CN107817349A - Urine protein marker of chronic pancreatitis and application thereof - Google Patents

Abstract

The application is related to urine protein marker of chronic pancreatitis and application thereof.Specifically, the application relates to the use of the purposes of Urine proteins mark that chronic pancreatitis disease model and mass spectral analysis obtain in the early diagnosis of people's chronic pancreatitis disease, course of disease monitoring, and the Urine proteins mark is selected from the chain C areas of Ig γ 1, core connects albumen 2, the microglobulins of β 2, IL-4 acceptor alpha subunit, phosphoglycerate mutase 1, dipeptidyl peptidase 2, secreting type pyrophosphoric acid protein 24, glycan core albumen, endothelial cell-selective adhesion molecule, nestin 2, serum amyloid sample p materials, nerve cell adhesion molecule 1 etc..In the application, differential protein early diagnoses for chronic pancreatitis in the urine of acquisition and successive treatment provides a kind of noninvasive and easy selection.

Description

Urine protein markers for chronic pancreatitis and their use

technical field

This application relates to clinical medicine, in particular to protein markers related to human chronic pancreatitis. Specifically, the present application relates to the use of protein markers related to the diagnosis and condition monitoring of human chronic pancreatitis obtained by using the rat chronic pancreatitis disease model and mass spectrometry analysis.

Background technique

Biomarkers are monitorable changes associated with physiological or pathological processes. The study of disease markers has always been a research focus and difficulty in recent years. Appropriate markers mean that they can indicate the occurrence and development of the disease earlier and more sensitively, and provide accurate information for the diagnosis, treatment and prognosis of the disease, which is more beneficial to the health of the patient.

The essence of biomarkers is change. Urine, as the excrement excreted by the body through the urinary system and urinary tract, enriches the changes in the whole body, is sensitive, and is not regulated by the homeostatic mechanism. Therefore, it changes early and can reflect the early changes of various diseases. These changes are potential disease markers (see Youhe G. Can urine be the gold mine for biomarker discovery? Sci China Life Sci 2013; 43). Urinary proteomics, as a novel, non-invasive analysis tool, can mine early disease-specific biomarkers in urine. Currently, more than 6,000 proteins can be identified in the depth of urinary proteomic analysis (Zhao, M et al. A comprehensive urinary proteome analysis: potential applications in disease biomarker discovery and validation. The Lancet, 2015.386: p. S63). It has great clinical application prospects in the research of disease markers.

Chronic pancreatitis (CP) is a chronic inflammatory disease that causes irreversible changes in pancreatic tissue and function due to various etiologies. Its clinical manifestation is mainly abdominal pain, and its pathological manifestations are mainly chronic inflammatory damage of pancreatic parenchyma and interstitial fibrosis (Tetsuhide Ito et al., Evidence-based clinical practice guidelines for chronic pancreatitis. 2015[J]. J Gastroenterol, 2016).

In recent years, the incidence rate has increased year by year, the etiology is complicated, and the disease is protracted, which seriously affects the quality of life of patients. At present, the clinical diagnosis of this disease, especially the early diagnosis, still faces many challenges. The diagnostic methods of chronic pancreatitis mainly include imaging examination, pancreatic function test and pancreatic biopsy. Imaging information cannot provide valuable diagnostic clues at an earlier stage of the disease. Pancreatic function tests have low sensitivity. Pancreatic biopsy is a definitive standard for the diagnosis of CP, but biopsy itself has certain defects. Therefore, there is an urgent need to develop noninvasive biomarkers.

Contents of the invention

In view of the above needs in the art, according to some embodiments of the present disclosure, a use of a protein identification reagent in the preparation of a reagent for diagnosing chronic pancreatitis is provided, wherein the identification reagent is specific to any one of the following or its combination:

CD48 antigen, Igγ-1 chain C region, L-lactate dehydrogenase B chain, Na(+)/H(+) exchange regulator nhe-rf3, Thy-1 membrane glycoprotein, T-kininogen 2, V Proton-type ATPase subunit S1, α2-mercaptoglycoprotein, α-2-mercaptoglycoprotein, β-2-microglobulin, aminoacylase-1A, interleukin-4 receptor α subunit, cysteine Protease Inhibitor-B, Nestin-2, Amyloid-βA4 Protein, Polymeric Immunoglobulin Receptor, Dipeptidyl Peptidase 2, Sortilin, Secreted Pyrophosphoprotein 24, Cadherin-2, Glutamine Aminopeptidase, glutamylcysteine synthetase catalytic subunit (GCLC), nucleocin-2, aflatoxin B1 aldehyde reductase 3, actin cytoplasmic 1, matrix-remodeling-associated Protein 8, methyldopa Aβ subunit, collagen C-endopeptidase enhancer 1, glycan protein-3, glycan core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate translocation Enzyme 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chloride intracellular channel protein 1, sodium-dependent neutral amino acid carrier B(0)AT3, endothelial cell selectivity Adhesion molecule, prothrombin, prostatic gland kallikrein-6, mitochondrial kynurenine/α-aminooxalate transaminase, cartilage oligomer matrix protein, neural cell adhesion molecule 1, growth inhibitory protein 6, double sugar chain Proteoglycan, carbonic anhydrase 1, sialic acid O-acetylesterase, fibronectin, serum amyloid substance p, alcohol dehydrogenase (coenzyme II(+)), neutral and basic amino acid transport protein rBAT, tissue alpha - Levofucosidase.

In a particular embodiment, a decreased expression level of a protein selected from the group consisting of CD48 antigen, L-lactate dehydrogenase B chain, Na(+)/ H(+) exchange regulator nhe-rf3, Thy-1 membrane glycoprotein, V-type proton ATPase subunit S1, α-2-thiol glycoprotein, aminoacylase-1A, interleukin-4 receptor α subunit , cystatin-B, nestin-2, amyloid-βA4 protein, dipeptidyl peptidase 2, sortilin, secreted pyrophosphoprotein 24, cadherin-2, glutaminyl peptide Enzyme, Glutamylcysteine Synthetase Catalytic Subunit, Aflatoxin B1 Aldehyde Reductase 3, Actin Cytoplasm 1, Matrix-Remodeling-Associated Protein 8, Methyldopa Aβ Subunit, Collagen C-endopeptidase enhancer 1, glycan protein-3, glycan core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phospholysine phosphohistidine sub Phosphate inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chloride intracellular channel protein 1, sodium-dependent neutral amino acid carrier B(0)AT3, endothelial cell selective adhesion molecule, prothrombin, mitochondrial kynurenine /α-aminooxalate transaminase, cartilage oligomeric matrix protein, neural cell adhesion molecule 1, growth inhibitory protein 6, biglycan, sialic acid O-acetylesterase, fibronectin, serum amyloid substance p, Alcohol dehydrogenase (coenzyme II(+)), neutral and basic amino acid transport protein rBAT, tissue α-L-fucosidase, and combinations thereof.

In a specific embodiment, an increased expression level of a protein selected from the group consisting of Igγ-1 chain C region, T-kininogen 2, β-2, as compared to healthy controls indicates that the subject has chronic pancreatitis - Microglobulin, Polymeric Immunoglobulin Receptor, Nucleonectin-2, Prostate Kallikrein-6, Carbonic Anhydrase 1 and combinations thereof.

In specific embodiments, healthy controls refer to individuals who do not suffer from chronic pancreatitis.

In another embodiment, use of an identification reagent selected from the following proteins in the preparation of a reagent for the early diagnosis of chronic pancreatitis in a subject is provided, wherein the identification reagent is specific to any one of the following or Its combination: Ig gamma-1 chain C region, nucleonectin-2, beta-2-microglobulin, interleukin-4 receptor alpha subunit, phosphoglycerate mutase 1, dipeptidyl peptidase 2, secretory Pyrophosphoprotein type 24, glycan core protein, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, neuronal cell adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphate Enzymes, cartilage oligomeric matrix proteins and combinations thereof.

In a specific embodiment, an increase in the expression level of a protein selected from the group consisting of the following early damage to chronic pancreatitis, as compared to the protein level in a healthy control: Igγ-1 chain C region, nucleonectin -2 and beta-2-microglobulin.

In a specific embodiment, a reduced expression level of a protein selected from the group consisting of interleukin-4 receptor alpha subunit, phosphoglycerate mutase, as compared to healthy controls indicates that the subject has chronic pancreatitis 1. Dipeptidyl peptidase 2, secreted pyrophosphoprotein 24, glycan core protein, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, nerve cell adhesion molecule 1, phospholysine Phosphohistidinephosphite inorganic pyrophosphate phosphatases, cartilage oligomer matrix proteins, and combinations thereof.

In specific embodiments, healthy controls refer to individuals who do not suffer from chronic pancreatitis.

In another embodiment, there is provided the use of an identification reagent in the preparation of a reagent for monitoring the severity of chronic pancreatitis in a subject, wherein the identification reagent is specific for a protein selected from the group consisting of β-2-microglobulin , T-kininogen 2, carbonic anhydrase 1, polyimmunoglobulin receptor, glycan core protein, phosphoglycerate mutase 1, dipeptidyl peptidase 2, endothelial cell selective adhesion molecule, Nestin-2, Serum Amyloid Substance P, Phosphoprotein Secretion 24, Neural Cell Adhesion Molecule 1, Phospholysine Phosphohistidine Phosphite Inorganic Pyrophosphate Phosphatase, Cartilage Oligomer Matrix Protein, CD48 Antigen, Glycans Protein-3, matrix-remodeling-associated protein 8, amyloid βA4 protein, chondroitin sulfate proteoglycan 4, Thy-1 membrane glycoprotein, fibronectin, α2-thiol glycoprotein, tissue α-levofucosidase , actin cytoplasmic 1, growth arrestin 6, prothrombin, quinone oxidoreductase, neutral and basic amino acid transport protein rBAT, methyldopa Aβ subunit, mitochondrial kynurenine/a-aminoethanedi Acid transaminases, and combinations thereof.

In a specific embodiment, an increased expression level of a protein selected from the group consisting of beta-2-microglobulin and T-kininogen is indicative of exacerbation of chronic pancreatitis in said subject, as compared to the protein level in a healthy control 2. Carbonic anhydrase 1, polyimmunoglobulin receptor; compared to healthy controls, a decrease in the expression level of a protein selected from the following in a subject indicates exacerbation of chronic pancreatitis in the subject: glycan core protein, Phosphoglycerate mutase 1, dipeptidyl peptidase 2, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, phosphoprotein secretion 24, neuronal cell adhesion molecule 1, phospholysine Phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage oligomer matrix protein, CD48 antigen, glycan protein-3, matrix-remodeling-associated protein 8, amyloid βA4 protein, chondroitin sulfate proteoglycan 4, Thy -1 membrane glycoprotein, fibronectin, α2-thiol glycoprotein, tissue α-L-fucosidase, actin cytoplasmic 1, growth arrestin 6, prothrombin, quinone oxidoreductase, neutral and alkaline Amino acid transport protein rBAT, methyldopa Aβ subunit, mitochondrial kynurenine/a-aminooxalate transaminase, and combinations thereof.

Identification reagents suitable for use in the present disclosure are mass spectrometric identification reagents, antibodies or antigen-binding fragments thereof. In specific embodiments, the antibody is a monoclonal antibody. The present disclosure has no limitation on the species source of the monoclonal antibody, and any antibody capable of binding to the above-mentioned proteins can be used.

In specific embodiments, antigen-binding fragments include, but are not limited to: Fab, Fab', (Fab')2, Fv, ScFv, diabodies, triabodies, tetrabodies, bi-scFv. Any antibody fragment that retains antigen binding activity is suitable for use in the present disclosure.

In specific embodiments, protein markers according to the present disclosure can be used for diagnosis and/or prognosis of chronic pancreatitis.

In a specific embodiment, the expression level is selected from nucleic acid level and protein level, especially protein level.

In specific embodiments, said subject is a mammal, preferably a human.

In specific embodiments, the expression level is determined in a urine sample.

In specific embodiments, said protein is urinary protein.

According to other embodiments, there is also provided a kit or chip for diagnosing chronic pancreatitis, which comprises, or consists of, an identification reagent for a protein selected from: CD48 antigen, Igγ -1 chain C region, L-lactate dehydrogenase B chain, Na(+)/H(+) exchange regulator nhe-rf3, Thy-1 membrane glycoprotein, T-kininogen 2, V-type proton ATPase Subunit S1, α2-mercaptoglycoprotein, α-2-mercaptoglycoprotein, β-2-microglobulin, aminoacylase-1A, interleukin-4 receptor α subunit, cysteine protease inhibitor- B. Nestin-2, amyloid-βA4 protein, polyimmunoglobulin receptor, dipeptidyl peptidase 2, sortilin, secreted pyrophosphoprotein 24, cadherin-2, glutaminyl peptidase , Glutamylcysteine Synthetase Catalytic Subunit, Nucleonectin-2, Aflatoxin B1 Aldehyde Reductase 3, Actin Cytoplasm 1, Matrix-Remodeling-Associated Protein 8, Methyldopa Aβ subunit, collagen C-endopeptidase enhancer 1, glycan protein-3, glycan core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phospholysine Phosphohistidine phosphite inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chloride intracellular channel protein 1, sodium-dependent neutral amino acid carrier B(0)AT3, endothelial cell selective adhesion molecule, prothrombin, Prostate gland kallikrein-6, mitochondrial kynurenine/α-aminooxalate transaminase, cartilage oligomeric matrix protein, neuronal cell adhesion molecule 1, growth inhibitory protein 6, biglycan, carbonic anhydrase 1 , sialic acid O-acetyl esterase, fibronectin, serum amyloid substance p, alcohol dehydrogenase (coenzyme Ⅱ (+)), neutral and basic amino acid transport protein rBAT, tissue α-levofucosidase and its combination.

In some embodiments, identification reagents for the above-mentioned proteins are included in the kit. In other embodiments, identification reagents for the above-mentioned proteins are immobilized on the chip. In some embodiments, the identification reagent is an antibody or antigen-binding fragment thereof.

In a specific embodiment, a kit or chip for early diagnosis of chronic pancreatitis is provided, which comprises or consists of an identification reagent for a protein selected from: Igγ-1 Chain C region, nucleonectin-2, β-2-microglobulin, interleukin-4 receptor α subunit, phosphoglycerate mutase 1, dipeptidyl peptidase 2, secreted pyrophosphoprotein 24, Glycan core protein, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, neural cell adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage oligomers matrix protein.

In other specific embodiments, a kit or chip for monitoring the severity of chronic pancreatitis in a subject is provided, which comprises an identification reagent selected from the following proteins, or is identified by the identification of the proteins selected from Reagent composition: β-2-microglobulin, T-kininogen 2, carbonic anhydrase 1, polyimmunoglobulin receptor, glycan core protein, phosphoglycerate mutase 1, dipeptidyl peptidase 2. Endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance P, phosphoprotein secretion 24, nerve cell adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage low Polymeric matrix protein, CD48 antigen, glycan protein-3, matrix-remodeling-associated protein 8, amyloid βA4 protein, chondroitin sulfate proteoglycan 4, Thy-1 membrane glycoprotein, fibronectin, α2-thiol sugar Protein, tissue α-L-fucosidase, actin cytoplasmic 1, growth arrestin 6, prothrombin, quinone oxidoreductase, neutral and basic amino acid transport protein rBAT, methyldopa Aβ subunit, mitochondria Kynurenine/a-aminooxalate transaminase.

According to some embodiments, there is provided a method for diagnosing chronic pancreatitis in a subject comprising the steps of:

1) Obtain a urine sample from the subject,

2) Optionally, isolating urinary protein from the urine sample,

3) Determine the expression level of the protein selected from the following in the subject's urine sample: CD48 antigen, Igγ-1 chain C region, L-lactate dehydrogenase B chain, Na(+)/H(+) exchange regulator nhe-rf3, Thy-1 membrane glycoprotein, T-kininogen 2, V-type proton ATPase subunit S1, α2-thiol glycoprotein, α-2-thiol glycoprotein, β-2-microglobulin, amino Acylase-1A, Interleukin-4 receptor alpha subunit, Cystatin-B, Nestin-2, Amyloid βA4 protein, Polymeric immunoglobulin receptor, Dipeptidyl peptidase 2, Sortilin, secreted pyrophosphoprotein 24, cadherin-2, glutaminyl peptidase, glutamyl cysteine synthetase catalytic subunit, nucleectin-2, aflatoxin B1 aldehyde reduction Enzyme 3, actin cytoplasm 1, matrix-remodeling-associated protein 8, methyldopa Aβ subunit, collagen C-endopeptidase enhancer 1, glycan-3, glycan core protein, Quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chloride intracellular channel protein 1, sodium Dependent neutral amino acid carrier B(0)AT3, endothelial cell selective adhesion molecule, prothrombin, prostate gland kallikrein-6, mitochondrial kynurenine/α-aminooxalate transaminase, cartilage oligomeric matrix protein, nerve cell adhesion molecule 1, growth inhibitory protein 6, biglycan, carbonic anhydrase 1, sialic acid O-acetyl esterase, fibronectin, serum amyloid substance p, alcohol dehydrogenase (coenzyme Ⅱ ( +)), neutral and basic amino acid transport protein rBAT, tissue α-L-fucosidase, and combinations thereof.

In specific embodiments, expression levels are determined using mass spectrometry methods, ELISA methods, or Western methods.

When mass spectrometry is used to determine the protein and its expression level, after the step of obtaining the urine sample, a digestion step may also be included. In a specific embodiment, proteins in the urine sample are digested with proteases.

According to some embodiments, there is provided a method for early diagnosis of chronic pancreatitis in a subject, comprising the steps of:

1) Obtain urine samples from subjects and healthy controls,

2) Determining the expression levels of proteins selected from the group consisting of Igγ-1 chain C region, nucleonectin-2, β-2-microglobulin, interleukin-4 receptor α in urine samples of subjects and healthy controls Subunits, phosphoglycerate mutase 1, dipeptidyl peptidase 2, secreted pyrophosphoprotein 24, glycan core protein, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, Neural cell adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage oligomer matrix protein, and combinations thereof,

3) comparing the expression level of the protein in the subject with the expression level of the protein in the healthy control,

4) Determining whether the subject has chronic pancreatitis.

According to some embodiments, there is provided a method for monitoring the severity of chronic pancreatitis in a subject comprising the steps of:

1) Obtain urine samples from subjects and healthy controls,

2) Determining the expression level of a protein selected from the group consisting of β-2-microglobulin, T-kininogen 2, carbonic anhydrase 1, polyimmunoglobulin in the subject's urine sample compared to healthy controls Receptors, Glycan Core Protein, Phosphoglycerate Mutase 1, Dipeptidyl Peptidase 2, Endothelial Cell Selective Adhesion Molecule, Nestin-2, Serum Amyloid Substance P, Phosphoprotein Secretion 24, Neuronal Cells Adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage oligomer matrix protein, CD48 antigen, glycan protein-3, matrix-remodeling-associated protein 8, amyloid βA4 protein, Chondroitin sulfate proteoglycan 4, Thy-1 membrane glycoprotein, fibronectin, α2-thiol glycoprotein, tissue α-L-fucosidase, actin cytoplasmic 1, growth arrestin 6, prothrombin, quinone oxidation Reductase, neutral and basic amino acid transport protein rBAT, methyldopa Aβ subunit, mitochondrial kynurenine/a-aminooxalate transaminase, and combinations thereof,

3) comparing the expression level of the protein in the subject with the expression level of the protein in the healthy control,

4) Determining whether the subject's chronic pancreatitis has progressed or worsened.

Description of drawings

Figure 1: Body weight monitoring results of rats in the chronic pancreatitis group and rats in the control group. ● represents the model group; ■ represents the normal control group. * indicates p<0.05; ** indicates p<0.01.

Figure 2A to Figure 2E: HE staining results of rat pancreas tissue sections in the control group (A), chronic pancreatitis model groups at day 14 (B), 21 (C), 28 (D) and 42 days (E).

Figure 3A to Figure 3D: Masson staining results of pancreatic tissue sections of rats in the control group (A), chronic pancreatitis model groups on day 21 (B), 28 (C) and 42 days (D).

Detailed ways

The present application will be further illustrated by the following non-limiting examples. Those skilled in the art know that many modifications can be made to the application without departing from the spirit of the application, and such modifications also fall within the scope of the application. Unless otherwise specified, all experimental materials used were obtained from commercial companies.

Example

Embodiment 1: Establishment of chronic pancreatitis rat model

Diethyldithiocarbamate (diethyldithiocarbamate, DDC) induced chronic pancreatitis rat model fibrosis can simulate the process of chronic pancreatitis disease (see Naoki Matsumura et al., Study onFree Radicals and Pancreatic Fibrosis-Pancreatic Fibrosis Induced by Repeated Injections of Superoxide Dismutase Inhibitor Panereas, 2001, 22; 53-57). Observe the overall changes of pancreatic fibrosis from normal, early fibrosis, fibrosis progression, and its pathological process and pathological features are similar to human chronic pancreatitis. Intraperitoneal injection of DDC induced pancreatic injury in rats in a gradual process with good reproducibility. Using this animal model to simulate the pathogenesis of chronic pancreatitis has important guiding significance for clinical early diagnosis and monitoring of disease progression.

In this application, the animal model of rat chronic pancreatitis is induced by intraperitoneal injection of sodium diethyldithiocarbamate (DDC). 21st day, 28th day) urine. Using this animal model to simulate chronic pancreatitis has important guiding significance for the early diagnosis, treatment and prognosis of clinical chronic pancreatitis.

1. Materials and Reagents

1) Instrument:

Rat metabolic cage: purchased from Beijing Jiayuan Xingye Technology Co., Ltd. Thermo orbitrap fusion lumos mass spectrometer: purchased from Thermo Fisher Scientific; Thermo EASY-Nlc1200 high-performance liquid chromatography: purchased from Thermo Fisher Scientific; MillliQ RG ultrapure water system: purchased from Millipore; C18 reversed-phase analytical column (RP Column, 0.1×150mm, 3μm, ): purchased from Michrom Bioresources.

2) Main reagents:

Diethyldithiocarbamate (DDC) was purchased from Sigma; deionized water was from MillliQ RG ultrapure water system; chromatographic grade acetonitrile, formic acid and methanol were produced by Fisher; iodoacetamide (IAA), carbonic acid Ammonium hydrogen and dithiothreitol (DTT) were purchased from Merck; sequencing-grade trypsin was purchased from Sigma.

3) Animals:

Male Wistar rats (body weight 180 to 200 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in a standard feeding environment.

2. Experimental method

1) Establishment of chronic pancreatitis rat model and sample collection:

Steps for establishing rat model of chronic pancreatitis: intraperitoneal injection with a sterile 1mL syringe, according to the weight of the rat, the amount of DDC injected is 500mg/kg.

Steps for establishing the rat model of the control group: intraperitoneal injection with a sterile 1mL syringe, and injecting 500 mg/kg of normal saline according to the weight of the rat.

Sample collection process: Before modeling, rats were placed in metabolic cages to collect normal urine, and rats were placed in metabolic cages to collect urine samples on the 7th, 14th, 21st, and 28th days during the modeling process .

2) Indicator detection:

Body weight monitoring: The body weight of the rats was measured before each injection of DDC.

Pancreatic histopathology: On the 7th, 14th, 21st, 28th, and 42nd day of modeling, some rats in Example 1 were euthanized and the rat pancreatic tissues were obtained. The pancreas was removed from each animal and fixed by immersion in 4% formaldehyde at 4 °C. After sectioning, H&E staining and Masson staining were used to observe the changes of pancreatic tissue.

Example 2

1. Urinary protein extraction and storage:

The urine was centrifuged at 2000g at 4°C for 30 minutes, the supernatant was taken, placed in a new EP tube, and centrifuged at 12000g at 4°C for 30 minutes; the supernatant was taken and stored at -80°C.

Urine protein was precipitated with ethanol, and the protein concentration was measured by Bradford method, followed by enzyme digestion on the membrane. See WisniewskiJR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nature methods 2009; 6:359-62. The BCA method was used to measure the concentration of peptides.

2. LC-MS/MS tandem mass spectrometry analysis:

Peptide samples were diluted to 0.5 μg/μl with 0.1% formic acid. Peptide samples were separated by Thermo liquid phase system EASY-nLC 1200 loading system. The elution time is 120 minutes, and the column flow rate is 0.3 μl/min. The elution gradient was 5% to 40% mobile phase B (mobile phase A: 0.1% formic acid; mobile phase B: 89.9% acetonitrile). The eluted peptides were analyzed using a ThermoOrbitrap Lumos mass spectrometer. In positive ion mode, the resolution of precursor and product ions is 120,000.

3. Database search:

All mass spectrometry results were searched in the database using mascot software. The database used is the Swiss-Prot ratdatabase. The search conditions are: trypsin digestion; 2 missed cleavage sites are allowed; cysteine is a fixed modification; methionine oxidation and protein N-terminal acetylation are variable modifications. Thermo orbitrap fusion lumos data mass spectrometry data retrieval allowable error: parent ion 0.05Da, product ion 0.05Da.

4. Relative quantitative analysis results:

All mass spectrometry results were analyzed by primary LC-MS/MS software for relative quantitative results. Software operations were used according to methods indicated in the literature (see Hauck SM, Dietter J, Kramer RL et al. Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry. Molecular & cellular proteomics: MCP2010; 9: 2292- 305).

5. Statistical analysis:

Statistical analysis was performed on the proteome data at different time points, and the proteins with a p-value less than 0.05 were selected as differential proteins with a fold change of more than 2 times compared with the number of spectra on day 0 before modeling.

6. Experimental results:

(1) Weight change

As shown in Figure 1, the body weight of the experimental group was lower than that of the control group from day 10, and there was a significant statistical difference between the experimental group and the control group (Figure 1).

(2) Pathological test results

As shown in Figures 2A to 2E, the results of HE staining showed: the control group showed normal tissue structure (Figure 2A); the DDC-induced chronic pancreatitis rat experimental group showed varying degrees of edema, widening, inflammatory cell infiltration, Pancreatic parenchymal necrosis, hemorrhage, pancreatic ductal wall thickening, and hypercellularity occurred in some cases. and deepened over time (Fig. 2B to 2E).

As shown in Figure 3, the results of Masson's trichrome staining showed that the control group showed normal tissue structure (Figure 3A); the experimental group of rats with DDC-induced chronic pancreatitis started at day 21, and the pancreatic lobule and septum had different degrees of fiber The tissue proliferated and the degree of pancreatic fibrosis progressed over time (Fig. 3B to 3D), indicating that the modeling was successful.

(3) The urine of the rats (n=3) in the control group and the chronic pancreatitis group was identified by mass spectrometry, and two technical repetitions were performed respectively. At the level of FDR less than 1%, 557 proteins were identified based on more than two peptides.

Compared with the number of protein quantification spectra on day 0, the differential proteins with a change fold of more than 2 times at other time points and a repeated measurement ANOVA p value of less than 0.05 were screened out, and the rat protein was converted into the corresponding human homogeneous protein using the Uniprot database source protein.

Compared with the control group, there were 21 differential proteins in the urine of the 14-day chronic pancreatitis group, 32 differential proteins in the urine of the 21-day chronic pancreatitis group, and 32 differential proteins in the urine of the 28-day chronic pancreatitis group. There are 40 differential proteins in rats.

Comparing the urine protein of the chronic pancreatitis model at different time points with that of the control group rats, after ANOVA statistical analysis and multiple change screening, there are 52 reliable differential proteins that are homologous to humans, namely: CD48 antigen, Igγ-1 Chain C region, L-lactate dehydrogenase B chain, Na(+)/H(+) exchange regulator nhe-rf3, Thy-1 membrane glycoprotein, T-kininogen 2, V-type proton ATPase subunit S1, α2-thiol glycoprotein, β-2-microglobulin, aminoacylase-1A, interleukin-4 receptor α subunit, cystatin-B, nestin-2, amyloid βA4 protein, polyimmunoglobulin receptor, dipeptidyl peptidase 2, sortilin, secreted pyrophosphoprotein 24, cadherin-2, glutaminyl peptidase, glutamyl cysteine synthetase Catalytic subunit, nucleonectin-2, aflatoxin B1 aldehyde reductase 3, actin cytoplasm 1, matrix-remodeling-associated protein 8, methyldopa Aβ subunit, collagen C-peptide chain Dicer enhancer 1, glycan protein-3, glycan core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphate Enzymes, chondroitin sulfate proteoglycan 4, chloride intracellular channel protein 1, sodium-dependent neutral amino acid carrier B(0)AT3, endothelial cell selective adhesion molecule, prothrombin, prostatic gland kallikrein-6, mitochondria Kynurenine/α-aminooxalate transaminase, cartilage oligomer matrix protein, neuronal cell adhesion molecule 1, growth inhibitory protein 6, biglycan, carbonic anhydrase 1, sialic acid O-acetylesterase, fiber Zonulin, serum amyloid substance p, alcohol dehydrogenase (coenzyme II (+)), neutral and basic amino acid transport protein rBAT, tissue α-L-fucosidase.

Compared with the healthy control group, there were 7 urinary proteins with increased expression levels in the chronic pancreatitis group, including Igγ-1 chain C region, T-kininogen 2, β-2-microglobulin, polyimmunoglobulin Receptors, Nucleonectin-2, Prostatic Gland Kallikrein-6, Carbonic Anhydrase-1.

Compared with the healthy control group, 45 urine proteins with reduced expression levels in the chronic pancreatitis group were CD48 antigen, L-lactate dehydrogenase B chain, Na(+)/H(+) exchange regulator nhe-rf3 , Thy-1 membrane glycoprotein, V-type proton ATPase subunit S1, α2-thiol glycoprotein, aminoacylase-1A, interleukin-4 receptor α subunit, cystatin-B, nest Catalyzed by protein-2, amyloid-βA4 protein, dipeptidyl peptidase 2, sortilin, secreted pyrophosphoprotein 24, cadherin-2, glutaminyl peptidase, glutamyl cysteine synthetase subunit, aflatoxin B1 aldehyde reductase 3, actin cytoplasm 1, matrix-remodeling-associated protein 8, methyldopa Aβ subunit, collagen C-endopeptidase enhancer 1, poly Glycoprotein-3, glycan core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4. Chloride intracellular channel protein 1, sodium-dependent neutral amino acid carrier B(0)AT3, endothelial cell selective adhesion molecule, prothrombin, mitochondrial kynurenine/α-aminoethanedioic acid transaminase, cartilage oligomer Matrix protein, neural cell adhesion molecule 1, growth inhibitory protein 6, biglycan, sialic acid O-acetyl esterase, fibronectin, serum amyloid substance P, alcohol dehydrogenase (coenzyme II (+)), Neutral and basic amino acid transport protein rBAT, tissue α-L-fucosidase.

Because on the 14th day after modeling, there was no significant difference in the pathological detection of the rats in the chronic pancreatitis group and the control group compared with the control group. Therefore, 14 differential proteins were screened out in the early stage of chronic pancreatitis (14 days after modeling) (Table 1). After conversion to human homologous proteins, the expression levels of Igγ-1 chain C region, nucleonectin-2 and β-2-microglobulin are increased in the early stage of chronic pancreatitis; interleukin-4 receptor α subunit, phosphoglycerate Estermutase 1, dipeptidyl peptidase 2, secreted pyrophosphoprotein 24, glycan core protein, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, neural cell adhesion molecule 1, The expression of phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase and cartilage oligomer matrix protein decreased.

Among them, there are 11 differential proteins, namely: phosphoglycerate mutase 1, dipeptidyl peptidase 2, secreted pyrophosphoprotein 24, glycan core protein, endothelial cell selective adhesion molecule, nestin- 2. Serum amyloid substance P, nerve cell adhesion molecule 1, phospholysine phosphohistidine phosphite inorganic pyrophosphate phosphatase, cartilage oligomer matrix protein, β-2-microglobulin in other patients with chronic pancreatitis There were also significant differences in the stages (Table 2), illustrating the reliability of these proteins as an early diagnosis of the disease.

Table 1. Urine protein markers for early diagnosis of chronic pancreatitis

Since the 21st day after modeling, the pathological Masson staining of rats in the chronic pancreatitis group showed obvious fibrosis compared with the control group. Therefore, the group of chronic pancreatitis on the 14th, 21st and 28th day was repeatedly identified to summarize the mass spectrometric differential proteins, which can be used to detect the progression of chronic pancreatitis (Table 2).

Increased expression levels of β-2-microglobulin and T-kininogen 2, carbonic anhydrase 1, polyimmunoglobulin receptor compared with protein levels in healthy controls;

Glycan core protein, phosphoglycerate mutase 1, dipeptidyl peptidase 2, endothelial cell selective adhesion molecule, nestin-2, serum amyloid substance p, Phosphoprotein Secretion 24, Neural Cell Adhesion Molecule 1, Phospholysine Phosphohistidine Phosphite Inorganic Pyrophosphate Phosphatase, Cartilage Oligomer Matrix Protein, CD48 Antigen, Glycan Protein-3, Matrix-Remodeling-Associated Protein 8. Amyloid βA4 protein, chondroitin sulfate proteoglycan 4, Thy-1 membrane glycoprotein, fibronectin, α2-thiol glycoprotein, tissue α-L-fucosidase, actin cytoplasm 1, growth inhibitory protein 6. The expression levels of prothrombin, quinone oxidoreductase, neutral and basic amino acid transport protein rBAT, methyldopa Aβ subunit, and mitochondrial kynurenine/a-aminooxalate transaminase decreased.

Table 2. Changes in protein expression levels during the progression of chronic pancreatitis

Claims (10)

1. indentifying substance is being prepared for diagnosing the purposes in subject in the reagent of chronic pancreatitis, wherein the indentifying substance It is specific to selected from any one of following or its combination：

CD48 antigens, Ig γ -1 chain C areas, LDH heartunit, Na (+)/H (+) exchange regulatory factor nhe-rf3, Thy-1 Membrane glycoprotein, T- Prokineticin 2s, V-type proton ATP enzyme subunit S1, α 2- sulfydryls glycoprotein, beta-2-microglobulin, aminoacylates Enzyme -1A, interleukin-4 receptor alpha subunit, cystatin-B, nestin -2, amyloid A4 albumen, poly Immunoglobulin receptor, dipeptidyl peptidase 2, sorting protein, secreting type pyrophosphoric acid protein 24, cadherin -2, glutamy amido Peptase, glutamylcysteine synthase catalytic subunit, core connect albumen -2, aflatoxin B1 aldehyde reductase 3, actin born of the same parents It is matter 1, matrix-reconstruct-GAP-associated protein GAP 8, ethyldopa A β subunits, collagen C- endopeptidases enhancer 1, glycan protein-3, poly- Sugared core protein, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phosphate lysine phosphoric acid group ammonia The neutral amino that sour phosphorous acid inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chlorine intracellular channel protein 1, sodium rely on Acid vectors B (0) AT3, endothelial cell-selective adhesion molecule, haemoglutinin, prostate gland kallikrein -6, mitochondria dog urinary ammonia Acid/α amino ethanedioic acids transaminase, cartilage oligomeric matrix albumen, nerve cell adhesion molecule 1, growth inhibition albumen 6, disaccharide Catenin polysaccharide, carbonic anhydrase 1, Sialate O-acetylesterase, fibronectin, serum amyloid sample p materials, alcohol dehydrogenase are (auxiliary Enzyme II (+)), neutral and basic amino acid transport protein matter rBAT, tissue α-left-handed fucosidase.

2. purposes according to claim 1, wherein：

Compared with normal healthy controls, the expression selected from following albumen, which reduces, indicates that the subject suffers from chronic pancreatitis： CD48 antigens, LDH heartunit, Na (+)/H (+) exchange regulatory factor nhe-rf3, Thy-1 membrane glycoprotein, V-type proton ATP enzyme subunit S1, α 2- sulfydryls glycoprotein, amino acylase-1 A, interleukin-4 receptor alpha subunit, cysteine proteinase Inhibitor-B, nestin -2, amyloid A4 albumen, dipeptidyl peptidase 2, sorting protein, secreting type pyrophosphoric acid protein 24, calcium glue Albumen -2, glutamy amido peptase, glutamylcysteine synthase catalytic subunit, aflatoxin B1 aldehyde reductase 3, flesh move Protein cytoplasmic 1, matrix-reconstruct-GAP-associated protein GAP 8, ethyldopa A β subunits, collagen C- endopeptidases enhancer 1, glycan egg In vain -3, glycan core albumen, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phosphate lysine Phosphohistidine phosphorous acid inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chlorine intracellular channel protein 1, sodium rely on Neutral amino acid carrier B (0) AT3, endothelial cell-selective adhesion molecule, haemoglutinin, mitochondria kynurenin/α amino second two Sour transaminase, cartilage oligomeric matrix albumen, nerve cell adhesion molecule 1, growth inhibition albumen 6, biglycan, saliva Liquid acid O- acetylesterases, fibronectin, serum amyloid sample p materials, alcohol dehydrogenase (codehydrogenase Ⅱ (+)), neutral and basic amine group Sour transport protein matter rBAT, tissue α-left-handed fucosidase, and combinations thereof；

Compared with normal healthy controls, the expression selected from following albumen, which improves, indicates that the subject suffers from chronic pancreatitis： Ig γ -1 chain C areas, T- Prokineticin 2s, beta-2-microglobulin, polymeric immunoglobulin receptor, core connect albumen -2, prostate gland kassinin kinin Discharge enzyme -6, carbonic anhydrase 1 and combinations thereof.

3. purposes of the indentifying substance in the kit early diagnosed for chronic pancreatitis in subject is prepared, the identification examination Agent is specific to selected from following albumen：

Ig γ -1 chain C areas, core connect albumen -2, beta-2-microglobulin, interleukin-4 receptor alpha subunit, the displacement of phosphoglycerol acid esters Enzyme 1, dipeptidyl peptidase 2, secreting type pyrophosphoric acid protein 24, glycan core albumen, endothelial cell-selective adhesion molecule, nest egg In vain -2, serum amyloid sample p materials, nerve cell adhesion molecule 1, phosphate lysine phosphohistidine phosphorous acid inorganic pyrophosphate phosphorus Sour enzyme, cartilage oligomeric matrix albumen and combinations thereof；

It is preferred that the indentifying substance is specific to selected from following albumen：Phosphoglycerate mutase 1, dipeptidyl peptidase 2, divide Secrete formed coke Phospoprotein 24, glycan core albumen, endothelial cell-selective adhesion molecule, nestin -2, serum amyloid sample p things Matter, nerve cell adhesion molecule 1, phosphate lysine phosphohistidine phosphorous acid inorganic pyrophosphate phosphatase, cartilage oligomeric base Matter albumen, beta-2-microglobulin, and combinations thereof.

4. purposes according to claim 3, wherein：

Compared to normal healthy controls, the expression selected from following albumen, which improves, indicates that the subject has chronic pancreatitis Earlier damage：Ig γ -1 chain C areas, core connect albumen -2 and beta-2-microglobulin；

Compared to normal healthy controls, the expression selected from following albumen, which reduces, indicates that the subject has chronic pancreatitis Earlier damage：Interleukin-4 receptor alpha subunit, phosphoglycerate mutase 1, dipeptidyl peptidase 2, secreting type pyrophosphoric acid albumen 24th, glycan core albumen, endothelial cell-selective adhesion molecule, nestin -2, serum amyloid sample p materials, nerve cell adhesion Molecule 1, phosphate lysine phosphohistidine phosphorous acid inorganic pyrophosphate phosphatase, cartilage oligomeric matrix albumen, and combinations thereof.

5. indentifying substance is being prepared for monitoring the purposes in subject in the kit of the chronic pancreatitis order of severity, the mirror Determine reagent to be specific to selected from following albumen：

Beta-2-microglobulin, T- Prokineticin 2s, carbonic anhydrase 1, polymeric immunoglobulin receptor, glycan core albumen, phosphoglycerol Acid esters mutase 1, dipeptidyl peptidase 2, endothelial cell-selective adhesion molecule, nestin -2, serum amyloid sample p materials, phosphorus egg White matter secretion 24, nerve cell adhesion molecule 1, phosphate lysine phosphohistidine phosphorous acid inorganic pyrophosphate phosphatase, cartilage Oligomeric matrix albumen, CD48 antigens, glycan protein-3, matrix-reconstruct-GAP-associated protein GAP 8, amyloid A4 albumen, chondroitin sulfate Fibroin polysaccharide 4, Thy-1 membrane glycoproteins, fibronectin, α 2- sulfydryls glycoprotein, tissue α-left-handed fucosidase, flesh move egg White kytoplasm 1, growth inhibition albumen 6, haemoglutinin, quinone oxidoreductase, neutrality and basic amino acid transport protein matter rBAT, methyl DOPA A β subunits, mitochondria kynurenin/a- amino ethanedioic acids transaminase, and combinations thereof.

6. purposes according to claim 5, wherein：

Compared to normal healthy controls, the expression selected from following albumen, which improves, indicates that the chronic pancreatitis of the subject is disliked Change：Beta-2-microglobulin, T- Prokineticin 2s, carbonic anhydrase 1 and polymeric immunoglobulin receptor；

Compared to normal healthy controls, the expression selected from following albumen, which reduces, indicates that the chronic pancreatitis of the subject is disliked Change：Glycan core albumen, phosphoglycerate mutase 1, dipeptidyl peptidase 2, endothelial cell-selective adhesion molecule, nest egg In vain -2, serum amyloid sample p materials, phosphoprotein secretion 24, nerve cell adhesion molecule 1, phosphate lysine phosphohistidine are sub- Phosphoric acid inorganic pyrophosphate phosphatase, cartilage oligomeric matrix albumen, CD48 antigens, glycan protein-3, matrix-reconstruct-related egg White 8, amyloid A4 albumen, chondroitin sulfate proteoglycan 4, Thy-1 membrane glycoproteins, fibronectin, α 2- sulfydryls glycoprotein, group Knit α-left-handed fucosidase, actin kytoplasm 1, growth inhibition albumen 6, haemoglutinin, quinone oxidoreductase, neutrality and alkalescence Amino acid transport proteins matter rBAT, ethyldopa A β subunits, mitochondria kynurenin/a- amino ethanedioic acid transaminases and its group Close.

7. purposes according to any one of claim 1 to 6, wherein：

The indentifying substance is selected from：Mass Spectrometric Identification reagent, antibody or its antigen-binding fragment；

It is preferred that the antibody is monoclonal antibody.

8. purposes according to any one of claim 1 to 6, wherein the albumen is urine albumen.

9. purposes according to any one of claim 1 to 6, wherein the subject is mammal, preferably people.

10. a kind of kit or chip, it includes indentifying substance；The indentifying substance is specific to selected from following albumen：

CD48 antigens, Ig γ -1 chain C areas, LDH heartunit, Na (+)/H (+) exchange regulatory factor nhe-rf3, Thy-1 Membrane glycoprotein, T- Prokineticin 2s, V-type proton ATP enzyme subunit S1, α 2- sulfydryls glycoprotein, α -2- sulfydryls glycoprotein, β -2- microballoons Albumen, amino acylase-1 A, interleukin-4 receptor alpha subunit, cystatin-B, nestin -2, amyloid β A4 albumen, polymeric immunoglobulin receptor, dipeptidyl peptidase 2, sorting protein, secreting type pyrophosphoric acid protein 24, cadherin- 2nd, glutamy amido peptase, glutamylcysteine synthase catalytic subunit, core connect albumen -2, aflatoxin B1 aldehyde reductase 3rd, actin kytoplasm 1, matrix-reconstruct-GAP-associated protein GAP 8, ethyldopa A β subunits, collagen C- endopeptidases enhancer 1, poly- Glycoprotein -3, glycan core albumen, quinone oxidoreductase, phosphoprotein secretion 24, phosphoglycerate mutase 1, phosphoric acid rely Propylhomoserin phosphohistidine phosphorous acid inorganic pyrophosphate phosphatase, chondroitin sulfate proteoglycan 4, chlorine intracellular channel protein 1, sodium according to Bad neutral amino acid carrier B (0) AT3, endothelial cell-selective adhesion molecule, haemoglutinin, prostate gland kallikrein -6, Mitochondria kynurenin/α amino ethanedioic acids transaminase, cartilage oligomeric matrix albumen, nerve cell adhesion molecule 1, growth suppression Albumen 6 processed, biglycan, carbonic anhydrase 1, Sialate O-acetylesterase, fibronectin, serum amyloid sample p materials, second Alcohol dehydrogenase (codehydrogenase Ⅱ (+)), neutral and basic amino acid transport protein matter rBAT, tissue α-left-handed fucosidase and its Combination；

The indentifying substance is selected from：Mass Spectrometric Identification reagent, antibody or its antigen-binding fragment；

It is preferred that the antibody is monoclonal antibody；

Described kit or chip are used to implement to be selected from following any one or combination：The early diagnosis of chronic pancreatitis, prison Survey the chronic pancreatitis order of severity of subject.