PDT对反义bcr-abl寡核苷酸k562细胞转染的影响

目的 :体外研究光动力治疗 (PDT)对b3a2型反义bcr-abl寡核苷酸 (ASO)慢性髓细胞性白血病 (CML)细胞株K5 6 2转染的影响 ,为PDT合并反义技术应用于CML患者治疗提供实验基础。方法 :半导体激光 ,波长 6 5 0nm ,剂量 9J cm2 垂直照射。光敏剂为血卟啉单甲醚(HMME) ,采用体外细胞培养技术 ,流式细胞仪检测PDT实验组与对照组荧光标记的反义bcr-abl寡核苷酸 (ASPO)K5 6 2细胞摄入率及荧光强度。结果 :①PDT可显著提高k5 6 2细胞对反义bcr-abl寡核苷酸的摄入 ,比直接转染增加转染率可达 4-1 0倍。且k5 6 2细胞对反义bcr-abl寡核苷酸的摄入和反义bcr-abl寡核苷酸浓度及作用时间有关。②光敏剂量浓度为 0 .9μmol·L- 1 时转染的效率最高 ,4小时点细胞内平均荧光强度最强 (P <0 .0 1 ) ,且细胞内平均荧光强度随ASPO浓度的增加而增高。但直接转染时 ,6小时才达到高峰。浓度为 0 .6 μmol·L- 1 ～ 0 .9μmol·L- 1 时K5 6 2细胞对荧光物质的摄入即达饱和。结论 :PDT可以增加反义bcr -abl寡核苷酸K5 6 2细胞转染率

The study of effects of photodynamic gene transfection of HMME-PDT to bcr-abl antisense oligonucleotides on k562 cells in vitro

Objective:To investigate the effects of photodynamic gene transfection of HMME-PDT to antisense oligonucleotides targeted b3a2-typed bcr-abl fusion gene on k562 cells in vitro and provide experimental supports for the utility of HMME-PDT in combination with b3a2-ASO in the gene therapy on K562 cells and the bone marrow purging of the patients with chronic myelogenous leukemia(CML).Methods:low intensity semiconductor laser,wave length:650nm,dosage:9J/cm 2 in vitro cell 1 culture,k562 cells are exposed to HMME-PDT and FAM-labelled b3a2-ASO simultaneously.the uptake of FAM-labelled b3a2-ASO are measured by flow cytometry(FCM).Results:The uptake of ASPO into k562 cells is greatly improved by photochemical gene transfection which increased the uptake of b3a2-ASO into k562 cells about 4-10 times compared with application of b3a2-ASO alone.Accumulation of b3a2-ASO was time and concentration dependent in the absence or presence of photochemical gene transfection.After 4-hour exposure to b3a2-ASO,the mean intracellular fluorescein intensity peak of k562 cells is reached to in the presence of photochemical gene transfection while it required 6-hour to reach to the peak of the mean intracellular fluorescein intensity in the absence of photochemical gene transfection.Conclusions:Photodynamic gene transfection of HMME-PDT increases uptake of ASPO on k562 cells.