重组起搏基因质粒构建生物起搏器的实验研究  

Recombinant plasmid pIRES2-EGFP-HCN2 improved pacing function in canine model of sick sinus syndrome

作　　者：牛萍[1] 黄从新[1] 赵月强[2] 杨波[1] 赵庆彦[1] 王腾[1] 范国华[3] 

机构地区：[1]武汉大学人民医院心内科,430060 [2]武汉大学人民医院整形外科,430060 [3]武汉大学人民医院心外科,430060

出　　处：《中华心血管病杂志》2006年第12期1126-1130,共5页Chinese Journal of Cardiology

摘　　要：目的研究旨在构建重组起搏基因质粒pIRES2-EGFP-HCN2,并检测其在体外心房肌细胞及犬病态窦房结综合征(病窦)模型体内的表达。方法对含mHCN2cDNA的PTR载体进行转化和扩增,将所得mHCN2基因定向克隆到真核表达载体pIRES2-EGFP中,进行双酶切来鉴定克隆的正确性。将重组质粒用电穿孔法转染心房肌细胞,同时直接注射至犬病窦模型的窦房结区域,体表心电图监测犬窦房结功能改善情况,并通过荧光显微镜及RT-PCR检测重组质粒pIRES2-EGFP-HCN2在体内外的表达。而对照组仅注射生理盐水。结果构建了重组质粒pIRES2-EGFP-HCN2。荧光显微镜下可见转染后的心房肌细胞呈绿色荧光,其搏动频率较未转染的细胞明显增快[(180±11)次/min与(140±14)次/min,P<0.05]。注射组织的冰冻切片显示绿色荧光蛋白,RT-PCR显示了mHCN2基因片段,体表心电图显示犬窦房结功能改善[(150±13)次/min与(105±17)次/min,P<0.05]。而对照组始终未见到绿色荧光蛋白表达及窦房结功能改善。结论成功构建了重组质粒pIRES2-EGFP-HCN2,并在体外心房肌细胞及犬病窦模型体内成功表达,为生物起搏的研究奠定基础。Objective To construct plasmid expressing pacemaker gene pIRES2-EGFP-HCN2 and study its effects in transfected atrial myocytes in vitro and in canine model of sick sinus syndrome （SSS）. Methods mI-ICN2 gene was isolated from PTR plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. Recombinant plasmids pIRES2-EGFP-HCN2 was transfected with by electroporation into neonatal atrial cardiomyocytes or injected to the sinoatrial （SA） region of canines with SSS induced by catheter and chemical ablation, pIRES2-EGFP-HCN2 expression was detected under fluorescence microscope and confirmed by reverse transcription-polymerase chain raction （RT-PCR）. Spontaneous beating rate in atrial cardiomyocytes was detected with light microscope. Results EGFP expression was seen in transfected atrial cardiomyocytes 24 to 48 hours after transfection and the spontaneous beating rate was significantly increased than that in non-transfected atrial cardiomyocytes [ （ 180 ± 11 ） bpm vs （ 140 ± 14） bpm, P 〈 0. 05 ]. Heart rate was significantly increased 24 hours post recombinant plasmids pIRES2-EGFP-HCN2 injection compared to saline injection in canines with SSS[ （ 150 ± 13 ） bpm vs （ 105 ± 17 ） bpm, P 〈 0. 05 ]. Green fluorescence was also detected in frozen SA tissue sections of canines injected with recombinant plasmids pIRES2-EGFP-HCN2 and the production amplified by RT-PCR was about 300 bp which is consistent with mHCN2 gene fragment. Conclusion The recombinant eukaryotic expression plasmid pIRES2-EGFP-HCN2 can improve pacing function in atrial myocytes and in canine model of SSS.

关 键 词：基因转移技术 病窦综合征 基因疗法 

分 类 号：R541.7[医药卫生—心血管疾病]