反义c-myc、增殖细胞核抗原基因片断抑制血管平滑肌细胞相关基因表达

Antisense oligonucleotides inhibit c myc and PCNA expression in the vascular smooth muscle cells

目的研究有效的防治血管术后再狭窄的方法，以及应用反义ｃｍｙｃ、增殖细胞核抗原（ＰＣＮＡ）基因片断来封闭其相应基因表达的可行性。方法设计并合成ｃｍｙｃ、ＰＣＮＡ正义、反义及四个碱基错配的反义寡核苷酸，经体外稳定性检测和细胞转染实验证实其有效性后，分别作用于体外培养的血管平滑肌细胞（ＶＳＭＣｓ），应用逆转录ＰＣＲ半定量测定及计算机图像分析的方法，观测其对ｃｍｙｃ、ＰＣＮＡ基因表达的影响。结果设计合成的寡核苷酸在血清培养基中１～５天未被完全降解；寡核苷酸能顺利进入ＶＳＭＣｓ内；１０μｍｏｌ／Ｌ浓度的反义ｃｍｙｃ、ＰＣＮＡ作用于ＶＳＭＣｓ后１～５天，其相应的ｃｍｙｃ和ＰＣＮＡ基因表达减弱，计算机图像分析表明其表达产物与对照组相比差异有显著意义（Ｐ＜０．０５），而正义和错配反义寡核苷酸无此效应（Ｐ＞０．０５）。

objective To study the methods for prevention and treatment of vascular restenosis. Methods We used two factors which result in hyperplasia of vascular smooth muscle cells (VSMCs) and applied antisense c myc, PCNA oligonucleotides to attenuate the expression of their related gene. The sense, antisense, and mismatched oligomers for c myc or PCNA were designed and synthesized. They were individually delivered to the cultured VSMCs after the oligomers were stable in culture medium for 5 days and could cross the plasma membranes into the cells. Using RT PCR and the image analysis system, we observed the changes of both c myc and PCNA gene expression in the VSMCs. Results Antisense c myc and PCNA at a concentration of 10 μmol/L were able to inhibit the expression of c myc and PCNA gene in VSMCs ( P <0.05),whereas the sense and the mismatched ones couldn′t inhibit the expression ( P >0.05). Conclusion The antisense technique is an effective method for the inhibition of VSMCs proliferation.