表达cdk5-siRNA重组腺相关病毒载体的构建及CDK在NPC神经变性中的作用
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摘要
目的
了解npc小鼠不同年龄阶段cdk活化所导致的神经元细胞骨架损害的程度。以便进一步探索NPC的发病机制和新的治疗策略。
方法
在小鼠出生后的1、2、3、4和5周处死所有同龄的小鼠,然后采用PCR的方法鉴定基因型,保留npc-/-小鼠和相应数量的同龄野生型小鼠(每年龄组至少4只)的脑组织做免疫组织化学染色,观察cdk活化所致的球状神经轴突出现的时间和数目。
结果
在3周npc小脑即可检测到cdk的异常活化所引起的球状神经轴突,且在小脑较脑干出现早、数量更多。球状神经轴突数目随着小鼠的年龄增大越来越多。
结论
①npc小鼠自3周龄开始出现神经元细胞骨架损害及cdk激活;②病变的严重程度随年龄增长而增加。
目的
构建能表达cdk5特异性siRNA(cdk5-siRNA)的II型重组腺相关病毒载体,体外观察其对cdk5基因的特异性沉默作用。
方法
设计一段在体内能转录出特异性cdk5-siRNA的寡核苷酸序列,人工合成该序列后采用基因克隆技术,将其克隆至通用RNA i表达质粒pSilencer-U6中,构建出质粒pSilencer-U6-cdk5-siRNA。同时将EGFP连入表达质粒pAAV-MCS,得到质粒pAAV-MCS-EGFP。然后通过PCR从pSilencer-U6-cdk5-siRNA中扩增出U6-cdk5-siRNA片段,并将其克隆至pAAV-MCS-EGFP,构建出表达质粒pAAV-MCS- EGFP-cdk5-siRNA-U6。重组质粒通过酶切,测序鉴定后与该系统中的控制质粒pAAV-RC﹑辅助质粒pHelper用磷酸钙法共转染HEK293细胞,包装得到表达cdk5-siRNA的II型重组腺相关病毒载体(rAAV2-cdk5-siRNA)。重组病毒纯化后通过斑点杂交法测定滴度,病毒感染体外培养的PC12细胞,Western blot检测其抑制cdk5表达的特异性和效果。
结果
各质粒构建正确,成功包装出重组腺相关病毒载体rAAV2-cdk5-siRNA,病毒滴度达2×1012v.g/ml,重组病毒能感染体外培养的PC12细胞,并能明显而特异性下调cdk5的表达。
结论
构建的重组腺相关病毒载体rAAV2-cdk5-siRNA能明显干扰细胞内cdk5的表达,为进一步探索cdk在神经变性过程中的地位和神经变性疾病基因治疗的可行性奠定了基础。
Objective
Investigate the impairment of neuronal cytoskeleton caused by activation of cdks in different aged npc mice ,in order to study the pathogenesis and explore the new therapies for NPC.
Methods
Sacrifice all sibling mice in one breeding cage at the age of 1、2、3、4 and 5 week, keep the npc-/- mice and same aged wild type mice as control (at least 4 mice per group). Use PCR for genotyping and immunohistochemistry for the study of spheroid caused by activation of cdk.
Results
Axonal spheroids caused by activation of cdk can be found as early as 3 weeks age in npc mice.The pathology is more obvious in the cerebellum compared to pons.The number of spheroids increased as the mice grow up.
Conclusion
①The impairment of neuronal cytoskeleton and cdk activation can be found in npc mice as early as 3 weeks age.②The severity of pathology increased along with the increase of age.
Objective
To construct the adeno-associated virus vector encoding the small interfering RNA(siRNA) specific for cyclin dependent kinase5(CDK5) gene and to observe its silencing effect on cdk5 gene in vitro,in order to explore the role of the CDK5 in the neurodegenerative disease.
Methods
The specific cdk5-siRNA sequence was cloned into the plasmid of pAAV-MCS-EGFP in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-MCS-EGFP-U6-cdk5-siRNA. The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell line (HEK293 cell) was co-transfected with the pAAV-MCS-cdk5-siRNA together with the control plasmid pAAV-RC and pHelper by phosphate-calcium deposit method. The titer was measured by dot hybridization. Infected the PC12 cell using recombinant adeno-associated virus, at 72h after infection, the expression of cdk5 was detected by Western-blot.
Results
All plasmids and the recombinant adeno-associated virus vector carrying the cdk5-siRNA sequences were constructed successfully, the viral titer was 4×1013,it can infect the PC12 cell and down-regulated the expression of the cdk5 gene in PC12 cell.
Conclusion
The recombinant adeno-associated virus vector rAAV-cdk5-siRNA can interfere with the expression of cdk5 gene significantly, which lays the basis for its application in the treatment of the neurodegenerative disease.
了解npc小鼠不同年龄阶段cdk活化所导致的神经元细胞骨架损害的程度。以便进一步探索NPC的发病机制和新的治疗策略。
方法
在小鼠出生后的1、2、3、4和5周处死所有同龄的小鼠,然后采用PCR的方法鉴定基因型,保留npc-/-小鼠和相应数量的同龄野生型小鼠(每年龄组至少4只)的脑组织做免疫组织化学染色,观察cdk活化所致的球状神经轴突出现的时间和数目。
结果
在3周npc小脑即可检测到cdk的异常活化所引起的球状神经轴突,且在小脑较脑干出现早、数量更多。球状神经轴突数目随着小鼠的年龄增大越来越多。
结论
①npc小鼠自3周龄开始出现神经元细胞骨架损害及cdk激活;②病变的严重程度随年龄增长而增加。
目的
构建能表达cdk5特异性siRNA(cdk5-siRNA)的II型重组腺相关病毒载体,体外观察其对cdk5基因的特异性沉默作用。
方法
设计一段在体内能转录出特异性cdk5-siRNA的寡核苷酸序列,人工合成该序列后采用基因克隆技术,将其克隆至通用RNA i表达质粒pSilencer-U6中,构建出质粒pSilencer-U6-cdk5-siRNA。同时将EGFP连入表达质粒pAAV-MCS,得到质粒pAAV-MCS-EGFP。然后通过PCR从pSilencer-U6-cdk5-siRNA中扩增出U6-cdk5-siRNA片段,并将其克隆至pAAV-MCS-EGFP,构建出表达质粒pAAV-MCS- EGFP-cdk5-siRNA-U6。重组质粒通过酶切,测序鉴定后与该系统中的控制质粒pAAV-RC﹑辅助质粒pHelper用磷酸钙法共转染HEK293细胞,包装得到表达cdk5-siRNA的II型重组腺相关病毒载体(rAAV2-cdk5-siRNA)。重组病毒纯化后通过斑点杂交法测定滴度,病毒感染体外培养的PC12细胞,Western blot检测其抑制cdk5表达的特异性和效果。
结果
各质粒构建正确,成功包装出重组腺相关病毒载体rAAV2-cdk5-siRNA,病毒滴度达2×1012v.g/ml,重组病毒能感染体外培养的PC12细胞,并能明显而特异性下调cdk5的表达。
结论
构建的重组腺相关病毒载体rAAV2-cdk5-siRNA能明显干扰细胞内cdk5的表达,为进一步探索cdk在神经变性过程中的地位和神经变性疾病基因治疗的可行性奠定了基础。
Objective
Investigate the impairment of neuronal cytoskeleton caused by activation of cdks in different aged npc mice ,in order to study the pathogenesis and explore the new therapies for NPC.
Methods
Sacrifice all sibling mice in one breeding cage at the age of 1、2、3、4 and 5 week, keep the npc-/- mice and same aged wild type mice as control (at least 4 mice per group). Use PCR for genotyping and immunohistochemistry for the study of spheroid caused by activation of cdk.
Results
Axonal spheroids caused by activation of cdk can be found as early as 3 weeks age in npc mice.The pathology is more obvious in the cerebellum compared to pons.The number of spheroids increased as the mice grow up.
Conclusion
①The impairment of neuronal cytoskeleton and cdk activation can be found in npc mice as early as 3 weeks age.②The severity of pathology increased along with the increase of age.
Objective
To construct the adeno-associated virus vector encoding the small interfering RNA(siRNA) specific for cyclin dependent kinase5(CDK5) gene and to observe its silencing effect on cdk5 gene in vitro,in order to explore the role of the CDK5 in the neurodegenerative disease.
Methods
The specific cdk5-siRNA sequence was cloned into the plasmid of pAAV-MCS-EGFP in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-MCS-EGFP-U6-cdk5-siRNA. The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell line (HEK293 cell) was co-transfected with the pAAV-MCS-cdk5-siRNA together with the control plasmid pAAV-RC and pHelper by phosphate-calcium deposit method. The titer was measured by dot hybridization. Infected the PC12 cell using recombinant adeno-associated virus, at 72h after infection, the expression of cdk5 was detected by Western-blot.
Results
All plasmids and the recombinant adeno-associated virus vector carrying the cdk5-siRNA sequences were constructed successfully, the viral titer was 4×1013,it can infect the PC12 cell and down-regulated the expression of the cdk5 gene in PC12 cell.
Conclusion
The recombinant adeno-associated virus vector rAAV-cdk5-siRNA can interfere with the expression of cdk5 gene significantly, which lays the basis for its application in the treatment of the neurodegenerative disease.
引文
1 Sturley SL, Patterson MC, Balch W, et al. The pathophysiology and mechanisms of NP-C disease. Biochim Biophys Acta, 2004, 1685(1-3):83-7.
2 Tseng, H. C.,Zhou, Y.,Shen, Y. and 11. Tsai, L. H. A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 523, 58-62 (2002).
3 German, D. C., et al. Selective neurodegeneration, without neurofibrillary tangles, in a mouse model of Niemann-Pick C disease. J Comp Neurol. 433, 415-25. (2001)
4 Nakamura, S., et al., Cyclin-dependent kinase 5 in Lewy body-like inclusions in anterior horn cells of a patient with sporadic amyotrophic lateral sclerosis. Neurology, 1997. 48(1): p. 267-70.
5 Bu, B., et al. Niemann-Pick disease type C yields possible clue for why cerebellar neurons do not form neurofibrillary tangles. Neurobiol Dis. 11, 285-97 (2002).
6 Borghi, R., et al., Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy. Neurology, 2002. 58(4): p. 589-92.
7 Nguyen, M.D., R.C. Lariviere, and J.P. Julien, Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron, 2001. 30(1): p. 135-47.
8 Nakamura, S., et al., Cyclin-dependent kinase 5 and mitogen-activated protein kinase in glial cytoplasmic inclusions in multiple system atrophy. J Neuropathol Exp Neurol, 1998. 57(7): p. 690-8.
9 Tseng, H. C.,Zhou, Y.,Shen, Y. and 11. Tsai, L. H. A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 523, 58-62 (2002).
10 Bu, B.,Li, J.,Davies, P. and Vincent, I. Deregulation of cdk5, hyperphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model. J Neurosci. 22, 6515-25 (2002).
11 Klunemann, H., B.B, Z.M and I.V. Cell cycle markers and neurofibrillary tangles in Niemann-Pick Type. Under review 2003.
12 Elleder M, Jirasek A: Neuropathology of various types of Niemann-Pick disease. Acta Neuropathol Suppl 1981, 7:201-203
13 Yang Y, Geldmacher DS, Herrup K: DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci 2001, 21:2661-2668.
14 Tomashevski A, Husseman JW, Zheng J-H, Vincent I: Constitutive Wee1 activity in adult brain neurons with M phase-type alterations in Alzheimer neurodegeneration. J Alzheimer's disease 2001, 3:195-206
15 Bu B, Li J, Davies P, Vincent I: Deregulation of cdk5, hyperphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model. J Neurosci 2002, 22:6515-6525
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2 Tseng, H. C.,Zhou, Y.,Shen, Y. and 11. Tsai, L. H. A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 523, 58-62 (2002).
3 German, D. C., et al. Selective neurodegeneration, without neurofibrillary tangles, in a mouse model of Niemann-Pick C disease. J Comp Neurol. 433, 415-25. (2001)
4 Nakamura, S., et al., Cyclin-dependent kinase 5 in Lewy body-like inclusions in anterior horn cells of a patient with sporadic amyotrophic lateral sclerosis. Neurology, 1997. 48(1): p. 267-70.
5 Bu, B., et al. Niemann-Pick disease type C yields possible clue for why cerebellar neurons do not form neurofibrillary tangles. Neurobiol Dis. 11, 285-97 (2002).
6 Borghi, R., et al., Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy. Neurology, 2002. 58(4): p. 589-92.
7 Nguyen, M.D., R.C. Lariviere, and J.P. Julien, Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron, 2001. 30(1): p. 135-47.
8 Nakamura, S., et al., Cyclin-dependent kinase 5 and mitogen-activated protein kinase in glial cytoplasmic inclusions in multiple system atrophy. J Neuropathol Exp Neurol, 1998. 57(7): p. 690-8.
9 Tseng, H. C.,Zhou, Y.,Shen, Y. and 11. Tsai, L. H. A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 523, 58-62 (2002).
10 Bu, B.,Li, J.,Davies, P. and Vincent, I. Deregulation of cdk5, hyperphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model. J Neurosci. 22, 6515-25 (2002).
11 Klunemann, H., B.B, Z.M and I.V. Cell cycle markers and neurofibrillary tangles in Niemann-Pick Type. Under review 2003.
12 Elleder M, Jirasek A: Neuropathology of various types of Niemann-Pick disease. Acta Neuropathol Suppl 1981, 7:201-203
13 Yang Y, Geldmacher DS, Herrup K: DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci 2001, 21:2661-2668.
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