Lentiviral Vector-mediated Downregulation Of Cdk4 On Neurodegeneration In Niemann-Pick Type C

Partâ… The construction and identification of Cdk4-siRNA of mouseObjective Previous studies have been showed that the activation of cdk4/cyclinD1 plays an important role in neuronal degeneration of npc-/- mice. Intracerebroventricularly injection of flavopiridol, a specific pharmacological inhibitors of cdk4 activity, can reduce the hyperphosphorylation of cytoskeleton and improved the behavior of npc mice. In this study, we used lentiviral vector mediated RNAi, silence cdk4 gene in vitro and in vivo. In this part, three expression vector of pSilencer-1.0-U6siRNA aim directly at cdk4 gene was constructted. Then transfection 293T cell and gained the best sequence of silencing for the construction of lentiviral vector.Methods (1) Design three shRNA sequences aim at cdk4 gene, synthesized sense strand and antisense strand which contain loop-stem structure, annealled to become a couple. (2) Insert the couple strands to Psilencer-1.0-U6siRNA vector(including enzyme point of Apaâ… a nd EcoRâ… ),after the promotor of U6. (3) Electrophoresis and sequencing of three recombinant plasmids. (4) The vector of Psilencer-1.0-U6 and three recombinant plasmids transfected HEK293 cell. After 72 hours, analyzed the expression of cdk4 by Western blotting and picked out the plasmid of best silencing.Results (1) The three purpose gene segment built in the recombinant plasmids correctly. (2) Psilencer-1.0-U6-cdk4-siRNA2 is of best effect of silencing, and the inhibition ratio is 71%.Conclusion Constructed three RNAi vector of cdk4, Psilencer-1.0-U6-cdk4-siRNA1, Psilencer-1.0-U6-cdk4-siRNA2 and Psilencer-1.0-U6-cdk4-siRNA3. Psilencer-1.0-U6- cdk4-siRNA2 showed the best effect of inhibition which established the groundwork for the further study of cdk4-related disease. Partâ…¡Construction and identification of RNAi lentiviral vector on murine cdk4 geneObjective It is thought that cdks(including the activation of Cdk4/CyclinD1-pRb- E2F1 passway) mediated cytoskeletal protein hyperphosphorylation lead eventually to development of hallmark cytoskeletal lesions and neurodegeneration in neurodegenerative disease. Use RNAi to silence the cdk4 gene may build good effect intervention and lentiviral vector can infect all kinds of cells, both G0 stage and dividing phase and its disturbing effect last for permanent. In this Part, constructted lentiviral vector of cdk4-SiRNA, verification its effct of silencing in vitro, it's a groundwork for the further experiment in vivo.Methods (1)Synthesized sequences of Cdk4-siRNA2 and a nonsense control. BamHâ… was insert into the sense strand and EcoRâ… into antisense strand. (2) After annealled, the double strands were inserted in the expression vector of pSIH1-H1-copGFP shRNA digested by BamHâ… and EcoRâ… . (3) Identification the recombinant plasmid by PCR and sequencing. (4) Transfected HEK293T cell by shuttle plasmid pSIH1-siRNA including the sequences of Cdk4-siRNA2 or a nonsense control and lentivirus package plasmids, generated lentivirus. (5) Purification, concentration and determination of the lentivirus. (6) Infection BV-2 cells. After 72h, infection rate was calculated and analyze the expression by RT-PCR and Western blotting.Results (1) The recombinant plasmids gained a 278bp fragment while no-load vector gained a 278bp fragment; the sequences of Cdk4-siRNA 2 and a nonsense control insreted in pSIH1-H1-copGFP correctly; (2) Transfected HEK293T cell by shuttle plasmid pSIH1-siRNA including the sequences of Cdk4-siRNA or a nonsense control and lentivirus package plasmids, generated lentivirus; (3) After concentration, the titer of Cdk4-siRNA lentivirus was 2 x 108ifu/ml, and the control lentivirus was 1x 108ifu/ml; (4) The infection rate was 90%. RT-PCR and Western-blotting showed that cdk4 of lentivirus-cdk4 decreased significant comparied to the lentivirus-control and blank in mRNA and protein. There was no significant difference between the lentivirus-control and blank. Conclusion (1) Constructted expression vector of Cdk4-siRNA 2 and a nonsense control; (2) Transfected HEK293T cell by shuttle plasmid pSIH1-siRNA and lentivirus package plasmids, generated high titer lentivirus; (3) Lentivirus take along Cdk4-siRNA infected BV-2 cell and silenced the cdk4 gene specificly.Partâ…¢Lentivirus with Cdk4-siRNA on Niemann-Pick Type C MouseObjecive Dysregulation of cyclin-dependent kinases (cdks) and cytoskeletal protein hyperphosphorylation characterizes a subset of human neurodegenerative diseases, such as Niemann-Pick Type C (NPC). It is thought that these cytoskeletal changes lead eventually to development of hallmark cytoskeletal lesions such as neurofibrillary tangles and axonal spheroids. The naturally occurring npc-1 mutant mouse mimics human NPC, in displaying activation of cdk5, mitotic cdc2, and cdk4, with concomitant cytoskeletal pathology and neurodegeneration. We availed of this model and Lentivirus-cdk4 to determine whether cdks are necessary for NPC neuropathology.Methods The lentivirus were infused intracerebroventricularly and observed for a 4-week period, initiated at a pathologically incipient stage. We observed the neuropathology and subsequent motor impairment in NPC by immunohistochemistry, Western-blotting and hanger-test.Results As a result, lentivirus-cdk4 can reduce the hyperphosphorylation of cytoskeleton and improved the behavior of npc mice.Conclusion Due to the low birth rate and the mortality of npc-/- mice, we have no enough number of mice to gain a certain conclusion until now, but we can complete the study as soon as possible. Part IV The characteristics of neurofibrillary tangles in brains of the patients with Niemann-Pick disease type C (NPC)Objective Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of many neurodegenerative diseases including Alzheimer's disease (AD), Niemann-Pick disease type C(NPC),frontotemporal dementia and progressive supranuclear palsy. Due to be seen frequently in senile patients, it is generally thought to be an age-related pathological change in the brain. But some research revealed that NFTs can be seen commonly in the brain of young patients with neurodegenerative disease. In this study, the spatial and temporal characteristics of neurofibrillary tangles (NFTs) in brains of the patients with Niemann-Pick disease type C (NPC) have been described.Methods In order to analyze formation of NFTs in NPC, the brains which have been collected from the patients with NPC aged from 7 months to 55 years old, have been investigated using antibodies against the protein tau and the specific proteins in mitotic phase.Results Typical NFTs have been earliest detected in the parahippocampus of a NPC patient aged 4 years. The number of NFTs were apparently age-dependant. Gradually, the hippocampus and other regions of the temporal lobes and the frontal lobes will be affected with the advance of age. Immunohistochemically, the NFTs took the shapes similar to NFTs seen in AD brains, without presence of senile plagues. Interestingly, mitosis-phase markers appeared in the degenerating NPC neurons prior to hyperphosphorylation of tau and formation of NFTs.Conclusion The formation of NFTs is not the result of aging, and there is no direct link between presence of senile plagues and formation of NFTs. Ectopic activation of cdc2/CyclinB1 kinase complex might be an early event leading to NFT formation. Inhibiting the activity of the kinase complex may have a potential role in slowing down the formation of NFTs.