腺病毒介导gax基因转染在培养的血管平滑肌细胞中的表达

The Expression of Gax Gene in Cultured Vascular Smooth Muscle Cells Mediated by Adenovirus Transfection

目的 以腺病毒介导gax基因转染血管平滑肌细胞 (VSMC) ,增强VSMC中 gax基因的表达。方法 采用位置特异性重组方法构建携带大鼠 gax基因表达序列的复制缺陷型 5型腺病毒载体 (Ad CMV gax) ,经 2 93细胞扩增 ,纯化制备高滴度病毒转染液 ;以病毒转染液常规转染VSMC后 ,应用RT PCR、流式细胞仪和免疫细胞化学等方法分别检测VSMC中 gaxmRNA和蛋白质的表达。 结果 流式细胞仪检测和免疫细胞化学染色均显示AdCMV gax转染VSMC后 ,VSMC的Gax蛋白表达率明显增高 ,转染后 2 4小时即可达 80 %左右 ,高水平的表达可维持 5天以上 ;AdCMV gax转染前 ,PDGF BB对 gax基因转录和翻译水平的表达均有下调作用 ,AdCMV gax转染后 ,无论有无PDGF BB刺激 ,VSMC中 gax基因的表达均比转染前显著增高。 结论 腺病毒载体可有效地介导 gax基因转染VSMC ,并且表达为蛋白质。这有利于进一步研究

Objective To study the overexpressing gax gene in vascular smooth muscle cells(VSMC) mediated by adenovirus transfection. Methods Replication defective adenovirus type 5 (Ad5) vector encoding the rat gax gene (AdCMV gax) was constructed via site specific recombination and amplified in 293 cells. The resulting adenovirus was purified for a high viral titer. The expression of gax mRNA and protein was evaluated by a series of methods such as reverse transcription polymerase chain reaction (RT PCR), flow cytometry and immunocytochemical technique after gax gene was transferred to VSMCs. Results When AdCMV gax was transfected to VSMCs, the expression of gax protein increased significantly in VSMCs, and reached 80% in 24 hours. High expression of gax protein sustained for over 5 days. Before AdCMV gax was transfected to VSMCs, gax mRNA and protein was down regulated by platelet derived growth factor (PDGF) BB. After AdCMV gax was transfected to VSMCs, the level of gax expression was significantly higher regardless of the stimulation of PDGF BB. Conclusion gax gene can be transfected to VSMC by AdCMV gax and translated to protein, which is advantageous to exploring the effects of gax gene on biological behaviour of VSMC.