CTLA-4基因多态性调节溃疡性结肠炎患者CTLA-4 mRNA稳定性和蛋白水平

目的 研究细胞毒T淋巴细胞相关抗原4(cytotoxic T-lymphocyte associated antigen 4,CTLA-4)基因3'非转录区(AT)n重复序列多态性对溃疡性结肠炎(ulcerative colitis,UC)患者CTLA-4mRNA稳定性和基因表达的影响.方法 采用实时定量PCR方法检测膜型CTLA-4(full length CTLA-4,flCTLA-4)和可溶性CTLA-4(soluble CTLA-4,sCTLA-4)mRNA表达,半衰期法分析其mRNA稳定性.免疫组化检测flCTLA-4蛋白表达.酶联免疫吸附实验测定sCTLA-4蛋白水平.荧光PCR-毛细管电泳技术检测300例UC患者和700名健康对照者CTLA-4基因(AT)n重复序列多态性.结果 活动期UC患者肠黏膜sCTLA-4 mRNA的表达显著低于缓解期UC患者(P=0.004).在UC患者中,(AT)n重复序列长等位基因携带者表达低水平的flCTLA-4和sCTLA-4 mRNA以及sCTLA-4蛋白(均P＜0.01).携带长等位基因的UC患者CTLA-4 mRNA的稳定性明显降低.UC患者CTLA-4基因(AT)n重复序列长等位基因携带者(≥116 bp)频率显著高于正常对照组(P＜0.01),且与广泛型结肠炎相关(P=0.008).结论 UC患者CTLA-4基因(AT)n重复序列多态性与CTLA-4基因表达水平相关,携带(AT)n重复序列长等位基因的UC患者CTLA-4 mRNA及蛋白的表达降低,提示CTLA-4基因在UC遗传免疫发病机制中起重要作用.

CTLA-4 gene polymorphism regulates CTLA-4 mRNA stability and protein level in patients with ulcerative colitis

Objective To investigate the effect of (AT)n repeat polymorphism of the 3'untranslated region in cytotoxic T-lymphocyte associated antigen 4 (CTLA-4)gene on CTLA-4 mRNA stability and full length (flCTLA-4) and soluble CTLA4 (sCTLA-4) expression in ulcerative colitis (UC). Methods flCTLA-4 mRNA in colonic biopsies and sCTLA-4 mRNA stability in peripheral blood mononuclear cells of UC patients were measured by quantitative PCR and half-life, respectively. The protein expression of flCTLA-4 in colonic biopsies and sCTLA-4 in sera of UC patients were determined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The polymorphism of CTLA-4 (AT)n repeats in 300 UC and 700 age and sex matched healthy controls was genotyped by fluorescent PCR. Results Among the UC patients, sCTLA-4 mRNA expression levels were decreased in active disease compared to non-active disease (P=0. 004). Carriers of the longer alleles of the (AT)n repeats expressed lower levels of flCTLA-4 and sCTLA - 4 mRNA and sCTLA - 4 protein than those of the shorter alleles in UC (all P＜0.01), and mRNA with long (AT)n repeat alleles has shorter half-life than mRNA with short alleles and, hence, are unstable. The frequency of long allele carriers of CTLA-4 (AT)n repeats was significantly higher in UC patients than in the healthy controls (22.0% vs. 6.3%, P＜0.01, OR=4.21, 95% CI: 2.79-6.33), and associated with extensive colitis (P=0. 008). Conclusion CTLA-4 gene expression levels were associated with (AT)n repeat polymorphisms in UC patients. The expression of CTLA-4 mRNA and protein were decreased in carriers of the longer alleles of the (AT)n repeats of CTLA-4 gene. This study suggests that CTLA-4 plays an important role in genetic risk and pathophysiology for UC in central China.