HER-2反义寡核苷酸对靶基因抑制作用的时相变化与微观分布研究  

Study on changes of inhibitory effect of HER-2-specific antisense phosphorothioate oligodeoxynucleotides on target gene and subcellular distribution

作　　者：李鹏[1] 尚明美[1] 王强[1] 付洁[1] 宋海峰[1] 刘秀文[1] 汤仲明[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所药理毒理研究室,北京100850

出　　处：《军事医学科学院院刊》2008年第5期401-405,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金资助项目(30572224)

摘　　要：目的：研究靶向HER-2基因、具有不同活性的反义硫代寡核苷酸（S-ODNs）在SK—BR-3细胞内的微观分布差异，以及与靶基因转录和翻译抑制作用的关系。方法：运用实时定量PCR和Western印迹方法检测反义寡核苷酸对细胞靶基因转录和翻译抑制的时相性变化，用激光扫描共聚焦显微镜（LsM）观察6-羧基荧光素标记S-ODNs在靶细胞内的分布。结果：反义S—ODNs能够在mRNA与蛋白水平抑制HER-2的表达。不同序列的抑制活性各不相同，但最大抑制强度均出现于转染后48～72h之间，此后靶基因表达逐渐恢复。高活性序列主要均匀分布于细胞核，且持续时间长，低活性序列呈点状分布于胞质，少量分布于细胞核且持续时间短。结论：活性不同的S-ODNs亚细胞分布有显著区别，且分布变化与对靶基因的抑制效应具有对应关系。Objective：To study the subcellular distribution of HER-2-specific antisense phosphorothioate oligodeoxynu- cleotides （S-ODNs） in breast cancer cell SK-BR-3 and their relationship with target gene transcription and translation inhibition effect. Methods： The expression of HER-2 at mRNA and protein level were determined by real-time quantitative PCR and Western blot, respectively. The subcellular distribution of FAM-labeled antisense S-ODNs was observed by laser- scanning microscope （LSM）. Results： S-ODNs inhibited the expression of HER-2 both at transcription and translation levels. The maximal inhibition started at 48 - 72 h after transfection although the effect of different sequences varied dramatically. S-ODNs with high efficacy were distributed in the nuclei uniformly with long duration. In contrast, S-ODNs with ion efficacy were agglomerately distributed in the cytoplasm, and to a very low extent in the nucleus and lasted a short time. Conclusion ： The subcellular distribution of S-ODNs with different inhibitory effect differed remarkably, and the distribution character of individual S-ODNs was correlated with its inhibitory efficacy.

关 键 词：HER-2 硫代反义寡核苷酸 实时定量PCR 亚细胞分布 转录 遗传 转染 细胞核 

分 类 号：Q78[生物学—分子生物学]