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大鼠Adrb3基因重组慢病毒干扰载体的构建及其对血管平滑肌细胞Adrb3表达的影响 预览

Construction of lentiviral vectors to deliver short hairpin RNA of rat Adrb3 gene and its effect on rat vascular smooth muscle cells
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摘要 目的构建大鼠Adrb3(rAdrb3)基因慢病毒干扰载体,筛选高效率rAdrb3基因干扰序列,观察其对大鼠血管平滑肌细胞(VSMC)Adrb3基因表达的影响,为进一步研究Adrb3在心力衰竭中的作用机制提供实验工具。方法设计2条rAdrb3基因shRNA寡核苷酸序列,构建慢病毒干扰质粒并进行测序鉴定。三质粒法共转染293T细胞包装慢病毒,测定慢病毒滴度。大鼠VSMC设置正常对照组(Normal组),空载慢病毒组(Lv-rAdrb3-shRNA-control组),携带rAdrb3基因慢病毒干扰载体1组(Lv-rAdrb3-shRNA-1组),携带rAdrb3基因慢病毒干扰载体2组(Lv-rAdrb3-shRNA-2组),感染5 d后,收集细胞。提取细胞总RNA和蛋白,Real-time PCR检测rAdrb3 mRNA表达,Western blot检测rAdrb3蛋白表达。结果构建2个携带rAdrb3基因慢病毒干扰载体,滴度均为2×10~8TU/mL。慢病毒感染大鼠VSMC 72 h后,感染效率可达80%。与Normal组比较,Lv-rAdrb3-shRNA-1、Lv-rAdrb3-shRNA-2组Adrb3 mRNA水平沉默效率为87.18%、65.27%(P<0.05);与Normal组比较,Lv-rAdrb3-shRNA-1、Lv-rAdrb3-shRNA-2组rAdrb3蛋白水平沉默效率为85.57%、70.04%(P<0.05)。结论成功构建并筛选出有效携带rAdrb3基因慢病毒干扰载体,可显著抑制大鼠VSMC Adrb3的表达。 Objective To construct recombinant lentiviral vectors to deliver short hairpin RNA of rat Adrb3(rAdrb3)gene and observe the effect of rAdrb3 gene expression on rat vascular smooth muscle cells(VSMC).Methods Two rAdrb3 gene shRNA oligonucleotide sequences were designed,and lentiviral interference plasmids were constructed and sequenced.The lentivirus was packaged by co-transfecting 293 T cells with three plasmids,and then the lentivirus titers were measured.Rat VSMC were randomly divided into 4 groups,Normal group,Lv-rAdrb3-shRNA-control group,Lv-rAdrb3-shRNA-1 group and Lv-rAdrb3-shRNA-2 group.The cells were collected at the fifth day after infection.Total RNA and protein were extracted.The expression of rAdrb3 mRNA on rat VSMC was detected by real-time PCR.The expression of rAdrb3 protein on rat VSMC was detected by Western blot assay.Results The recombinant lentivirus was successfully constructed and the titer after purification was 2×10~8 TU/mL.The infection efficiency of the recombinant lentivirus on rat VSMC was 80%.Compared with the Normal group,the silencing efficiencies of rAdrb3 mRNA were 87.18%and 65.27%for Lv-rAdrb3-shRNA-1 group and Lv-rAdrb3-shRNA-2 group(P<0.05).Compared with the Normal group,the silencing efficiecies of rAdrb3 protein were 85.57%and 70.04%for Lv-rAdrb3-shRNA-1 group and Lv-rAdrb3-shRNA-2 group(P<0.05).Conclusion The recombinant lentiviral vectors to deliver short hairpin RNA of rAdrb3 gene,which can significantly inhibit the expression of Adrb3 gene on rat VSMC,are constucted successfully.
作者 宋衍秋 毛用敏 秦勤 丛洪良 SONG Yan-qiu;MAO Yong-min;QIN Qin;CONG Hong-liang(Tianjin Cardiovascular Insititute,Tianjin 300222,China;Department of Cardiology,Tianjin Chest Hospital)
出处 《天津医药》 CAS 北大核心 2019年第2期122-126,226-227共6页 Tianjin Medical Journal
基金 天津市卫生局资助项目(2013KY33).
关键词 受体 肾上腺素能β3 RNA干扰 慢病毒 慢性心力衰竭 血管平滑肌细胞 receptors,adrenergic,beta-3 RNA interference lentivirus chronic heart failure vascular smooth muscle cells
作者简介 通讯作者:宋衍秋(1979),女,副研究员,博士,主要从事心血管疾病基础研究,E-mail:huozhebulei_2012@163.com.
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