CN105002172B - Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease - Google Patents
Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease Download PDFInfo
- Publication number
- CN105002172B CN105002172B CN201510463528.7A CN201510463528A CN105002172B CN 105002172 B CN105002172 B CN 105002172B CN 201510463528 A CN201510463528 A CN 201510463528A CN 105002172 B CN105002172 B CN 105002172B
- Authority
- CN
- China
- Prior art keywords
- eapp
- genes
- disease
- alzheimer
- product
- 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
- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 47
- 230000014509 gene expression Effects 0.000 title claims abstract description 38
- 238000003745 diagnosis Methods 0.000 title claims abstract description 25
- 101150010837 EAPP gene Proteins 0.000 title description 23
- 239000003153 chemical reaction reagent Substances 0.000 title description 7
- 101710155837 E2F-associated phosphoprotein Proteins 0.000 claims abstract description 90
- 108090000623 proteins and genes Proteins 0.000 claims description 50
- QQAHEGDXEXIQPR-UHFFFAOYSA-N 2-(ethylamino)-1-phenylpentan-1-one Chemical compound CCCC(NCC)C(=O)C1=CC=CC=C1 QQAHEGDXEXIQPR-UHFFFAOYSA-N 0.000 claims description 40
- 102100032262 E2F-associated phosphoprotein Human genes 0.000 claims description 40
- 102000004169 proteins and genes Human genes 0.000 claims description 32
- 239000003814 drug Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 230000002068 genetic effect Effects 0.000 claims description 7
- 238000011529 RT qPCR Methods 0.000 claims description 6
- 238000009396 hybridization Methods 0.000 claims description 6
- 238000003757 reverse transcription PCR Methods 0.000 claims description 5
- 238000007901 in situ hybridization Methods 0.000 claims description 4
- 238000003499 nucleic acid array Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 201000010099 disease Diseases 0.000 abstract description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 19
- 210000004369 blood Anatomy 0.000 abstract description 11
- 239000008280 blood Substances 0.000 abstract description 11
- 238000002474 experimental method Methods 0.000 abstract description 6
- 230000002265 prevention Effects 0.000 abstract description 3
- 238000002405 diagnostic procedure Methods 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 108020004999 messenger RNA Proteins 0.000 abstract description 2
- 239000003147 molecular marker Substances 0.000 abstract description 2
- 229940000406 drug candidate Drugs 0.000 abstract 1
- 239000002547 new drug Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 25
- 239000000047 product Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 230000006870 function Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 8
- 238000003208 gene overexpression Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000001890 transfection Methods 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
- 241000819233 Tribulus <sea snail> Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 206010025482 malaise Diseases 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 238000007619 statistical method Methods 0.000 description 5
- 229940047183 tribulus Drugs 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000003153 stable transfection Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 230000008485 antagonism Effects 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 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
- 206010029350 Neurotoxicity Diseases 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 206010044221 Toxic encephalopathy Diseases 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 230000002018 overexpression 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
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 206010000117 Abnormal behaviour Diseases 0.000 description 1
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 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
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 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
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010039966 Senile dementia Diseases 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
- 241000700605 Viruses Species 0.000 description 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 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 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
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 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
- 244000309466 calf Species 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000004927 clay Substances 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
- 238000013211 curve analysis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012502 diagnostic product Substances 0.000 description 1
- 239000003085 diluting agent 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
- 230000004064 dysfunction Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000001605 fetal effect Effects 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
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health 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
- 230000008676 import Effects 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
- 239000002502 liposome Substances 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
- 230000003340 mental effect Effects 0.000 description 1
- 238000002156 mixing Methods 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
- 210000005036 nerve Anatomy 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
- 230000000474 nursing effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 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
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 230000005180 public health Effects 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
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000952 spleen Anatomy 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
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000024891 symptom Diseases 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
- 238000003151 transfection method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of molecular marker EAPP genes for alzheimer ''s disease early diagnosis.Experiment is proved, the mRNA level in-site of EAPP genes in patients with Alzheimer disease blood is substantially less than normal population, therefore it may determine that subject whether there is the risk with Alzheimer disease by determining the expression of EAPP genes in subject's blood, or may determine that whether subject suffers from Alzheimer disease.EAPP genes, which can be used for preparing being applied to patients with Alzheimer disease or being applied to, suffers from the high crowd of risk of Alzheimer disease, for the generation for the treatment of Alzheimer disease or prevention Alzheimer disease.The present invention provides new diagnostic method for clinically diagnosis of alzheimer's disease and provides new drug candidate for treatment Alzheimer disease.
Description
Technical field
The present invention relates to biological technical field, diagnosis, treatment more particularly to people EAPP genes in Alzheimer disease
In purposes.
Background technology
Alzheimer disease (Alzheimer ' s disease, AD) is also known as senile dementia, is a kind of severe human's health
Nerve degenerative diseases.Clinic is characterized with progressive failure of memory, cognition dysfunction and abnormal behavior, with encephalatrophy disease
There is senile plaque expelling in contracting, intracerebral and neurofibrillary tangles, neuron loss are its pathological characters.
It is known that various types of dull-witted at least 32 kinds, and wherein AD just account for its incidence of disease 50~60% it
It is many.Found according to epidemiology survey, there is more than 25,000,000 AD patient in the current whole world.U.S. patient AD estimation in 2008 reaches
To 5,200,000, it is AD patient just to have 1 in 65 years old and over-65s (13%) crowd 8, just has within every 71 seconds 1 people to catch AD, arrives
Middle Ages then just have 1 people to turn into AD patient for every 33 seconds, and the direct cost for not being used to treat AD every year including nurse fees exceedes
148000000000 dollars.The elderly population sample for extracting the different living environments in town and country by the strict statistics methods of sampling in China is carried out
Statistical analysis, as a result shows, China AD illness rate is low unlike western countries, belongs to AD severely afflicated areas.AD patient is generally true
After examining, can averagely live 8~10 years, have up to 20 years, therefore the annual cost for being used to treating AD and rehabilitation nursing and to patient
Mental burden that family members bring so that AD has turned into the disaster that 21 century many families have to face, the society thus brought,
Economy, family and social public health problem also become increasingly conspicuous.
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 in early days 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 EAPP 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 EAPP 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 EAPP genes
Primer；The product of the use real-time quantitative PCR diagnosis of alzheimer's disease at least includes a pair of specific amplified EAPP genes
Primer；The product of the use immune detection diagnosis of alzheimer's disease includes：The antibody combined with EAPP protein-specifics；It is described
Included with the product of in situ hybridization diagnosis of alzheimer's disease：With the probe of the nucleic acid array hybridizing of EAPP genes；It is described to use core
The product of piece diagnosis of alzheimer's disease includes：Protein chip and genetic chip；Wherein, protein chip includes special with EAPP albumen
The antibody that the opposite sex is combined, genetic chip includes the probe with the nucleic acid array hybridizing of EAPP genes.
Preferably, the product includes chip, kit.
Present invention also offers application of the people EAPP genes in high-flux sequence platform.With high throughput sequencing technologies
Development, will turn into the structure of the gene expression profile of a people and very easily work.By contrasting Disease and normal person
The gene expression profile of group, 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 EAPP genes falls within the purposes of people's EAPP genes, equally in protection scope of the present invention
Within.
Present invention also offers people EAPP genes and its expression product in the medicine for preparing treatment Alzheimer disease
Using.
Further, the medicine includes：The medicines of the genes of EAPP containing someone, the carrier of carrier's EAPP genes or host are thin
Medicine that born of the same parents are formed, people EAPP pharmaceutical grade proteins or other can promote the medicine of EAPP gene expressions.The medicine of the present invention
It can be used for the missing or deficiency for supplementing endogenic people EAPP albumen, by improving the expression of people's EAPP albumen, so as to treat
The Alzheimer disease caused by people's EAPP hypoproteinosis.
The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid,
Clay, bacteriophage, virus etc..
In the present invention, term " host cell " includes prokaryotic and eukaryotic.Conventional prokaryotic host cell
Example includes Escherichia coli, hay bacillus etc..Conventional eukaryotic host cell includes yeast cells, insect cell and mammal
Cell.It is preferred that the host cell is eukaryotic, such as Chinese hamster ovary celI, COS cells.
Further, medicine of the invention also includes pharmaceutically acceptable carrier, and carrier, this kind of carrier is included (but not
It is limited to)：Diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, bright
Glue and polyvinylpyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium acid carbonate；Sorbefacient quaternary ammonium
Compound；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate
With magnesium, polyethylene glycol etc..
The medicine of the present invention, which imports tissue or the mode of cell, can be divided into external or internal mode.Vitro formats
Including the medicine of the medicine of the genes of EAPP containing someone or the protein of EAPP containing someone is imported in cell, then by cell transplantation or
Feed back in vivo.Internal mode includes directly noting the medicine of the medicine of the genes of EAPP containing someone or the protein of EAPP containing someone
Enter in in-vivo tissue.
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 chip of diagnosis of alzheimer's disease, the chip includes genetic chip, protein
Chip；The genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotides is visited
Pin includes the oligonucleotide probe for EAPP genes for being used to detect EAPP gene transcription levels；The protein-chip includes
Solid phase carrier and be fixed on solid phase carrier EAPP albumen specific antibody.
Further, the genetic chip can be used for multiple genes of the detection including people's EAPP genes (for example, and A Er
The related multiple genes of Ci Haimo diseases) expression.The protein-chip can be used for detection including people's EAPP albumen
Multiple protein (such as Ahl tribulus sea silent sickness related multiple protein) expression.By by multiple and A Erci
The mark of the silent disease in sea is detected simultaneously, is greatly improved the accuracy rate of diagnosis of Alzheimer disease.
The invention provides a kind of kit of diagnosis of alzheimer's disease, the kit includes gene detecting kit
With protein immunization detection kit；The gene detecting kit includes the reagent for being used to detect EAPP gene transcription levels；Institute
Stating protein immunization detection kit includes the specific antibody of EAPP albumen.
Further, the reagent is including the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip method
Reagent needed for during detection EAPP gene expression doses.Preference, the reagent include for EAPP genes primer and/
Or probe.Easily designed according to the nucleotide sequence of EAPP genes can be used for detect EAPP gene expression doses primer and
Probe.
Probe with the nucleic acid array hybridizing of EAPP genes can be that DNA, RNA, DNA-RNA chimera, PNA or other spread out
It is biological.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, is appointed
What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe
Degree can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to hybridization
Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than
30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequences
Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the EAPP albumen includes monoclonal antibody, polyclonal antibody.The EAPP albumen
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 EAPP albumen.Antibody for protein level
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, " EAPP genes " includes any function of people EAPP genes and people's EAPP genes etc.
The polynucleotides of jljl.EAPP genes include and EAPP genes (NC_ in current international public GenBank GeneBank
000014.9) DNA sequence dna has more than 70% homology, and coding identical function protein DNA sequence；
Preferably, the coded sequence of EAPP 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 EAPP genes is the DNA shown in SEQ ID NO.1
Sequence.
In the context of the present invention, EAPP gene expression products include the part of people EAPP albumen and people's EAPP albumen
Peptide.The partial peptide of the EAPP albumen contains the related functional domain of Ahl tribulus sea silent sickness.
" EAPP albumen " includes any function equivalent of people EAPP albumen and people's EAPP albumen.The function equivalent
It is allelic variant, natural prominent including people EAPP albumen conservative variation protein or its active fragment, or its reactive derivative
Variant, induced mutants, can be with the protein coded by the DNA of people EAPP DNA hybridization under high or low stringent condition.
Preferably, EAPP 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 EAPP 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 EAPP albumen
Albumen.Do not limited for the peptide or protein with EAPP protein fusions, as long as the fusion protein of gained retains EAPP albumen
Biological activity.
The EAPP 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 EAPP albumen.It is mutated in such modifying protein
Amino acid number is 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
The alleviation of disease, the complete healing 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 EAPP 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 EAPP genes can just represent the expression of EAPP genes in situ tumor tissue in serum.According to the present invention
Achievement in research understand, it is 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 EAPP genes.
The advantages of the present invention：
Present invention firstly discovers that EAPP gene expressions Ahl tribulus sea silent sickness is related, by detecting in subject's blood
EAPP 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-EAPP genes, compared to traditional detection means, gene diagnosis is more
In time, it is more special, sensitiveer, the early diagnosis of Alzheimer disease can be realized, so as to reduce the death of Alzheimer disease
Rate.
Brief description of the drawings
Fig. 1 displays detect expression of the EAPP genes in Alzheimer disease blood using QPCR；
Fig. 2 displays detect the situation of EAPP gene overexpressions using QPCR；
Fig. 3 shows that EAPP gene expressions cause the influence of nerve cell death to A β；
Fig. 4 shows that EAPP gene expressions cause the influence of nervous process denaturation to A β.
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 within 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 EAPP genes and GAPDH genes in Genbank, work is given birth to by Shanghai
Biotechnology Services Co., Ltd synthesizes.Specific primer sequence is as follows：
EAPP genes：
Forward primer is 5 '-CTCTGAGGATGAAGTGGAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-CGGTAAGACATTCTCTGATG-3 ' (SEQ ID NO.4).
GAPDH genes：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(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 EAPP gene expression amounts contained in Alzheimer patients serum
Significantly reduce, difference has statistical significance (P<0.05).
The EAPP gene expression plasmids of embodiment 2 are built
1st, by human nerve cell strain SH-SY5Y, with DMEM (high sugar) culture medium containing 10% calf serum 37 DEG C, 5%
CO2, relative humidity for 90% incubator in cultivate.Change within 2-3 days liquid 1 time, passed on using 0.25% trypsase conventional digestion.
2nd, the overexpression of EAPP genes
The structure of 2.1 EAPP expression vectors
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of EAPP genes, primer sequence is as follows：Just
It is 5 '-ATGAACCGGCTTCCGGAT-3 ' (SEQ ID NO.7) to primer, reverse primer is 5 '-
TTAGGAATGGCTTGCTAAAAC-3’(SEQ ID NO.8).From cDNA library (the clontech companies, article No. into Human fetal spleen：
638831) coded sequence of the EAPP genes of amplification total length in, above-mentioned cDNA sequence is double through restriction enzyme BamHI and XhoI
It is inserted into the eukaryotic expression vector pcDNA3.1 through restriction enzyme BamHI and XhoI double digestion, connects after digestion
The recombinant vector pcDNA3.1-EAPP of acquisition is used for subsequent experimental.
2.2 transfections
Cell is divided into two groups, respectively control group (transfection pcDNA3.1 empty carriers) and (transfection of EAPP overexpressions group
pcDNA3.1-EAPP).The transfection of carrier is carried out using liposome 2000, the instruction of specific transfection method to specifications is carried out.
PcDNA3.1 empty carriers and pcDNA3.1-EAPP working concentration are 0.5 μ g/ml.
2.3 QPCR are detected
Specific steps be the same as Example 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, difference between the two is examined using t, it is believed that work as P<Have when 0.05
It is statistically significant.
4th, result
As shown in Fig. 2 compared with transfecting the cell of pcDNA3.1 empty carriers, in the cell for transfecting pcDNA3.1-EAPP
EAPP mRNA level in-site is significantly raised, and difference has statistical significance (P<0.05).
The EAPP gene expressions of embodiment 3 cause the antagonism of nerve cell death to A β
1st, cell transfecting：According to embodiment 2 method to nerve cell strain R2L1 carry out pcDNA3.1-EAPP and
PcDNA3.1 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 pcDNA3.1, be added without 20 μM of A β 42；
Negative control group (pcDNA3.1+A β 42)：PcDNA3.1 is transfected, while adding 20 μM of A β 42 in culture medium；
EAPP gene overexpressions group (pcDNA3.1-EAPP+A β 42)：PcDNA3.1-EAPP is transfected, while adding in culture medium
Enter 20 μM of A β 42；
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 (pcDNA3.1+A β 42) OD value be 35%, EAPP gene overexpressions group (pcDNA3.1-EAPP+A β 42)
OD value be 75%, difference has statistical significance (P<0.05).Above-mentioned experiment shows that EAPP gene overexpressions can be short of money
The effect of the anti-neurotoxicities of A β 42.
The EAPP gene expressions of experimental example 4 cause the antagonism of nervous process denaturation to A β
1st, the cell line for building stable high expression EAPP genes is conventionally carried out.
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 pcDNA3.1, it is added without 1 μM of A β 42；
Negative control group (pcDNA3.1+A β 42)：Stable transfection pcDNA3.1, while adding 1 μM of A β 42 in culture medium；
EAPP gene overexpressions group (pcDNA3.1-EAPP+A β 42)：Stable transfection pcDNA3.1-EAPP, while culture medium
1 μM of A β 42 of middle addition；
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 (pcDNA3.1+A β 42) is 30%, EAPP gene overexpression groups
The cell process length of (pcDNA3.1-EAPP+A β 42) is 78%, and difference has statistical significance (P<0.05).Above-mentioned experiment
As a result show that EAPP gene overexpressions 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 for the present invention and its core concept.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 (6)
1. the application of people EAPP genes and its expression product in the product of diagnosis of alzheimer's disease is prepared, it is characterised in that
The coded sequence of EAPP genes is the DNA sequence dna shown in SEQ ID NO.1, and the expression product of EAPP genes is SEQ ID NO.2
Shown amino acid sequence.
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 EAPP genes and its expression product is with diagnosis of alzheimer's disease
Product.
3. application according to claim 2, it is characterised in that the product of the use RT-PCR diagnosis of alzheimer's disease is extremely
Include the primer of a pair of specific amplified EAPP genes less；The product of the use real-time quantitative PCR diagnosis of alzheimer's disease is at least
Include the primer of a pair of specific amplified EAPP genes；The product of the use immune detection diagnosis of alzheimer's disease includes：With
The antibody that EAPP protein-specifics are combined；The product of the use in situ hybridization diagnosis of alzheimer's disease includes：With EAPP genes
Nucleic acid array hybridizing probe；The product of the use chip diagnosis of alzheimer's disease includes：Protein chip and genetic chip；
Wherein, protein chip includes the antibody combined with EAPP protein-specifics, and genetic chip includes the nucleotide sequence with EAPP genes
The probe of hybridization.
4. the application according to any one of claim 1-3, it is characterised in that the product includes chip, kit.
5. the application of people EAPP genes and its expression product in the medicine for preparing treatment Alzheimer disease, it is characterised in that
The coded sequence of EAPP genes is the DNA sequence dna shown in SEQ ID NO.1, and the expression product of EAPP genes is SEQ ID NO.2
Shown amino acid sequence.
6. application according to claim 5, it is characterised in that the medicine includes：The medicine of the genes of EAPP containing someone, take
Medicine that carrier or host cell with people's EAPP genes are formed, people's EAPP pharmaceutical grade proteins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510463528.7A CN105002172B (en) | 2015-07-31 | 2015-07-31 | Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510463528.7A CN105002172B (en) | 2015-07-31 | 2015-07-31 | Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105002172A CN105002172A (en) | 2015-10-28 |
| CN105002172B true CN105002172B (en) | 2017-07-11 |
Family
ID=54375039
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510463528.7A Active CN105002172B (en) | 2015-07-31 | 2015-07-31 | Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105002172B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753954B (en) * | 2018-06-26 | 2022-11-18 | 中南大学湘雅医院 | Capture probe set of dementia-related gene, kit, library construction method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460853A (en) * | 2006-06-08 | 2009-06-17 | 柏林自由大学 | Assay for the diagnosis of alzheimer's disease based on the determination of the ratio of gamma-secretase Abeta cleavage products |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100099609A1 (en) * | 2008-07-28 | 2010-04-22 | Buck Institute For Age Research | eAPP AND DERIVATIVES FOR TREATMENT OF ALZHEIMER'S DISEASE |
-
2015
- 2015-07-31 CN CN201510463528.7A patent/CN105002172B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460853A (en) * | 2006-06-08 | 2009-06-17 | 柏林自由大学 | Assay for the diagnosis of alzheimer's disease based on the determination of the ratio of gamma-secretase Abeta cleavage products |
Non-Patent Citations (5)
| Title |
|---|
| Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer’s disease [amyloid precursor protein/presenilin 1 (PS1)];Noemi Esteras等;《European Journal of Neuroscience》;20120930;第36卷(第5期);第2609-2618页 * |
| AY869694;EBI;《EBI》;20050403;第1-3页 * |
| EAPP modulates the activity of p21 and Chk2;Peter Andorfer等;《Cell Cycle》;20110701;第10卷(第13期);第2077页摘要 * |
| EAPP, a Novel E2F Binding Protein That Modulates E2F-dependent Transcription;Michael Novy等;《Molecular Biology of the Cell》;20050216;第16卷(第5期);第2181-2190页 * |
| RNA-Seq Data Mining: Downregulation of NeuroD6 Serves as a Possible Biomarker for Alzheimer’s Disease Brains;Jun-ichi Satoh等;《Disease Markers》;20141208;第2014卷;第1-10页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105002172A (en) | 2015-10-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104975104B (en) | Alzheimer disease diagnosis and treatment marker and its application | |
| CN104962658B (en) | The application of MYOZ1 genes and its expression product in Parkinson's diagnosis and treatment | |
| CN105112552B (en) | Application of the IFT52 genes in diagnosis of osteoporosis | |
| KR102242639B1 (en) | Biomaker miRNA4668-5p for diagnosing liver fibrosis | |
| CN104962657B (en) | Application of the YAP1 genes in Alzheimer disease diagnosis and treatment | |
| CN105112550B (en) | MTUS1 genes as osteoporosis diagnosis and treatment target | |
| CN105002172B (en) | Detect application of the reagent of EAPP gene expressions in diagnosis and treatment Alzheimer disease | |
| CN105018484B (en) | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target | |
| WO2016173501A1 (en) | Filtering of protein molecular maker dkk-3 related to muscle atrophy, and application thereof | |
| CN110241208A (en) | Application of the TREM2 as the molecular marker of early diagnosis coronary heart disease | |
| CN105238872A (en) | Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma | |
| CN108048554A (en) | The molecular marker that THBD genes are diagnosed as parkinsonism | |
| CN105400888A (en) | Molecular marker for diagnosis and treatment of intracranial aneurysm | |
| CN108034714A (en) | Application of the ARHGAP26 genes in Parkinson's diagnostic tool is prepared | |
| CN105296657B (en) | Intracranial aneurysm diagnosis and treatment marker | |
| CN108929903A (en) | Marker and kit for chronic periodontitis screening, diagnosis, therapeutic evaluation | |
| CN105154540B (en) | The diagnosis and treatment target of PAEP gene and its expression product as osteoarthritis | |
| CN105200151B (en) | Purposes of the TCF21 genes as disease diagnosis marker | |
| CN105087821A (en) | Molecular marker for diagnosis and treatment of osteoporosis | |
| CN105506170B (en) | Purposes of the SAV1 gene as fibroid diagnosis and treatment marker | |
| CN105695621B (en) | Application of the REPS1 genes in diagnosis and treatment osteoarthritis product is prepared | |
| CN1712542B (en) | Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma | |
| CN107904304A (en) | Purposes of the DNASE2 as parkinsonism diagnosis 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 | |
| CN108220420A (en) | A kind of gene marker for being used to diagnose Parkinson |
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: Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment Effective date of registration: 20171212 Granted publication date: 20170711 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: 20170711 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: Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment Effective date of registration: 20181225 Granted publication date: 20170711 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: 20170711 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: 20191226 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 |