基于新型pGL4.10-RLUC-RAGE/CDH11载体构建抗老年性主动脉瓣钙化双报告中药筛选系统的研究

Calcified aortic valve disease (CAVD) belongs to” thoracic arthralgia”. Traditional Chinese medicine has an obvious advantage on CAVD, but it lacks effective screening system and scientific interpretation. The ingredients of Traditional Chinese medicines are very complicated, which makes it difficult for conventional drug screening systems to achieve rapid and efficientresult against CAVD, such as high- throughput analysis systems. Osteogenesis of valvular interstitial cells (VICs) is a key factor in the development of CAVD. Research of drugs target on RAGE/CDH11 have already been in clinical-trial step. Based on our newly constructed pGL4.10-RLUC dual- luciferase reporter gene system and theinvestigation of traditional Chinese medicine on VICs calcification, we hope to establish a new screening system for anti-CAVD drugs by pGL4.10-RLUC-RAGE/CDH11transmitted cells. Depend on this system, we can get effective therapeutic drugs by combining cell and animal experiments, using a series of molecular biological techniques such as PCR, Western-blot and gene reporter assay. The study could further demonstrate the effectiveness and mechanism of the selected drugs, explore the influence of traditional Chinese medicine on CAVD from macro to micro scope , and provide direct experimental evidence for the treatment of CAVD by traditional Chinese medicine.

老年性主动脉瓣钙化性疾病(CAVD)属胸痹症，中医药治疗CAVD具有一定优势，但缺乏药物有效性筛选及现代医学论证。中药成分复杂，讲究配伍的特性使得常规药物筛选系统如高通量等难以实现中药抗CAVD疾病药物的快速高效筛选。瓣膜间质细胞(VICs)成骨样变是CAVD发生发展的关键因素，针对VICs钙化的RAGE/CDH11靶点药物研究已进入临床试验阶段。基于课题组前期构建的pGL4.10-RLUC双荧光素酶报告基因载体系统及前期对VICs钙化中药干预机制的研究，我们希望通过建立新的pGL4.10-RLUC-RAGE/CDH11细胞稳转抗CAVD药物筛选体系，筛查出有效治疗药物，再结合细胞及动物试验，采用PCR、western-blot、报告基因等一系列分子生物学技术，进一步论证该筛选系统的有效性及作用机制，从宏观到微观探讨中医药对CAVD的影响，为中医药治疗CAVD提供直接实验证据。

老年性瓣膜病是一组累及多个瓣膜的退行性疾病，尤以主动脉瓣钙化所致的瓣口狭窄或关闭不全为甚。随着我国逐步进入高龄化社会，老年性主动脉瓣钙化(aortic valve calcification)的发病率逐年上升，50岁以上人群中瓣膜钙化检出率达12.5%，其中主动脉瓣受累占94.4%。目前，老年性主动脉瓣钙化性疾病(calcified aortic valve disease，CAVD)仅能采取手术或介入换瓣治疗策略，目前的置换瓣膜分生物瓣或机械瓣两种，生物瓣膜置换后需十年后再次开胸更换瓣膜，而机械瓣置换后需终生服用抗凝剂。瓣膜置换术费用高、风险大、恢复周期长，患者生活质量差。对有手术或介入禁忌症的患者，以及未达到手术条件但存在瓣膜钙化的病人，目前缺乏针对性治疗手段延缓或控制病程进展。因此，加强CAVD治疗药物的基础与临床研究，变被动手术为主动防控，提高CAVD治疗水平，具有深远意义。.项目在实施过程中，成功构建了单味中药治疗老年性瓣膜病药物筛选体系，并对CNKI中筛选出的靶向药物进行筛选，结果筛选出丹参、葛根等中药。细胞试验结果显示，丹参酮IIA及葛根可有效改善瓣膜间质细胞钙化及钙化相关蛋白RUNX2、Osteocalcin表达，该筛选系统对无荧光中药具有良好的筛选作用和准确性。动物实验结果显示，筛选中药葛根素及白藜芦醇可减轻主动脉瓣钙化进展，减少瓣叶增厚、瓣膜粘连导致的主动脉瓣狭窄的发生。.项目在实施过程中，一篇SCI投稿中，一篇北大中文核心论文修回中（见附件）。培养硕士生2名，其中1名拟于2023年毕业，毕业论文题目暂定为《消斑通脉方抗老年性主动脉瓣钙化机制研究》。申请发明专利1项，已公开，处于复审阶段（见附件）。该筛选系统初步显示了其有效性，虽然在稳定性和准确性方面，仍有待进一步提高，但如若能得到进一步资金支持，克服其稳定性和准确性相关的问题，将具有成果转化的可能和广阔的药物筛选应用空间，对促进老年性瓣膜病的中医药防治具有积极意义。