A method for constructing a kidney humanized mouse model

Abstract

The present application belongs to the field of biomedical technology, and relates to a construction method of an experimental animal model and a constructed experimental model, in particular to a method for constructing a kidney humanized mouse model based on human cells and a constructed kidney humanized mouse model. The construction method comprises the following steps: injecting mesenchymal stem cells, human umbilical vein endothelial cells and human renal tubular epithelial cells separated from urine into immunodeficient mice subcutaneously or subcapsularly to obtain a humanized kidney vascular unit mouse model communicating with the host. The model construction has the following characteristics: the cells used are derived from urine, have the characteristics of sufficient clinical source and convenient sampling, and the kidney source cells derived from urine can represent a unique individual alone, can be used to establish an individualized kidney humanized mouse model based on patients, and have the potential to be applied to individualized precision medical research; the model can form a functional human kidney vascular unit in mice, and has great application prospects in individualized precision medical treatment of kidney disease and human kidney specific virus infection research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and relates to a method for constructing an experimental animal model and the constructed experimental model, in particular to a method for constructing a kidney humanized mouse model based on human cells and the constructed kidney humanized mouse model. BACKGROUND

[0002] It is well known in the field of biomedical technology that animal models are essential for studying the pathogenesis of diseases, developing vaccines and drug screening, and are indispensable tools for preclinical research. In research practice, small animals such as mice and rats are widely used due to their small size, easy maintenance and operation, short breeding cycle, and easy genetic manipulation. However, due to the differences in physiological structure and metabolic system between animals and humans, traditional animal models often cannot accurately reflect the true condition of the human body. It is an urgent need in the biopharmaceutical industry to establish tissues and organs in animals that are more similar to human physiological characteristics. Humanized animal models are models that simulate the process of human diseases, pathophysiological changes, and the response of human cells and tissues to diseases at the level of living organisms by transplanting functional human genes, cells, and tissues into animals. As a living substitute model for human disease research, humanized animal models have been widely used in the study of the pathogenesis of tumors, infectious diseases, autoimmune diseases, and metabolic diseases, as well as drug screening.

[0003] It has been reported that in recent years, the global situation of kidney diseases, especially chronic kidney diseases, is becoming increasingly severe. It is estimated that the number of people suffering from chronic kidney disease worldwide has reached nearly 690 million, and the number of people dying from chronic kidney disease-related diseases worldwide each year exceeds 2.4 million, making it the sixth fastest-growing cause of death. At the same time, chronic kidney disease is also one of the important reasons for the increase in the incidence and mortality of other diseases such as cardiovascular disease, hypertension, diabetes, tuberculosis, and hepatitis.

[0004] Studies have shown that kidney disease can be caused by a variety of factors, resulting in irreversible damage to kidney structure and loss of function, including primary kidney disease and secondary kidney disease. Primary kidney disease may be related to single or multiple genetic inheritance, epigenetic factors, and other unknown causes; secondary kidney disease is mainly caused by hypertension, diabetes, or autoimmune diseases and other diseases. It can be inferred that there are significant differences in pathogenesis and treatment drug sensitivity among patients. From the perspective of precision medicine, it is necessary to study the pathogenesis of kidney disease and individualize the evaluation of treatment drugs.

[0005] With the development of genomic technology, the pathogenesis, diagnosis and treatment target research and drug screening based on individual genetic factors become the core goal of individualized precision medicine, and a suitable animal model is the key platform for individualized precision medicine. In the field of tumor, the patient-derived xenograft (PDX) model is widely used in the screening of precision medicine for tumor, because the model retains most of the characteristics of the individual primary tumor in histopathology, molecular biology and genetic level, and has good clinical efficacy prediction. However, in the field of kidney disease, there is still a lack of suitable individualized precision medicine animal model, which has become a technical bottleneck in the field of kidney precision medicine research.

[0006] Chronic kidney disease is one of the global public health problems, and also causes a huge burden to the health and economic fields of China. There is no effective drug for the treatment of the later stage of the disease in clinic; although kidney transplantation can be used as a treatment method, it is often accompanied by related viral infections (mainly BKV infection), which in turn causes a series of complications. The etiology of chronic kidney disease is complex, and modern medicine has developed to the stage of in-depth research on individualized precision medicine, but the related pathogenesis and treatment drug research still lack a suitable animal model. Studies have found that BKV virus infection mainly lurks in human urinary epithelial cells, especially human renal tubular epithelial cells, and once the function of the human immune system decreases, the virus can be reactivated to cause infection. The current mechanism of infection of the virus in the human body and the related receptors are not clear, and there is no particularly effective treatment method. At present, the research on BKV infection also lacks a suitable animal model, which has become a short board restricting the research progress in this field.

[0007] For the research of individualized precision medicine of kidney disease, the individualized kidney humanized mouse model based on human cells is the best animal model for research; especially the reconstructed human kidney vascular unit in mice is also an ideal animal model for BKV infection related research. Therefore, the construction of kidney humanized animal model has important significance for the research of kidney disease pathogenesis, precision medicine and individualized treatment drug screening.

[0008] Based on the current status of the prior art, the inventors of the present application provide a method for constructing a kidney humanized mouse model based on human cells and a kidney humanized mouse model constructed thereby. SUMMARY

[0009] The purpose of the present application is to provide a method for constructing a kidney humanized mouse model based on human cells and a kidney humanized mouse model constructed thereby based on the current status of the prior art.

[0010] The kidney epithelial cells isolated from the urine of an individual are cultured in vitro (2D or 3D culture), mixed with mesenchymal stem cells and human umbilical vein endothelial cells, and then transplanted into the subcutaneous tissue or under the renal capsule of a NOD-SCID mouse to construct a cell-based humanized kidney mouse model, which can be used to study the pathogenesis of related kidney diseases, screening of therapeutic drugs, and the mechanism of BK virus infection.

[0011] The meanings of the technical terms and scientific terms used in the present application are intended to be the same as those generally understood by those skilled in the art, including those technical changes or replacement of equivalent technologies that are obvious to those skilled in the art. The terms "include", "contain", "have", "contain" or "involve" and other variants thereof as used herein are inclusive or open, and do not exclude other unlisted elements or method steps.

[0012] The present application is realized by the following technical solutions, and the main technical points involved are as follows:

[0013] To construct the model, the construction method used by the present application is as follows:

[0014] The construction of the above-mentioned humanized kidney mouse model uses immunodeficient NOD-SCID mice as the construction object to construct human renal tubular epithelial cells (or 3D cultured tubuloids), mesenchymal stem cells and human umbilical vein endothelial cells, and then injects them into the subcutaneous tissue and under the renal capsule of the mouse to construct. Using immunodeficient mice (such as NOD-SCID mice) can avoid the failure of construction caused by immune rejection when human cells are injected into the mouse body.

[0015] Specifically, the construction method of the kidney humanized mouse model of the present application comprises the following steps: injecting mesenchymal stem cells, human umbilical vein endothelial cells and human renal tubular epithelial cells isolated from urine into the subcutaneous tissue or under the renal capsule of an immunodeficient mouse to obtain a humanized kidney vascular unit mouse model connected to the host.

[0016] More specifically, the construction method of the kidney humanized mouse model of the present application comprises,

[0017] Injecting renal tubular epithelial cells, primary mesenchymal stem cells and human umbilical vein endothelial cells into the subcutaneous tissue and under the renal capsule of NOD-SCID immunodeficient mice, and following the steps:

[0018] (1) Preparation of human renal tubular epithelial cells and tubuloids

[0019] It has been found that some living cells in the human urinary system are excreted with urine, including renal epithelial cells, podocytes and renal progenitor cells. These cells can be successfully isolated and expanded by cell culture technology.

[0020] Specifically, the urine of healthy people is collected, washed and centrifuged, and then cultured in a culture medium. The washing buffer used contains DMEM medium, HEPES (1M), GlutaMAX TM , Rho-kinase inhibitor (10mM), primocine (0.1mg / ml) and 1% penicillin-streptomycin. The primary medium is DMEM-F / 12 complete medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and REGM TM SingleQuots growth factors. The proliferation medium contains: REBM and REGM TM SingleQuots growth factors. The 3D culture medium contains ADMEM / F12 medium added with 1% penicillin-streptomycin, HEPES, GlutaMAX TM , N-acetylcysteine (1mM), 1.5% B27, 10% Rspo3 conditioned medium or FGF9 / FGF10 conditioned medium, EGF (50ng / ml), FGF-10 (100ng / ml), Rho-kinase inhibitor (10mM), A8301 (5μM), primocine (0.1mg / ml). The cell culture plate is pre-coated with laminin-521 (5mg / ml) to promote cell adhesion and proliferation.

[0021] (2) Immunofluorescence identification of human renal tubular epithelial cells and tubloids

[0022] Immunofluorescence identification of renal tubular epithelial cells, including immunofluorescence detection of epithelial cell adhesion molecule E-cadherin, ZO-1 tight junction, renal proximal tubular epithelial cell villin, and collecting duct GATA3.

[0023] (3) BK pseudovirus infection of human renal tubular epithelial cells

[0024] In one embodiment, the renal tubular epithelial cells are infected with BK pseudovirus in vitro. After 72 hours of infection, it can be observed that the cells can be infected with BK pseudovirus.

[0025] (4) Culture of primary mesenchymal stem cells and human umbilical vein endothelial cells

[0026] Primary mesenchymal stem cells and human umbilical vein endothelial cells are purchased commercially. The culture method is in accordance with the manufacturer's instructions. The primary mesenchymal stem cells are cultured in iCell primary mesenchymal stem cell serum-free medium (PriMed-iCell-012-SF, SAB Bio-Engineer) and passaged to 6-8 generations for use. Human umbilical vein endothelial cells are cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0027] (5) Injecting renal tubular epithelial cells, primary mesenchymal stem cells and human umbilical vein endothelial cells into NOD-SCID immunodeficient mice to obtain a humanized mouse model of kidney.

[0028] In an embodiment of the present application, human renal tubular epithelial cells are cultured in 2D, and then cultured in 3D in vitro to form tubloids.

[0029] In an embodiment of the present application, immunofluorescence identification of renal tubular epithelial cells and tubloids is included.

[0030] In an embodiment of the present application, renal tubular epithelial cells are infected with BK pseudovirus in vitro.

[0031] In an embodiment of the present application, in the step of injecting the human renal tubular epithelial cells, mesenchymal stem cells and human umbilical vein endothelial cells into immunodeficient mice subcutaneously, the amount of cells used for each mouse is 1.5 x 10 6 renal tubular epithelial cells, 1.5 x 10 6 mesenchymal stem cells and 1 x 10 6 human umbilical vein endothelial cells, mixed with 200 μl Matrigel matrix glue, injected subcutaneously into NOD-SCID mice to form human-mouse chimeric grafts.

[0032] In an embodiment of the present application, the present application also attempts to expose the kidney by surgery and transplant it under the kidney capsule of NOD-SCID mice to form human-mouse chimeric grafts.

[0033] In an embodiment of the present application, in order to identify whether the chimeric graft formed subcutaneously has human-derived vascular units, HE staining, immunohistochemical and immunofluorescence detection are performed on the human-derived graft, and whether the graft blood vessels are connected to the host blood circulation is verified.

[0034] In an embodiment of the present application, the present application performs HE staining and immunohistochemical detection on whether the human-mouse chimeric graft formed under the kidney capsule has vascular structure.

[0035] The injection method of the present application is selected from subcutaneous injection and subrenal capsule injection. Human umbilical vein endothelial cells have the ability to generate blood vessels in vivo; mesenchymal stem cells not only have the ability of multi-directional differentiation and can be differentiated into perivascular cells, but also have the paracrine function to promote the formation of renal tubular lumen of renal tubular epithelial cells and the formation of blood vessels of human umbilical vein endothelial cells.

[0036] In one of the embodiments, 1.5 x 10 6 renal tubular epithelial cells, 1.5 x 10 6 mesenchymal stem cells and 1 x 10 6 human umbilical vein endothelial cells are mixed and transplanted into the subcutaneous tissue of NOD-SCID mice together with 200 μl Matrigel matrix gel to form humanized vascular and tubular structures. The amount and ratio of the above three kinds of cells, the growth time in mice and the concentration of Matrigel matrix gel are also key factors affecting the formation of chimeric humanized mice.

[0037] In one of the embodiments, whether the 14-day graft formed in the subcutaneous tissue of mice has human vascular units and whether it is connected with the blood circulation of the host mice is verified.

[0038] In one of the embodiments, 1.0 x 10 6 mesenchymal stem cells and 7.5 x 10 5 human umbilical vein endothelial cells are mixed and transplanted into the subcutaneous tissue of NOD-SCID mice together with 200 μl Matrigel matrix gel to form humanized vascular and tubular structures.

[0039] The present application provides a method for constructing a kidney humanized mouse model and a kidney humanized mouse model constructed by the method. The kidney humanized mouse model constructed by the method has the following characteristics: the cells used are derived from urine, which has the characteristics of sufficient clinical source and convenient sampling; the kidney source cells derived from urine can represent a unique individual alone and can be used to establish an individualized kidney humanized mouse model based on patients, thus having the potential to be applied to individualized precision medical research; in addition, human umbilical vein endothelial cells are added to generate vascular structures; mesenchymal stem cells are added to facilitate their survival in vivo and form lumen structures. Therefore, the model can form functional human kidney vascular units in mice, and has great application prospects in individualized precision medical research of kidney disease and human kidney specific viral infection research. The model can be used as a preclinical animal model and applied to individualized precision medical research of chronic kidney disease, human kidney specific viral infection (such as polyomavirus BKV) research and related treatment drug screening and research and development. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1, kidney tubular epithelial cells were successfully isolated from urine, wherein,

[0041] Healthy adult urine 300-400ml in sterile culture flask, by centrifugation, washing, adding Primary Medium to 12-well plates (pre-coated with laminin-521) culture. And observe the 5th day, 7 days, 9 days, 11 days of cell growth. A: the 5th day of cell generation; B: the 7th day of cell generation; C: the 9th day of cell generation; D: the 11th day of cell generation.

[0042] Figure 2 , kidney tubular epithelial cells and tubuloids immunofluorescence identification, wherein,

[0043] The cells express epithelial markers: E-cadherin; renal proximal tubule villin protein: Villin; collecting duct marker: GATA3. Tubuloids also detect the expression of E-cadherin, ZO-1 (tight junction), GATA3 and Villin.

[0044] Figure 3 , BK pseudovirus can successfully infect human renal tubular epithelial cells in vitro, wherein,

[0045] A and B are uninfected groups; C and D are the results of BK pseudovirus infection for 72h.

[0046] Figure 4 , renal tubular epithelial cells, mesenchymal stem cells and human umbilical vein endothelial cells subcutaneously injected to form a graft.

[0047] Figure 5 , 14 days of grafts formed in the mouse subcutis were subjected to HE, immunohistochemistry and immunofluorescence to detect human-derived blood vessels and tubular units, wherein,

[0048] A: HE staining detected vascular structures (red arrows) and possible tubular structures (black arrows); B: immunohistochemistry HLA-A proved that the graft was of human origin; C and E: immunohistochemistry and immunofluorescence Cytokeratin (cytokeratin) proved the formation of tubular lumen structure; D: immunofluorescence: HLA-A and FITC-Dextran proved that the human-derived blood vessels were connected with the host blood circulation.

[0049] Figure 6 , kidney subcapsular transplantation surgery.

[0050] Figure 7 , 14 days of grafts formed under the kidney capsule.

[0051] Figure 8HE staining was performed on 14-day subcapsular kidney grafts, and human vascular units were detected by immunohistochemistry, wherein,

[0052] A: Vascular structure was observed by HE staining; B: Human vascular structure was detected by immunohistochemistry CD31. DETAILED DESCRIPTION

[0053] In order to facilitate the understanding of the present application, the following gives an embodiment of the present application, and the present application is described more fully with reference to the relevant drawings.

[0054] Example 1 Isolation and culture of human urine-derived renal tubular epithelial cells and tubloids culture

[0055] (1) First, collect 300-400 ml of urine from a healthy adult in a sterile culture bottle.

[0056] (2) In a biological safety cabinet, dispense it into a 50 ml centrifuge tube, centrifuge at 400 g / min at 4°C for 10 min, discard the supernatant, and leave about 1 ml, then move it all into a new 50 ml centrifuge tube.

[0057] (3) Add 15-20 ml of Washing Buffer, centrifuge at 200 g / min at 4°C for 10 min, discard the supernatant, and leave 0.2 ml.

[0058] (4) Then add 1 ml of Primary Medium to suspend and precipitate, transfer it to a 12-well plate (pre-coated with laminin-521), add 1 ml of Primary Medium, add 1 ml of Primary Medium every day for the next 3 days, on the 4th day, aspirate 4 ml of culture medium, and add 1 ml of REGM proliferation medium, then change half of the medium every day, culture for about 14 days, the cell density is 80%-90%, and mark it as P0 generation, as shown in Figure 1 .

[0059] (5) Next, the P0 generation cells are passaged at a ratio of 1:2. Wash 1-2 times with PBS, digest with 0.25% trypsin at 37°C in a 5% CO2 incubator for 4-5 min, then stop the digestion, centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and add new REGM proliferation medium to mark it as P1 generation. Then pass it to P3 generation according to this method.

[0060] (6) When the P0 generation cell density is 80%-90%, collect the renal epithelial cells; centrifuge the cells in a 1.5 ml EP tube, discard the supernatant, and resuspend the cells with an appropriate volume of culture medium.

[0061] (7), Transfer the cell suspension into Matrigel 3D culture system, use the cold gun head to suck the appropriate volume of Matrigel and cell suspension, mix well, pay attention to gentle, no bubbles. Then drop it into the 24-well plate: add 30 μl of Matrigel and 30 μl of cell suspension to each well (Note: the whole process is carried out on ice).

[0062] (8), Put the mixed suspension into 37°C carbon dioxide incubator, stand for 5 min, take it out from the incubator after the Matrigel solidifies, add 500 μl of 3D culture medium. Put it into 37°C carbon dioxide incubator, observe the cell state every day, and tubular structures (Tubloids) begin to appear after 3 days.

[0063] Example 2 Immunofluorescence identification of renal tubular epithelial cells and tubular structures (Tubloids)

[0064] First, the renal tubular epithelial cells isolated from urine are plated on 8-well NuncTM Lab-TekTM chamber coverslips at a plating density of 80%-90%, and the next day the renal tubular epithelial cells and tubular structures (Tubloids) are subjected to immunofluorescence staining. First, fix the cells with 4% tissue cell fixative at room temperature for 20 min, wash with PBS; add 0.5% Triton X-100 for 15 min, wash with PBS; block with 5% BSA at room temperature for 1 h, then incubate with the first antibody at 4°C overnight, wash with PBS, then add the fluorescent second antibody, incubate at room temperature in the dark for 1 h, wash with PBS; DAPI stain the cell nucleus, wash with PBS, and take pictures under a laser confocal microscope, as shown in Figure 2 .

[0065] Example 3 In vitro infection of renal tubular epithelial cells by BK pseudovirus

[0066] The cultured renal tubular epithelial cells are plated in 12-well plates to a confluence of about 80%. The next day, the BK pseudovirus packaged with the GFP fragment is added to the cells at a certain ratio, and after 72 hours of infection, the infection is observed under a fluorescent inverted microscope, and it is found that the virus can infect the primary cells, as shown in Figure 3 .

[0067] Example 4 Subcutaneous transplantation of renal tubular epithelial cells, mesenchymal stem cells and human umbilical vein endothelial cells

[0068] (1), Take out the P3 generation of renal tubular epithelial cells, P6-P8 generation of mesenchymal stem cells and human umbilical vein endothelial cells growing well in the CO2 incubator, place them in a biological safety cabinet, trypsinize, centrifuge, stain with trypan blue and count with a hemocytometer: take 1.5 x 10 6 ​6 Mesenchymal stem cells and 1 x 10 6 Human umbilical vein endothelial cells.

[0069] (2) Then mix the above three kinds of cells well with 200 μl Matrigel (Note: the pipette and pipette tip, syringe should be chilled in advance, Matrigel is thawed in advance at 4°C overnight, the whole process is operated on ice to prevent Matrigel from solidifying again).

[0070] (3) After 14 days, take out the mice from the isolation bag, and take out the graft from the subcutaneous tissue of the mice, as shown in Figure 4

[0071] Example 5 Verification of human-mouse vascular connectivity of subcutaneous graft

[0072] From the subcutaneous graft formed in Example 4, after 14 days, the mice were taken out from the isolation bag. 50 mg / ml, 0.2 ml of 200 KDa high molecular weight FITC-Dextran (a green intracellular tracer that can be injected into blood vessels, which can circulate in the blood vessels in vivo, if human-derived blood vessels are generated, the tracer can be detected in the graft) was injected through the tail vein, and the mice were sacrificed after 0.5-1 h to take out the graft. After 4% paraformaldehyde fixation of the graft, the morphology of the graft tissue was observed by HE staining: the formation of vascular lumen structure; human tubular structure was detected by immunohistochemistry and immunofluorescence: cytokeratin; at the same time, human leukocyte antigen (HLA-A) and FITC-Dextran were detected to determine whether there were human-derived blood vessels in the graft and whether they were connected with the blood circulation of the mice, as shown in Figure 5

[0073] Example 6 Subcutaneous transplantation of mesenchymal stem cells and human umbilical vein endothelial cells under kidney capsule

[0074] (1) The P6-P8 generation of mesenchymal stem cells and human umbilical vein endothelial cells cultured in a CO2 incubator were taken out and placed in a biological safety cabinet, trypsinized, centrifuged, stained with trypan blue, and counted with a hemocytometer: 1.5 x 10 6 Mesenchymal stem cells and 1 x 10 6 Human umbilical vein endothelial cells.

[0075] ​​(2), then mix the two kinds of cells with 20 μl Matrigel matrix glue (Note: the process of pipette and gun head, syringe need to be cold, Matrigel in advance on 4℃ ice overnight to melt, the whole process on ice to avoid Matrigel again solidification).

[0076] (3), NOD-SCID mice from aseptic isolator into sterile after the clean bench, after Averdin anesthesia, the mouse prone position, the skin of the mouse hair in the kidney area removed after iodine disinfection, exposure of the kidney (all surgical instruments: scissors, forceps, hemostatic forceps, suture needle, cotton swab, etc. are sterile). And use capillary glass tube carefully kidney capsule and kidney parenchyma prying gap (the process with cotton swab dipped in PBS on the kidney, keep the kidney moist), with 25ml Hamilton Syringe plus PE50 Flexible soft tube slowly injected into the mouse kidney capsule mixture (the whole operation process need to be as soon as possible). Then quickly suture the mouse wound, and in the wound scattered with sodium penicillin powder, the mouse is placed in a 37℃ thermostat, after recovery will be put into aseptic isolator, while injection of 0.1ml, 40 million units / ml of sodium penicillin to avoid mouse infection and death, such as Figure 6 shown (renal subcapsular transplantation).

[0077] (4), 14 days, the mice were killed and the graft was removed, as shown in Figure 7 (graft).

[0078] (5), the graft with 4% paraformaldehyde fixation, by HE staining to observe the morphology of the graft tissue; immunohistochemical identification of human vascular structure: platelet endothelial cell adhesion molecule (PECAM-1 or called CD31), as shown in Figure 8 .

Claims

1. A method for constructing a kidney humanized mouse model, characterized by, Isolating renal-derived cells from human urine, Human renal tubular epithelial cells in 2D culture or organoids cultured into tubuloids in 3D culture are co-injected with mesenchymal stem cells and human umbilical vein endothelial cells into immunodeficient mice to prepare a kidney humanized mouse model; the following steps are taken: (1) preparing human renal tubular epithelial cells and tubuloids, (2) immunofluorescence identification of human renal tubular epithelial cells and tubuloids, (3) BK pseudovirus infection of human renal tubular epithelial cells, (4) culture of primary mesenchymal stem cells and human umbilical vein endothelial cells, (5) injection of renal tubular epithelial cells, primary mesenchymal stem cells and human umbilical vein endothelial cells into NOD-SCID immunodeficient mice to obtain a kidney humanized mouse model.

2. The method for constructing a humanized mouse model of kidney according to claim 1, characterized in that, The human renal tubular epithelial cells are isolated from a single human individual; After isolation, they are cultured in vitro, and the process uses a specific initial culture medium and a proliferation culture medium for 2D culture or Matrigel-based 3D culture.

3. The method of claim 1, wherein the humanized kidney mouse model is constructed by, In the step of co-injecting human renal tubular epithelial cells or tubuloids cultured in 3D, mesenchymal stem cells and human umbilical vein endothelial cells into immunodeficient mice, the injection site is subcutaneous or subcapsular.

4. The method of claim 1, wherein the humanized kidney mouse model is constructed by, The immunodeficient mice are mice lacking T cell, B cell and NK cell functions.

5. The method of claim 1, wherein the humanized kidney mouse model is constructed by, The prepared kidney humanized mouse model can form humanized renal vascular units that communicate with the host mouse systemic circulation vessels.

6. The method of claim 1, wherein the humanized kidney mouse model is constructed by, The prepared kidney humanized mouse model is used for the preparation of individualized precision medical research products related to human kidney diseases.

7. The method of claim 1, wherein the humanized kidney mouse model is constructed by, The prepared kidney humanized mouse model is used for the preparation of human kidney-specific virus and related drug research products.

8. The method of claim 7, wherein the humanized kidney mouse model is constructed by, The human kidney-specific virus is BKV virus.

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