MECHANISMS OF LUNG BETA2AR FUNCTION IN TRANSGENIC MICE

β-激动剂是一种有效的支气管扩张剂，经常用于治疗哮喘等支气管痉挛肺疾病。在完整的肺中，β-激动剂介导的支气管扩张可能是由于许多不同类型的细胞上的β_2ARs被激活所致。然而，这些细胞类型中任何一种的B2AR信号的相对贡献尚不清楚。在体内描述这些细胞特异性效应的一种潜在方法是利用转基因选择性地过度表达并激活特定细胞上的Beta2AR。通过使用细胞特异性启动子来靶向受体表达，我们建议建立多个转基因小鼠系，使其在呼吸道上皮和气道平滑肌中过度表达Beta2AR。初步的体外和体内研究表明，气道上皮细胞和平滑肌之间存在显著的Beta2AR介导的通讯，在呼吸道上皮细胞中，Beta2AR介导的通讯功能的过度表达可降低支气管的过度活动。在气道平滑肌上的过度表达导致另一种表型，这允许评估关于气道功能的Beta2AR调节的每一条途径。利用分子生物学、药理学和生理学技术相结合的策略，我们建议使用转基因方法：(1)确定上皮细胞β_2AR在调节平滑肌功能中的作用；(2)选择性地描述气道平滑肌中的β_2AR信号和功能；以及(3)评估上皮和平滑肌β_2AR过表达对气道炎症和支气管高反应性的影响。应聘者已经完成了肺和重症护理的临床研究。在此期间，候选人使用基本的分子生物学技术开始研究Beta2AR基因的表达。这位候选人开始有兴趣继续这些研究，并决定搬到辛辛那提大学医学中心斯蒂芬·利格特的实验室。在获得更丰富的实验室经验后，候选人、导师和二级导师(Jeffrey Whitsett)开发了一项利用转基因来研究不同细胞类型上的Beta2AR如何调节呼吸道功能的计划。拟议的培训计划结合了各种药理学和生理学技术，与应聘者以前的经验有很大不同。学习这些额外的技术将有助于将候选人的技能提高到自主调查员的水平。为实现这一目标，已经创造了一种特别有利于候选人培训的环境。这包括利格特和惠特塞特博士的导师，以及一个由在培训内科医生科学家方面具有丰富经验的知名调查人员组成的咨询委员会。

Beta-agonists are potent bronchodilators that are frequently used in the treatment of bronchospastic lung diseases such as asthma. In the intact lung, Beta-agonists-mediated bronchodilation may result from the activation of Beta2ARs on a number of different cell types. However, the relative contribution of B2AR signaling from any one of these cell types is unclear. One potential approach to delineate these cell-specific effects in vivo is to use transgenesis to selectively over express, and thus activate, Beta2AR on specific cells. By using cell-specific promoters to target receptor expression, we propose to create multiple lines of transgenic mice that over express the Beta2AR in airway epithelium and in airway smooth muscle. Preliminary ex vivo and in vivo studies have indicated a significant Bets2AR- mediated communication between the epithelium and smooth muscle, in that over expression of the Beta2AR -mediated communication between the epithelium and smooth muscle, in airway epithelium decreases bronchial hyperactivity. Over expression on airway smooth muscle results in another phenotype, and this allows for assessment of each pathway with regard to Beta2AR regulation of airway function. Using a strategy that integrates molecular biological, pharmacological and physiological techniques, we propose to use such transgenic approaches to: (1) establish the role of epithelial cell Beta2ARs in regulating smooth muscle function; (2) selectively delineate Beta2AR signaling and function in airway smooth muscle; and (3) assess the effects of epithelial and smooth muscle Beta2AR over expression on airway inflammation and bronchial hyperresponsiveness. The candidate has completed a clinical fellowship in Pulmonary and Critical Care. During that time the candidate used basic molecular biological techniques to begin studies of Beta2AR gene expression. The candidate became interested in pursuing these studies further, and made the decision to move to the laboratory of Stephen Liggett at he University of Cincinnati Medical Center. After gaining more intensive laboratory experience, the candidate, mentor and secondary mentor (Jeffrey Whitsett) developed a plan utilizing transgenesis to study how Beta2AR on different cell types regulate airway function. The training plan proposed incorporates a variety of pharmacological and physiological techniques that represent a significant departure from the candidate's previous experience. Learning these additional techniques will serve to advance the candidate's skills to the level of autonomous investigator. To this aim, an environment has been created that is uniquely conducive to the candidate's training. This includes the mentor ship of Drs. Liggett and Whitsett, and an Advisory committee composed of established investigators with extensive experience in raining physicians scientists.