A biomarker for the diagnosis of liver fibrosis and use thereof

Abstract

The present application provides a group of plasma markers for liver fibrosis diagnosis and application thereof. Specifically, the present application provides a biomarker, which is matrix cell protein CCN5. The biomarker of the present application can be used for evaluating the diagnosis of liver fibrosis of a subject to be tested, early diagnosis of liver fibrosis, and diagnosis of the severity of liver fibrosis. The present application also provides a kit comprising the biomarker and application of the kit in the diagnosis of liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a biomarker for the diagnosis of liver fibrosis and its application. Background Technology

[0002] Liver fibrosis is a crucial pathological process in the development of various chronic liver diseases and is considered an early stage of cirrhosis. Major liver diseases include viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease. It is estimated that there are over 400 million people with chronic liver disease in my country. Most chronic liver diseases eventually lead to liver fibrosis, which is an essential pathological process in the progression of serious and fatal diseases such as cirrhosis and liver cancer. Early diagnosis and timely treatment of liver fibrosis are crucial for slowing disease progression. However, the burden of liver fibrosis is increasingly heavy, becoming a serious public health problem, making its prevention and treatment urgent. How to effectively prevent and treat liver fibrosis, especially the search for accurate plasma diagnostic biomarkers, has become a key focus of current research.

[0003] The incidence of liver fibrosis is on the rise, posing a serious threat to public health. Diagnosis of liver fibrosis requires a combination of medical history, symptoms, physical examination, and various laboratory tests. Plasma biomarkers are widely used in assessing the degree of fibrosis and predicting prognosis.

[0004] Currently, the detection of biomarkers for liver fibrosis mainly focuses on macromolecules, such as hyaluronic acid (HA), laminin (LN), type III procollagen (PCIII), and type IV collagen (IV-C). The detection methods for these biomarkers typically employ immunological approaches, and their specificity and accuracy need improvement, thus failing to fully meet clinical needs. Furthermore, the results of these biomarker tests are easily affected by multiple factors, including individual patient differences, sample preservation conditions, and medications.

[0005] Currently, the biomarkers for liver fibrosis are not clearly defined. Therefore, there is an urgent need to further explore plasma biomarkers that are highly specific, sensitive, and stable in order to promote the early diagnosis and personalized treatment of liver fibrosis. Summary of the Invention

[0006] The purpose of this invention is to provide a plasma biomarker for the diagnosis of liver fibrosis and its application.

[0007] In a first aspect of the invention, there is provided the use of a gene, mRNA, cDNA, protein, or a detection reagent for a biomarker for preparing a kit or reagent for (a) diagnosing liver fibrosis; (b) making an early diagnosis of liver fibrosis; and / or (c) diagnosing the severity of liver fibrosis; wherein the biomarker is CCN5.

[0008] In another preferred embodiment, the detection reagent or kit is used to detect the expression level of the biomarker in the sample to be tested.

[0009] In another preferred embodiment, the expression level includes protein expression level or mRNA expression level.

[0010] In another preferred embodiment, the CCN5 gene includes a wild-type CCN5 gene and a mutant CCN5 gene.

[0011] In another preferred embodiment, the liver fibrosis is selected from the group consisting of: viral hepatitis cirrhosis, alcoholic cirrhosis, non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease, cholestatic liver disease, drug-induced liver injury, and cardiac cirrhosis.

[0012] In another preferred embodiment, the biomarker is derived from blood, plasma, or serum samples.

[0013] In another preferred embodiment, an increase in the level (e.g., content) of each biomarker among the biomarkers, compared to a reference value, indicates that the subject has liver fibrosis.

[0014] In another preferred embodiment, the biomarker is used to assess a diagnosis of liver fibrosis in the subject.

[0015] A second aspect of the present invention provides a kit for the diagnosis of liver fibrosis, the kit comprising a detection reagent for detecting the genes, mRNA, cDNA, or proteins, or combinations thereof, of the biomarkers described in the first aspect of the present invention.

[0016] In another preferred embodiment, the detection reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0017] In another preferred embodiment, the detection reagent includes a nucleic acid reagent for detecting CCN5 mRNA or cDNA.

[0018] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0019] In another preferred embodiment, the diagnostic reagent or kit contains one or more detection reagents selected from the group consisting of:

[0020] (a) Specific antibodies against CCN5, specific binding molecules of CCN5; and / or

[0021] (b) Primers or primer pairs, probes or chips for specific amplification of CCN5 mRNA or CCN5 cDNA.

[0022] In another preferred embodiment, the detection reagent is coupled with or carries a detectable marker.

[0023] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0024] In another preferred embodiment, the specific antibody for CCN5 is a monoclonal antibody or a polyclonal antibody.

[0025] In another preferred embodiment, the expression level of CCN5 protein in the sample is detected by RT-PCR or immunohistochemistry.

[0026] In another preferred embodiment, the gene, mRNA, cDNA, or protein of CCN5 is derived from a subject suffering from liver fibrosis or chronic liver disease.

[0027] In another preferred embodiment, the subject is a human or a non-human mammal.

[0028] In another preferred embodiment, the detection is for an ex vivo sample; preferably, the ex vivo sample is selected from the group consisting of blood, plasma, serum, or combinations thereof.

[0029] In another preferred embodiment, the kit contains genes, mRNA, cDNA, and / or proteins of liver fibrosis biomarkers as controls or quality control products.

[0030] In another preferred embodiment, the kit further includes a label or instructions indicating that the kit is used for (a) assessing the risk of developing liver fibrosis and / or (b) evaluating the efficacy of treatment for liver fibrosis.

[0031] In another preferred embodiment, the specification describes a reference dataset of levels of biomarkers as described in the first aspect of the invention, derived from patients with liver fibrosis and / or healthy controls.

[0032] In another preferred embodiment, the detection of the liver fibrosis biomarkers can be quantitative.

[0033] In another preferred embodiment, the liver fibrosis is selected from the group consisting of: viral hepatitis cirrhosis, alcoholic cirrhosis, non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease, cholestatic liver disease, drug-induced liver injury, and cardiac cirrhosis.

[0034] In a third aspect of the present invention, a detection method is provided, comprising the steps of:

[0035] (a) Provide a test sample, said test sample being a blood, plasma, or serum sample;

[0036] (b) The expression level of CCN5 in the test sample was detected and denoted as C1; and

[0037] (c) Compare the CCN5 concentration C1 with the control reference value C0;

[0038] When C1 / C0 > 1, the subject of the test is judged to have a high risk of developing liver fibrosis or a high degree of liver fibrosis.

[0039] In another preferred embodiment, when C1 / C0 ≥ 1.5, preferably ≥ 2, or preferably ≥ 3, the subject is judged to have a high risk of developing liver fibrosis or a high degree of liver fibrosis.

[0040] In another preferred embodiment, the sample to be tested is plasma. If the test result for liver fibrosis of the subject meets the following conditions, it indicates that the subject has a high risk of developing liver fibrosis or a high degree of liver fibrosis: C1 ≥ 2 ng / ml, preferably ≥ 2.5 ng / ml, more preferably ≥ 3 ng / ml.

[0041] In another preferred embodiment, the method is an in vitro method.

[0042] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0043] In a fourth aspect of the invention, a diagnostic device for liver fibrosis is provided, the device comprising:

[0044] (a) An input module configured to input CCN5 data of a biological sample of an object;

[0045] (b) A processing module configured to process liver fibrosis biomarker data and provide an assessment of the risk of occurrence; wherein the processing includes comparing the expression level C1 of input CCN5 with a control reference value C0;

[0046] Specifically, when C1 is significantly higher than C0, it indicates a high risk of liver fibrosis or a high degree of liver fibrosis in the individual; when C1 is not significantly higher than C0, it indicates a low risk of liver fibrosis or a low degree of liver fibrosis in the individual; and

[0047] (c) Output module, which is configured to output the evaluation results.

[0048] In another preferred embodiment, the device further includes a detection module configured to detect the transcriptional or expression level of CCN5.

[0049] In another preferred embodiment, the device further includes a storage module configured to store pre-set threshold values ​​for markers and comparison result values.

[0050] In another preferred embodiment, the device further includes a control module configured to control the operation of the modules.

[0051] In another preferred embodiment, the detection module is selected from the group consisting of: ELISA analyzer, PCR sequencer, sequencer, or combinations thereof.

[0052] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can interact with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0053] Figure 1 The standard regression curve for the stromal cell protein CCN5 solution obtained in Example 1 is shown below. The compound name is stromal cell protein CCN5. The standard curve formula is f(x) = (-0.0136) + (1.5331)*x. The correlation coefficient (R) is 0.99932419, and the goodness of fit (R²) is... 2 = 0.9986.

[0054] Figure 2 This is a model diagram of the ROC curve used for testing with the receiver operating characteristic (ROC) method. Detailed Implementation

[0055] Through extensive and in-depth research, the inventors unexpectedly discovered a novel biomarker for the diagnosis of liver fibrosis. Specifically, this invention discovers a biomarker that can be used to assess the diagnosis, early diagnosis, and severity of liver fibrosis in test subjects. It possesses high sensitivity and specificity, and even surpasses existing diagnostic methods in accuracy, thus having significant application value. Based on this discovery, the inventors completed this invention.

[0056] the term

[0057] The terminology used in this invention has the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, some definitions and related terms are explained below:

[0058] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0059] The stromal cell protein CCN5 is used as an example for description.

[0060] As used in this article, the term "stromal cell protein CCN5" is abbreviated as CCN5, and "stromal cell protein CCN5" and "CCN5" are used interchangeably.

[0061] The term “approximately” as used herein refers to the value shown plus or minus 10% when referring to a quantitative measurement.

[0062] In one embodiment of the invention, the reference value refers to the reference value or normal value of a healthy control. Those skilled in the art will understand that, given a sufficiently large sample size, the range of normal values ​​(absolute values) for each biomarker can be obtained through testing and calculation methods. Therefore, the absolute values ​​of these biomarker levels can be directly compared with normal values ​​to evaluate the diagnosis of liver fibrosis or the early diagnosis of liver fibrosis. Statistical methods may also be used in this invention.

[0063] According to the present invention, the term "biomarker," also known as "biological marker," refers to a measurable indicator of an individual's biological state. Such biomarkers can be any substance in an individual, as long as they are related to a specific biological state (e.g., disease) of the individual being examined, such as nucleic acid markers (e.g., DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers, species markers (markers of species / genus), and functional markers (KO / OG markers), etc. Biomarkers, after measurement and evaluation, are frequently used to examine normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions, and are useful in many scientific fields.

[0064] According to the present invention, the term "individual" or "object" refers to an animal, particularly a mammal, such as a primate, and preferably a human.

[0065] According to the present invention, the term "plasma" refers to the liquid component of whole blood. Depending on the separation method used, plasma may be completely free of cellular components or may contain varying amounts of platelets and / or small amounts of other cellular components.

[0066] According to the present invention, terms such as “a,” “an,” and “this” refer not only to a singular number of individuals, but also to a general class that can be used to describe a particular implementation.

[0067] It should be noted that the explanations of the terminology provided herein are only to enable those skilled in the art to better understand the invention and are not intended to limit the invention.

[0068] Detection methods

[0069] According to the present invention, plasma CCN5 content data are obtained using a kit.

[0070] Reagent test kit

[0071] In this invention, the kit includes the biological markers described in the first aspect of this invention.

[0072] In another preferred embodiment, the biomarker described in the first aspect of the invention is used as a standard.

[0073] In another preferred embodiment, the kit further includes an instruction manual that describes a reference dataset of levels (e.g., concentrations) of biomarkers as described in the first aspect of the invention, derived from patients with liver fibrosis and / or healthy controls.

[0074] ROC-AUC

[0075] ROC-AUC is a method for evaluating the accuracy of a diagnostic model. The ROC curve is the Receiver Operating Characteristic curve, a graph plotting the false positive rate on the horizontal axis and the true positive rate on the vertical axis. It is a comprehensive indicator reflecting the sensitivity and specificity of continuous variables. AUC is the area under the ROC curve. An ROC-AUC value between 1.0 and 0.5 indicates a better diagnostic effect, with values ​​closer to 1. Values ​​between 0.5 and 0.7 indicate lower accuracy, values ​​between 0.7 and 0.9 indicate some accuracy, and values ​​above 0.9 indicate high accuracy. An AUC of 0.5 indicates that the diagnostic method is completely ineffective and has no diagnostic value. An AUC < 0.5 is unrealistic and rarely occurs in practice.

[0076] liver fibrosis

[0077] Liver fibrosis refers to the pathological process in which excessive deposition of extracellular matrix (ECM, such as collagen and fibronectin) occurs in the liver under chronic injury or inflammatory stimulation, leading to gradual hardening and scarring of the liver structure. It is a key link in the progression of chronic liver disease, and if not intervened in time, it may develop into cirrhosis. There are various causes of liver fibrosis, including, but not limited to, viral hepatitis cirrhosis, alcoholic cirrhosis, non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease, cholestatic liver disease, drug-induced liver injury, and cardiac cirrhosis. The METAVIR score is a test to determine the grade of liver inflammation and necrosis and the degree of fibrosis, including F1 = portal fibrosis without septa; F2 = portal fibrosis with a few septa; F3 = multiple septa without cirrhosis; and F4 = cirrhosis. The Child-Pugh score is one of the most commonly used methods to evaluate liver reserve function in patients with cirrhosis and has important predictive value for patient prognosis. A higher score indicates poorer liver reserve function.

[0078] CCN5

[0079] Cellular communication network factor 5 (CCN5, also known as WISP2) is a member of the CCN family of stromal cell proteins. It is a WNT1-induced signaling pathway protein containing tandem modules: 1) an insulin-like growth factor binding protein module (IGFBP); 2) a von Willebrand factor C repeat module (VWC); and 3) a platelet-reactive protein type 1 repeat module (TSP-1). Unlike other CCN family members, it lacks a module containing a cysteine ​​carboxyl terminus (CT). The amino acid sequence of the CCN5 protein used in this invention is shown in SEQ ID NO. 1 or 2, where SEQ ID NO. 1 is the human CCN5 protein and SEQ ID NO. 2 is the mouse CCN5 protein.

[0080] The proteins of this invention can be recombinant proteins, natural proteins, or synthetic proteins. They can be naturally purified products, chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the proteins of this invention can be glycosylated or non-glycosylated. The proteins of this invention may or may not include an initial methionine residue.

[0081] The main advantages of this invention include:

[0082] (a) This invention discloses for the first time a set of plasma biomarkers for the diagnosis of liver fibrosis, including stromal cell protein CCN5. Stromal cell protein CCN5 is being used for the first time in the diagnosis of liver fibrosis. Verification shows that the AUC of stromal cell protein CCN5 among the plasma biomarkers provided by this invention is above 0.85 when used to differentiate between patients with liver fibrosis and healthy individuals (in the ROC curve evaluation method, when the area under the ROC curve (AUC) is greater than 0.5, the closer it is to 1, the better the diagnostic effect. AUC between 0.5 and 0.7 has lower accuracy, AUC between 0.7 and 0.9 has some accuracy, and AUC above 0.9 has high accuracy).

[0083] (b) The stromal cell protein CCN5, a plasma biomarker provided by this invention, has an AUC of over 0.85 when used to diagnose and differentiate patients with early liver fibrosis.

[0084] (c) The stromal cell protein CCN5 in the plasma biomarkers provided by this invention has an AUC of over 0.85 when used to diagnose and differentiate the severity of liver fibrosis.

[0085] (d) The detection method of the present invention is simple to operate, has a short analysis time, accurate detection results, and a high reproducibility rate.

[0086] (e) The kit provided by this invention can be used for the diagnosis of liver fibrosis, improving diagnostic convenience and promoting the standardization of diagnostic methods.

[0087] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0088] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are commercially available products.

[0089] 1. Specimen source

[0090] After obtaining the patients' consent, plasma samples were collected from 183 patients with liver fibrosis. The age and sex of 50 healthy individuals were matched with those of patients with liver fibrosis. All blood samples were collected in the morning on an empty stomach.

[0091] 2. Reagents and Materials

[0092] CCN5 standard products, anticoagulant vacuum blood collection tubes, etc. are all commercially available reagents.

[0093] 3. Experimental Procedure

[0094] 3-1 Solution Preparation

[0095] 3.1.1 Preparation of standard curve solution:

[0096] Take eight 1.5 ml centrifuge tubes and label them 1, 2, 3, 4, 5, 6, 7, and Blank. Add 100 μl of standard (containing 10 ng of CCN5, diluted with phosphate buffer) / 900 μl of phosphate buffer to tube 1. Add 200 μl of standard / phosphate buffer to tubes 2 through Blank, respectively. Add 100 μl of standard CCN5 solution to tube S1 and mix on a vortex mixer. Then, pipette 200 μl and transfer it to tube S2. Repeat this serial dilution to tube S7 and mix well. Blank serves as a blank control.

[0097] 3-2 Plasma sample pretreatment and injection analysis:

[0098] Add 50 μL each of the standard working solution and the plasma sample to be tested to different wells of a 96-well plate. Add 100 μL of antibody diluent to the blank wells, and add 100 μL of antibody diluent containing CCN5 to each of the remaining wells. Mix well and incubate at 37°C for 50 minutes. Add 100 μL of SABC (Strept Avidin-Biotin Complex) working solution to each well, mix well, and incubate at 37°C for 30 minutes. Add 100 μL of pre-prepared TMB mixture (AB two-component 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system) to each well and incubate at 37°C in the dark for 10-20 minutes. Add 50 μL of TMB colorimetric stop solution to each well, mix well, and measure the absorbance at 450 nm using a microplate reader within 30 minutes.

[0099] Example 1

[0100] The levels of CCN5 were significantly increased in the liver tissue and plasma of patients with liver fibrosis.

[0101] 1.1 Among 183 patients with liver fibrosis (including 60 patients with liver fibrosis according to the METAVIR classification and 123 patients with cirrhosis), the plasma CCN5 level was significantly higher in patients with liver fibrosis compared with normal healthy individuals. Figure 1 A).

[0102] 1.2 Co-staining of liver tissue from patients with liver fibrosis with αSMA (myofibroblast marker) and CCN5, and co-staining with Desmin (resting fibroblast marker) and CCN5 from healthy individuals, revealed that the expression of CCN5 in myofibroblasts from patients with liver fibrosis was significantly increased. Figure 1 B).

[0103] The results suggest that fibroblast CCN5 levels are significantly elevated in the liver tissue of individuals with liver fibrosis. Furthermore, plasma CCN5 levels are significantly elevated in patients with liver fibrosis, suggesting that plasma CCN5 could serve as a potential biomarker for liver fibrosis.

[0104] Example 2

[0105] Plasma CCN5 levels are highly correlated with the progression of liver fibrosis.

[0106] 2.1 Logistic regression analysis revealed a positive correlation between plasma CCN5 levels and liver fibrosis. Figure 2 A).

[0107] 2.2 Co-staining of αSMA (myofibroblast marker) and CCN5 in tissues of patients with different degrees of liver fibrosis and co-staining of Desmin (resting fibroblast marker) and CCN5 in healthy individuals revealed that the expression of CCN5 in myofibroblasts of patients with liver fibrosis increased with the severity of fibrosis. Figure 2 B).

[0108] 2.3 Correlation analysis revealed a high correlation between plasma CCN5 levels and the severity of liver fibrosis, exhibiting a higher correlation (R = 0.80) compared to classic liver fibrosis detection methods (FIB-4 test, PRO-C3 serum levels, MELD score). Figure 2 CF).

[0109] Example 3

[0110] Plasma CCN5 can serve as a biomarker for the early diagnosis of liver fibrosis.

[0111] Diagnostic efficacy analysis of plasma CCN5 levels in normal individuals (50 cases) and individuals with mild liver fibrosis (30 cases) revealed that, compared with classic liver fibrosis detection methods (FIB-4 test, PRO-C3 serum content, MELD score), plasma CCN5 had a higher AUROC (Table 1), suggesting that plasma CCN5 has better diagnostic efficacy for the early diagnosis of liver fibrosis.

[0112] Table 1

[0113]

[0114] Example 4

[0115] Plasma CCN5 can serve as a biomarker for diagnosing the severity of liver fibrosis.

[0116] Diagnostic efficacy analysis of plasma CCN5 levels in healthy individuals (50 cases) and individuals with different degrees of liver fibrosis (based on METAVIR classification; F1, 30 cases; F2-3, 60 cases; F4, Child Pugh A (CP-A), 60 cases; Child Pugh B (CP-B), 63 cases) revealed that plasma CCN5 had a higher AUROC (Table 2) compared to classic liver fibrosis detection methods (FIB-4 test, PRO-C3 serum content, MELD score), suggesting that plasma CCN5 has better diagnostic efficacy in diagnosing the severity of liver fibrosis.

[0117] Table 2

[0118]

[0119] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a gene, mRNA, cDNA, protein, or a detection reagent for a biomarker for preparing a kit or reagent for (a) diagnosing liver fibrosis; (b) diagnosing early liver fibrosis; and / or (c) diagnosing the severity of liver fibrosis; wherein the biomarker is CCN5.

2. The use as described in claim 1, characterized in that, The detection reagent or kit is used to detect the expression level of the biomarker in the sample to be tested.

3. A kit for the diagnosis of liver fibrosis, the kit comprising a detection reagent for detecting genes, mRNA, cDNA, or proteins, or combinations thereof, of liver fibrosis biomarkers; wherein the biomarker is CCN5.

4. The kit according to claim 3, characterized in that, The detection reagents include antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

5. The reagent kit as described in claim 3, characterized in that, The kit contains one or more detection reagents selected from the group consisting of: (a) Specific antibodies against CCN5, specific binding molecules of CCN5; and / or (b) Primers or primer pairs, probes or chips for specific amplification of CCN5 mRNA or CCN5 cDNA.

6. The reagent kit as described in claim 3, characterized in that, The detection is for an ex vivo sample; preferably, the ex vivo sample is selected from the group consisting of blood, plasma, serum, or combinations thereof.

7. The kit according to claim 3, characterized in that, The kit contains genes, mRNA, cDNA and / or proteins of liver fibrosis biomarkers as controls or quality control products.

8. The reagent kit as described in claim 3, characterized in that, The liver fibrosis is selected from the following groups: viral hepatitis cirrhosis, alcoholic cirrhosis, non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease, cholestatic liver disease, drug-induced liver injury, and cardiac cirrhosis.

9. A method for detecting the risk of developing liver fibrosis or the degree of liver fibrosis, comprising the steps of: (a) Provide a test sample, said test sample being a blood, plasma, or serum sample; (b) The expression level of CCN5 in the test sample was detected and denoted as C1; and (c) Compare the CCN5 concentration C1 with the control reference value C0; When C1 / C0 > 1, the subject of the test is judged to have a high risk of developing liver fibrosis or a high degree of liver fibrosis.

10. A diagnostic device for liver fibrosis, the device comprising: (a) An input module configured to input CCN5 data of a biological sample of an object; (b) A processing module configured to process liver fibrosis biomarker data and provide an assessment of the risk of occurrence; wherein the processing includes comparing the expression level C1 of input CCN5 with a control reference value C0; Specifically, when C1 is significantly higher than C0, it indicates a high risk of liver fibrosis or a high degree of liver fibrosis in the individual; when C1 is not significantly higher than C0, it indicates a low risk of liver fibrosis or a low degree of liver fibrosis in the individual; and (c) Output module, which is configured to output the evaluation results.

Images