PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达  

Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells

作　　者：文扬[1] 朱亚琴[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院·口腔医学院口腔综合科、上海市口腔医学重点实验室、上海市口腔医学研究所、国家口腔疾病临床研究中心,上海200011

出　　处：《上海口腔医学》2017年第4期363-367,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81271134,81300867);高等学校博士学位点专项科研基金(博导类,20130073110013);上海高校高峰高原学科建设项目

摘　　要：目的:构建人PERK基因的shRNA慢病毒载体,检测其对人牙髓细胞(dental pulp cells,DPCs)PERK基因表达的抑制作用。方法:针对人PERK基因的cDNA序列,设计并合成针对人PERK基因的shRNA表达序列,将其连接到载体hU6-MCS-CMV-EGFP中。测序正确后,将构建的目的载体和包装质粒共转染293T细胞,72 h后收获并浓缩得到重组慢病毒颗粒。筛选最佳感染复数(multiplicity of infection,MOI),将病毒感染人牙髓细胞,通过实时荧光定量PCR(quantitative real-time PCR,RT-PCR)和蛋白质免疫印迹(Western blot)技术检测PERK基因mRNA和蛋白的表达水平,验证干扰效果。采用SPSS24.0软件包对数据进行统计学分析。结果:成功构建LV-PERK-RNAi慢病毒载体,病毒滴度为3×10~8 TU/mL,最适MOI=30;RT-PCR和Western印迹法检测显示,与空白对照组相比,PERK基因的mRNA和蛋白质表达水平显著下降,在mRNA水平的表达抑制率约为63%。结论:成功构建PERK干扰慢病毒表达载体,为后续的研究创造了条件。PURPOSE： To construct an expression vector of a small hairpin RNA （shRNA） targeting human PERK gene and to observe gene-sileneing effects of PERK in human dental pulp cells （DPCs）. METHODS： According to PERK gene cDNA sequence, shRNA was designed and synthesized, which was then annealed into hU6-MCS-CMV-EGFP vector. After identified by sequencing, hU6-MCS-CMV-EGFP vector and packaging vector were co-transfected into 293 T cells. 72 hours later, the recombinant lentiviruses were obtained after harvesting and concentrating. Then LV-PERK-RNAi vectors were transfected into DPCs at an appropriate multiplicity of infection. To verify the interference effect, real- time PCR and Western blot were used to detect expression levels of PERK mRNA and protein in the transfected DPCs. The data were analyzed with SPSS 24.0 software package. RESULTS： LV-PERK-RNAi vectors were successfully constructed with a high titer of 3x10s TU/mL. The results of RT-PCR and Western blot demonstrated that after infection with LV- PERK-RNAi vector at a multiplicity of infection of 30, the expression level of PERK gene in DPCs was significantly down-regulated compared with control group. At mRNA level, the interference rate was about 63%. CONCLUSIONS： An effective lentiviral shRNA expression vector targeting the PERK gene is successfully constructed and can be used for further study on the function of PERK gene.

关 键 词：PERK RNAI 慢病毒载体 

分 类 号：R781.3[医药卫生—口腔医学]