Cytokine storm mouse model as well as preparation method and application thereof

Abstract

The invention relates to the field of animal models, in particular to a cytokine storm mouse model and a preparation method thereof. The method comprises the following step: injecting one or more of IL-1beta, IL-6, TNF-alpha, IL-2 and IFN-gamma into a mouse to obtain the cytokine storm mouse model. According to the method, the original microenvironment of the mouse can be changed after five cytokines are inoculated into the body of the mouse, and the multi-cytokine storm mouse model established by the method has the advantages of short disease occurrence time, long and stable model maintenance time and better clinical manifestation effect of simulating related diseases of the cytokine release syndrome.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and in particular to a cytokine storm mouse model and a preparation method and application thereof. Background Art

[0002] Cytokine Release Syndrome (CRS), also known as Cytokine Storm, is an over-activation of the immune system caused by infection, drugs or certain diseases. Once it occurs, it can quickly cause single or multiple organ failure, and ultimately threaten life. Cytokines mainly include interferon (IFN), interleukin (IL), chemokines and tumor necrosis factor (TNF). These cytokines are secreted by certain immune cells. Some of them promote inflammation, while others inhibit inflammation. The normal human body maintains a state of balance. Proinflammatory factors can activate and recruit other immune cells, and immune cells can secrete more cytokines, activate and recruit more immune cells, thus forming a positive feedback loop. When the immune system is over-activated by infection, drugs, autoimmune diseases and other factors, it may secrete a large number of proinflammatory factors, leading to a positive feedback loop, breaking through a certain threshold and becoming out of control and over-amplified, eventually forming a cytokine storm.

[0003] Currently, CAR-T therapy or the new coronavirus are more common, causing severe cytokine storms in the body. One of the main causes of death from the new coronavirus is the cytokine storm caused by overactivation of the human immune system. However, there is no good animal model to simulate the cytokine storm. The more commonly used method is to use LPS to create a model, which can cause acute or chronic inflammatory reactions, but cannot fully simulate the cytokine storm.

[0004] The establishment of animal models plays an important role in simulating the pathogenesis of cytokine storm and drug development. However, there is currently no animal model that is consistent with the pathogenesis of cytokine storm and the response to therapeutic drugs in humans. Animal models that simulate cytokine storm, such as spontaneous mouse models, genetically engineered mouse models, and xenograft models, all have their own advantages and disadvantages, but they are relatively expensive or complicated to operate, and their applicability and promotion are not strong. Drug-induced animal models are inexpensive and simple to operate. Among them, the imiquimod-induced cytokine storm animal model is the most widely used. The length of its modeling time is similar to the early and late histopathology of human cytokine storm simulations, and is believed to be caused by the synergy of multiple mechanisms of action. However, there are also shortcomings such as model instability, short model maintenance time after modeling, and long-term application that can cause systemic inflammation and immune response. Therefore, establishing a new animal model that simulates cytokine storm or optimizing existing models is of great significance for the discovery of the cause of cytokine storm simulations and therapeutic drugs.

[0005] In the prior art, most cytokine storm mouse models are constructed using a single cytokine, but a cytokine storm is caused by multiple cytokines. The models obtained by the prior art have a poor simulation effect on the cytokine storm. Summary of the invention

[0006] In view of the defect of the lack of good animal models for simulating cytokine storm in the prior art, the present invention provides a method for preparing a cytokine storm mouse model, a cytokine storm mouse model prepared by the preparation method, and an application of the cytokine storm mouse model. The preparation method is relatively simple, and the prepared cytokine storm mouse model can fully simulate the cytokine storm, avoiding the conventional (LPS, etc.) modeling method to cause inflammatory response and serious damage to the organs of the model animal. And the cytokine storm mouse model obtained by the present invention is more in line with the actual situation of patients with new coronavirus, cytokine release syndrome, immune reconstitution inflammatory syndrome and secondary hemophagocytic lymphohistiocytosis, and can better simulate the clinical manifestations of these diseases.

[0007] In order to solve the above technical problems, the present invention provides a technical solution: a method for preparing a cytokine storm mouse model, the preparation method comprising the following steps:

[0008] One or more of IL-1β, IL-6, TNF-alpha, IL-2 and IFN-gamma are injected into mice to obtain the cytokine storm mouse model.

[0009] In a preferred embodiment of the present invention, the injection is intraperitoneal injection.

[0010] In a preferred embodiment of the present invention, the injection is a continuous injection. Preferably, the duration of the continuous injection is more than 3 days, such as 4 days, 5 days, 6 days, 7 days, 8 days, 9 days and 10 days.

[0011] In a preferred embodiment of the present invention, the mouse is a C57BL / 6 mouse.

[0012] In a preferred embodiment of the present invention, the dosage of one or more of IL-1β, IL-6, TNF-alph a, IL-2 and IFN-gamma is 50-200 ng / mouse / day. Preferably, the dosage of IL-1β, IL-6, TNF-alph a, IL-2 and IFN-gamma is 100 ng / mouse / day.

[0013] In order to solve the above technical problems, the present invention provides a technical solution: a cytokine storm mouse model prepared by the preparation method described in the present invention.

[0014] In order to solve the above technical problems, the present invention provides a technical solution: use of the cytokine storm mouse model described in the present invention in screening or studying drugs for treating cytokine storm.

[0015] In a preferred embodiment of the present invention, the application is to study the effects, adverse reactions and / or the absorption, distribution, metabolism and excretion of drugs for treating cytokine storm.

[0016] In a preferred embodiment of the present invention, the cytokine storm in the application is IL-1β, IL-6, TNF-alpha, IL-2 and IFN-gamma and the induced cytokine storm.

[0017] In a preferred embodiment of the present invention, the use is for non-therapeutic or diagnostic purposes.

[0018] In order to solve the above technical problems, the present invention provides a technical solution: application of the cytokine storm mouse model described in the present invention in studying the pathogenesis of cytokine storm.

[0019] In a preferred embodiment of the present invention, the use is for non-therapeutic or diagnostic purposes.

[0020] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0021] The reagents and raw materials used in the present invention are commercially available.

[0022] The positive and progressive effects of the present invention are:

[0023] 1. Compared with the animal model established by using only one cytokine in the prior art, the multi-cytokine storm mouse model established by this method has the advantages of shortening the onset of symptoms and maintaining the model for a longer and more stable period of time.

[0024] 2. The cytokine storm mouse model established by this method is more in line with the actual situation of patients with new coronavirus, cytokine release syndrome, immune reconstitution inflammatory syndrome and secondary hemophagocytic lymphohistiocytosis, and is better in simulating the clinical manifestations of these diseases.

[0025] 3. Compared with the cytokine animal models in the prior art, the animal model established by this method has more types of cytokines, higher expression levels, and relatively mild damage to tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the level of IL-1 in the serum of mice after five-factor modeling.

[0027] Figure 2 This is the level of IL-2 in the serum of mice after five-factor modeling.

[0028] Figure 3 This is the level of TNF-α in the serum of mice after five-factor modeling.

[0029] Figure 4 This is the level of IL-6 in the serum of mice after five-factor modeling.

[0030] Figure 5 This is the level of INF-γ in the serum of mice after five-factor modeling.

[0031] Figure 6 This is a picture of the liver section of mice after five-factor modeling.

[0032] Figure 7 This is a cross-sectional image of the spleen of mice after five-factor modeling.

[0033] Figure 8 This is a cross-sectional image of mouse kidney after five-factor modeling.

[0034] Fig. 9 This is a picture of the lung sections of mice after five-factor modeling.

[0035] Fig.10 This is a scan of the HE-stained pathological section. DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0037] Example 1: Cytokine Storm Modeling

[0038] (1) Experimental animals: Mice, C57BL / 6, SPF grade, male, 42-56 days old, were purchased from Hubei Provincial Experimental Animal Research Center.

[0039] (2) Experimental grouping: A five-factor combination method was used, Murine IL-1β (Cat. No. 211-11B-2UG, manufacturer PeproTech), Murine IL-6 (Cat. No. 216-16-2UG, manufacturer PeproTech), Murine TNF-alph a (Cat. No. 315-01A-20UG, manufacturer PeproTech), Murine IL-2 (Cat. No. 212-12-5UG, manufacturer PeproTech), Murine IFN-gamma (Cat. No. 315-05-20UG, manufacturer PeproTech), each reagent 100 ng / mouse / day, continuous injection (intraperitoneal injection) for 2 days, 3 days and 7 days, the next day after the injection, the mice were killed. Serum was collected for serum data testing.

[0040] (3) Results and analysis: For the data of ELISA test of serum for different cytokines, see Figure 1-5 (Control group, LPS treatment group, and five cytokines injected for 7 consecutive days). Figure 1-5 It can be seen that after two or three days of continuous treatment with the five factors, the damage to tissues and organs was not obvious, so the time was extended to 7 days, the shortest time of actual cytokine storm in clinical practice. After seven days of continuous treatment with the five factors, there was slight damage to tissues and organs, and the livers of some membrane-forming mice were normal without damage.

[0041] Example 2: Paraffin-embedded HE staining

[0042] (1) Specific reagents, consumables and experimental steps for paraffin embedding and HE staining: The experimental equipment used in the experiment is shown in Table 1, and the main experimental reagents used are shown in Table 2.

[0043] Table 1 Experimental equipment information

[0044] Pathology slicer German Leica RM 2016 rotary microtome Slicing knife Japanese Feather R35 Disposable Blade Tissue baking machine Wuhan Junjie JK-6 Biological Tissue Slice Baking Machine Slides and coverslips Jiangsu Shitai Experimental Equipment Co., Ltd. microscope Olympus BX53 biological microscope Dehydrator Wuhan Junjie JT-12J Computerized Biological Tissue Dehydrator Embedding machine Wuhan Junjie JB-P5

[0045] Table 2 Main experimental reagent information

[0046] Reagents factory Part Number Hematoxylin Sigma H9627 Eosin Y (water soluble) Sinopharm Group 71014544 Anhydrous ethanol Sinopharm Group 10009218 Xylene Sinopharm Group 10023418 hydrochloric acid Sinopharm Group 10011018 Paraffin embedding Sinopharm Group 69019361 Neutral gum Sinopharm Group 10004160

[0047] (2) Paraffin section HE staining experimental steps

[0048] 1. Tissue dehydration: Dehydrate the tissue with gradient alcohol, 75% alcohol (4h) - 85% alcohol (2h) - 90% alcohol (1.5h) - 95% alcohol (1h) - anhydrous ethanol I (0.5h) - anhydrous ethanol II (0.5h);

[0049] 2. Tissue transparency: Tissue blocks must be transparent after being dehydrated with alcohol. The transparent agent (xylene) can be mixed with the dehydrating agent and paraffin at the same time. After it replaces the dehydrating agent, the paraffin can smoothly penetrate into the tissue. Anhydrous ethanol: xylene (1:1) (10min) - xylene I (10min) - xylene II (10min)

[0050] 3. Wax dipping: After the tissue block has been made transparent, it is dipped into three cylinders of paraffin wax (60°C) in sequence, paraffin wax I (60°C) (1h) - paraffin wax II (60°C) (1h) - paraffin wax III (60°C) (1h)

[0051] The above three experimental steps are all completed in the biological tissue dehydrator (machine automatic program)

[0052] 4. Embedding: Embedding is to wrap the wax-soaked tissue block in a paraffin block. The temperature of the embedding wax should be slightly higher than the wax soaking temperature to ensure that the tissue block and the embedding paraffin are completely integrated.

[0053] 5. Slicing and baking: Use a Leica pathology slicer to slice the embedded wax block. Place the sliced ​​tissue slices in a 40°C water bath for spreading. Insert an anti-shedding slide into the water surface at an angle to pick up the slices. Make sure the slices are attached to the appropriate position on the slide and bake in a 60°C oven for 3 hours.

[0054] 6. Dewaxing of sections: Place the paraffin sections in sequence: xylene Ⅰ (20 min) - xylene Ⅱ (20 min) - xylene Ⅲ (20 min) - anhydrous ethanol Ⅰ (5 min) - anhydrous ethanol Ⅱ (5 min) - 95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min), and then soak in distilled water for 5 min.

[0055] 7. Stain the sections with Mayer's hematoxylin (the staining background is clean and no differentiation is required) for 5 minutes, and then wash with tap water until the blue color returns.

[0056] 8. Stain the sections with 1% water-soluble eosin solution for 5 minutes and then wash with tap water for 30 seconds.

[0057] 9.95% alcohol 30s

[0058] 10.95% alcohol 1min

[0059] 11. Anhydrous ethanol I 5min

[0060] 12. Anhydrous ethanol Ⅱ 5min

[0061] 13. Xylene 1 5min

[0062] 14. Xylene Ⅱ 5min

[0063] 15. After air drying, seal the slides with neutral gum and examine under a microscope.

[0064] (3) Results and interpretation: The slice results are as follows: Figure 6-9 As shown. By judging that the cell nucleus is blue, the cytoplasm, muscle fibers, collagen fibers and red blood cells are different degrees of red, the tissue morphology and structure can be observed. As can be seen from the figure, the cytokine storm mouse model obtained by the present invention is more in line with the actual situation of patients with new coronavirus, cytokine release syndrome, immune reconstitution inflammatory syndrome and secondary hemophagocytic lymphohistiocytosis, and can better simulate the clinical manifestations of these diseases.

[0065] Example 3: Further HE staining pathological detection

[0066] (1) Sample preparation: The samples to be tested are fixed with 4% paraformaldehyde. After the fixation is in good condition, they are trimmed, dehydrated, embedded, sliced, stained, and sealed in strict accordance with the SOP procedures of the pathological laboratory detection of this unit. The qualified samples are examined under the microscope. The image acquisition instruments are shown in Table 3.

[0067] Table 3 Image acquisition instruments

[0068]

[0069] (2) Slice scanning analysis: Microscopic examination / panoramic scanning analysis of the slices, including but not limited to textual description of congestion, congestion, hemorrhage, edema, deformation, necrosis, hyperplasia, fibrosis, degree of damage repair, etc., imaging of typical lesions and marking them with arrows in the report.

[0070] (3) Inspection results and analysis: The slice scanning results are as follows: Fig.10 As shown. Fig.10 As shown in 1-1, the liver tissue shows that the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The liver tissue shows a large number of hepatocyte granular degeneration, cell swelling, and fine red-stained granules in the cytoplasm, as shown by the yellow arrows; the hepatic plates are arranged regularly and neatly, and the hepatic sinusoids have no obvious expansion or compression; there are no obvious abnormalities in the portal areas between adjacent hepatic lobules; no obvious inflammatory infiltration is observed; among them, Fig.10 As shown in 1-2, liver tissue showed more granular degeneration of hepatocytes, cell swelling, and fine red-stained granules in the cytoplasm, as shown by the yellow arrow; individual hepatocytes showed dark-stained degeneration, as shown by the blue arrow; hepatocyte cords were irregularly arranged, and there was no obvious expansion or compression of the hepatic sinusoids, no obvious necrosis, and no obvious inflammatory cell infiltration; among them, Fig.10As shown in 1-3, the liver tissue shows that the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The hepatocytes are round and plump; the hepatic plates are arranged regularly and neatly, and the hepatic sinusoids have no obvious expansion or compression; there are no obvious abnormalities in the portal areas between adjacent hepatic lobules; no obvious inflammatory infiltration is observed; among them, Fig.10 As shown in Figure 2-1, the liver tissue shows that the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The hepatocyte cords are arranged irregularly, and there is no obvious expansion or compression of the hepatic sinusoids. The hepatocytes are round and plump, and no obvious necrosis and degeneration are observed. A small amount of inflammatory cell focal infiltration can be seen, as shown by the black arrows. Fig.10 As shown in Figure 2-2, the liver tissue shows that the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The hepatocytes are round and plump; the hepatic plates are arranged regularly and neatly, and the hepatic sinusoids have no obvious expansion or compression; there is no obvious abnormality in the portal area between adjacent hepatic lobules; a small amount of inflammatory cell infiltration can be seen, as shown by the black arrow; among them, Fig.10 As shown in Figure 2-3, compared with the normal group, the number of hepatocytes was significantly increased, and the hepatocytes were round and plump, without obvious necrosis and degeneration; the hepatic plates were arranged regularly and neatly, and the hepatic sinusoids were not significantly expanded or squeezed; there was no obvious abnormality in the portal area between adjacent hepatic lobules; a small amount of inflammatory cell infiltration was observed, as shown by the black arrows; Fig.10 As shown in Figure 3-1, the hepatocyte cords are arranged irregularly, and the hepatic sinusoids are not obviously expanded or squeezed; some hepatocytes are deeply stained and degenerated, as shown by the blue arrows; a necrotic degeneration can be seen in the tissue, and there is focal infiltration of some inflammatory cells, as shown by the black arrows; Fig.10 As shown in Figure 3-2, the liver tissue shows that the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The hepatocytes are arranged relatively loosely, and the hepatocytes are round and plump. There is no obvious abnormality in the portal area between adjacent hepatic lobules. A small amount of inflammatory cell infiltration can be seen, as shown by the black arrows. Fig.10 As shown in Figure 3-3, in the liver tissue, the hepatic lobule is centered on the central vein, surrounded by hepatocytes and hepatic sinusoids arranged roughly radially. The hepatocytes are round and plump; the hepatic plates are arranged regularly and neatly, and there is no obvious expansion or compression of the hepatic sinusoids; there is no obvious abnormality in the portal area between adjacent hepatic lobules; there is a focal infiltration of inflammatory cells, as indicated by the black arrow.

[0071] Example 4: A comparative experiment was carried out using a three-cytokine treatment group, i.e., mice were injected with TNF-α, IL-1β and IL-6, and the injection amount of each cytokine was 200 ng / ml. Other experimental conditions and methods were referred to Examples 1-3. The inflammatory effect on the mouse model and the cytokine concentration in the serum were lower than those of the mouse model injected with 100 ng / ml each of TNF-α, IL-1β, IL-6, IL-2 and IFN-γ.

[0072] In summary, the above experimental results show that the use of the present invention to treat IL-1β, IL-

[0073] 6. TNF-alpha, IL-2 and IFN-gamma were injected into mice at the same time, and endothelial cells were subjected to cytokine storm membrane formation. It was found that the combined treatment of the five factors had a stronger effect on the cells, and the cytokine level in the serum of the constructed cytokine storm mouse model was high, indicating that the induction of the mouse model obtained by the present invention has achieved better results, and the slice scanning results show that it can better simulate the actual conditions of patients with, for example, new coronavirus, cytokine release syndrome, immune reconstitution inflammatory syndrome and secondary hemophagocytic lymphohistiocytosis, and then simulate the clinical manifestations of these diseases, achieving unexpected technical effects.

[0074] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for preparing a cytokine storm mouse model, characterized in that: The preparation method comprises the following steps: One or more of IL-1β, IL-6, TNF-alpha, IL-2 and IFN-gamma are injected into mice to obtain the cytokine storm mouse model.

2. The preparation method according to claim 1, characterized in that The injection is intraperitoneal injection; or the injection is continuous injection.

3. The preparation method according to claim 2, characterized in that: The duration of the continuous injection is more than 3 days; preferably, the duration of the continuous injection is 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

4. The preparation method according to claim 1, characterized in that: The mice are C57BL / 6 mice.

5. The preparation method according to claim 1, characterized in that: The dosage of one or more of IL-1β, IL-6, TNF-alph a, IL-2 and IFN-gamma is 50-200 ng / mouse / day.

6. The preparation method according to claims 1-5, characterized in that: Mice were injected with five of IL-1β, IL-6, TNF-alpha, IL-2 and IFN-gamma simultaneously.

7. The preparation method according to claim 4, characterized in that: The dose of IL-1β, IL-6, TNF-alph a, IL-2 and IFN-gamma was 100 ng / mouse / day.

8. A cytokine storm mouse model prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the cytokine storm mouse model as claimed in claim 8 in screening or studying drugs for treating cytokine storm.

10. Use of the cytokine storm mouse model as claimed in claim 8 in studying the pathogenesis of cytokine storm.

Images