The Role of Dysfunctional CCK-1R in Cholelithogenesis

描述（由申请人提供）：本实验室的长期目标是鉴定、定位和评估病理生理相关胆结石（LITH）基因的致石作用;从根本上了解胆固醇结石的遗传机制;并探索小鼠和最终人类LITH基因的基因型和表型。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对环境因素的反应，从而形成胆固醇结石。数量性状基因座（QTL）分析是一种强大的遗传技术，可以识别主要的、通常是限速的遗传缺陷，并将其与主要基因突变引起的次要下游病理生理效应区分开来。对胆石病易感品系129 S3/SvlmJ小鼠和抗胆石病AKR/J小鼠的杂交后代进行了QTL分析，确定了胆石病易感品系中胆石病易感基因的亚群。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，即胆囊中功能障碍的胆囊收缩素-1受体（CCK-1 R）在接受致石饮食的129 S3/SvlmJ小鼠胆固醇结石的形成中起着关键作用。此外，在胆固醇结石患者中观察到胆囊排空功能对外源性给予CCK-8的反应异常，表明胆囊CCK-1 R基因的结构和功能改变可能与人类胆固醇结石的形成有关。然而，CCK-1 R基因突变的致石机制的鉴定仍然是一个具有挑战性的任务。本研究旨在通过对CCK-1 R同源小鼠和CCK-1 R基因敲除小鼠的病理生理学研究，探讨CCK-1 R功能障碍的致石作用。此外，我们将研究胆囊郁积对胆固醇结晶和胆石形成的病理生理学影响，以及这些小鼠胆囊动力障碍的基因治疗。在本申请中，申请人提出（i）阐明突变的CCK-1 R是否由于受体-G蛋白偶联的缺陷而导致胆囊停滞;（ii）确定由胆囊运动不足诱导的改变，所述改变是具有突变的CCK-1 R的小鼠中胆固醇快速结晶和胆结石形成的原因;以及（iii）探索慢病毒介导的小鼠CCK-1 R基因转移是否能预防胆囊运动功能减退小鼠胆固醇结石形成。由于人类和小鼠基因组之间的密切同源性，识别功能失调的CCK-1 R在小鼠中的致石作用可能阐明以前未知的，但病理生理学相关的人类胆固醇胆石症的遗传决定因素。

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.