CN105018484B - CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target - Google Patents
CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target Download PDFInfo
- Publication number
- CN105018484B CN105018484B CN201510463469.3A CN201510463469A CN105018484B CN 105018484 B CN105018484 B CN 105018484B CN 201510463469 A CN201510463469 A CN 201510463469A CN 105018484 B CN105018484 B CN 105018484B
- Authority
- CN
- China
- Prior art keywords
- crtap
- genes
- disease
- product
- alzheimer
- 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.)
- Active
Links
- 101000859758 Homo sapiens Cartilage-associated protein Proteins 0.000 title claims abstract description 86
- 230000014509 gene expression Effects 0.000 title claims abstract description 49
- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 42
- 238000003745 diagnosis Methods 0.000 title claims description 20
- 101150008104 CRTAP gene Proteins 0.000 claims abstract description 34
- 210000004369 blood Anatomy 0.000 claims abstract description 11
- 239000008280 blood Substances 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 48
- 102100027848 Cartilage-associated protein Human genes 0.000 claims description 36
- 102000004169 proteins and genes Human genes 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 15
- 108020004459 Small interfering RNA Proteins 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 230000002068 genetic effect Effects 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 8
- 239000007790 solid phase Substances 0.000 claims description 8
- 238000011529 RT qPCR Methods 0.000 claims description 5
- 230000003053 immunization Effects 0.000 claims description 5
- 238000002649 immunization Methods 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 108020005187 Oligonucleotide Probes Proteins 0.000 claims description 4
- 239000002751 oligonucleotide probe Substances 0.000 claims description 4
- 238000003757 reverse transcription PCR Methods 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 238000007901 in situ hybridization Methods 0.000 claims description 3
- 108091034117 Oligonucleotide Proteins 0.000 claims 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims 1
- 239000002777 nucleoside Substances 0.000 claims 1
- 125000003835 nucleoside group Chemical group 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 18
- 230000006870 function Effects 0.000 abstract description 11
- 238000011160 research Methods 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 3
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 239000003147 molecular marker Substances 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 20
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 125000003275 alpha amino acid group Chemical group 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 230000009368 gene silencing by RNA Effects 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 210000002569 neuron Anatomy 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108091030071 RNAI Proteins 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- MUKYLHIZBOASDM-UHFFFAOYSA-N 2-[carbamimidoyl(methyl)amino]acetic acid 2,3,4,5,6-pentahydroxyhexanoic acid Chemical compound NC(=N)N(C)CC(O)=O.OCC(O)C(O)C(O)C(O)C(O)=O MUKYLHIZBOASDM-UHFFFAOYSA-N 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 241000819233 Tribulus <sea snail> Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 206010025482 malaise Diseases 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 229940047183 tribulus Drugs 0.000 description 5
- 230000000692 anti-sense effect Effects 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 238000003153 stable transfection Methods 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 230000008485 antagonism Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000003499 nucleic acid array Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 2
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 206010029350 Neurotoxicity Diseases 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- 206010044221 Toxic encephalopathy Diseases 0.000 description 2
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102100027831 14-3-3 protein theta Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- -1 DNA-RNA chimera Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 241001635598 Enicostema Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010034719 Personality change Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 206010002022 amyloidosis Diseases 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 208000036815 beta tubulin Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000002247 constant time method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000012502 diagnostic product Substances 0.000 description 1
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000008684 selective degradation Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Abstract
The invention discloses CRTAP genes and its expression product can as alzheimer ''s disease early diagnosis molecular marker, you can to judge whether subject suffers from Alzheimer disease by detecting the CRTAP gene expression doses in subject's blood.According to the achievement in research of the present invention, can research and develop can suppress CRTAP gene expressions or can suppress the medicine of CRTAP gene expression product functions, so as to realize prevention and treatment clinically for Alzheimer disease.
Description
Technical field
The present invention relates to biological technical field, diagnosis, treatment more particularly to people CRTAP genes in Alzheimer disease
In purposes.
Background technology
Alzheimer disease (Alzheimer disease, AD), is called senile dementia, is a kind of central nervous system
Degenerative disease, insidious onset, the course of disease is in chronic progressive, is the most common type of senile dementia.It is mainly shown as progressive
Property the neuropsychic symptom such as memory disorders, cognition dysfunction, personality change and aphasis, have a strong impact on social, occupation with
Vital function.The AD cause of disease and pathogenesis is not yet illustrated, and characteristic pathological changes into amyloid beta and deposits the cell to be formed
Neurofibrillary tangles in the nerve cell of outer senile plaque expelling and Protein tau Hyperphosphorylationof formation, and neuron loss is with glue
Cell plastid hyperplasia etc..
Because the AD cause of disease and pathogenesis are still not clear, disease progression is reversed and prevented currently without effect method, because
This is main at present by early stage progress symptomatic treatment, and the AD patient of clinical diagnosis at present is basic all in middle and advanced stage, existing
Treatment be only capable of improve symptom, can not organize or reverse disease progress.Therefore, it is early diagnosed and intervened, energy
AD harm is substantially reduced, so as to mitigate the burden of society and family.
The content of the invention
In order to make up the deficiencies in the prior art, it can be used for Alzheimer disease early stage it is an object of the invention to provide one kind
The molecular marker of diagnosis.Compared to the diagnostic method of traditional Alzheimer disease, A Erci is diagnosed using gene marker
The silent disease in sea has promptness, specificity and sensitivity so that patient in disease early stage with regard to disease risks can be known, for wind
Danger height, takes corresponding prevention and treatment measure.
To achieve these goals, the present invention is adopted the following technical scheme that：
The invention provides a kind of people CRTAP genes and its expression product in the product of diagnosis of alzheimer's disease is prepared
Application.
Further, diagnostic products mentioned above include：Pass through RT-PCR, real-time quantitative PCR, immune detection, original position
The expression of hybridization or chip detection CRTAP genes and its expression product is with the product of diagnosis of alzheimer's disease.
Further, the product of the use RT-PCR diagnosis of alzheimer's disease at least includes a pair of specific amplified CRTAP bases
The primer of cause；The product of the use real-time quantitative PCR diagnosis of alzheimer's disease at least includes a pair of specific amplified CRTAP genes
Primer；The product of the use immune detection diagnosis of alzheimer's disease includes：The antibody combined with CRTAP protein-specifics；
The product of the use in situ hybridization diagnosis of alzheimer's disease includes：With the probe of the nucleic acid array hybridizing of CRTAP genes；It is described
Included with the product of chip diagnosis of alzheimer's disease：Protein chip and genetic chip；Wherein, protein chip includes and CRTAP
The antibody that protein-specific is combined, genetic chip includes the probe with the nucleic acid array hybridizing of CRTAP genes.
Preferably, the product includes chip, kit.
Present invention also offers application of the people CRTAP genes in high-flux sequence platform.With high throughput sequencing technologies
Development, the structure of the gene expression profile of a people will be turned into and very easily worked.By contrasting Disease and normal
The gene expression profile of crowd, the exception for easily analyzing which gene is related to disease.Therefore, people is known in high-flux sequence
The abnormal Ahl tribulus sea silent sickness correlation of CRTAP genes falls within the purposes of people's CRTAP genes, equally the protection model in the present invention
Within enclosing.
Present invention also offers people CRTAP genes and its expression product in the medicine for preparing treatment Alzheimer disease
Using.
The main active of " medicine for the treatment of Alzheimer disease " of the present invention includes suppressing CRTAP gene tables
The material reached, the material, and/or the active thing of suppression CRTAP gene expression products that suppress CRTAP gene expression product stability
Matter.
Further, the medicine for the treatment of Alzheimer disease of the present invention includes：CRTAP bases are suppressed by RNA interfering
Because of the double stranded RNA of expression, or tumor vaccine based on CRTAP antigen proteins or for suppressing CRTAP protein actives
Protein.
Present invention also offers a kind of pharmaceutical composition for being used to treat Alzheimer disease, described pharmaceutical composition is included
CRTAP genes and/or its expression product inhibitor.The inhibitor includes suppressing the material of CRTAP gene expressions, suppressed
The material of CRTAP gene expression product stability, and/or the material for suppressing CRTAP gene expression products activity.
Further, inhibitor of the present invention includes：Suppress the double-strand ribose core of CRTAP gene expressions by RNA interfering
Acid, or the tumor vaccine based on CRTAP antigen proteins or the protein for suppressing CRTAP protein actives.
Treatment Alzheimer is being prepared present invention also offers above-mentioned CRTAP genes and/or its expression product inhibitor
Application in medicine.
In the present invention, the RNA interference (RNA interference, RNAi) refers to be highly conserved during evolution
, induced by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Make
With RNAi technology can with specific depletion or close specific gene expression, the technology have been widely used for explore gene function and
The field of gene of communicable disease and malignant tumour.RNAi based on cell is screened in terms of functional gene research
With many advantages, RNAi methods can be used by being mainly manifested in most cell types, and is easier to lower or is sunk relatively
The expression of silent any target gene.
In order to ensure CRTAP genes can be rejected efficiently or silence, devised according to the mRNA sequence of CRTAP genes
SiRNA specific fragments.SiRNA design according to delivered general design principle (Elbashir et.al 2001,
Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al2004,
Ui-Tei et.al 2004), by online tool complete design, the online tool is：
SiRNASelectionProgram of Whitehead Institute (BingbingYuan et.al 2004,
http://jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTM RNAi Designer ofINVITROGEN
(winner of the 2004Frost＆Sullivan Excellence in Research Award, https://
rnaidesigner.invitrogen.com/sirna/).It is comprehensive two in order to further improve the validity of siRNA segments
The advantage of Photographing On-line instrument come be designed for screening siRNA segments.Finally, by sequence analysis (NCBI BLAST) come
SiRNA sequence is filtered, to improve the specificity of siRNA segments and reduce the effect of missing the target of RNAi interference.
The medicine of the present invention also includes pharmaceutically acceptable carrier, and carrier, this kind of carrier includes (but being not limited to)：It is dilute
Release agent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and poly- second
Alkene pyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium acid carbonate；Sorbefacient quaternary ammonium compound；Table
Face activating agent such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium, poly- second
Glycol etc..
The medicine of the present invention can also be with other treatment Alzheimer disease drug combination, multi-medicament is used in combination can be with
The success rate for the treatment of is mentioned significantly.
Present invention also offers a kind of product of diagnosis of alzheimer's disease, the product includes but is not limited to chip, examination
Agent box.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes being used to detect being directed to for CRTAP gene transcription levels
The oligonucleotide probe of CRTAP genes；The protein-chip includes solid phase carrier and is fixed on the CRTAP eggs of solid phase carrier
White specific antibody；The genetic chip can be used for detection including people's CRTAP genes multiple genes (for example, and Ah
The related multiple genes of Alzheimer's disease) expression.The protein-chip can be used for detecting that including people's CRTAP albumen exists
The expression of interior multiple protein (such as related multiple protein of Ahl tribulus sea silent sickness).By by multiple and A Er
The mark of Ci Haimo diseases is detected simultaneously, is greatly improved the accuracy rate of diagnosis of Alzheimer disease.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for being used to detect CRTAP gene transcription levels；The protein immunization detection kit includes CRTAP albumen
Specific antibody.Further, the reagent is including the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Reagent needed for during method detection CRTAP gene expression doses.Preference, the reagent is included for CRTAP genes
Primer and/or probe.Easily designed according to the nucleotide sequence information of CRTAP genes and can be used for detecting CRTAP gene tables
Up to horizontal primer and probe.
Probe with the nucleic acid array hybridizing of CRTAP genes can be DNA, RNA, DNA-RNA chimera, PNA or other
Derivative.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over there are different influences, the length of the probe is typically at least 14 base-pairs, most long not surpass typically
30 base-pairs are crossed, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequence
Row are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the CRTAP albumen includes monoclonal antibody, polyclonal antibody.The CRTAP eggs
White specific antibody include complete antibody molecule, any fragment of antibody or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the fragment can retain the binding ability with CRTAP albumen.For protein level
Antibody preparation when well known to a person skilled in the art and the present invention can use any method to prepare the antibody.
In the context of the present invention, " CRTAP genes " includes any work(of people CRTAP genes and people's CRTAP genes
The polynucleotides of energy equivalent.CRTAP genes include and CRTAP bases in current international public GenBank GeneBank
Because (NC_000003.12) DNA sequence dna has more than 70% homology, and coding identical function protein DNA sequence；
Preferably, the coded sequence of CRTAP genes includes following any DNA molecular：
(1) DNA sequence dna in sequence table shown in SEQ ID NO.1；
(2) under strict conditions with 1) the DNA sequence dna hybridization that limits and coding identical function protein DNA sequence；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical work(
Can protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of the CRTAP genes is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, CRTAP gene expression products include people CRTAP albumen and people's CRTAP albumen
Partial peptide.The partial peptide of the CRTAP albumen contains the related functional domain of Ahl tribulus sea silent sickness.
" CRTAP albumen " includes any functional equivalent of people CRTAP albumen and people's CRTAP albumen.Described function etc.
Jljl includes people CRTAP albumen conservative variation protein or its active fragment, or its reactive derivative, allelic variant, day
Right mutant, induced mutants, can be with the albumen coded by the DNA of people CRTAP DNA hybridization under high or low stringent condition
Matter.
Preferably, CRTAP albumen is the protein with following amino acid sequences：
(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；
(2) by the amino acid sequence shown in SEQ ID NO.2 is by the substitution of one or several amino acid residues and/or lacks
Lose and/or addition and with the amino acid sequence shown in SEQ ID NO.2 have identical function as the ammonia shown in SEQ ID NO.2
Protein derived from base acid sequence.The number of the amino acid of substitution, missing or addition is usually 1-50, preferably 1-30
It is individual, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2,
It is highly preferred that the homology with the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%,
98%th, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the CRTAP albumen is with the amino acid sequence shown in SEQ ID NO.2
The protein of row.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein.
Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or indivedual additions to amino acid sequence,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
By adding the fusion that the example for the protein that an amino acid or more amino acid are modified is CRTAP albumen
Albumen.Do not limited for the peptide or protein with CRTAP protein fusions, as long as the fusion protein of gained retains CRTAP eggs
White biological activity.
The CRTAP albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, as long as
Protein by modification remains able to retain the biological activity of CRTAP albumen.It is mutated in such modifying protein
Amino acid number be typically 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis of alzheimer's disease " both includes judging whether subject suffers from A Er
Ci Haimo is sick, also include judging that subject whether there is the risk with Alzheimer disease.
In the context of the present invention, " treatment Alzheimer disease " divides from the state change of disease, can include disease
Alleviation, the complete healing of disease of disease.
Because Beta 4 amyloids (Amyloid-beta is commonly abbreviated as A β) are considered as AD morbid substance.A β are certainly
Body polymerizing power is strong, forms the oligomer and fiber more stronger than A beta monomers toxicity.Therefore, a kind of gene or its expression product are judged
The function whether with treatment Alzheimer disease just can be by detecting whether gene or its expression product there is suppression A β to gather
The effect of collection, the effect with the effect for removing intracerebral A β and with the neurotoxicity for blocking A β judge.
In the embodiment of the present invention, extraction is the total serum IgE in serum to carry out CRTAP gene differential expressions
Research.Skilled person will appreciate that, the tumour cell in situ tumor tissue, which can be shed in blood, to be circulated, therefore
The expression of CRTAP genes can just represent the expression of CRTAP genes in situ tumor tissue in serum.According to this hair
Bright achievement in research is understood, total in tumor tissues or body fluid (including but is not limited to blood, urine, tissue fluid) by extracting
RNA is used equally for judging whether subject suffers from Alzheimer disease to detect the expression of CRTAP genes.
The advantages of the present invention：
Present invention firstly discovers that CRTAP gene expressions Ahl tribulus sea silent sickness is related, by detecting in subject's blood
CRTAP expression, it can be determined that whether subject, which suffers from Alzheimer disease or judge that subject whether there is, suffers from A Er
The risk of Ci Haimo diseases, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-CRTAP genes, compared to traditional detection means, gene diagnosis
More in time, it is more special, sensitiveer, the early diagnosis of Alzheimer disease can be realized, so as to reduce the dead of Alzheimer disease
Die rate.
Brief description of the drawings
Fig. 1 displays detect expression of the CRTAP genes in Alzheimer disease blood using QPCR；
Fig. 2 displays detect influences of the siRNA to CRTAP gene expressions using QPCR；
Fig. 3 shows that CRTAP gene pairs A β cause the influence of nerve cell death；
Fig. 4 shows that CRTAP gene pairs A β cause the influence of nervous process denaturation.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
The related gene marker of the screening Ahl tribulus sea silent sickness of embodiment 1
1st, the collection of peripheral blood sample
AD patient comes from Beijing 301 Hospital, and totally 60, age 53-84 Sui, all cases are diagnosed as AD, and it diagnoses mark
Quasi- reference Americanism medical diagnosis on disease statistic handbook third edition revised edition.Control crowd totally 50, selected from the conventional body of Beijing Hospital
Inspection crowd, it is all enter examination person exclude the diseases such as blood lipid metabolism, age 60-82 Sui.All research objects endorsed to the inspection
The informed consent form of survey project, and the detection there is provided peripheral blood for gene.
2nd, blood Total RNAs extraction
The extraction of blood total serum IgE is carried out using hundred Tyke blood rna extracts kits.
(1) the μ l (or 0.25g) of whole blood 250 are taken into RNase-Free Filter columns, 13000rpm is centrifuged 2 minutes, under collection
Liquid, adds 0.75ml lysates RLS.
(2) homogenised sample is acutely shaken to mixing, 5 minutes are incubated under the conditions of 15-30 DEG C so that ribosome divides completely
Solution.
(3) optional step：12,000rpm is centrifuged 10 minutes under conditions of 4 DEG C, carefully takes supernatant to be transferred to a new nothing
In the centrifuge tube of RNase.
(4) 0.2ml chloroforms are added per 1ml RLS.Sample tube cover is covered tightly, acutely vibrates 15 seconds and it is incubated 3 at room temperature
Minute.
(5) centrifuged 10 minutes in 4 DEG C of 12,000rpm, sample can be divided into three layers：Lower floor's organic phase, intermediate layer and upper strata without
The aqueous phase of color, RNA is present in aqueous phase.The capacity of aqueous layer is about the 60% of added RLS volumes, and aqueous phase is transferred to new pipe
In, carry out next step operation.
(6) 1 times of ethanol of volume 70% is added, overturns and mixes (now it is possible that precipitation), obtained solution and may
Precipitation is transferred in adsorption column RA (adsorption column is enclosed in collecting pipe) together.
(7) 10,000rpm are centrifuged 45 seconds, discard waste liquid, adsorption column is recovered into collecting pipe again.
(8) 500 μ l protein liquid removals RE are added, 12,000rpm centrifugations 45 seconds discard waste liquid.
(9) 700 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(10) 500 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(11) adsorption column RA is put back in sky collecting pipe, 12,000rpm centrifugations 2 minutes remove rinsing liquid as far as possible, in order to avoid drift
Residual ethanol suppresses downstream reaction in washing lotion.
(12) adsorption column RA is taken out, is put into a centrifuge tube without RNase, according to expected RNA yield in adsorbed film
Middle part adds water of the 50-80 μ l without RNase, and room temperature is placed 2 minutes, and eluent is collected in 12,000rpm centrifugations 1 minute.
3rd, QPCR is expanded
(1) design of primers
QPCR amplimers are designed according to the coded sequence of CRTAP genes and GAPDH genes in Genbank, given birth to by Shanghai
Work biotechnology Services Co., Ltd synthesizes.Specific primer sequence is as follows：
CRTAP genes：
Forward primer is 5 '-TCAAGGACTTCAAGGATT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-ATTCTTCAGGTCGTTCAA-3 ' (SEQ ID NO.4).
GAPDH genes：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.11)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.12).
(2) PCR reaction systems are prepared according to table 1：
Wherein, SYBR Green PCRs system is purchased from Invitrogen companies.
The PCR reaction systems of table 1
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green PCR systems | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as
Fluorescent marker, in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instruments, is analyzed by melt curve analysis and electrophoresis is true
Determine purpose band, Δ Δ CT methods carry out relative quantification.
4th, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that work as P<Have when 0.05
It is statistically significant.
5th, result
As a result as shown in figure 1, compared with normal population, the CRTAP gene expressions contained in Alzheimer patients serum
Amount is dramatically increased, and difference has statistical significance (P<0.05).
Embodiment 2 disturbs the expression of CRTAP genes
1st, siRNA designs synthesis
For CRTAP siRNA sequence：
siRNA1-CRTAP：
Positive-sense strand is 5 '-UUAUUUGCCUUGAAGUAAGCG-3 ' (SEQ ID NO.5)；
Antisense strand is 5 '-CUUACUUCAAGGCAAAUAAUC-3 ' (SEQ ID NO.6),
siRNA2-CRTAP：
Positive-sense strand is 5 '-UAUGGAAAGGUAGAAAUCCUU-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-GGAUUUCUACCUUUCCAUAGC-3 ' (SEQ ID NO.8),
siRNA3-CRTAP：
Positive-sense strand is 5 '-UCGUUCAACUUAUAAUAGGCA-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-CCUAUUAUAAGUUGAACGACC-3 ' (SEQ ID NO.10)
Negative control siRNA sequence (siRNA-NC)：
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO.13)；
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO.14).
Nerve cell strain R2L1 cells are pressed 1 × 104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2Training
Cell culture 24h in case is supported, in without DMEM culture mediums dual anti-, containing 10%FBS, is transfected according to lipofectamine 2000
The specification transfection of (being purchased from Invitrogen companies), experiment is divided into normal group, negative control group and experimental group (20nM), its
In, normal group is not transfection group, and the sequence of negative control group siRNA and CRTAP genes is without homology, and concentration is 20nM/ holes, together
When transfect respectively.
2nd, QPCR detects the transcriptional level of CRTAP genes
The extraction of 2.1 cell total rnas
Using TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification
Offer method extracts the total serum IgE of QBC939 cells.Specific method is：Cell is taken, the PBS for being 0.01M with concentration is rinsed 3 times, plus
Enter appropriate TRIzol reagents, room temperature is placed 5min cell lysis, dispensed after piping and druming is uniform with 1mL/ pipes to 1.5mL Eppendorf
Guan Zhong.Often pipe adds 0.2mL chloroforms, acutely shakes 15s, and room temperature places 2-3min, 4 DEG C, 12000r/min centrifugation 15min, will
Upper strata aqueous phase is moved in clean Eppendorf pipes, add 0.5mL isopropanols, gently mix, room temperature place 10min, 4 DEG C,
7500r/min centrifuges 10min.Supernatant is abandoned, 75% ethanol washing RNA precipitate, 7500r/min centrifugations 5min, drying at room temperature RNA is heavy
Form sediment, appropriate DEPC water is dissolved in after 5-10min.Mass fraction is complete for 1.0% agarose gel electrophoresis detection RNA samples
Property, the RNA of extraction is quantitative determined using Bio-Photometer.
2.2 reverse transcription step be the same as Examples 1.
2.3 QPCR amplification steps be the same as Examples 1.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, the difference between interference CRTAP gene expression panels and control group is adopted
Examined with t, it is believed that work as P<There is statistical significance when 0.05.
4th, result
As a result as Fig. 2 shows that compared with siRNA2-CRTAP, siRNA3-CRTAP, siRNA1-CRTAP can be more effective
Suppression CRTAP genes expression, difference has statistical significance (P<0.05) follow-up reality, is carried out using siRNA1-CRTAP
Test.
The CRTAP gene pairs A β of embodiment 3 cause the antagonism of nerve cell death
1st, cell transfecting：SiRNA1-CRTAP and siRNA- is carried out to nerve cell strain R2L1 according to the method for embodiment 2
NC transfection.
2nd, after transfection 24h, nerve cell strain R2L1 is cultivated in 96 orifice plates, cell density is 0.5 × 104Cells/well.
Cell is divided into following groups：
Group is not induced：Transfect siRNA-NC, be added without 20 μM of A β 42；
Negative control group (siRNA-NC+A β 42)：SiRNA-NC is transfected, while adding 20 μM of A β 42 in culture medium；
CRTAP gene interference group (siRNA-CRTAP+A β 42)：SiRNA-CRTAP is transfected, while adding 20 μ in culture medium
M Aβ42；
Every group of setting three wells.After 37 DEG C are incubated 20h, MTT is then added, 4h is incubated.Lysate is being added, 37 DEG C again
It is incubated at 12h, 600nm and reads OD value.OD value is higher, shows that cell survivaling number is more.
3rd, result
As a result as shown in figure 3, (comparing for convenience, 100%) this OD value organized being set to, the moon with not inducing group to be compared
Property control group (siRNA-NC+A β 42) OD value be 32%, CRTAP gene interference group (siRNA-CRTAP+A β 42) light
Density value is 91%, and difference has statistical significance (P<0.05).Above-mentioned experiment shows that interference CRTAP gene expressions can be short of money
The effect of the anti-neurotoxicities of A β 42.
The CRTAP gene pairs A β of experimental example 4 cause the antagonism of nervous process denaturation
1st, the cell line of stable low-expression CRTAP genes is built
SiRNA1-CRTAP design synthesis shRNA are selected, are inserted into shRNA expression vectors, this work is by Shang Haiji
Agate Pharmaceutical Technology Inc. completes, and is obtained from Shanghai Ji Ma companies and carries out stablizing low table after CRTAP gene shRNA expression vectors
Up to the structure of the cell line of CRTAP genes, construction method is known for those skilled in the art.
2nd, the nerve cell of stable transfection strain SH-SY5Y cultivate in 24 orifice plates, cell density for 0.5 ×
103Cells/ holes, add 10 μM of all-trans retinoic acid, 37 DEG C are cultivated 7 days in culture medium DMEM.Afterwards will
Cell is divided into three groups：
Group is not induced：Stable transfection shRNA-NC plasmids, it is added without 1 μM of A β 42；
Negative control group (shRNA-NC plasmid+A β 42)：Stable transfection shRNA-NC plasmids, while adding 1 μ in culture medium
M Aβ42；
CRTAP gene interference group (shRNA-CRTAP plasmid+A β 42)：Stable transfection shRNA-CRTAP plasmids, are trained simultaneously
Support and 1 μM of A β 42 is added in base；
Every group of setting three wells.It is incubated 5 days at 37 DEG C.Culture medium is removed, 4% paraformaldehyde is then added and fixes cell.With
Anti- Beta-tubulin antibody, carries out routine immunization histochemical staining.Taken pictures under 20 times of mirrors, measure cell process length.
3rd, result
As a result as shown in figure 4, (comparing for convenience with not inducing group to be compared, inducing the cell process average length of group
It is set to 100%), the cell process length of negative control group (shRNA-NC plasmid+A β 42) is 31%, CRTAP gene interference groups
The cell process length of (shRNA-CRTAP plasmid+A β 42) is 89%, and difference has statistical significance (P<0.05).Above-mentioned reality
Test result and show that interference CRTAP gene expressions being capable of nervous process denaturation caused by antagonism A β 42.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Claims (9)
1. the application of people CRTAP genes and its expression product in the product of diagnosis of alzheimer's disease is prepared；Characterized in that,
The coded sequence of people's CRTAP genes is as shown in SEQ ID NO.1.
2. application according to claim 1, it is characterised in that the product includes：By RT-PCR, real-time quantitative PCR,
The expression of immune detection, in situ hybridization or chip detection CRTAP genes and its expression product is with diagnosis of alzheimer's disease
Product.
3. the application of people CRTAP genes and its expression product in the medicine for preparing treatment Alzheimer disease, it is characterised in that
The coded sequence of people's CRTAP genes is as shown in SEQ ID NO.1.
4. a kind of product of diagnosis of alzheimer's disease, it is characterised in that the product can be by detecting CRTAP bases in blood
The expression of cause carrys out diagnosis of alzheimer's disease, and the coded sequence of CRTAP genes is as shown in SEQ IDNO.1.
5. product according to claim 4, it is characterised in that the product includes chip or kit；Wherein, the core
Piece includes genetic chip or protein-chip；The genetic chip includes solid phase carrier and is fixed on the few nucleosides of solid phase carrier
Acid probe, the oligonucleotide probe includes the oligonucleotides for CRTAP genes for being used to detect CRTAP gene transcription levels
Probe；The protein-chip includes solid phase carrier and is fixed on the specific antibody of the CRTAP albumen of solid phase carrier；It is described
Kit includes gene detecting kit and protein immunization detection kit；The gene detecting kit includes being used to detect
The reagent of CRTAP gene transcription levels；The protein immunization detection kit includes the specific antibody of CRTAP albumen.
6. kit according to claim 5, it is characterised in that the reagent include for CRTAP genes primer and/
Or probe.
7. a kind of pharmaceutical composition for being used to treat Alzheimer disease, it is characterised in that described pharmaceutical composition includes CRTAP
The inhibitor of gene and/or the inhibitor of CRTAP gene expression products, the coded sequence such as SEQ ID NO.1 institutes of CRTAP genes
Show.
8. pharmaceutical composition according to claim 7, it is characterised in that the inhibitor is for CRTAP genes
siRNA。
9. application of the inhibitor in the medicine for preparing treatment Alzheimer disease described in claim 7 or 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510463469.3A CN105018484B (en) | 2015-07-31 | 2015-07-31 | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510463469.3A CN105018484B (en) | 2015-07-31 | 2015-07-31 | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105018484A CN105018484A (en) | 2015-11-04 |
| CN105018484B true CN105018484B (en) | 2017-10-13 |
Family
ID=54408787
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510463469.3A Active CN105018484B (en) | 2015-07-31 | 2015-07-31 | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105018484B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048554B (en) * | 2017-12-29 | 2019-03-01 | 北京泱深生物信息技术有限公司 | The molecular marker that THBD gene is diagnosed as parkinsonism |
| CN115078570A (en) * | 2022-05-30 | 2022-09-20 | 郑州大学第一附属医院 | Application of taurin 639 thiocyanate amino acid modification in rapid mass spectrometry to Alzheimer's disease detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101454335A (en) * | 2006-05-19 | 2009-06-10 | 斯克里普斯研究所 | Treatment of protein misfolding |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010057020A2 (en) * | 2008-11-13 | 2010-05-20 | Modgene, Llc | Modification of amyloid-beta load in non-brain tissue |
| WO2011006214A1 (en) * | 2009-07-16 | 2011-01-20 | Peter Maccallum Cancer Institute | Method of detecting radiation exposure and adverse toxicity thereto |
| GB201112246D0 (en) * | 2011-07-15 | 2011-08-31 | Univ Birmingham | Diagnosis of alzheimer's disease |
-
2015
- 2015-07-31 CN CN201510463469.3A patent/CN105018484B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101454335A (en) * | 2006-05-19 | 2009-06-10 | 斯克里普斯研究所 | Treatment of protein misfolding |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105018484A (en) | 2015-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Brennan et al. | Genome-wide microRNA profiling of plasma from three different animal models identifies biomarkers of temporal lobe epilepsy | |
| CN104962658B (en) | The application of MYOZ1 genes and its expression product in Parkinson's diagnosis and treatment | |
| CN105543408B (en) | coronary heart disease early diagnosis marker | |
| JP2013178260A5 (en) | ||
| CN108611413A (en) | A kind of Parkinson's associated biomarkers and its application | |
| CN105567861B (en) | Purposes of the IFI27 as diagnosis of coronary heart disease marker | |
| CN104962657B (en) | Application of the YAP1 genes in Alzheimer disease diagnosis and treatment | |
| KR102242639B1 (en) | Biomaker miRNA4668-5p for diagnosing liver fibrosis | |
| CN104975104B (en) | Alzheimer disease diagnosis and treatment marker and its application | |
| CN105018484B (en) | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target | |
| CN105695622B (en) | Molecular marker of the PILRA genes as Diagnosis of osteoporosis | |
| CN105132550B (en) | Detect application of the reagent of MUC21 gene expressions in preparing osteoarthritis diagnosis and treatment product | |
| CN105400888B (en) | A kind of molecular marker of diagnosis and treatment intracranial aneurysm | |
| CN105400894B (en) | marker for diagnosis and treatment of intervertebral disc degenerative disease | |
| CN105331690B (en) | Application of the EPB41L4B gene in Parkinson's disease diagnosis and treatment | |
| CN108048554B (en) | The molecular marker that THBD gene is diagnosed as parkinsonism | |
| CN105296657B (en) | Intracranial aneurysm diagnosis and treatment marker | |
| TW201204393A (en) | Diagnostic agent and therapeutic agent of cancer | |
| CN105002172B (en) | Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease | |
| CN103083686B (en) | Application of DKK4 gene and coding protein thereof in preparation of medicament | |
| CN106729756A (en) | Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment | |
| CN107881240B (en) | The diagnosis and treatment marker of osteosarcoma | |
| CN102787163B (en) | Kit for detecting c.1671_1673del G mutation of OTOF gene | |
| CN105648103B (en) | Purposes of the VSIG10L gene as lung squamous cancer transfer diagnosis and treatment marker | |
| CN102965447A (en) | Alzheimer's disease (AD) lesion early-stage APOE-4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization screening kit, as well as screening method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: CRTAP gene and expression product thereof capable of serving as target for diagnosing and treating Alzheimer's disease Effective date of registration: 20171212 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2017990001155 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20181206 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2017990001155 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: CRTAP gene and expression product thereof capable of serving as target for diagnosing and treating Alzheimer's disease Effective date of registration: 20181225 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2018990001252 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20191206 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2018990001252 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191225 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Patentee after: Qingdao Yangshen biomedical Co., Ltd Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: Beijing Yang Shen biology information technology company limited |
|
| TR01 | Transfer of patent right |