c-myc基因mRNA核酶对血管平滑肌细胞增殖的抑制作用

目的：构建特异性切割c-myc基因mRNA的锤头状核酶（Ribozyme)真核表达载体。探讨核酶对血管平滑肌细胞（VSMCs)增殖的影响。方法：计算机分析大鼠c-myc基因mRNA的二级结构，选择第2029位点作为锤头状核酶的切割位点并设计相应的核酶。采用DNA合成仪合成核酶基因，以克隆技术将核酶基因构建人pGEM3Zf( )体外转录载体，以Sanger双脱氧末端终止法测定核酶基因的序列后，通过亚克隆将核酶基因构建入pcDNA3真核表达载体；核酶真核表达载体以阳离子脂质体LipofectAMINE作为介导，转染体外培养的第5代SD大鼠VSMCs，经G418筛选出细胞克隆，流式细胞仪测定VSMCs的细胞周期。结果：核酶基因准确克隆入pGEM3Zf( )载体，DNA序列测定表明所克隆入的核酶基因序列正确；经过亚克隆将核酶基因准确克隆入pcDNA3载体（pcDNA-Rz);pcDNA-Rz转染VSMCs经G418筛选出多个抗性细胞克隆，扩大培养获得转染细胞；细胞周期分析显示，pcDNA-Rz转染细胞的细胞周期有明显变化，S期和G2/M期比率明显减少，细胞生长阻滞于G0/G1期，细胞增殖指数明显降低。结论：特异性切割c-myc基因mRNA核酶对体外培养VSMCs的增殖具有明显的抑制作用。

Inhibition of vascular smooth muscle cell proliferation by specific c-myc mRNA cleaving hammerhead ribozyme

AIM: To investigate the effects of hammerhead ribozyme that specifically cleaves c myc mRNA on the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Based on the computer analysis of the secondary structure of c myc mRNA, nt 2029 in rat c myc oncogene was selected as the cleaving site for hammerhead ribozyme and the ribozyme was designed. With an automatic DNA synthesizer, two complementary DNA strands of the ribozyme were synthesized. The ribozyme gene was cloned into pGEM3Zf (+) vector and subcloned into eukaryotic expression pcDNA3 vector. The recombinant pcDNA Rz was transfected into the cultured rat VSMCs by lipofectAMINE mediated DNA transfection protocol and individual cell clones were selected by G418. RESULTS: The sequence of ribozyme gene inserted in pGEM3Zf (+) vector was proved to be perfectly correct. In vascular smooth muscle cells transfected with recombinant pcDNA Rz, flow cytometry analysis showed that the S phase and G2/M fractions decreased significantly and VSMCs proliferation stagnated in the G 0/G 1 phase. CONCLUSION: The results suggest that hammerhead ribozyme that specifically cleaves c myc mRNA can significantly inhibit the proliferation of VSMCs.