Lentivector-mediated RNAi efficiently downregulates expression of murine TNF-α gene in vitro and in vivo
Lentivector-mediated RNAi efficiently downregulates expression of murine TNF-α gene in vitro and in vivo
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DOI：
10.1007/s11596-009-0124-2
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发表时间：
2009-02
期刊：
影响因子：
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通讯作者：
Xue-zhen Wang;Ronghua Tang;Z. Xue;Feng Jiang;Min Zhang;B. Bu
中科院分区：
文献类型：
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作者：
Xue-zhen Wang;Ronghua Tang;Z. Xue;Feng Jiang;Min Zhang;B. Bu
为探讨肿瘤坏死因子-α（TNF-α）在C型尼曼匹克病（Niemann-Pick type C，NPC）中的作用，采用慢病毒介导的RNA干扰（Lentiviraldelivered RNA interference，RNAi）技术，在体外和NPC小鼠体内沉默小鼠TNF-α基因的表达。采用RT-PCR和ELISA方法检测2× 108 ifu/mL的TNF-α-siRNA慢病毒对BV-2细胞和星形胶质细胞的干扰效率。同时，将构建的Lenti-TNF-α-siRNA经侧脑室注入4周龄NPC小鼠体内，共4周，并将小鼠分为3组：Lenti-TNF-α-siRNA组（n=6）、对照慢病毒组（n =6）和未干预组（n=4）。通过免疫组化和实时荧光定量PCR检测靶基因的下调。Lenti-TNF-α-siRNA在体外可有效下调小鼠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。免疫组化和实时荧光定量PCR结果显示，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在体内外均能下调TNF-α基因的表达，为研究和治疗神经退行性疾病及TNF-α相关疾病提供了潜在的工具。
In order to explore the role of TNF-α in Niemann-Pick type C (NPC) disease, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of murine TNF-α genein vitroand in npc mice. Interference efficiency of the lentivirus expressing TNF-α-siRNA, previously constructed with the concentration of 2×108ifu/mL, was determined by RT-PCR and ELISA in BV-2 cells and astrocytes. At the same time, the constructed Lenti-TNF-α-siRNA was intracerebroven-tricularly infused into 4-week old npc mice for a 4-week period, and the mice were divided into 3 groups: Lenti-TNF-α-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-α-siRNA downregulated the expression of murine TNF-α gene efficientlyin vitroand 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-α-siRNA efficiently down-regulated the expression of murine TNF-α gene with the interference efficiency being 66.9%. TNF-α-siRNA down-regulated the expression of TNF-α genein vitroandin vivo, which provided a potential tool for studying and treating neurodegenerative diseases and TNF-α-related diseases.