【摘要】 目的:为进一步研究肌质网钙ATP酶转基因在心力衰竭中的质粒价值,并观察腺相关病毒对心肌细胞感染的动态过程和对外源基因的表达能力。方法:构建含肌质网钙ATP酶的腺相关病毒载体(recombinantade-no-associatedvirus-sarcoplasmicreticulumcalciumATPase2a,rAAV-SERCA2a),利用激光共聚焦显微镜观察了活体异硫氰酸荧光素标记的腺相关病毒(fluoresceinisothiocyanate-rAAV,FITC-rAAV)进入心肌细胞的过程,利用流式细胞仪检测含绿色荧光蛋白的腺相关病毒rAAV-GFP感染心肌细胞后绿色荧光蛋白表达细胞的百分率。结果:构建、生产的重组病毒rAAV-SERCA2a的滴度为7×1014v·g/L,并可以有效介导肌质网钙ATP酶基因转录;FITC-rAAV感染心肌细胞后,大约15min可见病毒吸附在细胞膜上,30min时可大部分进入细胞内;未加丁酸钠组腺相关病毒介导的绿色荧光蛋白表达效率最高达33.3%,加用丁酸钠组该数据为48.7%。结论:成功构建了含肌质网钙ATP酶的腺相关病毒载体,该病毒可有效介导目的基因的表达。在体外腺相关病毒能有效感染心肌细胞,感染后的心肌细胞能有效表达外源基因。更多还原

【Abstract】 AIM:To assess plasmid value of sarcoplasmic reticulum calcium ATPase(SERCA2a ) transgene in heart failure, and to observe the effect of recombinant adeno associated virus(rAAV) on dynamic process of cardiac myocytes infection and expression of exogenous genes. METHODS:A recombinant Adeno Associated Virus sarcoplasmic reticulum calcium ATPase (rAAV SERCA2a) was constructed, in which the SERCA2a cDNA was inserted under the control of CMV promoter.We observed the process of fluorescein isothiocyanate rAAV(FITC rAAV) transferring into the cardiac myocytes under the con focal microscope and detected the positive cell percent after the cardiac myocytes infected by rAAV2 GFP(containing green fluorescent protein) by using the flow cytometer. RESULTS: The titre of the constructed and produced rAAV SERCA2a was 7×1014 v·g/L and the rAAV SERCA2a could effectively mediate transcription of SERCA2a expression.After FITC rAAV infected myocardial cell,about 15 min were needed for rAAV to bind on cardiac myocytes, and they got into the cells in the following 30 min.The maximum GFP expression efficiency was 33.3%, while the data was changed to 48.7%in the group treated by sodium butyrate.更多还原