TL1A基因多态性对炎症性肠病粘膜固有层巨噬细胞功能的影响

The pathogenesis of inflammatory bowel disease(IBD) remains unclear. Recently it is thought that abnormalities of innate immunity play an important role in IBD by genome wide association study. Our preliminary study has shown that TL1A gene polymorphisms were associated with IBD. As one of main source of TL1A, the lamina propria macrophages also play an important role in the pathogenesis of IBD. However we don't know whether the polymorphisms of TL1A have an impact on the mRNA expression and protein level of TL1A, and on functions of lamina propria macrophage, and on pathogenesis of IBD and mechanisms. The present study aims to genotype TL1A gene polymorphisms in IBD patietns and healthy controls by using TaqMan/MGB probe, and to study effects of TL1A haplotype on TL1A mRNA and protein expressions at ELISA、RT-PCR and Western-blotting levels, and to take expression microarray, cell culture, flow cytometry, RT-PCR and other methods to investigate the impact of TL1A gene polymorphisms on the phenotypes and functions of lamina propria macrophages in both in vitro and in vivo(transgenic mice) and to clarify the role of TL1A gene on functions of macrophges in the pathogenesis of IBD.

炎症性肠病发病机制未明，近来的基因组学研究发现天然免疫功能异常起到重要作用。我们的前期研究发现TL1A基因多态性与IBD相关。作为TL1A主要来源之一的固有层巨噬细胞在IBD发病中也起着重要作用。但尚未明确TL1A基因多态性是否影响TL1A的mRNA和蛋白表达，也未明确TL1A基因多态性对固有层巨噬细胞功能的影响以及这种影响在IBD发病中的重要性和具体机制。本项目采用TaqMan/MGB探针法研究TL1A基因多态性与IBD的关系，采用ELISA、RT-PCR、Western-blotting等技术研究TL1A基因多态性对TL1A基因表达水平的影响，采用转基因小鼠、表达谱芯片、细胞培养、流式细胞术以及RT-PCR等技术，探讨TL1A基因型对肠固有层巨噬细胞表型特征和功能的影响，以阐明TL1A基因多态性对固有层巨噬细胞功能的影响在IBD发病中的作用机制。

肿瘤坏死样因子配体1A (TL1A）基因多态性与炎症性肠病（IBD）相关。作为TL1A主要来源之一的固有层巨噬细胞在IBD发病中也起着重要作用。本研究首先从基因水平上寻找中国汉族人特有的TNFSF15基因多态性位点，研究其与IBD的关系。发现rs6478108-rs6478109-rs7848647-rs7865494-rs7849487基因多态性与克罗恩病（CD）相关，由于5个SNP位点间存在强烈的连锁不平衡，只选择CD患者rs6478108位点基因型与各临床表型进行相关性分析，CD患者年龄、病变范围及疾病行为(狭窄、穿孔)等临床表型和基因型无明显相关。rs10114470基因多态性与CD相关，在CD患者中CC基因型高于正常对照组，与之对应C等位基因高于正常对照组；CD患者年龄、病变范围、疾病行为(狭窄、穿孔)等临床表型和基因型有相关性。 rs4263839基因多态性与溃疡性结肠炎（UC）相关。其次从局部和系统水平上探讨中国IBD患者TL1A，DR3和DcR3的mRNA和蛋白表达水平。结果表明，CD和UC患者的TL1A及DR3mRNA表达较对照组增加。这种血清及肠粘膜水平的改变与其基因型无关。最后我们构建了TL1A过表达的CAG-TNFSF15品系转基因小鼠，并获得国外实验室赠送的TL1A—/—基因敲除鼠，与野生型对照组小鼠同时用于构建葡聚糖硫酸钠（DSS）诱导性结肠炎，评估了造模后不同品系小鼠体重、DAI评分、结肠长度和组织学评分改变。通过实时定量PCR检测肠粘膜DR3，IL-1β，IL-10，caspase 1，TNF-α，IL-23a，P62，NF-ĸB，IFN-γ的mRNA表达水平，western blot检测DR3，NLRP3，caspase 1，P62，NF-ĸB的蛋白表达水平。CBA检测血清中IL-1β，IL-6，IL-10，TNF-a,IFN-r，IL-17a的表达水平。