低声压级水平次声治疗大鼠心肌纤维化中microRNA-29a调控TGF/Smad传导通路研究

Infrasound has been used for disease convalescence abroad. Our previous experiments indicated that infrasound of low decibel may ameliorate the ventricle remodeling in rats with myocardial infarction, which was proved by gene chip to be related with miRNA-29a downregulation, while its mechanism has not been clear. Myocardial fibrosis is an important factor of ventricle remodeling, therefore we presume that infrasound may inhibit myocardial fibrosis.In the course of myocardial fibrosis, the exudation maladjustment of MMPs/TIMPs and cardiac collagen appear,it is proved that TGF-β1/Smad3 conductive pathway is involved in myocardial fibrosis. Based on the above reasons, we suppose that miRNA-29a may directly or indirectly regulate the expression of MMPs/TIMPs and cardiac collagen by regulating TGF-β1/Smad3 conductive pathway in the course of myocardial fibrosis treated by infrasound of low decibel.In this experiment（in vivo and in vitro）, we will observe the effects of infrasound of low decibel on myocardial fibrosis in rats,and study the changes of conductive pathway of myocardial fibrosis, and investigate the mechanism of infrasound treatment with myocardial fibrosis by the clone of miRNA-29a,the construction of miRNA-29a carriers and the transfection of myocardial fibroblasts, and choose the idea infrasound treatment parameters. Our studies will establish the theory base of low decibel infrasound treatment in cardiovascular diseases convalescence.

在国外次声已用于疾病康复治疗。本课题组前期研究发现低声压级水平次声能改善心肌梗死大鼠心室重构，基因芯片初步证实次声这一作用与miRNA-29a下调有关，机制尚不清楚。心肌纤维化是心室重构的重要因素，因此，推测次声或能抑制心肌纤维化。当心肌纤维化发生时，MMPs/TIMPs及心肌胶原失调，已经证实TGF-β1/Smad3参与心肌纤维化信号传导。由此我们设想miRNA-29a可能通过调控TGF-β1/Smad3信号通路直接或间接调控MMPs/TIMPs及心肌胶原的分泌，在次声治疗心肌纤维化中发挥重要作用。本课题进行体外及体内实验，观察低声压级水平次声对大鼠心肌纤维化的作用；通过克隆miRNA-29a、构建miRNA-29a表达载体、转染心肌成纤维细胞，研究心肌成纤维细胞上述信号通路的变化，探索低声压级水平次声对心肌纤维化作用机制；筛选次声理想治疗参数，为次声用于心血管疾病康复治疗奠定基础。

目的：研究低声压级水平次声对大鼠心肌纤维化的作用及机制，评价低声压级水平次声康复治疗缺血性心脏病的临床意义。方法：利用次声治疗仪，通过离体培养的小鼠心肌成纤维细胞及整体大鼠心肌纤维化模型，采用细胞培养、病理学及分子生物学等技术开展实验研究。结果：在整体动物实验中，与对照组比较，低声压级水平次声组大鼠心功能明显改善，心肌组织及心肌成纤维细胞超微结构损伤明显改善，心肌组织中Collagen-Ⅰ/3及Ang Ⅱ合成明显减少，而NO分泌明显增加，在次声加药物组更加明显；与对照组比较，在转染miR-29a组及miR-29a+Infra组大鼠心梗后瘢痕面积均明显缩小，Collagen-1/3分泌明显减少，而在miR-29a+Infra组及miR-29a+Infra+Drugs组更加明显。在低氧培养的心肌细胞中，与脂质体转染miRNA-29a比较，在脂质体转染miRNA-29a+Infra组，TGF-β1、Collagen-Ⅰ/3、TIMP-1、Smad2/3的表达明显降低，MMP-1的表达明显增加，在miR-29a+Infra+Drugs组更加明显；在anti-miR-29a+Infra组，结果则相反。结论：低声压级水平次声能够明显改善大鼠心肌纤维化，其作用机制与低声压级水平次声激活miRNA-29a有关。低声压级水平次声通过miRNA-29a激活调控Smad2/3磷酸化过程而直接影响TGF-β1的分泌，减少Collagen-Ⅰ/3的合成；低声压级水平次声通过miRNA-29a激活直接影响细胞基质代谢，可明显使细胞基质中MMP-1生成增加，TIMP-1生成减少，从而明显减轻大鼠心肌纤维化，改善其心功能。低声压级水平次声与ACEI类药物联合用于治疗心肌纤维化，效果更明显。