摘要目的 通过质粒电穿孔法、腺病毒转染法(rAd)和9型腺相关病毒转染法(rAVV9)转染Gαi2羧基末端肽(Gαi2lp)基因至乳鼠心肌细胞,比较不同方法的转染效率、心肌毒性以及对胆碱类药物迷走神经效应的拮抗作用,探寻Gi3ctp基因功能表达的最佳转导途径.方法 构建重组质粒pDC316-Gαi2ctp-mCMV-EGFP(质粒电穿孔组)、重组腺病毒rAd-Gαi2ctp-mCMV-EGFP(rAd组)和重组9型腺相关病毒rAAV9-Gαi2ctp-IRES-EGFP(rAAV9组)以及未携带Gαi2ctp基因的空白对照组(n=10).3种方法转染携带免疫荧光蛋白(EGFP)的Gαi2tp目的基因至乳鼠心肌细胞,探寻最佳电转导参数和病毒最佳感染复数(MOI);比较最佳参数下不同方法转染情况和最大效率;AlamarBlue法测定各时间点还原比率评估心肌毒性;Western-blot法检测Cαi2ctp蛋白表达情况.最后以最适浓度的卡巴胆碱干预各组细胞,通过计数细胞整体搏动频率比较3种方法下Cαi2ctp基因的抗迷走神经效应差异.结果 3种方法最佳参数设置下Gαi2ctp-EGFP蛋白均成功表达且差异无统计学意义,转染效率分别于2、1.5、4 d达最高(54.2％±4.8％、100％和59.2％±4.4％),于第11 d转染效率分别为(30.1％±6.6％、12.0％±-3.4％和36.0％±6.1％);rAAV9组在全观察期l1d内细胞活性接近空白对照组(P＞0.05),质粒电穿孔组和rAd组细胞活性于3d后开始明显下降且后者较显著(P＜0.05).卡巴胆碱以最佳药效浓度200 μg/ml干预培养3d后心肌细胞,发现Gαi2ctp基因组较无基因组具有明显的抗卡巴胆碱迷走神经效应(P＜0.05),但各方法之间比较差异无统计学意义(P＞0.05).结论 质粒电穿孔法转染方式迅速、转染效率尚可,但对细胞破坏力大;rAd法转染效率最高可达100％,但表达持续时间较短,心肌毒性较大;rAAV9法转染起效较慢但能够稳定地长时间表达且生物安全性最好.3种方法皆可有效表达Gαi2ctp并发挥拮抗卡巴胆碱迷走神经变时效应.该实验为Gαi2ctp转染治疗心房颤动提供了理论支持和方法学依据.

abstractsObjective To explore the optimal transduction pathways of Gαti2ctp gene function expression by comparing the different way of transfection efficiencies,cardiotoxicity and the antagonism against choline effect tovagus through transfecting Gαi2ctp gene into neonatal rat cardiacmyocytes with the method of electroporation transfection,adenovirus transfection (rAd) and type 9 adeno-associated virus transfection (rAAV9).Methods The recombinant plasmid of pDC316-Gαi2ctp-mCMV-EGFP,the recombinant adenovirus of rAdGαi2ctp-mCMV-EGFP,type 9 adeno-associated virus rAAV9-Gαi2ctp-IRES-EGFP,and the corresponding empty control group were structured.Transfer Gαi2ctp gene by each method mentioned above was transferred into neonatal rat cardiacmyocytes.The maximum transfect-efficiency of the optimum parameter with different methods was compared.The reduction ratios at different time point and myocardial toxicity were evaluated by AlamarBlue staining.The expression of protein was detected by western-blot.Finally,neonatal rat cardiacmyocytes from each group were interfered with carbamylcholine(CCh) and its anticholinergic effect was compared.Results Under the relatively optimal parameter,the expresses of Gαi2ctp-EGFP protein in each group was successful with no statistical difference.The optimal transfection efficiencies of the 3 groups were 54.2％±4.8％、100％ and 59.2％±4.4％ respectively on the 2,1.5 and 4 d.The transfection efficiencies on the 11 d were 30.1％± 6.6％,12.0％±3.4％ and 36.0％±6.1％.During the observation period(11 days),the cell viability of rAAV9 group was similar with the control group (P＞0.05),the cell viability of electroporation group and rAd group went down after 3 days,and the latter was more significant(P＜0.05).After incubating the myocardial cells with the CCh in optimal concentration(200 μg/ml)for 3 days,the Gαi2ctp group was obvious resistant to the CCh (P＜0.05),with no statistical significance among different approaches.Conclusion The approach of electroporation is fast,efficient but destructive to the cell.The approach of rAd has high transfection efficiency (100％),but is unabiding with more poisonous.The approach of rAAV9 effects slowly but expresses more stably,durably with best biological safety.All the 3 approaches cantransfer Gαi2ctp into cardiacmyocytes effectively and can reduce the rate of cardiacmyocytes.