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目的:系统发育比较分析与二级结构模拟结合用于反义药物优化设计。方法:靶向自由能低的完全保守区域设计反义脱氧寡核苷酸(ODN)。体外评价ODN对人A549细胞和鼠B16-BL6细胞的增殖抑制效应。细胞原位杂交与RT-PCR方法检测靶mRNA表达。用裸鼠荷A549肿瘤和小鼠荷B16肿瘤模型评价ODN的体内活性。结果:3个ODN体外对A549细胞的IC_(50)值显著低于阳性对照ISIS3521。效果最佳的ODN AP1261可剂量依赖地抑制PKC-α mRNA的表达。AP1261在0.005-0.5mg·kg~(-1)·d~(-1)的剂量范围内可抑制A549和B16肿瘤的体内生长。相同剂量下AP1261对A549肿瘤体内生长的抑制率显著高于ISIS3521,而ISIS3521对鼠B16肿瘤的体内外生长无影响。结论:AP1261可能成为有效的抗癌药物或佐剂,系统发育比较分析与二级结构模拟相结合有助于反义药物设计。
OBJECTIVE: Phylogenetic analysis was used in combination with secondary structure simulation to optimize antisense drug design. Methods: Antisense oligonucleotide (ODN) was designed for the fully conserved regions targeting low free energy. The inhibitory effect of ODN on the proliferation of human A549 and murine B16-BL6 cells was evaluated in vitro. Cell in situ hybridization and RT-PCR method to detect the target mRNA expression. In vivo activity of ODN was evaluated using nude mouse bearing A549 tumors and mouse bearing B16 tumor models. Results: The IC50 values of A549 cells in three ODN groups were significantly lower than that of ISIS3521 positive control. The best-performing ODN AP1261 dose-dependently inhibited PKC-α mRNA expression. AP1261 inhibited the growth of A549 and B16 tumors in vivo in the dose range of 0.005-0.5 mg · kg -1 d -1. The inhibition rate of AP1261 on the growth of A549 tumor at the same dose was significantly higher than that of ISIS3521, while ISIS3521 had no effect on the growth of B16 tumor in vitro and in vivo. Conclusion: AP1261 may be an effective anticancer drug or adjuvant. The combination of phylogenetic analysis and secondary structure simulation is helpful for antisense drug design.