Zhang Wanguang, Chen Xiaoping, Qiu Fazu
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
J Huazhong Univ Sci Technolog Med Sci. 2003;23(4):387-91. doi: 10.1007/BF02829425.
To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE 2000 (LF2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by Western-blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.
探讨靶向生存素的反义RNA对肝癌细胞体外化疗敏感性的影响。采用逆转录聚合酶链反应（RT-PCR）扩增生存素mRNA结构区，并反向插入真核表达载体pcDNA3。用脂质体2000（LF2000）将反义表达质粒pcDNA3/生存素转染至HepG2细胞，同时加入低浓度的5-氟尿嘧啶（5-Fu）。转染后12 h、24 h、48 h，采用蛋白质免疫印迹法检测生存素蛋白，噻唑蓝比色法（MTT）检测细胞生长活性，流式细胞术检测细胞凋亡情况。转染后48 h采用定量分析法检测半胱天冬酶-3（caspase-3）活性。通过限制性内切酶消化和核苷酸测序验证反义RNA载体pcDNA3/生存素的构建。与正常组、5-Fu组和反义生存素组比较，反义生存素转染+5-Fu组细胞生长抑制率、凋亡指数和caspase-3活性均升高。生存素沉默后凋亡阈值降低，化疗敏感性增加。这些研究结果提示存在一个潜在的基因治疗新靶点。
