A method for establishing a mouse model of MHC haplotype compatible allogeneic hematopoietic cell transplantation

Abstract

The application discloses a method for establishing a mouse model of MHC haplotype compatible allogeneic hematopoietic cell transplantation, and belongs to the technical field of animal experiment model construction. The technical problem to be solved by the application is how to construct a mouse model which can be used to study the immune reconstruction mechanism and treatment effect evaluation of MHC haplotype compatibility and MHC full compatibility in bone marrow cell transplantation recipient mice. To solve the technical problem, the application provides a method for constructing a mouse model of MHC haplotype compatible allogeneic hematopoietic cell transplantation, which comprises transplanting bone marrow cells of a donor mouse into a recipient mouse, and the MHC haplotype of the donor mouse and the recipient mouse is compatible. The application sets a modeling condition, and the model establishment condition is evaluated by survival condition, graft-versus-host disease and rejection reaction observation, flow cytometry and histopathological section, so as to prove that the model is reliable and stable.

Description

Technical Field

[0001] This invention belongs to the technical field of animal experimental model construction, and particularly relates to a method for establishing a mouse model of MHC haploidentical allogeneic hematopoietic cell transplantation. Background Technology

[0002] Allogeneic hematopoietic cell transplantation (alloHCT) is an effective treatment strategy for hematologic malignancies. The efficacy of this therapy is related to the recipient's post-transplant immune function status; the quantity and quality of recipient immune cell reconstitution can influence the incidence and severity of early post-transplant complications and long-term survival. Multiple clinical studies have shown that the degree of major histocompatibility complex (MHC) incompatibility is associated with the level of post-transplant immune reconstitution. However, due to numerous factors affecting immune reconstitution in the clinical context, such as the graft source (bone marrow, peripheral blood, umbilical cord blood) and the number of hematopoietic cells, the use of immunosuppressants, and the severity of graft-versus-host disease, it is difficult to conclude from clinical evidence alone that MHC incompatibility is directly related to immune reconstitution. Therefore, establishing a pure MHC haploidentical allogeneic hematopoietic cell transplantation animal model for immune reconstitution monitoring is of great significance for elucidating these issues.

[0003] In normal organisms, T cell development and maturation are closely related to thymic function. Thymic cells carrying specific TCRs interact with their own peptide / MHC complex, inducing lymphoid progenitor cells to acquire MHC restriction and further differentiate into CD4 or CD8 single-positive mature T cells. MHC molecules play an irreplaceable and crucial role in this process. However, whether MHC incompatibility affects immune reconstitution after alloHCT by interfering with thymic function in transplant recipients remains unreported. Clinically, thymic sampling from transplant recipients is not feasible. In contrast, thymic sampling from mice is more convenient and allows for various treatments to reveal the impact of MHC incompatibility on thymic function and post-transplant immune reconstitution, as well as related mechanisms. Therefore, establishing a mouse model of MHC haploidentical allogeneic hematopoietic cell transplantation has significant research and application value. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to construct a mouse model that can be used to study the immune reconstitution mechanism and evaluate the therapeutic effect of MHC haplotype and MHC fully matched mice in bone marrow cell transplant recipients.

[0005] To address this technical problem, in a first aspect, the present invention provides a method for constructing a mouse model, the method comprising A1) and / or A2):

[0006] A1) Bone marrow cells from donor mice are transplanted into recipient mice irradiated with a sublethal dose to obtain an MHC haploidentical allogeneic hematopoietic cell transplantation mouse model, wherein the donor mice and the recipient mice are MHC haploidentical.

[0007] A2) Bone marrow cells from donor mice are transplanted into recipient mice irradiated with a sublethal dose to obtain a mouse model of MHC-matched allogeneic hematopoietic cell transplantation, wherein the donor mice and the recipient mice are MHC-matched.

[0008] Furthermore, in the above methods, in A1), the MHC molecules of the donor and recipient mice are hemiquoted and have the same CD45 molecular subtype; in A2), the MHC molecules of the donor and recipient mice are completely quotiented, but have different CD45 molecular subtypes.

[0009] In one embodiment of the present invention, the CD45 molecular subtype of the donor mouse and the recipient mouse described in A1) are the same, both being CD45.2.

[0010] In one embodiment of the present invention, the CD45 molecular subtype of the mouse donor (A2) is CD45.1, and the CD45 molecular subtype of the recipient mouse is CD45.2.

[0011] Furthermore, in the above method, the MHC of the mouse donor in A1) is H-2Kb (i.e., the MHC surface molecules of the mouse are H-2Kb), and the MHC of the mouse recipient is H-2Kb / d (i.e., the MHC surface molecules of the mouse are H-2Kb / d).

[0012] Furthermore, in the above method, the MHC of the mouse donor in A2) is H-2Kb (i.e., the MHC surface molecule of the mouse is H-2Kb), and the MHC of the mouse recipient is H-2Kb (i.e., the MHC surface molecule of the mouse is H-2Kb).

[0013] Furthermore, in the above method, the recipient mouse in A1) is a CB6F1 mouse, and the donor mouse is a C57BL / 6 mouse.

[0014] Furthermore, in the above method, the recipient mouse in A2) is a 129Sv mouse, and the donor mouse is a C57BL / 6 mouse.

[0015] Furthermore, the above method includes the following steps:

[0016] B1) Obtain mouse bone marrow cells;

[0017] B2) Treat the rats with sublethal doses of irradiation;

[0018] B3) Transplant the bone marrow cells obtained from B1) into the recipient mouse.

[0019] Furthermore, in the above method, B1) obtaining mouse bone marrow cells includes the following steps:

[0020] B1-1) Administer antibiotics to the rats;

[0021] B1-2) Obtain bone marrow cells from the femur and tibia of rats;

[0022] B1-3) After treating bone marrow cells with erythrocyte lysis buffer, the cell pellet was collected by centrifugation.

[0023] B1-4) Resuspend the cell pellet in DPBS solution to obtain mouse bone marrow cells.

[0024] Furthermore, in the above method, the sublethal dose irradiation includes the following steps: one day before transplantation, the recipient mouse is subjected to whole-body irradiation with 60Coγ in a γ-ray biological irradiator, with a total irradiation dose of 8Gy.

[0025] In one embodiment of the present invention, the sublethal dose irradiation is performed in two sessions, each with a dose of 4 Gy, and the interval between the two irradiations is 4 hours.

[0026] Furthermore, in the above method, B3) describes the transplantation method of transplanting the donor mouse bone marrow cells obtained in B1) into the recipient mouse via tail vein injection.

[0027] Furthermore, the above method further includes an evaluation method for the obtained mouse experimental model, the evaluation method comprising B4-1)-B4-3):

[0028] B4-1) Complete engraftment is defined as a rat bone marrow cell percentage exceeding 90%.

[0029] B4-2) Mixed implantation is performed when the proportion of donor mouse bone marrow cells is between 20% and 90%.

[0030] B4-3) Transplantation failure is defined as the proportion of donor mouse bone marrow cells being less than 20%.

[0031] Furthermore, in the above method, the proportion of donor bone marrow cells in the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model and the MHC fully identical allogeneic hematopoietic cell transplantation mouse model is higher than 90%.

[0032] Secondly, the present invention provides an application in which the mouse model prepared by the above method is used in any of the following:

[0033] C1) Application in the study of the mechanism of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation;

[0034] C2) Application in the preparation of products for evaluating the efficacy of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation.

[0035] Furthermore, in the above applications, the application described in C2) is any of the following:

[0036] C2-1) Application in the preparation of products for evaluating the reconstruction of immune cell subsets in peripheral blood of mouse models of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation;

[0037] C2-2) Application in the preparation of products for in vitro functional evaluation of T cells in mouse models of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation;

[0038] C2-3) Application in the preparation of products for evaluating the morphology and function of the thymus in mouse models of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation.

[0039] In this invention, both the donor and recipient mice can be 6-8 week old mice.

[0040] In this invention, both the donor and recipient mice can be female mice.

[0041] The beneficial effects of this invention are as follows:

[0042] This invention selects wild-type C57BL / 6 mice as donor mice for MHC haploidentical and MHC fully identified allogeneic hematopoietic cell transplantation, and CB6F1 mice as recipient mice for MHC haploidentical allogeneic hematopoietic cell transplantation. Simultaneously, wild-type 129Sv mice are selected as recipient mice for MHC fully identified hematopoietic cell transplantation, serving as a control for the haploidentical transplantation model. Modeling conditions are set, and the model establishment is evaluated through survival status, graft-versus-host disease, and rejection response observations, as well as flow cytometry and histopathological examination, to demonstrate the reliability and stability of this model. Attached Figure Description

[0043] Figure 1 This study describes the engraftment of peripheral blood cells from mice in each group 14 days after transplantation.

[0044] Figure 2 This study aimed to assess the reconstitution of peripheral blood immune cell subsets in each group of recipient mice at specified post-transplantation testing time points.

[0045] Figure 3 This study describes the functional reconstruction of spleen immune cells in each group of recipient mice after transplantation.

[0046] Figure 4 The results show the morphology and immunohistochemical results of the thymus in each group of transplanted mice. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] In the following examples, unless otherwise specified, the experiments were all repeated three times, and the results were averaged.

[0050] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0051] Streaming cytometry data were acquired and analyzed using Diva 7.0 software (BD Systems, USA). Statistical analysis and graphing were performed using SPSS 22.0 software (IBM SPSS Statistics, USA) and GraphPad Prism 7 (La Jolla CA, USA). Experimental results are expressed as mean ± SD. One-way ANOVA was used. P < 0.05 was considered statistically significant, P < 0.01 was considered statistically significant, and P < 0.001 was considered extremely significant.

[0052] Example 1: Method for establishing an experimental mouse model of MHC haploidentical allogeneic hematopoietic cell transplantation

[0053] 1.1 Laboratory Animals

[0054] All experimental mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the MHC molecules and CD45 gene subtypes of the experimental mice were verified to be correct.

[0055] The experimental animals were divided into two groups:

[0056] Group 1: MHC haplotype-matched mouse model construction mice, of which the donor mice were 6-8 week old SPF-grade female C57BL / 6 mice (H-2Kb, CD45.2) weighing 21-23g; the recipient mice were 6-8 week old SPF-grade female CB6F1 mice (H-2Kb / d, CD45.2);

[0057] Group 2: MHC fully homozygous mouse model constructed, with donor mice being 6-8 week old SPF-grade female C57BL / 6 mice (H-2Kb, CD45.1) weighing 21-23g; and recipient mice being 6-8 week old SPF-grade female 129Sv mice (H-2Kb, CD45.2) weighing 21-23g.

[0058] C57BL / 6 mice (H-2Kb, CD45.2) indicate that the MHC (major histocompatibility complex) molecule of this C57BL / 6 mouse is H-2Kb and carries the CD45.2 allele. CB6F1 mice (H-2Kb / d, CD45.2) indicate that the MHC molecule of this CB6F1 mouse is H-2Kb / d and carries the CD45.2 allele. C57BL / 6 mice (H-2Kb, CD45.1) indicate that the MHC molecule of this C57BL / 6 mouse is H-2Kb and carries the CD45.1 allele. 129Sv mice (H-2Kb, CD45.2) indicate that the MHC molecule of this 129Sv mouse is H-2Kb and carries the CD45.2 allele.

[0059] 1.2. Feeding mice with antibiotic water

[0060] One week prior to transplantation, CB6F1 mice and 129Sv mice were fed with sterile antibiotic solution as described in 1.1. The sterile antibiotic solution was a 0.5% penicillin-streptomycin mixture.

[0061] 1.3. Exposure to rat radiation

[0062] One day before transplantation, CB6F1 mice and 129Sv mice were irradiated whole-body in a 60Co γ-ray biological irradiator. The total irradiation dose was 8 Gy, which was completed in two sessions of 4 Gy each, with an interval of 4 hours.

[0063] 1.4. Procurement of mouse bone marrow cells

[0064] On the day of transplantation, two groups of donor mice, C57BL / 6 (H-2Kb, CD45.2) and C57BL / 6 (H-2Kb, CD45.1), were anesthetized and euthanized by cervical dislocation. The femurs and tibias were then separated. The femurs and tibias were placed in culture dishes, and bone marrow was expelled from the medullary cavity using a 1mL syringe filled with DPBS solution containing 2% FBS. This process was repeated several times to obtain a bone marrow cell suspension. The cell suspension was then filtered through a 70μm filter, and the collected filtrate was the donor mouse bone marrow cell suspension.

[0065] The DPBS solution containing 2% FBS consisted of 2% (v / v) fetal bovine serum, with the remainder being DPBS solution (excluding calcium, magnesium, and phenol red). The DPBS was purchased from Beijing Xigong Biotechnology Co., Ltd., catalog number CC010.

[0066] Centrifuge the mouse bone marrow cell suspension at 1500 rpm for 5 min and discard the supernatant. Add 1 mL of 1× erythrocyte lysis buffer to the cell suspension and incubate on ice for 8 min. Then centrifuge again to collect the cell pellet. Wash once more with DPBS and count the cells.

[0067] 1× Red blood cell lysis buffer was purchased from Beijing Solarbio Co., Ltd., product number R1010.

[0068] 1.5 Adjusting cell concentration and re-infusion

[0069] The concentration of donor mouse bone marrow cells was adjusted to 2.5 × 10⁻⁶ using sterile DPBS solution. 7 / mL. 200 μL of bone marrow cell suspension was injected into each of the irradiated CB6F1 and 129Sv recipient mice via the tail vein. The recipient mice were then fed again after the cell suspension injection, and the expression levels of H-2Kb and CD45.1 in the peripheral blood of the recipient mice after transplantation were detected by flow cytometry to assess the engraftment of the donor cells.

[0070] Example 2, Model Evaluation

[0071] 2.1 Evaluation of Implantation Efficiency in Mice After Transplantation

[0072] (1) Mouse data statistics

[0073] If a mouse dies within 5 days after transplantation, it is considered to be an irradiation-related death, and the mouse is removed from the group and no data is collected (the day of transplantation is defined as day 0 after transplantation).

[0074] (2) Starting 7 days after transplantation, 100 μL of blood was collected from the facial vein of the transplant recipient mice at 7, 14, 28, 45, 60, 90 and 120 days after transplantation. The expression levels of H-2Kb in the peripheral blood of CB6F1 and CD45.1 in the peripheral blood of 129Sv of the recipient mice were detected by flow cytometry to assess the engraftment of donor mice cells.

[0075] The testing was conducted in four groups:

[0076] Group 1: BV421-H-2Kb and PE-H-2Kd monoclonal antibodies were added to 10 test tubes of CB6F1 mouse venous blood;

[0077] Group 2: PE-CD45.1 and BV421-CD45.2 monoclonal antibodies were added to 129Sv mouse test tubes, for a total of 10 test tubes;

[0078] Group 3: CB6F1 mouse control tubes were supplemented with BV421-IG and PE-IG isotype monoclonal antibodies, totaling 10 test tubes;

[0079] Group 4: 10 test tubes containing PE-IG and BV421-IG isotype monoclonal antibodies were added to the 129Sv mouse control tubes.

[0080] The reaction was carried out at 4℃ in the dark for 30 min, followed by the addition of hemolysin to break down the red blood cells and incubation at room temperature in the dark for 20 min. The cells were washed twice with 1 mL of PBS buffer, resuspended in an appropriate amount of PBS, and then analyzed. The donor and recipient mice had different MHC class I antigen molecules. Donor mice (C57BL / 6) expressed H-2Kb or CD45.1. Pre-transplant bone marrow cells from recipient mice (CB6F1) expressed H-2Kb / d, while those from recipient mice (129Sv) expressed CD45.2. If bone marrow cells from donor mice (C57BL / 6) were successfully engrafted, peripheral blood cells from recipient mice (CB6F1) expressed H-2Kb, or peripheral blood cells from recipient mice (129Sv) expressed CD45.1.

[0081] The hemolysin was purchased from BD Company in the United States; product number: 555899.

[0082] BV421-H-2Kb monoclonal antibody was purchased from Biolegend; catalog number: 116525.

[0083] PE-H-2Kd monoclonal antibody was purchased from Biolegend; catalog number: 116608.

[0084] PE-CD45.1 monoclonal antibody was purchased from Biolegend; catalog number: 110708.

[0085] BV421-CD45.2 monoclonal antibody was purchased from Biolegend; catalog number: 109832.

[0086] The PE-IG isotype monoclonal antibody was purchased from Biolegend; catalog number: 400212.

[0087] BV421-IG isotype monoclonal antibody was purchased from Biolegend; catalog number: 400260.

[0088] Evaluation indicators:

[0089] 1. A positive rate of H-2Kb or CD45.1 >90% is defined as complete implantation;

[0090] 2. A positive rate of H-2Kb or CD45.1 <20% is defined as transplant failure;

[0091] 3. 20% to 90% are mixed implants.

[0092] A haploidentical MHC transplantation mouse model was established by transplanting C57BL / 6 (H-2Kb, CD45.2) bone marrow cells into CB6F1 mice (H-2Kb / d, CD45.2). A fully matched MHC transplantation mouse model was established by transplanting bone marrow cells from C57BL / 6 mice (H-2Kb, CD45.1) into 129Sv mice (H-2Kb, CD45.2), serving as a control for the haploidentical transplantation mouse model. The engraftment of donor cells after transplantation was detected by flow cytometry using H-2Kb and CD45.1 as markers. In CB6F1 mice, H-2Kb was used as a marker; for donor mice, the proportion of H-2Kb single-positive cells was greater than 90%, while for recipient mice, the proportion of H-2Kb and H-2Kd double-positive cells was greater than 90%. After transplantation, 129Sv mice were used as markers for CD45.1. If the mice were donor mice, the proportion of CD45.1 positive cells was greater than 90%, and if the mice were recipient mice, the proportion of CD45.2 positive cells was greater than 90%.

[0093] The results are as follows Figure 1 As shown, the chimerism rate of donor cells in all transplant recipient mice exceeded 90% 14 days post-transplantation and exhibited a stable and durable level of engraftment (defined as the percentage of donor-derived cells remaining almost unchanged with increasing recipient age within 120 days after alloHCT). None of the recipient mice developed graft-versus-host disease or rejection after transplantation and survived for more than 150 days (this experiment was repeated 6 times, with approximately 15-20 fully matched and haploidentical transplant recipients in each batch). The results indicate that the first group of CB6F1 mice became MHC haploidentical transplant recipients 14 days after transplantation, and the second group of 129Sv mice became MHC fully matched transplant recipients 14 days after transplantation.

[0094] 2.2 Flow cytometry detection of immune reconstitution in mouse peripheral blood

[0095] The content parameters of peripheral blood lymphocyte subsets in each group of mice at specified time points were determined by flow cytometry to evaluate the immune reconstitution status. The content parameters included γδT cells, NK cells, DC cells, CD3 T cells, CD4 T cells, and CD8 T cells.

[0096] (1) Starting 7 days after transplantation, 6 CB6F1 recipient mice and 6 129Sv recipient mice were randomly selected at 7, 14, 28, 45, 60, 90 and 120 days after transplantation and 100 μL of blood was collected from the facial vein. The day of transplantation was defined as day 0 after transplantation.

[0097] (2) Collect 1×10 whole blood 6 Cells to sterile flow cytometry tubes.

[0098] (3) Add 1 μL of surface flow cytometry antibody to each whole blood sample, gently mix and incubate at room temperature in the dark for 15 min.

[0099] (4) Add 2 mL of red blood cell lysis working solution to each flow cytometer, shake well for 10 seconds, and let stand at room temperature in the dark for 8 minutes.

[0100] (5) After centrifuging at 1300 rpm for 5 min and removing the supernatant, add 2 mL of PBS, centrifuge at 1500 rpm for 5 min and remove the supernatant.

[0101] (6) Surface staining of γδT cells, NK cells, DC cells, CD3 T cells, CD4 T cells and CD8 T cells was performed. After completing step (5), the cells were resuspended in 200 μL PBS and detected by flow cytometry.

[0102] The surface flow cytometry antibodies used and their sources of purchase are as follows:

[0103] Alexa 647-TCRγ / δ was purchased from Biolegend, item number: 116525;

[0104] Brilliant Violet 510 TM -NK-1.1 purchased from Biolegend, item number: 108738;

[0105] Brilliant Violet 650 TM -CD11c was purchased from Biolegend, item number: 117339;

[0106] FITC-CD3 purchased from Biolegend, item number: 100204;

[0107] PerCP / Cyanine5.5-CD4 was purchased from Biolegend, item number: 116012;

[0108] Brilliant Violet 510 TM -CD8 was purchased from Biolegend, item number: 100752;

[0109] Red blood cell lysis buffer was purchased from BD Laboratories, USA, catalog number: 555899.

[0110] The reconstitution of immune cell subsets in the peripheral blood of recipient mice was detected by flow cytometry at specific time points post-transplantation (7, 14, 28, 45, 60, 90, and 120 days post-transplantation). Results are as follows: Figure 2 As shown, Figure 2 (A) represents the proportion of γδT cells in PB. Figure 2(B) represents the proportion of NK cells in PB. Figure 2 (C) represents the proportion of DC cells in PB. Figure 2 (D) represents the proportion of CD3 T cells in PB. Figure 2 (E) represents the proportion of CD4 T cells in PB. Figure 2 (F) represents the proportion of CD8 T cells in PB. Figure 2 (G) is CD4 + / CD8 + In MHC haploidentical recipients, the reconstitution levels of innate immune cell subsets, such as NK cells, were lower than those in fully matched recipients. γδ T cell reconstitution levels were similar, while dendritic cell (DC) reconstitution levels were higher. The adaptive immune cell subset CD3 T cells were higher initially after transplantation but were subsequently surpassed by MHC fully matched recipients, while CD4 T cell reconstitution levels were similar to those in CD4+ recipients. + / CD8 + The T cell ratio was lower in recipient mice than in MHC-matched mice. These results indicate that MHC incompatibility leads to delayed T cell immune reconstitution in transplanted mice.

[0111] 2.3 Isolation of mouse spleen cells and in vitro detection of T cell function

[0112] (1) Isolation of mouse spleen cells

[0113] Six CB6F1 recipient mice and six 129Sv recipient mice were euthanized 45 days after transplantation.

[0114] ① Under aseptic conditions, the spleen was harvested and its membrane was removed. A 70μm filter was placed on a petri dish, and a small amount of 1×PBS was added (to ensure that the spleen and the obtained cells were in a liquid environment).

[0115] ② Place the spleen on a sieve and grind it using a syringe plunger (control the grinding force, keep the sieve suspended, and avoid grinding directly on the bottom of the dish, which could cause a large number of cells to die).

[0116] ③ After complete grinding, rinse the sieve with 1×PBS, collect the cell suspension, filter it through a filter, collect the filtrate, centrifuge at 2000 rpm for 10 min, and then discard the supernatant.

[0117] ④ Add 5 mL of red blood cell lysis buffer to resuspend the cells and lyse on ice for 5 min.

[0118] ⑤ Add 10 mL of pre-cooled 1×PBS, centrifuge at 1500 rpm for 5 min, and wash twice.

[0119] ⑥ Resuspend in 1×PBS and adjust the cell concentration to 1×10⁻⁶. 7 / mL.

[0120] (2) Treatment of mouse spleen cells with concanavalin A (hereinafter referred to as ConA)

[0121] Take a 96-well round-bottom cell culture plate and add 199 μL of complete culture medium + 1 μL of ConA working solution to each well to make the final ConA concentration 5 μg / ml (200-fold dilution). Incubate at 37°C in a 5% CO2 incubator.

[0122] (3) Detect the proliferation level of spleen T cells and the secretion of cytokines.

[0123] After 42 hours of culture, a protein transport inhibitor (a mixture of brevidin A and monensin) was added to the culture system. Six hours later, cells and their supernatant were collected, and flow cytometry was used to detect the proliferation levels (Ki67) of CD3 T cells, B cells, NK cells, and dendritic cells, as well as the production of cytokines (TNF-α, CD107a).

[0124] The specific method for detecting the proliferation (Ki67) levels and cytokine (TNF-α, CD107a) production of CD3 T cells, B cells, NK cells, and dendritic cells by flow cytometry is as follows: ① Stain the surface of B cells, CD3 T cells, NK cells, and dendritic cells, centrifuge at 1300 rpm for 5 min, remove the supernatant, then add 100 μL of fixative solution A, gently mix, and incubate at room temperature in the dark for 15 min. Add 2 mL of PBS, centrifuge at 1500 rpm for 5 min, and remove the supernatant. ② Add 100 μL of fixative solution B and 1 μL of intracellular flow cytometry antibody, gently mix, and incubate at room temperature in the dark for 15 min. ③ Add 2 mL of PBS, centrifuge at 1500 rpm for 5 min, remove the supernatant, resuspend the cells in 200 μL of PBS, and then perform flow cytometry analysis.

[0125] The types of intracellular flow cytometry antibodies and their sources of purchase are as follows:

[0126] The APC-Ki67 was purchased from Biolegend, part number: 652405;

[0127] PE-DAZZLE594-TNF-α was purchased from Biolegend, product number: 506346;

[0128] BV711-CD107a was purchased from Biolegend, item number: 121631;

[0129] The film-breaking agent was purchased from BD Company, USA, product number: 554714;

[0130] Concha pea lectin A was purchased from Sigma, product number: C5275;

[0131] The protein transport blocker was purchased from eBioscience, Inc., USA, catalog number: C5275.

[0132] The results are as follows Figure 3 As shown, B cells (CD19) in the spleen of MHC-fully-matched mice + The levels of CD107a and TNF-α secreted by CD3 T cells, NK cells, and pDCs were higher in mice than in MHC haplotype-matched mice (P<0.001). Figure 3 In mice with AD and FI), the levels of CD107a and TNF-α secreted by mDCs were lower than those in MHC haplotype-matched mice (P<0.001). Figure 3 (E and J). Furthermore, this study also examined the proliferation capacity of immune cells in the two groups. The results showed that the proliferation levels of spleen B cells, CD3 T cells, NK cells, and pDCs in MHC fully homozygous mice were also higher than those in MHC haploidentical mice (P<0.001). Figure 3 In mice with high mDC proliferation (MHC haplotype-associated mice), except that the proliferation level of mDCs was lower than that of MHC haplotype-associated mice (P<0.001), Figure 3 These results indicate that MHC haploidentical mice had impaired immune cell function compared to MHC fully identical mice.

[0133] 2.4. Histopathological examination of mouse tissues

[0134] Forty-five days post-transplantation, two CB6F1 recipient mice and two 129Sv recipient mice were sacrificed. Thymus tissue was harvested from the mice after sacrifice and fixed at room temperature in a tissue fixative (4% paraformaldehyde) for at least 24 hours. The fixed samples were dehydrated, embedded in paraffin, and sectioned to prepare 4μm thick paraffin sections. Hematoxylin-eosin (H&E) staining was performed, and the thymus tissue structure (the junction of the thymic cortex and medulla; the number of thymocytes and adipocytes) was observed under a microscope. Immunohistochemical staining (anti-mouse anti-FoxN1, anti-mouse anti-EpCAM, and anti-mouse anti-IL22BP) was also performed, and image analysis was conducted using the HPIAS.1000H high-resolution color pathological image measurement system.

[0135] The H&E staining method is as follows:

[0136] ① Dewaxing of paraffin sections: Immerse the paraffin sections in xylene for 20 minutes, replace with fresh xylene for 20 minutes, replace with anhydrous ethanol for 10 minutes, then immerse in anhydrous ethanol for 10 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and finally soak in double-distilled water for 5 minutes.

[0137] ② HE staining: After dewaxing, add hematoxylin staining solution to stain for 3-8 minutes, soak in tap water for 10 minutes to wash away excess staining solution, then soak the paraffin sections in 1% hydrochloric acid ethanol for 3 seconds, soak in tap water for 1 minute, then turn blue with 0.6% ammonia water, soak in tap water for 1 minute, and stain with eosin solution for 1-3 minutes.

[0138] ③ Dehydration, clearing, and mounting: Immerse the stained paraffin sections in 95% ethanol for 5 min x 2 times, anhydrous ethanol for 5 min x 2 times, and xylene for 5 min x 2 times in sequence. After slightly drying, mount with neutral resin and store at room temperature for microscopic examination.

[0139] The immunohistochemical staining method is as follows:

[0140] ① Place paraffin sections in fresh xylene and soak for 10 min × 3 times; after removing excess liquid, soak in anhydrous ethanol for 3 min × 3 times; after removing excess liquid, soak in 95% ethanol for 3 min × 2 times; after removing excess liquid, soak in 75% ethanol for 3 min × 2 times; rinse with distilled water for 1 min, then place in PBS buffer. ② Antigen retrieval. ③ Add an appropriate amount of endogenous peroxidase inhibitor and incubate at room temperature for 10 min; rinse with PBS buffer for 3 min × 3 times. ④ Depending on the tissue size, add 100 μL or an appropriate amount of primary antibody and incubate at 37℃ for 60 min; rinse with PBS buffer for 3 min × 3 times. ⑤ Add 100 μL or an appropriate amount of reaction enhancement solution and incubate at 37℃ for 20 min; rinse with PBS buffer for 3 min × 3 times. ⑥ Add 100 μL or an appropriate amount of enzyme-labeled goat anti-mouse / rabbit IgG polymer and incubate at 37℃ for 20 min; rinse with PBS buffer for 3 min × 3 times. ⑦ Add an appropriate amount of freshly prepared DAB staining solution and incubate at room temperature for 5-8 minutes. ⑧ Rinse with tap water, incubate with hematoxylin staining solution for 20 seconds; differentiate, rinse, and return to blue. ⑨ Dehydrate, clear, and mount. ⑩ Read the slide.

[0141] The primary antibodies used in the immunoassay combination staining and their sources of purchase are as follows:

[0142] ① The anti-FoxN1 was purchased from Bioss, product number: bs-6970R;

[0143] ② The anti-epCAM for rodents was purchased from Bioss, product number: bs-0593R;

[0144] ③ The anti-mouse anti-IL22BP was purchased from Bioss, product number: bs-2625R;

[0145] ④ Immunohistochemical secondary antibody: Universal two-step kit (mouse / rabbit enhanced polymer assay system) purchased from Zhongshan Jinqiao, catalog number: PV-9000. Main components: Reagent 1: endogenous peroxidase inhibitor reagent, 2: reaction enhancement solution, Reagent 3: enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0146] The results are as follows Figure 4 As shown, Figure 4 Image (A) shows the results of H&E staining. The upper image in (A) shows the junction of the thymic cortex and medulla, while the lower image shows the number of thymic cells and adipocytes. Figure 4 In the middle (B)-(D), the immunohistochemical staining results of FoxN1, EpCAM and IL22 in the thymus of transplanted mice are shown respectively. Figure 4 The results showed that, compared to fully matched recipients, the thymus morphology of haploidentical MHC recipients was incomplete and underdeveloped. Furthermore, the expression of FoxN1 and EpCAM, markers of thymic epithelial cells, was decreased in the thymus. The expression of IL22, which can signal thymic epithelial cells and promote their proliferation and survival, was also reduced in haploidentical MHC recipients. These results indicate that MHC incompatibility leads to thymic structural damage and delayed immune cell reconstitution in recipients after HCT. This finding is consistent with the delayed immune reconstitution observed in clinical transplant recipients due to MHC incompatibility and suggests that the thymus plays a role in this process.

[0147] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of mouse models in the preparation of products for evaluating the transplantation efficacy of MHC fully matched and / or haploidentical allogeneic hematopoietic cell transplantation; the transplantation efficacy includes the reconstruction of immune cell subsets in peripheral blood of mouse models, in vitro T cell function, and thymus function; The mouse models include the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model and the MHC fully identical allogeneic hematopoietic cell transplantation mouse model. The method for preparing the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model includes transplanting bone marrow cells from a donor mouse into a recipient mouse irradiated with a sublethal dose to obtain the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model, wherein the donor mouse and the recipient mouse are MHC haploidentical and have the same CD45 molecular subtype. The method for preparing the MHC-identical allogeneic hematopoietic cell transplantation mouse model includes transplanting bone marrow cells from a donor mouse into a recipient mouse irradiated with a sublethal dose to obtain the MHC-identical allogeneic hematopoietic cell transplantation mouse model. The donor mouse and the recipient mouse are MHC-identical, but have different CD45 molecular subtypes. The preparation method includes the following steps: B1) Obtaining mouse bone marrow cells; B2) Treat the rats with sublethal doses of irradiation; B3) Transplant the bone marrow cells obtained from B1) into the recipient mouse; in, B1) The process of obtaining mouse bone marrow cells includes the following steps: (1) Obtain bone marrow cells from the femur and tibia of rats; (2) After treating the bone marrow cells with red blood cell lysis buffer, the cell pellet was collected by centrifugation. (3) Resuspend the cell pellet in DPBS solution to obtain mouse bone marrow cells; Step (1) Bone marrow is blown out of the bone marrow cavity using a DPBS solution containing 2% fetal bovine serum by volume.

2. The application according to claim 1, characterized in that: The MHC molecules in the donor mice of the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model were H-2Kb, and the MHC molecules in the recipient mice were H-2Kb / d. The MHC molecules in the donor mice of the MHC-fully-identified allogeneic hematopoietic cell transplantation mouse model were H-2Kb, and the MHC molecules in the recipient mice were H-2Kb.

3. The application according to claim 2, characterized in that: The recipient mice in the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model were CB6F1 mice, and the donor mice were C57BL / 6 mice. The recipient mice in the MHC fully matched allogeneic hematopoietic cell transplantation mouse model were 129Sv mice, and the donor mice were C57BL / 6 mice.

4. The application according to claim 1, characterized in that: The sublethal dose irradiation includes the following steps: the recipient mouse is irradiated with 60Co γ-rays in a γ-ray biological irradiator one day before transplantation, with a total irradiation dose of 8 Gy.

5. The application according to claim 1, characterized in that: As described in B3), the transplantation method for transplanting the donor mouse bone marrow cells obtained in B1) into the recipient mouse is tail vein injection.

6. The application according to any one of claims 1-5, characterized in that: The proportion of donor bone marrow cells in the MHC haploidentical allogeneic hematopoietic cell transplantation mouse model and the MHC fully identical allogeneic hematopoietic cell transplantation mouse model is higher than 90%.

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