5'-FITC标记的PCNA反义寡核苷酸在人膀胱癌细胞BIU-87中的分布及稳定性分析

目的观察硫代修饰型及天然型PCNA反义寡核苷酸在脂质体介导下或直接转染人膀胱癌细胞BIU-87后在细胞内的分布及其稳定性。方法将异疏氰酸荧光素（5＇－FITC）标记的18mer硫代磷酸化修饰型及未修饰型PCNA反义寡核苷酸在脂质体介导下或直接转染人膀胱癌细胞BIU－87，应用荧光显微镜观察转染细胞从细胞内的时相分布。结果修饰型反义核酸直接转染细胞后30分钟，少数细胞胞浆荧光呈离散型、点状分布，4小时后有少数细胞胞核有强荧光聚积。在脂质体介导下，荧光细胞数目明显增加，4小时后绝大多数细胞迅速积聚于细胞核，4～8小时后，细胞核内荧光强度进一步增强，12小时后细胞荧光减弱甚至消失。而非修饰型反义核酸在直接转染或脂质体介导下均发现荧光在3小时后消失。结论脂质体增加PCNA修饰型反义寡核苷酸与膀胱癌细胞BIU－87结合的数量，同时使其在细胞核内分布聚积明显；PCNA反义寡核苷酸硫代修饰具有更好的稳定性。

The distribution and stability of fluorescent-labeled PCNA antisense oligomer in human bladder cancer cell BIU-87

Objective Fluorescent-labeled antisense oligomer that is hybridized to 18-bp sequences next to the start coden of human proliferating cell nucleus antigen (PCNA) gene was used to assess the intracellular distribution and stability of the oligonucleotides in the presence or absence of liposome (lipofectin). Method IUM 18-mer Phosphorothioate and unmodified antisense oligonucleotides conjugated with fluorescent 5' -isothiocyananate (5 '-FITC) were cncapsulated by lipofctin or without, and then added into human bladder cancer cell BIU-87 culture media. The celltular distribution was observed by fluorescence microcopy in fixed cells. Result In the abscnce of lipofectin, the oligonucleotide localized to discrete structures in the cytoplasm of the cells, resulting in a punctate fluorescence pattern . In the presence of lipofectin, cellular fluorescence was markedly increased and the oligonucleotide localized with the nucleus, as well as to discrete stuctures in the cytoplasm. Accumulation of the oligonucleotide in the presence in the presence of lipofectin was time -dependent. Conclusion Cationic lipids can increase the amount of oligonucleotide associated with cells and alter the intracellular distribution of the oligonucleotide .The phosphorotide. The phosphorothioate antisense oligonucleotide remained in the nuclei as well as cytoplasm more stable than unmodified .