In Vivo Investigations Using Microdialysis Sampling

炎症性肠病（IBD）是一种胃肠道慢性炎症，包括克罗恩病和溃疡性结肠炎。参与IBD炎症反应介导的主要酶是环氧合酶（COX）、脂氧合酶（LOX）和一氧化氮合酶（NOS）。这些酶被诱导和引起炎症的确切机制仍未完全了解。本提案的目的是开发结肠粘膜的体内微透析取样，与微量分析技术结合使用，以监测IBD动物模型中这三种酶系统的活性。作为该项目的一部分，将开发三种分析方法。首先，用9 -蒽基重氮甲烷（ADAM）或2-（2,3 -萘酰亚胺）乙基三氟甲烷磺酸盐（NE-OTf）衍生化后，用CE-LIF测定COX产物前列腺素E2和血栓素B2， LOX产物羟二碳四烯酸、5-HETE和15-HETE以及白三烯B4。其次，经萘-2,3-二甲醛/氰化物（NDA/CN）衍生后，用CE-LIF测定NOS和精氨酸酶产物瓜氨酸和鸟氨酸。第三，硝酸盐和亚硝酸盐将用于监测NOS活性，并在与2,3 -二氨基萘（DAN）衍生后直接通过CE-UV或CE-LIF测定。将采用2,4,6-三硝基苯磺酸对大鼠进行化学诱导，建立IBD动物模型。该模型的实现将涉及开发微透析探针植入方案，评估探针植入引起的任何组织反应，并验证探针位于炎症区域。然后，微透析取样和开发的分析方法将用于在动物发展为IBD时持续监测模型和对照动物中的酶活性。

Inflammatory bowel disease (IBD) is a chronic inflammation of the GI tract that includes Crohn's disease and ulcerative colitis. The major enzymes involved in mediating the inflammatory response in IBD are cyclooxygenase (COX), lipoxygenase (LOX) and nitric oxide synthase (NOS). The exact mechanisms by which these enzymes are induced and inflammation is caused are still not fully understood. The objective of this proposal is to develop in vivo microdialysis sampling in the colonic mucosa to be used in conjunction with microanalytical techniques to monitor the activity of these three enzyme systems in an animal model of IBD. Three analytical methods will be developed as part of this project. First, the COX products prostaglandin E2 and thromboxane B2 and the LOX products the hydroxyeicosatetraenoic acids, 5-HETE and 15-HETE, and leukotriene B4 will be determined by CE-LIF after derivitization with either 9anthryldiazomethane (ADAM) or 2-(2, 3-naphthalimino) ethyltrifluoromethanesulphonate (NE-OTf). Second, the NOS and arginase products citrulline and ornithine will be determined using CE-LIF after derivatization by naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN). Third, nitrate and nitrite will be used to monitor NOS activity and determined directly by CE-UV or by CE-LIF following derivatization with 2,3�diaminoaphtalene (DAN). The animal model of IBD to be used will be by chemical induction using 2,4,6-trinitrobenzene sulfonic acid in the rat. Implementation of this model will involve development of microdialysis probe implantation protocols, evaluation of any tissue response due to probe implantation, and verification that the probe is located in the inflamed region. Microdialysis sampling and the developed analytical methods will then be used to continuously monitor the enzyme activity in the model as the animal develops IBD and in control animals.