MicroRNA-29b参与调控钙化性主动脉瓣疾病进展的机制研究

Differentiations of aortic valve interstitial cells to osteoblast phenotype and heterotopic ossification in the valve are the main characteristics in calcific aortic valve disease (CAVD) and the key steps in the progression of the lesion. We found that the expression of microRNAs (miRNA-29b) increased significantly during the differentiation. Based on the previous experiments, the cell models with high or low expression of miRNA-29b were established in the present study. Induced by calcification, the cell models were detected by calcification related factors and calcium deposits determination, etc., to clear the influences of miRNA-29b expression on the differentiation of interstitial cells to osteoblast phenotype and ossification formation. Real-time PCR, Western-Blot and other techniques were used to detect the effect of up-regulation and down-regulation of miRNA-29b expression on activation state of TGF-β/Smad and Wnt/β-catenin signaling pathway. Luciferase gene detection was used to determine the interaction between miRNA-29b and target gene. And miRNA-29b antagomir was directly injected to CAVD mice model. The effectiveness of in vivo experiments was evaluated by several methods such as echocardiogram for trans-valvular velocity and calcium deposits determination, in order to provide a new therapeutic target for early prevention and intervention of CAVD.

主动脉瓣间质细胞向成骨细胞表型转化和瓣膜内异位骨化是钙化性主动脉瓣疾病(CAVD)的主要特征和病变进展的关键步骤。我们发现，瓣膜间质细胞向成骨细胞转化过程中microRNA-29b (miRNA-29b)显著升高。本课题将在前期基础上构建miRNA-29b高/低表达细胞,在钙化诱导下，通过钙化相关因子检测和钙沉积测定等方法，明确miRNA-29b表达水平对间质细胞表型转化和骨化形成影响；通过real-time PCR、Western-Blot等技术检测miRNA-29b上/下调表达对TGF-β/Smad和Wnt/β-catenin信号通路活化状态影响，通过荧光素酶基因检测系统验证miRNA-29b与靶基因相互作用；同时在CAVD小鼠模型中直接注射miRNA-29b antagomir (拮抗剂)，通过心超测定跨瓣流速和钙沉积定量等，评价在体实验有效性，为CAVD早期预防和干预提供新的靶点。

钙化性主动脉瓣疾病（CAVD）作为一种临床常见疾病，由于病理机制未能完全明确，目前缺乏有效的药物治疗策略。主动脉瓣间质细胞向成骨细胞表型转化和瓣膜内异位骨化是CAVD的主要特征。我们发现主动脉瓣间质细胞向成骨细胞转化过程中miRNA-29b显著升高。依托前期建立的钙化诱导模型和分离获得的人主动脉瓣间质细胞，我们在细胞水平调控miRNA-29b表达，利用茜素红染色、钙沉积定量、免疫荧光检测及qRT-PCR、Western-Blot等方法，发现下调miRNA-29b表达，可以抑制主动脉瓣间质细胞向成骨细胞转化；上调miRNA-29b表达，Smad3，Runx2，Wnt3，β-catenin和VEGFA表达增加，而TGF-β3表达下降（P<0.05）；在前期生物信息学软件预测基础上，我们通过双荧光素酶基因报告系统验证发现，相对于TGF-β1促成骨作用，TGF-β3作为负性调控因子，TGF-β3是miRNA-29b的直接作用靶基因。后续，借助CAVD小鼠模型，通过尾静脉注射miRNA-29b antagomir，我们进一步证实，拮抗miRNA-29b表达，可以上调TGF-β3表达水平，负性调控Wnt/β-catenin和Runx2/Smad3、VEGFA通路，抑制钙化形成。通过对miRNA-29b/TGF-β3调控钙化性主动脉瓣疾病进展的研究探索，我们从microRNA角度进一步揭示了CAVD的病理机制，为CAVD的早期药物干预和临床实验检测提供了可能。