Application of MUC1 as hepatic fibrosis marker

Abstract

The invention relates to an application of MUC1 as a hepatic fibrosis marker. Whether a patient suffers from hepatic fibrosis or not and the severity of the hepatic fibrosis are judged according to the expression level of the MUC1. And the hepatic fibrosis can be accurately and non-invasively diagnosed by utilizing the MUC1 expression level and combining with the corresponding diagnostic standard.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly to an application of MUC1 as a liver fibrosis biomarker. Background Art

[0002] Liver fibrosis is an important pathological process of liver diseases, usually caused by factors such as viral hepatitis, fatty liver, alcoholic liver disease, etc., manifested as the gradual destruction of the liver structure and the excessive proliferation and accumulation of fibrous tissues, and its pathological basis is the activation of hepatic stellate cells. With the progression of the disease, liver fibrosis may develop into cirrhosis and eventually lead to liver failure. Therefore, the early diagnosis of liver fibrosis is of great significance for disease intervention and treatment. Existing methods for diagnosing liver fibrosis include liver biopsy, imaging examinations (such as transient elastography, ultrasonic elastography, etc.), serum biomarker detection, etc. However, liver biopsy is an invasive examination, imaging examinations have certain limitations, and the sensitivity and specificity of serum biomarkers are often insufficient.

[0003] In this context, it is particularly important to find a non-invasive, sensitive, and specific biomarker for the early diagnosis of liver fibrosis. Summary of the Invention

[0004] Objective of the present invention: In order to overcome the defects of the prior art, the present invention provides an application of MUC1 as a liver fibrosis biomarker.

[0005] Technical solution of the present invention: An application of MUC1 as a liver fibrosis biomarker, which determines whether a patient has liver fibrosis and the severity thereof based on the expression level of MUC1.

[0006] Preferably, the expression level of MUC1 is obtained by detecting a sample using molecular biology methods or immunological detection methods, and the sample is the serum or liver tissue section of the patient.

[0007] Preferably, the molecular biology methods include real-time quantitative PCR (qPCR) and / or reverse transcription PCR (RT-PCR).

[0008] Preferably, the immunological detection methods include enzyme-linked immunosorbent assay (ELISA), Western Blot, and / or immunohistochemistry (IHC).

[0009] MUC1 is a transmembrane glycoprotein that is widely expressed on the surfaces of various epithelial cells and is distributed in tissues such as the gastrointestinal tract, respiratory tract, and liver. In the present invention, the expression level of MUC1 (mucin 1) in patients is detected to diagnose liver fibrosis. Specifically, by using the MUC1 expression levels in serum and liver tissue samples and combining with the corresponding diagnostic criteria, liver fibrosis can be accurately and non-invasively diagnosed. It aids in the early diagnosis of liver fibrosis, the assessment of the disease progression degree, and the monitoring of treatment effects. It has the characteristics of high sensitivity, high specificity, and simple operation, providing a new and effective tool for the early diagnosis of liver fibrosis and the evaluation of clinical treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the ROC curve graph of the control group and the liver fibrosis group in Specific Example 1 of the present invention;

[0011] Figure 2 It is the MUC1 immunohistochemical staining graph in Specific Example 2 of the present invention;

[0012] Figure 3 It is the protein level graph of MUC1 in Specific Example 3 of the present invention;

[0013] Figure 4 It is the mRNA level graph of MUC1 in Specific Example 4 of the present invention. SPECIFIC EMBODIMENTS

[0014] Next, the technical solutions in this embodiment will be clearly and completely described in conjunction with the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0015] The relevant detection experiments are as follows:

[0016] Example 1 - Quantitative Detection of Serum MUC1

[0017] 1.1 Sample collection: Collect serum samples from 20 patients with liver fibrosis and 20 healthy control groups;

[0018] 1.2 Plasma separation: Let the blood samples stand at room temperature for 30 min, then centrifuge at 1000 g for 10 min to separate the plasma;

[0019] 1.3 ELISA detection: Fix the MUC1-specific capture antibody on the ELISA plate; add the plasma sample to be tested and incubate for a certain time; perform a secondary incubation with the detection antibody, then add the enzyme-labeled secondary antibody and substrate, and quantify the MUC1 expression level through the intensity of color change;

[0020] 1.4 Standard curve establishment: A standard curve was established using MUC1 protein with known concentrations to determine the concentration of MUC1 in the samples to be tested;

[0021] 1.5 Plot the ROC curve between the Liver Fibrosis group and the Control group.

[0022] Experimental results: The AUC value was 0.8825, indicating that MUC1 has good diagnostic value for liver fibrosis patients. And when the serum MUC1 ≥ 36.85 U / mL, liver fibrosis can be diagnosed.

[0023] Example 2 - Immunohistochemical detection of MUC1 expression in liver tissue

[0024] 2.1 Sample collection: Collect liver tissue samples from 10 liver fibrosis mice and 10 healthy control groups;

[0025] 2.2 Section preparation: Embed the fixed liver tissue samples in paraffin and cut them into 4-μm thick sections;

[0026] 2.3 Immunohistochemical staining: Stain the sections with anti-MUC1 antibody, and the antibody dilution ratio is 1:100;

[0027] 2.4 After a series of washes and biotin-streptavidin complex treatment, use DAB chromogenic reagent for color development;

[0028] 2.5 Use an optical microscope to evaluate the staining results.

[0029] Experimental results: The cells with positive MUC1 expression showed brown, and the MUC1 expression in the liver fibrosis group was significantly increased compared with the two groups.

[0030] Example 3 - Western Blot detection of MUC1 protein expression

[0031] 3.1 Sample collection: Collect liver tissue samples from 10 liver fibrosis mice and 10 healthy control groups;

[0032] 3.2 Detect the expression level of MUC1 protein in liver tissue samples by Western Blot;

[0033] 3.21 Extraction and preparation of total protein in liver tissue samples:

[0034] (1) Lyse cell or tissue samples using RIPA lysis buffer or other suitable lysis buffers, and add protease and phosphatase inhibitors to prevent protein degradation;

[0035] (2) Determine the protein concentration by BCA protein quantification kit;

[0036] 3.22 Electrophoresis:

[0037] (1) Prepare an SDS-PAGE gel and select acrylamide with a suitable concentration according to the molecular weight of the target protein;

[0038] (2) Mix the protein sample with the loading buffer and heat it at 95 °C for 5 min to depolymerize the protein;

[0039] (3) Load the protein sample onto the SDS-PAGE gel and then perform electrophoresis separation;

[0040] 3.23 Transfer membrane and color development:

[0041] (1) Transfer the electrophoretically separated protein from the gel to a nitrocellulose membrane or a PVDF membrane;

[0042] (2) Wash the membrane with PBST (phosphate buffer containing 0.1% Tween-20) and block it in 5% skim milk powder or BSA to reduce non-specific binding;

[0043] (3) Incubate the membrane with the primary antibody against MUC1, and then incubate it with the secondary antibody labeled with horseradish peroxidase (HRP);

[0044] (4) Detect the specifically bound secondary antibody using chemiluminescent reagents and obtain the expression level of MUC1-related proteins in liver tissues by exposing the membrane to an X-ray film or capturing the signal using a gel imaging system.

[0045] Experimental results: The expression level of MUC1 protein in the liver fibrosis group (CCL4) was significantly higher than that in the control group (con) (p < 0.01).

[0046] Example 4 - Detection of the expression of the MUC1 gene by molecular biology methods

[0047] 4.1 Sample collection: Collect liver tissue samples from 10 mice with liver fibrosis and 10 healthy control groups;

[0048] 4.2 Detect the mRNA expression level of the MUC1 gene in liver tissue samples by qPCR;

[0049] 4.21 RNA extraction and purification:

[0050] (1) Extract total RNA from liver tissue or cell samples using an RNA extraction kit;

[0051] (2) Determine the purity and concentration of RNA by ultraviolet spectrophotometer and ensure that the A260 / A280 ratio of RNA is between 1.8 and 2.0;

[0052] (3) Detect the integrity of RNA by 1.7% agarose gel electrophoresis to ensure clear bands;

[0053] 4.22 Reverse transcription (usually using random primers, oligo(dT) or gene-specific primers for reverse transcription):

[0054] Reverse transcribe total RNA into cDNA using reverse transcriptase: Mix the RNA with primers, add dNTPs, reverse transcription buffer and reverse transcriptase, and perform the reaction according to the manufacturer's recommended conditions (usually incubate at 55 - 60 °C for 10 min, then denature at 95 °C for 5 min);

[0055] 4.23 Quantitative PCR (qPCR):

[0056] (1) Design specific primers for the MUC1 gene and perform qPCR reactions using SYBR Green or TaqMan probes;

[0057] (2) Set the reaction conditions in a qPCR instrument, usually 95 °C for 15 s for denaturation, 60 °C for annealing and extension for 1 min, for a total of 40 cycles;

[0058] (3) Compare the CT values to quantify the expression level of the MUC1 gene (usually requires normalization using a reference gene (β-actin)).

[0059] Experimental results: The expression level of the MUC1 gene in the liver fibrosis group (CCL4) was significantly higher than that in the control group (con). The expression level of MUC1-related genes in liver tissue can be detected using real-time quantitative PCR (qPCR) technology, providing data support for the early diagnosis and staging of liver fibrosis.

[0060] Establish a diagnostic criterion for liver fibrosis: Set the corresponding diagnostic threshold according to the expression level of MUC1. When the concentration of MUC1 in serum is higher than the normal range, liver fibrosis can be preliminarily diagnosed. The staging of liver fibrosis can be further evaluated based on the expression changes of MUC1. The overexpression of MUC1 is usually positively correlated with the severity of liver fibrosis.

[0061] As a biomarker for liver fibrosis, the expression of MUC1 is significantly increased in patients with liver fibrosis and can be distinguished from the healthy control group, with high sensitivity and specificity. By quantitatively detecting the expression level of MUC1, the severity of liver fibrosis can be evaluated, providing guidance for the treatment plan of patients. The change in MUC1 provides a reliable biomarker for the diagnosis of liver fibrosis, contributing to the early detection and timely intervention of liver fibrosis without invasive operations such as liver biopsy. It can be applied in a wide range of clinical settings, providing a convenient and effective tool for the early screening of liver diseases.

Claims

1. A use of MUC1 as a liver fibrosis marker, characterized in that: The presence and severity of liver fibrosis can be determined by the expression level of MUC1.

2. The use of MUC1 as a liver fibrosis marker according to claim 1, characterized in that: The expression level of MUC1 is obtained by detecting a sample using a molecular biological method or an immunological detection method, and the sample is a patient's serum or liver tissue section.

3. The use of MUC1 as a liver fibrosis marker according to claim 2, characterized in that: The molecular biological method includes real-time quantitative PCR and / or reverse transcription PCR.

4. The use of MUC1 as a liver fibrosis marker according to claim 2, characterized in that: The immunological detection methods include enzyme-linked immunosorbent assay, immunoblotting and / or immunohistochemistry.

Images