氮酮促进脂质体介导质粒DNA转染兔角膜内皮细胞及毒性的实验研究

Experimental study of azone promoting transfection of plasmid DNA mediated with liposomes and the influence to rabbit corneal endothelial cells

目的探讨氮酮(AZONE)促进脂质体介导质粒DNA转染兔角膜内皮细胞的作用及其对细胞活性的影响。方法采用细胞培养方法,利用绿色荧光蛋白基因做报告基因,配制不同转染液,脂质体联合pEGFP-N1、氮酮联合脂质体及pEGFP-N1、氮酮联合pEGFP-N1、单纯pEGFP-N1、单纯脂质体、单纯氮酮,分别处理兔角膜内皮细胞,荧光显微镜观察细胞中绿色荧光蛋白的表达。用RT-PCR方法检测EGFPmRNA表达情况。采用MTT法研究不同转染液对细胞活性的影响。结果单纯氮酮、单纯脂质体及对照组处理的细胞不表达绿色荧光蛋白。氮酮联合脂质体及pEGFP2N1对兔角膜内皮细胞的转染效率为42.6%,脂质体联合pEGFP2N1组的转染效率为22.4%,氮酮联合PEGFP-N1组的转染效率为7.2%,单组质粒组的转染效率为1%。氮酮加脂质体加PEGFP-N1组转染效率高于其他转染组(P<0.01)。各组转染液对角膜内皮细胞活性无影响。结论氮酮可促进脂质体介导质粒DNA转染兔角膜内皮细胞,在有效的转染浓度下对细胞无明显毒性,有望成为促进角膜内皮细胞基因转染的新型试剂。

Objective To study azone-promoting plasmid DNA transfection mediated with liposome and its influence on rabbit corneal endothelial cells （CECs）. Methods The following types of transfection agents were dispensed： liposome＋ DNA plasmid pEGFP- N1, azone ＋ liposome ＋ pEGFP-N1, azone＋ pEGFP-N1, pEGFP- N1, liposome only and azone only. Rabbit corneal endothelial cells were treated with the previously listed agents. A green fluorescence protein （GFP） gene as a report gene was detected by fluorescence microscopy, and EGFP mRNA expression was assessed by RT-PCR. The various transfection agents were added to the culture medium to test their influence on cell vitality using the MTT method. Results GFP was not detected in corneal endothelial cells treated with liposome or azone agents alone. The transfection rate of GFP was 42.6% in corneal endothelial cells treated with the agent azone ＋ liposome ＋ pEGFP-N1. With liposome ＋ pEGFP-N1, the rate of transfection in corneal endothelial cells was 22.4 %. The expression of GFP was 7.2 % in corneal endothelial cells treated with azone ＋ pEGFP-N1. With the agent azone ＋ liposome ＋ pEGFP-N1, a high transfection rate of the GFP gene was obtained. And no significant influence on the vitality of corneal endothelium cells was observed. Conclusion Azone may enhance the transfection rate of plasmid DNA mediated with liposome. Azone had no inhibitory effect on CECs in vitro with sufficient concentrations and may be a new agent for gene transfection.