抑制VEGF表达的锤头状核酶系统

The Hammerhead Ribozyme Mediated Repression of VEGF for Cancer Gene Therapy

目的: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 ribozyrae)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 2^nd strueture 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% （ lueiferase activity） as well as 66.3%, 27.0% and 30.0% （protein levd） of the control; The same set of ribozymes reduced the cotransfeeted VEGF-LUC RNA level down to 81.4% ,56.6% and 69.1% of the control in a transient transfeetion analysis and essentially abolished the endogenous VEGF RNA in the stable transfeeted setting. Conclusion： We have established three effective hammerhead ribozyme vector systems targeting ＋ 8. ＋36 and ＋ 71 of the VEGF RNA.