【摘要】 目的优化信号转导和转录活化因子5(signal transducers and activators of transcription,STAT5)诱骗寡核苷酸(decoyoli-godeoxynucleotide,decoyODNs)技术的实验条件,为阻断K562细胞STAT5信号转导途径,进而诱导细胞表型转变奠定基础。方法设计并合成STAT5decoyODNs、FAM荧光标记的decoyODNs和decoyODNs突变型(M-decoyODNs);SDS-PAGE检测不同退火缓冲液条件下decoyODNs双链形成率;倒置荧光显微镜下通过阳性细胞计数比较DEAE、lipofectin和DMRIE-C3种转染试剂对decoyODNs的转染效率;流式细胞仪(FCM)检测转染12、24和72h时FAM-decoyODNs摄取率和荧光强度,检测decoyODNs的稳定性;MTT实验检测decoyODNs对K562细胞活力的影响;RT-PCR检测decoyODNs对pim-1和c-myc基因表达的影响。结果退火缓冲液(10mmol/LTris,pH8.0,50mmol/LNaCl,1mmol/LEDTA)更有利于decoyODNs双链形成。DMRIE-C的转染效率(99.2±4.6)%高于DEAE和lipofectin(67.15±7.3)%、(83.2±3.8)%,P<0.05,并且DMRIE-C介导decoyODNs转染12、24、72h的转染效率均>98%。MTT实验发现DMRIE-C的细胞毒性低,细胞活性为94.23%,decoyODNs处理组能显著抑制K562细胞MTT还原能力,与其余组相比具有显著性差异(P<0.05);RT-PCR结果发现decoyODNs处理组pim-1基因下降了71.20%,c-myc下降了59.46%,与其余组相比有统计学意义(P<0.05)。结论建立了decoyODNs最佳的双链形成条件;DMRIE-C能显著增强decoyODNs转染K562细胞的效率;decoyODNs能抑制K562细胞活力,抑制STAT5靶基因的转录。更多还原

【Abstract】 Objective To optimize the experimental conditions for the application of signal transducers and activators of transcription 5 (STAT5) decoy oligodeoxynucleotide (ODNs) to reverse the malignant phenotype of human leukemia K562 cells.Methods STAT5 decoy ODNs and fluorescein phosphoramidite (FAM) labeled decoy ODNs were designed and synthesized in vitro.The effect of different annealing buffer on double strands formation rate of decoy ODNs was checked by SDS-PAGE.Then,the incorporation rates of FAM-decoy ODNs transfected by three reagent (DEAE,lipofectin and cation lipofectin DMRIE-C) were analyzed with fluorescent inversive microscope.The effects of time on transfection efficiency and stability of FAM-decoy ODNs to K562 cells were analyzed by flow cytometry (FCM) at 12,24 and 72 hours after transfection.MTT were used to check the cell viability of K562 cells.Semi-quantitative RT-PCR analysis of mRNA was performed to analysis pim-1 and c-myc gene expression.Results The annealing buffer (10mmol/L Tris,pH8.0,50mmol/L NaCl,1mmol/L EDTA) was best for decoy ODNs double strand forming.DMRIE-C with positive rate of 99.2%±4.6% was better than DEAE and lipofectin for decoy ODNs-K562 cell transfection(67.15%±7.3%,83.2%±3.8%,P<0.05,and the incorporation efficiency of FAM-decoy ODNs into K562 cells reached to 98% by DMRIE-C.MTT assay showed decoy ODNs could inhibit MTT reduction rate of K562 cells,compared with the other groups,(P<0.05),and DMRIE-C had little toxicity to cells.RT-PCR showed the expression of pim-1 reduced 71.20% and c-myc reduced 59.46%.Conclusion The annealing conditions for decoy ODNs was well optimized in this study.DMRIE-C can highly enhance the incorporation rate of decoy ODNs to K562 cells,and decoy ODNs can reduce K562 cells viability and STAT5 transcription of target gene.更多还原