人类超极化激活环核苷酸门控阳离子通道基因亚型4导入大鼠心脏构建生物起搏点的初步研究

Transfecting rat heart with human pacemaker gene in vivo to create a biological pacemaker

目的探讨利用人类超极化激活环核苷酸门控阳离子通道基因亚型4在大鼠心脏中构建生物起搏点的可能性、安全性和时效性。方法6周龄SD大鼠分为对照组（10只）和实验组（10只），分别经心外膜注射50山的腺病毒和人类超极化激活环核苷酸门控阳离子通道基因亚型4重组腺病毒（滴度为10^10～10^12）至左心室心尖部。术后3d内每天记录一次心电图，每次10min。术后第7天分离颈部双侧迷走神经干并进行双侧或单侧电刺激，同时记录体表心电图。随后在心尖部进行起搏标测。最后取出心脏进行病理学检查和免疫荧光检测。结果术后3d内心电图没有记录到室性心律失常事件。在术后第7天进行颈部迷走神经干电刺激时，实验组大鼠室性异搏节律的频率明显快于对照组（分别为69bpm±6bpm和41bpm±10bpm，P〈0．001）。心电图和起搏标测均证实实验组大鼠室性异搏节律起源于注射部位附近。免疫荧光检测证实注射局部人类超极化激活环核苷酸门控阳离子通道基因亚型4蛋白过度表达。1个月后，注射部位心脏组织未检测到该蛋白的表达。结论人类超极化激活环核苷酸门控阳离子通道基因亚型4导入心室可以使通道蛋白在局部过度表达，提高室性异位起搏点的频率。利用人类超极化激活环核苷酸门控阳离子通道基因亚型4在体内构建生物起搏器的长期效果有待于进一步研究。

Objective To test the ability and safety of injecting the hHCN gene into the ventricle of intact rats to create a novel biological pacemaker and to explore the duration of the hHCN over-expression in vivo. Methods Adenoviral constructs incorporating hHCN4 and green fluorescent protein （GFP） were subepicardially injected into the rats＇ left ventricular wall in situ （n = 10）, Control group was injected with adenoviral constructs of GFP alone （n = 10）, ECGs were recorded every day within the initial three days after operation. Up to seven days after injection, all rats were anesthetized and subjected to cervical vagal trunks stimulation to permit the emergence of escape rhythm. Then pace-mapping was performed by the hand-hold electrode. Finally, hearts were removed for histology and immunofluorescence study. Results Ventricular arrhythmic events were not found during the first three days. During vagal stimulation, the rate of spontaneous rhythm in the hHCN4-injected group was significantly more rapid than that in the control group （69 bpm ± 6 bpm vs 41 bpm ± 10 bpm, P 〈 0, 001 ）. Pace-mapping confirmed the escape rhythm in the hHCN4-injected group was originated near the injection site. Immunofluorescence study showed the overexpression of hHCN4 on the site of injection. However, the duration of hHCN overexpression was no more than one month after injection. Conclusion The overexpression of hHCN4 provides ventricular escape rhythm with the physiologically acceptable rate. Long-term feasibility of this approach needs to be tested.