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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 and evaluate a hammerhead ribozyrae system that inhibits the expression of vascular endothelial growth factor (VEGF). Methods: The secondary structure of VEGF121 gene was sequenced and its target was selected. The expressible hammerhead ribozyme (1-4) vector system targeting VEGF secreted peptide RNA and the VEGF-fluorochromease fusion gene Reporter plasmids were prepared; the specificity and efficiency of ribozyme for the cleavage of VEGF RNA transcribed in vitro by the in vitro test were evaluated; the cleavage efficiency of the ribozyme in the cells for VEGF RNA was assessed by transient co-transfection experiments and stable transfection experiments . RESULTS: The exposed regions (+8, +36 and +71 sites: ribozymes 1, 3 and 4) and non-exposed regions (+17 sites: ribozyme 2 ) Four sets of hammerhead ribozymes (1-4); in vitro cutting assays showed that ribozymes (1, 3 and 4) at the +8, +36 and +71 sites were effective in vitro VEGF RNA was cut specifically to 61.7%, 27.6% and 44.8% (fluorochromease activity) or 66.3%, 27.0% and 30.0% (protein level) of the control, respectively; The plasmids and VEGF-LUC template plasmids were co-transfected into SMMC-7721 hepatoma cells, and the levels of ribozymes 1, 3 and 4 VEGF-LUC were reduced to 81.4%, 56.6% and 69.1% of the control respectively. Stable expression of ribozyme targeting VEGF 1, 3 or 4 SMMC-7721 cell line, the level of endogenous VEGF RNA decreased to below 5% of the control level. Control untransfected, empty vector transfected, and SMMC-7721 cells transfected with ribozyme 2 (+17). Conclusion: The targeting of VEGF hammerhead ribozyme at the sites of VEGF +8, +36 and +71, respectively, can effectively inhibit the RNA level of VEGF.