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目的构建人血管内皮生长因子(VEGF)m RNA靶向小片段干扰RNA(si RNA)表达的慢病毒载体,并测定其对VEGFA基因的沉默率,选择效率最高的干扰序列。方法设计3种人VEGFA靶向的发夹样si RNA(KD 1、KD 2、KD 3),分别2条互补的寡核苷酸链,退火后连接入p GCSIL-GFP载体,转化扩增后得到重组载体p GCSIL-GFP-si VEGFA,进行测序。将重组载体与慢病毒包装辅助质粒p Helper 1.0和p Helper 2.0通过Lipofectamine 2000共同转染至细胞人胚肾细胞株(293 T),产生病毒后,再转染至人脐静脉内皮细胞(HUVEC),逆转录-聚合酶链反应法检测转染细胞VEGFA m RNA的表达水平,比较3种si RNA序列的效率。结果经过酶切鉴定与测序,p GCSIL-GFP-si VEGFA构建成功。转染至HVUEC后,细胞VEGFA m RNA表达水平均明显降低(KD 1:0.614±0.043;KD 2:0.334±0.030;KD 3:0.201±0.015),其中以KD 3为相对最有效的si RNA序列。结论成功构建了针对VEGFA m RNA的si RNA载体,并可以显著抑制内皮细胞VEGFA m RNA的表达。
Objective To construct a lentiviral vector targeting human hair microvascular endothelial growth factor (VEGF) m RNA targeting small interfering RNA (si RNA), and to determine the silencing rate and the selection efficiency of VEGFA gene. Methods Hairpin-like si RNAs (KD 1, KD 2, KD 3) targeted by VEGFA were designed and two complementary oligonucleotide strands were designed. After annealing, they were ligated into p GCSIL-GFP vector. After transformation and amplification The recombinant vector p GCSIL-GFP-si VEGFA was obtained and sequenced. The recombinant vector and the lentiviral packaging helper plasmids p Helper 1.0 and p Helper 2.0 were co-transfected into 293T cell line by Lipofectamine 2000 to generate the virus and then transfected into human umbilical vein endothelial cells (HUVECs) The expression of VEGFA m RNA in transfected cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the efficiency of three siRNA sequences was compared. Results After restriction enzyme digestion and sequencing, the construction of p GCSIL-GFP-si VEGFA was successful. After transfected into HVUEC, the expression level of VEGFA m RNA in cells was significantly decreased (KD 1: 0.614 ± 0.043; KD 2: 0.334 ± 0.030; KD 3: 0.201 ± 0.015), of which KD 3 was the most effective siRNA sequence . Conclusion The siRNA vector targeting VEGFA m RNA was successfully constructed and the expression of VEGFA m RNA in endothelial cells was significantly inhibited.