体外培养细胞摄取反义核酸的放射自显影研究

An autoradiographic study on antisense oligodeoxynucleotide uptake by cultured cells

用放射自显影观察了 NIH 3T3细胞和大鼠胸主动脉平滑肌细胞对[γ-(32)~P]ATP 标记未修饰反义核酸的摄取过程,并进行了同步与非同步细胞的比较。结果表明,未修饰反义核酸能迅速跨膜进入细胞,其摄入量与保温时间呈正相关;不同种类的细胞与不同周期时相的同种细胞对反义核酸的摄取能力不同。提示临床使用反义技术调控基因表达时,除用量外还须考虑细胞类型、作用时间、细胞时相和应用时机,才能获得最佳效果。

PURPOSE Unmodified antisense oligodeoxynucleotide (ODN) was synthesized and tested for its ability to enter into the cytosol of the NIH 3T3 and cultured SD male rat thoracic aorta smooth muscle cells.METHOD The antisense ODN was labeled at the 5' end with[γ-^(32)P] ATP.The uptake of the antisense ODN by both synchronized and unsynchronized cells was also compared.RESULTS Unmodified antisense ODN could rapidly enter into the cell through the cell membrane within a few seconds at 37℃ in Dulbecco's medium without serum.The amount of antisense ODN in both cells was proportional to the incubation time.Cell entry of antisense ODN was related to the cell types and the cell cycling events.CONCLUSIONS Using the antisense technology to modulate gene expression,the cell types,the time for uptake of the antisense ODN,and the cell cycling events together with dosage to be used should he considered.