【作者】 裴凤华；

【导师】 迟宝荣；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 背景及目的溃疡性结肠炎(Ulcerative colitis, UC)是原因不明的肠道慢性非特异性疾病,对其发病机制的研究目前集中在遗传因素,环境因素和免疫调节紊乱等方面。UC是多基因疾病,目前发现与UC易感性相关的基因有二十余种,分布于1-19号染色体上。维生素D具有重要的免疫调节和抗微生物的作用,可调控上皮细胞抑菌肽的产生,参与肠道微生态的平衡,调节多种细胞的生长和分化。维生素D受体(Vitamin D receptor,VDR)基因位于第12号染色体,是维生素D发挥作用的效应基因,在人体内具有重要生理作用。VDR基因具有SNP多态性,含有多个限制性内切酶位点,如FokⅠ、BsmⅠ、ApaⅠ、TaqⅠ、poly (A)和Cdx-2等,这些SNP位点多态性与多种疾病密切相关。国外有研究表明,VDR基因多态性与UC易感性相关。由于基因多态性与种族和地域有密切关系,在我国,VDR基因多态性是否与UC易感性相关,目前还没有报道。人β-防御素(human bata-defensin,hBD)是在人肠道上皮细胞表达的抗微生物多肽,是目前研究得最多的抑菌肽,具有强大的抗菌活性,同时参与机体的先天免疫和后天免疫。在肠道组织中,p-防御素表达的改变与炎症过程的相关性还不是十分清楚。目前对肠道组织p-防御素的表达情况研究的比较多,但是同时研究VDR和β-防御素在肠道组织表达的文献很少。本课题探讨VDR基因多态性与中国北方汉族UC发病的相关性及活动性UC结肠组织中VDR mRNA和β-防御素1(hBD-1) mRNA、β-防御素2 (hBD-2) mRNA的表达,为进一步揭示UC的发病机制提供依据。材料与方法应用PCR-限制性片段长度多态性(PCR-RFLP)方法,检测218例中国北方汉族人群UC患者和251例汉族健康对照者的VDR基因四个(ApaⅠ、TaqⅠ、BsmⅠ和Fok I)限制性内切酶位点的多态性。应用实时荧光定量PCR (Real-time PCR)方法检测30例活动性UC患者和32例正常对照者的肠道上皮组织中VDR mRNA、hBD-1 mRNA和hBD-2 mRNA的表达。结果VDR基因在中国北方汉族人群中具有多态性,四个位点基因型均符合Hardy-Weinberg平衡,表明本研究所纳入样本能够代表总体基因型分布规律。在VDR基因的四个多态性位点中,只有BamⅠ位点与UC易感性相关。与对照组相比,Bb基因型频率在UC组明显增加(28.4% vs.18.7%χ2=6.044, P=0.014; OR=1.739,95%CI=1.122-2.697),B等位基因频率在病例组比对照组明显增加(14.7% vs. 7.8%χ2=6.222, P=0.013; OR=1.670,95% CI= 1.113-2.506)。其余三个多态性位点(ApaⅠ、TaqⅠ和FokⅠ)各基因型及等位基因频率均与UC易感性无关。VDR基因多态性与UC临床表型无相关性。在活动性UC结肠粘膜中,VDR mRNA和hBD-2 mRNA表达均较正常粘膜升高(P<0.05), hBD-1 mRNA的表达在两组中无统计学意义。结论在中国北方汉族人群中,VDR基因Bsm I位点多态性与UC易感性具有相关性,与UC临床表型无相关性。VDR基因可能是通过上调hBD-2 mRNA在肠道的表达而影响UC的发病。更多还原

【Abstract】 Background and purposeUlcerative colitis (UC) is a chronic inflammation of the intestine. The etiopathogenesis of UC has not been clearly elucidated, however, its development is influenced by genetic, environmental and immunological factors.In its place UC is considered to be complex polygenic diseases. Several studies have demonstrated the identification of 20 gene locus located on chromosome 1-19 as a susceptibility gene in UC.Vitamin D is an important immune system regulator and antimicrobial. Many data identify vitamin D as a key regulator of gastrointestinal homeostasis and an important player in regulation of the innate immune response.These effects of vitamin D are thought to be mediated through the Vitamin D receptor(VDR), located on chromosome 12. Several polymorphic sites were found on the VDR gene, such as Fok I, Bsm I, Apa I, Taq I, poly (A) and Cdx2. The study found that VDR gene polymorphism has been associated with increased susceptibility to UC in overseas patients, but have not study in Chinese patients.Human bata-defensin (hBD) are antimicrobial peptides secreted by endothelial cells in intestinal tract as a component of the innate host defence. In the gastrointestinal tract, these peptides have bactericidal activity. This antimicrobial quality allows defensins to protect the host epithelium and stem cells from virulent pathogens and also help to regulate the number and composition of commensal microbiotal. The relationship between gut inflammation and the timing of defensin deficiency is unclear. There was not study about VDR and defensin at the same time. This study was designed to determine if polymorphisms of the VDR gene are associated with UC susceptibility in within north China Han people. Meanwhile, to offer the evidence of genetic mechanism in UC.Methods A total of 218 UC patients and 251 healthy controls were genotyped for VDR gene polymorphisms (Apa I, Taq I, Bsm I and Fok I) using PCR-RFLP assay. To study of expression of VDR mRNA、hBD-1 mRNA and hBD-2 mRNA in epithelial tissue of 30 patients with active UC and 32 controls using Real-time PCR assay. Results Among the four examined VDR gene polymorphisms, the Bsm I polymorphism showed a slightly higher distribution in our study population than that of previous studies. The increased frequency of the Bb genotype of the Bsm I VDR gene polymorphism was associated with UC as compared to healthy controls (28.4% vs.18.7%χ2=6.044, P=0.014, OR=1.739,95% CI=1.122-2.697). Moreover, Bsm I polymorphic allele (B) frequency was significantly increased in the UC cases, as compared to the healthy controls (14.7% vs.7.8%χ2=6.222, P=0.013; OR=1.670,95% CI=1.113-2.506). The other three VDR gene polymorphisms (Apa I, Taq I, and Fok I) were not associated with UC susceptibility. None of these four VDR polymorphisms had statistical association with clinicopatholgical parameters of these UC patients.The express of VDR mRNA and hBD-2 mRNA incresed in active UC mucosa compared with normal mucosa (P<0.05). However, the express of hBD-1 mRNA was not statistically significant in two groups.Conclusion This study demonstrated a probable association of the Bsm I polymorphism of the VDR gene with ulcerative colitis susceptibility in Han Chinese. None of VDR polymorphisms had statistical association with clinicopatholgical parameters of these UC patients. The mechanism of VDR gene may be increasing the expression of hBD-2 mRNA in occurrence of UC.更多还原