3条硫代寡核苷酸的亲和性与内皮细胞吸收的比较研究  

Comparison Investigation of Affinity of Three Series Phosphorothioate Oligonucleotide and Its Uptake in Endothelial Cells

作　　者：李黔宁[1] 戴光明[1] 郑健[1] 肖道虹[1] 邓志宽[1] 刘勇[1] 任萍[1] 周永琴[1] 刘立杰[1] 应大君[2] 

机构地区：[1]重庆第三军医大学新桥医院神经内科,400037 [2]第三军医大学基础部解剖教研室

出　　处：《中国微循环》2002年第6期325-328,共4页Journal of Chinese Microcirculation

基　　金：国家自然科学基金资助(NO39970269)

摘　　要：目的研究硫代寡核苷酸的亲和性及其在内皮细胞株的吸收与亚细胞分布。方法 利用电泳迁移分析法检测硫代寡核苷酸的亲和性;将同位素标记的硫代寡核苷酸与ECV304细胞直接孵化,24 F后测定内皮细胞的吸收率及亚细胞分布。结果3条硫代寡核苷酸(T21GTa、T14Gta及T15Gta)的亲和性(Kd值)分别为2.3×10-9、3.8×10-9及1.5×10-8(M).细胞吸收率分别为11.9％、10.8％、11.1％,核沉淀放射活性分别为77.25％、76.75％、69.32％。结论3条硫代寡核苷酸能够与内皮细胞TF基因SSRE的3个Egr-1/Spl位点结合,具有较好的透膜性、耐酶性及稳定性,能够透过内皮细胞膜和核膜,抵达核内靶基因部位。Objective To investigate affinity of three series of phosphorothioate oligonucleotide and their uptake and subcellular distribution in endothelial cells. Methods Electrophoretic Mobility Shift Analysis (EMSA) was utilized to assay the affinity of phosphorothioate oligonucleotide and cellular uptake and distribution of γ-32 P labelled phosphorothioate oligonucleotide by direct incubation after 24 hours were observed in ECV304 cells. Results Affinity (the Kd) of three series of phosphorothioate oligonucleotide (T21GTa,T14GTa and TISGTa) is 2.3 × 10-9, 3.8×10-9, 1.5× 10-8(M) respectively; Cellular uptake rate of radioactivity in ECV304 11. 9,10. 8, 11. 1 (% ) respectively; Distribution of radioactivity in the nuclear pellet hold 77.25, 76.75 , 69.32 (% ) respectively. Conclusions Three series of phosphorothioate oligonucleotide can bind to three Egr - 1/Spl site of SSRE in promoter of TF gene in endothelial cell and posses pretty penetration, bearenzyme and stability enough to reach target gene spot through endothelial cell membrane and nucleus membrane.

关 键 词：比较研究 硫代寡核苷酸 细胞吸收 亚细胞分布 

分 类 号：R329.27[医药卫生—人体解剖和组织胚胎学]