The Role of Dysfunctional CCK-1R in Cholelithogenesis

描述(申请人提供)：该实验室的长期目标是识别、定位和评估病理生理学相关的胆结石(LITH)基因的致石作用；在基础水平上了解胆固醇结石的遗传机制；并探索LITH基因在小鼠体内以及最终在人类中的基因类型和表型。对人类和老鼠的研究都清楚地表明，复杂的遗传基础决定了个体对环境因素产生胆固醇结石的易感性。作为一种强大的遗传技术，数量性状基因座(QTL)分析可以识别原发的、通常是限速的遗传缺陷，并将它们与由主基因突变引起的次要下游病理生理效应区分开来。我们对胆结石易感的129S3/SvlmJ小鼠和耐药的AKR/J小鼠的杂交后代进行了QTL分析，并确定了易感品系中存在的胆结石易感基因亚群。我们来自这些小鼠研究的分子和遗传学数据支持这样的观点，即胆囊中功能失调的CCK-1R在挑战致石饮食的129S3/SvlmJ小鼠的胆固醇结石形成中起关键作用。此外，在胆固醇结石患者中观察到外源性给予CCK-8导致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，对突变的CCK-1R基因的致石机制的鉴定仍然是一个具有挑战性的任务。这项应用将集中于通过系统地研究CCK-1R在一些“制造”的小鼠品系中的病理生理功能来鉴定功能失调的CCK-1R的致石作用，例如CCK-1R同源基因小鼠和CCK-1R基因敲除小鼠。此外，我们还将研究胆汁淤积对胆固醇结晶和胆结石形成的病理生理影响，以及对这些小鼠胆汁动力障碍的基因治疗。在本申请中，申请人提议：(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停滞；(Ii)确定携带CCK-1R突变的小鼠的胆固醇快速结晶和胆结石形成的原因是由胆囊动力低下引起的变化；以及(Iii)探索慢病毒介导的小鼠CCK-1R基因转移是否能防止胆囊运动低下的小鼠胆固醇结石的形成。由于人类和小鼠基因组之间的高度同源性，鉴定功能失调的CCK-1R在小鼠中的致石作用可能阐明先前未知的、但与人类胆固醇胆石症的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.