哮喘气道平滑肌细胞IP3R C末端突变及其功能变化的研究

The changes of Calcium homeostasis of airway smooth muscles cells(ASMC) of asthma could result in ASMC excessive contraction and airway hyperresponsiveness.Hence, the fine-tuning of cellular Ca2+ homeostasis is important and that is the mainly problem needed to be solved in asthma control research.In ASMC, the inositol trisphosphate receptors(IP3Rs) gated Ca2+ store is thought to be the major responsible for Ca2+ waves and global intracelluar Ca2+ concentration elevations for ASMC contraction. The discovery of IP3R was similar to that of Ryanodine receptors (RyRs). Our group found before that RyR2 C-terminal domain mutation could change its function of regulating Ca2+ homeostasis of cardiac cell.Whether the C-terminal mutatants of IP3R could change its function of regulating Ca2+ homeostasis of ASMC, there isn't related report so far. This project will use HEK293 cell line and ASMC which from rat and asthma patients as tool respectively, use with calcium bioluminescence probe, such as Fluo3,Fura2,D1ER, and detect with confocal microscope and patch clamp, to ivestigate the function of C-terminal mutatants of IP3R. The final results of this project will help to explain why excessive hypertrophy and hyperresponsiveness of ASMC from asthma patients and will help to improve asthma control.

哮喘气道平滑肌细胞（ASMC）内钙稳态发生改变，可引起ASMC过度收缩和异常高反应。如何使ASMC胞浆钙离子保持良好的调控、维持细胞正常生理功能，是目前哮喘治疗研究的难点。IP3R是ASMC中最主要的钙离子调节通道，与另一钙通道兰尼定受体（RyR）高度同源。项目组前期研究表明，IP3R C末端区部分氨基酸突变可改变其对HEK293细胞浆钙离子的调控功能；其是否像RyR2 突变体调节心肌细胞浆钙离子一样，调控ASMC中钙离子并影响其功能，目前还不清楚。本项目将在前期研究基础上，以HEK293、ASMC、哮喘大鼠和患者气道平滑肌为研究对象，借助Fluo3、Fura2、D1ER等钙离子生物荧光探针，用共聚焦显微镜及膜片钳等技术研究IP3R C末端突变与其调节胞浆内钙离子功能改变的关系，以明确ASMC中IP3R C末端结构变化对其功能的影响。力求明确其突变的关键位点，为治疗哮喘患者提供新途径。

支气管哮喘是以气道炎症、气道高反应性和气道重塑为特征的呼吸道疾病。其病理改变主要表现为平滑肌增厚和基底膜增厚及玻璃样变。其中气道平滑肌细胞增殖是导致气道重塑的主要构成因素之一，而平滑肌的收缩及增殖与细胞内的钙离子是密不可分的。多数细胞内质网上存在两种钙释放通道， 兰尼碱受体/钙通道(RyRs)和三磷酸肌醇受体/钙通道(IP3Rs)。在支气管平滑肌细胞中，IP3R1作为内质网上调节胞浆Ca2+浓度的关键通道，其活性受到复杂信号网络的精细调节。随着IP3R1结构和功能研究不断的深入，发现其第六跨膜区控制着Ca2+由内质网转移到胞浆，该区域的疏水氨基酸可能是调节Ca2+敏感性部位。本项目有望通过改变IP3R1的C-末端区氨基酸表达，进而下调其对钙离子的敏感性，并探讨三磷酸肌醇受体在慢性哮喘大鼠支气管肺组织中的表达差异及与气道重塑的关系。本项目成功构建pcDNA3.0-RFP-IP3R1双表达框质粒，完全符合预期设计 ；结果显示气管壁厚度及气道平滑肌厚度较对照组均明显增加，差异有统计学意义（P<0.01）；在ASMC内质网中，IP3Rs的3种亚型均有表达，并且在建立的哮喘鼠模型中，IP3R1的mRNA和蛋白较正常组明显上调，且与气管壁厚度及平滑肌层厚度呈正相关（r=0.945,0.929, P<0.01）， 推测IP3R1可能通过影响细胞内的钙离子浓度促进平滑肌细胞的增殖，加速了气道重塑的发生。为研究和治疗哮喘患者气道平滑肌细胞重塑提供理论基础。