原代培养大鼠海马神经元中滋补脾阴方药抗Aβ神经毒性的保护机制
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摘要
目的:阿尔茨海默病(Alzheimer’s disease,AD)是一种以进行性认知功能障碍为主要特征的神经退行性疾病。现代研究的热点认为β?淀粉样蛋白(amyloid-β-peptide,Αβ)的神经毒性在AD发病机制中起关键作用。Αβ能引起进行性突触功能异常和神经元丢失,最终导致AD中认知功能障碍和行为异常。体内外实验研究Αβ能直接对神经元产生毒性,或增加神经元对兴奋毒的敏感性。突触结构的高度动力学可塑性及突触传递中相应的结构改变是学习记忆的基础,90%的兴奋性突触主要分布于树突棘,树突棘的发育和结构变化受到调节突触活化的分子机制的影响。突触活化可诱导血清诱导激酶(serum-inducible kinase,SNK)靶向树突棘相关的Rap-特异性GTPase-活化蛋白( spine-associated Rap guanosine triphosphatase activating protein,SPAR),通过泛素-蛋白酶体通路引起SPAR降解。SPAR作为突触后蛋白通过调节Rap信号和肌动蛋白骨架重塑来发挥调节树突棘形态的作用。Rap活性和肌动蛋白动力学受到突触活化调节而且与突触可塑性有关,了解调节树突棘肌动蛋白骨架的重塑必然有助于揭示奠定学习记忆基础的突触可塑性的分子机制。尚无研究报道在SNK-SPAR途径中Αβ神经毒性是如何发挥作用,进而引起突触可塑性改变。同样,N-甲基-D-门冬氨酸受体(N-methyl-D-aspartate Receptor,NMDAR)参与突触发生、突触传递、突触可塑性、学习记忆等一系列进程。Αβ的神经毒性能引起谷氨酸细胞外堆积,NMDAR功能异常,钙稳态失衡。我们推测是否Αβ神经毒性、SNK-SPAR途径、NMDAR三者之间存在联系。
滋补脾阴方药(Zibu Piyin Recipe,ZBPYR)以滋补脾阴为立方原则,针对脾阴虚证而设,含药血清是根据血清药理学方法获得。既往临床和实验研究发现ZBPYR有脑保护作用,含药血清能明显抗谷氨酸兴奋毒,然而关于ZBPYR如何发挥神经保护作用调控突触和树突棘的分子机制我们还不十分清楚。
本课题的研究目的在于观察Αβ与SNK-SPAR途径、NMDAR之间的关系,探讨ZBPYR抗Αβ神经毒性保护神经元的作用机制,为AD和其他神经退行性疾病提供了新的研究思路和治疗手段。
方法:1.原代大鼠海马神经元培养。
2.通过脂质体转染将针对SNK的小干扰RNA( small interfering RNA,siRNA)导入神经元中,RT-PCR检测SNKmRNA沉寂情况以及SNK沉寂时SPARmRNA表达变化。
3.将干粉Αβ1-40配制成溶液,在37℃恒温箱中孵育72h后获得聚集态纤维状Αβ1-40。
4.RT-PCR观察神经元暴露于5μMΑβ1-40,在刺激不同时间点SNK、SPARmRNA表达变化。
5.以血清药理学方法获得ZBPYR含药血清;不同浓度的ZBPYR含药血清预处理Αβ1-40刺激的神经元,RT-PCR检测神经元中SNK、SPAR、NR1、NR2A、NR2BmRNA表达变化。
结果:1.siRNA转染入神经元后,与对照组和RNAi对照组相比,SNKmRNA表达下调,SPARmRNA表达上调,以SNK siRNA1效果最显著。
2.在Αβ1-40刺激神经元不同时间点中,与对照组相比,SNKmRNA表达呈上调趋势,SPARmRNA表达呈下调趋势,其中在Αβ1-40刺激2h时SNK、SPARmRNA表达变化达到高峰。
3.与对照组相比,Αβ1-40刺激神经元2h后,SNKmRNA表达上调,SPAR、NR1、NR2A、NR2BmRNA表达下调。
4.与Αβ1-40组相比,ZBPYR含药血清预处理组SNKmRNA表达下调,SPAR、NR1、NR2A、NR2BmRNA表达上调,以浓度2%ZBPYR含药血清效果最显著。
结论:1.siRNA对SNKmRNA表达有抑制作用,相应地SPARmRNA表达上调,表明SNK在SNK-SPAR途径中起关键作用。
2.Αβ1-40诱导SNK、SPARmRNA表达变化,且具有时间依赖性,提示Αβ1-40引起神经元损伤的过程与SNK-SPAR途径密切相关。
3.ZBPYR含药血清预处理可以明显干预SNK、SPAR、NMDAR亚基mRNA表达变化,提示ZBPYR含药血清预处理对Αβ损伤神经元有保护作用,以2%ZBPYR含药血清效果最显著;同时ZBPYR对Αβ损伤神经元的保护机制与抑制NMDAR过度激活从而干预SNK-SPAR途径有关。
Objective: Alzheimer’s disease (AD) is a neurodegenerative disorder that affects cognitive function. Many recent researches have focused on the toxic effect of amyloid-β-peptide (Αβ) which plays a central role in the pathogenesis of AD.Αβis considered to cause progressive synaptic degeneration and neuronal loss, thereby resulting in cognitive dysfunction and behavioral abnormalities in AD.Αβis directly toxic to neurons or potentialize neuronal vulnerability to excitatory neurotoxins, resulting in extensive neuronal loss and synapse dysfunction. Synapses are highly dynamic“plastic”structures, and corresponding dynamic changes in the efficiency of synaptic transmission are thought to represent the physic- ological basis of learning and memory. Dendritic spines are the major sites of synapse formation. The development and morphology changes of spines are considered as strong candidates for the cellular mechanism regulating synaptic activity. It was reported that serum-inducible kinase (SNK) was induced by synapse activity and targeted spine-associated Rap guanosine triphosphatase (GTPase) activating protein (SPAR). By the ubiquitin- proteasome pathway SPAR was degraded. SPAR is a multidomain postsynaptic protein that controls dendritic spines shape by regulating actin arrangement as well as the signalof small GTPase Rap. Actin dynamics and Rap activity are both regulated by synaptic activity and involved in synaptic plasticity. Understanding the mechanisms that control the actin cytoskeleton of dendritic spines may help to reveal the cellular basis of the synaptic plasticity that underlies learning and memory. There are no reports about what role does the toxic ofΑβplay in SNK-SPAR pathway and the dysfunction of synapse. What’s more, N-methyl-D-aspartate Receptors (NMDARs) are involved in a wide variety of processes in the central nervous system (CNS), including synaptogenesis, synaptic plasticity, memory and learning.Αβcould cause extracellular accumulation of glutamate and destabilize calcium homeostasis. We presume whether or not there is close relationship betweenΑβ-induced neurotoxicity, SNK-SPAR pathway and NMDAR.
Principle of nouring spleen yin recipe (Zibu Piyin Recipe, ZBPYR)is to nourish spleen yin, it is a prescription designed specially for deficiency of spleen-yin. ZBPYR serum was gained by the method of serum pharma- cology. Previous researches and clinic experiments reported that ZBPYR had significant effect on brain protection and could protect neurons from excitatory injury in vitro. However, the molecular protective mechanism of ZBPYR for synapses and dendritic spines has not been elucidated clearly.
The objective of this study is to observe the relationship between toxicity induced byΑβ, SNK-SPAR pathway and NMDAR in primary cultured rat hippocampal neurons, and to explore the mechanism of the protective effect of ZBPYR serum on neurons againstΑβtoxicity, and to supply a new therapy method for AD and other neurodegenerative disorders.
Methods: 1. The primary rat hippocampal neurons were cultured.
2. Small interfering RNA (siRNA) targeting for SNK was transfected by Lipofectamine2000 into neurons and then we detected the expression of SNK and SPARmRNA with RT-PCR method.
3.Αβ1-40 peptide was added to 1×PBS and incubated in the incubator for 72h, 37℃, and then aggregated fibrillarΑβ1-40 was obtained.
4. RT-PCR was used to detect SNK、SPAR mRNA expression by exposure to 5μMΑβ1-40 for different time intervals.
5. ZBPYR serum was gained by the method of serum pharmacology; RT-PCR was used to detect SNK, SPAR, NR1, NR2A and NR2B mRNA expression by exposure toΑβ1-40 and with different concentration of ZBPYR serum pretreatment.
Results: 1. After siRNA was transfected into neurons, SNK mRNA was down-regulated, and SPAR mRNA was up-regulated.
2. After exposed toΑβ1-40 for different time intervals, we found SNK mRNA was up-regulated and SPAR mRNA was down-regulated compared with the control group, and the changes of mRNA expression were significant at 2h.
3. After exposed toΑβ1-40 for 2h, we found SNK mRNA was up-regulated, and SPAR, NR1, NR2A, NR2B mRNA were down-regulated compared with the control group.
4. While with ZBPYR serum pretreatment, we observed SNK mRNA was down-regulated and SPAR, NR1, NR2A, NR2B mRNA were up- regulated compared withΑβ1-40 group, and the 2%ZBPYR serum had the best effect.
Conclusions: 1. siRNA could down-regulate the expression of SNKmRNA with corresponding change of SPARmRNA expression. It suggested that SNK played an important role in the SNK-SPAR pathway.
2.Αβ1-40 could induce the changes of SNK and SPAR mRNA expression and these changes had time dependent. It suggestedΑβ-induced neurotoxicity was related to SNK-SPAR pathway.
3. ZBPYR serum could protect neurons againstΑβneurotoxicity, and 2%ZBPTR serum had the best effect. What’s more, the protective effect of ZBPYR serum on neurons againstΑβtoxicity was performed by preventing over-activation of NMDAR and blocking of SNK-SPAR pathway.
滋补脾阴方药(Zibu Piyin Recipe,ZBPYR)以滋补脾阴为立方原则,针对脾阴虚证而设,含药血清是根据血清药理学方法获得。既往临床和实验研究发现ZBPYR有脑保护作用,含药血清能明显抗谷氨酸兴奋毒,然而关于ZBPYR如何发挥神经保护作用调控突触和树突棘的分子机制我们还不十分清楚。
本课题的研究目的在于观察Αβ与SNK-SPAR途径、NMDAR之间的关系,探讨ZBPYR抗Αβ神经毒性保护神经元的作用机制,为AD和其他神经退行性疾病提供了新的研究思路和治疗手段。
方法:1.原代大鼠海马神经元培养。
2.通过脂质体转染将针对SNK的小干扰RNA( small interfering RNA,siRNA)导入神经元中,RT-PCR检测SNKmRNA沉寂情况以及SNK沉寂时SPARmRNA表达变化。
3.将干粉Αβ1-40配制成溶液,在37℃恒温箱中孵育72h后获得聚集态纤维状Αβ1-40。
4.RT-PCR观察神经元暴露于5μMΑβ1-40,在刺激不同时间点SNK、SPARmRNA表达变化。
5.以血清药理学方法获得ZBPYR含药血清;不同浓度的ZBPYR含药血清预处理Αβ1-40刺激的神经元,RT-PCR检测神经元中SNK、SPAR、NR1、NR2A、NR2BmRNA表达变化。
结果:1.siRNA转染入神经元后,与对照组和RNAi对照组相比,SNKmRNA表达下调,SPARmRNA表达上调,以SNK siRNA1效果最显著。
2.在Αβ1-40刺激神经元不同时间点中,与对照组相比,SNKmRNA表达呈上调趋势,SPARmRNA表达呈下调趋势,其中在Αβ1-40刺激2h时SNK、SPARmRNA表达变化达到高峰。
3.与对照组相比,Αβ1-40刺激神经元2h后,SNKmRNA表达上调,SPAR、NR1、NR2A、NR2BmRNA表达下调。
4.与Αβ1-40组相比,ZBPYR含药血清预处理组SNKmRNA表达下调,SPAR、NR1、NR2A、NR2BmRNA表达上调,以浓度2%ZBPYR含药血清效果最显著。
结论:1.siRNA对SNKmRNA表达有抑制作用,相应地SPARmRNA表达上调,表明SNK在SNK-SPAR途径中起关键作用。
2.Αβ1-40诱导SNK、SPARmRNA表达变化,且具有时间依赖性,提示Αβ1-40引起神经元损伤的过程与SNK-SPAR途径密切相关。
3.ZBPYR含药血清预处理可以明显干预SNK、SPAR、NMDAR亚基mRNA表达变化,提示ZBPYR含药血清预处理对Αβ损伤神经元有保护作用,以2%ZBPYR含药血清效果最显著;同时ZBPYR对Αβ损伤神经元的保护机制与抑制NMDAR过度激活从而干预SNK-SPAR途径有关。
Objective: Alzheimer’s disease (AD) is a neurodegenerative disorder that affects cognitive function. Many recent researches have focused on the toxic effect of amyloid-β-peptide (Αβ) which plays a central role in the pathogenesis of AD.Αβis considered to cause progressive synaptic degeneration and neuronal loss, thereby resulting in cognitive dysfunction and behavioral abnormalities in AD.Αβis directly toxic to neurons or potentialize neuronal vulnerability to excitatory neurotoxins, resulting in extensive neuronal loss and synapse dysfunction. Synapses are highly dynamic“plastic”structures, and corresponding dynamic changes in the efficiency of synaptic transmission are thought to represent the physic- ological basis of learning and memory. Dendritic spines are the major sites of synapse formation. The development and morphology changes of spines are considered as strong candidates for the cellular mechanism regulating synaptic activity. It was reported that serum-inducible kinase (SNK) was induced by synapse activity and targeted spine-associated Rap guanosine triphosphatase (GTPase) activating protein (SPAR). By the ubiquitin- proteasome pathway SPAR was degraded. SPAR is a multidomain postsynaptic protein that controls dendritic spines shape by regulating actin arrangement as well as the signalof small GTPase Rap. Actin dynamics and Rap activity are both regulated by synaptic activity and involved in synaptic plasticity. Understanding the mechanisms that control the actin cytoskeleton of dendritic spines may help to reveal the cellular basis of the synaptic plasticity that underlies learning and memory. There are no reports about what role does the toxic ofΑβplay in SNK-SPAR pathway and the dysfunction of synapse. What’s more, N-methyl-D-aspartate Receptors (NMDARs) are involved in a wide variety of processes in the central nervous system (CNS), including synaptogenesis, synaptic plasticity, memory and learning.Αβcould cause extracellular accumulation of glutamate and destabilize calcium homeostasis. We presume whether or not there is close relationship betweenΑβ-induced neurotoxicity, SNK-SPAR pathway and NMDAR.
Principle of nouring spleen yin recipe (Zibu Piyin Recipe, ZBPYR)is to nourish spleen yin, it is a prescription designed specially for deficiency of spleen-yin. ZBPYR serum was gained by the method of serum pharma- cology. Previous researches and clinic experiments reported that ZBPYR had significant effect on brain protection and could protect neurons from excitatory injury in vitro. However, the molecular protective mechanism of ZBPYR for synapses and dendritic spines has not been elucidated clearly.
The objective of this study is to observe the relationship between toxicity induced byΑβ, SNK-SPAR pathway and NMDAR in primary cultured rat hippocampal neurons, and to explore the mechanism of the protective effect of ZBPYR serum on neurons againstΑβtoxicity, and to supply a new therapy method for AD and other neurodegenerative disorders.
Methods: 1. The primary rat hippocampal neurons were cultured.
2. Small interfering RNA (siRNA) targeting for SNK was transfected by Lipofectamine2000 into neurons and then we detected the expression of SNK and SPARmRNA with RT-PCR method.
3.Αβ1-40 peptide was added to 1×PBS and incubated in the incubator for 72h, 37℃, and then aggregated fibrillarΑβ1-40 was obtained.
4. RT-PCR was used to detect SNK、SPAR mRNA expression by exposure to 5μMΑβ1-40 for different time intervals.
5. ZBPYR serum was gained by the method of serum pharmacology; RT-PCR was used to detect SNK, SPAR, NR1, NR2A and NR2B mRNA expression by exposure toΑβ1-40 and with different concentration of ZBPYR serum pretreatment.
Results: 1. After siRNA was transfected into neurons, SNK mRNA was down-regulated, and SPAR mRNA was up-regulated.
2. After exposed toΑβ1-40 for different time intervals, we found SNK mRNA was up-regulated and SPAR mRNA was down-regulated compared with the control group, and the changes of mRNA expression were significant at 2h.
3. After exposed toΑβ1-40 for 2h, we found SNK mRNA was up-regulated, and SPAR, NR1, NR2A, NR2B mRNA were down-regulated compared with the control group.
4. While with ZBPYR serum pretreatment, we observed SNK mRNA was down-regulated and SPAR, NR1, NR2A, NR2B mRNA were up- regulated compared withΑβ1-40 group, and the 2%ZBPYR serum had the best effect.
Conclusions: 1. siRNA could down-regulate the expression of SNKmRNA with corresponding change of SPARmRNA expression. It suggested that SNK played an important role in the SNK-SPAR pathway.
2.Αβ1-40 could induce the changes of SNK and SPAR mRNA expression and these changes had time dependent. It suggestedΑβ-induced neurotoxicity was related to SNK-SPAR pathway.
3. ZBPYR serum could protect neurons againstΑβneurotoxicity, and 2%ZBPTR serum had the best effect. What’s more, the protective effect of ZBPYR serum on neurons againstΑβtoxicity was performed by preventing over-activation of NMDAR and blocking of SNK-SPAR pathway.
引文
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33. Gascón S, Deogracias R, Sobrado M, Roda JM, Renart J, Rodríguez-Pe?a A, Díaz-Guerra M. Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia. J Biol Chem. 2005;280(41):35018-27
34. Yuting Mao1, Shaoyun Zang, Jiping Zhang, Xinde Sun. Early chronic blockade of NR2B subunits and transient activation of NMDA receptors modulate LTP in mouse auditory cortex. Brain research. 2006; 1073-1074:131-38
35. Neema Agrawal, P.V.N.Dasaradhi. RNA Interference: Biology, Mechanism, and Applications. Microbiol Mol Biol Rev. 2003; 67(4):657-85
36. Weilin Wu, Emily Hodges, Jenny Redelius and Christer Hoèoèg. A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Nucleic Acids Research. 2004; 32(2):1-6
37. Anna M, Krichevsky, Kenneth S, Kosik. RNAi functions in cultured mammalian neurons. PNAS. 2002; 99(18):11926-29
38.张晓勤.RNAi下调β分泌酶表达治疗阿尔茨海默病的前景展望.国际神经病学神经外科学杂志.2006;33 (2 ):142-45
1. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990; 2(4):279-89
2. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995; 81(4):611-20
3. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391(6669):806-11
4. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes. 2001; 15(2):188-200
5. Zou GM, Wu W, Chen J, Rowley JD. Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells. Biol Cell. 2003; 95(6):365-71
6. Hammond SM. Dicing and slicing:the core machinery of the RNA interference pathway. FEBS Lett. 2005; 579(26):5822-29
7. Tebes SJ, Kruk PA. The genesis of RNA interference, its potential clinical applications, and implications in gynecologic cancer. Gynecol Oncol. 2005; 99(3): 736-41
8. Preall JB, Sontheimer EJ. RNAi:RISC Gets Loaded. Cell. 2005; 123(4):543-45
9. Zamore PD. RNA interference: listening to the sound of silence. Nat Struct Biol. 2001; 8(9):746-50
10. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000; 101(1):25-33
11. Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO. 2002; 21(21):5875-85
12. Wu W, Hodges E, Redelius J, H??g C. A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Nucleic Acids Res. 2004; 32(2):e17
13. Nguyen T, Menocal EM, Harborth J, Fruehauf JH. RNAi therapeutics: An update on delivery. Curr Opin Mol Ther. 2008; 10(2):158-67
14. Pancoska P, Moravek Z, Moll UM. Efficient RNA interference depends on global context of the target sequence:quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA. Nucleic Acids Res. 2004; 32(4):1469-79
15. Tebes SJ, Kruk PA. The genesis of RNA interference, its potential clinical applications, and implications in gynecologic cancer. Gynecol Oncol. 2005; 99(3):736-41
16. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004; 22(3):326-30
17. Chang CI, Hong SW, Kim S, Lee DK. A structure-activity relationship study of siRNAs with structural variations. Biochem Biophys Res Commun. 2007; 59(4): 997-1003
18. Micura R. Small interfering RNAs and their chemical synthesis. Angew Chem Int Ed Engl. 2002; 41(13):2265-69
19. Xia Y, Lin RX, Zheng SJ, Yang Y, Bo XC, Zhu DY, Wang SQ. Effective siRNA targets screening for human telomerase reverse transcriptase. World J Gastro- enterol. 2005; 11(16):2497-501
20. Myers JW, Jones JT, Meyer T, Ferrell JE Jr. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol. 2003; 21(3):324-28
21. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector- based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci. 2002; 99(8):5515-20
22. Luo Q, Kang Q, Song WX, Luu HH, Luo X, An N, Luo J, Deng ZL. Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. Gene. 2007; 395(1-2):160-69
23. Link CD. Invertebrate models of Alzheimer's disease. Genes Brain Behav. 2005; 4(3):147-56
24. Orlacchio A, Bernardi G, Orlacchio A, Martino S. RNA interference as a tool for Alzheimer's disease therapy. Mini Rev Med Chem. 2007; 7(11):1166-76
25. Kao SC, Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem. 2004; 279(3):1942-49
26. Nawrot B, Antoszczyk S, Maszewska M, Kuwabara T. Efficient inhibition of beta-secretase gene expression in HEK293 cells by tRNAVal-driven and CTE- helicase associated hammerhead ribozymes. Eur J Biochem. 2003; 270(19):3962-70
27. Shuxia Zhou, Hua Zhou, Peter J Walian, Bing K.Jap. CD147 is a regulatory subunit of theγ-secretase complex in Alzheimer’s disease amyloidβ-peptide production PNAS. 2005; 102(21):7499-7504
28. Feng X, Zhao P, He Y, Zuo Z. Allele-specific silencing of Alzheimer's disease genes:the amyloid precursor protein genes with Swedish or London mutations. Gene. 2006; 371(1):68-74
29. Xie Z, Romano DM, Tanzi RE. Effects of RNAi-mediated silencing of PEN-2, APH-1a, and nicastrin on wild-type vs FAD mutant forms of presenilin 1. J Mol Neurosci. 2005; 25(1):67-77
30. Huan-Min Luo, Hui Deng, Fei Xiao, Qin Gao. Down-regulation Amyloidβ-protein 42 Production by Interfering with Transcript of Presenilin 1 Gene with siRNA. Acta Pharmacologica Sinica. 2004; 25(12):1613-18
31. Victor M. Miller, Cynthia M.Gouvion. Targeting Alzheimer's disease genes with RNA interference:an efficient strategy for silencing mutant alleles. Nucleic Acids Res. 2004; 32(2):661-68
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8. Irvine GB, El-Agnaf OM, Shankar GM, Walsh DM. Protein aggregation in the brain-the molecular basis for Alzheimer's and Parkinson's Diseases. Mol Med. 2008; Mar 27
9. Anja Schneider, Walter Schulz-Schaeffer, Tobias Hartmann, Jorg B. Schulz, Mikael Simons. Cholesterol depletion reduces aggregation of amyloid-beta peptide in hippocampal neurons. Neurobiology of Disease. 2006; 23(3):573-77
10. Brikha R.Shrestha, Ottavio V. Vitolo, Powrnima Joshi, Tamar Lordkipanidze. Amyloidβpeptide adversely affects spine number and motility in hippocampal neurons. Mol. Cell. Neurosci. 2006; 33 (3):274-82
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26.宫晓洋.滋补脾阴方药对脾阴虚痴呆大鼠神经元突触的保护作用及其机制研究.学位论文.2007
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31. Suho Lee, Kyoungwoo Lee, Suha Hwang, Sung Hyun Kim, Woo Keun Song. SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP independent mechanism. EMBO. 2006; 25(20):4983-95
32. Oztürk OH, Kü?ükatay V, Y?nden Z, A?ar A, Ba?ci H, Deliba? N. Expressions of N-methyl-D-aspartate receptors NR2A and NR2B subunit proteins in normal andsulfite-oxidase deficient rat’s hippocampus:effect of exogenous sulfite ingestion. Arch Toxicol. 2006; 80(10):671-79
33. Gascón S, Deogracias R, Sobrado M, Roda JM, Renart J, Rodríguez-Pe?a A, Díaz-Guerra M. Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia. J Biol Chem. 2005;280(41):35018-27
34. Yuting Mao1, Shaoyun Zang, Jiping Zhang, Xinde Sun. Early chronic blockade of NR2B subunits and transient activation of NMDA receptors modulate LTP in mouse auditory cortex. Brain research. 2006; 1073-1074:131-38
35. Neema Agrawal, P.V.N.Dasaradhi. RNA Interference: Biology, Mechanism, and Applications. Microbiol Mol Biol Rev. 2003; 67(4):657-85
36. Weilin Wu, Emily Hodges, Jenny Redelius and Christer Hoèoèg. A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Nucleic Acids Research. 2004; 32(2):1-6
37. Anna M, Krichevsky, Kenneth S, Kosik. RNAi functions in cultured mammalian neurons. PNAS. 2002; 99(18):11926-29
38.张晓勤.RNAi下调β分泌酶表达治疗阿尔茨海默病的前景展望.国际神经病学神经外科学杂志.2006;33 (2 ):142-45
1. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990; 2(4):279-89
2. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995; 81(4):611-20
3. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391(6669):806-11
4. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes. 2001; 15(2):188-200
5. Zou GM, Wu W, Chen J, Rowley JD. Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells. Biol Cell. 2003; 95(6):365-71
6. Hammond SM. Dicing and slicing:the core machinery of the RNA interference pathway. FEBS Lett. 2005; 579(26):5822-29
7. Tebes SJ, Kruk PA. The genesis of RNA interference, its potential clinical applications, and implications in gynecologic cancer. Gynecol Oncol. 2005; 99(3): 736-41
8. Preall JB, Sontheimer EJ. RNAi:RISC Gets Loaded. Cell. 2005; 123(4):543-45
9. Zamore PD. RNA interference: listening to the sound of silence. Nat Struct Biol. 2001; 8(9):746-50
10. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000; 101(1):25-33
11. Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO. 2002; 21(21):5875-85
12. Wu W, Hodges E, Redelius J, H??g C. A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Nucleic Acids Res. 2004; 32(2):e17
13. Nguyen T, Menocal EM, Harborth J, Fruehauf JH. RNAi therapeutics: An update on delivery. Curr Opin Mol Ther. 2008; 10(2):158-67
14. Pancoska P, Moravek Z, Moll UM. Efficient RNA interference depends on global context of the target sequence:quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA. Nucleic Acids Res. 2004; 32(4):1469-79
15. Tebes SJ, Kruk PA. The genesis of RNA interference, its potential clinical applications, and implications in gynecologic cancer. Gynecol Oncol. 2005; 99(3):736-41
16. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004; 22(3):326-30
17. Chang CI, Hong SW, Kim S, Lee DK. A structure-activity relationship study of siRNAs with structural variations. Biochem Biophys Res Commun. 2007; 59(4): 997-1003
18. Micura R. Small interfering RNAs and their chemical synthesis. Angew Chem Int Ed Engl. 2002; 41(13):2265-69
19. Xia Y, Lin RX, Zheng SJ, Yang Y, Bo XC, Zhu DY, Wang SQ. Effective siRNA targets screening for human telomerase reverse transcriptase. World J Gastro- enterol. 2005; 11(16):2497-501
20. Myers JW, Jones JT, Meyer T, Ferrell JE Jr. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol. 2003; 21(3):324-28
21. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector- based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci. 2002; 99(8):5515-20
22. Luo Q, Kang Q, Song WX, Luu HH, Luo X, An N, Luo J, Deng ZL. Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. Gene. 2007; 395(1-2):160-69
23. Link CD. Invertebrate models of Alzheimer's disease. Genes Brain Behav. 2005; 4(3):147-56
24. Orlacchio A, Bernardi G, Orlacchio A, Martino S. RNA interference as a tool for Alzheimer's disease therapy. Mini Rev Med Chem. 2007; 7(11):1166-76
25. Kao SC, Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem. 2004; 279(3):1942-49
26. Nawrot B, Antoszczyk S, Maszewska M, Kuwabara T. Efficient inhibition of beta-secretase gene expression in HEK293 cells by tRNAVal-driven and CTE- helicase associated hammerhead ribozymes. Eur J Biochem. 2003; 270(19):3962-70
27. Shuxia Zhou, Hua Zhou, Peter J Walian, Bing K.Jap. CD147 is a regulatory subunit of theγ-secretase complex in Alzheimer’s disease amyloidβ-peptide production PNAS. 2005; 102(21):7499-7504
28. Feng X, Zhao P, He Y, Zuo Z. Allele-specific silencing of Alzheimer's disease genes:the amyloid precursor protein genes with Swedish or London mutations. Gene. 2006; 371(1):68-74
29. Xie Z, Romano DM, Tanzi RE. Effects of RNAi-mediated silencing of PEN-2, APH-1a, and nicastrin on wild-type vs FAD mutant forms of presenilin 1. J Mol Neurosci. 2005; 25(1):67-77
30. Huan-Min Luo, Hui Deng, Fei Xiao, Qin Gao. Down-regulation Amyloidβ-protein 42 Production by Interfering with Transcript of Presenilin 1 Gene with siRNA. Acta Pharmacologica Sinica. 2004; 25(12):1613-18
31. Victor M. Miller, Cynthia M.Gouvion. Targeting Alzheimer's disease genes with RNA interference:an efficient strategy for silencing mutant alleles. Nucleic Acids Res. 2004; 32(2):661-68