人心钠素基因裸质粒与脂质体介导转染在培养鼠心肌细胞中表达的研究

Expressions of Liposome-mediated and Naked Plasmid DNA Encoding Human Atrial Natriuretic Peptide in Cultured Rat Ventricular Myocytes

目的 采用脂质体介导转染法和裸质粒直接转染法 ,将构建的人心钠素 (hANF)真核表达质粒 (pCR3.hANF)导入培养鼠心室肌细胞中 ,观察和比较hANF基因在心肌细胞中的表达效率。方法 实验分为脂质体 /pCR3.hANF转染组、裸 pCR3.hANF转染组及pCR3对照组 ,通过RNA的提取、逆转录 -聚合酶链式反应、Southern杂交及放射免疫测定 ,检测表达效率。结果 (1)转染后 2 4h ,脂质体 / pCR3.hANF转染组和pCR3.hANF转染组hANFmRNA均有明显表达 (P值分别为 <0 0 1和 <0 0 5 ) ,且前者大于后者 (P <0 0 1) ;转染后 4 8h ,两组表达更为显著 (P值均 <0 0 1) ,且较转染后 2 4h明显增高 (P值均小于 0 0 5 ) ;(2 )转染后 4 8h ,脂质体 /pCR3.hANF转染组和 pCR3.hANF转染组培养液中ANF的浓度明显增高 (P值均小于 0 0 1) ;转染组间比较 ,前者hANF的表达也明显高于后者 (P <0 0 1)。结论 脂质体介导的hANF基因转染和裸hANF质粒直接转染均能显著提高hANF基因的表达效率 ;直接转染法更具方便性和实用性 ,有利于将来的临床应用。

Objective To investigate the expression efficiency of human atrial natriuretic peptide(hANF) gene, a eukaryotic expression plasmid containing hANF gene pCR3.hANF was transferred into the cultured rat ventricular myocytes by 2 ways of liposome mediated pCR3.hANF transfection, direct naked pCR3, and hANF transfection with pCR3 as control. Methods hANF gene expression of three groups (liposome/pCR3.hANF, naked pCR3.hANF, and pCR3 control) in ventricular myocytes was determined by radioimmunoassay and reverse transcription polymerase chain reaction followed by Southern blot analysis. Results (1) 24 hours after gene transfer, liposome/pCR3.hANF and naked pCR3.hANF resulted in a significant hANF mRNA expression( P <0 01 vs P <0 05, respectively) and with former higher than in the latter. (2) 48h after gene transfer, much higher expression of hANF mRNA was observed in the two groups than in control group(both P <0 01); ANF levels of culture mediums increased markedly in the two groups(both P <0 01) and the ANF levels in liposome/pCR3.hANF group was higher than in the naked pCR3.hANF group( P <0 01). Conclusions Liposome mediated transfection or high dose pCR3.hANF can significantly improve the expression efficacy. Direct delivery of naked pCR3.hANF is a more convenient and practical method for gene transfer.