Shox2介导HCN4基因修饰MSCs定向分化重建心脏生物起搏点的实验研究

Biological pacemaker therapy has been a hot spot and emphasis of cardiac electrophysiology study in recent years. With the help of national natural science foundation we have successfully cultivated pacing cells derived from MSCs transfected with HCN4 gene in vitro. A biological pace maker can be detected when these MSCs were transplanted to the ventricular muscle, but the pacing frequency was much lower than that of in vitro which will restrict the further study on this issue. Research has demonstrated that Shox2、Nkx2.5 and Tbx3 were closely related to the heart prenatal development and Shox2 especially plays an important role in the embryonic sinoatrial node development with its strict control to maintain the cytogene of sinoatrial node. In order to make a ideal pace maker with better pacing frequency and persistence time, we are gonging to transfect Shox2 gene to those MSCs which has already been transfected with HCN4 gene before transplanting them to right atrium where the sinoatrial node has been destroyed. The work will improve the study of biological pacemaker and facilitated its clinical application, which will make great theory value as well as potential clinical practice.

心脏生物起搏是近年来心脏电生理领域研究的重点与热点内容之一。本课题组在国家自然科学基金资助下，已成功将HCN4基因转染至犬骨髓间充质干细胞(MSCs)，并在体外诱导培养出心脏起搏样细胞；通过将HCN4基因修饰的MSCs移植至犬心室肌重建生物起搏点获得成功，但因起搏频率较体外培养的细胞搏动频率明显减慢，成为制约该项研究的关键问题。研究显示，Shox2、Nkx2.5及Tbx3均是与心脏胚胎发育密切相关的转录调控因子，其中Shox2基因对维持胚胎心脏起搏细胞基因表型、促进窦房结发育形成起重要调控作用。因此，本项目研究拟在体外先将Shox2基因导入经HCN4基因修饰的MSCs，再将其移植至窦房结毁损犬右心房外膜下，重建出起搏频率及持续时间均较理想的心脏生物起搏点。本研究成果将有助于提升心脏生物起搏研究的应用价值，促进生物起搏治疗缓慢性心律失常的临床应用进程。具有重要的理论意义与潜在的临床实用价值。

心脏“生物起搏”是近年来探索治疗缓慢性心律失常的热点。起搏电流(If)是窦房结细胞舒张期自动除极化的主要决定因素，HCN4是If产生及维持基本窦性心律的基因基础。Shox2在窦房结分化过程中具有重要作用，但作用及机理不清楚。研究结果：1.慢病毒Shox2、HCN4双基因转染犬MSCs：⑴免疫荧光：HCN4组细胞可见HCN4蛋白表达；Shox2组和Shox2-HCN4组细胞表达Shox2、HCN4、cTnT，其中Shox2-HCN4组表达更显著。⑵分子生物学：①HCN4、Tbx3和Cx45：与对照组比较，Shox2组中HCN4、Tbx3和Cx45均增加；Shox2-HCN4组Tbx3和Cx45增加较Shox2组更显著；HCN4组中可见Cx45的表达。②Nkx2.5及Cx43：与对照组比较，Shox2组Nkx2.5、Cx43表达下降；Shox2-HCN4组Nkx2.5、Cx43下降更为显著；HCN4组细胞中可见Cx43表达。⑶膜片钳：Shox2组、HCN4组、Shox2-HCN4组细胞均能记录到If电流；2.经EPCS诱导后各组细胞形态及功能变化：⑴形态学：免疫荧光结果显示：EPCS诱导后，Shox2组和Shox2-HCN4组细胞HCN4和cTnT的表达更显著。⑵分子生物学：①HCN4、Tbx3和Cx45：经EPCS诱导后，Shox2组HCN4、Tbx3和Cx45增加较诱导前更显著 (p <0.05)；Shox2-HCN4组中Tbx3和Cx45增加较诱导前更显著 (p <0.05)；HCN4组Cx45较诱导前明显增加 (p <0.05)。②Nkx2.5及Cx43：经EPCS诱导后，Shox2组Nkx2.5、Cx43均较诱导前明显下降 (p <0.05)；Shox2-HCN4组Nkx2.5及Cx43量下降较诱导前更加显著(p<0.05)； HCN4组Cx43较诱导前明显增加 (p <0.05)。⑶膜片钳：Shox2+EPCS组、HCN4+EPCS组、Shox2-HCN4+EPCS组均能记录到If电流。科学意义：1.双基因共转染；2. cMSCs转染Shox2后，能促进 cMSCs向心脏起搏样细胞分化；3. EPCS能够促进转染Shox2的cMSCs向起搏样细胞分化；4. Shox2-HCN4双基因共转染+EPCS诱导，有促进cMSCs向起搏样细胞方向分化的叠加效应。