VEGF特异性核酶对Hela细胞VEGF表达的调节

目的：构建VEGF特异性发夹状核酶基因真核表达载体，观察其对Lela细胞VEGF表达的调节。方法：将核酶基因克隆入pcDNA3中，构建了抗VEGF发夹状核酶真核表达载体pcDNA3-RZ,并进行了核酶的体外切割活性检测。将真核表达载体转染人宫颈癌Hela细胞，采用RNA斑点杂交、流式细胞仪免疫荧光及免疫细胞化学方法，检测Hela细胞中VEGFmRNA和VEGF蛋白的表达水平。结果：所构建的重组表达载体pcDNA3-RZ正确，体外切割检测该核酶具有较高的切割活性。转染了pcDNA-RZ的Hela细胞中VEGFmRNA和VEGF蛋白的表达水平明显下降。结论：成功地构建了抗VEGF发夹状核酶基因真核表达载体，该载体能明显抑制Hela细胞VEGF的表达。

Regulation of VEGF Expression by VEGF Specific Ribozyme in Hela Cells

Objective To Construct eukaryotic expression vector of anti-VEGF hairpin ribozyme(RZ) gene and investigate the expression inhibition of VEGF in Hela cells.Methods RZ gene was cloned into eukaryotic expression vector pcDNA3 to construct plasmid pcDNA3 RZ.Cleavage function of RZ was checked in vitro.Then this eukaryotic expression vector was introduced into Hela cells by lipofectin mediation;The VEGF expression of Hela cells before or after transfection were detected by RNA dot blot,flow cytometer immunofluorescence staining and immunocytochemical analysis.Results The fregment of the RZ gene were confirmed by the digesting of pcDNA3 RZ and analyzing of seguence.And the results showed that expression product of the RZ gene had higher cleavage activity.VEGF mRNA and VEGF expression were decreased distinctly in Hela RZ cells.Conclusion The successfully constructed pcDNA3 RZ expression vector can inhibite VEGF level in human cervical cancer Hela cells.