A method for constructing a scleroderma model of pbmc humanized mice

Abstract

This invention discloses a method for constructing a PBMC-derived humanized mouse scleroderma model. Human peripheral blood mononuclear cells are transplanted into immunodeficient mice, and a human immune system is reconstructed in the mice. Bleomycin is injected into the local skin of the mice in a round-point manner to induce fibrosis and form a scleroderma model. This application can more comprehensively simulate the complex pathological process of immune abnormalities and tissue fibrosis coexisting in human scleroderma, and provides a more realistic and systematic experimental platform for in-depth exploration of the immune mechanism of this disease.

Description

Technical Field

[0001] This invention relates to the field of animal disease model construction technology, and in particular to a method for constructing a PBMC humanized mouse scleroderma model. Background Technology

[0002] Scleroderma (systemic sclerosis) is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. Its pathogenesis is not fully understood, and clinical treatment options are limited. In preclinical research on scleroderma, animal models are crucial tools for exploring pathological mechanisms and evaluating the efficacy of new drugs. Currently, bleomycin-induced mouse skin fibrosis models are among the most commonly used scleroderma models. This model, achieved through repeated subcutaneous injections of bleomycin, induces fibrosis-like changes such as inflammatory cell infiltration, increased collagen deposition, and skin thickening at the injection site. However, traditional bleomycin models have two inherent limitations that severely restrict their application in scleroderma research.

[0003] 1. Inappropriate injection methods lead to poor model reliability: Traditional bleomycin models often employ single-site repeated injections, meaning bleomycin is injected daily at the same fixed location throughout the modeling period. Due to bleomycin's strong local toxicity, long-term, repeated injections at the same site can cause drug accumulation exceeding the tissue's tolerance limit, inducing nonspecific tissue necrosis, induration, blisters, and even ulceration. Tissue necrosis triggers a secondary inflammatory response, resulting in multiple pathological changes at the injection site, including fibrosis, necrosis, and reparative inflammation. This leads to inaccurate skin thickness measurements (thinning in necrotic areas and thickening in inflammatory edema areas do not accurately reflect the degree of fibrosis) and complicates the model's pathological mechanisms, making it difficult to distinguish whether observed changes originate from the fibrotic process of scleroderma itself or from nonspecific reactions caused by direct drug damage. Furthermore, local necrosis easily leads to mouse intolerance, weight loss, and even death, resulting in low modeling success rates, large intragroup variability, and difficulty in reproducing experimental results.

[0004] 2. Inappropriate mouse strain selection fails to meet the needs of scleroderma immunopathological research. One of the core characteristics of scleroderma is abnormal activation of the immune system. Numerous studies have shown that immune cells such as T cells, B cells, and monocytes play a crucial role in the initiation and progression of fibrosis. However, traditional bleomycin models typically use mice with normal immune systems, whose immune systems differ significantly from those of humans, and cannot mimic the pathogenic function of specific immune cell subsets in human scleroderma. More importantly, these models cannot be used to evaluate novel drugs targeting human immune cells because these drugs only recognize human targets and have no cross-reactivity with mouse targets. Although "humanized" mouse models constructed by transplanting human peripheral blood mononuclear cells (PBMCs) into immunodeficient mice have emerged in recent years, existing humanized scleroderma models mainly rely on PBMC transplantation itself to induce systemic inflammation, lacking specific induction of local fibrosis, and thus failing to simulate the core clinical manifestations of skin fibrosis in scleroderma. In addition, different immunodeficient mouse strains vary greatly in their ability to support human PBMC implantation and long-term survival. Common strains (such as nude mice) have short human cell survival time (only 2-4 weeks) due to residual NK cell activity, which cannot meet the requirements of a complete experimental cycle of continuous bleomycin induction (usually requiring 21-35 days) plus subsequent observation.

[0005] In summary, existing animal models of scleroderma have significant shortcomings in both the reliability of local fibrosis induction and the long-term stable reconstruction of the human immune system. There is an urgent need to develop an improved model that can realistically simulate skin fibrosis and efficiently support the long-term survival of human immune cells. Summary of the Invention

[0006] To overcome the aforementioned deficiencies of existing technologies, this patent provides a method for constructing a PBMC humanized mouse scleroderma model. This method can effectively address the problems that existing scleroderma animal models lack the participation of a functional human immune system, thus failing to realistically simulate the immunopathological characteristics of scleroderma as an autoimmune disease, and have significant limitations in evaluating novel therapies targeting human immune cells or signaling pathways.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for constructing a PBMC-derived humanized mouse scleroderma model involves transplanting human peripheral blood mononuclear cells into immunodeficient mice and reconstructing the human immune system within the mice; bleomycin is administered to the local skin of the mice via spot injections to induce fibrosis, thus forming a scleroderma model.

[0008] In a preferred embodiment, the immunodeficient mouse is a female BRGSF strain mouse, aged 6-10 weeks.

[0009] In a preferred embodiment, the peripheral blood mononuclear cells are derived from healthy individuals or patients with scleroderma.

[0010] In a preferred embodiment, on day N post-transplantation, the chimerism rate and number of human T cells and / or B cells in the mouse are detected. When the chimerism rate of human T cells and / or B cells in the mouse is greater than a threshold, it is determined that humanized immune reconstitution is successful.

[0011] In a preferred embodiment, bleomycin is injected subcutaneously at at least one injection site in a fixed skin area at time intervals M.

[0012] In a preferred embodiment, the fixed skin area is a marked area after shaving the back, and there are multiple injection sites located at the edge of the marked area. The multiple sites are evenly distributed at the edge of the marked area, and one site is selected for injection each day in turn.

[0013] In a preferred embodiment, the marking area is circular or rectangular, and there are four injection points located at the four corners of the square marking area or on the circumference of the circular marking area.

[0014] In a preferred embodiment, the duration of continuous administration of bleomycin is 21 hours. 35 days.

[0015] In a preferred embodiment, 9. the method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that the bleomycin is bleomycin phosphate buffer with a concentration of 100... 300 μg / mL, preferably 200 μg / mL, with an injection volume of 0.05 μg / mL. 0.15 mL, preferably 0.1 mL.

[0016] In a preferred embodiment, the scleroderma model is validated by detecting skin thickness, tissue fibrosis, inflammatory cell infiltration, and / or collagen deposition status in model mice.

[0017] The technical effects and advantages of this invention are as follows: 1. This application adopts a rotating injection method, which sets multiple evenly distributed injection sites within a fixed skin marking area and selects different sites in turn for subcutaneous injection. This reduces the impact of the injection itself on skin lesions, makes the skin thickness measurement results truly reflect the degree of fibrosis, provides a clear interpretation of the pathological mechanism, has good tolerance in mice, significantly improves the success rate of modeling, greatly reduces the coefficient of variation within groups, and the experimental results have excellent reproducibility.

[0018] 2. This application selects BRGSF strain immunodeficient mice as recipients to reduce xenogeneic rejection and extend the survival time of human immune cells from 2-4 weeks to 8-12 weeks, enabling efficient immune reconstitution and fully covering the entire cycle of bleomycin injection for 21-35 days and subsequent observation, providing a sufficient experimental window for dynamic monitoring of the role of human immune cells in the fibrosis process.

[0019] 3. This application addresses the unreliability of local fibrosis induction through "rotational injection" and the issue of long-term reconstruction of the human immune system through the "BRGSF strain." Together, they construct a novel animal model that can realistically simulate the fibrosis process of scleroderma and support the long-term participation of human immune cells, providing an ideal preclinical experimental platform for the study of the immune mechanism of scleroderma and the development of drugs targeting human immune cells. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the method flow of this application; Figure 2 This is a schematic diagram of the change curve of skin thickness in the modeling area of ​​the mouse scleroderma model obtained by the method of this application. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] like Figure 1 , Figure 2 As shown, the method for constructing a humanized mouse scleroderma model using PBMCs involves transplanting human peripheral blood mononuclear cells into immunodeficient mice and reconstructing a human immune system within the mice. The immunodeficient mice are female BRGSF strain mice, aged 6-10 weeks. Immunodeficient mice lack functional T cells, B cells, and / or NK cells, and cannot recognize and eliminate xenogeneic human PBMCs. This method enables efficient implantation and long-term survival of human PBMCs, providing a sufficient experimental window (28 days of induction + subsequent observation) for observing the dynamic participation of human immune cells in the bleomycin-induced fibrosis process. This is a technical effect that is difficult to achieve with other immunodeficient strains (such as nude mice) or immunosuppressive regimens.

[0023] Peripheral blood mononuclear cells are derived from healthy individuals or scleroderma patients. In this embodiment, cells from scleroderma patients are preferred. More specifically, cells from scleroderma patients who have not received immunosuppressive therapy are used. By using PBMCs from scleroderma patients, these disease-specific immune abnormalities are introduced into a mouse model to simulate the immunopathological characteristics of scleroderma.

[0024] On day N after transplantation, the chimerism rate and number of human T cells and / or B cells in mice are detected. In this embodiment, day 14 is preferred. Then, the chimerism rate is used to distinguish which mice have successfully completed the construction of the human immune system. The mice that have successfully completed the construction are classified / stratified by the number of mice to balance the differences between groups in subsequent experiments.

[0025] Bleomycin was administered to the local skin of mice to induce fibrosis via a rotating injection method. Bleomycin was subcutaneously injected at least one injection site in the fixed skin area at intervals M. In this embodiment, the interval was 24 hours. After the interval, the fixed skin area was the marked area after shaving the back. There were multiple injection sites located at the edge of the marked area. The multiple sites were evenly distributed at the edge of the marked area. One site was selected for injection each day in rotation. The marked area was circular or square, and there were four injection sites. The four injection sites were located at the four corners of the square marked area or on the circumference of the circular marked area. In this embodiment, the marked area was preferably rectangular, and the four corners of the rectangle were used as injection sites.

[0026] In this application, point-by-point injection refers to a procedure in which multiple predetermined injection sites within a fixed skin marking area are selected sequentially (e.g., rotating daily) for subcutaneous injection. This method differs from continuous injection at the same site, thus avoiding local tissue necrosis and achieving a uniform distribution of fibrosis within the marked area.

[0027] In the technical field described in this application, single-site injection is commonly used. However, long-term single-site injection can easily cause non-specific tissue necrosis and severe inflammation, which is a serious drawback in the construction of scleroderma models. It directly affects the measurement of core indicators, leads to model instability, and hinders the clinical translation and evaluation of drugs. The site rotation strategy adopted in this application, through uniform drug administration and reduced local damage, constructs a more stable, pure, and realistic disease model platform, laying a solid foundation for subsequent drug screening and mechanism research.

[0028] Bleomycin 21 was administered continuously. A scleroderma model was established after 35 days. Bleomycin was administered as bleomycin phosphate buffer at a concentration of 100%. 300 μg / mL, preferably 200 μg / mL, with an injection volume of 0.05 μg / mL. 0.15 mL, preferably 0.1 mL, is used to validate the scleroderma model by detecting skin thickness, tissue fibrosis, inflammatory cell infiltration, and / or collagen deposition status in model mice.

[0029] Example 1 like Figure 1 As shown, a method for constructing a PBMC humanized mouse scleroderma model includes the following steps: Material preparation Laboratory animals: Purchase 30 animals of 6 years old 8 weeks old, weighing 18 20g of female BRGSF immunodeficient mice were acclimatized for one week in an SPF-grade environment.

[0030] PBMC separation: 20 mL of peripheral blood was collected from each of three healthy volunteers and anticoagulated with heparin. Ficoll was used. PBMCs were isolated using PaquePLUS density gradient centrifugation, washed twice with PBS, and finally resuspended in PBS containing 2% fetal bovine serum to adjust the cell concentration to 1×10⁻⁶. 8 cells / mL, keep on ice until ready to use.

[0031] Reagents: Bleomycin hydrochloride, sterile phosphate buffer, isoflurane, mouse shaver, sterile marker pen, 1mL insulin syringe, paraformaldehyde, paraffin, hematoxylin Eosin staining kit, Masson's trichrome staining kit, anti-human CD45 FITC, Anti-human CD3 PE, Anti-human CD19 APC flow cytometry antibody, anti- α SMA antibody, anti Collagen I antibody, etc.

[0032] Construction of a humanized mouse model of PBMC Cell transplantation: Thirty mice were randomly divided into a model group and a control group. In the model group, mice were anesthetized with isoflurane inhalation and then slowly injected via the tail vein with 100 μL of LPBMC suspension containing 1 × 10⁻⁶ cells / mL. 7 Each cell. Control group mice were injected with an equal volume of PBS.

[0033] Human immune reconstitution monitoring and grouping: On day 14 post-transplantation, approximately 50 μL of blood was collected from all mice via the tail vein, and total human CD45 in peripheral blood was detected by flow cytometry. + White blood cells, CD3 + T cells and CD19 + Percentage of B cells. Human CD45 was selected. +Mice in the model group with a cell chimerism rate greater than 15% proceeded to the next stage and were re-randomized according to their chimerism rate into a model induction group and a model control group. The original PBS control group remained unchanged.

[0034] Induction of scleroderma model Region marking: On day 28 post-transplantation, the backs of all mice in the model induction group, model control group, and PBS control group were shaved. Using a sterile marking pen, a 1cm × 1cm square area was marked on the shaved skin in the center of the back of each mouse. The four vertices of this square were clearly marked as injection sites A, B, C, and D.

[0035] Bleomycin injection: A bleomycin solution with a concentration of 200 μg / mL was prepared using sterile PBS. Starting from day 1, mice in the model induction group were injected subcutaneously daily: following the order A→B→C→D, a horn site was selected each day, and 0.1 mL of bleomycin solution was slowly injected subcutaneously using an insulin syringe inserted vertically into that site, resulting in a daily dose of 20 μg bleomycin per mouse. Mice in the model control group and PBS control group were injected with the same volume of sterile PBS at the same site. Injections were given once daily for 28 consecutive days.

[0036] Model Validation The model was evaluated 48 hours after the last injection.

[0037] Skin thickness measurement: Using digital vernier calipers, the skin fold thickness at the center point of the marked square area on the back of each group of mice was measured blindly by the same operator. Three measurements were taken, and the average value was recorded. Results are as follows: Figure 1 As shown, the skin thickness of the model-induced group (corresponding to curve 3) increased significantly from D7, reaching a peak at D28, and was significantly thicker than that of the model control group (corresponding to curve 4) and the PBS control group (the trend was consistent with curve 4); Sample Collection and Processing: After measurement, mice were euthanized, and full-thickness skin tissue from the marked area was completely excised. One portion was immediately fixed in 4% paraformaldehyde for 24 hours, followed by paraffin embedding and sectioning. The other portion was rapidly frozen. 80℃.

[0038] Histopathological analysis: H&E staining: Paraffin sections were stained with H&E and observed under a light microscope. The dermis of mice in the model-induced group showed significant thickening, with diffuse infiltration of numerous inflammatory cells such as lymphocytes and monocytes. The model control group and PBS control group showed only mild or no inflammatory cell infiltration.

[0039] Masson trichrome staining: Masson staining showed that the density of collagen fibers in the dermis of mice in the model-induced group was significantly increased and the arrangement was disordered, while the collagen fibers in the control group were fine and neatly arranged.

[0040] Immunohistochemical analysis: Immunohistochemical staining was performed on paraffin sections. Anti-... α SMA antibody staining showed that α-matrix was present in the dermis of the model-induced group. The number of SMA-positive myofibroblasts was significantly higher than that in the control group. (Using anti-) CollagenI antibody staining showed that the deposition area and intensity of CollagenI in the dermis of the model-induced group were significantly higher than those of the control group.

[0041] Molecular biological assays: Total RNA was extracted from frozen skin tissue, reverse transcribed, and then analyzed using qRT. PCR was used to detect the expression of fibrosis-related genes. The results showed that the mRNA expression levels of ACTA2, COL1A1, and TGFB1 in the skin tissue of the model-induced group were significantly upregulated, at 3.5-fold, 4.2-fold, and 2.8-fold respectively compared with the model control group.

[0042] Example 2 The PBMCs were derived from three scleroderma patients who had not received immunosuppressive therapy, and the rest were the same as in Example 1.

[0043] Compared with Example 1, the mouse PBMC group showed a more significant increase in skin thickness, human autoantibodies were detectable in serum, and more human CD3 was infiltrated in the skin tissue. + T cells.

[0044] Comparative Example 1 Single-point injection was used, and everything else was the same as in Example 1.

[0045] Comparative Example 2 Single-point injection was used, and everything else was the same as in Example 2.

[0046] The single-point injection sites of mice in Comparative Examples 1 and 2 were examined, and it was found that significant skin necrosis and large variations in skin thickness were observed in Comparative Examples 1 and 2 after single-point injection. No necrosis was observed in the round-point injection group, and the fibrosis was uniform.

[0047] Comparative Example 3 This embodiment uses a nude mouse; all other aspects are the same as in Embodiment 2.

[0048] On day 21, the proportion of human cells in nude mice decreased to <5%, and most could not complete the 28-day induction; on day 28, there was still about 10% chimerism, but it was lower than that of BRGSF; the chimerism rate of the BRGSF group was stable at >15% until day 42.

[0049] A humanized mouse model of scleroderma using PBMCs was successfully constructed using the method described in this application. This model not only exhibits typical fibrotic features such as skin thickening and excessive collagen deposition, but also shows significant inflammatory infiltration mediated by human immune cells and high expression of fibrosis-related molecular markers, comprehensively mimicking the core pathological changes of human scleroderma. The method used to construct this model is stable and reliable, and is suitable for studying the pathogenesis of scleroderma and for preclinical evaluation of targeted drugs.

[0050] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0051] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a PBMC humanized mouse scleroderma model, characterized in that, Human peripheral blood mononuclear cells were transplanted into immunodeficient mice to reconstruct the human immune system in the mice; bleomycin was administered to the local skin of the mice via spot injection to induce fibrosis, thus creating a scleroderma model.

2. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, The immunodeficient mice were female BRGSF strain mice, aged 6-10 weeks.

3. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, The peripheral blood mononuclear cells were derived from healthy individuals or patients with scleroderma.

4. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, On day N after transplantation, the chimerism rate and number of human T cells and / or B cells in mice were measured. When the chimerism rate of human T cells and / or B cells in mice was greater than the threshold, humanized immune reconstitution was considered successful.

5. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, Bleomycin was administered subcutaneously at at least one injection site in a fixed skin area at time interval M.

6. The method for constructing a PBMC humanized mouse scleroderma model according to claim 5, characterized in that, The fixed skin area is the marked area after shaving the back. There are multiple injection sites located at the edge of the marked area. The multiple sites are evenly distributed at the edge of the marked area, and one site is selected for injection each day in turn.

7. The method for constructing a PBMC humanized mouse scleroderma model according to claim 6, characterized in that, The marking area is circular or rectangular, and there are four injection points located at the four corners of the square marking area or on the circumference of the circular marking area.

8. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, The duration of continuous administration of bleomycin was 21 hours. 35 days.

9. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, The bleomycin mentioned is bleomycin phosphate buffer with a concentration of 100%. 300 μg / mL, preferably 200 μg / mL, with an injection volume of 0.05 μg / mL. 0.15 mL, preferably 0.1 mL.

10. The method for constructing a PBMC humanized mouse scleroderma model according to claim 1, characterized in that, The scleroderma model was validated by detecting skin thickness, tissue fibrosis, inflammatory cell infiltration, and / or collagen deposition status in model mice.

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