【作者】 沈冰冰；

【导师】 钱家鸣； 孙钢； 朱峰；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 炎症性肠病(IBD)是一组病因不明的慢性肠道疾病,主要包括溃疡性结肠炎(UC)和克罗恩病(CD)。近年在我国病例报告逐年增加,呈明显上升趋势。目前IBD的病因和发病机制尚不清楚,研究认为遗传、环境、免疫三者相互作用参与IBD发病,并可能决定临床表型。近年国内外学者对IBD遗传因素的研究发现基因突变除与疾病易感性相关外,与某些疾病表型也相关,如NOD2突变在回肠CD为多。作为第一个CD易感基因,NOD2的发现具有划时代的意义,它提示IBD发病与肠道细菌及自然免疫相关。DEFB1和ATG16L1属免疫防御功能相关基因,有研究发现DEFB1多态性与CD相关,在我国尚无相关报道。ATG16L1作为新近确定的CD易感基因,在高加索人群研究中得到较好的重复,而在我国尚无相对大样本的研究。除遗传因素之外,一些特异性抗体可能作为IBD与免疫反应有关的表现而受到重视,包括抗中性粒细胞胞浆抗体(Antineutrophil ctyoplasmic antibodies,p-ANCA)、抗酿酒酵母菌抗体(Anti-saccharomyces cerevisiae antibodies,ASCA)、抗胰外分泌腺抗体(Pancreatic antibodies,PAB)和抗小肠杯状细胞抗体(Goblet cellautoantibodies,GAB)等,四种抗体检测在国外广泛用于IBD的诊断,但在中国人群中报道较多的为p-ANCA和ASCA,二者联合检测诊断IBD敏感性较低,且不同中心的研究结果不甚一致,而PAB和GAB国内报道较少,且上述抗体与临床表型的关系报道尚有争议。IBD为异质性疾病,基于遗传学异质性和血清学异质性的研究可准确界定IBD临床分型,有助于治疗方案的细化和预后的判定。因此本研究从IBD遗传易感性和相关抗体检测着手,立足于国内现有试验条件,研究旨在:1.探讨自然免疫相关基因DEFB1和ATG16L1单核甘酸多态性与IBD遗传易感性的关系以及与IBD临床表型的可能关联;2.探讨p-ANCA、ASCA、PAB和GAB联合检测对IBD诊断及鉴别诊断的应用价值以及与IBDI临床表型的可能关联。第一部分中国汉族人群ATG16L1和DEFB1基因多态性与炎症性肠病遗传易感性的关联性研究方法:本研究采用医院为基础的病例—对照研究设计,对247例无血缘关系的我国汉族IBD患者(150例UC,97例CD)以及200例正常对照者,应用聚合酶链反应—限制性片段长度多态性(Polymerase chain reaction—restricted fragment lengthpolymophism,PCR-RFLP)方法和部分样本(10%)测序方法,检测DEFB1基因(rs11362和rs1800972位点)和ATG16L1基因(rs2241880和rs1045095位点)多态性,并分析上述基因多态性与IBD临床特征的关系。结果:1、DEFB1基因rs11362多态与UC患病风险增加相关,携带rs11362GG基因型个体患UC风险是携带AA和GA基因型个体的2.04倍(95%CI:1.32-3.06,P=0.0013)。2、单体型分析表明携带DEFB1基因rs11362和rs1800972G-C单体型个体患UC风险是携带A-C单体型个体的1.621倍(95%CI:1.010-2.610,P=0.035);3、基因型与表型关联分析表明DEFB1基因rs11362多态与UC临床表型相关,诊断年龄≤25岁UC患者携带DEFB1基因rs11362位点GG基因型显著高于与正常对照(P>=0.001,OR=3.29,95%CI:1.56-7.09),病变累及全结肠者携带GG基因型显著高于正常对照(P=0.003,OR=2.27,95%CI:1.31-3.91),重度结肠炎患者携带GG基因型显著高于正常对照(P=0.009,OR=2.55,95%CI:1.24—5.24);小结:DEFB1基因单核甘酸多态性不仅与我国汉族人群UC易感性相关,而且与发病早、重度及广泛结肠炎相关。第二部分中国人炎症性肠病血清学标志物联合检测的临床意义方法:本研究对217例IBD患者(UC108例,CD109例)、59例非IBD肠道疾病患者以及112例正常对照者,应用ELISA法检测ASCA,IIF法检测p-ANCA、PAB和GAB。结果:1.不同抗体在IBD中分布:UC中p-ANCA和GAB阳性率分别为60.2%和30.6%,均高于CD、疾病对照和正常对照,CD中ASCA和PAB阳性率分别为34.9%和41.3%,均高于UC、疾病对照和正常对照;p-ANCA阴性UC中仅有13.6%(6/44)病人GAB阳性,ASCA阴性CD中34.3%(24/70)病人PAB阳性;2.四种抗体联合检测对IBD诊断的意义:p-ANCA和GAB单项检测诊断UC敏感性分别为59.3%和27.8%,二者联合检测诊断UC敏感性可提高至64.8%;ASCA和PAB单项检测诊断CD敏感性均为35.8%,二者联合检测诊断CD敏感性可提高至57.8%;3.四种抗体联合检测对IBD鉴别诊断的意义:ASCA+p-ANCA-对于CD和UC的鉴别诊断敏感性57.8%,特异性96.3%,联合PAB和GAB检测特异性和阳性预测值均为100%,但敏感性仅为12.8%;p-ANCA+ASCA-对于UC和CD的鉴别诊断敏感性50%,特异性94.5%,联合GAB和PAB检测特异性和阳性预测值均达100%,但敏感性仅为19.4%;4.四种抗体检测在结肠CD和UC鉴别诊断中的意义:ASCA+p-ANCA-诊断结肠CD敏感性、特异性分别为60%和96.3%,阳性预测值为28.6%,联合PAB和GAB检测虽特异性和阳性预测值均提高至100%,而敏感性仅为0.9%。p-ANCA+ASCA-鉴别UC的敏感性和特异性50%和94.5%,阴性预测值为28.9%,联合GAB和PAB检测特异性和阳性预测值均达100%,但敏感性仅为16.7%;5.四种抗体检测在IBD与非IBD肠病的鉴别诊断中的意义:各抗体单项检测诊断IBD的特异性基本在95%以上,其中GAB可达100%;对于联合检测而言,PAB和GAB组合诊断IBD的特异性96.6%,高于p-ANCA和ASCA组合;6.四种抗体与IBD临床特征的关系:未发现UC病人p-ANCA和GAB、CD病人ASCA与临床特征有关。诊断年龄≤40组CD病人血清PAB阳性率为43.2%,诊断年龄>40岁组CD病人PAB阳性率20%,二者比较差异显著(P=0.018);有肠外表现者CD病人血清PAB阳性率明显高于无肠外表现者(58.8%vs 31.5%,P=0.031)。小结:1、多数UC患者可检测到p-ANCA和ASCA,多数CD患者可检测到ASCA和PAB;2、p-ANCA联合GAB检测可提高UC诊断的敏感性,ASCA联合PAB检测可提高CD诊断的敏感性;3、四种抗体联合检测较p-ANCA和ASCA更有助于IBD的鉴别诊断,同时亦有助于结肠CD和UC的鉴别;4、GAB检测有助于IBD与非IBD肠道疾病的鉴别;5、PAB与CD发病早、肠外表现有关。结论:1、DEFB1基因单核甘酸多态性不仅与我国汉族人群UC易感性相关,而且与发病早、重度及广泛结肠炎相关;2、ATG16L1基因单核甘酸多态性与我国汉族人群IBD无关;3、p-ANCA、ASCA、PAB和GAB四种抗体联合检测较p-ANCA和ASCA两种抗体联合检测更有助于IBD的鉴别诊断,同时亦有助于结肠CD和UC的鉴别;4、PAB与CD发病早、肠外表现有关。更多还原

【Abstract】 Inflammatory bowel disease(IBD) is a group of chronic intestinal diseases including of Crohn’s disease(CD) and ulcerative colitis(UC).The incidence of IBD shows a significant increase tendency in recent years.The causes and pathogenesis of IBD are not very clear so far.It is reported that the interaction between genetic factors, environmental factors and immune factors are involved in pathogenesis and its clinical phenotype of IBD.Recent studies on genetic factors of IBD found that gene mutation is not only related to disease susceptibility,also associated with disease phenotype,such as NOD2 mutation is frequently observed in ileum type of CD.The discovery of NOD2,the first CD susceptible gene,is a groundbreaking event.It suggests that the onset of IBD is associated with enteric bacteria and natural immunity.DEFB1 and ATG16L1 gene are associated with immunity defense.The genetic variations in the DEFB1 was associated with CD in Hungary populations.But there is no study on the related with IBD in Han population of China.The relative large-sample research about the relationship between IBD and ATG16L1,as a CD susceptibility gene identified recently,was not found in China.Besides of genetic factors,some specific antibodies would arouse concerns since they are evidence that IBD is related to immune reaction.Antineutrophil cytoplasmic antibodies(p-ANCA),Anti-saccharomyces cerevisiae antibodies(ASCA),Pancreatic antibodies(PAB)and Goblet cell autoantibodies(GAB) were included.These four antibodies have been widely used for the diagnosis of IBD abroad.Lots of studies were focus on p-ANCA and ASCA in China.The results of these studies showed that both p-ANCA and ASCA have some diagnostic value with low sensitivity.There was a few of studies on PAB and GAB in China.The association of PAB and GAB with clinical phenotype is controversial.IBD is heterogeneity disease.Studies based on genetic heterogeneity and serum heterogeneity could help to precisely define IBD clinical type,to make detail therapy plan and determine prognosis.In this study,the authors investigate IBD genetic susceptibility and antibodies detection based on current experimental condition.It aims to:1.Investigating the relationship between single nucleotide polymorphism of natural immunity related genes DEFB1 & ATG16L1,IBD genetic susceptibility,and IBD clinical phenotype.2.Investigating application value of combined detection of p-ANCA、ASCA、PAB and GAB in diagnosis and differential diagnosis of IBD,and the relationship with IBD clinical phenotype.Part 1.The Study of DEFB1 And ATG16L1 Gene Polymorphism with Genetic Susceptibility of Inflammatory Bowel Disease in Han Population of ChinaMethods:247 IBD patients(including 97 with CD and 150 with UC) and 200 healthy individuals of Chinese Han population were collected.Totally four SNPs of DEFB1(rs11362,rs1800972)and ATG16L1(,rs2241880,rs1045095)gene were genotyped by Polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP).10%of objective was selected to determine the sequence to confirm the PCR-RFLP.Genotype-phenotype analysis was performed with respect to disease susceptibility stratified by clinical feature.Results:The frequency of allele G of SNP site rs11362 in patients with UC is much higher than in controls(70 vs 60%,P=0.012).The distribution of genotype of AA,AG,GG of SNP rs11362 in UC patients has the significant difference,comparing with controls(P =0.006).It was found that multivariate-adjusted odds ratio(OR;95%confidence interval)for GG genotype compared with AA and AG were 2.04(95%CI:1.32-3.06). Haplotype analysis found that the haplotype G-C for the SNP site rs11362-rs1800972 were at significantly increased risk for UC(OR:1.621,95%CI:1.010-2.610).In subgroup analysis,GG genotype frequencies between UC patients with early onset of disease(age at diagnosis≤25,lower quartile)and controls showed significant difference (P=0.001,OR=3.29,95%CI:1.56-7.09).A significantly higher frequency of the GG genotype of SNP site rs11362 was observed among UC patients with extensive colitis(P=0.003,OR=2.27,95%CI:1.31-3.91) and severe course of disease behaviors(P= 0.009,OR=2.55,95%CI:1.24-5.24) respectively,as compared with healthy controls.No significant differences were noted in the DEFB1 gene SNP site rs1800972 and in the ATGI6LI gene SNP site rs2241880,rs1045095 polymorphisms among the patients with CD,UC and the control subjects.No association of above 3 SNP genotypes with disease subgroups in UC and CD was observed.Summary:These results indicated that rs11362 polymorphism in the DEFB1 might be associated with the risk for UC,but not for CD.It may also determine disease phenotype such as:early onset,severe degree and extensive colitis.The association of rs2241880 and rs1045095 polymorphism of ATGI6LI with IBD was not observed.Part 2.The Clinical Significance of Combined Measurement of Serological Marker in Inflammatory Bowel Disease in Chinese PopulationsMethods:A total of 217 patients with IBD(UC 108,CD 109),59 patients with non-IBD intestinal disease controls(DC) and 112 healthy controls(HC) were recruited in our research.ASCA were detected by enzyme-linked immunosorbent(ELISA) assay. p-ANCA,PAB and GAB were measured by indirect immunofluorescence(IIF) assay.Results:1.The distribution of these antibodies in IBD:The prevalence of p-ANCA in UC,CD,DC and HC group was 60.2%,5.5%,3.4%and 3.4%,respectively.The prevalence of GAB in UC,CD,DC and HC group was 30.6%,3.7%,0%and 1.8%, respectively.The prevalence of p-ANCA and GAB in UC group was statistically different compared with that in CD or DC(P＜0.05).The prevalence of ASCA in CD,UC,DC and HC group was 34.9%,13%,10.2%and 16.1%,respectively.The prevalence of PAB in CD,UC,DC and HC group was 41.3%,6.5%,3.4%and 0%,respectively.The prevalence of ASCA and PAB in CD group was statistically different compared with that in CD or DC(P＜0.05).Among the UC patients with negative for p-ANCA,13.6%(6/44) are positive for GAB.Among the CD patients negative for ASCA,34.3%(24/70) are positive for PAB.2.The significance of combined marker in diagnosis of IBD:The sensitivity of p-ANCA or GAB alone to diagnose UC was 59.3%and 27.8%,the sensitivity was increased to 64.8%.The sensitivity of ASCA or PAB alone to diagnose CD was 35.8%.If combination them, the sensitivity was increased to 57.8%;3.The significance of combined marker in the differentiation of IBD:The sensitivity,specificity of combination of positive ASCA and negative p-ANCA to diagnose CD was 57.8%and 96.3%respectively.If combined with PAB and GAB,the specificity and PPV increased to 100%,but the sensitivity was 12.8%for CD.The sensitivity and specificity of combination of positive p-ANCA and negative ASCA to diagnose UC was 50%and 94.5%, respectively.If combined with PAB and GAB,the specificity and PPV increased to 100%,but the sensitivity was 19.4%for UC;4.The significance of combined marker in the differentiation between colonic CD and UC:The sensitivity,specificity and PPV of combined of p-ANCA and ASCA for differentiation between type of colonic CD and UC were 60%,96.3%and 28.6%,respectively.If combined with PAB and GAB, the specificity and PPV were 100%,but the sensitivity was 0.9%for type of colonic CD.The sensitivity,specificity and PPV of combined of p-ANCA and ASCA for differentiation of UC from type of colonic CD were 50%,94.5%and 28.9%,respectively.The specificity and PPV were 100%,and the sensitivity was 16.7%for UC,when combined with PAB and GAB.5.The significance of combined marker in the differentiation between IBD and DC:The specificity of these antibodies alone for differentiation between IBD and DC were above 95%.The specify of combined of PAB and GAB was higher,compared combination of p-ANCA and ASCA.6.An association of these markers with clinical feature:No statistically significant association of p-ANCA and GAB with clinical feature were seen in patients with UC,as well as ASCA in patients with CD.The percentage of PAB positivity was different between CD patients with≤40 and＞40 age at diagnosis(43.2%vs 20%,P=0.018).The percentage of PAB positivity was different between CD patients with EIM and without EIM(58.8%vs 31.5%,P=0.031).Summary:p-ANCA and GAB were specific for UC,while ASCA and PAB were specific for CD.Compared with these antibodies alone,it improved the sensitivity in diagnosis of UC if the combination of p-ANCA and GAB.Meanwhile the combination of ASCA and PAB,it improved the sensitivity in diagnosis of CD.If the combination of p-ANCA,ASCA,PAB and GAB,it improved the differentiation between UC and CD,expecially in isolated colonic disease,compared with the combination of p-ANCA and ASCA.GAB alone is useful for differentiation between IBD and non-IBD intestinal disorders.Antibody response to PAB was associated with early onset and extensive colitis in Chinese populations.Expression of p-ANCA and GAB was not associated with disease behavior of UC.ASCA was not associated with disease behavior of CD.Conclusion:1.These results suggested that polymorphisms of the DEFB1 gene were associated with an increased risk of UC,but not for CD.It may also determine disease phenotype such as:early onset,severe degree and extensive colitis.2.The association of rs2241880 and rs1045095 polymophism of ATG16L1 gene with IBD was not observed.3.The combination of p-ANCA,ASCA,PAB and GAB improved the differentiation between UC and CD,particularly in isolated colonic disease,compared with the combination of p-ANCA and ASCA testing4.Antibody response to PAB was associated with early onset and extensive colitis in Chinese populations,p-ANCA and GAB expression was not associated with disease behavior of UC.ASCA was not associated with disease behavior of CD.更多还原