血管紧张素Ⅱ受体1反义寡核苷酸转染心肌细胞

Time Course of the Transfection of Angiotensin Ⅱ Receptor 1 Antisense Oligonucleotides to Cardiac Myocytes

目的 探讨血管紧张素Ⅱ 1型受体反义寡核苷酸转染心肌细胞的可行性。方法 应用显微荧光技术观察反义寡核苷酸在原代培养的心肌细胞内的转染效率 ,及其在细胞内的时相性分布 ;MTT法检测转染复合物对心肌细胞活力的影响。结果 寡核苷酸单独转染时 ,30分钟开始进入心肌细胞 ;6 0分钟进入细胞核 ,转染效率达峰值为 30 % ;12 0分钟部分被降解 ;4小时后大部分降解。脂质体的介导能显著提高反义寡核苷酸对心肌细胞的转染效率 ,6 0分钟达峰值为 6 0 % ,并且能改变寡核苷酸在细胞内的分布 ,4小时后仍能使寡核苷酸稳定于细胞核内 ;MTT法证实转染复合物对心肌细胞的活力无显著性影响。结论 反义寡核苷酸能成功的转染心肌细胞 ;脂质体的包裹能提高反义寡核苷酸转染效率、延长其作用时间 ,并改变寡核苷酸在细胞内的分布 ,使其长时间的稳定在细胞核内。本研究浓度的转染复合物对心肌细胞无明显的细胞毒性。

Objective To evaluate the time course of transfection of the antisense oligonucleotides of angiotensin Ⅱ receptor 1 to the cardiac myocytes. Methods The distribution of FITC labeled antisense oligonucleotides and the efficiency of the transfection in the cardiac myocytes were detected by microfluorescent technology. The poison effects of transfection on cardiac myocytes were detected by MTT. Results When transfecting alone, the antisense olignucleotides entered into cardiac myocytes at 30 minutes, into the nucleus at 60 minutes. While at 120 minutes, parts of oligonucleotides were degraded. Four hours later all of the antisens olignucleotides disappeared. The maximum efficiency of transfection was about 30 percent. While the delivery of oligfectamin enhanced the transfected efficiency to 60 percent, and it also change the distribution of antisense oligonucleotides in the cell. Four hours later most part of antisense oligonucleotides was still found in nucleus. No cytotoxity were found. Conclution Antisense oligonucleotides alone transfect cardiac myocytes succeedly. However the delivery of oligfectamin could prompt the transfected efficiency, alert the distribution of antisense oligonucleotides, and make it stay in nucleus more stably. Transfection complex used in our study do no harm to cardiac myocytes.