WO2024125665A1 - 用于肝纤维化早期诊断的蛋白和检测试剂盒 - Google Patents

用于肝纤维化早期诊断的蛋白和检测试剂盒 Download PDF

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WO2024125665A1
WO2024125665A1 PCT/CN2024/071065 CN2024071065W WO2024125665A1 WO 2024125665 A1 WO2024125665 A1 WO 2024125665A1 CN 2024071065 W CN2024071065 W CN 2024071065W WO 2024125665 A1 WO2024125665 A1 WO 2024125665A1
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biological sample
endostatin
liver fibrosis
protein
cathepsin
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French (fr)
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徐平
金芳
武军驻
戴二黑
常蕾
高慧英
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北京谱峰源生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of molecular diagnosis and treatment, and in particular to proteins and reagents for early detection and diagnosis of liver cirrhosis.
  • Liver cirrhosis is a common disease with a high incidence rate. Its early pathological manifestation is liver fibrosis. Patients with liver cirrhosis are usually accompanied by a variety of serious complications, such as varicose bleeding, ascites, renal failure, etc. Moreover, once a patient is diagnosed with liver cirrhosis, the chance of developing liver cancer will also increase greatly (Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008; 371: 838-851.).
  • liver cirrhosis In the early stage of liver fibrosis, the patient's clinical symptoms are not obvious, and there is a lack of effective clinical diagnostic methods. Once the patient is diagnosed, it has often developed into the stage of liver cirrhosis.
  • the existing methods for diagnosing liver cirrhosis such as imaging and serological indicators (four liver fibrosis items: serum type III procollagen amino-terminal peptide, serum laminin, hyaluronic acid, serum type IV collagen), vary greatly among different patients with liver fibrosis and cirrhosis, and cannot provide fast and accurate diagnostic results; histopathological testing through liver puncture is the "gold standard" for the diagnosis of liver cirrhosis.
  • the treatment of cirrhosis also faces challenges.
  • the clinical treatment strategy for cirrhosis is mainly aimed at the cause of the disease.
  • virus-induced fibrosis is treated with antiviral agents such as interferon and nucleoside (acid) analogs (Mallet V, Gilgenkrantz H, Serpaggi J, Verkarre V, Vallet-Pichard A, Fontaine H, et al.
  • antiviral agents such as interferon and nucleoside (acid) analogs (Mallet V, Gilgenkrantz H, Serpaggi J, Verkarre V, Vallet-Pichard A, Fontaine H, et al.
  • Fat V interferon and nucleoside (acid) analogs
  • ECM family proteins such as elastin are believed to have an impact on ECM remodeling.
  • the CTSS protein of the cathepsin family is composed of 331 amino acids and can cleave collagen (Wilkinson RDA, Williams R, Scott CJ, Burden RE. Cathepsin S: Therapeutic, diagnostic, and prognostic potential. Biol. Chem. 2015; 396: 867-882.), which is closely related to the development of liver cirrhosis.
  • CTSS cathepsin S protein
  • liver fibrosis and cirrhosis Since the treatment of liver fibrosis and cirrhosis is more effective in early detection than in late stages, and the current diagnostic methods are not very sensitive and need to be confirmed by liver tissue puncture, it causes pain to patients and is difficult to confirm in the early stages of liver fibrosis.
  • One object of the present invention is to provide the use of cathepsin S protein (CTSS) and endostatin (endostatin) in detection reagents related to liver fibrosis.
  • CTSS cathepsin S protein
  • endostatin endostatin
  • Another object of the present invention is to provide a detection reagent for early diagnosis of peripheral blood samples related to liver fibrosis.
  • a reagent for specifically detecting protein expression is used in the preparation of a product for detecting a biological sample associated with liver fibrosis, wherein the protein is one or two selected from cathepsin S protein and endostatin; wherein when the cathepsin S protein content of a healthy person is 16.91 ⁇ 2.54ng/mL, a sample with a cathepsin S protein content ⁇ 31.61 ⁇ 9.49ng/mL in the biological sample is determined to be a biological sample associated with early liver fibrosis; and/or
  • a sample having an endostatin content of ⁇ 289.62 ⁇ 166.78 ng/mL in the biological sample is determined to be a biological sample associated with early liver fibrosis.
  • the biological sample of the present invention is peripheral blood.
  • the biological sample is serum.
  • the application of the present invention further comprises determining the biological sample having a cathepsin S protein content of ⁇ 34.61 ⁇ 12.45 ng/mL as a biological sample associated with liver fibrosis; and/or
  • the biological sample with an endostatin content of ⁇ 356.60 ⁇ 172.33 ng/mL is determined to be a biological sample associated with liver fibrosis.
  • the reagent comprises a protein for detecting cathepsin S and/or vascular
  • the reagent for detecting the expression level of endostatin is preferably an enzyme-linked immunosorbent assay reagent.
  • the two proteins of the present invention can be used as detection marker molecules respectively. When their content in the blood exceeds a certain value, they can independently judge the relationship between the detected biological sample and liver fibrosis. More preferably, the two proteins are combined as detection marker molecules because the combination of the two proteins shows better detection sensitivity and effectiveness than a single protein.
  • a detection reagent is used to detect protein expression in a biological sample, the detection reagent comprising: a reagent for detecting protein expression, the detection reagent comprising at least one of the following reagents:
  • the biological sample is peripheral blood
  • a sample having a cathepsin S protein content of ⁇ 31.61 ⁇ 9.49 ng/mL in the biological sample is determined to be a biological sample associated with early liver fibrosis;
  • the sample with the endostatin content ⁇ 289.62 ⁇ 166.78 ng/mL in the biological sample is determined to be a biological sample associated with early liver fibrosis.
  • a sample having a cathepsin S protein content of ⁇ 34.61 ⁇ 12.45 ng/mL in the biological sample is determined to be a biological sample associated with liver fibrosis;
  • the biological sample having an endostatin content of ⁇ 356.60 ⁇ 172.33 ng/mL is determined to be a biological sample associated with liver fibrosis.
  • the preferred reagent for detecting protein expression is enzyme-linked immunosorbent assay (ELISA).
  • the detection reagents of the present invention may be the following types of reagents:
  • the reagent may include an antibody that recognizes the CTSS protein and/or endostatin antigen epitope, may also include a complex formed by coupling the CTSS and/or endostatin antibody to a fluorescent probe, and may also include primers for amplifying the CTSS and/or endostatin encoding gene from the sample to be tested.
  • the detection reagent of the present invention may further include a reagent for detecting whether a polypeptide having an amino acid sequence such as SEQ ID NO.1 (LVALNSPLSGGMR) is present in the biological sample.
  • the polypeptide is a cleavage site of CTSS, which produces known endostatin, and the presence of CTSS protein and/or endostatin in the biological sample can be determined by detecting whether the polypeptide is present in the biological sample.
  • the present invention also provides a detection kit, the container of the kit contains
  • the reagents for detecting CTSS and/or endostatin protein or gene may be provided together with the manufacturing, use and sales information of the drugs or biological products reviewed by the government drug administration agency.
  • a detection kit comprising 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:
  • a sample having a cathepsin S protein content of ⁇ 31.61 ⁇ 9.49 ng/mL in the biological sample is determined as a biological sample associated with early liver fibrosis;
  • a sample having an endostatin content of ⁇ 289.62 ⁇ 166.78 ng/mL in the biological sample is determined to be a biological sample associated with early liver fibrosis.
  • the biological sample having a cathepsin S protein content of ⁇ 34.61 ⁇ 12.45 ng/mL is determined to be a biological sample associated with liver fibrosis;
  • the biological sample with an endostatin content of ⁇ 356.60 ⁇ 172.33 ng/mL is determined to be a biological sample associated with liver fibrosis.
  • the present invention also provides a method for early diagnosis of liver fibrosis, by detecting the expression levels of cathepsin S protein (CTSS) and endostatin (endostatin) in the peripheral blood of the diagnosed subject, and using the expression levels of the proteins in healthy people as a reference to diagnose whether liver fibrosis exists.
  • CTSS cathepsin S protein
  • endostatin endostatin
  • the present invention reveals the close connection between cathepsin S (CTSS) protein and endostatin and liver fibrosis, provides a method for detecting the expression of CTSS protein and endostatin using peripheral blood as a biological sample, and determines a criterion for determining early liver fibrosis and protein content of liver fibrosis using healthy people as a reference, which can be used for early non-invasive diagnosis of liver fibrosis.
  • CTSS cathepsin S
  • CTSS is upregulated in cirrhotic tissues.
  • CTS family proteins are upregulated in cirrhotic tissues as a whole, with CTSS having the highest upregulation.
  • CL refers to cirrhotic tissues
  • NL refers to normal tissues.
  • Figure 3 Distribution of CTSS content in human serum.
  • Figure 4 Distribution of CTSS content in human serum of patients with liver fibrosis of different etiologies.
  • Figure 5 Comparison of CTSS levels in human serum of patients at early stages of liver fibrosis and healthy volunteers.
  • Figure 6 Distribution of CTSS content in human serum of patients with liver fibrosis at different stages.
  • Figure 7 ROC curves of ALT, GP73 and IV collagen in patients with liver fibrosis.
  • Figure 8 ROC curve of CTSS in patients with liver fibrosis.
  • Figure 9 ROC curve of CTSS in patients with liver fibrosis.
  • Figure 10 ROC curves of ALT, GP73 and IV collagen in patients with early liver fibrosis.
  • FIG. 11 CTSS cleaves collagen 18 in vitro to produce endostatin.
  • FIG. 12 Time series analysis of CTSS cleaving collagen 18 to produce endostatin in vitro.
  • FIG. 13 CTSS cleaves collagen 18 to generate LVALNSPLSGGMR.
  • Figure 14 Endostatin levels are significantly elevated in patients with liver fibrosis.
  • Figure 15 Distribution of endostatin content in human serum.
  • Figure 16 Comparison of endostatin levels in human serum of patients with early liver fibrosis and healthy volunteers
  • Figure 17 Distribution of endostatin levels in human serum of patients with liver fibrosis at different stages.
  • Figure 18 ROC curves of CTSS and endostatin in patients with liver fibrosis.
  • Figure 19 Comparison of the changing trends of endostatin and CCTSS levels in the human serum of patients with early (S1) liver fibrosis stage and those of healthy volunteers.
  • Example 1 Discovery of high expression of CTSS in human liver fibrosis tissue
  • the protein of TCL was quantified by calculating the gray value using SDS-PAGE short gel.
  • CL1 the whole protein of 3 patients with cirrhosis
  • CL2 the whole protein of 3 patients with cirrhosis
  • the whole protein of 5 normal liver tissues (NL) was also taken and mixed in equal amounts, and marked as NL.
  • the decolorized and dried particles were treated with 12.5 ng/ ⁇ L 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.) was digested at 37°C for 14 hours. The samples after trypsin digestion were extracted with an extraction solution (5% formic acid, 50% acetonitrile) to obtain trypsin-digested liver fibrosis tissue and normal tissue total cell proteome samples.
  • the trypsin-digested CL1, CL2, and NL were labeled with different labeling reagents using the iTRAQ-4plex kit (AB Sciex, USA) according to the instructions. Among them, the CL1 sample was labeled with 114; the CL2 sample was labeled with 115; and the NL sample was divided into two equal parts and labeled with 116 and 117, respectively, as technical replicates (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.).
  • Liquid chromatography-mass spectrometry (LC-MS) (LTQ-Orbitrap Velos mass spectrometer, Thermo Fisher Scientific, USA) analysis method is as follows: 1 ⁇ g sample was loaded on a 3 ⁇ m C18 self-packed reversed phase separation column (75 ⁇ m i.d. ⁇ 15 cm) and analyzed with a 100-min liquid phase gradient. The liquid chromatography separation condition was acetonitrile concentration from 0-35%.
  • Mass spectrometry analysis conditions maximum ionization voltage 2.0 kV, scanning range 400-1800 m/z, automatic gain control (AGC) 1 ⁇ 106, maximum ion injection time 150 ms, and the resolution of the first scan at 400 m/z was 30000.
  • the second spectrum scanning mode was higher energy collision induced dissociation (Higher Energy Collision Induced Dissociation, HCD), 40% collision energy, automatic gain control 30000, dynamic exclusion 35 s.
  • HCD Higher Energy Collision Induced Dissoci
  • the obtained mass spectrometry raw files were searched by MaxQuant (v1.5.8.3) software for positive and negative libraries.
  • the search parameters were set as follows: parent ion error range of 20ppm, maximum missed cleavage sites of 2, minimum amino acid length of 7, and false discovery rate (FDR) less than 1.0%. Protein quantification was based on the abundance of reporter ions of unique peptides.
  • cathepsin S cathepsin S family proteins were identified to be upregulated in cirrhotic tissues as a whole, among which cathepsin S (CTSS) was upregulated by more than 1.5 times ( Figure 1 ), and the secondary spectrum of CTSS matched well, indicating that the identification of CTSS was correct ( Figure 2 ).
  • Example 2 CTSS can be used for non-invasive diagnosis of early liver fibrosis
  • the degree of fibrosis in human liver fibrosis patients is based on METAVIR (F) (An algorithm for the grading 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.) were used for determination.
  • the determination indicators are shown in Table 1.
  • Serum samples of human patients with liver fibrosis were collected from hospitals, totaling 164 cases, and the degree of liver fibrosis was diagnosed by experienced pathologists based on the patients' own liver puncture samples. Serum samples of healthy volunteers were donated, totaling 30 cases.
  • the serum level of CTSS was measured by Abcam's ELISA kit (Abcam, ab155427), and the results are shown in Figure 3.
  • the CTSS content in the serum of healthy volunteers was 16.91 ⁇ 2.54ng/mL (mean ⁇ SD)
  • the CTSS content in the serum of patients with liver fibrosis was 34.61 ⁇ 12.45ng/mL (mean ⁇ SD).
  • the measurement of serum CTSS can easily distinguish between patients with liver fibrosis and healthy people.
  • liver fibrosis 164 patients with fibrosis were grouped according to the cause of disease, and their serum CTSS was compared with that of healthy volunteers. It was found that regardless of the cause of liver fibrosis, the mean CTSS in the blood of patients with hepatitis B (HBV), hepatitis C (HCV), drug-induced liver fibrosis (DILI), non-alcoholic fatty liver disease (NAFLD) and other types of liver fibrosis were 1.93, 2.04, 2.88, 2.15 to 2.11 times higher than the mean serum CTSS of healthy people, respectively, and the levels were extremely significant (p ⁇ 0.01), which is a universal characteristic (Figure 4).
  • HBV hepatitis B
  • HCV hepatitis C
  • DILI drug-induced liver fibrosis
  • NAFLD non-alcoholic fatty liver disease
  • ALT Alanine aminotransferase
  • GP73 Golgi protein 73
  • type IV collagen are commonly used markers of liver fibrosis.
  • the receiver operating characteristic curve (ROC curve) analysis was performed using ALT, GP73 and IV collagen in the blood of all 164 patients with liver fibrosis. The areas under the curve were 0.76, 0.643 and 0.928, respectively ( Figure 7), which were lower than the area under the curve of the ROC curve made by CTSS (0.951) ( Figure 8). This shows that CTSS is significantly better than existing liver fibrosis indicators such as ALT, GP73 and IV collagen as an indicator of liver fibrosis.
  • the ROC curve analysis of ALT showed an area under the curve of 0.818, a sensitivity of 71.9%, and a specificity of 80% (Figure 10);
  • the ROC curve analysis of GP73 showed an area under the curve of 0.539, a sensitivity of 78.1%, and a specificity of 50% (Figure 10);
  • the ROC curve analysis of 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), and the predictive diagnostic value was significantly lower than that of CTSS.
  • CTSS is the best marker molecule for the diagnosis of early (S1) liver fibrosis.
  • Example 3 CTSS can cleave collagen 18 to produce endostatin
  • 0.6 ⁇ M recombinant collagen 18 was reacted with a reaction system (50mM sodium acetate pH 5.5, 2mM dithiothreitol (DTT) and 5mM EDTA) with or without 20nM human CTSS at 37°C for 2 hours.
  • the reaction products were subjected to SDS-PAGE electrophoresis, and after blotting and transfer, WB analysis was performed with collagen 18 antibody or endostatin antibody.
  • Example 4 Endostatin can be used for early non-invasive diagnosis of liver fibrosis
  • the total cell proteins of livers of 5 patients with liver fibrosis and 3 healthy people were electrophoresed by SDS-PAGE gel electrophoresis, and the electrophoresis products were transferred to membranes.
  • Western blot analysis with anti-collagen 18 and anti-endostatin was performed, and it was found that the content of endostatin in the liver tissues of patients with liver fibrosis was significantly higher than that in healthy controls.
  • Serum samples from 164 patients with liver fibrosis and 30 healthy volunteers were analyzed.
  • the serum level of endostatin was measured using the ELISA kit (Cat#SEA542Hu) of Cloud-Clone Company, and the results are shown in Figure 15.
  • the endostatin content in the serum of healthy volunteers was 147.65 ⁇ 73.50ng/mL (mean ⁇ SD), and the endostatin content in the serum of patients with liver fibrosis was 356.60 ⁇ 172.33ng/mL (mean ⁇ SD).
  • the measurement of serum endostatin can easily distinguish between patients with liver fibrosis and healthy people.
  • CTSS and endostatin combination were used in 32 patients with early (S1) liver fibrosis.
  • the area under the ROC curve analysis was as high as 0.989, which was higher than the area under the ROC curve of 0.979 ( Figure 18) for early (S1) liver fibrosis using only CTSS, indicating that the combined use 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.

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Abstract

通过揭示组织蛋白酶 S（CTSS）蛋白和血管内皮抑制素（endostatin）与肝纤维化之间的紧密联系，提供了以外周血为生物样本检测 CTSS 蛋白和血管内皮抑制素的表达量，并确定了以健康人为参照判定早期肝纤维化和肝纤维化的蛋白含量判定标准，可早期无创诊断肝纤维化的应用。

Description

用于肝纤维化早期诊断的蛋白和检测试剂盒 技术领域
本发明涉及分子诊断和治疗领域，具体涉及肝硬化的早期检测和诊断的蛋白和试剂。
背景技术
肝硬化是一种常见的、发病率较高的疾病，其早期病理表现为肝纤维化。肝硬化患者通常伴随多种严重的并发症，如静脉曲张出血、肝腹水、肾衰竭等。并且，患者一旦确诊为肝硬化，发展成肝癌的几率也将大大增加(Schuppan D,Afdhal NH.Liver cirrhosis.Lancet.2008；371:838-851.)。
在早期肝纤维化阶段，患者临床症状不明显，又缺乏有效的临床诊断方法，患者一旦确诊往往已经发展到肝硬化阶段。目前已有的肝硬化诊断方法，比如影像学和血清学指标(肝纤四项：血清III型前胶原氨基端肽，血清层粘连蛋白，透明质酸，血清Ⅳ型胶原)在不同肝纤维化和肝硬化患者中变异较大，并不能提供快速准确的诊断结果；通过肝穿刺进行的组织病理学检测是肝硬化确诊的“金标准”。然而穿刺对患者造成的风险较大(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.)。目前，临床缺乏快速准确又无创的肝纤维化/肝硬化诊断方法。
肝硬化的治疗同样面临挑战。临床肝硬化治疗策略主要是针对病因的治疗。比如病毒诱发的纤维化通过干扰素、核苷(酸)类似物等进行抗病毒治疗(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 in chronic hepatitis C.Ann.Intern.Med.2008；149:399-403.)，在早期纤维化阶段可得到较好的治疗效果。然而，在肝硬化阶段，临床上主要通过抑制并发症进行治疗，并不能达到治疗肝硬化的目的(Tsochatzis EA,Bosch J,Burroughs AK.Liver cirrhosis.In:The Lancet.2014.p.1749-1761.)。因此，病理机制不明是造成肝硬化诊断和治疗困难的主要原因。已有研究表明，细胞外基质(Extracellular Matrix，ECM)重塑失调导致的细胞外基质在肝组织表面大量沉积是肝硬化的重要病理机制(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.)。如弹性蛋白等ECM家族蛋白，被认为具有影响ECM重 塑过程的重要酶类，而组织蛋白酶家族的CTSS蛋白由331个氨基酸组成，能酶切胶原(Wilkinson RDA,Williams R,Scott CJ,Burden RE.Cathepsin S:Therapeutic,diagnostic,and prognostic potential.Biol.Chem.2015；396:867-882.)，和肝硬化发生发展密切相关。但限于技术条件，组织蛋白酶家族的CTSS蛋白在哪个氨基酸残基切割胶原蛋白并不清楚。
发明专利申请CN201811239261.3为申请人之前的研究，通过定量蛋白质组学技术和免疫组化实验发现组织蛋白酶S蛋白(CTSS)在肝硬化组织中上调表达；通过将CTSS抗体和荧光探针偶联注射到CCl4诱导的肝硬化小鼠模型中，在CCl4诱导产生的肝硬化小鼠的肝脏观测到显著荧光亮度；用CCl4诱导处理野生型(WT)和CTSS-/-型小鼠，发现CTSS敲除组肝硬化小鼠模型肝硬化程度显著缓解。因此，确定了CTSS蛋白与肝硬化之间存在密切的关联。但在检测上仍然需要依赖肝脏组织的提供。
由于肝纤维化和肝硬化的治疗在早期的发现和治疗效果比晚期好，而目前的诊断方法灵敏度不高，需要通过肝组织穿刺进行确认，给患者造成痛苦且难以在肝纤维化的早期确证。
发明内容
本发明的一个目的是提供组织蛋白酶S蛋白(CTSS)和血管内皮抑制素(endostatin)在与肝纤维化相关的检测试剂中的应用。
本发明的另一个目的是提供一种检测试剂，用于与肝纤维化有关的外周血样品的早期诊断。
根据本发明的一个方面，一种特异性检测蛋白表达的试剂在制备检测与肝纤维化相关的生物样品的产品中的应用，其中所述蛋白为选自组织蛋白酶S蛋白和血管内皮抑制素中的一种或二种；其中在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或
在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品。
本发明所述的生物样品为外周血。优选的是所述生物样品为血清。
本发明所述的应用，进一步地，将所述生物样品中组织蛋白酶S蛋白含量≥34.61±12.45ng/mL的样品判定为与肝纤维化相关的生物样品；和/或
将所述生物样品中血管内皮抑制素含量≥356.60±172.33ng/mL的样品判定为与肝纤维化相关的生物样品。
本发明的所述的应用，其中所述试剂包含检测组织蛋白酶S蛋白和/或血管 内皮抑制素表达量的试剂，优选的是酶联免疫检测试剂。
本发明的两个蛋白组织蛋白酶S蛋白(CTSS)和血管内皮抑制素(endostatin)可以分别作为检测标志分子，其在血液中含量超过一定量值时，各自独立地判断所检测的生物样本与肝纤维化的关系，更优选的是，将二个蛋白组合作为检测标志分子，因为二个蛋白的组合显示出比单一蛋白更好的检测灵敏度和有效性。
根据本发明的另一个方面，一种检测试剂，用于检测生物样本中的蛋白表达，所述检测试剂包括：检测蛋白表达量的试剂，所述检测试剂至少包括下述试剂之一：
检测组织蛋白酶S蛋白表达量的试剂，以及
检测血管内皮抑制素表达量的试剂；
所述生物样本为外周血；并且
在所述检测组织蛋白酶S蛋白表达量的试剂中，在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或
在所述检测血管内皮抑制素表达量的试剂中，在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品。
进一步地，其中在所述检测组织蛋白酶S蛋白表达量的试剂中，将所述生物样品中组织蛋白酶S蛋白含量≥34.61±12.45ng/mL的样品判定为与肝纤维化相关的生物样品；和/或
在所述检测血管内皮抑制素表达量的试剂中，将所述生物样品中血管内皮抑制素含量≥356.60±172.33ng/mL的样品判定为与肝纤维化相关的生物样品。
优选的检测蛋白表达量的试剂为酶联免疫试剂。
在一般的应用中，本发明所述的检测试剂可以是下述类型的试剂：
抗体、引物、探针、测序文库、核酸芯片(基因芯片)、蛋白质芯片或上述的组合。
因此该试剂可以包含识别CTSS蛋白和/或血管内皮抑制素抗原表位的抗体，也可以包含与CTSS和/或血管内皮抑制素抗体偶联荧光探针形成的复合物，还可以包含从待测样品中扩增CTSS和/或血管内皮抑制素编码基因的引物。
本发明所述的检测试剂可以进一步包括检测所述生物样品中是否存在氨基酸序列如SEQ ID NO.1(LVALNSPLSGGMR)所示的多肽的试剂。该多肽为CTSS的切割位点，产生已知的血管内皮抑制素，可以通过检测所述生物样品中是否存在该多肽而判断所述生物样品中CTSS蛋白和/或血管内皮抑制素的存在。
在上述诊断试剂的基础上，本发明也提供检测试剂盒，试剂盒容器内装有 用以检测CTSS和/或血管内皮抑制素蛋白或基因的试剂，与之同时提供的可以是经政府药物管理机构审核的、有关药品或生物制品的制造、使用及销售信息。
根据本发明的再一方面，提供一种检测试剂盒，其中所述试剂盒包含检测组织蛋白酶S蛋白和/或血管内皮抑制素表达量的试剂，以及试剂盒说明书，其中所述试剂盒说明书中包含下述信息：
在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或
在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品。
进一步地，将所述生物样品中组织蛋白酶S蛋白含量≥34.61±12.45ng/mL的样品判定为与肝纤维化相关的生物样品；和/或
将所述生物样品中血管内皮抑制素含量≥356.60±172.33ng/mL的样品判定为与肝纤维化相关的生物样品。
本发明还提供一种早期诊断肝纤维化的方法，通过对来自诊断对象的外周血中组织蛋白酶S蛋白(CTSS)和血管内皮抑制素(endostatin)表达水平的检测，以健康人的所述蛋白的表达水平为参考，诊断是否存在肝纤维化。
本发明通过揭示组织蛋白酶S(CTSS)蛋白和血管内皮抑制素(endostatin)与肝纤维化之间的紧密联系，提供了以外周血为生物样本检测CTSS蛋白和血管内皮抑制素的表达量，并确定了以健康人为参照判定早期肝纤维化和肝纤维化的蛋白含量判定标准，可早期无创诊断肝纤维化的应用。
附图说明
图1：CTSS在肝硬化组织中上调表达。CTS家族蛋白在肝硬化组织中整体上调，CTSS上调幅度最高，“CL”为肝硬化组织，“NL”为正常组织。
图2：CTSS的MS2质谱谱图。
图3：人血清中CTSS的含量分布。
图4：不同病因肝纤维化患者人血清中CTSS的含量分布。
图5：早期肝纤维化阶段患者人血清中CTSS含量与健康志愿者的比较。
图6：不同阶段肝纤维化维化患者人血清中CTSS的含量分布。
图7：肝纤维化维化患者ALT、GP73和IV胶原的ROC曲线。
图8：肝纤维化维化患者CTSS的ROC曲线。
图9：肝纤维化维化患者CTSS的ROC曲线。
图10：早期肝纤维化维化患者ALT、GP73和IV胶原的ROC曲线。
图11：CTSS体外切割胶原18产生血管内皮抑制素endostatin。
图12：CTSS体外切割胶原18产生血管内皮抑制素的时间序列分析。
图13：CTSS切割胶原18产生LVALNSPLSGGMR。
图14：肝纤维化患者血管内皮抑制素含量显著升高。
图15：人血清中血管内皮抑制素的含量分布。
图16：早期肝纤维化阶段患者人血清中血管内皮抑制素含量与健康志愿者的比较
图17：不同阶段肝纤维化维化患者人血清中血管内皮抑制素的含量分布。
图18：肝纤维化维化患者CTSS和血管内皮抑制素的ROC曲线。
图19：早期(S1期)肝纤维化阶段患者人血清中血管内皮抑制素和CCTSS含量与健康志愿者的含量变化趋势比较。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明可以通过以下实例更容易地理解，但本发明并不仅限于这些实例。
实施例1、人肝纤维化组织中CTSS高表达的发现
人肝硬化和正常肝样本来自肝硬化手术治疗的患者，由临床取材，并进一步由病理科医生通过组织的病理学分析。
取50mg新鲜肝组织放入预冷研钵中，加液氮研磨，收集粉末到1.5mL Eppendorf管中。加入300μL细胞裂解液，超声细胞破碎仪裂解5-8min至溶液澄清透明，得到细胞裂解液(Total cell lysate，TCL)，4℃离心机最大转速离心5min，分装。
利用SDS-PAGE短胶通过计算灰度值对TCL进行蛋白定量。为减少个体差异对定量蛋白质组数据的影响，分别取3例肝硬化患者的全蛋白，混样，标记为“CL1”；另外取3例肝硬化患者的全蛋白，混样，标记为“CL2”。5例正常肝组织全蛋白(NL)也分别取等量蛋白进行混匀，标记为NL。
分别取“CL1”、“CL2”和“NL”等3组样品各100μg全蛋白，用SDS-PAGE短胶进行样品净化(Systematical optimization of reverse-phase chromatography for shotgun proteomics.J Proteome Res.2009,8:3944-50.)。SDS-PAGE胶经考马斯亮蓝染色、脱色后，染色区域的胶片分别切成1立方毫米的胶粒，进一步脱色、脱水(Systematical optimization of reverse-phase chromatography for shotgun proteomics.J Proteome Res.2009,8:3944-50.)。
对脱色、干燥的胶粒分别用12.5ng/μL的胰蛋白酶(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.)在37℃消化14小时。胰酶消化后的样品经抽提液(5％甲酸，50％乙腈)抽提，得到胰酶消化的肝纤维化组织和正常组织总细胞蛋白组样品。
用iTRAQ-4plex试剂盒(AB Sciex，美国)按照说明书对胰酶消化的CL1、CL2、NL分别进行不同标签试剂的标记。其中，CL1样品用114标记；CL2样品用115标记；NL等分为二，分别用116和117标签标记，作为技术重复(Down-regulation of RIP3potentiates cisplatin chemoresistance by triggering HSP90-ERK pathway mediated DNA repair in esophageal squamous cell carcinoma,Cancer Letters,2018,418:97-108.)。
为了检测标记效率，从4组样品中各取出1μL混合，用脱盐小柱脱盐后进行质谱鉴定(Down-regulation of RIP3potentiates cisplatin chemoresistance by triggering HSP90-ERK pathway mediated DNA repair in esophageal squamous cell carcinoma,Cancer Letters,2018,418:97-108.)。
液相色谱-质谱联用(LC-MS)(LTQ-Orbitrap Velos质谱仪，Thermo Fisher Scientific，美国)分析方法如下：取1μg样品上样3μm C18自主填充的反相分离柱(75μm i.d.×15cm)，以100分钟的液相梯度进行分析。液相色谱分离条件是乙腈浓度从0-35％。质谱分析条件：最高电离电压2.0kV，扫描范围400-1800m/z，自动增益控制(automatic gain control，AGC)1×106，最大离子注入时间150ms，一级扫描在400m/z的分辨率为30000。二级谱扫描模式为高能碰撞诱导解离(Higher Energy Collision Induced Dissociation，HCD)，40％碰撞能量，自动增益控制30000，动态排除35s。
将获得质谱原始raw文件通过MaxQuant(v1.5.8.3)软件进行正反库搜库，搜库参数设置为：母离子误差范围为20ppm，最大漏切位点为2，氨基酸长度最小为7，假阳性率(false discovery rate，FDR)小于1.0％。蛋白定量的依据为unique肽段的报告离子(reporter ion)的丰度。
我们将差异蛋白差值标准设为肝硬化肝(CL)/正常肝(NL)(ratio CL/NL)比例大于1.5倍或者小于-1.5倍且p value<0.05，共鉴定到201个上调差异蛋白和85个下调表达蛋白。
在上调的差异蛋白中鉴定到组织蛋白酶(CTS)家族蛋白在肝硬化组织中整体上调，其中组织蛋白酶S(CTSS)上调超过1.5倍(图1)，并且CTSS的二级谱匹配较好，提示CTSS鉴定正确(图2)。
实施例2、CTSS可进行肝纤维化早期无创诊断
人肝纤维化患者纤维化程度根据METAVIR(F)(An algorithm for the grading  of activity in chronic hepatitis C.The METAVIR Cooperative Study Group,Hepatology,1996,24:289-293.)和中国2000(病毒性肝炎的防治指南,中华肝脏病杂志,2000,8:324-329.)判定。判断指标见表1。
表1.肝纤维化病理分期评估标准对照表
人肝纤维化患者血清样本来自医院，共164例，其肝纤维化程度由患者本身肝穿刺样本，由有经验的病理科医生诊断。健康志愿者血清样本来自捐献，共30例。
血清水平的CTSS以Abcam公司的ELISA试剂盒(Abcam,ab155427)进行测定，结果见图3。其中健康志愿者血清中CTSS含量为16.91±2.54ng/mL(mean±SD)，肝纤维化患者的血清中CTSS含量为34.61±12.45ng/mL(mean±SD)，两组之间具有极显著差异(p<0.01)，且纤维化患者血清的CTSS是健康志愿者的2倍，可借助血清CTSS的测量容易区分肝纤维化患者人群和健康人群。
将164例纤维化患者根据病因进行分组，将其血清CTSS与健康志愿者的血清CTSS进行比较，发现无论什么病因所致的肝纤维化，乙肝(HBV)、丙肝(HCV)、药物性损伤引发的肝纤维化(DILI)、非酒精性脂肪肝(NAFLD)和其它类型肝纤维化患者血液的CTSS的均值比健康人群的血清CTSS均值分别高了1.93、2.04、2.88、2.15倍到2.11倍，且极显著(p<0.01)，具有普遍性特点(图4)。
早期肝纤维化易被逆转，治愈，因此具有临床治疗价值。但现有的技术都难于判断早期肝纤维化。将164例纤维化患者中经肝穿刺这一金标准检验的32例早期(S1期)肝纤维化的患者血样单独取出，并与健康志愿者血样同时进行CTSS分析，发现早期(S1期)肝纤维化患者血样中CTSS值为31.61±9.49ng/mL(mean±SD)，比健康志愿者的16.91±2.54ng/mL(mean±SD)提高了1.87倍(图5)，统计学差异显著。
为比较人肝纤维化不同阶段CTSS含量的分布，将164例纤维化患者根据患 者肝纤维化不同阶段进行分组，将其血清CTSS与健康志愿者的血清CTSS进行比较，发现无论S1、S2、S3还是S4，肝纤维化患者血液的CTSS的均值比健康人群的血清CTSS均值分别高了1.87、2.50、2.36倍到1.81倍，且极显著(p<0.01)(图6)。
丙氨酸转氨酶(ALT)是肝损伤的标志分子；高尔基体蛋白73(GP73)和IV型胶原是肝纤维化常用标志分子。分别以所有164例肝纤维化患者血液的ALT、GP73和IV胶原做受试者工作特征曲线(ROC曲线)分析，曲线下面积分别为0.76，0.643和0.928(图7)，均低于CTSS所做的ROC曲线的曲线下面积(0.951)(图8)。表明以CTSS作为肝纤维化判断指标，显著优于ALT、GP73和IV胶原等现有的肝纤维化指标。
以32例早期(S1期)肝纤维化患者血液的CTSS做ROC曲线分析，其曲线下面积高达0.969，灵敏度为96.9％，特异性为93.3％(图9)，具有良好的预测性能。与此对应，以ALT做ROC曲线分析，曲线下面积为0.818，灵敏度为71.9％，特异性为80％(图10)；以GP73做ROC曲线分析，曲线下面积为0.539，灵敏度为78.1％，特异性为50％(图10)；以IV型胶做ROC曲线分析，曲线下面积为0.922，灵敏度为81.3％，特异性为93.3％(图10)，预测诊断价值均显著低于CTSS。CTSS是用于早期(S1期)肝纤维化诊断最优的标志分子。
实施例3、CTSS可以切割胶原18产生血管内皮抑制素
以0.6μM的重组胶原18分别与加和不加20nM的人CTSS的反应体系(50mM pH 5.5的乙酸钠、2mM二硫苏糖醇(DTT)和5mM的EDTA)溶液中在37℃反应2小时。反应产物经SDS-PAGE电泳，印迹转膜后以胶原18的抗体或血管内皮抑制素(endostatin)抗体进行WB分析，发现加有CTSS的反应中新生成了显著量的血管内皮抑制素，但未加CTSS的反应管中没有血管内皮抑制素产生；且加有CTSS产生血管内皮抑制素的管中，其胶原18的量显著减少(图11)，证明CTSS可特异切割胶原18，产生血管内皮抑制素。
为证实这个体外反应的特异性和效率，我们以上述相同的体系进行CTSS切割胶原反应，并在反应开始后15和60分钟分别取样，进行SDS-PAGE分离，电泳产物进行银染。发现凝胶电泳银染胶图清晰(图12)；反应后15分钟即可见血管内皮抑制素的条带，且该条带随着时间反应时间延长到60分钟而显著增强；而随着反应的进行，60kD处胶原18的量逐渐减少，进一步证实了CTSS切割胶原18反应的特异高效性。
为确定CTSS切割胶原18的确切位点，我们将SDS-PAGE凝胶电泳分离的血管内皮抑制素胶条与40mM甲醛和20mM氰基硼氢化钠溶液于37℃反应4小时，并以0.1M Tris溶液(pH 6.8)终止反应。标记后的蛋白以12.5ng/μL胰 蛋白酶按照上述蛋白质组学样品制备技术进行胶内消化、肽段提取个LC-MS分析。我们发现来自18型胶原的肽段LVALNSPLSGGMR(SEQ ID NO.1)的L被特异标记，是CTSS的切割位点，产生已知的血管内皮抑制素。以化学合成的L位标记的肽段L*VALNSPLSGGMR，证实质谱碎裂谱图与我们发现的谱图完全一致，证实是LVALNSPLSGGMR(图13)。以现有公知的技术定量CTSS切割胶原18新生的包括切割位点的肽段，也可以判断早期肝纤维化。
实施例4、血管内皮抑制素可进行肝纤维化早期无创诊断
分别取50mg新鲜的人肝纤维化患者和健康人肝组织，放入预冷研钵中，加液氮研磨，收集粉末到1.5mL Eppendorf管中。加入300μL细胞裂解液，超声细胞破碎仪裂解5-8min至溶液澄清透明，得到细胞裂解液(Total cell lysate，TCL)，4℃离心机最大转速离心5min，分装。
将5例肝纤维化患者和3例健康人的肝脏总细胞蛋白利用SDS-PAGE凝胶电泳，电泳产物进行印迹转膜。分别以抗胶原18、抗血管内皮抑制素进行WB分析，发现肝纤维化患者的肝组织中血管内皮抑制素含量显著高于健康对照。
以164例人肝纤维化患者血清样本和30例健康志愿者血清样本进行分析。血清水平的血管内皮抑制素以云克隆公司的ELISA试剂盒(Cat#SEA542Hu)进行测定，结果见图15。其中健康志愿者血清中血管内皮抑制素含量为147.65±73.50ng/mL(mean±SD)，肝纤维化患者的血清中血管内皮抑制素含量为356.60±172.33ng/mL(mean±SD)，两组之间具有极显著差异(p<0.01)，且纤维化患者血清的血管内皮抑制素是健康志愿者的2.42倍，可借助血清血管内皮抑制素的测量容易区分肝纤维化患者人群和健康人群。
早期肝纤维化易被逆转，治愈，因此具有临床治疗价值。但现有的技术都难于判断早期(S1期)肝纤维化。将164例纤维化患者中经肝穿刺这一金标准检验的32例早期(S1期)肝纤维化的患者血样单独取出，并与健康志愿者血样同时进行血管内皮抑制素分析，发现早期(S1期)肝纤维化患者血样中血管内皮抑制素值为289.62±166.78ng/mL(mean±SD)，比健康志愿者的147.65±73.50ng/mL(mean±SD)提高了1.96倍(图16)，统计学差异显著。
为比较人肝纤维化不同阶段CTSS含量的分布，将164例纤维化患者根据患者肝纤维化不同阶段进行分组，将其血清血管内皮抑制素与健康志愿者的血清血管内皮抑制素进行比较，发现无论S1、S2、S3还是S4，肝纤维化患者血液的血管内皮抑制素的均值比健康人群的血清CTSS均值分别高了1.96、2.30、1.78倍到2.80倍，且极显著(p<0.01)(图17)，表明血管内皮抑制素也可有效指针早期肝纤维化。
以32例早期(S1期)肝纤维化患者血液的CTSS和血管内皮抑制素组合使 用，做ROC曲线分析，其曲线下面积高达0.989，高于仅用CTSS的早期(S1期)肝纤维化ROC分析曲线下面积的0.979(图18)，表明组合使用CTSS和血管内皮抑制素可更为有效地预测和诊断早期肝纤维化。CTSS和血管内皮抑制素是用于早期肝纤维化诊断的有效标志分子。
进一步比较了CTSS和血管内皮抑制素在早期(S1期)肝纤维化患者血样中对应关系，我们根据血清中血管内皮抑制素和CTSS的浓度，计算他们相对正常值(健康对应的血清含量)的Z-score值。如果Z-score大于0表示高于正常值，Z-score小于0表示低于正常值。发现7个早期(S1期)纤维化患者的血管内皮抑制素血清含量低于正常值(结果图在视觉上中显示6个点，原因是有2个患者的值非常接近，导致两个点重合)，而所有患者的CTSS血清含量均高于正常值(图19)。因此单以患者血管内皮抑制素血清含量判断肝纤维化可能存在一定风险，组合使用更为可靠。

Claims (13)

  1. 一种特异性检测蛋白表达的试剂在制备检测与肝纤维化相关的生物样品的产品中的应用，其特征在于所述蛋白为选自组织蛋白酶S蛋白和血管内皮抑制素中的一种或二种，所述生物样品为外周血；
    其中在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或
    在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品。
  2. 如权利要求1所述的应用，其特征在于，其中将所述生物样品中组织蛋白酶S蛋白含量≥34.61±12.45ng/mL的样品判定为与肝纤维化相关的生物样品；和/或
    将所述生物样品中血管内皮抑制素含量≥356.60±172.33ng/mL的样品判定为与肝纤维化相关的生物样品。
  3. 如权利要求1-2任一项所述的应用，其特征在于其中所述试剂包含检测组织蛋白酶S蛋白和/或血管内皮抑制素表达量的试剂。
  4. 如权利要求3所述的应用，其特征在于，其中所述检测组织蛋白酶S蛋白和/或血管内皮抑制素表达量的试剂为酶联免疫试剂。
  5. 如权利要求1所述的应用，其特征在于，所述试剂中将组织蛋白酶S蛋白和血管内皮抑制素组合用于检测。
  6. 如权利要求1所述的应用，其特征在于，所述生物样本为血清。
  7. 一种检测试剂，用于检测生物样本中的蛋白表达，其特征在于所述检测试剂至少包括下述试剂之一：
    检测组织蛋白酶S蛋白表达量的试剂，以及
    检测血管内皮抑制素表达量的试剂；
    所述生物样本为外周血；并且
    在所述检测组织蛋白酶S蛋白表达量的试剂中，在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或在所述检测血管内皮抑制素表达量的试剂中，在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品。
  8. 如权利要求7所述的检测试剂，其特征在于其中在所述检测组织蛋白酶S蛋白表达量的试剂中，将所述生物样品中组织蛋白酶S蛋白含量 ≥34.61±12.45ng/mL的样品判定为与肝纤维化相关的生物样品；和/或
    在所述检测血管内皮抑制素表达量的试剂中，将所述生物样品中血管内皮抑制素含量≥356.60±172.33ng/mL的样品判定为与肝纤维化相关的生物样品。
  9. 如权利要求7所述的检测试剂，其中所述检测蛋白表达量的试剂为酶联免疫试剂。
  10. 如权利要求7所述的检测试剂，其特征在于进一步包括检测所述生物样品中是否包含氨基酸序列如SEQ ID NO.1所示的多肽的试剂。
  11. 一种检测试剂盒，含有权利要求7-10任一项的检测试剂。
  12. 一种早期诊断肝纤维化的方法，包括用来自诊断对象的生物样品进行选自组织蛋白酶S蛋白和血管内皮抑制素中的一种或二种的表达水平的检测，其中在健康人的组织蛋白酶S蛋白含量为16.91±2.54ng/mL的情况下，将所述生物样品中组织蛋白酶S蛋白含量≥31.61±9.49ng/mL的样品判定为与早期肝纤维化相关的生物样品；和/或
    在健康人的血管内皮抑制素含量为147.65±73.50ng/mL的情况下，将所述生物样品中血管内皮抑制素含量≥289.62±166.78ng/mL的样品判定为与早期肝纤维化相关的生物样品；
    所述生物样品为外周血。
  13. 如权利要求12所述的方法，其中进一步包括检测所述生物样品中是否包含氨基酸序列如SEQ ID NO.1所示的多肽。
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