The Role of Dysfunctional CCK-1R in Cholelithogenesis

描述（由申请人提供）：该实验室的长期目标是识别、定位和估计与病理生理相关的胆结石（LITH）基因的产石作用；在基本水平上了解胆固醇胆结石的遗传机制；探索LITH基因在小鼠和最终在人类中的基因型和表型。对人类和小鼠的研究清楚地表明，复杂的遗传基础决定了个体在环境因素的影响下发生胆固醇胆结石的易感性。数量性状位点（QTL）分析是一种强大的遗传技术，可以识别原发性的，通常是限制性的遗传缺陷，并将它们与原发性基因突变引起的继发性下游病理生理效应区分开来。我们对胆结石易感小鼠129S3/SvlmJ与耐药小鼠AKR/J杂交后代进行了QTL分析，确定了易感菌株所具有的胆结石易感基因亚群。我们从这些小鼠研究中获得的分子和遗传数据支持这样的观点，即胆囊功能失调的胆囊收缩素-1受体（CCK-1R）在129S3/SvlmJ小鼠致石饮食中胆固醇胆结石的形成中起关键作用。此外，在胆固醇结石患者中观察到外源性CCK-8引起的胆囊排空功能异常，这表明胆囊CCK-1R基因的结构和功能改变可能参与了人类胆固醇结石的形成。然而，鉴定突变的CCK-1R基因的成岩机制仍然是一项具有挑战性的任务。本应用程序将通过系统地研究CCK-1R在一些“人造”小鼠品系（如CCK-1R同源小鼠和CCK-1R敲除小鼠）中的病理生理功能，重点确定功能失调的CCK-1R的造石作用。此外，我们还将研究胆囊淤积对胆固醇结晶和胆囊结石形成的病理生理影响，以及胆囊运动障碍的基因治疗。在本申请中，申请人提出(i)阐明突变的CCK-1R是否由于受体- g蛋白偶联缺陷而导致胆囊淤积；（ii）在CCK-1R突变小鼠中，确定胆囊功能低下导致的胆固醇快速结晶和胆结石形成的改变；（iii）探索慢病毒介导的小鼠CCK-1R基因的转移是否能阻止胆囊功能低下小鼠胆固醇胆石的形成。由于人类和小鼠基因组之间的密切同源性，鉴定小鼠功能失调的CCK-1R的产石作用可能阐明以前未知的但与人类胆固醇胆石症病理生理相关的遗传决定因素。

DESCRIPTION (provided by applicant): The long-term objectives of this laboratory are to identify, localize, and estimate the lithogenic effects of pathophysiologically relevant gallstone (LITH) genes; understand at a fundamental level the genetic mechanisms of cholesterol gallstones; and explore the genotypes and phenotypes of LITH genes in mice and eventually in humans. Studies on both humans and mice have clearly demonstrated that a complex genetic basis determines the individual predisposition to develop cholesterol gallstones in response to environmental factors. A powerful genetic technique, quantitative trait locus (QTL) analysis can identify primary, usually rate- limiting genetic defects and discriminate them from secondary downstream pathophysiologic effects caused by mutations of the primary genes. We performed a QTL analysis in intercross progeny of gallstone-susceptible 129S3/SvlmJ mice and resistant AKR/J mice, and determined the subset of gallstone susceptibility genes possessed in the susceptible strain. Our molecular and genetic data from these mouse studies support the notion that dysfunctional cholecystokinin-1 receptor (CCK-1R) in the gallbladder plays a critical role in the formation of cholesterol gallstones in 129S3/SvlmJ mice challenged to a lithogenic diet. Furthermore, abnormalities in gallbladder emptying function in response to exogenously administered CCK-8 have been observed in patients with cholesterol gallstones, suggesting that altered structure and function of the gallbladder CCK-1R gene could be involved in the formation of cholesterol gallstones in humans. However, the identification of the lithogenic mechanisms of the mutated CCK-1R gene still remains a challenging task. This application will be focused on identifying the lithogenic effects of dysfunctional CCK-1R by systematically studying its pathophysiological functions in some "manufactured" mouse strains such as CCK-1R congenic mice and CCK-1R knockout mice. Also, we will investigate pathophysiological effects of gallbladder stasis on cholesterol crystallization and gallstone formation, as well as gene therapy of gallbladder dysmotility in these mice. In this application, the applicant proposes to (i) elucidate whether the mutated CCK-1R results in gallbladder stasis due to a defect in receptor-G protein coupling; (ii) determine the alterations induced by gallbladder hypomotility that account for rapid cholesterol crystallization and gallstone formation in mice with the mutated CCK-1R; and (iii) explore whether lentivirus-mediated transfer of the mouse CCK-1R gene prevents cholesterol gallstone formation in mice with gallbladder hypomotility. Due to the close homology between human and mouse genomes, the identification of lithogenic effects of dysfunctional CCK-1R in mice may elucidate previously unknown but pathophysiologically relevant genetic determinants of cholesterol cholelithiasis in humans.