FEM探讨剪切力刺激诱导瓣膜钙化机制研究

Shear force stimulation is considered as one important trigger for heart valve calcification. Previous researches show that signal pathways related to Bone Morphogenetic Protein 2(BMP2), a member of TGF-β1 super families, such as the classical one (BMP2/Smad1) and the non-classical one (BMP2/Msx2) may play a key role in this process. In recent years, the emergence of tissue-engineered model and computer assisted three-dimensional analysis technology provide new methods for the study of valve-mechanics response mechanism. This project aimed to explore these potential mechanisms with the help of the analysis technology of finite element methods (FEM). A stenosis tissue engineering heart valve model will be manufactured and put in a bioreactor to exert shear force on it. The specific shear force distribution and strength on the valvular leaflets surface will be evaluated by CT scan and FEM. The calcification indicator as well as the level of transcription and translation of BMP2 related signal pathways in different parts of leaflets will be tested to reveal the effect of these signal pathways. PEG hydrogel with control release ability will be employed to release blockers to inhibit related pathways and further verify its role in the shear force induced valvular calcification. This project may depict a clear signal transduction processes of mechanics induced calcification, clarify valvular mechanical response mechanism, pinpoint promising therapeutic targets, and provide theoretical basis of new control strategies for valvular calcification.

剪切力刺激是心脏瓣膜钙化的重要始动因素，前期研究发现骨形态发生蛋白2（BMP2）作为力学刺激关联因子TGF-β1超家族成员，其经典（BMP2/Smad1）和非经典（BMP2/Msx2）信号通路可能在此过程中发挥关键作性用。近年出现的组织工程化模型和计算机软件三维分析技术，为瓣膜力学响应机制研究开拓了新的思路。本项目拟通过生物反应器施加剪切力，诱导组织工程化瓣膜狭窄模型发生钙化，结合有限元方法（FEM）及CT扫描重建分析，测算瓣叶表面局部具体剪切力种类、分布及强度，同时检测相应部位钙化程度及BMP2相关信号通路激活程度，进一步使用PEG水凝胶控释阻断剂，抑制相关通路，验证其在剪切力诱导瓣膜钙化中的作用，从而探明BMP2相关信号通路在剪切力刺激诱发瓣膜钙化发生中的可能作用，寻找潜在的钙化相关靶点，为制定瓣膜钙化防治策略提供理论基础。

项目组通过人瓣膜CT数据，构建了二瓣化畸形瓣膜的FEM模型，经由力学分析及生物学检测结果比对，提出震荡剪切力为该型瓣膜中诱发钙化的关键力学刺激类型，其刺激强度为26.8±5.1 ~ 29.9±4.3 dyn•cm-2。依照此结果构建组织工程化二叶瓣模型，在生物反应器刺激下（流量6L/min），FEM分析证实能模拟获得与二瓣化畸形瓣膜局部相似的震荡剪切力环境，并诱发钙化发生及TGF-b，BMP，Notch等重要细胞信号通路激活。项目组进一步依据基因芯片和文献报道，首次观察到CCN3蛋白这一细胞外基质蛋白，在异常力学刺激下的瓣叶组织中表达有明显改变。经由小鼠水平模型及组织工程化瓣膜模型验证，CCN3缺失将加速力学刺激下的瓣叶钙化，而CCN3高表达有助于延缓这一病理过程。离体细胞学实验在小鼠和人细胞水平均证实了这一现象，同时提示CCN3的这一生物学效应主要依赖其阻滞力学刺激激活TGF-β1和BMP2，对抗二者经由Smad依赖通路（Smad2）或Smad非依赖通路（ERK1/2，Akt）诱导的瓣膜间质细胞成骨样转化及钙化改变效应。上述研究成果提示CCN3可能是瓣膜钙化，尤其是力学刺激诱发的瓣膜钙化的关键调控靶点之一，调控其蛋白水平具有临床药理应用潜力。