Construction method and application of humanized immune reconstruction animal model with NK cell differentiation

Abstract

The invention relates to a construction method and application of a humanized immune reconstruction animal model with NK cell differentiation, and the method comprises the following steps: injecting exogenous IL-2 and IL-15 cytokines into the humanized immune reconstruction animal model, so that the model animal reconstructs NK cells, thereby obtaining the humanized immune system animal model with NK cell and ADCC capability. The humanized immune reconstruction model animal is further perfected through the method, so that the animal has a more complete immune system; by promoting induced differentiation of the NK cells, the proportion of human-derived NK cells with a killing effect is increased in an immune system, the deficiency of NK cell immunity is made up, and the ADCC effect mediated by the NK cells is remarkably enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and in particular relates to a method for constructing and applying a humanized immune reconstruction animal model with NK cell differentiation. Background Art

[0002] Immunotherapy is a revolutionary cancer treatment method. The human immune response needs to be carried out in vivo. Due to ethical restrictions, humans cannot be used directly as experimental subjects. Rodents are small animal models widely used in experimental research. Currently, most studies on the development, differentiation and function of the immune system come from these models. Although they have broad prospects, they face a fundamental challenge in the process of transforming them from preclinical research to clinical application: there are fundamental differences between the mouse and human immune systems. Due to the existence of species specificity, the conclusions obtained from rodent models cannot be directly derived from the human immune system. Therefore, an animal model that can be applied to human immune responses, the humanized immune system mouse, came into being. This animal model is an important tool for understanding the human immune system and its immune response.

[0003] At present, the main way to relatively completely construct myeloid cells and lymphoid cells in humanized immune system mice is to implant human hematopoietic stem cells in super-severe immunodeficient mice so that the mice have a relatively complete human immune system. Specifically, Mamoru Ito (founder of CIEA NOG mice) of CIEA in Japan transplanted huHSC-CD34+ into NOG-EXL mice to reconstruct the humanized immune system model huHSC-NOG-EXL, which will present a more complete human immune system, and will be more conducive to studying the interaction between the human immune system and tumors in anti-tumor drugs, and conducting research on inflammation and allergic reactions and immunotoxicity. However, studies have shown that the lack of NK cells in this model has severely limited research in the field of NK cell immunity. Therefore, it is urgently needed to reconstruct a preclinical model of the human immune system with NK cells using the humanized immune system mouse model huHSC-NOG-EXL. This model is expected to change preclinical research by providing a more accurate characterization of human immune responses. The construction of such a model requires not only the successful integration of human NK cells into the mouse host, but also the coordination of their activation and function. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for constructing a humanized immune reconstruction animal model with NK cell differentiation and its application.

[0005] The technical solution adopted by the present invention is: a method for constructing a humanized immune reconstruction animal model with NK cell differentiation, injecting human cytokines hIL-2 and hIL-15 into a humanized immune system model animal to obtain a humanized immune reconstruction animal model with NK cell differentiation.

[0006] Preferably, the human cytokines hIL-2 and hIL-15 are injected intraperitoneally.

[0007] Preferably, the model animal is a mouse, and hIL-2 is injected once a day for 5-7 consecutive days, with 4-6 μg each time; hIL-15 is injected once every 5 days, for a total of 3 injections, with 2-4 μg each time.

[0008] Preferably, the specific steps are as follows:

[0009] Step 1: Prepare conventional humanized immune reconstruction model animals;

[0010] Step 2: preparing hIL-2 and hIL-15 cytokine solutions;

[0011] Step 3: Inject cytokine solution into the conventional humanized immune reconstruction model animal in an exogenous manner to obtain a humanized immune system animal model with NK cell and ADCC capabilities.

[0012] Preferably, female NOG-EXL mice aged 6 to 8 weeks are selected and subjected to whole-body irradiation myeloablation. Within 5h-24h after irradiation, CD34-positive human hematopoietic stem cells are injected into the tail vein of the mice. After 8 weeks, the proportion of human immune cells in the peripheral blood of the humanized immune reconstructed mice is detected. When the human immune cells in the peripheral blood account for about 20% of the total immune cells, the conventional humanized immune reconstructed mice are considered to be successfully constructed.

[0013] Preferably, flow cytometry is performed on the humanized immune reconstructed model animals injected with cytokines. When the proportion of NK cells in the peripheral blood of the model animals accounts for no less than 5% of all human leukocytes, it proves that the humanized immune reconstructed animal model with NK cell differentiation is successfully constructed.

[0014] Method for constructing a humanized immune reconstructed animal model with NK cell differentiation A humanized immune reconstructed animal model with NK cell differentiation is constructed.

[0015] Preferably, the model animal is NOG-EXL mouse.

[0016] Application of humanized immune reconstitution animal models with NK cell differentiation in tumor immunotherapy research for non-disease diagnosis and treatment purposes.

[0017] Application of humanized immune reconstitution animal models with NK cell differentiation in NK cell research.

[0018] The advantages and positive effects of the present invention are: the humanized immune reconstruction model animal is further improved by the method of the present invention, so that it has a more complete immune system; by promoting the induced differentiation of NK cells, the proportion of human NK cells with killing effects in the immune system is increased, which makes up for the lack of NK cell immunity and significantly enhances the ADCC effect mediated by NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Human leukocyte population in the blank control group 4 weeks after exogenous cytokine injection;

[0020] Figure 2 Four weeks after exogenous cytokine injection, human leukocyte population in the medium-dose cytokine group;

[0021] Figure 3 Human leukocyte population in the high-dose cytokine group 2 weeks after exogenous cytokine injection;

[0022] Figure 4 Statistical chart of the proportion of human leukocytes in the blank control group and the medium-dose cytokine group 4 weeks after exogenous cytokine injection;

[0023] Figure 5 Four weeks after exogenous cytokine injection, the human NK cell population in the blank control group;

[0024] Figure 6 Four weeks after exogenous cytokine injection, human NK cell population in the medium-dose cytokine group;

[0025] Figure 7 Two weeks after exogenous cytokine injection, human NK cell population in the high-dose cytokine group;

[0026] Figure 8 Statistical chart of the proportion of human leukocytes in the blank control group and human NK cells in the medium-dose cytokine group 4 weeks after exogenous cytokine injection;

[0027] Fig. 9 Four weeks after the injection of exogenous cytokines, the standard lactate dehydrogenase (LDH) assay was used to detect the killing effect of peripheral blood cells of the constructed mouse model on SKBR3 cells in vitro. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0029] The present invention relates to a method for constructing and applying a humanized immune reconstruction animal model with NK cell differentiation. By injecting exogenous IL-2 and IL-15 cytokines into the humanized immune reconstruction animal model, NK cells are reconstructed in the model animal, thereby obtaining a humanized immune system animal model with NK cells and ADCC capabilities.

[0030] Taking model mice as an example, exogenous cytokines hIL-2 and hIL-15 are artificially supplemented by intraperitoneal injection to promote the induction and differentiation of NK cells in humanized immune reconstructed mice. After supplementing with exogenous human cytokines, the level of NK cell induction and differentiation in humanized immune reconstructed mice has been greatly improved, and they have corresponding functions. The method for constructing a humanized immune reconstructed mouse model with NK cell differentiation specifically includes the following steps:

[0031] Step 1: Prepare conventional humanized immune reconstructed mice; extract hematopoietic stem cells from healthy human umbilical cord blood, select 6-8 week old female NOG-EXL mice, perform whole body irradiation myeloablation, the radiation dose for each mouse is 1.5Gy, within 5h-24h after irradiation, inject CD34 positive human hematopoietic stem cells into the tail vein of the mice, the injection dose is 5×10 4 cells;

[0032] Eight weeks after the injection of hematopoietic stem cells derived from healthy human umbilical cord blood, the proportion of human immune cells in the peripheral blood of the humanized immune reconstructed mice was detected. When the human immune cells in the peripheral blood accounted for about 20% of the total immune cells, the conventional humanized immune reconstructed mice were considered to have been successfully constructed.

[0033] Step 2: Prepare hIL-2 and hIL-15 cytokine solutions; dissolve cytokines hIL-2 and hIL-15 in pure water to form solutions, and store them in aliquots to obtain hIL-2 and hIL-15 cytokine solutions, which are stored at -80°C.

[0034] Step 3: exogenously injecting the hIL-2 cytokine solution and the hIL-15 cytokine solution prepared in step 2 into the conventional humanized immune reconstitution mice obtained in step 1, respectively, to obtain a humanized immune reconstitution mouse model with NK cell differentiation;

[0035] Specifically, first take out the dissolved cytokine solution and dilute it to 200 μl with PBS; inject the dissolved and diluted cytokine solution intraperitoneally, hIL-2 is injected once a day for 5-7 consecutive days, and each mouse is injected with 4-6 μg each time; preferably, it is injected continuously for 7 days, and each mouse is injected with 5 μg each time; hIL-15 is injected once every 5 days, for a total of 3 injections, and each mouse is injected with 2-4 μg, preferably 2.5 μg; hIL-2 and hIL-15 cytokines can be injected sequentially or simultaneously.

[0036] Step 4: Identification of humanized immune reconstructed mouse model with NK cell differentiation; flow cytometry detection was performed on the humanized immune reconstructed mouse model with NK cell differentiation injected with cytokines. When the proportion of NK cells in the peripheral blood of the model mice accounted for about 5% of all white blood cells, the model was successfully constructed. The cells in the peripheral blood of the model mice were identified to be able to effectively induce the ADCC effect mediated by the Fc segment of the monoclonal antibody.

[0037] The prepared model is characterized by promoting the differentiation of NK cells. It increases the proportion of human NK cells with killing effects in the human immune system which is mainly composed of T lymphocytes and B lymphocytes and a small amount of myeloid cells, makes up for the lack of NK cell immunity, and significantly enhances the NK cell-mediated ADCC effect. It can effectively kill tumor cells and can be used for the research or application of tumor immunotherapy. Similarly, this animal model can also be used for the research or application of diseases that can be treated by NK cells.

[0038] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0039] Example 1: Construction of a humanized immune system mouse model of NK cell maturation and differentiation

[0040] 1.1 Preparation of conventional humanized immune reconstitution mice

[0041] Conventional human immune reconstructed mice were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Hematopoietic stem cells were extracted from healthy human umbilical cord blood, centrifuged, resuspended and counted with PBS, and placed on ice to maintain cell activity. Female NOG-EXL mice aged 6 to 8 weeks were selected, fixed on a mouse fixer, and the tail was disinfected. 0.2 mL of cell suspension was drawn with 1 mL of sterile injection and injected into the mouse through the tail vein.

[0042] After 8 weeks, the peripheral blood of the humanized immune reconstructed mice was collected and the proportion of human immune cells in the peripheral blood of the mice was detected by flow cytometry. When the human immune cells in the peripheral blood accounted for about 20% of the total immune cells, the conventional humanized immune reconstructed mice were considered to be successfully constructed and could be used to construct humanized immune reconstructed mice with NK cell maturation and differentiation.

[0043] 1.2 Construction of humanized immune reconstitution mice with NK cell maturation and differentiation

[0044] The purchased cytokine hIL-2 and hIL-15 dry powders were fully centrifuged at 12000rpm / min for 2min to prevent the dry powder from sticking to the wall; pure water was added to dissolve them so that the cytokine concentration was 100ug / ml. After complete dissolution, they were divided and stored in a -80℃ refrigerator. Before reconstructing the NK cells of humanized immune reconstructed mice, the dissolved cytokine solution was taken out, the corresponding dose was taken according to the required mass, and diluted to 200μl with PBS.

[0045] The dissolved and diluted cytokines were injected intraperitoneally, and a blank control group, a medium-dose cytokine group, and a high-dose cytokine group were set up. Specifically, in the medium-dose cytokine group, each mouse was injected with 5μg hIL-2 and 2.5μg hIL-15 each time; in the high-dose cytokine group, each mouse was injected with 10μg hIL-2 and 5μg hIL-15 each time; and the blank control group was injected with PBS. hIL-2 was injected once a day for 7 consecutive days; hIL-15 was injected once every 5 days, for a total of 3 injections. After 4 weeks of stimulation by cytokines IL-2 and IL-15, NK cells will differentiate in the humanized immune reconstruction mice. Among them, the mice in the high-dose cytokine group died one after another from the third week after injection. Each injection of 10μg hIL-2 and 5μg hIL-15 is a dangerous dose for model mice and should not be used.

[0046] 1.3 Identification of humanized immune reconstitution mice with NK cell differentiation and maturation

[0047] Four weeks after the injection of exogenous cytokines, the proportion of human leukocytes in the peripheral blood of the mice was detected by flow cytometry. Figure 1-3 As shown, Figure 1 The blank control group is a human leukocyte population; Figure 2 The human leukocyte population is in the medium-dose cytokine group. The mice in the high-dose cytokine group began to die from the third week after injection. Therefore, the proportion of human leukocytes in the high-dose cytokine group was tested two weeks after injection. The results are as follows: Figure 3 The percentage of human leukocytes in the blank control group and the medium-dose cytokine group was statistically analyzed, and the results are shown in Figure 4As shown in the results of each test, after stimulation by exogenous cytokines IL-2 and IL-15, the proportion of human immune cells in the peripheral blood of humanized immune reconstructed mice increased significantly, indicating that exogenous cytokines IL-2 and IL-15 can promote the proliferation of human leukocytes.

[0048] Furthermore, the mouse human NK cell population was tested, and the results were as follows Figure 5-7 As shown, Figure 5 The blank control group is human NK cell population. Figure 6 The human NK cell population is in the medium-dose cytokine group. Figure 7 This is the human NK cell population in the high-dose cytokine group 2 weeks after exogenous cytokine injection; the proportion of human NK cell population in the blank control group and the medium-dose cytokine group was statistically analyzed, and the results are as follows Figure 8 As shown, after stimulation by exogenous cytokines IL-2 and IL-15, the proportion of human NK cells in the peripheral blood of the humanized immune reconstructed mice of the present invention increased significantly, and exogenous cytokines IL-2 and IL-15 can promote the differentiation of human NK cells.

[0049] From the identification results, it can be seen that the medium-dose cytokine group successfully constructed a humanized immune system mouse model of NK cell maturation and differentiation, and the injection dose of 5μg hIL-2 and 2.5μg hIL-15 was more suitable for constructing the mouse model.

[0050] Example 2: Application of a humanized immune system mouse model of NK cell maturation and differentiation in the study of tumor immunotherapy

[0051] The killing effect of peripheral blood cells of the constructed mouse model on SKBR3 cells in vitro was detected by standard lactate dehydrogenase (LDH) experiment.

[0052] After the SKBR3 cells in the T25 flask are fully grown, they are digested with 0.5% trypsin and counted with a hemocytometer. The cells are inoculated into a 96-well plate, 200ul / well, 10,000 cells / well, and the cell density does not exceed 80-90% when tested. The growth medium is aspirated from the 96-well plate, the cells are washed once with PBS, the medium is replaced with a low-serum medium containing 1% serum, and the adaptive culture is performed for 1 hour; 1.28, 32, 160, 800, 40000ng / ml of trastuzumab monoclonal antibody are added and incubated at 37°C for 30 minutes.

[0053] Add 100 μL of fresh mouse blood to the centrifuge tube, add 2 mL of red blood cell lysis buffer, and mix well. Lyse the centrifuge tube at 4°C for 5 minutes, and the blood sample will slowly become transparent. Centrifuge at 300g for 5 minutes, discard the supernatant, and carefully aspirate the supernatant with a pipette to obtain a white cell pellet. Add 2 mL of PBS, centrifuge at 300g for 5 minutes (to wash off excess lysis buffer), discard the supernatant and aspirate the supernatant with a pipette. Add an appropriate amount of PBS or similar physiological buffer, resuspend the cells into a single cell suspension for subsequent experiments.

[0054] Add 250,000 cells to each well as the experimental group and incubate at 37°C for 2 hours; aspirate the supernatant, centrifuge at 400g for 10 minutes, take the supernatant, and add it to the corresponding 96-well plate. Add 20μL of 10% Triton X-100 solution to the cells as maximum release. Add 20μL of Assay Buffer to the cells as spontaneous release, and add 20μL of Assay Buffer to the wells without cells in 1% low serum medium as background control. Adjust the volume of the medium of the well plate cells to make the final volume 200ul; incubate at 37°C with CO 2 Incubate in an incubator for 3 hours. Centrifuge the 96-well plate at 400g for 5 minutes. Transfer 100 μL of cell supernatant to a new 96-well assay plate. Add 100 μL of LDH Reaction Solution to each well. Incubate the plate at 37°C for up to 30 minutes. Read the absorbance at 565 nm using a microplate reader.

[0055] The background A565 level was subtracted from all wells, and the killing rate was calculated according to the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100.

[0056] The results are as follows Fig. 9 As shown, peripheral blood cells of the humanized immune reconstructed mouse model injected with low cytokine can kill SKBR3 tumor cells with high HER2 expression in vitro through ADCC mediated by Trastuzumab monoclonal antibody, and the killing efficiency increases with the increase of antibody concentration.

[0057] The above results indicate that NK cell immunity was successfully induced in this model. Furthermore, the above experiments also demonstrate that the humanized immune reconstructed mice with mature NK cells of the present invention can be used in in vivo experiments related to tumor immunity.

[0058] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for constructing a humanized immune reconstruction animal model with NK cell differentiation, characterized in that: Humanized immune system model animals were injected with human cytokines hIL-2 and hIL-15 to obtain a humanized immune reconstruction animal model with NK cell differentiation.

2. The method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to claim 1, characterized in that: Human cytokines hIL-2 and hIL-15 were injected intraperitoneally.

3. The method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to claim 3, characterized in that: The model animals are mice. hIL-2 is injected once a day for 5-7 consecutive days, with 4-6 μg each time. hIL-15 is injected once every 5 days for a total of 3 injections, with 2-4 μg each time.

4. The method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to claim 1, characterized in that: The specific steps are as follows: Step 1: Prepare conventional humanized immune reconstruction model animals; Step 2: preparing hIL-2 and hIL-15 cytokine solutions; Step 3: Inject cytokine solution into the conventional humanized immune reconstruction model animal in an exogenous manner to obtain a humanized immune system animal model with NK cell and ADCC capabilities.

5. The method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to claim 4, characterized in that: Female NOG-EXL mice aged 6 to 8 weeks were selected and subjected to whole-body irradiation myeloablation. Within 5h-24h after irradiation, CD34-positive human hematopoietic stem cells were injected into the tail vein of the mice. After 8 weeks, the proportion of human immune cells in the peripheral blood of the humanized immune reconstructed mice was detected. When the human immune cells in the peripheral blood accounted for about 20% of the total immune cells, the conventional humanized immune reconstructed mice were considered to be successfully constructed.

6. The method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to claim 4, characterized in that: Flow cytometry was performed on the humanized immune reconstruction model animals injected with cytokines. When the proportion of NK cells in the peripheral blood of the model animals accounted for no less than 5% of all human leukocytes, it proved that the humanized immune reconstruction animal model with NK cell differentiation was successfully constructed.

7. A humanized immune reconstitution animal model with NK cell differentiation constructed by the method for constructing a humanized immune reconstitution animal model with NK cell differentiation according to any one of claims 1 to 6.

8. The humanized immune reconstitution animal model with NK cell differentiation according to claim 7, characterized in that: The model animal was NOG-EXL mouse.

9. Use of the humanized immune reconstructed animal model with NK cell differentiation as described in claim 7 or 8 in tumor immunotherapy research for non-disease diagnosis and treatment purposes.

10. Use of the humanized immune reconstitution animal model with NK cell differentiation according to claim 7 or 8 in NK cell research.

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