摘要
目的构建人HIF-1α基因RNAi重组腺病毒Ad.HIF-1α,并观察其对人肺腺癌细胞SPCA-1的影响。方法采用酶切、连接、转化等方法,利用AdEasy系统构建插入HIF-1αRNAi片段的重组腺病毒Ad.HIF-1α。氯化铯梯度离心法纯化病毒。病毒功能滴度采用传导293细胞的方法,以2MOIAd.HIF1α传导SPCA-1细胞,48h后应用流式细胞术检测绿色荧光蛋白(GFP)表达和HIF-1α蛋白表达。HIF-1αmRNA表达用荧光定量PCR检测。结果酶切和卡那霉素抗性筛选证实,重组穿梭载体pAdTrack.HIF-1α和腺病毒质粒pAd.HIF-1α均构建正确。pAd.HIF-1α导入293细胞3d,约15%的细胞表达GFP,最终所获病毒滴度为5.0×1010TU/mL。Ad.HIF-1α转导SPCA-1细胞48h,GFP表达率为92%;HIF-1αmRNA和蛋白表达分别下降89%和87%。结论成功构建人HIF-1α基因RNAi腺病毒,为通过阻断HIF-1α基因治疗肺癌提供初步实验依据。
Objective To construct the RNA interference （RNAi） recombinant adenovirus vector targeting at human hypoxia-inducible transcription factor lot （HIF-1α） and to evaluate its effect on human lung adenocarcinoma cell line SPCA-1. Methods The recombinant adenovirus Ad was constructed. HIF-1α inserted with HIF-1α RNAi fragment via AdEasy system. The virus was purifed by CsCI gradient centrifuge. The functional titer of recombinant adenovirus was measured by transfection test in HEK 293 cells. SPCA-1 cells were transducted with 2 multiplicity of infection （MOI） Ad. HIF1α in vitro, the expression rate of green fluorescence protein （GFP） was recorded by flow cytometry, HIF-1α mRNA and protein level was measured by Real-Time RT-PCR and flow cytometry. Results The recombinant shuttle plasmid PAdTrack. HIF-1α and adenovirus plasmid Ad. HIF-1α were all correct shown by enzyme digestion confirmation. The plasmid pAd.HIF-1α was transducted into HEK293 cells, 15% GFP expression were seen after 3 days. The final titers of recombinant adenovirus were 5.0 × 10^10 TU/mL. SPCA-1 cells was transducted by Ad. HIF-1α in vitro for 48 h ,GFP expression rate was 92% , HIF-1α mRNA and protein level decreased 89% and 87%, respectively. Conclusion RNAi adenovirus vector of human HIF-1α gene has been successfully constructed, which could facilitate the research on HIF-1α gene related gene therapy for lung cancer.
出处
《基础医学与临床》
CSCD
北大核心
2007年第11期1246-1250,共5页
Basic and Clinical Medicine
基金
山东省卫生厅课题(2005HW139)
关键词
肺癌
乏氧诱导因子-1Α
RNA干扰
腺病毒
基因治疗
lung neoplasm
hypoxia-inducible transcription factor 1α
RNA interference
Adenovirus
Gene therapy
