新型纳米靶向载体系统介导RhoA-siRNA干扰成骨分化构建抗钙化心脏瓣膜及其抗钙化机制研究

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 RhoA which is a key gene for osteogenic differentiation, inducing valvular calcification.but targeted intervention of cardiac valvular calcification by delivering calcification related gene RhoA-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) , PEG-PCL), for the purpose of specific targeting RhoA slience;and construct minimally invasive implantable anti calcification heart valve with RhoA-siRNA- PEG-PCL 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 RhoA targeting slience to promote heart valve anti-calcification 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RhoA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RhoA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，对RhoA进行特异性靶向沉默；利用RhoA-siRNA-PEG-PCL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RhoA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。

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RhoA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RhoA靶向干预心脏瓣膜钙化的研究未见报道，其机制也不清楚；我们使用含无机磷酸盐的成骨分化培养基（具有更高的钙化潜能）来构建瓣膜间质细胞（VICs）的钙化模型，通过RhoA表达的siRNA沉默研究刺激VICs钙化的机制，结果表明，RhoA/ROCK-1信号通路可能是参与瓣膜钙化的重要信号通路，且RhoA是其中重要的一环。同时应用聚乙二醇-聚己内酯（PEG-PCL）对猪主动脉瓣进行去细胞处理制备组织工程心脏瓣膜支架，成功制备具有良好生物学性能和力学性能的主动脉瓣支架仿生材料，为构建组织工程心脏瓣膜支架提供一种新的去细胞方法。我们借助iRNA技术并结合PEG-PCL纳米粒进行靶向干扰，为瓣膜钙化的研究提供一些理论基础。