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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 To observe the intracellular distribution and stability of modified and non-modified PCNA antisense oligonucleotides after cationic liposome-mediated or direct transfection of human glioblastoma multiforme BT325 cells Its mechanism Methods Fluorescein isothiocyanate fluorescein (5’-FTTC) labeled 18mer phosphorothioate modified and unmodified PCNA antisense oligonucleotides were either liposome-mediated or directly transfected into human pleomorphic glue Glioblastoma cells BT325, the fluorescence microscope dynamic observation of fluorescence in the transfected cells within the phase distribution. Results After the modified antisense oligonucleotides were directly transfected into the cells for 30min, the fluorescence appeared discrete dot-like distribution in the cytoplasm. After 6h, the fluorescence of the cytoplasm was strong but only a few nuclei had fluorescence accumulation. Under the condition of liposome, the number of fluorescent cells increased obviously, and almost all of the nuclei appeared fluorescence after 2h, and the fluorescence in nucleus decreased gradually after 24h. However, unmodified antisense oligodeoxynucleotides were found to disappear after a few hours either by direct transfection or by liposomes. Conclusion The cationic liposome DOSPER can not only promote the entry of PCNA antisense oligonucleotide into BT325 nucleus, but also its antisense oligonucleotide complex with antisense oligonucleotide to antisense oligonucleotide Has a certain protective effect; phosphorothioate modification can increase the stability of PCNA antisense oligonucleotide in BT325.