基于纳米载体的siRNA靶向干预成骨分化构建抗钙化组织工程心脏瓣膜及其机制研究

Valve calcification is a major cause of bioprosthetic valve failure, also is the main obstacles of bioprosthetic valve research; Our previous study suggested that endothelialization on decellularized valve scaffolds by loading VEGF165 (vascular endothelial growth factor 165) with nano delivery system on the surface of the scaffold can delay valve calcification, but unfortunately they are intend to calcification ultimately,Other studies have shown that valvular interstitial cells into osteoblast-like phenotype play a vital role in calcification of valve, the result is up-regulated the expression of Runx2 which is a key gene for osteogenic differentiation, inducing valvular calcification.but targeted intervention of cardiac valvular calcification by delivering calcification related gene Runx2-siRNA with nano vector has not been reported and its mechanism is not very clear; the project intends to adopt siRNA (small interfering RNA,) technology, using non viral vector nano delivery system poly (ethylene glycol) -poly (-caprolactone) -poly (ethylene, imine, PEG-PCL-PEI), for the purpose of specific targeting Runx2 science;and construct minimally invasive implantable anti calcification heart valve with PEG-PCL-PEI-Runx2-siRNA nanoparticles modified Valve and shape memory alloy mesh stent.Then in vivo and vitro anti calcification research will carry out in the model of minimally invasive animal ; our research aim at exploring the mechanism of Runx2 targeting slience to promote heart valve anticalcification in vitro and in vivo , and provide a novel idea for the research of heart valve anti calcification .

瓣膜钙化是生物瓣衰败的主要原因，也是生物瓣研究的主要瓶颈；我们前期研究发现：利用纳米载体负载VEGF165对去细胞瓣进行内皮化以覆盖瓣膜钙化位点，虽能延缓瓣膜钙化，但瓣膜最终还是走向钙化；研究认为成骨分化关键基因Runx2的表达上调促使心脏瓣膜间质细胞向成骨细胞表型转化是心脏瓣膜钙化的重要机制；但纳米载体负载钙化相关基因Runx2靶向干预心脏瓣膜钙化的研究未见报道，其机制也不清楚；本项目拟采用siRNA技术，借助非病毒纳米载体PEG-PCL-PEI，对Runx2进行特异性靶向沉默；利用PEG-PCL-PEI-Runx2-siRNA纳米粒修饰的去细胞瓣和记忆合金网状支架构建可微创植入的抗钙化心脏瓣膜，并进行微创动物模型体内外抗钙化的研究；旨在探讨Runx2特异性靶向沉默促进心脏瓣膜体内外抗钙化的机制，为心脏瓣膜抗钙化研究提供新的思路。

心脏瓣膜病是一种严重危害人类健康的世界性公共问题，心脏瓣膜置换是治疗瓣膜病的有效方法，然而目前临床所采用的机械瓣和生物瓣均有严重缺陷，其中心脏瓣膜钙化是生物瓣衰败的主要原因，因此，如何对心脏瓣膜实现有效的抗钙化成了当务之急。我们前期研究发现：利用纳米载体负载VEGF165对去细胞瓣进行内皮化以覆盖瓣膜钙化位点，虽能延缓瓣膜钙化，但瓣膜最终还是走向钙化；本课题推测：成骨分化关键基因Runx2的表达上调促使心脏瓣膜间质细胞向成骨细胞表型转化是心脏瓣膜钙化的重要机制。因此本研究通过可控释PEG-PCL-PEI-Runx2-siRNA纳米粒的制备及优化、包封率和载药量的测定、生物性能检测，Runx2-siRNA体外可控释放度及心脏瓣膜细胞对其摄取效率的研究及抑制瓣膜细胞成骨分化机制。并从体内体外构建可控释PEG-PCL-PEI-Runx2-siRNA纳米粒修饰的去细胞猪主动脉瓣，通过聚电解质层层技术，将PEG-PCL-PEI-Runx2-siRNA纳米粒固定到到去细胞瓣的复合材料，探索最佳反应条件及体内体外控释以及抗钙化机制等。通过研究已成功构建基于基因治疗的靶向干预心脏瓣膜钙化的新型纳米载体系统(PEG-PCL-PEI-Runx2-siRNA)，并利用PEG-PCL-PEI-Runx2-siRNA 纳米粒对新型心脏瓣膜进行抗钙化修饰，从而在基因水平上为新型组织心脏瓣膜抗钙化修饰提供理论支持。此外，探索RhoA/ROCK-1通过BMP-2/Smad1/5/9信号通路参与瓣膜间质细胞成骨分化的研究，对瓣膜骨化的具体机制提出新研究视角。研究结果可望为靶向心脏瓣膜抗钙化，进而提高心脏瓣膜置换的临床综合治疗效果提供新的思路和实验依据。