Proteins and test kits for early diagnosis of liver fibrosis

Abstract

The present application discloses the close relationship between cathepsin S (CTSS) protein, endostatin and liver fibrosis, provides the expression of CTSS protein and endostatin by taking peripheral blood as a biological sample, and determines the protein content determination standard of early liver fibrosis and liver fibrosis by taking healthy people as a reference, and can early non-invasive diagnose liver fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnosis and treatment, in particular to proteins and reagents for early detection and diagnosis of liver cirrhosis. BACKGROUND

[0002] Liver cirrhosis is a common and high-incidence disease, and its early pathological manifestation is liver fibrosis. Liver cirrhosis patients usually have multiple serious complications, such as variceal hemorrhage, liver ascites, renal failure, etc. Moreover, once a patient is diagnosed with liver cirrhosis, the probability of developing liver cancer will also be greatly increased (Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008; 371: 838-851.).

[0003] In the early stage of liver fibrosis, the clinical symptoms of patients are not obvious, and there is also a lack of effective clinical diagnosis methods. Once a patient is diagnosed, he or she has usually developed to the stage of liver cirrhosis. The existing liver cirrhosis diagnosis methods, such as imaging and serological indicators (liver fibrosis four indicators: serum type III procollagen amino-terminal peptide, serum laminin, hyaluronic acid, serum type IV collagen), have large variations in different liver fibrosis and liver cirrhosis patients, and cannot provide rapid and accurate diagnosis results. The histopathological detection through liver biopsy is the "gold standard" for the diagnosis of liver cirrhosis. However, the biopsy causes great risk to the patient (Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: Diagnosis, grading and staging. Hepatology. 1994; 19: 1513-1520; Rosenberg WMC, Voelker M, Thiel R, Becka M, Burt A, Schuppan D, et al. Serum markers detect the presence of liver fibrosis: A cohort study. Gastroenterology. 2004; 127: 1704-1713.). At present, there is a lack of rapid, accurate and non-invasive liver fibrosis / liver cirrhosis diagnosis method in the clinic.

[0004] The treatment of cirrhosis also faces challenges. Clinical treatment strategies for cirrhosis primarily target the underlying cause. For example, virus-induced fibrosis can be treated with antiviral therapy using interferon and nucleoside (acid) analogs (Mallet V, Gilgenkrantz H, Serpaggi J, Verkarre V, Vallet-Pichard A, Fontaine H, et al. Brief communication: The relationship of regression of cirrhosis to outcome inchronic hepatitis C. Ann. Intern. Med. 2008; 149:399–403.), achieving good treatment results in the early stages of fibrosis. However, in the cirrhosis stage, clinical treatment mainly focuses on suppressing complications, which does not necessarily achieve the goal of curing cirrhosis (Tsochatzis EA, Bosch J, Burroughs AK. Liver

[0005] Cirrhosis. In: The Lancet. 2014. p. 1749–1761. Therefore, the unclear pathological mechanism is the main reason for the difficulty in diagnosing and treating cirrhosis. Previous studies have shown that the excessive deposition of extracellular matrix (ECM) on the liver surface due to dysregulation of ECM is an important pathological mechanism of cirrhosis (Iredale JP, Thompson A, Henderson NC. Extracellular matrix degradation in liver fibrosis: Biochemistry and regulation. Biochim. Biophys. Acta-Mol. Basis Dis. 2013; 1832: 876–883.). Proteins in the ECM family, such as elastin, are considered to be important enzymes affecting the ECM remodeling process. The cathepsin S protein, composed of 331 amino acids, can cleave collagen (Wilkinson RDA, Williams R, Scott CJ, Burden RE. Cathepsin S: Therapeutic, diagnostic, and prognostic potential. Biol. Chem. 2015; 396:867–882.), and is closely related to the development and progression of cirrhosis. However, due to technological limitations, it is unclear at which amino acid residue the cathepsin S protein cleaves collagen.

[0006] The application patent application CN201811239261.3 is the previous research of the applicant. Through quantitative proteomics technology and immunohistochemical experiments, it is found that cathepsin S protein (CTSS) is up-regulated in cirrhotic tissues. By injecting the CTSS antibody and fluorescent probe into the CCl4-induced cirrhotic mouse model, a significant fluorescence brightness is observed in the liver of the CCl4-induced cirrhotic mouse. By inducing the wild type (WT) and CTSS - / - mice with CCl4, it is found that the degree of cirrhosis of the CTSS knockout group of cirrhotic mouse models is significantly alleviated. Therefore, it is determined that there is a close correlation between CTSS protein and cirrhosis. However, the detection still needs to rely on the provision of liver tissue.

[0007] Since the treatment of liver fibrosis and cirrhosis is better in the early stage and the treatment effect is better than in the late stage, the current diagnosis method has low sensitivity, and needs to be confirmed by liver tissue puncture, which causes pain to the patient and is difficult to confirm in the early stage of liver fibrosis. SUMMARY

[0008] An object of the present application is to provide the use of cathepsin S protein (CTSS) and endostatin in a detection reagent related to liver fibrosis.

[0009] Another object of the present application is to provide a detection reagent for early diagnosis of peripheral blood samples related to liver fibrosis.

[0010] According to one aspect of the present application, the use of a reagent for specifically detecting protein expression in the preparation of a product for detecting a biological sample related to liver fibrosis, wherein the protein is one or both selected from cathepsin S protein and endostatin; wherein the content of cathepsin S protein in the healthy person is 16.91±2.54 ng / mL, and the sample with a content of cathepsin S protein in the biological sample ≥ 31.61±9.49 ng / mL is determined as a biological sample related to early liver fibrosis; and / or

[0011] The content of endostatin in the healthy person is 147.65±73.50 ng / mL, and the sample with a content of endostatin in the biological sample ≥ 289.62±166.78 ng / mL is determined as a biological sample related to early liver fibrosis.

[0012] The biological sample according to the present application is peripheral blood. Preferably, the biological sample is serum.

[0013] The application further determines that the sample with the cathepsin S protein content in the biological sample being greater than or equal to 34.61 ± 12.45 ng / mL is a biological sample related to liver fibrosis; and / or

[0014] The sample with the endostatin content in the biological sample being greater than or equal to 356.60 ± 172.33 ng / mL is determined to be a biological sample related to liver fibrosis.

[0015] The application further determines that the sample with the cathepsin S protein content in the biological sample being greater than or equal to 34.61 ± 12.45 ng / mL is a biological sample related to liver fibrosis; and / or

[0016] The two proteins cathepsin S protein (CTSS) and endostatin of the application can be used as detection markers, respectively, and when the content of each of the two proteins in blood exceeds a certain amount, the detected biological sample is independently determined to be related to liver fibrosis, and more preferably, the two proteins are combined as detection markers, because the combination of the two proteins shows better detection sensitivity and effectiveness than a single protein.

[0017] According to another aspect of the application, a detection reagent for detecting protein expression in a biological sample, the detection reagent comprising: a reagent for detecting the expression amount of a protein, the detection reagent comprising at least one of the following reagents:

[0018] a reagent for detecting the expression amount of cathepsin S protein, and

[0019] a reagent for detecting the expression amount of endostatin;

[0020] The biological sample is peripheral blood; and

[0021] In the reagent for detecting the expression amount of cathepsin S protein, in the case that the cathepsin S protein content of a healthy person is 16.91 ± 2.54 ng / mL, the sample with the cathepsin S protein content in the biological sample being greater than or equal to 31.61 ± 9.49 ng / mL is determined to be a biological sample related to early liver fibrosis; and / or

[0022] In the reagent for detecting the expression amount of endostatin, in the case that the endostatin content of a healthy person is 147.65 ± 73.50 ng / mL, the sample with the endostatin content in the biological sample being greater than or equal to 289.62 ± 166.78 ng / mL is determined to be a biological sample related to early liver fibrosis.

[0023] Further, in the reagent for detecting the expression amount of cathepsin S protein, the sample with the content of cathepsin S protein in the biological sample ≥ 34.61 ± 12.45 ng / mL is determined as the biological sample related to liver fibrosis; and / or

[0024] In the reagent for detecting the expression amount of endostatin, the sample with the content of endostatin in the biological sample ≥ 356.60 ± 172.33 ng / mL is determined as the biological sample related to liver fibrosis.

[0025] Preferably, the reagent for detecting the expression amount of protein is an enzyme-linked immunoassay reagent.

[0026] In general application, the detection reagent of the present application can be the following types of reagents:

[0027] antibodies, primers, probes, sequencing libraries, nucleic acid chips (gene chips), protein chips, or combinations thereof.

[0028] Therefore, the reagent can comprise antibodies recognizing the antigenic epitopes of CTSS protein and / or endostatin, can comprise complexes of fluorescent probes coupled to the antibodies of CTSS and / or endostatin, and can comprise primers for amplifying the genes encoding CTSS and / or endostatin from the sample to be detected.

[0029] The detection reagent of the present application can further comprise a reagent for detecting the presence of a polypeptide with the amino acid sequence shown in SEQ ID NO. 1 (LVALNSPLSGGMR) in the biological sample. The polypeptide is the cleavage site of CTSS, which produces the known endostatin, and the presence of CTSS protein and / or endostatin in the biological sample can be determined by detecting the presence of the polypeptide in the biological sample.

[0030] On the basis of the above-mentioned diagnostic reagent, the present application also provides a detection kit, wherein the kit container contains a reagent for detecting CTSS and / or endostatin protein or gene, and the information related to the manufacture, use and sale of the drug or biological product approved by the government drug administration agency can be provided at the same time.

[0031] According to another aspect of the present application, a detection kit is provided, wherein the kit comprises a reagent for detecting the expression amount of cathepsin S protein and / or endostatin, and a kit instruction, wherein the kit instruction comprises the following information:

[0032] In the case that the cathepsin S protein content of healthy people is 16.91±2.54 ng / mL, the sample with the cathepsin S protein content ≥31.61±9.49 ng / mL in the biological sample is determined as the biological sample related to early liver fibrosis; and / or

[0033] In the case that the endostatin content of healthy people is 147.65±73.50 ng / mL, the sample with the endostatin content ≥289.62±166.78 ng / mL in the biological sample is determined as the biological sample related to early liver fibrosis.

[0034] Further, the sample with the cathepsin S protein content ≥34.61±12.45 ng / mL in the biological sample is determined as the biological sample related to liver fibrosis; and / or

[0035] The sample with the endostatin content ≥356.60±172.33 ng / mL in the biological sample is determined as the biological sample related to liver fibrosis.

[0036] The present application discloses the close relationship between cathepsin S (CTSS) protein and endostatin and liver fibrosis, provides the expression amount of CTSS protein and endostatin by taking peripheral blood as a biological sample, and determines the protein content determination standard for early liver fibrosis and liver fibrosis by taking healthy people as a reference, so as to early and non-invasively diagnose liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 : CTSS is up-regulated in cirrhotic tissues. The CTS family proteins are up-regulated in cirrhotic tissues as a whole, and the up-regulation of CTSS is the highest. "CL" is cirrhotic tissue, and "NL" is normal tissue.

[0038] Figure 2 : MS2 mass spectrum spectrum of CTSS.

[0039] Figure 3 : Content distribution of CTSS in human serum.

[0040] Figure 4 : Content distribution of CTSS in human serum of patients with liver fibrosis of different causes.

[0041] Figure 5 : Comparison of CTSS content in human serum of patients with early liver fibrosis stage and healthy volunteers.

[0042] Figure 6 : Content distribution of CTSS in human serum of patients with liver fibrosis of different stages.

[0043] Figure 7 ROC curve of ALT, GP73 and IV collagen in liver fibrosis patients.

[0044] Figure 8 ROC curve of CTSS in liver fibrosis patients.

[0045] Figure 9 ROC curve of CTSS in liver fibrosis patients.

[0046] Figure 10 ROC curve of ALT, GP73 and IV collagen in early liver fibrosis patients.

[0047] Figure 11 CTSS in vitro cleaves collagen 18 to produce endostatin.

[0048] Figure 12 Time sequence analysis of CTSS in vitro cleaving collagen 18 to produce endostatin.

[0049] Figure 13 CTSS cleaves collagen 18 to produce LVALNSPLSGGMR.

[0050] Figure 14 Endostatin content in liver fibrosis patients is significantly increased.

[0051] Figure 15 Endostatin content distribution in human serum.

[0052] Figure 16 Comparison of endostatin content in human serum between early liver fibrosis patients and healthy volunteers

[0053] Figure 17 Endostatin content distribution in human serum of liver fibrosis patients at different stages.

[0054] Figure 18 ROC curve of CTSS and endostatin in liver fibrosis patients.

[0055] Figure 19 Comparison of endostatin and CCTSS content in human serum between early liver fibrosis patients (S1 stage) and healthy volunteers. DETAILED DESCRIPTION

[0056] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0057] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0058] The present application can be more readily understood through the following examples, but the present application is not limited to these examples. Example 1 The discovery of high expression of CTSS in human liver fibrosis tissues

[0059] Human cirrhotic and normal liver samples were obtained from patients undergoing surgical treatment for cirrhosis, and were further analyzed by pathologists through histopathology of the tissues.

[0060] Take 50 mg of fresh liver tissue into a pre-cooled mortar, grind with liquid nitrogen, and collect the powder into a 1.5 mL Eppendorf tube. Add 300 μL of cell lysis solution, and lyse for 5-8 min with an ultrasonic cell disruptor until the solution is clear and transparent to obtain a total cell lysate (TCL). Centrifuge at 4°C at maximum speed for 5 min, and aliquot.

[0061] Quantify the protein in TCL by calculating the gray value of SDS-PAGE short gel. To reduce the influence of individual differences on the quantitative proteomic data, take 3 whole proteins from cirrhotic patients, mix, and label as "CL1"; take another 3 whole proteins from cirrhotic patients, mix, and label as "CL2". Take equal amounts of whole proteins from 5 normal liver tissues (NL) and mix, and label as NL.

[0062] Take 100 μg of whole proteins from each of the "CL1", "CL2", and "NL" groups, and purify the samples by SDS-PAGE short gel (Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res. 2009, 8: 3944-50.). After staining the gel with Coomassie brilliant blue and destaining, cut the gel film in the stained area into 1 cubic millimeter gel particles, and further destain and dehydrate (Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res. 2009, 8: 3944-50.).

[0063] The decolorized and dried micelles were digested with 12.5 ng / μL trypsin (Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies. Protein Expr Purif. 2015, 116: 120-126.) at 37°C for 14 hours. The samples after trypsin digestion were extracted with extraction solution (5% formic acid, 50% acetonitrile) to obtain trypsin-digested liver fibrosis tissue and normal tissue total cell protein group samples.

[0064] The trypsin-digested CL1, CL2, and NL were labeled with different label reagents according to the instructions of the iTRAQ-4plex kit (AB Sciex, USA). Among them, the CL1 sample was labeled with 114; the CL2 sample was labeled with 115; the NL was divided into two, and was labeled with 116 and 117 labels, respectively, as technical repeats (Down-regulation of RIP3 potentiates cisplatin chemoresistance by triggering HSP90-ERK pathway mediated DNA repair in esophageal squamous cell carcinoma, Cancer Letters, 2018, 418: 97-108.).

[0065] In order to detect the labeling efficiency, 1 μL of each of the four groups of samples was mixed, desalted with a desalting column, and then subjected to mass spectrometry identification (Down-regulation of RIP3 potentiates cisplatin chemoresistance by triggering HSP90-ERK pathway mediated DNA repair in esophageal squamous cell carcinoma, Cancer Letters, 2018, 418: 97-108.).

[0066] Liquid chromatography-mass spectrometry (LC-MS) (LTQ-Orbitrap Velos mass spectrometer, Thermo Fisher Scientific, USA) analysis method as follows: 1 μg sample was loaded on 3 μm C18 self-packed reversed-phase separation column (75 μm i.d. x 15 cm), and analyzed with a 100-minute liquid chromatography gradient. The liquid chromatography separation condition was acetonitrile concentration from 0-35%. Mass spectrometry analysis conditions: maximum ionization voltage 2.0 kV, scan range 400-1800 m / z, automatic gain control (AGC) 1 x 106, maximum ion injection time 150 ms, primary scan resolution at 400 m / z 30000. Secondary spectrum scanning mode was Higher Energy Collision Induced Dissociation (HCD), 40% collision energy, automatic gain control 30000, dynamic exclusion 35 s.

[0067] The obtained mass spectrum raw file was searched by MaxQuant (v1.5.8.3) software, and the search parameters were set as follows: parent ion error range 20 ppm, maximum missed cleavage site 2, minimum amino acid length 7, false discovery rate (FDR) less than 1.0%. Protein quantification was based on the abundance of unique peptide segment reporter ions.

[0068] We set the difference protein difference standard as the ratio of cirrhotic liver (CL) / normal liver (NL) (ratio CL / NL) greater than 1.5 times or less than -1.5 times and p value <0.05, and identified 201 up-regulated differential proteins and 85 down-regulated proteins.

[0069] Among the up-regulated differential proteins, cathepsin (CTS) family proteins were identified to be up-regulated in cirrhotic tissues as a whole, among which cathepsin S (CTSS) was up-regulated by more than 1.5 times ( Figure 1 ), and the secondary spectrum matching of CTSS was good, indicating that CTSS was correctly identified ( Figure 2 ).

[0070] Example 2 , CTSS can be used for non-invasive diagnosis of early liver fibrosis

[0071] The degree of liver fibrosis in human patients was determined according to METAVIR (F) (An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group, Hepatology, 1996, 24: 289-293.) and China 2000 (Guidelines for the prevention and treatment of viral hepatitis. Chinese Journal of Hepatology, 2000, 8: 324-329.). The judging indicators are shown in Table 1.

[0072] Table 1. Comparison table of pathological staging evaluation criteria for liver fibrosis

[0073]

[0074] The serum samples of human liver fibrosis patients came from hospitals, a total of 164 cases, and the degree of liver fibrosis of the patients was diagnosed by liver biopsy samples of the patients themselves by experienced pathologists. The serum samples of healthy volunteers came from donations, a total of 30 cases.

[0075] The serum level of CTSS was determined by an ELISA kit of Abeam Company (Abeam, ab155427), and the results are shown in Figure 3 . Among them, the CTSS content in the serum of healthy volunteers was 16.91 ± 2.54 ng / mL (mean ± SD), and the CTSS content in the serum of liver fibrosis patients was 34.61 ± 12.45 ng / mL (mean ± SD), with a very significant difference (p < 0.01) between the two groups, and the serum CTSS of the fibrosis patients was 2 times that of the healthy volunteers. The measurement of serum CTSS can easily distinguish the liver fibrosis patient population from the healthy population.

[0076] The 164 cases of fibrosis patients were grouped according to the cause, and the serum CTSS of the patients was compared with that of the healthy volunteers. It was found that no matter what cause of liver fibrosis, the mean of CTSS in the blood of patients with hepatitis B (HBV), hepatitis C (HCV), drug-induced liver injury (DILI), non-alcoholic fatty liver (NAFLD) and other types of liver fibrosis was 1.93, 2.04, 2.88, 2.15 to 2.11 times higher than that of healthy people, and very significant (p < 0.01), with the characteristics of universality Figure 4 ).

[0077] Early-stage liver fibrosis is easily reversed and cured, thus possessing clinical therapeutic value. However, current techniques struggle to accurately diagnose early-stage liver fibrosis. In a study of 164 fibrosis patients, blood samples from 32 patients with early-stage (S1 stage) liver fibrosis, identified using liver biopsy (the gold standard), were collected separately and analyzed simultaneously with blood samples from healthy volunteers. The results showed that the CTSS value in the blood samples of early-stage (S1 stage) liver fibrosis patients was 31.61±9.49 ng / mL (mean±SD), which was 1.87 times higher than the 16.91±2.54 ng / mL (mean±SD) in healthy volunteers. Figure 5 The statistical difference was significant.

[0078] To compare the distribution of total CTSS levels at different stages of liver fibrosis, 164 fibrosis patients were grouped according to their stage of liver fibrosis. Their serum CTSS levels were compared with those of healthy volunteers. The results showed that, regardless of whether the stage was S1, S2, S3, or S4, the mean CTSS levels in the blood of fibrosis patients were 1.87, 2.50, 2.36, and 1.81 times higher than those in healthy individuals, respectively, and this difference was highly significant (p<0.01). Figure 6 ).

[0079] Alanine aminotransferase (ALT) is a marker of liver injury; Golgi protein 73 (GP73) and type IV collagen are commonly used markers of liver fibrosis. Receiver operating characteristic (ROC) curves were performed on the blood ALT, GP73, and type IV collagen levels of all 164 patients with liver fibrosis. The areas under the curves were 0.76, 0.643, and 0.928, respectively. Figure 7 All of these values ​​are lower than the area under the ROC curve calculated by CTSS (0.951). Figure 8 This indicates that using CTSS as an indicator of liver fibrosis is significantly superior to existing liver fibrosis indicators such as ALT, GP73, and IV collagen.

[0080] ROC curve analysis of CTSS in the blood of 32 patients with early-stage (S1 stage) liver fibrosis showed an area under the curve (AUC) as high as 0.969, with a sensitivity of 96.9% and a specificity of 93.3%. Figure 9 It exhibits good predictive performance. Correspondingly, ROC curve analysis using ALT showed an area under the curve of 0.818, a sensitivity of 71.9%, and a specificity of 80%. Figure 10 ROC curve analysis using GP73 showed an area under the curve of 0.539, a sensitivity of 78.1%, and a specificity of 50%. Figure 10 ROC curve analysis using type IV gel showed an area under the curve of 0.922, a sensitivity of 81.3%, and a specificity of 93.3%. Figure 10), the predictive diagnostic value is significantly lower than CTSS. CTSS is the optimal marker molecule for early (S1) liver fibrosis diagnosis.

[0081] Example 3 , CTSS can cut collagen 18 to produce endostatin

[0082] The reaction system (50 mM pH 5.5 sodium acetate, 2 mM dithiothreitol (DTT) and 5 mM EDTA) solution with 0.6 μM recombinant collagen 18 and 20 nM human CTSS or without CTSS was reacted at 37°C for 2 hours. The reaction product was subjected to SDS-PAGE electrophoresis, and after blotting and transferring to the membrane, WB analysis was performed with collagen 18 antibody or endostatin antibody. It was found that a significant amount of endostatin was newly produced in the reaction with CTSS, but no endostatin was produced in the reaction tube without CTSS; and the amount of collagen 18 in the tube with CTSS producing endostatin was significantly reduced Figure 11 ), proving that CTSS can specifically cut collagen 18 to produce endostatin.

[0083] To confirm the specificity and efficiency of this in vitro reaction, we performed the collagen cutting reaction by CTSS in the same system as above, and sampled at 15 and 60 minutes after the start of the reaction, subjected to SDS-PAGE separation, and the electrophoretic product was silver stained. It was found that the silver staining gel of gel electrophoresis was clear Figure 12 ); the endostatin band was visible at 15 minutes after the reaction, and the band was significantly enhanced as the reaction time was prolonged to 60 minutes; and as the reaction proceeded, the amount of collagen 18 at 60 kD gradually decreased, further confirming the specificity and high efficiency of the CTSS cutting collagen 18 reaction.

[0084] To determine the exact site of CTSS cutting collagen 18, we reacted the endostatin gel strip separated by SDS-PAGE gel electrophoresis with 40 mM formaldehyde and 20 mM sodium cyanoborohydride solution at 37°C for 4 hours, and terminated the reaction with 0.1 M Tris solution (pH 6.8). The labeled protein was subjected to in-gel digestion with 12.5 ng / μL trypsin according to the above proteomics sample preparation technique, and LC-MS analysis. We found that the peptide LVALNSPLSGGMR (SEQ ID NO. 1) from collagen 18 was specifically labeled, which was the cutting site of CTSS, producing known endostatin. The chemically synthesized L-labeled peptide L*VALNSPLSGGMR confirmed that the mass spectrum fragmentation spectrum was completely consistent with the spectrum we found, confirming that it was LVALNSPLSGGMR Figure 13). The quantification of the CTSS-cleaved collagen 18 neoepitope-containing peptide fragment including the cleavage site by the existing known technology can also determine the early liver fibrosis.

[0085] Example 4 Endostatin can be used for non-invasive diagnosis of early liver fibrosis

[0086] Take 50 mg of fresh liver tissue from liver fibrosis patients and healthy people respectively, put them into a pre-cooled mortar, grind them with liquid nitrogen, and collect the powder into a 1.5 mL Eppendorf tube. Add 300 μL of cell lysis solution, and lyse for 5-8 min with an ultrasonic cell disruptor until the solution is clear and transparent. Centrifuge at 4°C at maximum speed for 5 min, and aliquot.

[0087] Use SDS-PAGE gel electrophoresis to separate the total cell proteins of liver tissues from 5 liver fibrosis patients and 3 healthy people, and perform blotting and membrane transfer on the electrophoresis products. Perform WB analysis with anti-collagen 18 and anti-endostatin respectively, and find that the endostatin content in the liver tissues of liver fibrosis patients is significantly higher than that of healthy controls.

[0088] Analyze 164 serum samples from liver fibrosis patients and 30 serum samples from healthy volunteers. The serum level of endostatin is determined by the ELISA kit of Cloud Clone Company (Cat# SEA542Hu), and the results are shown in Figure 15 . The endostatin content in the serum of healthy volunteers is 147.65 ± 73.50 ng / mL (mean ± SD), and the endostatin content in the serum of liver fibrosis patients is 356.60 ± 172.33 ng / mL (mean ± SD). There is a very significant difference between the two groups (p < 0.01), and the serum endostatin of fibrosis patients is 2.42 times that of healthy volunteers. The measurement of serum endostatin can easily distinguish liver fibrosis patients from healthy people.

[0089] Early liver fibrosis is easy to reverse and cure, so it has clinical treatment value. However, existing technologies are difficult to determine early (S1) liver fibrosis. Take 32 serum samples from early (S1) liver fibrosis patients among the 164 fibrosis patients, which are detected by the gold standard of liver biopsy, and analyze the endostatin in the serum samples at the same time as the healthy volunteer blood samples. The endostatin value in the serum samples of early (S1) liver fibrosis patients is 289.62 ± 166.78 ng / mL (mean ± SD), which is 1.96 times higher than that of healthy volunteers (147.65 ± 73.50 ng / mL (mean ± SD)) ( Figure 16 ).

[0090] To compare the distribution of CTSS content in different stages of human liver fibrosis, 164 fibrosis patients were grouped according to different stages of liver fibrosis, and their serum endostatin was compared with that of healthy volunteers. It was found that, regardless of S1, S2, S3 or S4, the mean value of serum endostatin in liver fibrosis patients was 1.96, 2.30, 1.78 to 2.80 times higher than that in healthy people, and extremely significant (p<0.01) Figure 17 ), indicating that endostatin can also be an effective indicator of early liver fibrosis.

[0091] ROC curve analysis was performed on the combination of CTSS and endostatin in 32 early (S1) liver fibrosis patients, and the area under the curve was as high as 0.989, higher than the area under the curve of ROC analysis of early (S1) liver fibrosis using only CTSS, 0.979 Figure 18 ), indicating that the combination of CTSS and endostatin can more effectively predict and diagnose early liver fibrosis. CTSS and endostatin are effective marker molecules for the diagnosis of early liver fibrosis.

[0092] Further comparison of the relationship between CTSS and endostatin in early (S1) liver fibrosis patients showed that, according to the concentration of endostatin and CTSS in serum, we calculated their Z-score value relative to the normal value (healthy corresponding serum content). If Z-score is greater than 0, it means higher than normal value, and Z-score less than 0 means lower than normal value. It was found that the serum content of endostatin in 7 early (S1) fibrosis patients was lower than the normal value (the result is shown visually in 6 points, because 2 patients have very close values, resulting in two points overlapping), while the serum content of CTSS in all patients was higher than the normal value Figure 19 ). Therefore, there may be some risk in determining liver fibrosis only by the serum content of endostatin in patients, and the combination is more reliable.

Claims

1. The application of a reagent for specifically detecting protein expression in serum in the preparation of products for detecting early liver fibrosis, characterized in that, The proteins mentioned are cathepsin S protein and vascular endostatin. Among them, when the cathepsin S protein content in healthy individuals is 16.91±2.54 ng / mL, biological samples with a cathepsin S protein content ≥31.61±9.49 ng / mL are classified as biological samples associated with early liver fibrosis; and Given that the endostatin content in healthy individuals is 147.65±73.50 ng / mL, biological samples with an endostatin content ≥289.62±166.78 ng / mL are considered biological samples associated with early liver fibrosis. The early liver fibrosis mentioned above refers to stage S1 liver fibrosis.

2. The application as described in claim 1, characterized in that, Samples with a serum cathepsin S protein content ≥34.61±12.45 ng / mL were identified as biological samples associated with early stage S1 liver fibrosis; and Samples with an endostatin content ≥356.60±172.33 ng / mL were identified as biological samples associated with early stage S1 liver fibrosis.

3. The application as described in any one of claims 1-2, characterized in that, The reagents mentioned therein include those for detecting the expression levels of cathepsin S protein and endostatin.

4. The application as described in claim 3, characterized in that, The reagents used to detect the expression levels of cathepsin S protein and endostatin are enzyme-linked immunosorbent assay (ELISA) reagents.

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