Biomarkers for diagnosing the progression of chronic hepatitis B cirrhosis and their applications

Abstract

The present invention relates to the field of medicine, and in particular, to biomarkers for diagnosing the progression of chronic hepatitis B cirrhosis and their applications. The present invention can assist in the diagnosis of the progression of the disease in HBV infected patients by detecting DBH and inflammatory factors in the plasma of HBV infected patients. The peripheral blood test is convenient, fast and low-cost. Especially in areas with backward medical conditions or when there are a large number of examiners for screening, the detection of plasma DBH and inflammatory factors is a simple and easy means for long-term monitoring of hepatitis B patients. It can quickly and accurately diagnose chronic hepatitis B cirrhosis, has strong specificity and high sensitivity, and has great clinical value and economic value.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to biomarkers for diagnosing the progression of chronic hepatitis B cirrhosis and applications thereof. Background Art

[0002] Hepatitis B is a major public threat worldwide. The 69th World Health Assembly adopted a global strategy for eliminating viral hepatitis, requiring all countries to achieve the goal of basically eliminating viral hepatitis by 2030. my country is a country with a high incidence of hepatitis B. To achieve this goal, it is necessary to raise the level of prevention, diagnosis and treatment of hepatitis B to a higher level. Hepatitis B virus (HBV) is a hepatotropic virus. The target cells infected are hepatocytes. After infection, it can cause cellular and humoral immune responses, stimulate autoimmune responses and immune regulation disorders, and cause hepatocyte damage. After HBV infects the human body, it can also inhibit the body's immune function, causing the infection to prolong and then become chronic. Long-term chronic inflammation will lead to cirrhosis and liver cancer. Cirrhosis is a major disease that threatens human health. If the progression of the disease cannot be stopped in a timely and effective manner, it will lead to portal hypertension, hepatic encephalopathy, liver failure, and even hepatocellular carcinoma. Cirrhosis poses a great threat to human health, but its pathological process is reversible. Therefore, finding diagnostic markers for the progression of hepatitis B disease is beneficial for identifying and reversing the pathological development process of inflammation-cancer transformation before hepatocellular carcinoma, and has great application value.

[0003] Liver biopsy has always been considered the gold standard for evaluating the degree of liver histological inflammation and liver fibrosis. It can understand the nature and degree of liver parenchymal damage and analyze the location of the disease. However, there are many contraindications for liver puncture. The invasiveness of liver biopsy limits its routine clinical application. Moreover, liver biopsy can only reflect the degree of liver lesions within the range of the sample, and the analysis of the liver as a whole is poor.

[0004] Currently, non-invasive imaging technologies such as transient elastic imaging (FibroScan) have received widespread attention. However, this examination is affected by the patient's body shape, such as the amount of abdominal fat, the presence of ascites, the intercostal space, etc., and the equipment is expensive, so it has not yet been popularized in resource-limited areas. Based on the above problems, there is an urgent need for a highly sensitive and specific biomarker and a diagnostic product made with it that can replace imaging examinations or puncture biopsies for monitoring the progression of hepatitis B disease.

[0005] In recent years, researchers have discovered more and more potential markers of liver fibrosis and cirrhosis from peripheral blood. Non-invasive detection indicators based on routine laboratory indicators have been used in clinical practice and for the evaluation and staging of liver fibrosis. Serum C-IV (type IV collagen), PC-III (procollagen III n-terminal propeptide), LN (laminin), and HA (hyaluronic acid) are considered to be direct markers for evaluating liver fibrosis. PⅢNP, HA, and LN are mainly related to the severity of liver inflammation, and CIV is mainly related to the balance of liver fibrosis and degradation, but the accuracy of diagnosing liver fibrosis is not ideal. The ratio of aspartate aminotransferase to alanine aminotransferase (AST / ALT), AST and platelet ratio index (APRI), and liver fibrosis 4-factor index (FIB-4) are some newly developed indicators that combine serum biomarkers to evaluate the degree of liver damage associated with hepatitis virus infection.

[0006] Realistic solutions

[0007] Liver puncture biopsy: When the patient's history of HBV infection is confirmed, liver tissue puncture biopsy is the gold standard for confirming the degree of liver inflammation and whether there is liver fibrosis. It can understand the nature and degree of liver parenchymal damage, stage inflammation and fibrosis, and diagnose early liver fibrosis.

[0008] Transient elastography (FibroScan), transient elastography technology is an ultrasonic elastography technology. The ultrasonic transducer on the probe emits ultrasonic waves to track shear waves and measure their speed. The greater the liver hardness, the faster the speed and the greater the elasticity value, thereby obtaining data on the degree of liver fibrosis, detecting liver tissue hardness and evaluating liver fibrosis stages.

[0009] Four liver fibrosis items: including PⅢNP, CIV, HA and LN are related to liver fibrosis and have been carried out in clinical laboratories for many years, but there is no appropriate cut-off value for diagnosing liver fibrosis in chronic hepatitis B (CHB).

[0010] AST / platelet ratio index (APRI): Its calculation method is: APRI = (AST / ULN) × 100 / PLT (109 / L), which is recommended by the World Health Organization as the most useful non-invasive tool for assessing cirrhosis in resource-limited settings.

[0011] The liver fibrosis-4 (FIB-4) index is calculated by the formula FIB-4 = (age × AST) / (platelet count × square root of ALT). Recent studies have shown that APRI and FIB-4 have similar efficacy in diagnosing liver fibrosis.

[0012] Disadvantages of existing technology:

[0013] (1) The traditional hepatitis B two-pair half test combined with HBV-DNA testing can assist clinical staging of the hepatitis B disease progression, but it cannot accurately reflect the pathological progression of the liver, nor can it judge the severity of the disease.

[0014] (2) A biopsy can directly reflect the degree of pathological damage to the liver, but it is an invasive procedure that causes greater damage. There is a risk of damaging the liver capsule and causing massive bleeding during the puncture process. Various contraindications need to be excluded before puncture. Patients with severe anemia, hypertension, bleeding tendency, infection of the punctured area or adjacent organs, etc. are not suitable for liver puncture. In addition, the results are easily affected by the puncture site, sample size, and the observer himself. There is a misdiagnosis rate and certain limitations. It is not suitable for early diagnosis and initial screening of the disease. Repeated testing is not suitable during disease progression, and long-term dynamic monitoring of the disease is impossible. The sampling site has a great influence on the test results, which are easily affected by the experience and subjectivity of the pathologist, which is not conducive to dynamic observation of the disease and quantitative evaluation of indicators.

[0015] (3) Ultrasound examinations such as transient elastography (FibroScan) and imaging examinations are non-invasive and widely used in clinical practice, but they are difficult to detect small lesions and are affected by many factors. For example, liver elasticity and hardness testing is greatly affected by obesity, fatty liver, intercostal space size, liver inflammation, edema, and cholestasis. Pregnant women cannot be tested. In addition, its accuracy depends to a certain extent on the operator's experience and skills. Different instruments may also lead to differences in diagnosis and treatment decisions. In addition, the equipment is expensive and difficult to popularize in resource-limited areas.

[0016] (4) Common laboratory indicators for diagnosing liver damage include alanine aminotransferase (ALT), aspartate amino transferase (AST), gamma-glutamine transferase (GGT) and serum albumin (ALB), among which AST and ALT are more sensitive indicators of liver damage. However, commonly used laboratory indicators in clinical practice cannot accurately predict liver fibrosis and can only serve as a reminder and auxiliary diagnosis.

[0017] (5) In recent years, in order to better perform non-invasive assessment of liver fibrosis, the clinical practice has included non-invasive indicators for assessing the degree of liver fibrosis, namely the FIB-4 index, AAR and APRI. The three indicators need to be calculated based on the patient's age and biochemical indicators to determine the degree of fibrosis. However, the above non-invasive indicators need to be calculated according to the formula based on serum indicators such as patient age, platelet count, hyaluronic acid, and liver enzyme content. Multiple indicators are needed to assist in the diagnosis of liver fibrosis. A single indicator in the formula cannot well indicate the degree of liver fibrosis, and there is a certain probability of error in the formula calculation process. Combining with imaging examinations may better ensure the accuracy of liver fibrosis diagnosis. In addition, when the above single serological indicator or laboratory indicator combined model is used to evaluate HBV-related liver fibrosis, its accuracy has not been determined.

[0018] The present invention takes advantage of the blood test, and the objectivity of the detection index is better than the above method. It is relatively convenient and fast, which is conducive to improving the compliance of long-term follow-up patients, and is of great significance for the early diagnosis and treatment of the disease and the prediction of disease outcome. It also has low requirements for equipment and operation, which is conducive to the routine implementation of grassroots hospitals, realizing comprehensive prevention and control of hepatitis B-related diseases, and reducing the social medical burden. Summary of the invention

[0019] In order to solve the above problems, the present invention first provides a biomarker for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, wherein the biomarker comprises one or more of DBH, TNF-α, IL-6, and IL-1β.

[0020] In certain embodiments, the biomarker is one or more of DBH, TNF-α, IL-6, and IL-1β.

[0021] In certain embodiments, the biomarkers are DBH, TNF-α, IL-6, and IL-1β.

[0022] The present invention also provides a use of the above marker in preparing a product for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis.

[0023] In certain embodiments, the product comprises an antibody, a primer, a probe, or a kit.

[0024] The present invention also provides a use of the above marker in diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, wherein the use is for non-diagnostic or non-therapeutic purposes;

[0025] The application includes the following:

[0026] (a) Diagnosis of chronic hepatitis B and / or progressive cirrhosis;

[0027] (b) Screening of drugs for the treatment of chronic hepatitis B and / or liver cirrhosis;

[0028] (c) Assist in the construction of cell or animal models of chronic hepatitis B and / or liver cirrhosis;

[0029] Optionally, the use is for the purpose of scientific research.

[0030] The present invention also provides a primer set for detecting the above markers, wherein the primer pair sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0031] The present invention also provides a kit for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, wherein the kit comprises the above-mentioned primer set.

[0032] The present invention also provides a use of the primer set as described above in preparing a kit for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis.

[0033] Finally, the present invention provides a method for diagnosing the progression of chronic hepatitis B and / or cirrhosis, which comprises the step of using the above-mentioned primers or the above-mentioned kit to detect the above-mentioned biomarkers in the sample to be tested, wherein the application is for non-diagnostic purposes, and optionally, the application is for the purpose of scientific research; optionally, the sample is an in vitro sample.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] The present invention can assist in the diagnosis of the disease progression of HBV infected patients by detecting DBH and inflammatory factors in the plasma of HBV infected patients. The peripheral blood test is convenient, fast and low-cost. Especially in areas with backward medical conditions or when there are a large number of examiners for screening, the detection of plasma DBH and inflammatory factors is a simple and easy means for long-term monitoring of hepatitis B patients. It can quickly and accurately diagnose chronic hepatitis B cirrhosis, has strong specificity and high sensitivity, and has great clinical and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , 3 Heat map of the expression trends of high-abundance genes in 46 cases of HCC tissues and adjacent cirrhotic liver tissues, and the expression differences of DBH mRNA in 46 cases of HCC tissues and adjacent cirrhotic liver tissues.

[0037] Figure 2 Analysis of the levels and correlation of DBH, TGFβ1, Hyp, and HBx in normal plasma (N), plasma of patients with chronic hepatitis B (HB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC).

[0038] Figure 3, chronic HBV carrier group, hepatitis B group, cirrhosis group and healthy control group, and their correlation with DBH.

[0039] Figure 4 Comparison of receiver operating curves for the differentiation of chronic HBV carriers from hepatitis B using serum TNF-α, IL-6, IL-1β and DBH alone.

[0040] Figure 5 Comparison of receiver operating curves of the combination of serum TNF-α, IL-6, IL-1β and DBH and APRI and FIB-4 in differentiating chronic HBV carriers from hepatitis B.

[0041] Figure 6 Comparison of receiver operating curves for serum TNF-α, IL-6, IL-1β and DBH alone in differentiating hepatitis B from cirrhosis.

[0042] Figure 7 Comparison of receiver operating curves of the combination of serum TNF-α, IL-6, IL-1β and DBH and APRI and FIB-4 in differentiating hepatitis B from cirrhosis.

[0043] Figure 8 Comparison of receiver operating curves for serum TNF-α, IL-6, IL-1β and DBH in distinguishing compensated and decompensated cirrhosis.

[0044] Fig. 9 Comparison of receiver operating curves for the combination of serum TNF-α, IL-6, IL-1β and DBH and APRI and FIB-4 in differentiating compensated and decompensated stages of liver cirrhosis.

[0045] Fig.10 , Western Blot was used to detect the expression level of HBx protein in three groups of LO2 hepatocyte cell lines.

[0046] Fig.11 , three Changes in morphology and expression of DBH and α-SMA in LX-2 cells cultured with conditioned medium of group A after 48 h of culture; a, changes in LX-2 cell morphology; b, changes in α-SMA and DBH mRNA expression; c, changes in α-SMA and Col1A1 protein expression; **P<0.01.

[0047] Fig.12 , CCK8 were used to detect the proliferation activity of LX-2 cells cultured in three groups of conditioned medium.

[0048] Fig.13, changes in mRNA expression of α-SMA, Col1A1, and DBH in LX-2 cells after stimulation with rhTGFβ1 **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0049] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0050] Terminology explanation:

[0051] Dopamine beta hydroxylase (DBH): Dopamine beta hydroxylase (DBH) is a copper-dependent monooxygenase that is mainly found in the adrenal glands and liver. It can convert dopamine into norepinephrine (and then produce epinephrine) and plays a key role in the biosynthesis pathway of catecholamines (including norepinephrine, epinephrine and dopamine).

[0052] Chronic hepatitis B: A chronic inflammatory disease of the liver caused by hepatitis B virus (HBV) infection. According to the course of the disease, it can be divided into three stages: chronic hepatitis B virus carrier, chronic hepatitis B, and hepatitis B cirrhosis.

[0053] Inflammatory factors: Inflammatory cytokines refer to various cytokines involved in inflammatory responses. For example, tumor necrosis factor α (TNF-α): mainly produced by activated monocytes / macrophages, can kill and inhibit tumor cells, promote neutrophil phagocytosis, resist infection, cause fever, and induce acute phase protein synthesis in hepatocytes; interleukin 6 (IL-6): IL-6 is a cytokine in the chemokine family; interleukin IL-1β (IL-1β) is an important mediator of inflammatory responses and participates in a variety of cell activities, including cell proliferation, differentiation and apoptosis.

[0054] The Youden Index is also called the correct diagnosis index, and its value range is between (0-1). The closer it is to 1, the better the diagnostic accuracy. In this application, the diagnostic standard is set at the maximum correct diagnosis index.

[0055] The receiver operating characteristic (ROC) reflects the balance between sensitivity and specificity. The area under the ROC curve is an important indicator of test accuracy. The larger the area under the ROC curve, the greater the diagnostic value of the test.

[0056] Sensitivity (true positive rate): The percentage of people who are actually sick but are correctly diagnosed as sick according to the test standards. The higher the sensitivity, the better. The ideal sensitivity is 100%.

[0057] Specificity (true negative rate): The percentage of people who are actually disease-free but are correctly judged as disease-free according to the test standards. The higher the specificity, the better. The ideal specificity is 100%.

[0058] Statistical methods: SPSS25.0 and GraphPadPrism 9.4.0 software were used to perform statistical analysis and present the results of serum quantitative data of the enrolled subjects. Shapiro-Wilk was used to test the normality of data distribution, t-test was used to compare the data between two groups with normal distribution, and multi-factor analysis of variance (ANOVA) was used to compare the data between multiple groups with normal distribution. The Mann-Whitney U test was used to compare the data between two groups with non-normal distribution, and the Kruskal-Wallis H test was used to compare the data between multiple groups with non-normal distribution. In this application, the diagnostic standard is set at the maximum Youden index, which is used to calculate the AUC, sensitivity and specificity for evaluating the diagnostic efficacy of potential biomarkers. The statistical result p<0.05 is considered to be statistically significant.

[0059] Example 1: Detection of DBH expression in liver cirrhosis and liver cancer tissues by gene chip and Realtime-PCR

[0060] Gene chip technology: Through liquid chromatography-mass spectrometry and microarray technology, high-density DNA fragments are attached to solid surfaces such as membranes and glass slides in a certain order or arrangement through high-speed robots or in situ synthesis. DNA probes labeled with isotopes or fluorescence (probe sequences are shown in Table 1) are used to conduct a large number of studies on gene expression and monitoring based on the principle of base complementary hybridization.

[0061] (1) Three pairs of HBsAg-positive liver cancer tissues (T) and adjacent liver cirrhosis tissues (Peritumoral), numbered T1, T2, T3, P1, P2, and P3, were used to study and screen the differential gene expression using gene chip technology.

[0062] (2) 46 cases of peritumoral liver cirrhosis tissues and hepatocellular carcinoma (HCC) tissues were collected, and the expression level of DBH in the collected liver tissues was detected by Realtime-PCR;

[0063] (3) The difference in DBH expression levels detected in each group was calculated, and when P < 0.05, it indicated a significant difference.

[0064] Results Figure 1The chip results suggest that a group of differentially expressed mRNAs were found between liver cancer tissue (T) and adjacent cirrhotic tissue (Peritumoral), among which DBH mRNA was upregulated 24 times in cirrhotic tissue, with a low P value, indicating a high possibility of inter-group differences. Realtime PCR test data of collected hepatitis B cirrhosis and liver cancer tissues showed that DBH mRNA in the hepatitis B cirrhosis group was higher than that in the liver cancer group, indicating that there was a significant difference in the gene expression of DBH between the liver cirrhosis group and the liver cancer group, which could distinguish the disease status. Moreover, the expression of DBH decreased with the occurrence of liver cancer. Therefore, continuous detection of DBH in HBV-infected patients has a certain predictive effect on the deterioration of the disease.

[0065] Table 1 Probes

[0066]

[0067] Example 2. Detection of the levels of DBH, TNF-α, IL-6, and IL-1β in the plasma of the chronic HBV carrier group (ASC), chronic hepatitis B group (CHB), and hepatitis B cirrhosis group (LC)

[0068] The key protein DBH was quantitatively verified by enzyme-linked immunosorbent assay (ELISA) in the validation cohort subjects. The ELISA kit is a commercial kit with a sensitivity of 1.00 ng / mL and a linear range of 6.25-200.00 ng / mL. Standard wells and sample wells are set on the enzyme-labeled coated plate, and 50 μl of standards of different concentrations are added to each standard well. 50 μl of the sample to be tested is added to the sample well, and nothing is added to the blank well. Follow the instructions for the ELISA kit. Zero the blank well and measure the absorbance of each well at a wavelength of 450 nm. The measurement process is strictly in accordance with the actual instructions.

[0069] ELISA method steps:

[0070] (1) Move the reagents to room temperature (18-25°C) and equilibrate for more than 30 minutes.

[0071] (2) Collect the culture supernatant of the experimental and control cells using a sterile tube, centrifuge at 3000 rpm / min for 20 min at 4°C, and carefully collect the supernatant.

[0072] (3) Before the experiment, design the method of adding samples to the standard wells and the sample wells. Set up three replicate wells for each sample, add 50 μL of the standard or sample to each well, shake to mix, cover with a plate sticker and incubate at 37°C for 30 min.

[0073] (4) Washing the plate: Discard the liquid and wash the plate 5 times. Fill each well and soak for 30 seconds each time. Turn the plate upside down to shake off the cleaning solution.

[0074] (5) Add 50 μL of enzyme labeling working solution to each well, cover with a new plate sticker, and incubate at 37°C for 15 min.

[0075] (6) Washing the plate: Discard the working solution and wash the plate 5 times, filling each well with the solution and soaking for 30 seconds each time, then spin-drying.

[0076] (7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop the color at 37°C in the dark for 10 min. Add 50 μL of stop solution to each well in the order of substrate addition to terminate the reaction.

[0077] (8) Termination: After adding the stop solution (reaction termination), measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader within 15 minutes.

[0078] New patient plasma samples were collected again and divided into four groups: 18 chronic HBV carriers (ASC), 10 chronic hepatitis B (CHB), 26 hepatitis B cirrhosis (LC), and 15 plasma samples from healthy people who came to the hospital for physical examination during the same period as a healthy control group (N). Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of DBH and HBx, TGFβ1, Hyp, TNF-α, IL-6, and IL-1β in plasma. The correlation between the indicators was analyzed and the ROC curve was drawn to evaluate the diagnostic efficacy of DBH and various inflammatory indicators. APRI and FIB-4 refer to the AST and platelet ratio index score and the liver fibrosis 4-factor index, respectively. The calculation formula is: APRI = [AST level / upper limit of normal (ULN) × 100] / platelet count (× 109 / L); FIB-4 = age (years) × AST (U / L) / [platelet count (× 109 / L) × ALT (U / L)1 / 2]. The basic information and clinical data of the patients are shown in Table 2.

[0079] Table 2 Basic information and liver function indicators of patients in the healthy control group, chronic HBV carrier group, hepatitis B group, and cirrhosis group

[0080]

[0081] Note: Compared with hepatitis B carriers, a p<0.05; compared with hepatitis B, b p<0.05; compared with liver cirrhosis, c p<0.05; ns: no significant difference

[0082] The results showed that: (1) the level of DBH in the plasma of hepatitis B patients was 7.5 times that of the normal control group, reaching a peak in the cirrhosis stage and showing a downward trend in the hepatocellular carcinoma stage. Correlation analysis showed that the level of DBH in the plasma of hepatitis B patients was positively correlated with the expression of HBx protein, and that the level of DBH was significantly positively correlated with the levels of TGFβ1 and Hyp, indicators of liver fibrosis and collagen production (see Figure 2 ).

[0083] (2) The human tumor necrosis factor α kit (Shanghai ELISA Biologicals), human interleukin 6 kit (Shanghai ELISA Biologicals), and human interleukin 1β kit (Shanghai ELISA Biologicals) were used to detect the protein levels of inflammatory factors TNF-α, IL-6, and IL-1β in the collected serum, and the correlation between TNF-α, IL-6, IL-1β, and DBH was calculated. Standard wells and sample wells were set on the enzyme-labeled plate, and 50 μl of standard of different concentrations was added to each standard well. 50 μl of the sample to be tested was added to the sample well, and nothing was added to the blank well. The ELISA kit instructions were followed. The blank well was zeroed, and the absorbance of each well was measured at a wavelength of 450 nm. The measurement process was strictly carried out in accordance with the actual instructions. The results are as follows. Figure 3 shown.

[0084] (3) Effectiveness of the four indicators in distinguishing chronic HBV carriers from hepatitis B

[0085] When diagnosed based on a single indicator, DBH had the largest AUC (=0.6722, P=0.1372) for distinguishing chronic HBV carriers from hepatitis B, TNF-α had the highest sensitivity (80.0%), and IL-1β had the highest specificity (94.4%); the AUC (=0.7333, P<0.05) for distinguishing chronic HBV carriers from hepatitis B was higher than any single indicator, with a sensitivity of 80.0% and a specificity of 72.2%. The AUC of DBH combined with the four indicators of inflammatory factors for distinguishing HBV carriers from hepatitis B was higher than that of FIB-4. The results are shown in Figure 4-Figure 5 , Table 3.

[0086] Table 3. The diagnostic efficacy of serum TNF-α, IL-6, IL-1β and DBH levels in the differential diagnosis of chronic HBV carriage and hepatitis B

[0087] index Area under the curve (95% CI) Sensitivity (%) Specificity (%) Youden Index Cutoff value P-value TNF-α 0.6667(0.4674～0.8659) 80.0 61.1 0.4111 31.87 0.1503 IL-6 0.5778(0.3495～0.8061) 40.0 83.3 0.2333 28.55 0.5021 IL-1β 0.5778(0.3304～0.8251) 40.0 94.4 0.3444 43.43 0.5021 DBH 0.6722(0.4433～0.9012) 60.0 83.3 0.4333 91.52 0.1372 Four joint 0.7333(0.5404～0.9262) 80.0 72.2 0.5222 0.3988 ＜0.05 APRI 0.8389(0.6699～1.000) 80.0 88.9 0.6889 0.2948 ＜0.05 FIB-4 0.5667(0.3327～0.8007) 60.0 61.1 0.2111 1.100 0.5651

[0088] (4) The efficacy of the four indicators in differentiating hepatitis B from cirrhosis: When diagnosed based on a single indicator, IL-6 had the highest AUC (=0.8346, P < 0.05), followed by TNF-α (=0.7308, P < 0.05). IL-1β had the highest sensitivity (96.2%), and TNF-α and DBH had the same specificity, both as high as 100.0%. The AUC (=0.8577, P < 0.05) of the four indicators combined to differentiate hepatitis B from cirrhosis was higher than that of any single indicator, with a sensitivity of 80.8% and a specificity of 80.0%. Moreover, the AUC of the four indicators combined to differentiate hepatitis B from cirrhosis was higher than that of APRI and FIB-4, reflecting good differential diagnostic efficacy. The results are shown in Figure 6-Figure 7 , Table 4.

[0089] Table 4. The diagnostic efficacy of serum TNF-α, IL-6, IL-1β and DBH levels in the differential diagnosis of hepatitis B and liver cirrhosis

[0090] index Area under the curve (95% CI) Sensitivity (%) Specificity (%) Youden Index Cutoff value P-value TNF-α 0.7308(0.5702～0.8914) 57.7 100.0 0.5769 30.91 ＜0.05 IL-6 0.8346(0.6948～0.9744) 69.2 90.0 0.5923 23.72 ＜0.05 IL-1β 0.6654(0.4598～0.8710) 96.2 40.0 0.3615 44.23 0.1288 DBH 0.6769(0.5032～0.8506) 46.2 100.0 0.4615 80.96 0.1042 Four joint 0.8577(0.7132～1.000) 80.8 80.0 0.6077 0.7320 ＜0.05 APRI 0.7731(0.6231～0.9231) 69.2 90.0 0.5923 0.5321 ＜0.05 FIB-4 0.8346(0.6999～0.9693) 80.8 80.0 0.6077 1.361 ＜0.05

[0091] (5) The effectiveness of the four indicators in distinguishing compensated cirrhosis from decompensated cirrhosis

[0092] When diagnosed based on a single indicator, the AUC of IL-6 for distinguishing compensated and decompensated cirrhosis was the largest (=0.7368, P=0.0686), and the AUC of IL-1β (=0.7293, P=0.0779) was similar. The sensitivity of TNF-α and IL-6 (100.0%) was the highest, and the specificity of IL-1β (89.5%) was the highest. The AUC of the four indicators combined for distinguishing compensated and decompensated cirrhosis (=0.9323, P<0.05) was higher than that of any single indicator, with a sensitivity of 85.7% and a specificity of 100.0%. Compared with APRI and FIB-4, the combination of the four indicators had the best AUC, sensitivity, and specificity. The results are shown in Figure 8-9 , Table 5.

[0093] Table 5. The diagnostic efficacy of serum TNF-α, IL-6, IL-1β and DBH levels in the differential diagnosis of compensated and decompensated cirrhosis

[0094]

[0095]

[0096] Example 3: Cell experiment

[0097] An in vitro HBV-related liver fibrosis cell model was established to study the expression changes of DBH before and after HSC activation at the cellular level. LX-2 is an immortalized human hepatic stellate cell line and a good cell model for liver fibrosis research. Hepatitis B virus X protein (HBx) has been shown to promote HSC activation. Therefore, this part of the study used the culture supernatant of the LO2 cell line that stably expressed HBx protein as a conditioned medium to intervene in the hepatic stellate cell line LX2 to construct an HBV-related liver fibrosis cell model.

[0098] On the other hand, recombinant human TGF-β1 (rhTGFβ1) was used to treat LX-2 cells to study the dose- and time-dependent relationship of DBH-AS1 in HSC activation. Cell RNA was extracted and the cell culture supernatant was retained. The expression of DBH and hepatic stellate cell activation marker a-SMAmRNA in cells was detected by RT-PCR, and the secretion of DBH protein in the culture supernatant was detected by ELISA. Gene interference experiments were used to interfere with the expression of DBH in HSC cells, and the role of DBH in HSC activation was verified by CCK8 cell viability experiments.

[0099] CCK-8 cell viability assay method:

[0100] LX2 cells were grown at 1.5 × 10 3 The cells were inoculated in 96-well plates, and a control group and an experimental model group were set up respectively, with 5 replicate wells in each group.

[0101] 24 hours after the cells adhered, 10 μL of CCK-8 solution was added to each well and incubated in a 37°C, 5% CO2 incubator for 2 hours. The wavelength of the microplate reader was set to 450 nm, and the absorbance (A) value was measured to calculate the cell survival rate. Cell survival rate (%) = [A (experimental group) - A (blank)] / [A (control) - A (blank)] × 100%

[0102] Western blot experimental method:

[0103] (1) The total protein content was determined using the BCA protein concentration assay kit.

[0104] (2) Adjust the protein concentration to 1ug / ul, the loading volume to 20ul, and the protein amount to 20ug / well.

[0105] (3) Denature in a 100°C water bath for 5 minutes.

[0106] (4) Load the sample after cooling.

[0107] (5) Run the glue at 80 volts for 25 minutes and at 160 volts for 50 minutes.

[0108] (6) Electroporation: 200 mA for 2 hours.

[0109] (7) Blocking: Block with 5% skim milk TBST for 2 hours.

[0110] (8) Primary antibody: dilute the antibody at 1:1000 in 5% BSA in TBST and incubate overnight at 4°C.

[0111] (9) Wash with TBST three times, 4 min each time.

[0112] (10) Secondary antibody: dilute the antibody at 1:10,000 in 5% skim milk in TBST and incubate at room temperature for 2 hours.

[0113] (11) Wash with TBST 4 times, 4 min each time.

[0114] (12) Color development: Prepare the luminescent solution at a ratio of 1:1, cover the membrane surface, and expose after 2 minutes of color development. Image acquisition and analysis.

[0115] Fluorescence real-time quantitative PCR (RT-PCR) method:

[0116] After the LX2 cell model was successfully constructed, it was washed with PBS, and the cells were lysed by adding 1mL Trizol to each 6-well plate. Chloroform was added and centrifuged at 4°C and 12000r / min for 15min to extract RNA. The upper liquid phase was precipitated with isopropanol, and each tube was washed with 1mL 75% ethanol (prepared with DEPC water) and the supernatant was discarded. The precipitated RNA was dried at room temperature and dissolved in DEPC water, and the RNA concentration was determined by Nanodrop. cDNA was synthesized according to the instructions of the reverse transcription kit, and 2μL was taken for RT-PCR. RT-PCR used the SYBR Green dye method to prepare a total system of 10μL, with β-actin as the internal reference gene, denaturation at 95℃ for 20s, annealing at 60℃ for 20s, extension at 72℃ for 50s, and a total of 40 cycles. The primers required in this experiment were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the sequences are shown in Table 6.

[0117] Table 6 Primer sequences of human related genes

[0118]

[0119]

[0120] result:

[0121] (1) Construction of LO2 stably transfected cells (LO2-HBx) that stably express the HBx gene. It can be seen that except for the cells in the LO2-HBx group, the cells in the control group and the blank group did not express HBx protein ( Fig.10 ).

[0122] (2) Effects of three groups of conditioned medium on the activation of LX-2 cells: The morphology of LX-2 cells in the three co-culture systems of LX-2 / LO2-HBx, LX-2 / LO2-con, and LX-2 / Mock was observed. After 48 hours of conditioned culture, the morphology showed obvious differentiation. Among them, the LX-2 cells in the LX-2 / LO2-HBx group showed obvious cell morphological changes, including cell shrinkage, significant elongation of cell processes, and a decrease in intracellular lipid droplets ( Fig.11 a), the expression levels of α-SMA and Col1A1 mRNA (F values ​​were 144.7 and 76.68, both P values ​​< 0.01) and protein (F values ​​were 234.1 and 5287, both P values ​​< 0.001) were increased ( Fig.11 b, c).

[0123] (3) Effects of three groups of conditioned medium on the proliferation of LX-2 cells. The proliferation activity of LX-2 cells in the LX-2 / LO2-HBx group was higher than that of LX-2 cells in the LX-2 / LO2-con group after 48 hours of conditioned culture (F=8.499, P<0.05). Fig.12 ).

[0124] (4) Recombinant human TGFβ1 (rhTGFβ1) stimulated LX-2 cells to detect changes in DBH expression: LX-2 cells were divided into four groups and treated with rhTGFβ1 concentrations of no treatment (0 ng / ml), 5 ng / ml, 10 ng / ml, and 15 ng / ml, respectively. After 48 h of culture, RNA was extracted from the four groups of cells to detect the expression levels of α-SMA, Col1A1, and DBH mRNA in the cells. The results showed that with the increase in rhTGFβ1 stimulation concentration, the expression of α-SMA (F=1794, P<0.01), Col1A1 (F=91.3, P<0.01), and DBH (F=2501, P<0.01) increased, reaching a peak at rhTGFβ1 10 ng / ml. When the rhTGFβ1 concentration reached 15 ng / ml, the expression of α-SMA, Col1A1, and DBH began to decline from the peak ( Fig.13 ).

[0125] In summary, the present invention has compared the differentially expressed genes of cancer tissues and adjacent cirrhotic liver tissues of hepatitis B-related hepatocellular carcinoma patients through gene chip detection, among which DBH mRNA has significant differences in inter-group comparisons, and the expression level in cirrhotic liver tissue is 24 times that of liver cancer tissue. The present invention has expanded the tissue sample volume to verify the chip results, and the expression of DBH mRNA in cirrhotic liver tissue is significantly upregulated in 46 pairs of liver cancer and cirrhotic liver tissues. At the same time, the present invention found that the content of DBH in the plasma of hepatitis B patients is 7.5 times that of the plasma of the normal control group, and the content reaches a peak in the cirrhosis stage, and a downward trend appears in the hepatocellular carcinoma stage. Through correlation analysis, it is found that the DBH content in the plasma of hepatitis B patients is positively correlated with the expression of HBx protein, and the DBH content is significantly positively correlated with the content of liver fibrosis and collagen production indicators TGFβ1 and Hyp. Cell experiments have found that HBx or TGFβ1 can induce DBH upregulation in hepatic stellate cells, indicating that DBH is related to the activation of hepatic stellate cells. More importantly, the expression of DBH in the patient's plasma is positively correlated with the development of hepatitis B-cirrhosis, but DBH expression decreases in the liver cancer stage. This finding is mutually confirmed by the results of gene chip experiments and tissue verification. In addition, the present invention also found that DBH has a strong correlation with the level of inflammatory factors in plasma. The detection of combined inflammatory factors has good diagnostic efficacy for diagnosing and predicting the development of hepatitis B-cirrhosis, and also has good diagnostic efficacy for distinguishing compensated and decompensated cirrhosis patients. The diagnostic efficacy is better than the existing evaluation combination modes APRI and FIB4. Therefore, the present invention illustrates that the expression of DBH in liver tissue and plasma of patients with hepatitis B cirrhosis increases significantly when hepatitis B progresses to the cirrhosis stage. The mechanism of increase is related to the activation of hepatic stellate cells after HBV infection. HBx and TGFβ1 can upregulate the expression of DBH. It is also found that DBH is related to the inflammatory response of hepatitis B patients. Combining DBH with plasma inflammatory factors of hepatitis B patients is beneficial to conveniently and preliminarily screening the disease progression of hepatitis B patients, and can improve the evaluation level of the severity of hepatitis B disease development.

[0126] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biomarker for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, characterized in that: The markers include one or more of DBH, TNF-α, IL-6, and IL-1β.

2. The biomarker according to claim 1, characterized in that The biomarker is one or more of DBH, TNF-α, IL-6, and IL-1β.

3. The biomarker according to claim 1, characterized in that The biomarkers are DBH, TNF-α, IL-6 and IL-1β.

4. Use of a marker as claimed in any one of claims 1 to 3 in the preparation of a product for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis.

5. The use according to claim 4, characterized in that The product comprises an antibody, a primer, a probe or a kit.

6. Use of the marker according to claim 1 in diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, wherein the use is for non-diagnostic or non-therapeutic purposes; The application includes the following: (a) Diagnosis of chronic hepatitis B and / or progressive cirrhosis; (b) Screening of drugs for the treatment of chronic hepatitis B and / or liver cirrhosis; (c) Assist in the construction of cell or animal models of chronic hepatitis B and / or liver cirrhosis; Optionally, the use is for the purpose of scientific research.

7. A primer set for detecting the marker according to claim 1, characterized in that: The primer pair sequences are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

8. A kit for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, characterized in that: The kit comprises the primer set according to claim 7.

9. Use of the primer set according to claim 7 in the preparation of a kit for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis.

10. A method for diagnosing the progression of chronic hepatitis B and / or liver cirrhosis, characterized in that: It comprises the step of using the primers described in claim 7 or the kit described in claim 8 to detect the biomarkers described in any one of claims 1 to 3 in the sample to be tested, and the application is for non-diagnostic purposes, optionally, the application is for the purpose of scientific research; optionally, the sample is an in vitro sample.

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