CAJ | 학술논문

目的构建有效抑制酪氨酸激酶受体B(TrkB)基因的RNA干扰慢病毒表达载体.方法根据大鼠TrkB基因的不同部位设计4对短发卡RNA (short hairpin RNA,shRNA)寡核苷酸片段,克隆到慢病毒干扰载体pXZRNAi 1.0中,构建shRNA慢病毒表达载体pXZRNAi-shTrkB-1,2,3,4.获得较高滴度的慢病毒颗粒后感染大鼠神经干细胞,应用实时荧光PCR和Western blot方法检测对TrkB的干扰效果.结果酶切鉴定和测序结果提示慢病毒载体的寡核苷酸链插入正确.包装浓缩慢病毒颗粒的滴度为8.6×105 cfu/ml,对神经干细胞感染效率可达80％.与未感染组相比,转染pXZRNAi-shTrkB-3和4的神经干细胞中TrkB mRNA表达水平分别为(66.7±5.5)％和(76.8±4.9)％,TrkB蛋白的表达水平分别为(68.5±4.3)％和(78.2±5.1)％,均差异有统计学意义(P＜0.05).结论成功构建可高效沉默TrkB基因的pXZRNAi-shTrkB慢病毒载体,为研究TrkB在神经干细胞中的作用提供了有力工具.
목적 구건유효억제락안산격매수체B(TrkB)기인적RNA간우만병독표체재체.방법 근거대서TrkB기인적불동부위설계4대단발잡RNA (short hairpin RNA,shRNA)과핵감산편단,극륭도만병독간우재체pXZRNAi 1.0중,구건shRNA만병독표체재체pXZRNAi-shTrkB-1,2,3,4.획득교고적도적만병독과립후감염대서신경간세포,응용실시형광PCR화Western blot방법검측대TrkB적간우효과.결과 매절감정화측서결과제시만병독재체적과핵감산련삽입정학.포장농축만병독과립적적도위8.6×105 cfu/ml,대신경간세포감염효솔가체80％.여미감염조상비,전염pXZRNAi-shTrkB-3화4적신경간세포중TrkB mRNA표체수평분별위(66.7±5.5)％화(76.8±4.9)％,TrkB단백적표체수평분별위(68.5±4.3)％화(78.2±5.1)％,균차이유통계학의의(P＜0.05).결론 성공구건가고효침묵TrkB기인적pXZRNAi-shTrkB만병독재체,위연구TrkB재신경간세포중적작용제공료유력공구.
Objective To construct a tyrosine kinase B(TrkB) targeted RNA interference (RNAi) lentiviral vector.Methods Four oligonucleotides targeting rat TrkB gene were synthesized and cloned into lentiviral vector pXZRNAi 1.0 to construct recombinant lentiviral vectors pXZRNAi-shTrkB-1,2,3,4.Neural stem cells prepared from rat hippocampus were infected with these high-titer viruses.Real-time PCR was employed to detect the TrkB mRNA expression and western blot was used to assess the gene silencing efficacy of these recombinants.Results Enzyme digestion and DNA sequencing results demonstrated that these shRNAs were correctly inserted into lentiviral vectors and the four recombinants were constructed successfully with the titer of 8.6 × 105cfu/ml.The infection efficiency of the letivirus on neural stem cells reached 80％.Compared with the uninfection group,the expression levels of TrkB mRNA in neural stem cells decreased significantly after transfected with pXZRNAi-shTrkB-3 and 4((66.7 ± 5.5) ％ and(76.8 ± 4.9) ％ respectively,P ＜ 0.05) ; and the protein expression levels were also significantly decreased ((68.5 ± 4.3)％ and (78.2 ± 5.1)％ respectively,P ＜ 0.05).Conclusion The lentiviral vectors for TrkB have been successfully constructed with high yield of lentivirus,which provides versatile method for assessing gene function in neural stem cells.