Construction method and application of a radiotherapy combined with immunotherapy mouse liver injury model

Abstract

The application discloses a kind of radiotherapy combined immunotherapy mouse liver injury model construction method and application, the construction method includes: using pentobarbital drug is anesthetized to mouse by intraperitoneal injection;Fix mouse exposure chest and abdomen;Depilating and marking liver position;Fixed front incisor and tail;In anesthetized state, the light of simulation machine is positioned at the liver area of mouse and illuminated, and the liver area is irradiated field, 100 μL of PD-1 antibody diluent is injected intraperitoneally once 1 day before whole liver irradiation, is injected once every 3 days, obtain radiotherapy combined immunotherapy mouse liver injury model, as animal model is used for screening the drug for preventing and treating liver injury caused by radiotherapy combined immunotherapy, including anti-CD20 antibody and its preparation, can prevent and relieve liver injury, and play an active role in liver disease treatment, further explore the important reference basis that anti-CD20 treatment is applied in liver disease treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a construction method and application of a radiotherapy combined with immunotherapy mouse liver injury model. BACKGROUND

[0002] The combination of radiotherapy and immunotherapy shows a significant anti-tumor effect in the treatment of malignant tumors, and has been approved by the US Food and Drug Administration (FDA) for the treatment of various cancers. However, the treatment-related adverse reactions, especially liver injury, caused by this combined treatment strategy need to be highly valued, which limits its wide application in the clinic. Studies have found that adverse reactions including liver injury are closely related to the presence of autoantibodies, suggesting the role of B cells, especially in tertiary lymphoid structures, which are ectopic lymphoid organs similar to germinal centers, usually appearing in chronic inflammatory areas such as tumor tissues. In addition, previous studies have further revealed that hepatocellular carcinoma patients who are positive for autoantibodies (ANA+) have adverse reactions when receiving immunotherapy, which are related to the activation of B cells and the release of autoantibodies. Therefore, depleting B cells may be able to reverse adverse reactions, especially liver injury.

[0003] Currently, there are limited treatment options for liver injury caused by radiotherapy and immunotherapy, and the effect is not ideal, and new treatment strategies need to be developed to more effectively manage these adverse reactions. Anti-CD20 antibodies have shown their potential in regulating B cell activity and reducing autoantibody production in other autoimmune diseases. Therefore, exploring their role in combination therapy provides a new solution to treat adverse reactions. SUMMARY

[0004] To solve the problem of liver injury caused by immune activation in existing tumor treatment, the main purpose of the present application is to provide a construction method of a radiotherapy combined with immunotherapy mouse liver injury model.

[0005] Another purpose of the present application is to provide the application of anti-CD20 antibodies and their preparations in the preparation of drugs for preventing and treating liver injury caused by radiotherapy combined with immunotherapy.

[0006] To achieve the above purpose, the present application adopts the following technical solutions:

[0007] The present application provides a construction method of a radiotherapy combined with immunotherapy mouse liver injury model, comprising the following steps:

[0008] (a) Anesthesia: anesthetize the mouse by intraperitoneal injection of pentobarbital drug;

[0009] (b) Fix the mouse: the mouse is in supine position, the limbs are spread out and fixed on a plate to expose the chest and abdomen;

[0010] (c) Hair removal and marking: remove the hair on the chest and abdomen of the mouse, and mark the liver position with a marker pen;

[0011] (d) Fix the front teeth and tail: fix the front teeth and tail of the mouse to ensure that the mouse remains stable during the radiotherapy treatment;

[0012] (e) Irradiation preparation: turn off the light in the examination room under anesthesia, and position and illuminate the light of the simulator on the liver area of the mouse;

[0013] (f) Irradiation treatment: use the simulator to perform the irradiation field on the liver area, and use 12eMV electron lines for whole liver irradiation, with a dose of 8 gray (Gy) per time, for a total of 3 times;

[0014] (g) Intraperitoneal injection of 100 μL of PD-1 antibody diluent once a day, and injection once every 3 days, to obtain a radiotherapy combined with immunotherapy mouse liver injury model.

[0015] Preferably, in step (a), the dose of the pentobarbital drug is 50 mg / kg of the mouse body weight.

[0016] Preferably, in step (c), the marking of the liver position is located below the xiphoid process, and the upper position is 0.5-1 cm below the xiphoid process, avoiding the stomach and duodenum.

[0017] Preferably, the PD-1+RT combined treatment group is injected with 250 μg of a depleting anti-CD20 antibody into the tail vein of the mouse 5 days before the injection of the PD-1 antibody diluent, and the sample is taken on day 21, to obtain a B cell-depleted radiotherapy combined with immunotherapy mouse liver injury model.

[0018] The present application also provides a radiotherapy combined with immunotherapy mouse liver injury model as an animal model for screening drugs for preventing and treating liver injury caused by radiotherapy combined with immunotherapy, wherein the radiotherapy combined with immunotherapy mouse liver injury model is obtained by the construction method of the radiotherapy combined with immunotherapy mouse liver injury model according to any one of claims 1 to 4.

[0019] Preferably, the drug includes an anti-CD20 antibody and a preparation thereof.

[0020] The present application also provides the use of an anti-CD20 antibody and a preparation thereof in the preparation of a drug for preventing and treating liver injury caused by radiotherapy combined with immunotherapy, which is verified by constructing the radiotherapy combined with immunotherapy mouse liver injury model, and shows that the B cell-depleting agent, anti-CD20 antibody, can effectively reverse the liver injury caused by radiotherapy combined with immunotherapy, thereby providing a new solution for adverse reactions in tumor treatment.

[0021] Compared with the prior art, the present application has the beneficial effects that:

[0022] 1) Anti-CD20 treatment can prevent the onset of liver injury and alleviate the disease: By injecting a depleting CD20 antibody, B cells in the mouse body can be effectively depleted, thereby reducing the occurrence of liver injury and alleviating the symptoms of the disease.

[0023] 2) Anti-CD20 treatment can reduce the infiltration of CD8+ T cells and CD4+ T cells in liver tissue: After anti-CD20 treatment, the infiltration of CD8+ T cells and CD4+ T cells in liver tissue is significantly reduced, indicating that anti-CD20 treatment can reduce the aggregation of immune cells in the liver and inflammatory response.

[0024] 3) Anti-CD20 treatment reduces the activation state and cytotoxic activity of CD8+ T cells: After anti-CD20 treatment, CD8+ T cells in liver tissue exhibit lower activation state and reduced cytotoxic activity, which may help reduce damage to hepatocytes.

[0025] In summary, anti-CD20 treatment can prevent and alleviate liver injury by depleting B cells, reducing immune cell infiltration, and reducing the activation state and cytotoxic activity of CD8+ T cells, and plays an active role in the treatment of liver disease, which provides important reference for further exploring the application of anti-CD20 treatment in the treatment of liver disease. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A schematic diagram of the process for constructing a radiotherapy combined with immunotherapy mouse model in the examples.

[0027] Figure 2 HE staining results of a radiotherapy combined with immunotherapy mouse liver injury model in the examples.

[0028] Figure 3 Immunofluorescence analysis results of a radiotherapy combined with immunotherapy mouse liver injury model in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0029] Figure 4 TUNEL detection analysis results of a radiotherapy combined with immunotherapy mouse liver injury model under the intervention of different drug combinations in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0030] Figure 5 Masson staining analysis results of a radiotherapy combined with immunotherapy mouse liver injury model under the intervention of different drug combinations in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0031] Figure 6 Liver function and autoantibody expression results of the liver injury model of radiotherapy combined with immunotherapy mice under different drug combination interventions in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0032] Figure 7 Results of immunofluorescence CD20 analysis of the liver injury model of radiotherapy combined with immunotherapy mice in the examples.

[0033] Figure 8 Results of B cells having pro-inflammatory characteristics of the liver injury model of radiotherapy combined with immunotherapy mice under different drug interventions in the examples.

[0034] Figure 9 Schematic diagram of the liver injury model of radiotherapy combined with immunotherapy mice induced by radiotherapy combined with anti-PD-1 inhibitor treatment for B cell depletion in the examples.

[0035] Figure 10 Results of injecting anti-CD20 to make the B cells in the peripheral blood of mice exhausted for about 30 days in the examples.

[0036] Figure 11 Histological and clinical remission results of liver injury caused by anti-CD20 administration in the examples; (A) hematoxylin-eosin staining of liver sections, magnification 200x; (B) CD4, CD8 and CD20 staining representative figure, magnification 200x; (C) liver function ALT, AST values Anti-CD20 treatment reduces T cell activation and cytotoxic activity.

[0037] Figure 12 Effects of anti-CD20 on T cell activation status and cytotoxic activity in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0038] Figure 13 Effects of anti-CD20 on T cell activation status and cytotoxic activity in the examples; * represents P<0.05; ** represents P<0.01; *** represents P<0.001. DETAILED DESCRIPTION

[0039] In order to make the technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below in combination with the examples and drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application.

[0040] Example 1

[0041] 1. Constructing a radiotherapy combined with immunotherapy mouse liver injury model:

[0042] 1) Mouse grouping:

[0043] Randomly divided into 4 groups, 5 mice in each group: a) IgG (control group); b) PD-1 (immunotherapy group); c) RT+IgG (radiotherapy group); d) RT+PD-1 (combined therapy group).

[0044] 2) Drug and radiotherapy intervention:

[0045] According to the corresponding group, drugs and radiotherapy were administered, as follows:

[0046] IgG control group: mice were given IgG antibody diluent, intraperitoneal injection, once every 3 days, 100 μL of mixed solution per mouse, a total of 5 injections.

[0047] PD-1 immunotherapy group: mice were given PD-1 antibody diluent, and the control group mice were given IgG antibody diluent, intraperitoneal injection, once every 3 days, 100 μL of mixed solution per mouse, a total of 5 injections.

[0048] RT+IgG radiotherapy group:

[0049] (1) Anesthesia: pentobarbital was used to anesthetize mice by intraperitoneal injection, with a dose of 50 mg / kg body weight.

[0050] (2) Fix the mouse: the mouse was placed in a supine position, and the limbs were spread out and fixed on a board using a self-made magic paste to expose the chest and abdomen.

[0051] (3) Hair removal and marking: use a hair removal machine to remove the hair on the chest and abdomen of the mouse, and use a marker pen to mark the position of the liver, which is generally located below the xiphoid process, 0.5-1 cm above the xiphoid process, avoiding the stomach and duodenum.

[0052] (4) Fix the front teeth and tail: fix the front teeth and tail of the mouse to ensure that the mouse remains stable during radiotherapy.

[0053] (5) Irradiation preparation: in the anesthetized state, turn off the light in the examination room, and position the light of the simulator machine on the liver area of the mouse and illuminate it.

[0054] (6) Irradiation therapy: use a simulator machine (Nuclear SimuLix-HQ) to irradiate the liver area with 12eMV electron beam, with a dose of 8Gy per irradiation, a total of 3 times.

[0055] (7) The mice were subjected to whole liver irradiation of 8 Gy three times, and 100 μL of immunoglobulin G antibody diluent was injected intraperitoneally once one day before whole liver irradiation, once every 3 days.

[0056] RT+PD-1 combination therapy group: the mice were subjected to whole liver irradiation of 8 Gy three times, and 100 μL of PD-1 antibody diluent was injected intraperitoneally once one day before whole liver irradiation, once every 3 days.

[0057] This study followed the ethical guidelines and animal experiment ethical norms, and obtained the relevant experimental license and approval.

[0058] 2. Establish a B cell-depleted radiotherapy combined immunotherapy mouse model:

[0059] In order to deplete B cells in mice, 250 μg of depleting CD20 antibody was injected into the tail vein of mice 5 days before injecting the PD-1 antibody diluent, to establish a B cell-depleted radiotherapy combined immunotherapy mouse liver injury model, and the sample was taken on day 21.

[0060] 1) Collect serum:

[0061] Mouse eyeball blood was placed in a 1.5 mL EP tube. The whole blood sample was placed at room temperature for 2 h or at 4°C overnight, then centrifuged at 1000 g for 20 min, and the upper light yellow serum sample was placed in a frozen tube, frozen in liquid nitrogen, and stored at -80°C. Repeated freezing and thawing should be avoided.

[0062] 2) Preparation of liver cell suspension:

[0063] (1) The mice were sacrificed by cervical dislocation, sterilized in 75% alcohol for 2-3 s, and the liver was taken out and placed in a dish containing hanks.

[0064] (2) Remove the fat, connective tissue and blood of the mouse, and transfer it to another dish containing hanks.

[0065] (3) Cut the organs into small pieces (about 1 mm 2 ) with surgical scissors, grind the slides, and transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 min.

[0066] (4) According to the amount of mouse tissue or cells, add 2 mg / mL collagenase type IV (about 3-5 mL), and digest in a 37°C water bath for 30 min, gently shake once every 5 min, or use a pipette to blow once, to facilitate cell separation.

[0067] (5) Add 3-5 mL of serum-containing culture medium to stop the digestion of collagenase type IV.

[0068] (6) Filter the culture medium through a 100-mesh sieve to remove undigested large tissue pieces.

[0069] (7)Centrifuge the culture solution again for 5 min, and discard the supernatant.

[0070] (8)Add 5 mL of serum-free culture solution to disperse the cells, centrifuge again, and discard the supernatant.

[0071] (9)Add 1 mL of red blood cell lysis solution to the bottom layer of the precipitate, and let stand at room temperature for 3 min. Then, add 5 mL of serum-free culture solution, centrifuge at 1000 rpm for 5 min, and discard the red supernatant.

[0072] (10)Add 1-2 mL of serum-containing culture solution (depending on the cell amount), and count the cells using a hemocytometer.

[0073] 3) ALT / AST detection:

[0074] After the mice are anesthetized at a specific time point after treatment, take the blood from the inner side using a disposable micro blood collection pipette, and use the automatic biochemical instrument Chemray 240 to detect ALT and AST.

[0075] 4) Hematoxylin-eosin staining:

[0076] (1) Baking: Place the tissue section in a 65°C oven for 45 min.

[0077] (2) De-waxing: Place the baked paraffin section in xylene 1 and 2 for 8 min each.

[0078] (3) Hydration: Soak the glass slide in anhydrous ethanol 1, anhydrous ethanol 2, 95% ethanol, 85% ethanol, 70% ethanol, and 50% ethanol for 5 min each, and then wash with PBS for 2 times, 2 min each.

[0079] (4) Nucleus staining: Place the glass slide in a wet box, and add hematoxylin dye for 40 s. Then, quickly differentiate the glass slide with 1% hydrochloric acid alcohol, and immediately place it in running water for slow rinsing and returning to blue.

[0080] (5) Eosin staining: Place the glass slide in eosin dye, and let stand at room temperature for 5 min, and then rinse with running water.

[0081] (6) Dehydration: Place the stained glass slide in 50% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and xylene for 5 min each.

[0082] (7) Mounting: Add neutral balsam to the glass slide, and cover it with a cover glass. Then, observe and take a photo under a normal microscope.

[0083] 5) Immunohistochemistry:

[0084] (1) The baking, dewaxing and hydration process is consistent with the HE staining step.

[0085] (2) Inactivation of catalase: Drop 3% solution on the slide, stand at room temperature for 10 min, then wash with PBS for 3 times, 3 min each time.

[0086] (3) Membrane breaking: According to the distribution of the stained index in the cells, if the nucleus index is stained, membrane breaking operation is needed, incubate with 0.5% Triton-100 at room temperature for 10 min, then wash with PBS for 3 times, 3 min each time.

[0087] (4) Blocking: Incubate with 5% goat serum at 37°C for 30 min.

[0088] (5) Primary antibody incubation: Draw a circle around the tissue on the slide with a hydrophobic pen to prevent the antibody from flowing out. According to the dilution ratio of each antibody, dilute the antibody with the immunohistochemical primary antibody diluent, then add 500 uL per well, and incubate overnight at 4°C.

[0089] (6) Secondary antibody incubation: The next day, take it out and wash it with PBS for 3 times, 3 min each time. Prepare goat anti-secondary antibody with PBS at a ratio of 1:100, incubate at room temperature for 1 h.

[0090] (7) DAB color development: After the secondary antibody incubation is complete, wash with PBS for 3 times, 3 min each time. Dilute the A and B liquids in the DAB color development kit into working solution at a ratio of 1:1, drop them on the slide, and observe the staining depth under the microscope, and record the time. The remaining samples are developed according to the same time.

[0091] (8) Nucleus staining, dehydration, and mounting are consistent with the HE staining steps.

[0092] (9) Observe the tissue under the microscope and collect images for analysis.

[0093] 6) Masson staining:

[0094] (1) Baking and dewaxing of paraffin sections: This is a pretreatment step to fix the tissue on the section and remove the paraffin.

[0095] (2) Soak the section in Masson A liquid overnight and then wash with water: Soak the section in Masson A liquid, usually overnight, then rinse with water.

[0096] (3) Soak in a dye solution made by mixing Masson B and Masson C liquids at an equal ratio, and soak for 1 min, then rinse with water: Soak the section in a dye solution made by mixing Masson B and Masson C liquids at an equal ratio, soak for 1 min, then rinse with water.

[0097] (4) 1% hydrochloric acid alcohol differentiation, water washing: the slice is soaked in 1% hydrochloric acid alcohol for differentiation, and then washed with water.

[0098] (5) Immersion in Masson D liquid for 6 min, water washing: the slice is soaked in Masson D liquid for 6 min for staining, and then washed with water.

[0099] (6) Immersion in Masson E liquid for 1 min, direct immersion in Masson F liquid for 30 s, the slice is soaked in Masson E liquid for 1 min for staining, and then directly transferred into Masson F liquid for 30 s.

[0100] (7) 1% glacial acetic acid rinsing differentiation, sequential immersion in two cylinders of anhydrous ethanol dehydration: the slice is soaked in 1% glacial acetic acid for differentiation, and then sequentially immersed in two cylinders of anhydrous ethanol for dehydration.

[0101] (8) Transparent mounting: the slice is sequentially placed in the third cylinder of anhydrous ethanol for 5 min, then placed in xylene for 5 min for transparent treatment, and finally mounted with neutral balsam.

[0102] (9) Microscope examination, image acquisition and analysis: the stained slice is observed under a microscope, and image acquisition and analysis are performed, and the results show that the collagen fibers are blue, and the muscle fibers, cytoplasm, cellulose, keratin and red blood cells are red.

[0103] 7) ANA detection:

[0104] (1) Sample dilution: the serum and plasma samples are diluted by 1:200 with a sample diluent for detection, and the specific operation is as follows: 5 μL of sample is added to 45 μL of sample diluent (1:10 dilution) and mixed, and then 15 μL of the above diluent is added to 285 μL of sample diluent (1:20 dilution) and mixed. After two steps, the sample diluted by 1:200 is obtained.

[0105] (2) The various reagents are equilibrated at room temperature (18-25°C) for at least 30 min, and the reagent preparation standards, washing solution working solution, biotin label working solution, and horseradish peroxidase labeled avidin working solution are prepared.

[0106] (3) Sample addition: standard wells and sample wells are set up, and 100 μL of standard or sample is added to each well, respectively, and gently shaken to mix, and a new plate cover is covered, and incubated at 37°C for 2 h.

[0107] (4) Discard the liquid and spin dry without washing.

[0108] (5) Add 100 μL of biotin label working solution to each well, cover a new plate cover, and incubate at 37°C for 1 h.

[0109] (6) Discard the liquid in the hole, spin dry, wash the plate 3 times, each time soak for 2 min, 200 μL / hole, spin dry.

[0110] (7) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each hole, cover the new plate sticker, and incubate at 37°C for 1 h.

[0111] (8) Discard the liquid in the hole, spin dry, wash the plate 5 times. Each time soak for 2 min, 200 μL / hole, spin dry.

[0112] (9) Add 90 μL of substrate solution to each hole in turn, and develop color at 37°C for 15-30 min.

[0113] (10) Add 50 μL of termination solution to each hole in turn to terminate the reaction.

[0114] (11) Within 5 min after the reaction is terminated, measure the optical density (OD value) of each hole in turn at 450 nm wavelength using an enzyme marker.

[0115] 8) Flow cytometry analysis:

[0116] The following antibodies or reagents were used for flow cytometry analysis: anti-mouse CD45 (clone 30-F11), anti-mouse CD19 (6D5), anti-mouse CD3 (145-2C11), anti-mouse CD8 (53-6.7), anti-mouse CD4 (RM4.5), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-B220 (RA3-6B2), anti-TNF-a (MP6-XT22), anti-IFN-g (XMG1.2), anti-granzyme B (NGZB), anti-CD107a (ebio14B), collected on a FACSAria III flow cytometer (BD Biosciences), and analyzed using Flowjo software (TreeStar Ashland, OR).

[0117] 9) Statistical analysis:

[0118] (1) The immunofluorescence pictures and immunohistochemistry pictures were analyzed using Image-ProPlus6.0 (MediaCybemetics, INC., Rockville, MD, USA) software.

[0119] (2) Statistical analysis was performed using SPSS 23.0 (IBM Corp., Chicago, IL, USA), R software version 4.0.5 (R Core Team, Vienna, Austria), and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA).

[0120] Results and Characterization

[0121] 1. Establish mouse liver injury models under different drug combination interventions

[0122] Mice were divided into four groups and treated with drugs and radiation therapy.

[0123] IgG group (100 μL of diluted IgG antibody was injected intraperitoneally once every 3 days in the morning);

[0124] PD-1 group (100 μL of PD-1 antibody dilution was injected intraperitoneally once every 3 days in the morning);

[0125] The RT+IgG group (mice were irradiated with 8 Gy of the whole liver three times, and injected intraperitoneally with 100 μL of diluted immunoglobulin G (IgG) solution once every 3 days one day before the whole liver irradiation);

[0126] The RT+PD-1 group (mice were irradiated with 8 Gy of whole liver three times, and injected intraperitoneally with 100 μL of PD-1 antibody dilution once on the morning of the day before whole liver irradiation, and injected once every 3 days).

[0127] Mice were sacrificed on day 21 after the start of treatment, and their livers were analyzed. Figure 1 As shown.

[0128] 2. HE staining analysis of liver under different drug combination interventions

[0129] Further observation and HE staining analysis of liver tissue under different drug combinations yielded the following results: Figure 2 As shown, under the microscope, the liver lobules in the IgG group were clearly demarcated and regularly arranged. The central vein was located in the center of the lobule, surrounded by hepatocytes and sinusoids arranged in a roughly radial pattern. The hepatic cords were regularly and neatly arranged, and there was no obvious dilation or compression of the sinusoids. There were no obvious abnormalities in the portal areas between adjacent lobules, and no obvious inflammatory changes were observed. Under the microscope, mild granular degeneration of hepatocytes, cell swelling, and loose, pale cytoplasm were observed in the PD-1 group. Under the microscope, moderate granular degeneration of hepatocytes, cell swelling, and loose, pale cytoplasm were widely observed in the RT+IgG group. The hepatic cords were regularly and neatly arranged, and there was no obvious dilation or compression of the sinusoids. There were no obvious abnormalities in the portal areas between adjacent lobules, and there were scattered inflammatory changes. In contrast, in the combined treatment group, severe granular degeneration of hepatocytes, cell swelling, and obvious scattered inflammatory cell infiltration were widely observed.

[0130] 3. Immunofluorescence analysis of the liver under different drug combination interventions

[0131] Immunofluorescence analysis was used to detect infiltrating immune cells in liver tissue, and the results were as follows: Figure 3As shown, the number of RT+PD-1 group was found to be more than the RT group, and the RT group was also increased compared to the IgG group. In addition, the infiltration of CD8+ T cells was more in the PD-1 group than in the IgG group. These results suggest that cytotoxic CD8-positive T cells can be a key mediator of acute liver toxicity, and the quantitative analysis also found this phenomenon.

[0132] 4. Immunohistochemical analysis of liver microenvironment under different drug combination intervention

[0133] 1) TUNEL detection of apoptosis in the liver

[0134] RT+PD-1 treatment was confirmed to promote apoptosis in the liver by TUNEL staining Figure 4 , which further supports the potential role of radiotherapy in promoting liver cell apoptosis, especially in combination with immune checkpoint inhibitors such as PD-1 antibodies, which is particularly prominent in liver cancer immunotherapy.

[0135] 2) Collagen deposition in the liver

[0136] When the liver is damaged, hepatocytes release cytokines and chemical signals to activate hepatic stellate cells (HSCs). HSCs are interstitial cells in the liver with active proliferation and collagen fiber secretion capacity. During liver injury, activated HSCs transform into mature fibroblasts, which secrete excess collagen. Collagen is a structural protein that plays an important supportive and structural function in liver tissue. However, excessive collagen deposition leads to liver fibrosis, a process in which healthy cells in the liver tissue are replaced by fibrous tissue. Therefore, the relationship between liver damage and liver fibrosis is closely related, and collagen fiber deposition is one of the core events in the process of liver fibrosis. The results of the study indicate that mice receiving RT treatment have collagen deposition in the liver compared to mice in the IgG or PD-1 groups. In addition, RT+PD-1 has more collagen deposition Figure 5 than the RT group.

[0137] 5. B cells secrete pro-inflammatory cytokines and autoantibodies in a mouse model of liver injury

[0138] It has been found in humans that anti-PD-1 treatment in liver cancer patients can cause autoimmune manifestations due to the release of autoantibodies ANA. By ELISA, it was found that the ALT and AST of the RT+PD-1 group were significantly higher than those of the IgG group, the RT group, and the PD-1 group, and in addition, the RT+PD-1 group had more autoantibodies ANA than the PD-1 group, and the RT group also increased compared to the PD-1 group Figure 6), fully suggesting that radioimmunotherapy causes normal tissue damage and induces autoantibody release. To understand whether B cells of autoantibody origin play an active role in the pathogenesis of the mouse liver damage model, the mouse B cell secretion was first studied, and the results showed that the number of RT+PD-1 group was more than that of the RT group, and the RT group also increased compared with the IgG group Figure 7 ), fully suggesting that B cells in normal tissue damage play a role therein.

[0139] In addition, compared with the RT group of mice, the proportion of IFN-γ and TNF-α produced by B cells in the liver of mice treated with radiotherapy combined with immunotherapy was significantly higher. The RT group also increased compared with the IgG group Figure 8 ), B cells producing INF-γ and TNF-α exist in the liver of radioimmunotherapy mice, and the results show that B cell activation and secretion of proinflammatory cytokines contribute to the pathogenesis of radiotherapy combined with immunotherapy.

[0140] 6、Anti-CD20 therapy can prevent disease onset and relieve

[0141] In order to deplete B cells in mice, 250 μg of depleting CD20 antibody was injected into the tail vein of mice 5 days before PD-1 to establish a B cell-depleted radioimmunotherapy liver damage model until the 21st day of sampling Figure 9 First, it was determined whether the B-depleted mouse model was successfully established, and B cell quantitative analysis at each drug injection time point after B cell depletion was detected, and it was found that injection of anti-CD20 could deplete peripheral blood B cells in mice for about 30 days Figure 10 Therefore, B cell depletion can be maintained for 21 days in the radioimmunotherapy model. It can be seen that in the radiotherapy combined with immunotherapy mouse liver damage model, liver cell inflammation cells increase, and fatty degeneration increases. However, after anti-CD20 treatment, the mouse inflammatory cell infiltration is reduced, the fatty degeneration is weakened, and the liver cord structure is less messy. In addition, the infiltration of CD4+T, CD8+T, CD20 cells in the liver tissue is reduced, and ALT and AST are also reduced after anti-CD20 treatment Figure 11

[0142] 7、Anti-CD20 therapy reduces T cell activation and cytotoxic activity

[0143] For CD8+T cells that play a major role in killing, anti-CD20 treatment significantly reduced CD8+T cell infiltration in liver tissue compared with the control group. Similarly, anti-CD20 treatment significantly reduced helper T cells CD4+T in liver tissue Figure 12 ), consistent with the previous immunohistochemical results Figure 11 B).

[0144] ​In the model of radiotherapy combined with immunotherapy-induced liver injury in mice, effector CD8+T cells, mainly cytotoxic T cells (Tc), were responsible for hepatocyte lysis and parenchymal destruction. Further analysis of T cell functional markers, granzyme B and CD107a, showed that the number of RT+PD-1 group was more than that of RT group, and the number of RT group was more than that of IgG group. Then, the effect of B cell depletion on the killing ability of CD8+T cells in the model was studied Figure 13 B), CD8+T cells isolated from anti-CD20 mouse liver also showed significantly reduced CD107a and granzyme B, suggesting that anti-CD20 can reduce the occurrence of liver injury.

[0145] In summary, the present application establishes a B cell-depleted radiotherapy combined with immunotherapy mouse model, and through a series of intervention measures and detection methods, reveals the important role of B cells in liver injury. The results show that the use of anti-CD20 antibody can significantly reduce liver injury and reduce the activation and cytotoxic activity of T cells, providing a new strategy for the prevention and treatment of radiotherapy combined with immunotherapy-induced liver injury.

[0146] The above describes the preferred embodiments of the present application, but the present application should not be limited to the content disclosed in the embodiments. Therefore, any equivalent or modification made without departing from the spirit of the present application falls within the scope of the present application.

Claims

1. A method for constructing a mouse liver injury model using radiotherapy combined with immunotherapy, characterized in that, Includes the following steps: (a) Anesthesia: Mice were anesthetized by intraperitoneal injection of pentobarbital; (b) Fixing the mouse: The mouse is placed in a supine position with its limbs spread out and fixed on a board to expose the chest and abdomen; (c) Hair removal and marking: Hair was removed from the chest and abdomen of the mice, and the liver was marked with a marker. (d) Fixing the anterior incisors and tail: Fixing the anterior incisors and tail of the mouse to ensure stability during radiotherapy; (e) Irradiation preparation: Under anesthesia, turn off the lights in the examination room and position the simulator light on the liver area of ​​the mouse and illuminate it; (f) Irradiation therapy: The liver area was irradiated using a simulator, and the whole liver was irradiated with 12 eMV electron beams. The dose of each irradiation was 8 Gy, and a total of 3 irradiations were performed. (g) One day before whole liver irradiation, 100 μL of PD-1 antibody dilution was injected intraperitoneally once, and once every 3 days to obtain a mouse liver injury model of radiotherapy combined with immunotherapy. Five days before administering PD-1 antibody dilution, mice were injected with 250 μg of depleted anti-CD20 antibody via the tail vein. The liver injury model in mice, characterized by B cell depletion from radiotherapy combined with immunotherapy, was obtained by collecting samples on day 21.

2. The method for constructing a mouse liver injury model using radiotherapy combined with immunotherapy according to claim 1, characterized in that, In step (a), the dosage of the pentobarbital drug is 50 mg / kg mouse body weight.

3. The method for constructing a mouse liver injury model using radiotherapy combined with immunotherapy according to claim 1, characterized in that, In step (c), the marker for the liver location is located below the xiphoid process.

4. The application of a mouse liver injury model induced by radiotherapy combined with immunotherapy as an animal model in screening drugs for the prevention and treatment of liver injury caused by radiotherapy combined with immunotherapy, wherein the mouse liver injury model induced by radiotherapy combined with immunotherapy is obtained by the method for constructing the mouse liver injury model induced by radiotherapy combined with immunotherapy as described in any one of claims 1 to 3.

5. The application according to claim 4, characterized in that, The drug includes anti-CD20 antibodies and their preparations.

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