三种反义c-myc RNA的瞬时表达对血管平滑肌细胞的体外生物学效应的影响

Biological effect of transient expression of 3 antisense c-myc RNA on the vascular smooth muscle cells in vitro

目的 了解癌基因c-myc分子中各个区域的作用，找出最佳的反义封闭靶区，为c-myc表达增高相关疾病的基因治疗提供依据。方法 系统地构建了人类3种反义c-myc重组逆转录病毒表达载体（aM1、aM2和aM3），分别载有c-myc第1、2和3外显子的反义片段，经脂质体转染大鼠主动脉平滑肌细胞，并于转染后48 h采用流式细胞仪对细胞进行瞬时表达检测。结果 与对照组相比，aM2使c-myc表达量减少了37.6%，aM1为对照组的98.8%，而aM3却使c-myc表达增高21.7%；与此同时，aM1和aM2组增殖细胞核抗原（PCNA）表达分别下降了36.0%和30.9%，而aM3组PCNA表达增高。DNA分析则表明3种载体均使处于G0/G1期的细胞比例略为增高（aM2>aM1>aM3），S 期比例略下降（aM1>aM2>aM3），但尚未有DNA合成量的明显改变。结论 反义基因的导入在瞬时表达时可影响细胞中靶蛋白及其他生长相关蛋白的表达，但对细胞周期和DNA合成的影响尚小。

objective To investigate the role of different regions in c-myc molecule and find out the best target region for gene therapy of c-myc overexpression-related proliferative diseases. Methods Three antisense retrovirus expression vectors for human c-myc exon 1, 2 and 3 had been systemically constructed, and respectively named aM1, aM2 and aM3. The 3 vectors were transfected into primary cultured rat arterial smooth muscle cells with lipofectamine, and subsequent transient expression assay were performed 48 h later by way of flow cytometric analysis. Results Compared with control, aM2 remarkably reduced Myc expression (by 37.6%), while aM1 only slightly reduced the expression down to 98.8%, and in contrast, aM3 elevated Myc expression by 21.7%. Simultaneously, PCNA expression in aM1 and aM2 group were reduced by 36.0% and 30.9% respectively, while that in aM3 group were increased. DNA analysis indicated that all 3 antisense c-myc slightly increased the cell ratio of G0/G1(aM2>aM1>aM3) and decreased the proportions of cells in S phase (aM1>aM2>aM3). However, little change was observed in DNA synthesis. Conclusion Transient expression of the transfected antisense c-myc molecules can change the expression of cellular target proteins and other growth-related proteins, but has little effect on cell cycle and DNA synthesis.