聚苯乙烯微塑料对质粒DNA的吸附及其转染效应研究

Study on the adsorption and transfection effects of polystyrene microplastics on plasmid DNA

微塑料(MPs)长期存在于自然环境中,可通过呼吸和食物摄入的方式危害人体健康.微塑料不仅本身具有生物毒性,还能吸附多种组分构成复合污染物.为了探究微塑料对质粒DNA的吸附特性及介导的质粒转染效果.本研究利用琼脂糖凝胶电泳和透射电子显微镜表征聚苯乙烯微塑料(PS-MPs)对质粒DNA(pcDNA 3.1-EGFP)的吸附作用.微塑料及其质粒DNA吸附复合物转染人食管上皮细胞EC109后,利用RTqPCR检测EC109细胞中源自质粒pcDNA 3.1-EGFP的eGFP基因及新霉素抗性基因(neoR)的转录,利用共聚焦显微镜观察EC109细胞中源自质粒pcDNA 3.1-EGFP的绿色荧光蛋白表达,并利用显微镜和CCK-8实验检测了微塑料及其质粒DNA吸附复合物对食管上皮细胞EC109的毒性效应.结果显示,聚苯乙烯微塑料可以有效吸附质粒DNA,且聚苯乙烯微塑料对质粒DNA的吸附效果与微塑料的粒径呈负相关,与微塑料的浓度呈正相关.微塑料对质粒DNA的吸附作用平衡时间约为12 h.聚苯乙烯微塑料-质粒DNA复合物呈串聚现象.转染细胞中检测出细胞中eGFP基因和抗性基因(neoR)的有效转录及绿色荧光蛋白的表达,细胞的形态及活性无显著改变.研究表明,聚苯乙烯微塑料有效吸附质粒DNA,并可介导质粒DNA对真核细胞的转染.

Microplastics(MPs)exist in the natural environment for a long time,and can endanger human health through respiration and food intake.Microplastics not only have biological toxicity,but also can adsorb various components to form complex pollutants.To explore the adsorption characteristics of microplastics on plasmid DNA and the effect of plasmid transfection mediated by microplastics.In this study,the adsorption of polystyrene microplastics(PS-MPs)on plasmid DNA(pcDNA 3.1-EGFP)was characterized by agarose gel electrophoresis and transmission electron microscopy.After the microplastics and its plasmid DNA adsorption complex were transfected into human esophageal epithelial cells EC109,RT-qPCR was used to detect the intracellular transcription of eGFP gene and neomycin resistance gene(neoR)derived from plasmid pcDNA 3.1-EGFP in EC109 cells.Moreover,the expression and cell distribution of green fluorescent protein derived from plasmid pcDNA 3.1-EGFP were observed under confocal microscope.The toxic effects of microplastics and their plasmid DNA adsorption observed under confocal microscope.The toxic effects of microplastics and their plasmid DNA adsorption complexes on EC109 cells were examined by microscope and CCK-8 test.The results showed that polystyrene microplastics could effectively adsorb plasmid DNA,and the adsorption effect of polystyrene microplastics on plasmid DNA was negatively correlated with the particle size of microplastics,and positively correlated with the concentration of microplastics.The equilibrium time of microplastics adsorption on plasmid DNA is about 12 hours.Polystyrene microplastics-plasmid DNA complex showed a cluster phenomenon.The effective transcription of eGFP gene and resistance gene(neoR)and the expression of green fluorescent protein were detected in the transfected EC109 cells,and there was no significant change in cell morphology and activity.This study shows that polystyrene microplastics can effectively adsorb plasmid DNA and mediate the transfection of plasmid DNA into eukaryotic cells.