Timepolymerase chain reaction (PCR) system.SPSS10.01software was used for statistical analysis. The chi-square test orFisher’s exact test was applied to compare the distribution of allele and genotype frequencies between SLE patients and normal controls. Stata7.0software was used toHardy-Weinberg equilibrium test. Haplotype analysis was assessed using SHEsissoftware. P values were calculated based on two-sided tests, with a statisticalsignificance defined as P value <0.05.Results(1) Association of rs16900617and SLE The genotype frequencies of AA, AG, GGwere86.4%,13.4%,0.2%in SLE patients and77.9%,20.3%,1.8%in controls. Theallele frequencies of A, G were93.1%,6.9%in SLE patients and88.0%,12.0%incontrols. There were significant differences between SLE patients and controls aboutgenotype (AG vs. AA: OR=0.59,95%CI0.44-0.81, P=0.001; GG vs. AA: OR=0.08,95%CI0.01-0.65, P=0.006; AG+GG vs. AA: OR=0.55,95%CI0.41-0.75, P <0.001) and allele frequencies (G vs. A: OR=0.54,95%CI0.41-0.72, P <0.001).The allele frequencies of A, G were94.0%,6.0%and the genotype frequencies ofAA, AG, GG were88.0%,12.0%,0%in lupus nephritis (LN) patients. They were93.2%,6.8%,86.6%,13.2%,0.2%respectively in non-LN patients. There were nosignificant differences between LN patients and non-LN patients about genotype (AGvs. AA: OR=0.90,95%CI0.52-1.55, P=0.698; GG vs. AA: OR=1.003,95%CI0.997-1.008, P=1.000; AG+GG vs. AA: OR=0.88,95%CI0.51-1.52, P=0.651) andallele frequencies (G vs. A: OR=0.87,95%CI0.52-1.48, P=0.612).(2) Association of rs16900627and SLE The allele frequencies of A, G were80.7%,19.3%and the genotype frequencies of AA, AG, GG were64.6%,32.2%,3.2%in SLEpatients. They were84.2%,15.8%,72.3%,23.9%,3.8%respectively in controls. Therewere significant differences between SLE patients and controls about genotype (AG vs.AA: OR=1.51,95%CI1.17-1.93, P=0.001; AG+GG vs. AA: OR=1.43,95%CI1.13-1.82, P=0.003) and allele frequencies (G vs. A: OR=1.28,95%CI1.04-1.58, P =0.019).The allele frequencies of A, G were77.1%,22.9%and the genotype frequenciesof AA, AG, GG were58.4%,37.3%,4.2%in LN patients. They were82.2%,17.8%,67.5%,29.5%,3.1%respectively in non-LN patients. There were significant differencesabout allele (G vs. A: OR=1.37,95%CI1.00-1.87, P=0.046) and genotypefrequencies (AG+GG vs. AA: OR=1.47,95%CI1.02-2.13, P=0.039) between LNpatients and non-LN patients.(3) Haplotype analysis Haplotypes were constructed with the two variants(rs16900617, rs16900627) and the frequencies of haplotypes were analyzed. The resultsrevealed that two haplotypes, AG and GA, were significantly associated with SLE (OR=1.37,95%CI1.11-1.70, P=0.003; OR=0.60,95%CI0.45-0.79, P=0.0003).(4) Association of RIP2gene polymprphisms and clinical features of SLE patients Nosignificant association was detected between the distribution of allele and genotypefrequencies of RIP2gene polymorphisms and clinical manifestation, such as arthritis,malar rash, oral ulcer and so on (P>0.05).Conclusion There are significant differences between SLE patients and normalcontrols regarding the SNPs of rs16900617, rs16900627and haplotypes constructedwith the two variants. The RIP2gene polymorphisms may be associated withsusceptibility to SLE in the Chinese population. Objective To compare the serum RIP2levels between patients with SLE and normalcontrols, patients with and without nephritis, active and inactive SLE patients. Toanalyze the association of serum RIP2level with laboratory results and clinical profiles.Additionally, to explore the role of RIP2in the occurrence and development of SLE.Methods80SLE patients were in-and out-patients recruited from Department ofRheumatology and Immunology of Anhui Provincial Hospital and the First AffiliatedHospital of Anhui Medical University. Diagnosis of SLE was established according tothe1997revised criteria of American College Rheumatology.76normal controls wererecruited from healthy volunteers. Patients and controls were age-, sex-matched. Thedemographic and clinical data were collected by questionnaire. Disease activity wasevaluated using the SLE Disease Activity Index (SLEDAI) score. After obtaining theinformed consent, blood samples from all studied subjects were collected. Serum RIP2levels were detected by Enzyme-linked Immunosorbent Assay (ELISA). The data wereanalyzed by SPSS10.01software. All P values were calculated based on two-sided tests,with a statistical significance defined as P value <0.05.Results(1) Comparisons of serum RIP2level between different groups There were nosignificant differences of serum RIP2level between patients with SLE and normalcontrols (2.725(1.901,3.876) ng/mL vs.2.627(1.906,3.309) ng/mL, Z=-1.009, P=0.313), patients with and without nephritis (2.684(1.906,3.529) ng/mL vs.2.970(1.897,4.258) ng/mL, Z=-1.353, P=0.176), active and inactive SLE patients (2.813(2.038,3.876) ng/mL vs.2.022(1.491,3.825) ng/mL, Z=-1.550, P=0.121). (2) Correlations between serum RIP2level and SLEDAI Correlation analysis betweenserum RIP2level and SLEDAI showed no significant association (r=0.094, P=0.409).(3) Association of serum RIP2level and clinical, laboratory data of SLE patientsSignificant association was found between serum RIP2level and optic nerve injury (Z=-2.096, P=0.023). As for laboratory features, serum RIP2level was significantlyassociated with anti-RNP antibodies (Z=-2.469, P=0.014), ESR (Z=-2.218, P=0.027) and CRP (Z=-1.967, P=0.049).Conclusion In conclusion, this study showed no significant differences of serum RIP2level between patients with SLE and normal controls, patients with and withoutnephritis, active and inactive SLE patients. However, we detected significant associationof serum RIP2level with part of clinical and laboratory features.更多还原