小鼠TNF-α基因RNAi慢病毒载体的构建及体外研究 |
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引用本文: | 王雪贞,曾莉,降风,唐荣华,张旻,卜碧涛.小鼠TNF-α基因RNAi慢病毒载体的构建及体外研究[J].卒中与神经疾病,2009,16(1):3-6. |
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作者姓名: | 王雪贞 曾莉 降风 唐荣华 张旻 卜碧涛 |
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作者单位: | 1. 华中科技大学同济医学院附属同济医院神经内科,武汉,430030 2. 内蒙古自治区医院神经内科 |
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摘 要: | 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-siRNA的慢病毒载体并进行体外研究。方法设计3个TNF-α-siRNA序列和1个无关对照序列,克隆到酶切的pSIH1-H1-copGFP siRNA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-siRNA;筛选出的pSIH-siRNA、pSIH-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-siRNA的沉默效果。结果成功构建携带TNF-α-siRNA的慢病毒载体,滴度达2×10^8ifu/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-siRNA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。
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关 键 词: | 慢病毒载体 TNF-α 神经系统变性疾病 RNA干扰 |
Construction of lentivector-mediated RNAi and it efficiently downregulates expression of murine TNF-α gene in vitro |
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Institution: | Wang Xuezhen, Zeng Li, Jiang Feng,et al. (Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030) |
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Abstract: | 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. |
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Keywords: | Lentiviral vector TNF-α Neurodegenerative disease RNA interference |
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