【作者】 李攀；

【导师】 钱家鸣；

【作者基本信息】 北京协和医学院 ， 内科学， 2011， 博士

【摘要】 背景及目的：炎症性肠病(IBD)主要包括溃疡性结肠炎(UC)和克罗恩病(CD)。目前公认IBD的病因是以遗传易感性为基础、环境因素参与,粘膜免疫系统对肠腔内抗原物质的异常免疫应答而造成的肠道损伤。此外,研究发现IBD患者易合并骨代谢异常。维生素D既往以调节钙磷代谢的作用而众所周知,近年来研究发现其还具有抗感染和免疫调节的作用。国外已有大量的基础研究证实维生素D参与了IBD的发病,并发现IBD患者存在维生素D的缺乏,血清25(OH)D水平与疾病活动程度相关,但国内目前尚无这方面的研究。另外,关于维生素D对IBD骨代谢异常的影响,国外研究结果并不一致,而国内仅有少数这方面的报道。维生素D通过与维生素D受体结合而发挥作用,目前研究发现VDR具有基因多态性,且其与IBD的易感性相关。另有研究发现维生素D可影响DEFB1、TLR4和IRF5的表达,且已有国外研究证实它们的基因多态性与IBD的易感性相关。国内目前尚无IRF5、DEFB1基因多态性与IBD易感性的报道,且无上述基因多态性关联作用的报道。由于IBD为多基因复杂疾病,单个基因多态性对疾病的影响可能较为微弱。因此,本研究旨在：1、探讨中国人血清25(OH)D水平与IBD疾病活动程度的关系,同时一并分析血清25(OH)D水平与IBD骨密度的关系及IBD患者发生骨密度异常的危险因素。2、探讨维生素D作用通路中VDR、DEFB1、TUR4、IRF5基因多态性与IBD易感性的关系,并拟对阳性位点之间的关联作用进行分析。第一部分维生素D与炎症性肠病临床特征的相关性研究方法：收集78例UC患者、57例CD患者及122例正常对照人群的临床资料和血清,所有样本均进行血清25(OH)D的检测(测量单位为ng/ml), IBD患者进行骨密度的检测。结果：1、UC组、CD组、正常对照组血清25(OH)D平均值分别为10.65±4.87,10.79±5.14,12.87±4.40。各组间25(OH)D比较结果：UC组VS对照组,P=0.017；CD组VS对照组,P=0.020. UC VS CD, P=0.874。2、UC组按疾病活动程度分类,血清25(OH)D平均值分别为：缓解期14.38±4.71,轻度10.81±4.70,中度10.05±4.17,重度6.91±3.76。分别进行两两比较,缓解期与各活动期、轻度与重度之间的差异均具有统计学意义(P<0.05)。3、CD组采用Harvey Bradshaw Score评分对疾病活动程度进行分类,血清25(OH)D平均值分别为：0-4分12.28±4.60,5-8分7.99±3.43,>8分17.9±14.00。因>8分组仅2人,故仅对0-4分和5-8分组进行比较,P<0.01。4、IBD并发低骨量/骨质疏松的危险因素分析：UC组：激素累积量(OR=2.821,95%CI1.365-5.831,P=0.005)和血清25(OH)D水平(OR=0.866,95%C10.757-0.992,P=0.038)是其危险因素。2、CD组：激素累积量(OR=3.362,95%CI1.218～9.821,P=0.019)和BMI(OR=0.651,95%CI0.457～0.929, P=0.018)是其危险因素。结论：1、UC和CD患者血清25(OH)D水平均较正常人群降低。2、UC患者血清25(OH)D水平与疾病活动程度呈负相关；CD患者中度活动期(简化CDAI评分4-8分)血清25(OH)D水平显著低于缓解期(简化CDAI评分0-4分)。3、多因素分析显示激素累积量是UC和CD患者发生骨量减少/骨质疏松共同的危险因素,此外,25(OH)D水平是UC患者发生骨量减少/骨质疏松的危险因素,BMI是CD患者发生骨量减少/骨质疏松的危险因素。第二部分维生素D作用通路中相关炎性因子(VDR, DEFB1, TLR4, IRF5)基因多态性与IBD遗传易感性的研究方法：收集300例UC患者,158例CD患者及302正常对照人群的临床资料和血清。采用质谱Sequenom法对VDR, DEFB1、IRF5、TLR4四个基因共14个位点的基因型进行检测。结果：1、IRF5基因rs3807306位点的A等位基因可增加UC的易感性(P=0.007)。2、IRF5基因rs3807306位点的AA纯合基因型(P=0.028)和rs4728142位点的AA纯合基因型(P=0.008)可增加UC的易感性。3、IRF5基因区域的GGATT单体型(P=0.0002)是UC的危险单体型。4、携带IRF5基因rs4728142位点(P=0.011)的A/A纯合基因型或TLR4基因rs7037117位点(P=0.038)的A/A纯合基因型、rs1927907位点(P=0.046)的A/A纯合基因型的UC患者肠道病变程度相对较轻。5、IRF5基因区域的GTACC单体型是CD的危险单体型(P=0.0223)。6、携带IRF5基因rs2004640(P=0.013)位点G/G纯合基因型的CD患者更易出现结肠的受累,携带DEFB1基因rs2978880位点(P=0.006)C/C纯合基因型的CD患者肠道手术的风险相对更高。结论：1、IRF5基因的rs3807306位点的多态性与UC的易感性相关,rs4728142位点的多态性可能有增加UC易感性的风险,但这两个位点与CD的易感性无关。2、VDR基因rs11574143、rs4760648、rs1544410位点、DEFB1基因rs1799946、rs2741136、rs2978880、rs2741108位点,TLR4基因rs7037117、rs1927907位点及IRF5基因rs2004640、rs1874328、rs7808907位点的多态性与UC和CD的易感性均无关。3、IRF5基因rs4728142位点、TLR4基因rs7037117、rs1927907位点的多态性与UC的临床表型相关。IRF5基因rs2004640位点、DEFB1基因rs2978880位点的多态性与CD的临床表型相关。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 pathogenesis of IBD is reported associated with genetic factors, environmental factors and abnormal immune factors. In addition, IBD patients are at higher risk of developing osteoporosis and osteopenia than the general population. The role of vitamin D in calcium, phosphorus, and bone metabolism has been recognized since many years ago. Recently, it has been discovered that vitamin D can play a role in autoimmune diseases, infectious diseases and tumor diseases. Many basic research have confirmed there is a link between vitamin D and pathogenesis of IBD. Some clinical study abroad found vitamin D insufficiency is common in IBD patients and serum25(OH) vitamin D level correlate with the activity of the disease. However, there is no similar study in China. Results from different researches about impact of vitamin D on bone metabolism in IBD were not consistent and there was only one single-center study of small samples about that in China. Vitamin D exert its effect by binding to vitamin D receptor. Vitamin D gene polymorphisms had been shown related to the susceptibility of many autoimmune diseases, including IBD. Vitamin D can directly and indirectly influence the expression of DEFB1, TLR4, and IRF5. All of these gene polymorphisms also had been proved associated with IBD in Caucasian population. There were only few reports studied whether the TLR4and VDR gene were associated with IBD in Chinese Han population and no reports about association study among these gene in IBD. Therefore, in our study we aim to:1. Comparing serum25(OH) vitamin D levels in patients with IBD and matched controls, to evaluate disease characteristics that correlate with low25(OH) vitamin D level and the consequence of low25(OH) vitamin D level on bone mineral density. Meanwhile, we analyzed risk factors for abnormal bone metabolism.2. Investigating the relationship between single nucleotide polymorphisms of vitamin D working pathway related genes VDR, DEFB1, TLR4and IRF5and IBD genetic susceptibility, as well as clinical phenotype.Part1. The Study of Correlation between Serum25(OH) Vitamin D Levels and Clinical Characteristics of IBD.Methods:135IBD patients (78UC and57CD) and122controls were collected. Serum25(OH) vitamin D levels were tested in all of participants (the unit of measurement was ng/ml). Spine and femoral neck bone mineral density were tested in IBD patients.Results:1. Serum25(OH) vitamin D level in IBD group were significantly lower than controls.(UC vs controls:10.65±4.87vs12.87±4.40, P=0.017; CD vs controls:10.79±5.14vs12.87±4.40, P=0.020).2. Serum25(OH) vitamin D level in UC group classified by disease activity:remission,14.38±4.71; mild activity,10.81±4.70; moderate activity,10.05±4.17; severe activity,6.91±3.76. The comparison of serum25(OH) vitamin D level between every two groups showed:the disparities between remission and each activity as well as between mild activity and severe activity were significantly, P<0.05.3. Serum25(OH) vitamin D level in CD group classified by Harvey Bradshaw Score:0-4score,12.28±4.60;5-8score,7.99±3.43;>8score,17.9±14.00. There were only two persons scored>8, so this group was not involved in comparison. The comparison of serum25(OH) vitamin D level between0-4score and5-8score group was significantly, P<0.01.4. Multivariate analysis for osteoporosis or osteopenia in IBD showed:The total doses of glucocorticoids (OR=2.821,95%CI1.365±5.831, P=0.005) and serum25(OH) vitamin D insufficiency (OR=0.866,95%CI0.757～0.992, P=0.038) were risk factors for osteoporosis or osteopenia in UC. The total doses of glucocorticoids (OR=3.362,95%CI1.218±9.821, P=0.019) and low BMI (OR=0.651,95%CI0.457±0.929, P=0.018) were risk factors for osteoporosis or osteopenia in CD.Conclusions:1. Serum25(OH) vitamin D level in both UC and CD group were significantly lower than controls.2. Serum25(OH) vitamin D level in UC correlated negatively with the severity of disease activity; Serum25(OH) vitamin D levels in CD were lower in those scored by5-8than0-4.3. Multivariate analysis for osteoporosis or osteopenia in IBD showed:The total doses of glucocorticoids were common risk factors for osteoporosis or osteopenia both in UC and in CD. However, serum25(OH) vitamin D insufficiency and low BMI were respectively risk factor for osteoporosis or osteopenia in UC and in CD.Part2. The Study of Vitamin D Working Pathway Related Genes:VDR, DEFB1, TLR4and IRF5with Genetic Susceptibility of IBD in Han Population of China.Methods:300UC patients,158CD patients,302healthy controls of Chinese Han population were included in our study. Fourteen tag SNPs of four genes were genotyped by Sequenom MassARRAY method.Results:1. The frequency of allele A in rs3807306site (P=0.007) of IRF5gene in UC patients was higher than in controls.2. The frequency of AA genotype both in rs3807306site (P=0.028) and rs4728142site (P=0.008) of IRF5gene were higher than in controls.3. Haplotypes GGATT (P=0.0002) in IRF5gene was related to disease susceptibility of UC.4. A significantly higher frequency of AA genotype in rs4728142site (P=0.011) of IRF5gene, AA genotype in rs7037117site (P=0.038) and in rs1927907site (P=0.046) of TLR4were observed among UC patients with mild activity.5. Haplotypes GTACC (P=0.0223) in IRF5gene is related to disease susceptibility of CD.6. A significantly higher frequency of GG genotype in rs2004640site (P=0.013) of IRF5and rs2978880site (P=0.006) of DEFB1were respectively observed among CD patients with colon involved and with surgery.Conclusions:1. rs3807306polymorphism of IRF5was associated with disease susceptibility of UC and rs4728142polymorphism of IRF5might be associated with the risk for UC. However, both these sites were not associated with CD.2. rs11574143、 rs4760648、rs1544410sites of VDR、rs1799946、rs2741136、rs2978880、rs2741108sites of DEFB1, rs7037117、rs1927907sites of TLR4and rs2004640、rsl874328、rs7808907sites of IRF5were not observed associated with genetic susceptibility of both UC and CD.3. rs4728142site of IRF5, rs7037117, rs1927907sites of TLR4and rs2978880site of DEFB1were observed associated with IBD phenotype.更多还原