靶向人血管内皮生长因子A基因小片段干扰RNA慢病毒载体的构建、比较及鉴定  

Construction and Identification of Lentiviral Vector-mediated Small Interfering RNA Targeting Human Vascular Endothelial Growth Factor A Gene

作　　者：姜小飞[1] 叶芬[1] 石理[1] 陈曦[1] 朱卫民[1] 张业明[1] 

机构地区：[1]珠海市人民医院心内科,广东珠海519000

出　　处：《华西医学》2016年第1期1-6,共6页West China Medical Journal

基　　金：广东省医学科研基金(A 2012608)~~

摘　　要：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-mediated gene-targeted small interfering RNA（siRNA）vector to vascular endothelial growth factor（VEGF）,and choose the RNAi with the highest silence efficiency to VEGFA gene.Methods Three kinds of VEGFA gene-targeted hairpin siRNA was designed（KD 1,KD 2,KD 3）,then two complementary oligo nucleotide strand were synthesized and inserted into pGCSIL-GFP vector.After annealing,the recombined vector pGCSIL-GFP-siVEGFA was gotten,which was digested by restrictive enzyme and sequenced,and was co-transfected with the pHelper 1.0 and pHelper 2.0 into 293 T cells by Lipofectamine 2000.After that,the new vector was transfected into human umbilical vein endothelial cells（HUVECs）,and the mRNA expression level of VEGFA gene in cells was detected by RT-PCR.Then we compared the mRNA expression level of VEGFA gene of the 3 groups.Results pGCSIL-GFP-siVEGFA was built successfully,and all the siRNA could silence the expression of VEGFA mRNA in the HUVECs,and the relative expressions of VEGFA mRNA to the control group were 0.614 ± 0.043（KD 1）,0.334 ± 0.030（KD 2）,and 0.201 ±0.015（KD 3） respectively.Conclusion We＇ve successfully constructed the siRNA vector for VEGFA mRNA,which can obviously suppress the expression of VEGFA mRNA.

关 键 词：RNA干扰 血管内皮生长因子 慢病毒 人脐静脉内皮细胞 

分 类 号：R373[医药卫生—病原生物学]