Biomarkers and their use in the assessment of liver damage in end-stage liver disease

Abstract

The present application relates to the field of medical immunology technology, and more particularly to biomarkers and their application in the assessment of liver damage in end-stage liver disease. In the present application, the proportion of CCR7+CD8+ T cells in CD8+ T cells is used as a marker for the assessment of liver damage in end-stage liver disease, which can reflect the liver immune status of end-stage liver disease, especially the assessment of the degree of liver damage, has high specificity and sensitivity, and provides a scientific basis for the development of clinical individualized treatment plan, and has good practical application value. At the same time, while reflecting the liver damage process of the patient, it can also reflect the immune status of the patient's liver, and can provide certain theoretical basis for slowing down the disease progression of subsequent biliary atresia and other end-stage liver disease patients with liver damage.

Description

Technical Field

[0001] This invention relates to the field of medical immunology, and more particularly to biomarkers and their application in assessing liver damage in end-stage liver disease. Background Technology

[0002] End-stage liver disease is the final outcome of chronic liver disease progressing to cirrhosis, decompensation, and chronic liver failure, characterized by portal hypertension or complications related to hepatocellular failure. Orthotopic liver transplantation is the only treatment option for patients with end-stage liver disease.

[0003] Currently, the clinical assessment of liver damage in end-stage liver disease mainly relies on serum biochemical indicators, imaging, and liver histopathological examination. However, these methods have limitations such as insufficient sensitivity, high invasiveness, or inability to dynamically monitor the condition. Furthermore, these methods cannot assess the patient's liver immune status or provide timely guidance for clinical medication. In addition, biliary atresia is one of the main causes of end-stage liver disease in children. Current assessments and prognoses of liver damage due to biliary atresia largely depend on liver biopsy and clinical indicators (plasma direct bilirubin, alanine aminotransferase, aspartate aminotransferase, total bile acid levels, etc.), but these methods all have certain limitations. First, liver biopsy has specific requirements for sample collection and can only reflect the local liver damage. Second, clinical indicators have low specificity and cannot predict the progression of liver damage. Moreover, none of the above conventional methods can assess the patient's liver immune status.

[0004] Therefore, there is an urgent need to discover a biomarker that can not only assess and predict the progression of liver damage in end-stage liver disease, but also reflect the liver's immune capacity in patients with end-stage liver disease. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide biomarkers and their application in the assessment of liver damage in end-stage liver disease.

[0006] This invention provides the application of the proportion of CCR7+CD8+ T cells in CD8+ T cells as a biomarker in the preparation of products for assessing liver injury in end-stage liver disease.

[0007] Furthermore, the liver injury in the end-stage liver disease includes at least one of: biliary atresia, progressive familial intrahepatic cholestasis, maple syrup diabetes mellitus, and / or glycogen storage disease.

[0008] In this invention, the population with end-stage liver disease and liver injury includes children (including infants and newborns), adults, and the elderly. This invention focuses on biliary atresia, one type of end-stage liver injury, demonstrating that the proportion of CCR7+CD8+ T cells in CD8+ T cells can serve as a biomarker for liver injury caused by biliary atresia. Furthermore, significant differences in the proportion of CCR7+CD8+ T cells in CD8+ T cells were also found between patients and controls in other end-stage liver injuries (such as progressive familial intrahepatic cholestasis type 3, maple syrup diabetes, and glycogen storage disease). Therefore, this indicates that the proportion of CCR7+CD8+ T cells in CD8+ T cells can serve as a biomarker for end-stage liver injury, used to assess and predict the progression of liver injury in end-stage liver disease.

[0009] This invention provides a biomarker for assessing liver damage in end-stage liver disease, namely the proportion of hepatic CCR7+CD8+ T cells in CD8+ T cells.

[0010] The present invention provides combined biomarkers, including at least one of the proportion of CD3+ T cells in lymphocytes, the proportion of CCR7+CD4+ T cells in CD4+ T cells, the proportion of CD8+ T cells in CD3+ T cells, and the biomarkers described in the present invention.

[0011] Furthermore, the proportion of CD3+ T cells in lymphocytes is derived from liver tissue;

[0012] The proportion of CCR7+CD4+ T cells in CD4+ T cells originates from liver tissue;

[0013] The proportion of CD8+ T cells in CD3+ T cells was derived from peripheral blood.

[0014] The combined biomarkers described in this invention also include other liver disease-related biomarkers.

[0015] Other liver disease-related biomarkers include serum biomarkers, tissue biomarkers, and / or imaging biomarkers.

[0016] The serum and / or tissue markers include liver function-related indicators; these liver function-related indicators include: albumin (Alb), prealbumin (PAB), globulin, total protein (TP), prothrombin time (PT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), cholinesterase (ChE), cholesterol (Chol), bile acids (TBA), hyaluronic acid (HA), laminin (LN), type IV collagen (CIV), type III procollagen (PⅢP), degree of hepatocyte degeneration and necrosis (e.g., hepatocyte ballooning degeneration, fragmented necrosis, bridging necrosis, etc.), degree of liver fibrosis, inflammatory activity (according to the grade of inflammation, such as G0-G4), and changes in liver tissue structure.

[0017] The imaging biomarkers include those based on CT and MRI examinations: liver morphology and density / signal, liver vascularity, characteristic features of liver cancer, elastography, and liver tissue stiffness characteristics.

[0018] This invention uses flow cytometry to detect the level of CCR7+CD8+ T cells in liver tissue of patients with end-stage liver disease, and assesses liver damage in patients with biliary atresia based on their expression levels. Results showed that in liver samples, the proportion of CCR7+CD8+ T cells was significantly negatively correlated with liver AST and TBA levels; the proportion of CCR7+CD8+ T cells in peripheral blood was positively correlated with ALP levels; however, in lymph node samples, the level of CCR7+CD8+ T cells was not correlated with liver-related indicators.

[0019] In addition, this invention also explored whether the expression of other cell types could serve as biomarkers for end-stage liver injury, such as the proportion of CD3+ T cells (the proportion of CD3+ T cells in the liver was negatively correlated with TBA levels), the proportion of CD4+ T cells (no correlation with liver function indicators), the proportion of CCR7+CD4+ T cells (the proportion of CCR7+CD4+ T cells was negatively correlated with AST and TBA), and the proportion of CD8+ T cells (the proportion of CD8+ T cells in liver samples was not correlated with liver function indicators; the proportion of CD8+ T cells in lymph node samples was not correlated with liver function indicators; the proportion of CD8+ T cells in peripheral blood samples was positively correlated with GGT levels). Although the expression of these cell proportions showed significant differences between BA patients and the Non-BA group, their correlation with liver function indicators was lower than that of the proportion of CCR7+CD8+ T cells. Furthermore, based on this, the correlation between the proportions of CCR7+CD4+ T cells and CCR7+CD8+ T cells, which were highly correlated with liver function indicators, and liver injury was further investigated. The results showed that the proportions of CCR7+CD4+ T cells and CCR7+CD8+ T cells, which were highly correlated with liver function indicators, and liver injury were significantly different. The proportion of T cells was not correlated with the liver fibrosis score, while the proportion of CCR7+CD8+ T cells was negatively correlated with the liver fibrosis score. The liver fibrosis score can more directly reflect the degree of liver damage, suggesting that the proportion of CCR7+CD8+ T cells is more suitable as an indicator for assessing liver damage.

[0020] In addition to the aforementioned proportions of CD3+ T cells, CD4+ T cells, CCR7+CD4+ T cells, and CD8+ T cells, this invention also explored whether other cell types or other cell proportions or serum markers could serve as markers of end-stage liver injury. The results showed that the proportion of CCR7+CD8+ T cells in CD8+ T cells had the best individual specificity in assessing the degree of liver injury in end-stage liver disease and a better correlation with liver function-related indicators.

[0021] This invention provides reagents for detecting or targeting the biomarkers and / or combined biomarkers described in this invention.

[0022] The present invention provides a kit comprising the reagents and auxiliary materials described herein, wherein the auxiliary materials include at least one of enzymes, washing solutions, culture media, lymphocyte separation media, and / or lysis media.

[0023] The present invention provides an immune assessment system or device for assessing the degree of liver damage in end-stage liver disease, which is based on the expression level of CCR7+CD8+ T cells in CD8+ T cells.

[0024] The assessment criteria include: the proportion of CCR7+CD8+ T cells in CD8+ T cells is negatively correlated with the degree of liver damage in end-stage liver disease; as the proportion decreases, it indicates that the patient's liver damage has worsened.

[0025] The evaluation system or apparatus described in this invention can be understood as a semi-automatic or automated mechanical or electronic device loaded with reagents and other auxiliary materials for detecting or targeting the biomarkers and / or combined biomarkers described in this invention, for indirectly or directly outputting results of the biomarkers and / or combined biomarkers described in this invention, or the results can be directly associated with clinical diagnostic conclusions.

[0026] This invention also provides a method for assessing liver damage in end-stage liver disease. This method assesses liver damage in end-stage liver disease based on the proportion of CCR7+CD8+ T cells among CD8+ T cells in the patient's liver tissue. A decreased proportion of CCR7+CD8+ T cells is negatively correlated with the severity of liver damage in end-stage liver disease; a decrease in the proportion indicates worsening liver damage.

[0027] This application used flow cytometry to examine 30 patients with biliary atresia (BA group), 20 patients with non-choleretic diseases (Non-BA group), 29 RRV-induced biliary atresia mouse models (experimental group), and 17 wild-type mice (control group). Flow cytometry analysis of the percentage of CCR7+CD8+ T cells revealed that the percentage of CCR7+CD8+ T cells in the BA group was significantly lower than that in the Non-BA group, and the percentage in the experimental group was also significantly lower than that in the control group. Furthermore, the percentage of CCR7+CD8+ T cells in the BA group was significantly negatively correlated with the patients' plasma AST and TBA levels; a decrease in the percentage indicated worsening liver damage.

[0028] This invention provides a method for assessing the degree of liver damage in end-stage liver disease by exploring the level of CCR7+CD8+ T cells (the proportion of CCR7+CD8+ T cells among CD8+ T cells). This method can assess and predict the progression of liver damage in patients and also reflects the patient's hepatic immune capacity. Furthermore, compared to other liver damage assessment indicators, the detection of CCR7+CD8+ T cells has better individual specificity.

[0029] By detecting or targeting the biomarkers or combined biomarkers described in this invention, or by utilizing the assessment methods described in this invention, not only can liver damage in patients be assessed, but the patient's liver immune status can also be further understood, allowing for timely adjustments to treatment strategies. This invention can also be combined with existing clinical scoring systems (such as the Metavir score) to further improve the accuracy of assessing the degree of liver damage.

[0030] This invention is applicable to the assessment of liver damage progression in end-stage liver disease. By detecting CCR7+CD8+ T cells, the degree of liver damage and hepatic immune capacity can be determined in a timely manner, allowing for timely adjustments to the treatment plan. Furthermore, this method can be combined with other assessment tools to provide a reference for personalized treatment. With the development of detection technology, this method is expected to be widely used in clinical practice, becoming a tool for assessing liver damage in end-stage liver disease.

[0031] In summary, this invention provides an assessment method for liver injury in end-stage liver disease based on the CCR7+CD8+ T cell level. By detecting the proportion of CCR7+CD8+ T cells in the patient's liver tissue, it assesses the degree of liver injury and the liver's immune capacity. This method is simple to operate, has higher specificity and personalization, and can provide a reliable means of assessing liver injury in clinical practice, as well as a scientific basis for the development of individualized treatment plans.

[0032] This invention uses the ratio of CCR7+CD8+ T cells to CD8+ T cells as a biomarker to assess liver damage in end-stage liver disease. This biomarker can reflect the liver's immune status in end-stage liver disease, especially in assessing the degree of liver damage. It has high specificity and sensitivity, and provides a scientific basis for the development of personalized clinical treatment plans, thus having good practical application value. At the same time, it can reflect the patient's liver immune status while reflecting the progression of liver damage, which can provide a certain theoretical basis for slowing the progression of disease in patients with biliary atresia and other end-stage liver diseases. Attached Figure Description

[0033] Figure 1 The percentage of CCR7+CD8+ T cells in patients with biliary atresia is shown. A represents the flow cytometry gating strategy of CCR7+CD8+ T cells; B represents the significantly lower expression of CCR7+CD8+ T cells in the BA group compared to the Non-BA group.

[0034] Figure 2 The percentage of CCR7+CD8+ T cells in RRV-induced biliary atresia mice is shown. In this figure, A represents the flow cytometry gating strategy of CCR7+CD8+ T cells, and B represents the significantly lower expression of CCR7+CD8+ T cells in the RRV group compared to the control group.

[0035] Figure 3The spatial distribution of CCR7+CD8+ T cells in the liver tissue of patients with biliary atresia is shown. A shows the immunofluorescence staining results of liver tissue from patients with biliary atresia; B shows that the mean fluorescence intensity of CD8+ T cells expressed in the liver tissue of the BA group was higher than that of the Non-BA group; C shows that the co-localization of CCR7 and CD8+ T cells in the liver tissue of the BA group was lower than that of the Non-BA group; D shows that the mean fluorescence intensity (MFI) of TUNEL+ cells in the BA group was higher than that in the Non-BA group; E shows that the proportion of CCR7+CD8+ T cells and the MFI of TUNEL+ cells were negatively correlated.

[0036] Figure 4 The spatial distribution of CCR7+CD8+ T cells in the liver tissue of RRV-induced biliary atresia mice is shown. A represents the immunofluorescence staining results of liver tissue from RRV mice; B shows that the mean fluorescence intensity of CD8+ T cells expressed in the liver tissue of the RRV group was higher than that in the control group; C shows that the co-localization of CCR7 and CD8+ T cells in the liver tissue of the RRV group was lower than that in the control group; D shows that the mean fluorescence intensity (MFI) of TUNEL+ cells in the RRV group was higher than that in the control group; E shows that the proportion of CCR7+CD8+ T cells and the MFI of TUNEL+ cells were negatively correlated.

[0037] Figure 5 The fitted curves of the correlation analysis between the percentage of CCR7+CD8+ T cells in the liver and liver function indicators in patients with biliary atresia are shown.

[0038] Figure 6 The table shows the proportion of CCR7+CD8+ T cells in the liver of other end-stage liver diseases, where A represents Alagille; B represents PFIC3 (progressive familial intrahepatic cholestasis type 3); C represents MSUD (maple syrup diabetes mellitus); and D represents GSD (glycogen storage disease).

[0039] Figure 7 This indicates the proportion of CCR7+CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient;

[0040] Figure 8 The figures show the proportions of CD8+ and CD4+ T cells in lymphocytes before and after optimization of the mouse liver lymphocyte extraction method. A represents the flow cytometry gating strategy before optimization; B represents the flow cytometry gating strategy after optimization; C represents the proportion of CD8+ T cells in lymphocytes after optimization being higher than before optimization; and D represents the proportion of CD4+ T cells in lymphocytes after optimization being higher than before optimization.

[0041] Figure 9 The proportion of CD3+ T cells in the liver of the BA group was higher than that of the Non-BA group;

[0042] Figure 10The proportion of CD4+ T cells in the liver of the BA group was higher than that of the Non-BA group;

[0043] Figure 11 The expression of CD4+ T cells gradually increased in the liver, lymph nodes, and peripheral blood of the same BA patient;

[0044] Figure 12 The proportion of CCR7+CD4+ T cells in the liver of the BA group was lower than that of the Non-BA group;

[0045] Figure 13 The proportion of CCR7+CD4+ T cells gradually increases in the liver, lymph nodes, and peripheral blood of the same BA patient;

[0046] Figure 14 The proportion of CD8+ T cells in the liver of the BA group was higher than that of the Non-BA group;

[0047] Figure 15 The proportion of CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient gradually decreased. Detailed Implementation

[0048] This invention provides biomarkers and their application in assessing liver injury in end-stage liver disease. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0049] This invention proposes a method for assessing liver damage in end-stage liver disease based on CCR7+CD8+ T cells. This method assesses the progression of liver damage by detecting the level of CCR7+CD8+ T cells in the patient's liver tissue. It also provides information on the patient's hepatic immune status, which can help adjust subsequent treatment plans.

[0050] In some specific embodiments of the present invention, the above detection method is flow cytometry, comprising:

[0051] S1. Separate and extract liver lymphocytes from the patient;

[0052] S2. The percentage of CCR7+CD8+ T cells among CD8+ T cells was obtained by flow cytometry;

[0053] S3. Determine the patient's liver immune status and assess the extent of liver damage based on the test results.

[0054] CCR7+CD8+ T cells refer to the CD8+ T cell subset that expresses the CCR7 molecule, and their level is closely related to the degree of liver injury caused by biliary atresia.

[0055] In the flow cytometry analysis involved in this application, the proportion of CCR7+CD8+ T cells is the number of CCR7+CD8+ T cells / the number of CD8+ T cells × 100%.

[0056] In this invention, the percentage of CCR7+CD8+ T cells, the proportion of CCR7+CD8+ T cells, or the proportion of CCR7+CD8+ T cells among CD8+ T cells have equivalent representative significance.

[0057] In this invention, the proportion of CD3+ T cells in lymphocytes is referred to as the CD3+ T cell ratio; where lymphocytes here refers to the total number of lymphocytes obtained in step S1.

[0058] The proportion of CD4+ T cells to CD3+ T cells, or simply CD4+ T cell ratio;

[0059] The proportion of CCR7+CD4+ T cells in CD4+ T cells is simply referred to as the CCR7+CD4+ T cell ratio.

[0060] The proportion of CD8+ T cells among CD3+ T cells is simply referred to as the CD8+ T cell ratio.

[0061] In some embodiments of the present invention, patients diagnosed with biliary atresia are recruited as research subjects.

[0062] In some embodiments of the present invention, the anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, and anti-CCR7 antibody, CD3+CD8+CD4-CCR7+ represent CCR7+CD8+ T cells, and the proportion of CCR7+CD8+ T cells in liver tissue is detected. The expression of CCR7 in CD8+ T cells is detected by antibody staining combined with flow cytometry.

[0063] In some embodiments of the present invention, correlation analysis was used to assess the relationship between CCR7+CD8+ T cell levels and liver injury caused by biliary atresia. According to the analysis results, a decrease in the proportion of CCR7+CD8+ T cells indicated worsening liver damage in patients.

[0064] A specific embodiment is a method for assessing liver damage in patients with biliary atresia by detecting CCR7+CD8+ T cell levels, specifically:

[0065] Study population: Clinical specimens included were from patients admitted to the hepatobiliary surgery department between 2021 and 2024. This included 30 patients diagnosed with liver biliary atresia (BA) as the experimental group (BA), and 20 patients with non-choleretic diseases requiring liver transplantation, including hepatoblastoma (liver tissue >1 cm from the tumor), hemangioma, and portal vein stenosis, as the control group (Non-BA). Liver, lymph node, and peripheral blood samples were collected from BA patients, and liver tissue was collected from Non-BA patients as clinical samples for this study.

[0066] Sample Collection: Collected liver tissue samples from patients were placed in sterile phosphate-buffered saline (PBS). After washing, the samples were cut into pieces <1 mm³. These pieces were digested with collagenase IV (Sigma-Aldrich) and DNase I (Roche). Cells were collected and centrifuged. The cell pellet was resuspended in 10 mL of 35% Percoll and centrifuged at 2000 rpm for 30 min at 4 °C to isolate liver mononuclear cells (MNCs) for flow cytometry.

[0067] Flow cytometry assay: 1640 medium containing 2% fetal bovine serum was added to freshly extracted liver mononuclear cells (MNCs) and mixed thoroughly. 100 μL of the cell mixture was then added to flow cytometry tubes, ensuring each tube contained 10 cells. 6 Cells were collected. After centrifugation and removal of the supernatant, anti-CD3, anti-CD4, anti-CD8, and anti-CCR7 antibodies were added for flow cytometry staining. The cells were incubated at room temperature for 30 minutes. Cell analysis was performed using a flow cytometer.

[0068] Data were analyzed using flow cytometry software (such as FlowJo). The proportion of CCR7+CD8+ T cells (CD3+CD8+CD4-CCR7+) to total CD8+ T cells (CD3+CD8+CD4-) was determined, and the percentage of CCR7+CD8+ T cells was recorded.

[0069] CCR7+CD8+ T cell level assessment: The test results show the percentage of CCR7+CD8+ T cells, and as the percentage decreases, it indicates that the patient's liver damage has worsened.

[0070] Integration with Clinical Scoring Systems: The method of this invention can be used in conjunction with existing liver injury scoring systems (such as the Metavir score). By comparing the results of CCR7+CD8+ T cell detection with the clinical score, the accuracy of the assessment can be improved. If a patient has low expression of CCR7+CD8+ T cells and the clinical score indicates severe liver injury, the patient's liver injury can be more clearly confirmed, allowing for more aggressive treatment measures.

[0071] The abbreviations used in this application are as follows: BA, Biliary Atresia; KPE, Kasaiportoenterostomy, Kasai portojejunostomy for the treatment of biliary atresia; CCR7, CC chemokine receptor type 7; RRV, Rhesus rotavirus type A; TBIL, Total bilirubin; DBIL, Direct bilirubin; IBIL, Indirect bilirubin; ALT, Alanine aminotransferase; AST, Aspartate Transaminase; GGT, Gamma-glutamyl transferase; ALP, Alkaline phosphatase; LDH, Lactate dehydrogenase; CHE, Cholinesterase; TBA, Total Bile acid, total bile acid.

[0072] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0073] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0074] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0075] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0076] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0077] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0078] Example 1: The proportion of CCR7+CD8+ T cells can assess the degree of liver damage in end-stage liver disease.

[0079] I. Flow cytometry analysis of the proportion of CCR7+CD8+ T cells in patients with biliary atresia

[0080] Samples to be tested: Thirty patients with biliary atresia (BA group) and 20 patients without cholestasis (Non-BA group) were included in the study according to the inclusion and exclusion criteria. Clinical laboratory indicators of the BA group are shown in Table 1.

[0081] Table 1. Laboratory indicators of liver function in the BA group

[0082]

[0083] Materials: Anti-CD3 (APC-Cy7) antibody, anti-CD4 (BV650) antibody, anti-CD8 (BV510) antibody and anti-CCR7 (PE) antibody were purchased from BD Pharmingen; human lymphocyte separation medium (Cedarlane) was purchased from Beijing Dakowei Biotechnology Co., Ltd.

[0084] Experimental methods:

[0085] Collected liver tissue samples from patients were placed in sterile phosphate-buffered saline (PBS). After washing, the samples were cut into pieces <1 mm³. These pieces were digested with collagenase IV (Sigma-Aldrich) and DNase I (Roche). Cells were collected and centrifuged. The cell pellet was resuspended in 10 mL of 35% Percoll and centrifuged at 2000 rpm for 30 min at 4°C to isolate liver mononuclear cells (MNCs) for flow cytometry.

[0086] Take 100 μL containing 10 6MNC cells were incubated with 1 μL each of anti-CD3 (APC-Cy7), anti-CD4 (BV650), anti-CD8 (BV510), and anti-CCR7 (PE) antibodies. After incubation at room temperature for 30 min, 1 mL of PBS was added to each cell, and the cells were centrifuged at 500g for 5 min. After washing once, 200 μL of PBS was added for suspension, and the cells were flow cytometry analyzed.

[0087] Results analysis: The percentage of CCR7+CD8+ T cells in patients with biliary atresia is shown in the table below. Figure 1 , Figure 1 A in the figure represents the flow cytometry gating strategy of CCR7+CD8+ T cells in this example. Figure 1 The results of B showed that the expression of CCR7+CD8+ T cells in the BA group was significantly lower than that in the Non-BA group, and the proportion of CCR7+CD8+ T cells in the liver of the Non-BA group was 11.88% (95% CI of mean: 6.959%, ​​16.79%), while the proportion of CCR7+CD8+ T cells in the liver of the BA group was lower than this range.

[0088] II. Flow cytometry detection of CCR7+CD8+ T cell proportion in an RRV-induced biliary atresia mouse model

[0089] Test samples: 29 RRV-induced biliary atresia mice (experimental group) and 17 wild-type BALB / c mice (control group).

[0090] Materials: Anti-CD45 (FITC) antibody, anti-CD4 (APC) antibody, anti-CD8 (BV510) antibody and anti-CCR7 (PE) antibody were purchased from BD Pharmingen; mouse lymphocyte separation medium was purchased from Cytiva.

[0091] Experimental methods:

[0092] Collected liver tissue samples were placed in sterile phosphate-buffered saline (PBS). After washing, the samples were ground in a culture dish using a 10 mL syringe, and the cells were collected and centrifuged. The cells were resuspended in 2 mL of mouse lymphocyte separation Ficoll and centrifuged at a gradient (800 g, room temperature, ascending speed 3, descending speed 3, 20 min) to isolate liver mononuclear cells (MNCs) for flow cytometry.

[0093] Take 100 μL containing 10 6MNC cells were incubated with 1 μL each of anti-CD45 (FITC), anti-CD4 (APC), anti-CD8 (BV510), and anti-CCR7 (PE) antibodies. After incubation at room temperature for 30 min, 1 mL of PBS was added to each cell. The cells were centrifuged at 500 g for 5 min, washed once, and then suspended in 200 μL of PBS. The cells were then analyzed by flow cytometry.

[0094] Results analysis: The percentage of CCR7+CD8+ T cells in RRV mice is shown in the figure. Figure 2 , Figure 2 A in the figure represents the flow cytometry gating strategy of CCR7+CD8+ T cells in this example. Figure 2 The results from B showed that the expression of CCR7+CD8+ T cells in the experimental group was significantly lower than that in the control group.

[0095] III. Spatial Distribution of CCR7+CD8+ T Cells in Liver Tissue with Biliary Atresia

[0096] Samples to be tested: liver tissue sections from patients with biliary atresia (BA group) and patients with non-choleretic diseases (Non-BA group).

[0097] Materials: Rabbit anti-mouse / human CCR7 (purchased from Abcam), Rat anti-mouse / human CD8 (purchased from NOVUS Biologicals), Goat anti-Rabbit AF790 and Donkey anti-RatDylight650 (purchased from Invitrogen), TUNEL apoptosis kit (purchased from Absin), DAPI (purchased from Beyotime), and sodium citrate antigen retrieval solution (purchased from Solarbio).

[0098] Experimental methods:

[0099] Liver biopsy samples were fixed overnight in 4% paraformaldehyde, then processed and embedded in paraffin. 5µm thick paraffin sections were dewaxed, heat-induced antigen retrieval with sodium citrate antigen retrieval solution (50×, Solarbio), and blocked with 5% bovine serum albumin. Sections were incubated overnight in a humidified chamber at 4°C in the dark using primary antibodies (rabbit anti-mouse / human CCR7 (1:50), Abcam); rat anti-mouse / human CD8 (1:100), NOVUS Biologicals). After washing with PBS, sections were incubated for 1 hour at room temperature in the dark with secondary antibodies (goat anti-rabbit AF790 (1:500), Invitrogen; donkey anti-rat Dylight650 (1:500), Invitrogen) followed by nuclear staining with DAPI (Beyotime). Apoptosis staining was performed using the TUNEL apoptosis detection kit (green fluorescence, Absin). Immunofluorescence images and analysis were acquired using a rotating disk microscope (Nikon).

[0100] Results analysis: Spatial distribution of CCR7+CD8+ T cells in the liver tissue of patients with biliary atresia was shown in... Figure 3 , Figure 3 A in the image shows the immunofluorescence staining results of liver tissue from a patient with biliary atresia. Figure 3 The mean fluorescence intensity (MFI) of liver CD8+ T cells in the BA group was higher than that in the Non-BA group. Figure 3 The C-value in the liver tissue of the BA group showed a lower degree of co-localization of CCR7 and CD8+ T cells compared to the Non-BA group. Figure 3 The mean fluorescence intensity (MFI) of TUNEL+ cells in the BA group was higher than that in the Non-BA group. Figure 3 The proportion of CCR7+CD8+ T cells and the MFI of TUNEL+ cells were negatively correlated (r=-0.4963 (-0.7699 to -0.06861)) (P=0.022).

[0101] IV. Spatial distribution of CCR7+CD8+ T cells in liver tissue of RRV-induced biliary atresia mice

[0102] Test samples: Liver tissue sections from RRV-induced biliary atresia mice and liver tissue sections from wild-type control mice.

[0103] Materials and experimental methods were the same as before regarding the spatial distribution of CCR7+CD8+ T cells in the liver tissue of patients with biliary atresia.

[0104] Results analysis: Spatial distribution of CCR7+CD8+ T cells in the liver tissue of RRV-induced biliary atresia mice is shown in the figure. Figure 4 , Figure 4 The A in the image shows the immunofluorescence staining results of RRV mouse liver tissue. Figure 4In the B group, the mean fluorescence intensity (MFI) of CD8+ T cells in the liver was higher than that in the control group. Figure 4 The C-value in the liver tissue of the RRV group showed a lower degree of co-localization of CCR7 and CD8+ T cells compared to the control group. Figure 4 The mean fluorescence intensity (MFI) of TUNEL+ cells in the RRV group was higher than that in the control group. Figure 4 The proportion of CCR7+CD8+ T cells and the MFI of TUNEL+ cells were negatively correlated (r=-0.8359 (-0.9563 to -0.4734)) (P=0.0013).

[0105] V. Correlation analysis of the proportion of CCR7+CD8+ T cells in the liver and liver function indicators in patients with biliary atresia.

[0106] Table 2. Correlation between liver function indicators and the proportion of hepatic CCR7+CD8+ T cells in patients

[0107]

[0108] By collecting laboratory liver function indicators and clinical data from patients with biliary atresia, a correlation analysis was conducted on the proportion of CCR7+CD8+ T cells and various liver function indicators.

[0109] Results analysis: Table 2 shows the correlation analysis results between CCR7+CD8+ T cells and various liver function indicators. The percentage of CCR7+CD8+ T cells was negatively correlated with plasma AST and TBA levels. Figure 5 The fitted curves show the correlation between the percentage of CCR7+CD8+ T cells and liver function indicators.

[0110] VI. Flow cytometry detection of the proportion of CCR7+CD8+ T cells in the liver of other end-stage liver diseases

[0111] The samples to be tested were: Alagille (1 case); PFIC3 (progressive familial intrahepatic cholestasis type 3) (2 cases); MSUD (maple syrup diabetes mellitus) (1 case); Glycogen storage disease (GSD) (2 cases); and control samples (3 cases each).

[0112] Materials and methods were the same as those used in the previous flow cytometry analysis to detect the proportion of CCR7+CD8+ T cells in patients with biliary atresia.

[0113] Results analysis: Figure 6 The proportion of CCR7+CD8+ T cells in the liver of other end-stage liver diseases showed a decreasing trend compared with the control group. Among them, A is Alagille, B is PFIC3 (progressive familial intrahepatic cholestasis type 3), C is MSUD (maple syrup diabetes mellitus), and D is GSD (glycogen storage disease).

[0114] In summary, this application provides a method based on CCR7+CD8+ T cells, which uses flow cytometry to detect the level of CCR7+CD8+ T cells in liver tissue. Combined with a clinical scoring system, this method can effectively assess liver damage in patients with biliary atresia and end-stage liver disease. This method has high specificity and sensitivity, and provides a scientific basis for the development of personalized clinical treatment plans.

[0115] Example 2: Selection of Sample Tissue

[0116] Samples from the BA group and the Non-BA group are as shown in Example 1.

[0117] 1. An investigation into the proportion of CCR7+CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient.

[0118] The proportion of CCR7+CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient was analyzed, such as... Figure 7 It can be seen that the proportion of CCR7+CD8+ T cells in the liver, lymph nodes and peripheral blood of the same BA patients gradually increased (14 cases).

[0119] Based on the above results, a correlation analysis was subsequently performed on CCR7+CD8+ T and liver function indicators in the liver, lymph nodes, and peripheral blood of different patients.

[0120] The correlation analysis results of the proportion of CCR7+CD8+ T cells in lymph nodes and liver function indicators in BA patients are shown in Table 3. After excluding age, gender, and disease duration, there was no correlation between the proportion of CCR7+CD8+ T cells in lymph nodes and various liver function indicators in BA patients (all were in the same disease duration). The correlation analysis results of the proportion of CCR7+CD8+ T cells in peripheral blood and liver function indicators in BA patients are shown in Table 4. After excluding age, gender, and disease duration, the proportion of CCR7+CD8+ T cells in peripheral blood and ALP levels in BA patients were positively correlated (all were in the same disease duration).

[0121] Table 3. Correlation analysis results of lymph node CCR7+CD8+ T cell ratio and liver function indicators in BA patients

[0122]

[0123] Table 4. Correlation analysis results of peripheral blood CCR7+CD8+ T cell ratio and liver function indicators in BA patients

[0124]

[0125] The correlation analysis results of the proportion of CCR7+CD8+ T cells in the liver and liver function indicators in BA patients are shown in Table 2. As can be seen from Tables 2 to 4, the proportion of CCR7+CD8+ T cells in the liver has a higher correlation with liver injury-related indicators (such as AST and TBA).

[0126] Example 3: Optimization of Mouse Liver Lymphocyte Extraction Method

[0127] The method for extracting mouse liver lymphocytes before optimization is as follows:

[0128] (1) Take mouse liver tissue, place the obtained mouse tissue in a 10cm culture dish, rinse the surface blood with 1×PBS, then cover with nylon membrane, add the pre-prepared 2%FBS+DMEM culture medium, grind with a 10mL syringe until the cells are completely released, and collect the cell suspension filtered with nylon membrane.

[0129] (2) Centrifuge the collected cell suspension (500g, 4℃, 5min) and discard the supernatant;

[0130] (3) Add the red blood cell lysis buffer directly, mix well, react for 5 min, add 2% FBS + DMEM medium to stop the reaction (1:1), centrifuge (500g, 4℃, 5 min), discard the supernatant; resuspend the precipitate for later use. (The final precipitate is considered as the extracted lymphocytes)

[0131] The optimized method for extracting mouse liver lymphocytes is as follows:

[0132] Steps (1) and (2) remain unchanged;

[0133] (3) Resuspend the cells in 2% FBS+DMEM medium and add them to preheated Ficoll medium for separating mouse liver lymphocytes (1:1). Centrifuge at a gradient (800g, room temperature, ascending speed 3, descending speed 3, 20min). After centrifugation, aspirate the middle white membrane layer and wash the cells with 2% FBS+DMEM medium. Then centrifuge again (500g, 4℃, 5min) and resuspend. (The white membrane layer is the extracted lymphocytes.)

[0134] like Figure 8 A in the figure shows the flow cytometry gating scheme for CD4+ and CD8+ T cells in mouse liver lymphocytes before optimization; Figure 8 B in the figure shows the optimized flow cytometry gating scheme for CD4+ and CD8+ T cells in mouse liver lymphocytes; Figure 8 As shown in C, the proportion of CD8+ T cells in lymphocytes increased after optimization compared to before optimization; Figure 8 As shown in Figure D, the proportion of CD4+ T cells among lymphocytes increased after optimization compared to before optimization. All examples used were normal wild-type Balb / c mice.

[0135] Example 4: Analysis of other cell proportions as assessment indicators of liver injury in end-stage liver disease

[0136] Samples from the BA group and the Non-BA group are as shown in Example 1.

[0137] I. The proportion of CD3+ T cells in lymphocytes (abbreviated as CD3+ T cell ratio)

[0138] 1. Expression levels of hepatic CD3+ T cells in the BA group and the Non-BA group

[0139] like Figure 9 The results showed that the proportion of CD3+ T cells in the liver was higher in patients with liver bronchitis (20 cases in non-BA and 30 cases in BA).

[0140] 2. Correlation analysis results of the proportion of CD3+ T cells in the liver and liver function indicators in patients with liver bronchitis (BA).

[0141] After excluding age, sex, and disease duration, the proportion of CD3+ T cells and TBA levels were negatively correlated.

[0142] Table 5. Correlation between liver function indicators and the proportion of CD3+ T cells in the liver

[0143]

[0144] II. The proportion of CD4+ T cells in CD3+ T cells (abbreviated as CD4+ T cell ratio)

[0145] 1. Expression level of hepatic CD4+ T cells in BA patients in BA group and Non-BA group

[0146] like Figure 10 As shown, the proportion of CD4+ T cells in the liver of patients with BA was higher than that in the non-BA group (20 cases in non-BA and 29 cases in BA).

[0147] 2. Expression of CD4+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient.

[0148] like Figure 11 As shown, the proportion of CD4+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient gradually increased (14 cases).

[0149] 3. Correlation analysis results of the proportion of CD4+ T cells in the liver and liver function indicators in patients with liver bronchitis (BA).

[0150] After excluding age, gender, and disease duration, there was no correlation between the proportion of CD4+ T cells in the liver of patients with liver bronchitis (BA) and various liver function indicators.

[0151] Table 6. Correlation between liver function indicators and the proportion of CD4+ T cells in the liver

[0152]

[0153] III. The proportion of CCR7+CD4+ T cells in CD4+ T cells (referred to as the CCR7+CD4+ T cell ratio)

[0154] 1. Expression level of the proportion of CCR7+CD4+ T cells in the liver of BA patients in the BA group and Non-BA group

[0155] like Figure 12 As shown, the proportion of CCR7+CD4+ T cells in the liver of BA patients was lower than that in the non-BA group (20 cases in Non-BA and 29 cases in BA).

[0156] 2. Expression of CCR7+CD4+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient.

[0157] like Figure 13 As shown, the proportion of CCR7+CD4+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient gradually increased (14 cases).

[0158] 3. Correlation analysis results of the proportion of CCR7+CD4+ T cells in the liver and liver function indicators in patients with liver cancer (BA).

[0159] As shown in Table 7, after excluding age, sex, and disease duration, the proportion of CCR7+CD4+ T cells was negatively correlated with AST and TBA. However, experiments revealed that in acute liver failure, low expression of CCR7 in CD8+ T cells exacerbated liver damage. This was not the case for CD4-related cells. Therefore, the proportion of CCR7+CD8+ T cells in CD8+ T cells is more accurate for assessing liver damage in end-stage liver disease.

[0160] Table 7. Correlation between liver function indicators and the proportion of hepatic CCR7+CD4+ T cells in patients

[0161]

[0162] IV. The proportion of CD8+ T cells among CD3+ T cells (referred to as the CD8+ T cell ratio)

[0163] 1. Expression level of hepatic CD8+ T cells in BA patients in BA group and Non-BA group

[0164] like Figure 14 As shown, the proportion of CD8+ T cells in the liver of patients with BA was higher than that in the non-BA group (20 cases in non-BA and 30 cases in BA).

[0165] 2. Expression levels of CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patients (14 cases).

[0166] like Figure 15 As shown, the proportion of CD8+ T cells in the liver, lymph nodes, and peripheral blood of the same BA patient gradually decreased (14 cases).

[0167] 3. Correlation analysis results of the proportion of CD8+ T cells in the liver and liver function indicators in patients with liver BA

[0168] After excluding age, gender, and disease duration, there was no correlation between the proportion of CD8+ T cells in the liver of patients with liver bronchitis and various liver function indicators.

[0169] Table 8. Correlation between the proportion of CD8+ T cells in the liver and liver function indicators in BA patients

[0170]

[0171] 4. Correlation analysis results of lymph node CD8+ T cell ratio and liver function indicators in BA patients

[0172] As shown in Table 9, after excluding age, gender, and disease duration, there was no correlation between the proportion of CD8+ T cells in lymph nodes and various liver function indicators in BA patients (all were in the same disease duration).

[0173] Table 9. Correlation between the proportion of CD8+ T cells in lymph nodes and liver function indicators in BA patients

[0174]

[0175] 5. Correlation analysis results of peripheral blood CD8+ T cell ratio and liver function indicators in BA patients

[0176] As shown in Table 10, after excluding age, gender, and disease duration, the proportion of peripheral blood CD8+ T cells and GGT levels in BA patients were positively correlated (all within the same disease duration).

[0177] Table 10. Correlation between peripheral blood CD8+ T cell percentage and liver function indicators in BA patients

[0178]

[0179] Example 5: Further analysis of the proportion of CCR7+CD4+ T cells and CCR7+CD8+ T cells as biomarkers of end-stage liver disease.

[0180] Correlation analysis of the proportion of CCR7+CD8+ T cells in the liver of patients with liver fibrosis (BA) with plasma cytokine levels (IL-1β, IFN-γ, TNF-α, IL-6, IL-8, IL-10, IL-17A) and liver fibrosis scores showed that the expression of CCR7+CD8+ T cells in the liver of BA patients was significantly negatively correlated with the level of the inflammatory factor TNF-α, and also negatively correlated with the BA liver fibrosis score, indicating that the degree of liver fibrosis increases with the decrease in the proportion of CCR7+CD8+ T cells in the liver. Combined with the correlation analysis of the proportion of CCR7+CD8+ T cells in the liver of BA patients and liver function, it was found that the proportion of CCR7+CD8+ T cells was negatively correlated with plasma AST and TBA levels. In conclusion, the proportion of CCR7+CD8+ T cells in the liver of BA patients can be used as an indicator for assessing liver fibrosis.

[0181] Table 11. Proportion of CCR7+CD8+ T cells and plasma cytokine levels

[0182] Correlation with liver fibrosis-related indicators

[0183]

[0184] Correlation analysis of the proportion of CCR7+CD4+ T cells in the liver of patients with liver fibrosis (BA) and plasma cytokine levels (IL-1β, IFN-γ, TNF-α, IL-6, IL-8, IL-10, IL-17A) with liver fibrosis scores showed that the expression of CCR7+CD4+ T cells in the liver of BA was negatively correlated with the level of the inflammatory cytokine IL-1β, but not with the liver fibrosis score. Although the proportion of CCR7+CD4+ T cells was negatively correlated with the levels of AST, TBA, and IL-1β, it cannot be used as an independent indicator for assessing liver fibrosis or liver injury and needs to be evaluated in conjunction with other biomarkers.

[0185] Table 12. Proportion of CCR7+CD4+ T cells and plasma cytokine levels

[0186] Correlation with liver fibrosis-related indicators

[0187]

[0188] The liver fibrosis score can more directly reflect the degree of liver damage, so the ratio of liver CCR7CD8+ T cells was chosen as an indicator to assess liver damage.

[0189] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of the proportion of CCR7+CD8+ T cells in CD8+ T cells as a biomarker in the preparation of products for assessing liver injury in end-stage liver disease; The proportion of CCR7+CD8+ T cells in CD8+ T cells is derived from peripheral blood and / or liver; The liver damage in end-stage liver disease includes: At least one of the following: biliary atresia, progressive familial intrahepatic cholestasis, maple syrup diabetes mellitus, and / or glycogen storage disease.

2. A combined biomarker for assessing liver injury in end-stage liver disease, characterized in that, This includes at least one of the following: the proportion of CD3+ T cells in lymphocytes, the proportion of CCR7+CD4+ T cells in CD4+ T cells, the proportion of CD8+ T cells in CD3+ T cells, and the proportion of CCR7+CD8+ T cells in peripheral blood and / or liver in CD8+ T cells.

3. The combined marker according to claim 2, characterized in that, The proportion of CD3+ T cells in lymphocytes was derived from liver tissue; The proportion of CCR7+CD4+ T cells in CD4+ T cells originates from liver tissue; The proportion of CD8+ T cells in CD3+ T cells was derived from peripheral blood.

4. The combined marker according to claim 3, characterized in that, It also includes other liver disease-related biomarkers; Other liver disease-related biomarkers include serum biomarkers, tissue biomarkers, and / or imaging biomarkers.

5. An immunoassay system and / or detection device for assessing the degree of liver damage in end-stage liver disease, characterized in that, The expression level of CCR7+CD8+ T cells in CD8+ T cells was assessed or detected. The proportion of CCR7+CD8+ T cells in CD8+ T cells is derived from peripheral blood and / or liver; The liver injury in the end-stage liver disease includes at least one of the following: biliary atresia, progressive familial intrahepatic cholestasis, maple syrup diabetes mellitus, and / or glycogen storage disease.

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