5’-FITC标记的PCNA反义寡核苷酸在人多形性胶质母细胞瘤细胞BT325中的分布和稳定性分析  

Distribution and stability of FTTC - labeled PCNA antisense oligonucleotide in human multifonn glioblastoma cell BT325

作　　者：李美华[1] 朱贤立[1] 赵洪洋[1] 

机构地区：[1]同济医科大学附属协和医院神经外科

出　　处：《中国临床神经外科杂志》2000年第3期176-177,共2页Chinese Journal of Clinical Neurosurgery

摘　　要：目的 观察修饰型和非修饰型PCNA反义寡核苷酸在阳离子脂质体介导下或直接转染人多形性胶质母细胞瘤细胞BT325后在细胞内的分布及其稳定性,探讨其机理。方法 将异硫氰酸荧光素(5’-FTTC)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多形性胶质母细胞瘤细胞BT325,应用荧光显微镜动态观察荧光在转染细胞内的时相分布。结果 修饰型反义寡核苷酸直接转染细胞30min后,荧光在胞浆内呈现离散型点状分布,6h后胞浆荧光较强但仅有极少数细胞核有荧光积聚。在脂质体介导下,荧光细胞数目明显增加,2h后几乎所有细胞核内均出现荧光,持续24h后核内荧光逐渐减弱。而未修饰型反义寡苷酸在直接转染或脂质体介导下均发现荧光在数小时后消散。结论 阳离子脂质体DOSPER不仅能促进PCNA反义寡核苷酸进入BT325细胞核,而且其与反义寡核苷酸形成的脂质体/反义寡核苷酸复合物对反义寡核苷酸有一定的保护作用;硫代磷酸化修饰能增加PCNA反义寡核苷酸在BT325中的稳定性。Objective FTTC - labeled PCNA antisense oligonucleotide was used to assess the intracellular distribution and stability of the oligonucleotide in the presence or absence of lipozome(DOSPER).Methods 18- mer phosphorothioate and unmodified antisense oligonucleotides conjugated with 5'-FTTC were encapsulated by DOSPER or without, and then added into human multiform glioblastoma cell BT325 culture media. The intracellular distribution was observed by fluorescence microscopy in fixed cells. Results In the absence of liposome, the oligonucleotide localized to discrete structure in the cytoplasm of cells, resulting in a punctate fluorescence patten. In the presence of liposome, cellular fluorescence markedly increased and the obgonucleotide localized to the nucleus as well as to discrete structures in the cytoplasm. Accmulation of the oligonucleotide in the nucleus in the presence of liposome was time-dependent. Conclusion Cationic lipid(DOSPER)can increase the amount of oligonucleotide associated with BT325 cells and alter the intracellular distribution of the oligonucleotide. The phosphorothioate antisense oh'gonucleotide remained more stable than unmodified in the nuclei as well as cytoplasm.

关 键 词：反义寡核苷酸 PCNA 细胞内分布 

分 类 号：R739.41[医药卫生—肿瘤] R730.264[医药卫生—临床医学]