Construction method and evaluation method of IDILI drug sensitivity model
By combining the administration of TLR1/2 agonist and immune checkpoint inhibitors, a drug-sensitive model that can simulate the pathogenesis of IDILI was constructed, which solved the problem of difficulty in reproducing the pathogenesis of IDILI in the prior art, and achieved efficient and accurate IDILI simulation.
Patent Information
- Application Number
- CN202411669541.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-02-28
AI Technical Summary
The prior art is difficult to effectively reproduce and simulate the pathogenesis of specific liver injury (IDILI) in animal models, and the existing IDILI animal models cannot accurately reflect the immune-mediated properties of IDILI.
By combining the administration of the TLR1/2 agonist Diprovocim and the immune checkpoint inhibitors anti-PD1 and anti-CTLA4, an IDILI drug-sensitive model was constructed, which significantly increased the inflammation level in the liver of mice and increased the proportion of immune cells in the liver.
The constructed IDILI drug-sensitive model can significantly increase the inflammation level and immune cell ratio in the liver without causing liver damage to mice, simulate the complex immune process during the onset of IDILI, and has the advantages of high modeling success rate, short cycle and relatively simple operation.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of animal models, and in particular to a method for constructing and evaluating an IDILI drug sensitivity model. Background Art
[0002] Idiosyncratic drug-induced liver injury (IDILI) is an unpredictable liver disease that occurs only in a small number of exposed individuals. Because IDILI exhibits different incubation periods, the onset time ranges from days to weeks, making it difficult to reproduce in animal models. Currently, there are more than 1,000 drugs that have the potential to cause IDILI, but clinically only an exclusionary diagnosis can be made through the RUCAM causal relationship assessment method, and the treatment is still timely discontinuation of the drug or the use of corticosteroids. IDILI has gradually become one of the most challenging liver diseases faced by hepatologists.
[0003] Due to the lack of confidence in phase I and phase II clinical trials in identifying and evaluating IDILI, a relatively rare adverse event, IDILI generally only appears in phase III, or even discovered after the drug is launched. In recent years, IDILI has gradually become the main reason for drug withdrawal from the market. This not only increases the cost of drug research and development, but also increases the safety risk of clinical drug use. Therefore, preclinical toxicity studies to evaluate the risk of candidate drugs inducing IDILI are of great significance for new drug development. Since the pathogenesis of IDILI is not clear, establishing an IDILI animal model remains a major problem. So far, there have been many attempts to develop in vitro assays to predict the risk of drug-induced IDILI, but in vitro tests cannot replicate the complexity of IDILI. Most of the IDILI animal models that have been studied are acute toxicity caused by high-dose drugs and cannot reflect the immune-mediated characteristics of IDILI.
[0004] At present, the most commonly used IDILI animal models include inflammatory stress models and suppressed immune tolerance models. The inflammatory stress model created by administering lipopolysaccharide (LPS) 2 hours in advance is prone to LPS tolerance, and the modeling process is too short, involving only innate immune responses, and cannot simulate the complex immune process in the IDILI process. - / - The mouse immune checkpoint inhibitor anti-CTLA4 antibody modeling method only causes mild liver damage and cannot simulate the liver failure that occurs clinically; and gene knockout mice are expensive.
[0005] In summary, it is of great significance to establish a reliable and practical preclinical sensitive model of drug-specific hepatotoxicity. Summary of the invention
[0006] The present invention aims to solve the technical problems existing in the prior art. To this end, the present invention provides a method for constructing and evaluating an IDILI drug sensitivity model.
[0007] The technical solution adopted by the present invention to solve its technical problem is:
[0008] In a first aspect, a method for constructing an IDILI drug sensitivity model is provided, comprising the following steps:
[0009] Step S1, selecting modeling subjects and grouping: 2n SPF-grade Balb / c mice were selected as modeling subjects and randomly divided into a Ctrl group and a CPI+TLR1 / 2 group, with n mice in each group;
[0010] Step S2, drug preparation: prepare TLR1 / 2 agonist Diprovocim and CPI; wherein CPI is anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo) respectively diluted to 5 mg / ml by PBS, and used immediately after preparation;
[0011] Step S3, drug administration: on days 1 and 5, mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of Diprovocim, and mice in the Ctrl group were given an equal amount of the matching solution; on days 2, 4, and 6, mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo), and mice in the Ctrl group were given an equal amount of PBS;
[0012] Step S4, sample collection: on the 0th and 3rd days, blood from the retro-orbital venous plexus of the two groups of mice was collected respectively, and serum was obtained and stored for later use; on the 7th day, blood from the eyeballs of the two groups of mice was obtained respectively, and serum was obtained and stored for later use, and then the livers of the two groups of mice were obtained respectively.
[0013] Preferably, in step S1, the mice are selected to be 6 to 8 weeks old and weigh 17 to 22 grams, and are grouped and numbered after adaptive feeding for 1 week.
[0014] Preferably, in step S2, the TLR1 / 2 agonist Diprovocim is prepared as follows: Diprovocim is prepared in advance with DMSO as a 10 mg / ml stock solution, and then diluted to a 0.5 mg / ml application solution according to a ratio of 45% PEG300: 5% Tween80: 45% saline: 5% stock solution, and the solution is prepared and used immediately.
[0015] Preferably, in step S3, the mixed solution is prepared according to the ratio of 45% PEG300: 5% Tween80: 45% physiological saline: 5% DMSO.
[0016] Preferably, in step S4, after obtaining the livers of the two groups of mice on the 7th day, they are first rinsed with physiological saline, and the surface moisture is absorbed with filter paper, and the liver tissue with a size of 0.5 cm × 0.5 cm in the middle of the left lobe of the liver is cut and immersed in 4% paraformaldehyde tissue fixative for histopathological evaluation; another portion of the liver tissue is taken and placed in a cryopreservation tube and stored in a -80°C refrigerator for standby use; the remaining liver tissue is immersed in a PBS solution and placed in a 4°C refrigerator for standby use.
[0017] In a second aspect, a method for evaluating an IDILI drug sensitivity model constructed by the method for constructing an IDILI drug sensitivity model is provided, comprising evaluating liver damage:
[0018] The serum of the two groups of mice was obtained by sampling blood from the retro-ocular venous plexus on the 0th day and the 3rd day, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected by using a kit to understand the baseline level of liver function of the mice and the changes of transaminases in the body after two days of administration;
[0019] On the 7th day, the ocular blood serum of the two groups of mice was obtained, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected using a kit;
[0020] On the 7th day, the livers of the two groups of mice were obtained, and the liver tissues fixed with 4% polyformaldehyde were embedded in paraffin, sliced, and stained with hematoxylin-eosin to observe the pathological manifestations of liver tissue.
[0021] Preferably, the evaluation method of the IDILI drug sensitivity model also includes evaluating the level of intrahepatic inflammation and oxidative stress: on the 7th day, extracting liver tissue proteins from the two groups of mice for Western Blot experiments, respectively, and detecting the expression of inflammatory factors IL-1β, IL-6, TNF-α, IL-12α and NLRP3 in the two groups of mice to reflect the level of intrahepatic inflammation; detecting the levels of intrahepatic GSH, SOD and MDA in the liver of the two groups of mice by a kit to reflect the level of intrahepatic oxidative stress.
[0022] Preferably, the evaluation method of the IDILI drug sensitivity model also includes evaluating the level of intrahepatic immune cells: performing flow cytometry on the liver tissues of the two groups of mice obtained on the 7th day to detect the changes in the proportion of major immune cells, supplemented by Western Blot method to detect the expression of some immune cell markers, and checking CD4 by immunofluorescence. + T cells and CD8 + T cell infiltration.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] 1. The method for constructing an IDILI drug-sensitive model provided by the present invention, compared with the existing IDILI animal model modeling method, is mainly based on the pathogenesis of IDILI, and is combined with the TLR1 / 2 agonist Diprovocim and the immune checkpoint inhibitors anti-PD1 and anti-CLTA4 for administration. It can significantly increase the level of inflammation in the liver of mice and increase the proportion of immune cells in the liver without causing liver damage to mice, so as to construct a sensitive model susceptible to specific hepatotoxic drugs. The preparation of this sensitive model is innovative, and the modeling success rate is high, the cycle is short, and the operation is relatively simple. Compared with the existing modeling method, it has great advantages and feasibility;
[0025] 2. The constructed IDILI drug-sensitive model can be used for the detection of idiosyncratic hepatotoxicity of new drugs in preclinical settings. The administration of drugs with idiosyncratic hepatotoxicity in this sensitive model can significantly increase the incidence of IDILI and simulate the complex immune process in the pathogenesis of IDILI. Furthermore, the mechanism by which the drug causes IDILI can be further explored in the IDILI model. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work, among which:
[0027] Figure 1 The present invention provides a graph of serum liver function levels and liver tissue pathological manifestations of two groups of mice during the administration period;
[0028] Figure 2 is a graph of inflammation levels in the livers of two groups of mice provided by the present invention;
[0029] Figure 3 is a diagram showing the degree of oxidative stress in the liver of two groups of mice provided by the present invention;
[0030] Figure 4 is a graph of adaptive immune cell levels in the liver of two groups of mice provided by the present invention;
[0031] Figure 5 is a graph of the levels of innate immune cells in the livers of two groups of mice provided by the present invention;
[0032] Figure 6 It is an experimental instrument table used in the present invention;
[0033] Figure 7 It is the experimental reagent table used in the present invention. DETAILED DESCRIPTION
[0034] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.
[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.
[0036] Embodiment: A method for constructing an IDILI drug sensitivity model comprises the following steps:
[0037] Step S1, selecting modeling subjects and grouping: 2n SPF-grade Balb / c mice aged 6 to 8 weeks and weighing 17 to 22 grams were selected as modeling subjects. After one week of adaptability (raising in a standard environment, free drinking water and food, and raising and operating in strict accordance with the ethical requirements of scientific research animals), they were randomly divided into a Ctrl group and a CPI+TLR1 / 2 group, with n mice in each group; preferably, 16 mice were selected in this embodiment, with 8 mice in each of the Ctrl group and the CPI+TLR1 / 2 group.
[0038] Step S2, drug preparation: preparing TLR1 / 2 agonist Diprovocim and CPI; wherein: the preparation method of TLR1 / 2 agonist Diprovocim is: Diprovocim is prepared in advance with DMSO to a 10 mg / ml mother solution, and then diluted to a 0.5 mg / ml application solution according to the ratio of 45% PEG300: 5% Tween80: 45% saline: 5% mother solution, and the solution is prepared and used immediately;
[0039] CPI is anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo) diluted with PBS to 5 mg / ml respectively, and is prepared and used immediately.
[0040] Step S3, drug administration: on days 1 and 5, mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of Diprovocim, and mice in the Ctrl group were given an equal amount of a mixed solution, wherein the mixed solution was prepared according to a ratio of 45% PEG300: 5% Tween80: 45% saline: 5% DMSO; mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo) on days 2, 4, and 6, and mice in the Ctrl group were given an equal amount of PBS;
[0041] Step S4, sample collection:
[0042] On the 0th and 3rd day, blood from the retro-orbital venous plexus of the two groups of mice was collected and serum was obtained and stored for later use. When collecting blood from the retro-orbital venous plexus, the two groups of mice were anesthetized by inhalation of isoflurane. Before use, the 1.0 mm glass capillary was broken into 2.0-2.5 cm capillary segments. After the mice were anesthetized, the left thumb and index finger were used to press the sides of the mouse's neck, and the mouse's neck was tightly held from the back to make the retro-orbital venous plexus congested; the right hand made the glass capillary segment form an angle of 45° with the mouse's eyeball surface, and pierced from the inner corner of the eye, with the bevel of the needle facing the eyeball first, and then turned 180 degrees after piercing so that the bevel faced the posterior border of the orbit. The piercing depth was about 2-3 mm. When resistance was felt, the advancement was stopped to allow the blood to flow naturally into the procoagulant blood collection tube. About 5-6 drops of blood (100ul) were collected.
[0043] On the 7th day, the eyeball blood of the two groups of mice was obtained and the serum was obtained and stored for later use. The specific method for obtaining eyeball blood serum is as follows: 100ul / mouse of pentobarbital was intraperitoneally injected into the mouse, and the mouse was grabbed after anesthesia, and the mouse whiskers were cut with scissors to prevent blood contamination; the mouse was fixed with one hand, and the eyeball skin on the blood collection side was gently pressed to make the eyeball congested and protruding; the mouse head was fixed, and the eyeball was quickly removed with tweezers, and the centrifuge tube was used to collect the eyeball blood at the bottom; the collected blood was placed at room temperature for 30 minutes, centrifuged at 3500r / min for 15 minutes, the supernatant was aspirated, and the serum was stored in a 4℃ refrigerator for later use;
[0044] After obtaining eyeball blood, the livers of the two groups of mice were obtained respectively, and the livers of the two groups of mice were processed as follows: first rinsed with physiological saline, the surface moisture was absorbed with filter paper, and the 0.5 cm × 0.5 cm liver tissue in the middle of the left lobe of the liver was cut and immersed in 4% paraformaldehyde tissue fixative for histopathological evaluation; another portion of liver tissue was put into a cryovial and stored in a -80°C refrigerator for later use; the remaining liver tissue was immersed in PBS solution and placed in a 4°C refrigerator for later use.
[0045] It is necessary to evaluate the IDILI drug sensitivity model constructed by the above method, thereby providing an evaluation method for the IDILI drug sensitivity model constructed by the construction method of the IDILI drug sensitivity model, including evaluating liver damage, evaluating the level of intrahepatic inflammation and oxidative stress, and evaluating the level of intrahepatic immune cells;
[0046] First, evaluate liver damage: The liver damage of mice was evaluated by testing serum biochemical indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and observing liver tissue pathology. Specifically:
[0047] The serum of the two groups of mice was obtained by sampling blood from the retro-ocular venous plexus on the 0th day and the 3rd day, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected by using a kit to understand the baseline level of liver function of the mice and the changes of transaminases in the body after two days of administration;
[0048] On the 7th day, the ocular blood serum of the two groups of mice was obtained, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected using a kit;
[0049] On the 7th day, the livers of the two groups of mice were obtained, and the liver tissues fixed with 4% polyformaldehyde were embedded in paraffin, sliced, and stained with hematoxylin-eosin (ie, HE staining) to observe the pathological manifestations of liver tissues.
[0050] As attached Figure 1 As shown, according to the experimental results, the liver damage is as follows:
[0051] (1) On days 3 and 7, the levels of ALT and AST in the serum of mice in the CPI+TLR1 / 2 group did not increase significantly compared with the baseline levels; compared with the Ctrl group, there was no significant increase in ALT and AST (P>0.05).
[0052] (2) On the 7th day, the liver tissues were paraffin-sectioned and stained with HE. Observation under an optical microscope revealed that compared with the Ctrl group, there was no obvious hepatocyte death in the CPI+TLR1 / 2 group, and only a small number of inflammatory cells infiltrated.
[0053] In summary, the administration method of CPI+TLR1 / 2 group mice did not cause obvious liver damage.
[0054] Secondly, the level of inflammation and oxidative stress in the liver was assessed. Specifically:
[0055] On the 7th day, liver tissue proteins were extracted from the two groups of mice for Western Blot experiments, and the expressions of inflammatory factors IL-1β, IL-6, TNF-α, IL-12α and NLRP3 in the two groups of mice were detected to reflect the level of intrahepatic inflammation; the levels of intrahepatic GSH, SOD and MDA in the two groups of mice were detected by kits to reflect the level of intrahepatic oxidative stress.
[0056] The specific operation method of the Western Blot experiment is as follows:
[0057] 1. Protein extraction, protein denaturation and protein concentration determination:
[0058] 20-30 mg of liver tissue was cut, and 10 times the mass of liver tissue lysis buffer (RIPA:PMSF=100:1) was added, and grinding beads (3 mm in diameter) were added for grinding (60 Hz, 60 s, twice) to obtain tissue homogenate;
[0059] The tissue homogenate was lysed on ice for 1-1.5 h and the supernatant was collected by centrifugation (13000 r / min, 15 min);
[0060] The supernatant protein concentration was detected by BCA method;
[0061] Dilute with PBS according to the protein concentration, add 5X loading buffer, and denature the protein in a metal bath (100°C, 10 min);
[0062] Store protein samples at -20°C.
[0063] 2. SDS-PAGE electrophoresis:
[0064] Prepare the corresponding separation gel (10% and 12%) according to the molecular weight; after the separation gel solidifies, add 5% concentrated gel and insert the comb;
[0065] Sample loading: Calculate the corresponding sample volume according to the protein concentration (to ensure the total amount of protein is consistent);
[0066] Electrophoresis: 80V, 30min, 120V, 60min;
[0067] Transfer: fast transfer 400mA, 30min;
[0068] Blocking: Rapid blocking solution for 20 min;
[0069] Wash with TBST for 2 hours for 5 minutes (shaking speed 100);
[0070] Apply primary antibody: incubate at 4°C for 16-18h;
[0071] Wash with TBST for 6 hours for 5 minutes (shaking speed 100);
[0072] Apply secondary antibody: incubate at room temperature for 1 hour;
[0073] Wash with TBST for 6 hours for 5 minutes (shaking speed 100);
[0074] development.
[0075] As attached Figure 2 and attached Figure 3 As shown, according to the experimental results, the levels of inflammation and oxidative stress in the liver are as follows:
[0076] (1) On the 7th day, liver tissue proteins were extracted for Western Blot experiments. The results showed that compared with the Ctrl group, the expression of inflammatory factors IL-1β (P<0.001), IL-6 (P<0.001), TNF-α (P<0.05), and IL-12α (P<0.05) in the liver of the CPI+TLR1 / 2 group mice was significantly increased; and the expression of NLRP3, a major regulatory factor in the development of inflammation, was also significantly increased in the CPI+TLR1 / 2 group mice (P<0.01).
[0077] (2) The test kit revealed that: compared with the Ctrl group, although the GSH level in the liver of the CPI+TLR1 / 2 group mice decreased significantly (P<0.01), the average content was 43.03μmol / gprot, which was still within the normal range; and the SOD content in the liver of the CPI+TLR1 / 2 group mice increased significantly (P<0.001), indicating that although the oxygen free radicals in the liver of the CPI+TLR1 / 2 group mice increased, the liver's oxygen free radical scavenging ability did not weaken. This can be confirmed by the fact that the MDA content can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage, but the MDA in the liver of the CPI+TLR1 / 2 group mice did not increase significantly, indicating that the liver cells of the CPI+TLR1 / 2 group mice did not show obvious damage.
[0078] Taken together, the results showed that the administration of CPI+TLR1 / 2 group mice could increase the level of inflammation in the liver without causing obvious liver damage.
[0079] Finally, the level of immune cells in the liver was evaluated. Specifically:
[0080] The liver tissues of the two groups of mice obtained on the 7th day were analyzed by flow cytometry to detect the changes in the proportion of major immune cells, and the expression of some immune cell markers was detected by Western Blot. + T cells and CD8 + T cell infiltration.
[0081] The specific operation method of the flow cytometry is as follows:
[0082] 1. Extraction of liver cells:
[0083] Digestion of liver with digestive enzymes: Tear the liver tissue into pieces, add digestive enzymes, and digest in a 37°C water bath for 30 min;
[0084] Preparation of single cell suspension: Grind liver fragments with a 70 μm mesh and rinse the mesh with PBS containing 2% FBS to prepare liver single cell suspension;
[0085] Sedimentation of hepatocytes: centrifugation at 500 r / min for 5 min (9; 9), aspirate the supernatant;
[0086] Sedimentation of hepatocyte parenchyma: centrifugation at 1000 g for 10 min (9; 9), and the sediment was retained;
[0087] Immune cell separation: add 40% percoll solution, mix well and centrifuge at 1200g for 20min (6;2);
[0088] Washing: aspirate the supernatant, add pre-cooled PBS (4°C), centrifuge at 400g for 5min (9:9), repeat twice, and add PBS to resuspend the precipitate.
[0089] 2. Dyeing:
[0090] Block, room temperature for 15 min;
[0091] Live and dead staining: 10 min at room temperature;
[0092] Remove the block and stain for live or dead cells: add PBS and mix well, centrifuge at 400g for 5 minutes, and remove the supernatant;
[0093] Staining: Add the corresponding flow cytometry antibody, mix gently by pipetting, and incubate at room temperature in the dark for 30 minutes;
[0094] Lysis of red blood cells: Add red blood cell lysis buffer, mix well, and lyse at room temperature for 10 minutes;
[0095] Stop red cell lysis: add PBS containing 2% FBS, mix well, centrifuge at 400g for 5min, and remove the supernatant;
[0096] Re-hang the machine.
[0097] As attached Figure 4 and attached Figure 5 As shown, according to the experimental results, the levels of intrahepatic immune cells are as follows:
[0098] The results of flow cytometry of liver tissues showed that compared with the Ctrl group, the CD4 + T cells increased (P<0.01), but CD8+ T cells decreased significantly (P<0.0001);
[0099] The results of immunofluorescence showed that compared with the Ctrl group mice, the CD4 + T cells and CD8 + T cell infiltration increased significantly, with CD4 + T cells were the main ones. Meanwhile, NK cells and macrophages also increased significantly (P<0.01), and macrophages mainly included M1 and M2 macrophages.
[0100] Western Blot experiments showed that the expression of INOS protein, a marker of M1 macrophages, was significantly increased (P<0.05), while the expression of Arg1 protein, a marker of M2 macrophages, was significantly decreased (P<0.05), indicating that the macrophages in the liver of mice in the CPI+TLR1 / 2 group were mainly pro-inflammatory M1 macrophages.
[0101] In summary, the administration of CPI+TLR1 / 2 to mice can significantly increase the levels of major immune cells involved in innate and adaptive immune responses without causing liver damage. + T cells, but this model does not make CD8 + The number of T cells increased significantly.
[0102] The test instruments and test methods used in the above experiments are as shown in the attached manual. Figure 6 and attached Figure 7 shown.
[0103] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for constructing an IDILI drug sensitivity model, characterized in that: The following steps are involved: Step S1, selecting modeling subjects and grouping: 2n SPF-grade Balb / c mice were selected as modeling subjects and randomly divided into a Ctrl group and a CPI+TLR1 / 2 group, with n mice in each group; Step S2, drug preparation: prepare TLR1 / 2 agonist Diprovocim and CPI; wherein CPI is anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo) respectively diluted to 5 mg / ml by PBS, and used immediately after preparation; Step S3, drug administration: on days 1 and 5, mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of Diprovocim, and mice in the Ctrl group were given an equal amount of the matching solution; on days 2, 4, and 6, mice in the CPI+TLR1 / 2 group were intraperitoneally injected with 5 mg / kg of anti-PD1-mAb (in vivo) and anti-CTLA4-mAb (in vivo), and mice in the Ctrl group were given an equal amount of PBS; Step S4, sample collection: on the 0th and 3rd days, blood from the retro-orbital venous plexus of the two groups of mice was collected respectively, and serum was obtained and stored for later use; on the 7th day, blood from the eyeballs of the two groups of mice was obtained respectively, and serum was obtained and stored for later use, and then the livers of the two groups of mice were obtained respectively.
2. The method for constructing an IDILI drug sensitivity model according to claim 1, characterized in that: In step S1, the mice are selected to be 6 to 8 weeks old and weigh 17 to 22 grams, and are grouped and numbered after adaptive feeding for 1 week.
3. The method for constructing an IDILI drug sensitivity model according to claim 1, characterized in that: In step S2, the TLR1 / 2 agonist Diprovocim is prepared as follows: Diprovocim is prepared in advance with DMSO to a 10 mg / ml stock solution, and then diluted to a 0.5 mg / ml application solution according to a ratio of 45% PEG300: 5% Tween80: 45% saline: 5% stock solution, and the solution is used immediately after preparation.
4. The method for constructing an IDILI drug sensitivity model according to claim 3, characterized in that: In step S3, the mixed solution is prepared according to the ratio of 45% PEG300: 5% Tween80: 45% physiological saline: 5% DMSO.
5. The method for constructing an IDILI drug sensitivity model according to claim 1, characterized in that: In step S4, after obtaining the livers of the two groups of mice on the 7th day, they were first rinsed with physiological saline, and the surface moisture was absorbed with filter paper. The liver tissue with a size of 0.5 cm × 0.5 cm in the middle of the left lobe of the liver was cut and immersed in 4% paraformaldehyde tissue fixative for histopathological evaluation; another portion of the liver tissue was placed in a cryopreservation tube and stored in a -80°C refrigerator for later use; the remaining liver tissue was immersed in a PBS solution and placed in a 4°C refrigerator for later use.
6. A method for evaluating an IDILI drug sensitivity model constructed by the method for constructing an IDILI drug sensitivity model according to any one of claims 1 to 5, characterized in that: Assessment of liver damage includes: The serum of the two groups of mice was obtained by sampling blood from the retro-ocular venous plexus on the 0th day and the 3rd day, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected by using a kit to understand the baseline level of liver function of the mice and the changes of transaminases in the body after two days of administration; On the 7th day, the ocular blood serum of the two groups of mice was obtained, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the two groups of mice were detected using a kit; On the 7th day, the livers of the two groups of mice were obtained, and the liver tissues fixed with 4% polyformaldehyde were embedded in paraffin, sliced, and stained with hematoxylin-eosin to observe the pathological manifestations of liver tissue.
7. The method for evaluating the IDILI drug sensitivity model according to claim 6, characterized in that: It also includes assessment of the level of inflammation and oxidative stress in the liver: On the 7th day, liver tissue proteins were extracted from the two groups of mice for Western Blot experiments, and the expressions of inflammatory factors IL-1β, IL-6, TNF-α, IL-12α and NLRP3 in the two groups of mice were detected to reflect the level of intrahepatic inflammation; the levels of intrahepatic GSH, SOD and MDA in the two groups of mice were detected by kits to reflect the level of intrahepatic oxidative stress.
8. The method for evaluating the IDILI drug sensitivity model according to claim 6, characterized in that: It also includes assessment of intrahepatic immune cell levels: The liver tissues of the two groups of mice obtained on the 7th day were analyzed by flow cytometry to detect the changes in the proportion of major immune cells, and the expression of some immune cell markers was detected by Western Blot. + T cells and CD8 + T cell infiltration.