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转录因子Oct-4和Survivin是细胞增殖的关键调控因子,构建针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体Ad5-Dual-shRNA,并研究其对肝癌细胞及移植瘤的生长抑制作用。合成Oct-4和Survivin基因的shRNA序列,插入腺病毒穿梭载体pDC312,含有shRNA的穿梭载体与腺病毒骨架载体pBHGloxdeltaE13Cre共转染HEK293细胞,经Cre/LoxP位点特异性重组获得重组腺病毒Ad5-Dual-shRNA;腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1,经Western blotting检测Oct-4和Survivin基因的表达情况,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐染色法(MTT实验)和裸鼠荷瘤实验检测对肿瘤细胞生长的影响。研究结果显示,双靶向重组腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1能够有效沉默Oct-4与Survivin基因的表达,并且在MTT实验和裸鼠荷瘤试验中都显示出较单一靶向的shRNA腺病毒载体Ad5-Surv-shRNA、Ad5-Oct4-shRNA具有更为明显的肿瘤细胞生长抑制作用。实验结果表明,特异性双靶向shRNA腺病毒载体Ad5-Dual-shRNA是一种更为高效的靶向肿瘤基因治疗载体。
Transcription factors Oct-4 and Survivin are key regulators of cell proliferation. The double-targeting shRNA adenovirus vector Ad5-Dual-shRNA targeting Oct-4 and Survivin gene was constructed and its growth inhibitory effects on hepatoma cells and xenografts were studied . The shRNA sequence of Oct-4 and Survivin gene was synthesized and inserted into adenoviral shuttle vector pDC312. The shuttle vector containing shRNA and adenoviral backbone vector pBHGloxdeltaE13Cre were co-transfected into HEK293 cells. Recombinant adenovirus Ad5- The expression of Oct-4 and Survivin gene was detected by Western blotting. The expression of Survivin gene was detected by Western blotting. The expression of EHBH- , 5-diphenyl tetrazolium bromide staining (MTT test) and nude mice tumor test on tumor cell growth. The results showed that double-targeting recombinant adenovirus Ad5-Dual-shRNA transfected hepatocellular carcinoma cell line EHBH-H1 can effectively silence the expression of Oct-4 and Survivin gene, and both in MTT experiment and nude mice bearing test showed a more single Targeted shRNA adenovirus vector Ad5-Surv-shRNA, Ad5-Oct4-shRNA has more obvious inhibition of tumor cell growth. The experimental results show that the specific double-targeting shRNA adenovirus vector Ad5-Dual-shRNA is a more efficient target gene therapy vector.