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国家自然科学基金(30670737)

作品数:5 被引量:3H指数:1
相关作者:卜碧涛张旻王伟张莉魏佳军更多>>
相关机构:华中科技大学更多>>
发文基金:国家自然科学基金武汉市青年科技晨光计划更多>>
相关领域:医药卫生更多>>

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Rab7与NPC1在小胶质细胞及小鼠小脑普肯耶细胞的定位
2009年
目的:观察Rab7与NPC1蛋白在小胶质细胞及小鼠小脑普肯耶细胞的定位及其与溶酶体的关系。方法:采用特异性识别Rab7、NPC1的抗体和溶酶体的特异性表面标记进行免疫荧光细胞化学染色及免疫荧光组织化学染色,在激光共聚焦显微镜下观察。结果:Rab7和NPC1表达于小胶质细胞及小鼠小脑普肯耶细胞溶酶体,Rab7与NPC1共定位于小胶质细胞及npc+/+与npc-/-小鼠小脑普肯耶细胞细胞质内,两者分布有良好的相关性。结论:Rab7对NPC1蛋白的正常功能可能有重要影响。
张莉张旻姜亚平卜碧涛郝又国
关键词:免疫荧光共定位
The Neuroprotective Effects of Cyclin-dependent Kinase-5 Inhibition in Mice with Niemann-Pick Disease Type C
2009年
In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 (cdk-5) inhibition in mice with Niemarm-Pick disease type C (NPC) (npc^-/-), recombinant adeno-associated virus (rAAV) carrying the small interfering RNA (siRNA) specific for cdk-5 gene was injected into 3-day-old npc^-/- mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc^-/- mice and non-surgery age-matched npc^-/- mice were employed as controls (n=6-10/group). From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc^-/- mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc^-/- mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc^-/- mice, and significantly attenuated the hyperphosphorylation oftau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.
郝又国潘邓记张旻徐金枝李琳娟魏佳军王雪真
Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA被引量:1
2008年
Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.
魏佳军张旻卜碧涛张苏明徐金枝
C型Niemann-Pick病神经原纤维缠结的形成与特征
2008年
目的探讨C型尼曼-皮克病(NPC)脑部神经原纤维缠结(NFT)形成的时相特征。方法以17例年龄7个月至55岁的NPC患者为研究对象,采用tau蛋白和有丝分裂期相关抗体进行免疫组织化学染色和银染,分析患者脑内NFT形成的特点。结果最早可在4岁的患者海马旁回发现典型的NFT形成,随年龄增长数量逐渐增多。在形态上与阿尔茨海默病(AD)所见高度相似,但未发现老年斑。在NPC中,有丝分裂期磷酸化表位早于tau蛋白过度磷酸化及NFT形成。结论NFT形成并非老龄化过程的结果,且与老年斑的存在与否并无关联。cdc2/cyclinB1可能是NFT形成的关键性早期事件,针对其活性的抑制剂对于早期干预NPC NFT的形成有重要意义。
张旻降风魏佳军王雪贞王伟Inez Vincent卜碧涛
关键词:尼曼-皮克病神经原纤维缠结TAU蛋白质类磷酸化
Lentivector-mediated RNAi Efficiently Downregulates Expression of Murine TNF-α Gene in vitro and in vivo被引量:2
2009年
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 gene in vitro and in npc mice. Interference efficiency of the lentivirus expressing TNF-α-siRNA, previously constructed with the concentration of 2 x 108 ifu/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 intracerebroventricularly 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 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-α-siRNA efficiently down-regulated the expression of murine TNF-α gene with the interference efficiency being 66.9%. TNF-α-siRNA downregulated the expression of TNF-α gene in vitro and in vivo, which provided a potential tool for studying and treating neurodegenerative diseases and TNF-α-related diseases.
王雪贞唐荣华薛峥降风张旻卜碧涛
关键词:LENTIVIRUS
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