HCN4和Cx45基因联合转染骨髓间充质干细胞构建生物起搏细胞治疗缓慢型心律失常的实验研究

Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) is the most predominant isoform in sinoatrial node which is the normal pace making region of the heart. Extensive research toward creating a biological pacemaker by enhancement of HCN4 expression has been performed. We have demonstrated that mesenchymal stem cells (MSCs) transfected with HCN4 genes were capable of increasing the spontaneous beating rate of co-cultured cardiac myocytes in our early study.However, the activaton of HCN4 needs long time,and it's response to hyperpolarization is slow.Furthermore ,there are poor communication between the biological pacemaker cells and implanted cardiomyocytes. These disadvantages make it difficult to meet the requirements of clinical application using biological pacemaker transfected with HCN4. In native sinus node, the connection between pacemaker cells and cardiomyocytes is mainly through connexin 45(Cx45). Cx45 plays an important role in bridge between adjacent cardiomyocytes and sinoatrial node cells.So our idea is, to generate biological pacemaker with MSCs transduced with HCN4 via transfection with a lentiviral vector. These MSCs are cocultured with neonatal rat ventricular myocytes, and while the pacemaker current is generated ,the pacemaker current conduction is promoted.The biological pacemaker constructed in this way will be reexamined in animal experiment,which lay the foundation for clinical application.

HCN4是哺乳动物窦房结等自律性高的区域优势表达的HCN通道亚型，我们早期研究证实，经HCN4基因修饰的骨髓间充质干细胞可使共培养的乳鼠心肌细胞搏动频率增加。但HCN4激活所需时间长，对超极化反应慢，构建的生物起搏细胞与植入周围心肌细胞沟通不良，这些缺点使得单纯HCN4转染构建的生物起搏细胞难以满足临床实际运用的要求。窦房结与周围心肌组织通过缝隙链接蛋白（Cx）形成联系，以Cx45为主。Cx45在窦房结细胞与邻近心肌细胞之间发挥了重要的桥梁作用。我们设想，以慢病毒和脂质体为载体，将HCN4基因和Cx45基因共同转染至骨髓间充质干细胞，产生起搏电流的同时促进起搏电流的传导，构建长期有效的生物起搏细胞，在动物实验中进行验证，为临床应用打下基础。

超极化激活环核苷酸门控阳离子通道（HCN）基因为If电流形成的分子基础。该研究以慢病毒和脂质体为载体，将HCN4和Cx45基因共同转染至骨髓间充质干细胞(MSCs)，和心肌细胞共培养，构建具有自律的长效的心脏生物起搏器模型。本研究发现：①慢病毒载体成功介导HCN4基因转染MSCs，重建起搏离子通道，为生物起搏模型的构建奠定了基础。hHCN4基因改造的MSCs可表达hHCN4基因,在转hHCN4的MSCs中，记录到时间和电压依赖性的超极化激活的内向电流，该电流对4mmol/LCsCl敏感，符合起搏电流的特征，证明MSCs上已经成功表达了有功能的起搏离子通道hHCN4；②表达有起搏离子通道的MSCs与乳鼠心室肌细胞共培养，成功构建具有自律性的心脏生物起搏器模型，且缝隙连接通道在该模型中具有重要意义；③脂质体2000成功介导CX45基因转染MSCs，构建成细胞膜上稳定表达CX45的细胞株CX45+MSCs；④HCN4基因转染CX45+MSCs，与心肌细胞共培养，形成了一个自律性更高的生物起搏器模型；⑤该生物起搏器模型中，心肌细胞的自发动作电位频率显著高于对照组；动作电位4相自动去极化速度（VDD）也明显提高，心肌细胞自发性收缩频率增加。免疫双标证实心肌细胞与CX45+MSCs相接触部位有CX43与CX45的表达。⑥在动物模型HCN4＋CX45＋MSCs组中，免疫荧光染色检测到HCN通道蛋白和Cx45蛋白的高表达，心率加快。本研究提供了构建生物起搏细胞的一种新的技术方案，为以后临床上治疗缓慢性心律失常贡献了一种可行策略。