荧光标记的反义寡核苷酸在人视网膜色素上皮细胞中的分布研究

The distribution and stability of fluorescent-labeled EGFR antisense oligonucleotide in human retinal pigment epithelium

目的 观察表皮生长因子受体（ＥＧＦＲ）反义寡核苷酸（ＡＳＯ）转染人视网膜色素上皮细胞（ＲＰＥ）后的分布。方法 将荧光素标记的硫代修饰型及非修饰型ＥＧＦＲ ＡＳＯ直接或在脂质体介导下转染人 ＲＰＥ，荧光显微镜观察细胞内的分布。结果 非修饰型 ＡＳＯ在直接转染或脂质体介导下均发现荧光在４ｈ后消失。修饰型直接转染 ８ｈ后消失。脂质体介导下，细胞内荧光数目明显增加，细胞核内荧光强度增强，１４～１６ｈ后细胞荧光消失。结论 脂质体增加修饰型 ＡＳＯ与ＲＰＥ结合的数量，延长在细胞内停留时间，同时使其在细胞核内分布聚积明显；硫代修饰ＡＳＯ具有更好的稳定性。

Objective Fluorescent-labeled antisense oligonucleotide hybridized to 21-bp sequences on the start codon of human epidermal growth factor receptor(EGFR) gene was used to assess the intracellular distribution and stability of the oligonucleotides in the presence or absence of liposome(lipofectin). Methods 21 -mer phosphorothioate and unmodified antisense oligonucleotides conjugated with fluorescent 5' -isothiocyanate(5' -FITC ) were encapsulated with or without lipofectin,and then added into human retinal pigment epithelium culture media. The cellular distribution of the transfection was observed by fluorescence microscopy in fixed cells. Results In the presence or absence of lipofectin,the unmodified antisense oligonucleotide disapperared 4 h later after transfected. In the absence of lipofectin, the phosphorothioate oligonucleotide disapperared 8 h later after transfected. In the presence of lipofectin,cellular fluorescence of the phosphorothioate oligonucleotide was markedly increased, the oligonucleotide localized with the nucleus,The oligonucleotide disapperared 14 - 16 h later after transfected. Accumulation of the oligonucleotide in the nucleus in the presence of lipofectin was time dependent. Conclusion Cationic lipids can increase the transfaction efficiency of oligonucleotide in RPE cells and alter the intracellular distribution of the oligonucleotide. The phosphorothioate antisense oligonucleotide remained in the nuclei as well as cytoplasm longer than unmodified.