Wang Xuezhen, Tang Ronghua, Xue Zheng, Jiang Feng, Zhang Min, Bu Bitao
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Huazhong Univ Sci Technolog Med Sci. 2009 Feb;29(1):112-7. doi: 10.1007/s11596-009-0124-2. Epub 2009 Feb 18.
In order to explore the role of TNF-alpha in Niemann-Pick type C (NPC) disease, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of murine TNF-alpha gene in vitro and in npc mice. Interference efficiency of the lentivirus expressing TNF-alpha-siRNA, previously constructed with the concentration of 2 x 10(8) ifu/mL, was determined by RT-PCR and ELISA in BV-2 cells and astrocytes. At the same time, the constructed Lenti-TNF-alpha-siRNA was intracerebroventricularly infused into 4-week old npc mice for a 4-week period, and the mice were divided into 3 groups: Lenti-TNF-alpha-siRNA (n=6), control lentivirus (n=6), and NPC mice without any intervention (n=4). By using immunohistochemistry and real-time PCR, the down-regulation of the target genes was detected. The Lenti-TNF-alpha-siRNA downregulated the expression of murine TNF-alpha gene efficiently in vitro and the interference efficiency was 66.7%. Lentivirus could be expressed stably for long-term in the npc mice brain. Immunohistochemistry and real-time PCR revealed that, as compared with non-intervention group and Lenti-control group, Lenti-TNF-alpha-siRNA efficiently down-regulated the expression of murine TNF-alpha gene with the interference efficiency being 66.9%. TNF-alpha-siRNA down-regulated the expression of TNF-alpha gene in vitro and in vivo, which provided a potential tool for studying and treating neurodegenerative diseases and TNF-alpha-related diseases.
为探究肿瘤坏死因子-α（TNF-α）在尼曼-匹克C型（NPC）病中的作用，采用慢病毒介导的RNA干扰（RNAi）技术在体外及NPC小鼠体内沉默小鼠TNF-α基因的表达。通过RT-PCR和ELISA法在BV-2细胞和星形胶质细胞中测定先前构建的浓度为2×10⁸ifu/mL的表达TNF-α-siRNA的慢病毒的干扰效率。同时，将构建好的Lenti-TNF-α-siRNA脑室内注入4周龄的NPC小鼠体内，持续4周，将小鼠分为3组：Lenti-TNF-α-siRNA组（n = 6）、对照慢病毒组（n = 6）和未进行任何干预的NPC小鼠组（n = 4）。采用免疫组化和实时PCR检测靶基因的下调情况。Lenti-TNF-α-siRNA在体外能有效下调小鼠TNF-α基因的表达，干扰效率为66.7%。慢病毒可在NPC小鼠脑内长期稳定表达。免疫组化和实时PCR结果显示，与未干预组和Lenti-对照组相比，Lenti-TNF-α-siRNA能有效下调小鼠TNF-α基因的表达，干扰效率为66.9%。TNF-α-siRNA在体外和体内均下调了TNF-α基因的表达，为研究和治疗神经退行性疾病及TNF-α相关疾病提供了一种潜在工具。
