负载siRNA-BMP-2的PEG-PCL纳米载体介导瓣膜间质细胞成骨分化和M2型巨噬细胞极化构建抗钙化心脏瓣膜及其抗钙化机制研究

Valvular calcification is the main cause of bioprosthetic valve failure and obstacles of bioprosthetic valve research. Our previous study found that covering valvular calcification site and introduction of anti-calcification factors could delay valve calcification, but eventually valvular calcification could not be prevented. Research believes that transformation of valvular stromal cells to osteoblasts promoted by up-regulated BMP-2 expression, destruction of the endothelial cell layer on the valvular surface,activation of inflammatory cells are important mechanism of valvular calcification,the study of valvular anti-calcification using these mechanism has not been reported, and its mechanism is not clear. According to this, we put forward the hypothesis that the modification of decellularized valve by nanocarriers loaded with siRNA-BMP-2 can not only achieve anti-calcification of the valve, but also induce the polarization of macrophages to M2 type macrophages to promote valvular endothelialization,and achieve synergistic anti-calcification for heart valve with them.This project intends to silence BMP-2 in target using siRNA and PEG-PCL nanocarriers,a new type of composite valve scaffold material will be constructed via decellularized valve covalently modified by siRNA-BMP-2-PEG-PCL nanoparticles,using RT-PCR, immunohistochemistry, minimally invasive animal models and other methods, the mechanism of anti-calcification, endothelialization and synergistic anti-calcification of novel composite valve stent materials will be explored from the molecular, cellular and in vivo levels to provide new insights into the study of anti-calcification of heart valves.

瓣膜钙化是生物瓣衰败的主要原因和生物瓣研究的主要瓶颈。我们前期研究发现：覆盖瓣膜钙化位点和引入抗钙化因子，虽能延缓瓣膜钙化，但瓣膜最终还是走向钙化。研究认为上调BMP-2表达促进瓣膜间质细胞向成骨细胞转化、瓣膜表面内皮层破坏、炎症细胞激活是瓣膜钙化的重要机制，利用这些机制进行瓣膜抗钙化研究未见报道，其机制也不清楚。据此我们提出假说：负载siRNA-BMP-2的纳米载体修饰去细胞瓣以实现瓣膜抗钙化和诱导巨噬细胞向M2型巨噬细胞极化以促进瓣膜内皮化，并实现两者协同抗钙化。本项目拟利用siRNA和PEG-PCL纳米载体靶向沉默BMP-2,利用siRNA-BMP-2-PEG-PCL纳米粒共价修饰去细胞瓣构建一种新型复合瓣膜支架材料，以RT-PCR、免疫组化、微创动物模型等方法，从分子、细胞和在体水平，探讨新型复合瓣膜支架材料抗钙化、内皮化及协同抗钙化的机制，为瓣膜抗钙化研究提供新思路。

瓣膜钙化是生物瓣衰败的主要原因和生物瓣研究的主要瓶颈。我们前期研究发现：覆盖瓣膜钙化位点和引入抗钙化因子，虽能延缓瓣膜钙化，但瓣膜最终还是走向钙化。研究认为上调BMP-2表达促进瓣膜间质细胞向成骨细胞转化、瓣膜表面内皮层破坏、炎症细胞激活是瓣膜钙化的重要机制，利用这些机制进行瓣膜抗钙化研究未见报道，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和BMP-2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和BMP-2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。随后我们进行了拓展研究，利用钛纳米管及肝素水凝胶涂层的钛纳米管诱导M2型巨噬细胞极化，覆盖钙化位点实现协同抗钙化，也为抗钙化的协同治疗提供一些理论基础。