CN110376271A - The screening and application thereof of the relevant urine protein marker of Crohn disease - Google Patents
The screening and application thereof of the relevant urine protein marker of Crohn disease Download PDFInfo
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Abstract
The present invention relates to the screenings and application thereof of the relevant urine protein marker of Crohn disease.In particular it relates to which the reagent for establishing the method for the animal model for screening urine protein marker relevant to Crohn disease, the urine protein marker relevant with Crohn disease that the animal model established using the above method is obtained and the detection marker is being prepared for diagnosing the purposes in subject in the kit of Crohn disease.
Description
Technical field
The present invention relates to clinical medicine domains；Specifically the present invention relates to the relevant urine protein marks of people's Crohn disease
Remember object.Specifically, the present invention relates to the use of the rat model of Crohn disease and mass spectral analysis urine protein omics technology obtains
Obtain the purposes of urine protein marker relevant to people's Crohn disease and the urine protein marker.
Background technique
Inflammatory bowel disease is a kind of agnogenic chronic nonspecific bowl inflammatory diseases, clinical manifestation have abdominal pain,
Bloody stool and indigestion etc..Inflammatory bowel disease mainly includes Crohn disease and ulcerative colitis.Epidemiologic data is shown, scorching
Disease property enteropathy is higher in Europe, north America region disease incidence, but is in recent years to increase trend year by year in the disease incidence of China, referring to
Ng SC, Shi HY, Hamidi N, et al. Worldwide incidence and prevalence of inflammatory
bowel disease in the 21st century:A systematic review of population-based
studies.The Lancet,2017；With Kaplan GG, Ng SC.Globalisation of inflammatory bowel
disease:Perspectives from the evolution of inflammatory bowel disease in the
uk and china.The Lancet Gastroenterology&Hepatology,2016,1:307-316。
The diagnosis of inflammatory bowel disease at present is mainly according to clinical manifestation, enteron aisle endoscope, iconography and pathological tissue biopsy
Determined, is diagnosed under the premise of excluding other causes of disease such as infection.Crohn disease is similar with ulcerative colitis clinical manifestation,
But there are great differences in terms of histology and endoscopy, therefore endoscopy joint pathological biopsy is to identify to examine at this stage
It is disconnected, and the important evidence of assessment intestinal inflammatory degree and activity, referring to Tontini GE, Vecchi M, Pastorelli
L, et al. Differential diagnosis in inflammatory bowel disease colitis:State of
the art and future perspectives.World journal of gastroenterology,2015,21:21-
46；Gomollon F, Dignass A, Annese V, et al., third edition european evidence-based consensus
on the diagnosis and management of crohn's disease 2016:Part 1:Diagnosis and
medical management.Journal of Crohn's&colitis,2017,11:3-25；With Dignass A,
Eliakim R, Magro F, et al. Second european evidence-based consensus on the
diagnosis and management of ulcerative colitis part 1:Definitions and
diagnosis.Journal of Crohn's&colitis,2012,6:965-990.But still there is patient's lesion of 5%-15%
It is indefinite only to involve colon antidiastole, therein 80% final development is Crohn disease or ulcerative colitis, referring to Nuij
VJ, Zelinkova Z, Rijk MC, et al. Phenotype of inflammatory bowel disease at
diagnosis in the netherlands:A population-based inception cohort study(the
delta cohort).Inflammatory bowel diseases,2013,19:2215-2222；Burisch J,
Pedersen N, Cukovic-Cavka S, et al. East-west gradient in the incidence of
inflammatory bowel disease in europe:The ecco-epicom inception cohort.Gut,
2014,63:588-597；With Ng SC, Tang W, Ching JY, et al. Incidence and phenotype of
inflammatory bowel disease based on results from the asia-pacific crohn's and
colitis epidemiology study.Gastroenterology,2013,145:158-165e152.And endoscopy
And pathological biopsy have the shortcomings that it is invasive, time-consuming, laborious, can not repeat the operation several times.So needing to find a kind of noninvasive, simple
List, hypersensitivity, high specific biological marker.
Urine is the discharged waste that organism metabolism generates, not by the stringent regulation of homeostasis mechanism, so urine can
With sensitive, reaction disease phase body early variation, i.e. urine is the important next of searching disease marker more better than blood
Source, referring to Gao Y.Urine-an untapped goldmine for biomarker discovery? Sci China
Life Sci,2013,56:1145-1146；With Wu J, Gao Y.Physiological conditions can be
reflected in human urine proteome and metabolome.Expert review of proteomics,
2015,12:623-636；With Li M, Zhao M, Gao Y.Changes of proteins induced by
anticoagulants can be more sensitively detected in urine than in plasma.Sci
China Life Sci,2014,57:649-654.Furthermore also have the advantages that can a large amount of, continuity, non-invasive collection for urine.
According to Urinary Protein Biomarker Database (6/29/2017 updates), there are 924 urine candidate protein labels
Object is reported related to 159 kinds of diseases.Except urologic disease, urine can also reflect each systemic disease state of whole body, for example, brain
Portion's disease, heart disease, metabolic disease.Forgive information abundant in urine, to urinary system, so that entire airframe systems disease
The research of sick marker all has important directive significance.
The inflammatory bowel disease animal model of chemical reagent induction is widely used in laboratory research.Wherein 2,4,6- trinitro-
Benzene sulfonic acid sodium salt (TNBS) is a kind of enteritis animal model of haptens induction, and the immune response of primary activation Th1 type causes inflammatory thin
Born of the same parents' infiltration and inflammatory reaction, referring to Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse
models of acute and chronic intestinal inflammation.Nature protocols,2017,12:
1295-1309；Elson CO1, Beagley KW, Sharmanov AT, et al. Hapten-induced model of
murine inflammatory bowel disease:Mucosa immune responses and protection by
tolerance.J Immunol,1996,157(5):2174-2185.Compare in terms of immunology and histopathology, TNBS is lured
The enteritis animal model and mankind's Crohn disease led are closely similar, referring to Kiesler P, Fuss IJ, Strober
W.Experimental models of inflammatory bowel diseases.Cellular and molecular
gastroenterology and hepatology,2015,1:154-170。
Summary of the invention
It is carried out in this experiment using urine protein group of the protein technique to the TNBS enteritis animal model induced
High-throughput depth identification, excavates the special biomarker of disease in urine.
On the one hand, it establishes the present invention provides a kind of for screening urine protein marker relevant to Crohn disease
The method of animal model, the described method comprises the following steps:
I) TNBS ethanol solution is injected by bowel lavage or is obtained respectively by bowel lavage saline injection with Crohn disease
Rat model and control rats model；
Ii) the urine of the rat model and control rats model with Crohn disease obtained in collection step i)；With
Iii) the rat model and control rats mould with Crohn disease by being collected in mass spectral analysis authentication step ii)
Protein spectrum in type urine.
Further, method according to the present invention, wherein collecting bowel lavage in step ii) injects TNBS ethanol solution
Or pass through the 2nd or 7 day after bowel lavage saline injection rat urine.
Further, method according to the present invention further comprises step iv) comparison step iii) in obtain
Protein spectrum in rat model and control rats model urine with Crohn disease, obtains the protein of differential expression.
On the other hand, the present invention provides it is a kind of by bowel lavage inject that TNBS ethanol solution obtains with Crohn disease
Purposes of the rat model in the animal model that preparation is used to screen urine protein marker relevant to Crohn disease.
Purposes according to the present invention, wherein by identification with the protein in the rat model urine of Crohn disease
Spectrum obtains urine protein marker relevant to Crohn disease.
On the other hand, the present invention provides for detecting one or more urine albumen obtained according to the method for the present invention
The reagent of matter marker is in preparation for diagnosing the purposes in subject in the kit of Crohn disease.
Purposes according to the present invention, wherein the urine that one or more methods according to the present invention obtain
Protein label is selected from ribalgilase pancreas γ type (RNS1G), neutrophil gelatinase-associated lipocalin
(NGAL), Matrix metalloproteinase 8 (MMP8), two-N- acetylcholinesterases (DIAC), fructosediphosphate aldolase B
(ALDOB), glyceraldehyde-3-phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium dependence
Neutral amino acid transporter B (S6A18), dihydrolipoic acid dehydrogenase, mitochondria (DLDH), lysosomal associated membrane glycoprotein 1
(LAMP1), beta-Mannosidase (MANBA), neuroglia former (NPTN), Glutamate-cysteine ligase adjust subunit
(GSH0), gamma glutamyl transpeptidase 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homologue
(CK054), Collectrin (TMM27) and carbonic anhydrase 1 (CAH1).
Purposes according to the present invention, wherein the reagent is used to detect the urine protein spectrum of subject, the urine
Liquid protein spectrum by ribalgilase pancreas γ type (RNS1G), neutrophil gelatinase-associated lipocalin (NGAL),
Matrix metalloproteinase 8 (MMP8), two-N- acetylcholinesterases (DIAC), fructosediphosphate aldolase B (ALDOB), glycerol
Aldehyde -3- phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium dependence neutral amino acid turn
Transport protein B (S6A18), mitochondria dihydrolipoic acid dehydrogenase (DLDH), lysosomal associated membrane glycoprotein 1 (LAMP1), β-sweet dew
Glycosidase (MANBA), neuroglia former (NPTN), Glutamate-cysteine ligase adjust subunit (GSH0), gamma-glutamyl
Transpeptidase 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homologue (CK054),
Collectrin (TMM27) and carbonic anhydrase 1 (CAH1) composition.
Purposes according to the present invention, wherein in urine protein spectrum, compared with the control group, ribalgilase
Pancreas γ type (RNS1G), neutrophil gelatinase-associated lipocalin (NGAL), Matrix metalloproteinase 8 (MMP8),
Two-N- acetylcholinesterases (DIAC), mitochondria dihydrolipoic acid dehydrogenase (DLDH), lysosomal associated membrane glycoprotein 1
(LAMP1), beta-Mannosidase (MANBA) and neuroglia former (NPTN) up-regulation；And fructosediphosphate aldolase B
(ALDOB), glyceraldehyde-3-phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium dependence
Neutral amino acid transporter B (S6A18), Glutamate-cysteine ligase adjust subunit (GSH0), gamma-glutamyl turns peptide
Enzyme 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homologue (CK054), Collectrin
(TMM27) it is lowered with carbonic anhydrase 1 (CAH1).
Purposes according to the present invention, wherein the subject is people.
Detailed description of the invention
Fig. 1 shows the changes of weight of the rat of control rats and TNBS induction.Wherein, p < 0.01 * p < 0.05, * *.
Fig. 2A-Fig. 2 B shows rat colon pathological change.Fig. 2A: it substantially sees；Fig. 2 B:HE dyeing (Con: control group,
TNBS: experimental group).
Specific embodiment
In following experiment using the Crohn disease enteritis animal model of protein technique high flux screening TNBS induction
In urine protein group, find the special urine protein marker of Crohn disease.
1. material and reagent
1) instrument:
Orbitrap Fusion Lumos Tribird mass spectrograph is purchased from Thermo Scientific company；Metabolism of rat
Cage: it is purchased from the good Asource industry in Beijing Science and Technology Ltd..
2) main agents:
Chromatographic mass spectrometry grade water, acetonitrile, formic acid and methanol are bought from Fisher company；Iodacetyl ammonium (IAA), ammonium hydrogen carbonate,
Urea, dithiothreitol (DTT) (DTT), 2,4,6- trinitrobenzene sulfonic acid (TNBS) are bought from Sigma company；Gold pancreatin from
The purchase of Promega company；TMT (Tandem Mass Tags) isotope labeling reagent box is bought from Thermo Pierce company.
3) animal:
Male Wistar rat (weight 180-200g) raises (room purchased from dimension company, tonneau China in feeding standard environment
22 ± 1 DEG C of temperature, humidity 65-70%).
2. experimental method
1) foundation of animal model and urine specimen are collected
It weighs before experiment to Wistar rat, 24 rats is randomly divided into two groups.Experimental group (n=14): rat
Etherization after fasting 24 hours, with polyethylene catheter (being about 15cm, diameter 2mm) by the enteric cavity of the light and slow insertion 8cm of anus
It is interior, the 150 μ l of 50%TNBS ethanol solution of 100mg/kg is quickly injected (in 5s).Anus is clutched after injection, extracts rat tail
It is inverted 3 minutes, comes into full contact with medical fluid with colon.Control group (n=10): being similarly processed with experimental group, perfusion 150 in enteron aisle
μ l physiological saline, rather than TNBS.The weight of experimental group rat and control rats after measurement modeling daily.
Urine specimen is collected: after modeling, being placed in collect in rat fixator by experimental group and control rats within the 2nd, 7 day and be urinated
Liquid.And 3 rats are put to death respectively, it takes colon site formalin to fix, HE dyeing pathological analysis is done after paraffin embedding.
2) acetone precipitation extracts protein in urine
Acetone precipitation urine protein method is referring to the methods of Thongboonkerd, referring to Thongboonkerd V,
Mcleish KR, Arthur JM, et al. Proteomic analysis of normal human urinary proteins
isolated by acetone precipitation or ultracentrifugation.Kidney Int,2002,62:
9.The centrifugation of 8ml urine: 3500g/min, 4 DEG C, 30min take supernatant, 12000g/min, 4 DEG C of centrifugation 30min.Supernatant 5ml is taken, is added
Enter the acetone of 20mL pre-cooling, 4 DEG C of precipitates overnights.12000g/min, 4 DEG C of centrifugation 30min, stay precipitating to be air-dried at room temperature.Cracking is added
About 500 μ l of liquid piping and druming dissolution precipitating, ultrasonic 3min.12000g/min, 4 DEG C of centrifugation 30min, leave and take supernatant, and Bradford method is surveyed
- 80 DEG C of protein concentration save backup.
3) urine protein pancreatin digests
Each sample takes 200 μ g urine proteins, digests by the methods of Wisniewski, referring to Wisniewski JR, Zo μ
Gman A, Nagaraj N, et al. Universal sample preparation method for proteome
analysis.Nat Methods,2009,6:359-363.DTT final concentration 4.5mM, 37 DEG C, 60min reduction is added；IAA is added
Final concentration 10mM, room temperature are protected from light, 30min is alkylated；Pancreatin is added according to mass ratio (urine protein: pancreatin=50:1),
37 DEG C, water-bath, 16 hours.Peptide fragment Oasis HLB pillar desalination is recycled after enzymatic hydrolysis, and is lyophilized into powder, it is standby in -20 DEG C of storages
With.
4) TMT (Tandem Mass Tags) isotope labelling peptide fragment and offline high ph-values reverse-phase chromatography pre-separation polypeptide
TMT labelled reagent (totally 9: 126,127,127N, 128,128N, 129,129N, 130,131) is separately added into 41
μ l acetonitrile, be vortexed concussion 5min, and it is spare to place 10min.Will enzymatic hydrolysis freeze-drying after 9 peptide fragment samples (control group n=3 (1-3),
Second day group n=3 (4-6), the 7th day group n=3 (7-9)), it is dissolved in 100mM triethylamine carbonate (TEAB) solution respectively, is vortexed
Shake 5min.Each sample takes 70 μ g peptide fragments, is added separately in TMT reagent (sample 1,127 is added in 126 labelled reagents to be added
Sample 2, and so on), be vortexed concussion 3min, and room temperature reflects 1h.Drain sample.
Utilize the 400 μ g of mixed polypeptide after high ph-values reversed phase chromatography separation TMT label.A phase (pH 10) 1mM ammonium hydroxide, B phase
(pH 10) 90% acetonitrile, 2mM ammonium hydroxide.60min separates gradient, 0.7ml/min flow velocity.1 part of component is collected per minute, is collected altogether
60 parts.Gradient is as follows:
It is mixed according to the 1st, 21 and 41 part with 0.1% aqueous formic acid sample dissolution after the centrifugation freeze-drying of all components sample
First sample is synthesized, the 2nd, 22 and 42 are mixed into second sample, and 60 component samples are mixed into 20 samples with this rule
Product.To subsequent mass spectral analysis.
5) efficient liquid phase tandem mass spectrometry (LC-MS/MS) is analyzed
A) label-free: by step 3 peptide fragment be dissolved in 0.1% formic acid, each sample takes 500ng peptide fragment to carry out chromatography
It separates (Thermo EASY-nLC 1200): elution time 60min, column flow rate 0.3 μ L/min, gradient 4%-
28% Mobile phase B (+20% water of+79.9% acetonitrile of 0.1% formic acid).Under reversed-phase column (C18, long 15cm, internal diameter 50um) elution
Polypeptide is identified with Orbitrap Fusion Lumos mass spectrograph.Spray voltage 2.1kV, 300 DEG C of ion transfer tube temperature.
Level-one full scan: 350-1550m/z, 120 000 resolution ratio.Second level scanning be data dependence acquisition mode, circulation time 3s, most
High speed mode.Other parameters: HCD fragmentation pattern, 30% fragmentation energies, 30 000 resolution ratio, scanning starting 110m/z.Each
Sample carries out 3 technology repetitive identifieds.
B) TMT label is quantitative: 20 parts of polypeptide samples of the step 4 after high ph-values reverse-phase chromatography pre-separation are dissolved in 0.1%
Formic acid.Each sample about 500ng peptide fragment carries out chromatographic isolation (Thermo EASY-nLC 1200): elution time 60min, color
0.3 μ L/min of column flow rate is composed, gradient is 4%-35% Mobile phase B (+20% water of+79.9% acetonitrile of 0.1% formic acid).Reverse phase
Polypeptide under column (C18, long 15cm, internal diameter 50um) elution is identified with Orbitrap Fusion Lumos mass spectrograph.It is spraying
Voltage 2.1kV, 300 DEG C of ion transfer tube temperature.Level-one full scan: 350-1550m/z, 60 000 resolution ratio；Second level scanning:
HCD fragmentation, 40% fragmentation energies, 60 000 resolution ratio, scanning starting 110m/z.Each sample carries out 2 technology repetitive identifieds.
C) PRM (Parallel Reaction Monitoring) targeting is quantitative:
Establish the acquisition of spectrogram library data: by polypeptide sample (control group n=8, experimental group the after urine specimen to be verified enzymatic hydrolysis
2 days n=11, the 7th day n=11 of experimental group), every group is selected 4 samples at random, and each sample takes the unification mixing of 1 μ g peptide fragment group
Sample.750ng mixing peptide fragment is taken to carry out chromatographic isolation (Thermo EASY-nLC 1200): elution time 90min, chromatographic column stream
0.3 μ L/min of speed, gradient are 4%-28% Mobile phase B (+20% water of+79.9% acetonitrile of 0.1% formic acid).Reversed-phase column
Polypeptide under (C18, long 25cm, internal diameter 50um) elution is identified with Orbitrap Fusion Lumos mass spectrograph.Spraying electricity
Press 2.1kV, 300 DEG C of ion transfer tube temperature.Level-one full scan: 350-1550m/z, 60 000 resolution ratio.Second level scanning is number
According to dependence acquisition mode, circulation time 3s, highest speed mode.Other parameters: HCD fragmentation, 30% fragmentation energies, 30 000 points
Resolution, scanning starting 110m/z.Carry out 6 technology repetitive identifieds.
PRM target data acquisition: by urine specimen to be verified enzymatic hydrolysis after polypeptide sample (control group n=8, experimental group the 2nd day
N=11, the 7th day n=11 of experimental group), each sample takes 750ng peptide fragment to carry out chromatographic isolation (Thermo EASY-nLC respectively
1200): elution time 90min, 0.3 μ L/min of column flow rate, gradient be 4%-28% Mobile phase B (0.1% formic acid+
+ 20% water of 79.9% acetonitrile).Polypeptide Orbitrap Fusion under reversed-phase column (C18, long 25cm, internal diameter 50um) elution
Lumos mass spectrograph is identified.Spray voltage 2.1kV, 300 DEG C of ion transfer tube temperature.Level-one full scan: 350-1550m/z,
60 000 resolution ratio；Second level scanning: isolation window width 2m/z, HCD fragmentation, 30% fragmentation energies, 30 000 resolution ratio.Second level
Targeting quantitative peptide fragment retention time window is 4min, details such as table 1.
6) data are analyzed
A the Orbitrap Fusion Lumos data (raw formatted file) acquired) label-free: are introduced directly into use
Progenesis LC-MS software (version: 4.1).Software automatically selects most suitable data as reference, to other spectra counts
According to the correction for carrying out retention time.Selection band 2+, the parent ion of 3+, 4+ charge number carry out subsequent quantitation.By all general peak datas
Mgf formatted file is exported, is carried out searching library with Mascot software (version 2 .4.0).Database used is Swissprot
Database (data were ended on May 3rd, 2013).Search library condition: digestion mode is pancreatin digestion, allows 2 mistake enzyme sites；
The fixed modification for being modified to cysteine, albumen n end acetylation modification；Parent ion mass deviation is 0.02Da, product ion mass
Deviation 10ppm.After search complete, p value is set as 0.01, Mascot marking and is set as 30, exports xml formatted file, and import
progenesis.Quantitative analysis is carried out based on the peak intensity for identifying polypeptide.
B) TMT label is quantitative: the Orbitrap Fusion Lumos data (raw formatted file) acquired are introduced directly into
Data retrieval and quantitative analysis (TMT in Q Exactive series instrument are carried out with Proteome Discoverer (version 2 .1)
The instrument parameter optimization of paced work process and 2.1 software data processing method of Proteome Discoverer).Data used
Library is Swissprot database (data were ended on May 3rd, 2013).Sequest search condition: pancreatin digestion；Allow
Most 2 leakages enzyme sites；On the fixed ureidomethy for being modified to cysteine, peptide fragment nitrogen end and lysine residue
229.163Da mass increases；Parent ion mass deviation is 10ppm, product ion mass deviation 0.05Da.Quantitative approach selects TMT
It reports ion quantitative approach, exports the intensity of initial report ion.
C) PRM targeting is quantitative:
The data processing of PRM uses Skyline software, referring to MacLean B, Tomazela DM, Shulman N, et al.
Skyline:An open source document editor for creating and analyzing targeted
proteomics experiments.Bioinformatics,2010,26:966-968.It establishes spectrogram library: building library matter for 6
Spectrum initial data, which merges, searches library with PD2.1 software: pancreatin digestion；Allow most 2 leakages enzyme sites；Fixation is modified to cysteine
Ureidomethy, protein nitrogen it is terminated acetylated modification and methionine oxidation be modified to variable modification；Parent ion mass deviation
For 10ppm, product ion mass deviation 0.02Da.Pdresult is imported into Skyline together with original document, protein FDR is set
It is 1%.Screen quantitative peptide fragment: the protein sequence fasta text for 77 differential expressions that discovery phase interested is screened
Part imports Skyline and general picture library, and 11 protein not identified in general picture library are deleted.Retain each protein specific
Peptide fragment at least 2, peptide segment length is 8-18 amino acid, retains the peptide fragment that ureidomethyization modification occurs for cysteine, removes
Signal peptide.Peptide fragment retention time window is set as 4min, by the m/z and retention time window of corresponding 320 peptide fragments of 65 protein
Mouth export is table 1.
Collected PRM initial data is imported into Skyline.Screen quantitative daughter ion: parent ion valence state+2/+3, son from
Sub- valence state+1, daughter ion b, y, p type, fragment ion is from third ion to penultimate ion, fragment ion masses
Deviation 0.02Da.The peak area of manual extraction each peptide secondary fragment ions, and export.It is mentioned with Progenesis QI software
+ 2-+5 ion peak areas in the general figure of level-one is taken, correction PRM targets quantitative all proteins.
Experimental result
1. rat clinical manifestation and changes of weight
Rats in normal control group is active, hair luster, and stool and urine is normal.TNBS induces enteritis group rat in modeling the 2nd day
Appearance activity reduction, crissum are filthy, and excrement quality is abdominal distension, loose stools, and is persistently weighed out to pus and blood stool.TNBS induces enteritis group
Rat body weight was significantly lower than control group (Fig. 1) since the 2nd day.
2. colon pathological analysis result
In the 7th natural gift other places 3 rats of dead experimental group, 3 rats of control group.Colon progress formalin is taken to fix, stone
Wax embedding, HE dye pathological analysis.Control group is substantially seen: intestinal wall thickness is normal, the cleaning of pleat unity and coherence in writing, and mucous membrane is smooth (Fig. 2A).
Histopathology (HE dyeing): enteric epithelium is complete, result is clear, intrinsic once goblet cell is abundant, body of gland queueing discipline, and nothing is filled
Blood, water oedema, no ulcer (Fig. 2 B).Substantially see within experimental group the 7th day: colon part intestinal wall thickens, contrafluxion, it is seen that obvious to burst
Ulcer (Fig. 2A).Histopathology (HE dyeing): colon holostrome generally thickens, and mucous layer missing, goblet cell is lost, and body of gland is broken
It is bad, there are a large amount of center granulocytes, lymphocyte, Eosinophil Infiltration, it is seen that hyperblastosis (Fig. 2 B).
3. urine differential expression protein screens (non-targeted quantitative analysis)
From 3 periods of rat, ((control group refers to the control rats after processing 7 days, i.e. physiology to control group n=3
Rat after saline enema 7 days), the 2nd day n=3 of experimental group, the 7th day n=3) 9 urine specimens.Firstly, by non-marked
Quantitative proteomics analyze and identify.Screening protein condition is level of the FDR less than 1%, and each protein at least 1
The special peptide fragment of item identifies 599 protein altogether.The protein screening criteria of differential expression: protein abundance changes multiple
In 2 times or more, p value is less than 0.05.Secondly, 9 samples are analyzed and identified by TMT label quantitative proteomics.Screen egg
White matter condition is level of the FDR less than 1%, and each protein at least 1 special peptide fragment identifies 1543 eggs altogether
White matter.The protein screening criteria of differential expression: protein abundance changes multiple at 1.2 times or more, includes at least 2 specificity
Peptide fragment, p value is less than 0.05.Corresponding people's homologous protein is converted by rat protein using Uniprot database simultaneously.
Compared with the control group, label-free and the quantitative two methods of label identify altogether 33 differences to 2nd day experimental group
The protein of different expression, there is 16 protein expression up-regulations, and 17 protein expressions are lowered.7th day experimental group and control group phase
Than label-free and the quantitative two methods of label have the protein for identifying 48 differential expressions altogether, there is 20 protein tables
Up to up-regulation, 28 protein expressions are lowered.One shares 77 protein expressions and becomes in experimental group the 2nd day, the 7th day urine
Change, it is verified in more urine specimens using PRM targeting quantitative analysis tech.
4. urine differential protein verifying (PRM targets quantitative analysis)
Quantitative analysis tech is targeted using PRM, the urine protein of 77 differential expressions (is compareed in 30 urine specimens
Group n=8, the 2nd day n=11 of experimental group, the 7th day n=11) in verified, 63 protein can be by accurate quantitative analysis.Wherein,
Protein abundance variation is greater than 1.5 times, and the protein of p < 0.05 shares 30.Compared with the control group, become within experimental group the 7th day
The protein of change has 18, wherein 8 protein expression up-regulations, 10 protein expressions are lowered, such as table 1.This 18 difference tables
The urine protein reached can be used as the urine protein marker of diagnosis Crohn disease.
1. Crohn disease urine protein marker of table
Claims (10)
1. a kind of method for establishing the animal model for screening urine protein marker relevant to Crohn disease, the side
Method the following steps are included:
I) TNBS ethanol solution is injected by bowel lavage or is obtained respectively by bowel lavage saline injection big with Crohn disease
Mouse model and control rats model；
Ii) the urine of the rat model and control rats model with Crohn disease obtained in collection step i)；With
Iii) the rat model and control rats model urine with Crohn disease by being collected in mass spectral analysis authentication step ii)
Protein spectrum in liquid.
2. according to the method described in claim 1, wherein collecting bowel lavage injection TNBS ethanol solution in step ii) or passing through filling
2nd or 7 day rat urine after intestines saline injection.
3. method according to claim 1 or 2 further comprises step iv) comparison step iii) in obtain suffer from
Protein spectrum in the rat model and control rats model urine of Crohn disease, obtains the protein of differential expression.
4. by bowel lavage inject TNBS ethanol solution obtain the rat model with Crohn disease preparation for screen and gram
Purposes in the animal model of the relevant urine protein marker of sieve grace disease.
5. purposes according to claim 4, wherein by identification with the albumen in the rat model urine of Crohn disease
Mass spectrum obtains urine protein marker relevant to Crohn disease.
6. the urine protein label obtained for detecting one or more methods according to any one of claim 1-3
The reagent of object is in preparation for diagnosing the purposes in subject in the kit of Crohn disease.
7. purposes according to claim 6, wherein described one or more according to any one of claim 1-3
It is related that the urine protein marker that method obtains is selected from ribalgilase pancreas γ type (RNS1G), neutrophil leucocyte gelatinase
Lipocalin protein (NGAL), Matrix metalloproteinase 8 (MMP8), two-N- acetylcholinesterases (DIAC), fructose diphosphate aldehyde
Contracting enzyme B (ALDOB), glyceraldehyde-3-phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium
Dependence neutral amino acid transporter B (S6A18), dihydrolipoic acid dehydrogenase, mitochondria (DLDH), lysosomal associated membrane sugar
Albumen 1 (LAMP1), beta-Mannosidase (MANBA), neuroglia former (NPTN), Glutamate-cysteine ligase adjust sub-
Base (GSH0), gamma glutamyl transpeptidase 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homology
Object (CK054), Collectrin (TMM27) and carbonic anhydrase 1 (CAH1).
8. purposes according to claim 6 or 7, wherein the reagent is used to detect the urine protein spectrum of subject, institute
Urine protein spectrum is stated by ribalgilase pancreas γ type (RNS1G), neutrophil gelatinase-associated lipocalin
(NGAL), Matrix metalloproteinase 8 (MMP8), two-N- acetylcholinesterases (DIAC), fructosediphosphate aldolase B
(ALDOB), glyceraldehyde-3-phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium dependence
Neutral amino acid transporter B (S6A18), mitochondria dihydrolipoic acid dehydrogenase (DLDH), lysosomal associated membrane glycoprotein 1
(LAMP1), beta-Mannosidase (MANBA), neuroglia former (NPTN), Glutamate-cysteine ligase adjust subunit
(GSH0), gamma glutamyl transpeptidase 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homologue
(CK054), Collectrin (TMM27) and carbonic anhydrase 1 (CAH1) composition.
9. purposes according to claim 8, wherein in urine protein spectrum, compared with the control group, ribonucleic acid
Enzyme pancreas γ type (RNS1G), neutrophil gelatinase-associated lipocalin (NGAL), Matrix metalloproteinase 8
(MMP8), two-N- acetylcholinesterases (DIAC), mitochondria dihydrolipoic acid dehydrogenase (DLDH), lysosomal associated membrane sugar egg
White 1 (LAMP1), beta-Mannosidase (MANBA) and neuroglia former (NPTN) up-regulation；And fructosediphosphate aldolase B
(ALDOB), glyceraldehyde-3-phosphate dehydrogenase (G3P), neutral and basic amino acid transport albumen Rbat (SLC31), sodium dependence
Neutral amino acid transporter B (S6A18), Glutamate-cysteine ligase adjust subunit (GSH0), gamma-glutamyl turns peptide
Enzyme 1 (GGT1), adenylate kinase isozyme 1 (KAD1), ester hydrolase C11 or f54 homologue (CK054), Collectrin
(TMM27) it is lowered with carbonic anhydrase 1 (CAH1).
10. according to the described in any item purposes of claim 6-9, wherein the subject is people.
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