反义寡核苷酸抑制血管平滑肌细胞bFGF基因表达

Sequence-specific Inhibitory Effects of Gene Expression on Antisense Basic-fibroblast-growth-factor Oligodeoxynucleotides in Vascular Smooth Muscle Cells

目的:探讨转染反义脱氧寡核苷酸(AODN)对血管平滑肌细胞(VSMCs)碱性成纤维细胞生长因子(bFGF)基因表达的影响机制。方法:使用原位杂交和免疫组化技术检测体外培养的VSMCs中bFGFmRNA和蛋白表达;利用脂质体分别将设计合成浓度为5μmol/L反义(反义组)、正义(正义组)及随机(随机组)脱氧寡核苷酸导入体外培养的VSMCs;应用逆转录PCR和WesternBlot半定量测定及计算机图像分析的方法,观测寡核苷酸对bFGF基因表达的影响。结果:体外培养的大鼠VSMCs均有bFGFmRNA和蛋白表达,反义bFGF寡核苷酸作用于VSMCs后1～5d,bFGFmRNA表达和蛋白表达均明显减弱,反义组与对照组比较,mRNA、蛋白表达在第1～5d表达差异均有统计学意义;而正义、随机组无此效应,与单纯脂质体和空白对照组相比差异无统计学意义(P>0.05)。结论:反义bFGF寡核苷酸对血管平滑肌细胞bFGF基因表达的抑制作用具有序列特异性。

Objective： To investigate the effect mechanism of gene expression of antisense oligodeoxynucleotides（ODN） on the basic fibroblast growth factor（bFGF） that was in the vascular smooth muscle cells（VSMCs）. Methods： The expession of bFGF mRNA and protein in the VSMCs cultured in vitro was detected by in situ hybridization and immunocytochemistry. The designed synthesized sense,antisense, and random ODN that had a density of 5 μmol/L was delivered to culture the VSMCs in vitro respectively by using liposomes.The effect of ODN on bFGF gene expression was studied by using RT-PCR,semiquatitive Western Blot and computer image analysis. Results： The VSMCs of rats cultured in vitro could express bFGF mRNA and protein. The expression of bFGF mRNA and protein was inhibited significantly after bFGF AODN exerting on the VSMCs one to five days,the difference of mRNA and protein expression one to five days later in the AODN group had statistical significance compared with the control group. However, SODN and RODN couldn＇t have these results and the difference was not statistically significant compared with the liposomes and empty group （P 〉 0.05）. Conclusion： The habitory effect of bFGF AODN on gene expression of VSMCs bFGF has sequence-specificity.