小鼠TNF-α基因RNAi慢病毒载体的构建及体外研究

Construction of lentivector-mediated RNAi and it efficiently downregulates expression of murine TNF-α gene in vitro

目的为探讨TNF-α在C型Niemann-Pick病(NPC)神经变性中的作用,构建携带小鼠TNF-α-si RNA的慢病毒载体并进行体外研究。方法设计3个TNF-α-si RNA序列和1个无关对照序列,克隆到酶切的pSI H1-H1-copGFP si RNA载体;重组质粒转染293T细胞,通过实时定量PCR检测筛选出干扰效果最佳的TNF-α-si RNA;筛选出的pSI H-si RNA、pSI H-negative分别与慢病毒包装质粒共转染293T细胞生成慢病毒;慢病毒感染BV-2细胞和星形胶质细胞,采用RT-PCR以及Elisa检测TNF-α-si RNA的沉默效果。结果成功构建携带TNF-α-si RNA的慢病毒载体,滴度达2×108ifu/L;慢病毒感染BV-2和星形胶质细胞,表达GFP且可下调TNF-α表达。结论TNF-α-si RNA能明显下调TNF-α的表达,为研究和治疗神经变性疾病以及TNF-α相关疾病提供了有力的工具。

Objective To study the function of TNF-α in Niemann-Pick type C diseases, and to use lentiviral-delivered RNA interference （RNAi） to silenceexpression of murine TNF-α gene invitro. Methods Three TNF-α-siRNA of mouse and a negative sequence were designed and cloned into linearized pSIH1-HI-copGFP siRNA Vector. Above recombinants were transfected by lipofectamine into 293T cells. The gene silencing efficacy of the 3 targets was compared by realtime PCR. The optimized pSIH1- TNF-msiRNA2 and pSIH-negative were co-transfected into 293T cells with the TNF-α-siRNA was determined by RT-PCR in BV-2 cells and astrocytes. Results Successfully constructed Lentivector-mediated -siRNA , and the tite is 2 × 10^8 ifu/L. RNAi efficiently down regulated expression of murine TNF-α gene invitro. Conclusions TNF-α-siRNA down regulated expression of TNF-α gene in vitro, this may provide a potential tool for studying and treating Neurodegen erative diseases and TNF-α-related diseases.