摘要目的 构建干扰大鼠耳蜗前体细胞(CPCs)β-catenin基因的短发夹RNA(shRNA)重组慢病毒载体,并对构建的载体进行鉴定.方法 用聚合酶链式反应(PCR)方法扩增β-catenin基因,根据β-catenin基因设计shRNA oligo构建干扰载体,利用Gateway技术构建携带β-catenin基因的shRNA慢病毒载体,将构建好的慢病毒载体与辅助质粒pLV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5共转染293FT细胞,收集病毒上清获得病毒颗粒,经浓缩后用4种不同浓度(0.0、5.0、10.0、20.0μl)的重组慢病毒瞬时感染大鼠CPCs,同时构建shRNA对照组,采用实时荧光PCR定量检测(qPCR)和免疫印迹法(Western blot)从基因和蛋白水平鉴定shRNA-β-catenin慢病毒载体对β-catenin的抑制效应.结果 成功构建shRNA-β-catenin重组慢病毒载体,shβ-catenin和shβ-catenin-对照的病毒滴度分别为5.05×107和4.34×107.体外实验显示,4种不同浓度重组慢病毒转染的CPCs中的β-catenin蛋白量随着转染病毒浓度的增加而减少,各组间差异有统计学意义(P＜0.05),转染shβ-catenin的CPCs表达的β-catenin mRNA的量明显低于shβ-catenin-对照组,两组间差异有统计学意义(P＜0.05).结论 本次包装的β-catenin重组慢病毒载体具有较高的感染效力,慢病毒载体的成功构建为研究β-catenin在CPCs向听觉毛细胞的分化机制提供了一定的技术支持.

abstractsObjective To construct a recombinant short-hairpin RNA (shRNA) lentiviral vector targeting the β-catenin gene in cochlear precursor cells (CPCs) in mice,and to investigate its inhibitory effect.Methods PCR was used for the multiplication of the β-catenin gene,and shRNA oligo was designed based on the β-catenin gene to construct an interference vector.Gateway Technology was used to construct shRNA lentiviral vector which carried the β-catenin gene,and then 293FT cells were transfected with the constructed lentiviral vector and helper plasmids pLV/helper-ST3,pLV/helper-ST4,and pLV/helper-SL5.The virus supernatant was collected to obtain viral particles,and then mouse CPCs were transiently infected with the recombinant lentivirus with four different concentrations (0,5,10,and 20 μl).The shRNA control group was established.Quantitative real-time PCR and Western blot were used to investigate the inhibitory effect of shRNA β-catenin lentiviral vector on β-catenin.Results The recombinant shRNA β-catenin lentiviral vector was successfully constructed,and the virus titers of shβ-catenin and shβ-catenin-control were 5.05×107 and 4.34×107,respectively.The results of in vitro experiments showed that in CPCs transfected with four different concentrations of recombinant lentivirus,the content of β-catenin protein gradually decreased with the increase in concentration,and there was a significant difference between groups (P＜0.05);the CPCs transfected with shβ-catenin had significantly lower mRNA expression of β-catenin than those in the shβ-catenin-control group (P＜0.05).Conclusion The constructed lentiviral vector targeting the β-catenin gene has a high infection efficiency,and the successful construction of lentiviral vectors provides a technical support for analyzing the role of β-catenin in the differentiation of CPCs into auditory hair cells.