CN116990395A - 一种基于粪便的阿尔兹海默症生物标志物及其应用 - Google Patents
一种基于粪便的阿尔兹海默症生物标志物及其应用 Download PDFInfo
- Publication number
- CN116990395A CN116990395A CN202210448068.0A CN202210448068A CN116990395A CN 116990395 A CN116990395 A CN 116990395A CN 202210448068 A CN202210448068 A CN 202210448068A CN 116990395 A CN116990395 A CN 116990395A
- Authority
- CN
- China
- Prior art keywords
- alzheimer
- disease
- sample
- biomarker
- early diagnosis
- 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.)
- Pending
Links
- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 90
- 239000000090 biomarker Substances 0.000 title claims abstract description 42
- 210000003608 fece Anatomy 0.000 title abstract description 11
- 238000013399 early diagnosis Methods 0.000 claims abstract description 36
- ZCKYOWGFRHAZIQ-UHFFFAOYSA-N dihydrourocanic acid Chemical compound OC(=O)CCC1=CNC=N1 ZCKYOWGFRHAZIQ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims description 50
- 238000001819 mass spectrum Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 239000012488 sample solution Substances 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 10
- 230000002550 fecal effect Effects 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000007405 data analysis Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000011282 treatment Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- 239000002207 metabolite Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 6
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 3
- 239000004380 Cholic acid Substances 0.000 description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 3
- 229960002471 cholic acid Drugs 0.000 description 3
- 235000019416 cholic acid Nutrition 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- -1 imidazole propionate (Imidazole Propionate) Chemical compound 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000019260 propionic acid Nutrition 0.000 description 3
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 2
- 102000000018 Chemokine CCL2 Human genes 0.000 description 2
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 2
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 2
- 102000003810 Interleukin-18 Human genes 0.000 description 2
- 108090000171 Interleukin-18 Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 description 1
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- KXGVEGMKQFWNSR-OFYXWCICSA-N 3beta,12alpha-dihydroxy-5beta-cholan-24-oic acid Chemical compound C([C@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-OFYXWCICSA-N 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- DGABKXLVXPYZII-UHFFFAOYSA-N Hyodeoxycholic acid Natural products C1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DGABKXLVXPYZII-UHFFFAOYSA-N 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 208000026680 Metabolic Brain disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 206010034719 Personality change Diseases 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229960002997 dehydrocholic acid Drugs 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- DGABKXLVXPYZII-SIBKNCMHSA-N hyodeoxycholic acid Chemical compound C([C@H]1[C@@H](O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DGABKXLVXPYZII-SIBKNCMHSA-N 0.000 description 1
- 206010027175 memory impairment Diseases 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 229940096998 ursolic acid Drugs 0.000 description 1
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
本发明涉及一种基于粪便的阿尔兹海默症生物标志物及其应用。所述阿尔兹海默症生物标志物包括丙酸咪唑。本发明首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本,将其作为阿尔兹海默症生物标志物,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断,有助于无创快速检测,且具备及时、方便、高特异性及高灵敏度的特点。
Description
技术领域
本发明属于生物技术领域,涉及一种基于粪便的阿尔兹海默症生物标志物及其应用。
背景技术
阿尔兹海默症(Alzheimer disease,AD),是一种发生于老年期的进行性发展的中枢神经系统退行性变性疾病,以渐进性记忆障碍及认知功能下降和日常生活能力丧失为特征,伴随人格改变等神经精神症状。由于阿尔兹海默症发病机制尚未完全明确,加上其早期症状比较隐秘,阿尔兹海默症患者容易被漏诊或错诊,因此寻找高灵敏度、高准确度的生物标志物,对于阿尔兹海默症的诊断和药物干预具有重要意义。
目前对于AD的诊断主要依靠记忆量表、PET以及脑脊液、血液中Aβ、磷酸化tau等病理指标的水平检测,然而这些诊断指标在临床中的检测结果仍存在一定争议,而且对于AD发病早期的症状尚缺乏有效的检测证据。
CN106062563A公开了一种用于阿尔兹海默症的早期诊断的生物标志物及方法,所述AD生物标志物是至少四种选自脑源性神经营养因子(BDNF)、胰岛素样生长因子-1(IGF-1)、肿瘤生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)、白介素18(IL-18)和单核细胞趋化蛋白-1(MCP-1)中的生物标志物,通过分析其表达水平,能够辅助AD的早期诊断。
宿主和肠道菌群在代谢食物物质的过程中产生大量的代谢物,肠道菌群的多样性以及丰度的改变会对机体中小分子代谢物的种类和浓度产生重要影响,有数据显示AD患者肠道菌群组成与健康同龄人不同,且越来越多证据表明各种代谢途径的紊乱可能介导AD的病理发生和发展,AD中的菌群失衡可能与机体代谢紊乱的发生有重要关联,而机体外周代谢改变又可能通过血液循环进一步导致中枢神经系统代谢紊乱。
综上所述,筛选新的AD生物标志物,有助于扩充AD早期诊断的判断依据,可与其他标志物检测相互结合,提高AD诊断的准确性,有助于疾病的早期预警、病理分型以及发展阶段的预测评估等。
发明内容
针对现有技术的不足和实际需求,本发明提供一种基于粪便的阿尔兹海默症生物标志物及其应用,本发明基于靶向代谢组学分析技术对人粪便代谢物进行定性定量分析,将粪便中丙酸咪唑作为阿尔兹海默症标志物,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断,且具备及时、方便、无创、高特异性及高灵敏度的特点。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种基于粪便的阿尔兹海默症生物标志物,所述阿尔兹海默症生物标志物包括丙酸咪唑(Imidazole Propionate)。
本发明基于靶向代谢组学分析技术对粪便代谢物进行定性定量分析,检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本,将其作为阿尔兹海默症生物标志物,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断。
第二方面,本发明提供如第一方面所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
第三方面,本发明提供一种阿尔兹海默症早期诊断模型,所述阿尔兹海默症早期诊断模型的输入变量包括第一方面所述的阿尔兹海默症生物标志物的质谱峰强度值。
优选地,所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数,所述差异表达倍数的计算公式如方程式(1)所示:
优选地,阿尔兹海默症阳性的判断标准为所述差异表达倍数≥1.66。
本发明中,通过对正常粪便样本和AD粪便样本中阿尔兹海默症生物标志物的质谱峰强度值进行充分对比分析,并进行理性设计,构建了一种阿尔兹海默症早期诊断模型,所述模型以阿尔兹海默症生物标志物的质谱峰强度值为输入变量,以差异表达倍数为输出变量,能够快速输出结果,且充分表征阿尔兹海默症生物标志物水平异常的样本,从而辅助阿尔兹海默症早期诊断。
第四方面,本发明提供一种阿尔兹海默症早期诊断装置,所述装置包括如下单元:
样本配制单元,用于执行包括以下步骤:
用于将待测样本配制成可用于液相色谱仪分离的待测样本溶液;
检测单元,用于执行包括以下步骤:
利用所述液相色谱仪分离所述待测样本溶液,利用质谱仪对分离后样本进行检测,进行数据处理,测定样本中第一方面所述的阿尔兹海默症生物标志物的质谱峰强度值;
分析单元,用于执行包括以下步骤:
将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行数据分析,输出样本对应的差异表达倍数,并判断是否为阿尔兹海默症阳性。
本发明的阿尔兹海默症早期诊断装置中,各单元间有效配合,简单高效,能够快速完成样本处理、检测及获得差异表达倍数,同时以经过合理设计的判断标准进行阿尔兹海默症阳性评估,对于阿尔兹海默症早期诊断具有重要意义。
优选地,所述待测样本包括粪便样本。
优选地,所述待测样本溶液的配制方法包括将待测样本加入甲醇-乙腈水溶液中,离心并收集上清液,得到所述待测样本溶液。
优选地,所述待测样本溶液的配制方法包括以下步骤:
(1)取待测样本加入预冷甲醇/乙腈/水溶液中,混合并超声25~35min(例如可以是26min、27min、28min、29min或32min),置于-20~-15℃(例如可以是-19℃、-18℃、-16℃或-17℃)静置5~15min(例如可以是6min、7min、8min、9min、10min、12min或14min),于0~4℃(例如可以是1℃、2℃或3℃)、12000~16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心15~25min(例如可以是16min、17min、18min、19min、20min、21min、22min、23min或24min),取上清进行真空干燥,得到预处理样本;
(2)将所述预处理样本加入80~120μL乙腈水溶液中复溶,涡旋,于0~4℃、12000~16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心10~20min(例如可以是11min、12min、13min、14min、15min、16min、17min、18min或19min),取上清液,得到所述待测样本溶液。
优选地,所述甲醇/乙腈/水溶液中甲醇、乙腈和水的体积比为(1~2):(1~2):1包括但不限于1.2:2:1、1.2:1:1、2:2:1、1.4:1.5:1、1.6:1.2:1、1.8:2:1、1.9:1.8:1或1.1:1.4:1。
优选地,所述乙腈水溶液中乙腈和水的体积比为(1~2):1,包括但不限于1.1:1、1.2:1、1.3:1、1.5:1、1.6:1、1.7:1、1.8:1或1.9:1。
优选地,所述液相色谱仪包括超高效液相色谱仪。
优选地,所述超高效液相色谱仪包括Agilent 1290Infinity LC超高效液相色谱仪。
优选地,所述质谱仪包括三重四级杆质谱仪。
优选地,所述三重四级杆质谱仪包括AB 5500/6500Q-trap质谱仪(AB SCIEX)。
优选地,所述数据处理包括:
使用MultiQuant软件对MRM原始数据进行峰提取,计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值,作为质谱峰强度值。
作为优选的技术方案,所述阿尔兹海默症早期诊断装置包括如下单元:
样本配制单元,用于执行包括以下步骤:
将待测样本配制成可用于液相色谱仪分离的待测样本溶液;
检测单元,用于执行包括以下步骤:
利用所述液相色谱仪分离所述待测样本溶液,利用质谱仪对分离后样本进行检测,使用MultiQuant软件对MRM原始数据进行峰提取,计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值,作为质谱峰强度值;
分析单元,用于执行包括以下步骤:
将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析,输出样本对应的差异表达倍数,并判断是否为阿尔兹海默症阳性。
本发明中,对粪便样本中阿尔兹海默症生物标志物水平进行检测,可以作为一种诊断依据,与其他检测结果结合,辅助阿尔兹海默症早期诊断,预期可以提高阿尔兹海默症诊断的准确性,但并不单独作为能够100%诊断阿尔兹海默症的诊断指标。
第五方面,本发明提供第一方面所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
即以第一方面所述的阿尔兹海默症生物标志物作为靶点筛选治疗和/或预防阿尔兹海默症的药物。
与现有技术相比,本发明具有以下有益效果:
本发明首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本,将其作为阿尔兹海默症生物标志物,并提供阿尔兹海默症早期诊断模型和装置,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断,有助于无创快速检测,且具备及时、方便、高特异性及高灵敏度的特点。
附图说明
图1为AD模型小鼠和野生型小鼠的粪便样本中丙酸咪唑水平图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
本发明实施例中实验仪器和试剂包括:
AB 5500/6500Q-trap质谱仪(AB SCIEX);
Agilent 1290Infinity LC超高压液相色谱仪(Agilent);
低温高速离心机(Eppendorf5430R);
色谱柱:Waters,ACQUITY UPLC BEH Amide 1.7μm,2.1mm×100mmcolumn;Waters,ACQUITY UPLC BEH C181.7μm,2.1mm×100mm column;
乙腈(Merck,1499230-935);
乙酸铵(Sigma,70221);
甲醇(Fisher,A456-4);
氨水(Sigma,221228);
甲酸铵(Sigma,70221);
甲酸(Sigma,00940);
同位素标准品(Cambridge Isotope Laboratories)。
实施例1
本实施例对9月龄AD模型小鼠(APP/PS1转基因小鼠,由南京大学模式动物研究所提供)与野生型(WT)小鼠的粪便样本进行代谢物定性定量分析。
分别采集在相同条件下培养的(小鼠饲养在无特定病原体的环境中,12小时光照/黑暗循环,温度保持在24℃,提供灭菌饮用水和标准饲料)10只AD模型小鼠和9只野生型小鼠的粪便,采用超高效液相色谱-质谱联用分别检测粪便样本中的β-鼠胆酸、牛磺-β-鼠胆酸、熊果酸、7-脱氢胆酸、脱氧胆酸、甘氨石胆酸-3-硫酸盐、异脱氧胆酸、猪去氧胆酸和ω-鼠胆酸水平,具体方法包括:
(1)取粪便样本加入预冷甲醇/乙腈/水溶液(体积比为2:2:1),涡旋混合,低温超声30min,置于-20℃静置10min,于4℃、14000×g离心20min,取上清进行真空干燥,得到预处理样本;
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水=1:1)中复溶,涡旋,于4℃、14000×g离心15min,取上清液,以备进样分析;
(3)采用Agilent 1290Infinity LC超高效液相色谱系统(UHPLC)依次经HILIC和C18色谱柱进行分离,HILIC色谱柱柱温25℃;流速0.3mL/min;进样量2μL;流动相组成包括:A相:按体积比将90%水混合10%乙腈并加入终浓度为2mM(mmol/L)的甲酸铵,B相:甲醇中加入0.4%(体积百分比)甲酸;梯度洗脱程序如下:0~1.0min,85%B相;1.0~3min,B相从85%线性变化至80%;3~4min,80%B相;4~6min,B相从80%线性变化至70%;6~10min,B从70%线性变化至50%;10~12.5min,B相维持50%;12.5~12.6min,B相从50%线性变化至85%;12.6~18min,B相维持在85%;
C18色谱柱柱温40℃,流速0.4mL/min,进样量2μL;流动相组成A相:水中加入终浓度为5mM的乙酸铵和0.2%(体积百分比)氨水,B相:按体积比混合99.5%乙腈和0.5%氨水;梯度洗脱程序如下:0~5min,B相从5%线性变化至60%;5~11min,B相从60%线性变化至100%;11~13min,B相维持在100%;13~13.1min,B相从100%线性变化至5%;13.1~16min,B相维持在5%;整个分析过程中样本置于4℃自动进样器中;样本队列中插入同位素标准品(来自于Cambridge Isotope Laboratories),用于监测和评价系统的稳定性及实验数据的可靠性;
(5)采用AB 6500QTRAP质谱仪(AB SCIEX)对步骤(4)超高效液相色谱系分离后的样本质谱分析,ESI源条件如下sheath gas temperature,350℃;dry gas temperature,350℃;sheath gas flow,11L/min;dry gas flow,10L/min;capillary voltage,4000Vor-3500V in positive or negative modes,respectively;nozzle voltage,500V;andnebulizerpressure,30psi,采用MRM模式监测,使用MultiQuant软件对MRM原始数据进行峰提取,得到各物质的峰面积和相对应的同位素标准品内标峰面积的比值,作为这些代谢物归一化后的峰强度值,以峰强度平均值作图,结果如图1所示,随后,基于各个样本归一化后的代谢物峰强度值,使用OPLS-DA分析去检测不同组别代谢物组成的差异,并筛选变量重要性(VIP)大于1的代谢物,然后,使用秩和检验对不同组别间的代谢物进行差异分析,获得p值小于0.05的差异代谢物,最后将筛选VIP>1和p<0.05的组间差异代谢物作为阿尔兹海默症生物标志物。
结果如图1及表1所示,AD模型小鼠粪便样本中丙酸咪唑水平显著高于野生型小鼠,表明可将粪便中丙酸咪唑作为阿尔兹海默症生物标志物,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断。
表1
注:**为p<0.01。
实施例2
本实施例对实施例中阿尔兹海默症生物标志物丙酸咪唑进行分析,由组氨酸产生的丙酸咪唑(imidazole propionate),是一种重要的微生物代谢物,会促进胰岛素抵抗的发展并最终导致2型糖尿病(T2DM),这些结果提示粪便代谢物水平变化可能反映AD相关代谢通路的异常,对临床早期诊断具有重要意义。
综上所述,本发明首次检测到阿尔兹海默症粪便样本中丙酸咪唑水平显著高于正常粪便样本,将粪便中丙酸咪唑作为阿尔兹海默症生物标志物,并提供阿尔兹海默症早期诊断模型和装置,通过检测粪便中丙酸咪唑水平能够辅助阿尔兹海默症早期诊断,有助于无创快速检测,且具备及时、方便、高特异性及高灵敏度的特点。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (10)
1.一种基于粪便的阿尔兹海默症生物标志物,其特征在于,所述阿尔兹海默症生物标志物包括丙酸咪唑。
2.如权利要求1所述的基于粪便的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
3.一种阿尔兹海默症早期诊断模型,其特征在于,所述阿尔兹海默症早期诊断模型的输入变量包括权利要求1所述的阿尔兹海默症生物标志物的质谱峰强度值;
所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数。
4.根据权利要求3所述的阿尔兹海默症早期诊断模型,其特征在于,所述差异表达倍数的计算公式如方程式(1)所示:
5.根据权利要求4所述的阿尔兹海默症早期诊断模型,其特征在于,阿尔兹海默症阳性的判断标准为所述差异表达倍数≥1.66。
6.一种阿尔兹海默症早期诊断装置,其特征在于,所述装置包括如下单元:
样本配制单元,用于执行包括以下步骤:
用于将待测样本配制成可用于液相色谱仪分离的待测样本溶液;
检测单元,用于执行包括以下步骤:
利用所述液相色谱仪分离所述待测样本溶液,利用质谱仪对分离后样本进行检测,进行数据处理,测定样本中权利要求1所述的阿尔兹海默症生物标志物的质谱峰强度值;
分析单元,用于执行包括以下步骤:
将检测到的阿尔兹海默症生物标志物质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析,输出样本对应的差异表达倍数,并判断是否为阿尔兹海默症阳性。
7.根据权利要求6所述的装置,其特征在于,所述待测样本包括粪便样本。
8.根据权利要求6或7所述的装置,其特征在于,所述数据处理包括:
使用MultiQuant软件对MRM原始数据进行峰提取,计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值,作为质谱峰强度值。
9.根据权利要求6-8任一项所述的装置,其特征在于,所述装置包括如下单元:
样本配制单元,用于执行包括以下步骤:
将待测样本配制成可用于液相色谱仪分离的待测样本溶液;
检测单元,用于执行包括以下步骤:
利用所述液相色谱仪分离所述待测样本溶液,利用质谱仪对分离后样本进行检测,使用MultiQuant软件对MRM原始数据进行峰提取,计算阿尔兹海默症生物标志物的峰面积和内标峰面积的比值,作为质谱峰强度值;
分析单元,用于执行包括以下步骤:
将检测到的阿尔兹海默症生物标志物的质谱峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行数据分析,输出样本对应的差异表达倍数,并判断是否为阿尔兹海默症阳性。
10.权利要求1所述的基于粪便的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210448068.0A CN116990395A (zh) | 2022-04-26 | 2022-04-26 | 一种基于粪便的阿尔兹海默症生物标志物及其应用 |
PCT/CN2022/098168 WO2023206739A1 (zh) | 2022-04-26 | 2022-06-10 | 一种基于粪便的阿尔兹海默症生物标志物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210448068.0A CN116990395A (zh) | 2022-04-26 | 2022-04-26 | 一种基于粪便的阿尔兹海默症生物标志物及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116990395A true CN116990395A (zh) | 2023-11-03 |
Family
ID=88517127
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210448068.0A Pending CN116990395A (zh) | 2022-04-26 | 2022-04-26 | 一种基于粪便的阿尔兹海默症生物标志物及其应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN116990395A (zh) |
WO (1) | WO2023206739A1 (zh) |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6679309B2 (ja) * | 2012-08-29 | 2020-04-15 | カリフォルニア インスティチュート オブ テクノロジー | 自閉症スペクトラム障害の診断および処置 |
GB2511525A (en) * | 2013-03-05 | 2014-09-10 | Randox Teoranta | Methods and Compositions for the Diagnosis of Alzheimer's Disease |
US20140357525A1 (en) * | 2013-03-26 | 2014-12-04 | Duke University | Markers for alzheimer's disease and mild cognitive impairment and methods of using the same |
GB201308077D0 (en) * | 2013-05-03 | 2013-06-12 | Univ Nottingham Trent | Biomarkers |
EP2899543A1 (en) * | 2014-01-28 | 2015-07-29 | Predemtec GmbH | Biomarker and methods for early diagnosis of Alzheimer's disease |
CN106501409B (zh) * | 2016-10-26 | 2019-09-24 | 王喜军 | 一种基于老年痴呆症的尿液代谢标志物鉴定方法 |
WO2018157014A1 (en) * | 2017-02-24 | 2018-08-30 | Duke University | Metabolic biomarkers for the identification and characterization of alzheimer's disease |
JP6978122B2 (ja) * | 2017-03-31 | 2021-12-08 | アキシャル セラピューティクス,インク. | 自閉症および関連障害の治療および予防のための腸管選択的隔離剤 |
CN109576362B (zh) * | 2018-12-28 | 2020-09-04 | 青岛泱深生物医药有限公司 | 阿尔茨海默诊治用标志物fam170a |
CN109709235A (zh) * | 2019-02-25 | 2019-05-03 | 马红华 | 阿尔茨海默病或轻度老年认知障碍的早期诊断、预测生物标志物组合、应用及其测定方法 |
US20220163538A1 (en) * | 2019-04-05 | 2022-05-26 | Arizona Board Of Regents On Behalf Of Arizona State University | Metabolites as diagnostics for autism spectrum disorder in children with gastrointestinal symptoms |
WO2021083977A1 (en) * | 2019-10-28 | 2021-05-06 | Agent | Biomarkers and uses thereof for diagnosing the silent phase of alzheimer's disease |
CN112852916A (zh) * | 2021-02-19 | 2021-05-28 | 王普清 | 肠道微生态的标志物组合、辅助诊断模型及其应用 |
-
2022
- 2022-04-26 CN CN202210448068.0A patent/CN116990395A/zh active Pending
- 2022-06-10 WO PCT/CN2022/098168 patent/WO2023206739A1/zh unknown
Also Published As
Publication number | Publication date |
---|---|
WO2023206739A1 (zh) | 2023-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Matysik et al. | Metabolomics of fecal samples: a practical consideration | |
Petrick et al. | An untargeted metabolomics method for archived newborn dried blood spots in epidemiologic studies | |
Ryan et al. | Recent and potential developments in the analysis of urine: a review | |
JP6082328B2 (ja) | 胆汁酸同時分析方法 | |
EP2284540A1 (en) | Method of diagnosing organ failure | |
JP6199952B2 (ja) | 健康的な老化のためのバイオマーカーとしてのフェニルアセチルグルタミン | |
Courant et al. | Metabolomics as a potential new approach for investigating human reproductive disorders | |
WO2011157655A1 (en) | Use of bile acids for prediction of an onset of sepsis | |
JP6170128B2 (ja) | 健康的な老化のためのバイオマーカーとしてのp−クレゾール硫酸塩 | |
CN110220987B (zh) | 胆汁酸联合标志物在制备用于预测或诊断糖尿病的检测试剂或检测物的用途 | |
CN109507337A (zh) | 一种基于血尿中代谢产物预测甘地胶囊治疗糖尿病肾病机制的新方法 | |
JP6208736B2 (ja) | 健康的な老化に関するバイオマーカーとしての1−o−アルキル−2−アシルグリセロホスホコリン(pc−o)40:1 | |
Liang et al. | The fecal metabolome is associated with gestational diabetes mellitus | |
Chowdhury et al. | NMR-based metabolomics as a significant tool for human nutritional research and health applications | |
CN112748249A (zh) | 新生儿胆道闭锁诊断标志物的应用 | |
CN116990395A (zh) | 一种基于粪便的阿尔兹海默症生物标志物及其应用 | |
CN117030893A (zh) | 长链脂肪酸分类标志物组合在制备诊断糖尿病的检测产品中的应用 | |
CN116990396A (zh) | 一种阿尔兹海默症生物标志物及其应用 | |
WO2023035465A1 (zh) | 一种阿尔兹海默症生物标志物牛磺酸及其应用 | |
CN112180013B (zh) | 用于心肌梗死诊断的肠道微生物代谢标志物组合物及其检测方法和应用 | |
WO2023035468A1 (zh) | 一种阿尔兹海默症生物标志物l-缬氨酸及其应用 | |
CN109596748B (zh) | 一种检测甘地胶囊治疗糖尿病肾病患者代谢产物的方法 | |
Laurent et al. | A targeted UHPLC-MS/MS method to monitor lipidomic changes during a physical effort: optimization and application to blood microsamples from athletes | |
WO2023035467A1 (zh) | 一种阿尔兹海默症生物标志物肌苷及其应用 | |
CN112946150A (zh) | 一种快速检测人体尿液中高香草酸、香草扁桃酸、5-羟基吲哚乙酸及肌酐的方法及试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |