CN101361984B - Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 - Google Patents
Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 Download PDFInfo
- Publication number
- CN101361984B CN101361984B CN2008102222769A CN200810222276A CN101361984B CN 101361984 B CN101361984 B CN 101361984B CN 2008102222769 A CN2008102222769 A CN 2008102222769A CN 200810222276 A CN200810222276 A CN 200810222276A CN 101361984 B CN101361984 B CN 101361984B
- Authority
- CN
- China
- Prior art keywords
- sequence
- gene
- dpys12
- expression
- sirna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 230000004766 neurogenesis Effects 0.000 title abstract description 5
- 230000001737 promoting effect Effects 0.000 title abstract description 4
- 101150066838 12 gene Proteins 0.000 title 1
- 101150068576 Dpys gene Proteins 0.000 title 1
- 230000002401 inhibitory effect Effects 0.000 title 1
- 230000004069 differentiation Effects 0.000 claims abstract description 42
- 210000001671 embryonic stem cell Anatomy 0.000 claims abstract description 39
- 239000002243 precursor Substances 0.000 claims abstract description 32
- 108020004459 Small interfering RNA Proteins 0.000 claims abstract description 11
- 101150090723 Dpysl2 gene Proteins 0.000 claims abstract 8
- 108090000623 proteins and genes Proteins 0.000 claims description 50
- 230000014509 gene expression Effects 0.000 claims description 43
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 43
- 230000001537 neural effect Effects 0.000 abstract description 37
- 239000013604 expression vector Substances 0.000 abstract description 9
- 208000012902 Nervous system disease Diseases 0.000 abstract description 4
- 230000001276 controlling effect Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 230000000452 restraining effect Effects 0.000 abstract 3
- 229940079593 drug Drugs 0.000 abstract 2
- 239000004480 active ingredient Substances 0.000 abstract 1
- 239000004055 small Interfering RNA Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 20
- 101100310657 Mus musculus Sox1 gene Proteins 0.000 description 12
- 238000001890 transfection Methods 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 11
- 108010088225 Nestin Proteins 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 102000008730 Nestin Human genes 0.000 description 9
- 210000005055 nestin Anatomy 0.000 description 9
- 238000003753 real-time PCR Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000003153 stable transfection Methods 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 6
- 210000000653 nervous system Anatomy 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 239000003550 marker Substances 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 238000000889 atomisation Methods 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 102000029749 Microtubule Human genes 0.000 description 3
- 108091022875 Microtubule Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000004688 microtubule Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 101150103927 Numb gene Proteins 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 102000013008 Semaphorin-3A Human genes 0.000 description 2
- 108010090319 Semaphorin-3A Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003596 drug target Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000000020 growth cone Anatomy 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 238000009343 monoculture Methods 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 201000000980 schizophrenia Diseases 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 101100344902 Drosophila melanogaster skd gene Proteins 0.000 description 1
- 102100020767 Dystrophin-related protein 2 Human genes 0.000 description 1
- 101000941893 Felis catus Leucine-rich repeat and calponin homology domain-containing protein 1 Proteins 0.000 description 1
- 101000930818 Homo sapiens Dihydropyrimidinase Proteins 0.000 description 1
- 101001053503 Homo sapiens Dihydropyrimidinase-related protein 2 Proteins 0.000 description 1
- 101000931797 Homo sapiens Dystrophin-related protein 2 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101000713102 Mus musculus C-C motif chemokine 1 Proteins 0.000 description 1
- 102000017099 Myelin-Associated Glycoprotein Human genes 0.000 description 1
- 108010013731 Myelin-Associated Glycoprotein Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 208000036752 Schizophrenia, paranoid type Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- UELITFHSCLAHKR-UHFFFAOYSA-N acibenzolar-S-methyl Chemical compound CSC(=O)C1=CC=CC2=C1SN=N2 UELITFHSCLAHKR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000028600 axonogenesis Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000013016 learning Effects 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000003959 neuroinflammation Effects 0.000 description 1
- 230000007514 neuronal growth Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 208000002851 paranoid schizophrenia Diseases 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 102000000568 rho-Associated Kinases Human genes 0.000 description 1
- 108010041788 rho-Associated Kinases Proteins 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了Dpys12基因抑制或促进早期神经发生的药物中的应用。该应用中的抑制或促进早期神经发生为抑制或促进胚胎干细胞向神经前体细胞分化。所述抑制早期神经发生的药物以可表达Dpys12基因的小干扰RNA的真核表达载体为有效成分。所述促进早期神经发生的药物以可表达Dpys12基因的真核表达载体为有效成分。本发明为调控胚胎干细胞向神经前体细胞分化的效率提供了一条有效的途径,为神经系统疾病的研究和治疗提供重要的基础。
Description
技术领域
本发明涉及Dpys12基因在制备抑制或促进早期神经发生的药物中的应用。
背景技术
神经系统控制着生物个体的感觉、认知、学习和记忆等多种机能,神经系统的功能失调特别是神经退行性疾病(如Alzheimer′s disease,Parkinson′s disease)目前在人群中有着较高的发病比例,严重的威胁着人类健康。利用干细胞技术将胚胎干细胞分化为功能性的神经细胞为治疗神经退行性疾病提供了希望(Lee,J.P.,M.Jeyakumar,et al.(2007)."Stem cells act through multiple mechanisms to benefit micewith neurodegenerative metabolic disease."Nat Med13(4):439-47)。
神经发生(Neurogenesis)是胚胎发育早期的重要事件,受到多种信号通路(如FGF,BMPs,Wnt等)、多个基因在时间和空间上的精确调控。尽管近年来成体神经系统中也被发现存在神经发生过程,但胚胎期的早期神经发生仍在神经系统的发育中起主导作用。究竟哪些基因/蛋白参与了胚胎期的神经发生已成为该领域的研究热点,其研究成果将对治疗神经系统疾病提供直接或间接的药物靶点。胚胎干细胞因具有发育的全能性因此可以通过体外诱导分化进而形成神经细胞,该体系为寻找神经发生的相关因子提供了合适的研究模型。
Dpys12(也称DRP2、Crmp2、Ulip2)是一个在神经系统高度表达的基因,研究表明其在Semaphrorin3A介导的神经细胞轴突生长及导向过程中起作用,在生长锥塌陷(growth cone collapse)过程中还可通过调控微管系统的组装及Numb蛋白介导的内吞过程中起作用(Fukata,Y.,T.J.Itoh,et al.(2002)."CRMP-2binds to tubulinheterodimers to promote microtubule assembly."Nat Cell Biol4(8):583-91;Nishimura,T.,Y.Fukata,et al.(2003)."CRMP-2regulates polarized Numb-mediated endocytosis foraxon growth."Nat Cell Biol5(9):819-26)。近来的研究还表明外周T淋巴细胞中Dpys12的高表达与病毒引起的神经感染相关(Vuaillat,C.,M.Varrin-Doyer,et al.(2008)."High CRMP2expression in peripheral T lymphocytes is associated withrecruitment to the brain during virus-induced neuroinflammation."J Neuroimmunol193(1-2):38-51),并且该基因表达蛋白的超磷酸化是AD及某些肿瘤的表征(Cole,A.R.,W.Noble,et al.(2007)."Collapsin response mediator protein-2hyperphosphorylation is an early event in Alzheimer′s disease progression."J Neurochem103(3):1132-44),其单核苷酸多态性(SNP)更是与精神分裂症(schizophrenia)相关(Nakata,K.,H.Ujike,et al.(2003)."The human dihydropyrimidinase-related protein2gene on chromosome 8p21 is associated with paranoid-type schizophrenia."BiolPsychiatry53(7):571-6)。但迄今为止,该基因/蛋白在神经系统中的功能尚未完全揭示,目前研究多集中在特定的神经系统中,对于其在胚胎期的神经发生的过程中的作用尚不明确。
胚胎干细胞是从早期胚胎中分离出来的具有分化成个体所有组织和细胞潜能的多能性干细胞,在体外通过适当的诱导分化,可形成不同胚层的多个类型的细胞。利用胚胎干细胞向神经细胞分化已成为研究神经发生的分子机制的良好研究模型并且如何提高由胚胎干细胞向特定功能性神经细胞分化的能力与效率已成为治疗神经系统疾病的一个重要议题之一。2003年,Austin Smith领导的研究小组建立了无血清单层贴壁培养诱导胚胎干细胞成为Sox1阳性神经前体细胞的方法,这些神经前体细胞可进一步被诱导分化为神经元和神经胶质细胞,操作简捷且诱导效率高等特点使该方法成为了研究早期神经分化的良好系统(Ying,Q.L.,M.Stavridis,et al.(2003)."Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture."Nat Biotechnol21(2):183-6)。
发明内容
本发明的目的是提供Dpys12基因在制备抑制或促进早期神经发生的药物中的应用。
本发明所提供的Dpys12基因在制备抑制或促进早期神经发生的药物中的应用,即可表达Dpys12基因的小干扰RNA的真核表达载体或Dpys12基因的小干扰RNA在制备抑制早期神经发生的药物中的应用;可表达Dpys12基因的的真核表达载体在制备促进早期神经发生的药物中的应用。
所述抑制或促进早期神经发生为抑制或促进胚胎干细胞向神经前体细胞分化。
所述抑制早期神经发生的药物以可表达Dpys12基因的小干扰RNA的真核表达载体为有效成分。
所述小干扰RNA为对应靶标序列为序列表中序列1的DNA序列的小干扰RNA和/或靶标序列为序列表中序列2的DNA序列的小干扰RNA。所述靶标序列为序列表中序列1的DNA序列的小干扰RNA的编码序列为序列表中序列3所示的核苷酸序列;所述靶标序列为序列表中序列2的DNA序列的小干扰RNA的编码序列为序列表中序列4所示的核苷酸序列。
所述可表达Dpys12基因的小干扰RNA的真核表达载体的出发载体为pll3.7。
所述促进早期神经发生的药物以可表达Dpys12基因的真核表达载体为有效成分。
本发明通过实验发现Dpys12基因的表达可促进胚胎干细胞向神经前体细胞分化,通过Dpys12基因的过表达或者利用Dpys12小干扰RNA干扰Dpys12表达活性可促进或抑制胚胎干细胞向神经前体细胞分化的效率,为神经系统疾病的研究和治疗提供重要的基础。通过抑制Dpys12来抑制早期神经分化,可以作为神经系统以外干细胞移植治疗中的药物靶点之一,使移植的干细胞避免向神经细胞分化从而增加目的分化细胞的纯度,从而增强治疗效果。本发明提供的Dpys12小干扰RNAi载体可作为抑制离体胚胎干细胞向神经前体细胞分化的药物,为研究早期神经分化机制的科学研究提供生物材料。
附图说明
图1为Dpys12基因在小鼠胚胎干细胞向神经前体细胞分化过程中的表达检测
图2为稳定转染Dpys12基因RNA干涉载体的胚胎干细胞细胞株的建立
图3为Dpys12RNAi抑制胚胎干细胞向神经前体细胞的分化
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法
实施例1、基因Dpys12在促进早期神经发生中的应用
一、利用胚胎干细胞向神经前体细胞分化体系检测Dpys12基因在早期神经发生过程中的表达情况
利用由胚胎干细胞向神经前体细胞分化的体系,以Sox1和Nestin为神经前体细胞的标志基因,探讨了Dpys12基因在神经分化过程中的表达情况。以小鼠胚胎干细胞系R1(可通过ATCC组织购得,Number:SCRC-1036TM)按照文献(Ying,Q.L,M.Stavridis,et al.(2003)."Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture."Nat Biotechnol21(2):183-6)报道的方法进行神经分化,用Sox1和nestin为标志蛋白和神经前体分化的情况,具体方法如下所述:
1)在小鼠胚胎干细胞系R1神经分化的第1,3,5天(将开始更换无血清诱导分化培养基的时间记为0天)分别提取分化细胞的RNA,进行反转录成cDNA,用Real-Time PCR检测Sox1(GenBank Number,GI:164663796)(神经前体细胞分子标记之一)的表达情况,Real-Time PCR引物序列为F1:5’-TTACTTCCCGCCAGCTCTTC-3’,R1:5’-TGATGCATTTTGGGGGTATCTCTC-3’。以未进行神经分化小鼠胚胎干细胞系R1进行如上相同的Real-Time PCR结果作为对照(图1中A中的ES)。结果如图1中的A所示,结果表明,小鼠胚胎干细胞系R1神经分化天数增加,Sox1的表达量也不断增加。而对照的Sox1的表达量最低,表明神经前体分化过程成功。图1中A的1d、3d、和5d分别为Real-time PCR检测小鼠胚胎干细胞系R1分化1、3、和5天神经前体标志基因Sox1的表达。
2)同时在小鼠胚胎干细胞系R1神经分化第5天通过免疫荧光技术用鼠抗Nestin(神经前体细胞分子标记之一)的抗体(Chemicon International公司购得)检测Nestin的表达情况。具体方法为:将六孔板中培养的神经分化第5天的小鼠胚胎干细胞系R1细胞先用pH为7.4的PBS缓冲液润洗一遍,之后用浓度为0.4g/L的多聚甲醛溶液交联20分钟,然后依次用pH为7.4的PBS润洗,0.2g/L的Triton-X-100溶液处理5分钟,用浓度为0.3g/L的BSA溶液处理20分钟,一抗(抗nestin抗体,购自Chemicon International公司,使用浓度按抗体说明书)染1小时,荧光二抗(FITC抗兔二抗,购自Jackson ImmunoResearch公司,使用浓度按抗体说明书)染1小时,洗去未结合抗体后用封片剂及盖玻片封片并在荧光显微镜下观察。结果如图1中B所示,结果表明,神经分化第5天的细胞有明显的Nestin抗原表达,神经前体分化过程成功。图1中B中左侧为细胞在明视野下的图像,右侧为相同细胞在经抗Nestin抗体进行免疫荧光染色后所显示的荧光信号图像。
3)通过Real-time PCR检测Dpys12基因(GenBank Number,GI:162287190)在未分化小鼠胚胎干细胞系R1(图1中C中的ES)及小鼠胚胎干细胞系R1神经分化第1,3,5天时的表达情况,引物序列为F2:5’-CAGAATGGTGATTCCCGGAGG-3’,R2:5’-CAGCCAATAGGCTCGTCCC-3’。Real-time PCR检测小鼠胚胎干细胞系R1分化1、3、和5天Dpys12基因的表达分别如图1中C的1d、3d、和5d所示。
上述实验结果表明,将上述小鼠胚胎干细胞系R1向神经前体细胞诱导分化的过程中Sox1(图1中A)和Nestin(图1中B)表达量增加,结果表明胚胎干细胞已经成功向神经前体细胞分化;在此分化过程中Dpys12基因的表达量不断增高(图1中C),表明Dpys12基因可能作为早期神经发生中的一个效应因子调控该生理过程。图1中A为Real-time PCR检测分化不同时期(天数)神经前体标志基因Sox1的表达;B为免疫荧光染色检测分化第5天神经前体标志基因nestin的表达;C为神经分化过程中不同时期(天数)Dpys12基因的表达变化。
二、Dpys12基因的RNA干扰(RNAi)表达载体在胚胎干细胞中表达
1、Dpys12基因的RNA干扰(RNAi)表达载体的构建
按照文献(Mimura,F.,S.Yamagishi,et al.(2006)."Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism."J BiolChem281(23):15970-9;Vincent,P.,Y.Collette,et al.(2005)."A role for the neuronal protein collapsin response mediator protein2in T lymphocyte polarizationand migration."J Immunol175(11):7650-60)合成两个靶向Dpys12基因的RNAi序列,以排除RNAi方法上的潜在脱靶效应,并将其装入带有嘌呤霉素抗性的RNAi表达载体pll3.7构建得到Dpys12基因(GenBank Number,GI:162287190)的RNAi表达载体,Dpys12基因的RNAi表达载体具体构建方法为:
1)Dpys12基因的siRNA(小干扰RNA)的编码基因的合成
直接合成引物F3和R3(上海生工公司),F3和R3为互补序列,基因(GenBank Number,GI:162287190)小干扰RNA(命名为siRNA1)的编码序列;
正向引物F3:5’-TGATGGGTTGATCAAGCAAATTCAAGAGATTTGCTTGATCAACCCATCTTTTTTC-3’(序列表中序列3);反向引物R3:5’-TCGAGAAAAAAGATGGGTTGATCAAGCAAATCTCTTGAATTTGCTTGATCAACCCATCA-3’。
该互补序列最终翻译得到的小干扰RNA序列对应的靶标Dpys12序列为:5’-GATGGGTTGATCAAGCAAA-3’(序列表中序列1)。
直接合成引物F4和R4(上海生工公司),F4和R4为互补序列,基因(GenBank Number,GI:162287190)小干扰RNA(命名为siRNA2)的编码序列:
正向引物F1:-5’-TACTCCTTCCTCGTGTACATTTCAAGAGAATGTACACGAGGAAGGAGTTTTTTTC-3’(序列表中序列4);反向引物R4:5’-TCGAGAAAAAAACTCCTTCCTCGTGTACATTCTCTTGAAATGTACACGAGGAAGGAGTA-3’。
该互补序列最终翻译得到的小干扰RNA序列对应的靶标Dpys12序列为:5’-ACTCCTTCCTCGTGTACAT-3’(序列表中序列2)。
2)Dpys12基因的RNA干扰(RNAi)表达载体的构建
首先将步骤1)所示的合成好的RNA干扰引物F3和R3,F4和R4分别退火,分别形成互补配对的双链DNA,F3/R3,F4/R4退火形成的双链DNA都包括一个XhoI酶切位点和一个平末端,以备和酶切后的RNA干扰表达进行连接。
将RNA干扰表达载体pll3.7(可通过http://www.addgene.org/获得)用XhoI和HpaI进行酶切后,分别与上述得到的F3/R3或F4/R4退火形成的双链DNA连接,分别得到可表达siRNA1或siRNA2的重组表达载体,用XhoI和XbaI双酶切鉴定后测序确认插入片段是否正确。将验证表明正确的可表达siRNA1重组表达载体命名为Dpys12-siRNA1,将验证表明正确的可表达siRNA1重组表达载体命名为Dpys12-siRNA2。
2、Dpys12基因表达量被稳定下调的胚胎干细胞株的获得
将Dpys12基因的RNAi表达载体Dpys12-siRNA1或Dpys12-siRNA2分别用脂质体Lipofectamine2000(Invitrogen)转染到R1胚胎干细胞系,对于六孔板中培养的R1细胞,每孔细胞用8ul脂质体和4ug质粒,转染过程按照转染试剂说明书操作。转染48小时后将细胞按照1:3比例传代,并在24小时后更换含有1μg/ml嘌呤霉素的干细胞培养液筛选约10天得到稳定的转染Dpys12-siRNA1细胞株或Dpys12-siRNA2细胞株。
同时筛选以靶向荧光素酶(Luciferase)蛋白序列的RNAi稳定细胞株作为对照。具体方法如下所述:合成对照小干扰RNA的编码序列:正向引物:5-TGCGACCAACGCCTTGATTGTTCAAGAGACAATCAAGGCGTTGGTCGCTTTTTTC-3;反向引物:5-TCGAGAAAAAAGCGACCAACGCCTTGATTGTCTCTTGAACAATCAAGGCGTTGGTCGCA-3
该正向引物序列和反向引物序列为互补序列为对照小干扰RNA的编码序列,该序列编码的小干扰RNA的靶向序列为:5’-GCGACCAACGCCTTGATTG-3’。
将合成得到的编码序列(上述正向引物和反向引物)退火得到双链DNA,然后将该DNA插入到pll3.7的HapI和XhoI酶切位点之间得到重组载体,将该重组载体进行酶切和测序鉴定,将鉴定表明含有上述对照小干扰RNA的编码序列的重组载体命名为Control-RNAi质粒。
将Control-RNAi质粒用脂质体Lipofectamine2000(Invitrogen)转染到R1胚胎干细胞系,用1μg/ml嘌呤霉素的干细胞培养液筛选约10天后得到稳定转染的对照细胞株。
用Real-time PCR方法检测Dpys12基因在转染Dpys12-siRNA1细胞株或Dpys12-siRNA2细胞株中的RNAi效果,具体方法为将培养中的对照细胞株及转染Dpys12-siRNA1细胞株或Dpys12-siRNA2细胞株分别用Trizol(Invitrogen)处理并提取RNA,经反转录后按照步骤一的步骤3)的方法检测Dpys12基因的mRNA表达水平。结果用△△Ct法(Livak,K.J.and T.D.Schmittgen(2001)."Analysis of relativegene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))Method."Methods25(4):402-8.)处理后得到相对mRNA的表达量。
结果如图2所示,结果表明转染Dpys12-siRNA1细胞株(图2中的D1-RNAi)和Dpys12-siRNA2细胞株(图2中的D2-RNAi),Dpys12基因表达量比对照细胞株(图2中的ctrl-RNAi)明显降低,该转染Dpys12-siRNA1细胞株(图2中的D1-RNAi)和Dpys12-siRNA2细胞株(图2中的D2-RNAi)即为受到Dpys12基因的小干扰RNA干扰后Dpys12基因表达量降低的细胞株。图2中,D1-RNAi和D2-RNAi分别为稳定转染了Dpys12-siRNA1的细胞株和稳定转染了Dpys12-siRNA2的细胞株;Ctr1-RNAi为稳定转染了Control-RNAi质粒的细胞株。
3、Dpys12稳定RNAi后对神经前体细胞分化的影响
将上述获得的对照RNAi转染细胞株与稳定转染的转染Dpys12-siRNA1细胞株和Dpys12-siRNA2细胞株同时按照步骤一的方法进行神经分化诱导,分别提取分化第1,3,5天的cDNA,按照步骤一的步骤1)的方法检测Sox1的表达,同时在第5天按照步骤一的步骤2)的方法检测Nestin的表达。
结果如图3中A和B所示,与对照组RNAi相比,上述Dpys12RNAi稳定转染细胞株中Sox1(图3中A)和Nestin(图3中B)的表达量明显降低,表明干扰了Dpys12基因的表达,可抑制胚胎干细胞向神经前体细胞分化,即Dpys12基因是胚胎干细胞向神经前体细胞分化的必需因子,Dpys12的表达可促进胚胎干细胞向神经前体细胞分化,利用Dpys12基因的干扰RNA或可表达其干扰RNA的表达载体可抑制胚胎干细胞向神经前体细胞分化。图3中A为Real-time PCR检测对照细胞株(Ctr1-RNAi)与两个转染Dpys12-siRNA1细胞株(D1-RNAi)和Dpys12-siRNA2细胞株(D2-RNAi)细胞株在神经分化过程中Sox1的表达;图3中B为免疫荧光检测对照细胞株(Ctrl-RNAi)与转染Dpys12-siRNA1细胞株(D1-RNAi)和Dpys12-siRNA2细胞株(D2-RNAi)在神经分化第5天时nestin的表达情况。
序列表
<160>4
<210>1
<211>19
<212>DNA
<213>人工序列
<220>
<223>
<400>1
<210>2
<211>19
<212>DNA
<213>人工序列
<220>
<223>
<400>2
<210>3
<211>55
<212>DNA
<213>人工序列
<220>
<223>
<400>3
<210>4
<211>55
<212>DNA
<213>人工序列
<220>
<223>
<400>4
Claims (4)
1.Dpysl2基因在制备抑制胚胎干细胞向神经前体细胞分化的药物中的应用;所述药物以可表达Dpysl2基因的小干扰RNA的真核表达载体或Dpysl2基因的小干扰RNA为有效成分;所述小干扰RNA为靶标序列为序列表中序列1的DNA序列的小干扰RNA和/或靶标序列为序列表中序列2的DNA序列的小干扰RNA。
2.根据权利要求1所述的应用,其特征在于:所述靶标序列为序列表中序列1的DNA序列的小干扰RNA的编码序列为序列表中序列3所示的核苷酸序列;所述靶标序列为序列表中序列2的DNA序列的小干扰RNA的编码序列为序列表中序列4所示的核苷酸序列。
3.根据权利要求1或2所述的应用,其特征在于:所述可表达Dpysl2基因的小干扰RNA的真核表达载体的出发载体为pll3.7。
4.Dpysl2基因在制备促进胚胎干细胞向神经前体细胞分化的药物中的应用;所述药物以可表达Dpysl2基因的真核表达载体为有效成分。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2008102222769A CN101361984B (zh) | 2008-09-12 | 2008-09-12 | Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2008102222769A CN101361984B (zh) | 2008-09-12 | 2008-09-12 | Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101361984A CN101361984A (zh) | 2009-02-11 |
CN101361984B true CN101361984B (zh) | 2010-08-11 |
Family
ID=40388709
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2008102222769A Expired - Fee Related CN101361984B (zh) | 2008-09-12 | 2008-09-12 | Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101361984B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104450602B (zh) * | 2013-09-17 | 2020-08-07 | 中国科学院遗传与发育生物学研究所 | 非人哺乳动物神经精神疾病动物模型及其制备方法和用途 |
CN113599522B (zh) * | 2021-08-03 | 2022-09-20 | 深圳市北科生物科技有限公司 | Kdm6作为靶标在制备用于提高早期神经外胚层分化效率的药物中的应用 |
CN114990163A (zh) * | 2022-03-31 | 2022-09-02 | 中海峡(福建)细胞生物科技有限公司 | 用于干细胞基因修饰的慢病毒载体及其构建方法和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1331332A (zh) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | 一种新的多肽——人二氢嘧啶酶相关蛋白-1(drp-1)9.35和编码这种多肽的多核苷酸 |
WO2006021810A1 (en) * | 2004-08-27 | 2006-03-02 | Proteome Sciences Plc | Methods and compositions relating to alzheimer's disease |
-
2008
- 2008-09-12 CN CN2008102222769A patent/CN101361984B/zh not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1331332A (zh) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | 一种新的多肽——人二氢嘧啶酶相关蛋白-1(drp-1)9.35和编码这种多肽的多核苷酸 |
WO2006021810A1 (en) * | 2004-08-27 | 2006-03-02 | Proteome Sciences Plc | Methods and compositions relating to alzheimer's disease |
Non-Patent Citations (5)
Title |
---|
FRANZEN B, ET AL..Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteome.《Proteomics》.WILEY-VCH Verlag GmbH & Co. KGaA,2003,第 3 卷(第 10 期),1920-1929. * |
Fumiaki Mimura,et al..Myelin-associated Glycoprotein Inhibits Microtubule Assembly by a Rho-kinase-dependent Mechanism.《THE JOURNAL OF BIOLOGICAL CHEMISTRY》.The American Society for Biochemistry and Molecular Biology, Inc.,2006,第 281 卷(第 23 期),15970-15979. * |
Peggy Vincent, et al..A Role for the Neuronal Protein Collapsin Response Mediator Protein 2 in T Lymphocyte Polarization and Migration.《The Journal of Immunology》.The American Association of Immunologists, Inc.,2005,第 175 卷(第 11 期),7650-7660. * |
杨国锋,王鲁宁,纪建国,何思志,朱明伟,王青松.Tau蛋白病的蛋白质组研究.《中华内科杂志》.2005,第 44 卷(第 5 期),374-377. * |
王进,王蔚蔚.成年脑内神经发生及影响因素.《周口师范学院学报》.2008,第 25 卷(第 2 期),87-89. * |
Also Published As
Publication number | Publication date |
---|---|
CN101361984A (zh) | 2009-02-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Antón-Bolaños et al. | Developmental interactions between thalamus and cortex: a true love reciprocal story | |
Lanzuolo et al. | Memories from the polycomb group proteins | |
Ming et al. | Adult neurogenesis in the mammalian central nervous system | |
Nakagawa et al. | Regulation of neurogenesis in adult mouse hippocampus by cAMP and the cAMP response element-binding protein | |
Ge et al. | Dividing glial cells maintain differentiated properties including complex morphology and functional synapses | |
Young et al. | An Fgfr3‐iCreERT2 transgenic mouse line for studies of neural stem cells and astrocytes | |
Ang et al. | Diverse lncRNA mechanisms in brain development and disease | |
Liu et al. | Role of miRNAs in neuronal differentiation from human embryonic stem cell—derived neural stem cells | |
Ahmad et al. | Müller glia: a promising target for therapeutic regeneration | |
Liu et al. | Developmental origins of brain tumors | |
Kokovay et al. | The incredible elastic brain: how neural stem cells expand our minds | |
Jørgensen et al. | REST selectively represses a subset of RE1-containing neuronal genes in mouse embryonic stem cells | |
Matsubara et al. | Regulation of adult mammalian neural stem cells and neurogenesis by cell extrinsic and intrinsic factors | |
Gonçalves et al. | Human adipose tissue‐derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells | |
Gherzi et al. | KSRP controls pleiotropic cellular functions | |
Liao et al. | Direct conversion of cord blood CD34+ cells into neural stem cells by OCT4 | |
Bofill-De Ros et al. | Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges | |
Su et al. | LncRNA AW112010 promotes mitochondrial biogenesis and hair cell survival: implications for age-related hearing loss | |
CN101361984B (zh) | Dpys12基因在制备抑制或促进早期神经发生的药物中的应用 | |
Sun et al. | miR‐30c and semaphorin 3A determine adult neurogenesis by regulating proliferation and differentiation of stem cells in the subventricular zones of mouse | |
Reeve et al. | Targeted activation of primitive neural stem cells in the mouse brain | |
Perri et al. | A focus on regulatory networks linking MicroRNAs, transcription factors and target genes in neuroblastoma | |
Ronzoni et al. | Guide cells support muscle regeneration and affect neuro-muscular junction organization | |
Pratt et al. | Loss of neuronal Tet2 enhances hippocampal-dependent cognitive function | |
Cheng et al. | Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)‐associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100811 Termination date: 20140912 |
|
EXPY | Termination of patent right or utility model |