建立和评估抑制血管内皮生长因子（VEGF)表达的锤头状核酶（hammerhead ribozyme）技术系统。 方法: 对VEGF121基因RNA序列进行二级结构分析，选择靶点；设计并构建针对VEGF 的分泌肽RNA的可表达锤头状核酶（1 4）载体系统和VEGF-荧光色素酶融合基因报告质粒；通过试管内切割实验等方法评估核酶对于试管内转录得到的VEGF RNA切割特异性和效率；通过瞬时共转染实验和稳定转染实验评估核酶在细胞内对于VEGF RNA的切割效率。 结果: 设计和构建了对VEGF RNA二级结构水平上的暴露区（+8,+36和+71位点：核酶1，3和4）和非暴露区（+17位点：核酶2）4个锤头状核酶（1-4）的两套质粒；试管内切割检测表明，针对+8, +36和+71位点的核酶（1，3和4）可以有效地在试管内对VEGF RNA进行特异性切割，使其水平分别降至对照的61.7%, 27.6%和44.8%(荧光色素酶活性)或66.3%,27.0%和30.0%(蛋白质水平)；将核酶表达质粒与VEGF-LUC模板质粒瞬时共转染入SMMC-7721肝癌细胞，核酶1，3和4 VEGF-LUC水平分别降低到对照的81.4%，56.6%和69.1%；稳定表达针对VEGF的核酶1， 3或4的SMMC-7721细胞株中，内源性VEGF RNA的水平降至对照水平的5％以下。对照未转染的、转染空载体的和转染了核酶2（+17）的SMMC-7721细胞。结论: 分别针对VEGF+8， +36和+71位点锤头状核酶可有效地抑制VEGF的RNA水平。

Objective: To establish a novel and robust hammerhead ribozyme system against the Vascular Endothelial Growth Factor (VEGF) for anti-cancer gene therapy. Methods： The structural analysis of the 2ndstructure of the VEGF RNA and the vector construction of hammerhead ribozymes (1-4) against VEGF leader region(+1 to+75); The in vitro analyses of ribozyme mediated specific cleavage; The in cell evaluation of the ribozyme mediated cleavage of the VEGF RNA. Results：Ribozymes targeting +8, +36, or +71 (Rz1,3 and 4) of the exposed region or +17 (Rz2) of the unexposed region of VEGF RNA were constructed in pGVal and pFB retroviral vector systems; Rz1,3 and 4, but not Rz2, specifically cleaved the VEGF RNA and brought the VEGF RNA level down to 61.7%, 27.6% and 44.8%(luciferase activity)as well as 66.3%, 27.0% and 30.0%(protein level) of the control; The same set of ribozymes reduced the co-transfected VEGF-LUC RNA level down to 81.4%，56.6% and 69.1% of the control in a transient transfection analysis and essentially abolished the endogenous VEGF RNA in the stable transfected setting. Conclusion: We have established three effective hammerhead ribozyme vector systems targeting +8, +36 and +71 of the VEGF RNA.

上海市科委(04DZ14006)；国家基金委(30450001)；973项目（2004CB518804）；863项目(2002AA2Z3352)