9型腺相关病毒转染乳鼠心肌细胞的可行性及安全性

Feasibility and safety of adeno-associated virus serotype 9 transfection of neonatal rat cardiomyocytes

背景：9型腺相关病毒对心肌细胞具有更好的靶向转染,是目前研究基因治疗心脏疾病的理想载体。目的：探索9型腺相关病毒体外转染乳鼠心肌细胞的可行性及对乳鼠心肌细胞的毒性评估。方法：分离培养原代乳鼠心肌细胞,以携带荧光蛋白基因的9型腺相关病毒为载体,将分离纯化的乳鼠心肌细胞分为正常培养组、无病毒转染组、病毒转染组。结果与结论：第1周细胞搏动频率及百分率正常培养组和病毒转染组与无病毒转染组比较,差异无显著性意义（P〉0.05）,但正常培养组高于病毒转染组（P〈0.05）;3周后各组比较,差异无显著性意义（P〉0.05）。经9型腺相关病毒转染的心肌细胞24h开始有表达,4~6d荧光最强,3组细胞72h内不同时间点还原比率均接近于1.0。说明9型腺相关病毒能成功有效地转染乳鼠心肌细胞,对乳鼠心肌细胞无明显毒性作用。

BACKGROUND： Transfection of cardiomyocytes is better achieved using adeno-associated virus serotype 9; adeno-associated virus serotype 9 is an ideal carrier for gene therapy research on heart disease at present. OBJECTIVE： To explore the feasibility of adeno-associated virus serotype 9 transfection of neonatal rat cardiomyocytes and to assess its toxicity on neonatal rat cardiomyocytes. METHODS： The primary cultured neonatal rat cardiomyocytes were isolated and cultured. Based on adding adeno-associated virus serotype 9 carrying enhanced green fluorescent protein or not, the myocardiocytes were divided into three groups： control group, non transfection group and transfection group. RESULTS AND CONCLUSION： There was no significant difference in beat frequency and percentage of pulsating cells among control group, non transfection group and transfection group in the 1st week （P 0.05）. However, the beat frequency and percentage of pulsating cells in control group was larger than that in the transfection group （P 0.05）. There still was no significant difference in beat frequency and percentage of pulsating cells among the three groups in the 3rd week （P 0.05）. Cells in the transfection group began to express myocardial cells in 24 hour after transfection and reached the maximum fluorescence intensity on the 4th to the 6th days. The reduction ratios of three groups were close to 1.0 at different times within 72 hours. These findings indicate that adeno-associated virus serotype 9 can be effectively transfected into neonatal rat cardiomyocytes without significant toxicity.