An alzheimer disease "bone-brain" axis 3D cell model and a construction method and application thereof

Abstract

The application relates to the technical field of cell model construction, and discloses an Alzheimer disease 'bone-brain' axis 3D cell model and a construction method and application thereof. The method is based on the pathogenesis evolution theory of 'kidney deficiency brewing toxin-toxin hiding in bone marrow-injurying brain marrow', a conditioned medium is prepared by co-culturing human peripheral blood mononuclear cells and bone marrow mesenchymal stem cells in a Transwell chamber, and the conditioned medium is used to intervene in the co-culture system of human microglial cells and neurons, so that the in-vitro interaction of the peripheral immunity, bone marrow and central nervous system is simulated. The model construction method is simple, has high repeatability and long-term stability, effectively fills the blank of the 'peripheral-bone marrow-central' interaction research model, and provides a reliable in-vitro model and technical support for the pathogenesis research of Alzheimer disease and high-throughput screening and evaluation of drugs.

Description

Technical Field

[0001] This invention belongs to the field of cell model construction technology, and relates to a 3D cell model of the "bone-brain" axis of Alzheimer's disease, its construction method and application. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease. Early clinical manifestations include memory loss, followed by diffuse cortical damage such as impaired calculation, orientation, and language abilities. Late-stage AD is often accompanied by psychiatric symptoms, severely impacting patients' quality of life and imposing a heavy socioeconomic burden. Current AD research strategies primarily focus on β-amyloid (Aβ) plaques or phosphorylated Tau protein in the brain. While anti-Aβ / Tau monoclonal antibodies have shown potential in clearing pathological proteins, significant challenges remain in improving cognitive function and ensuring safety (such as microvascular complications and bleeding risk). Recent preventative clinical studies have failed to demonstrate that intervention targeting Aβ protein alone can prevent AD, further revealing the complexity of AD's pathological mechanisms and the limitations of single-target intervention strategies. Given the limited efficacy of current conventional clinical treatments, exploring long-acting drugs or efficient brain delivery pathways has become an important direction in AD treatment.

[0003] Furthermore, traditional AD drug development relies excessively on transgenic animal models. However, this development model has several inherent flaws, mainly in the following three aspects: First, it lacks economic efficiency and timeliness. Transgenic AD model mice have a long breeding cycle and high maintenance costs, making it difficult to meet the needs of large-scale drug screening and high-throughput development. Second, transgenic mice ignore the core variable of aging. Drug development targeting toxins such as Aβ in animal models often involves forcibly overexpressing toxic proteins, ignoring aging as a major risk factor. Third, the transgenic mouse model is limited. Modern medical research shows that bone marrow immunosenescence is a key pathogenic factor in AD, while traditional Chinese medicine theory states that "the kidney governs bones and produces marrow," and that immunosenescence caused by kidney deficiency is closely related to the pathogenesis of AD. However, existing animal models cannot simulate the unique peripheral immunosenescence environment in humans, especially the immune state related to kidney deficiency, in response to drugs, resulting in extremely low clinical conversion rates.

[0004] Alzheimer's disease (AD) may not be an isolated brain disease, but rather closely related to the interaction of multiple organs throughout the body caused by aging. Current technologies lack a research system capable of simulating the dynamic interaction between peripheral immune aging and the brain environment. Therefore, developing cost-effective, efficient, and reproducible in vitro bone-brain axis cell models, their construction methods, and applications are urgent technical problems that need to be solved by those skilled in the art. Summary of the Invention

[0005] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a 3D cell model of the "bone-brain" axis in Alzheimer's disease, its construction method, and its applications. Specifically, this invention provides a method for constructing a 3D cell model of the "bone-brain" axis in Alzheimer's disease, which follows the pathogenesis of "kidney deficiency leading to toxin accumulation - toxin lurking in the bone marrow - damage to the brain marrow," thereby solving the current problem of lacking cell models capable of simulating the interaction between peripheral immune aging and the brain environment.

[0006] To address the aforementioned technical problems, this invention discloses a 3D cell model of the Alzheimer's disease "bone-brain" axis, its construction method, and its applications. The specific technical solution is as follows: In a first aspect, the present invention provides a method for constructing a 3D cell model of the Alzheimer's disease "bone-brain" axis, comprising the following steps: Step 1: Induce human peripheral blood mononuclear cells to differentiate into macrophages; Step 2: Co-culture human bone marrow mesenchymal stem cells and macrophages obtained in Step 1, filter, and obtain cell supernatant; Step 3: Culture human microglia; Step 4: Culture human neuroblastoma cells; Step 5: Discard the culture supernatant of human microglia obtained in Step 3, replace it with conditioned medium, and co-culture it with human neuroblastoma cells obtained in Step 4. Collect the human neuroblastoma cells to obtain the Alzheimer's disease "bone-brain" axis 3D cell model. The conditioned medium is a culture medium containing the cell supernatant obtained in Step 2.

[0007] In step one, the human peripheral blood mononuclear cells are THP-1 cells; the induction of differentiation is performed by inducing differentiation with 50-200 ng / mL phorbol ester for 12-24 h. In some embodiments of the present invention, the induction of differentiation is performed by inducing differentiation with 100 ng / mL phorbol ester for 24 h. In other embodiments of the present invention, THP-1 cells in the logarithmic growth phase are resuspended in complete THP-1 cell culture medium containing 100 ng / mL phorbol ester for induction of differentiation into macrophages. The complete THP-1 cell culture medium is RPMI Medium 1640 medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 100 mg / mL amphotericin B.

[0008] In steps two and five, a Transwell system is used for co-culturing. The Transwell system includes an upper chamber and a lower chamber, with a permeable membrane at the bottom of the upper chamber having a pore size of 0.4 μm. In some embodiments of the invention, the Transwell system includes at least one of a 6-well plate, a 12-well plate, a 24-well plate, or a 96-well plate, wherein the Transwell system in step two is a 6-well plate, and the Transwell system in step five is a 24-well plate. The co-culturing time is 12–24 h, and the culture temperature is 30–37 °C. In some embodiments of the invention, the co-culturing time is 24 h, and the culture temperature is 37 °C.

[0009] In step two, the human bone marrow mesenchymal stem cells (hBMSCs) are seeded in the lower chamber, and the macrophages are seeded in the upper chamber for co-culture. In some embodiments of the present invention, the seeding density of the human bone marrow mesenchymal stem cells (hBMSCs) is 4~8×10⁻⁶. 4 / cm 2 The macrophage seeding density, calculated based on the human peripheral blood mononuclear cell seeding density in step one, is 40 × 10⁻⁶. 4 / mL.

[0010] The culture medium for macrophages contains 10-30 ng / mL Aβ1-40 oligomers; in some embodiments of the present invention, the culture medium for macrophages contains 20 ng / mL Aβ1-40 oligomers.

[0011] The aforementioned filtration process involves filtering the human bone marrow mesenchymal stem cell culture system seeded in the lower chamber, with a filter membrane pore size of 0.2~0.3 μm.

[0012] In some embodiments of the present invention, in step two, the hBMSCs are hBMSCs in the logarithmic growth phase, and the hBMSCs are cultured using hBMSC complete cell culture medium, which is a human bone marrow mesenchymal stem cell culture system. The hBMSC complete cell culture medium is MEM-α medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 100 mg / mL amphotericin B. The macrophages are cultured using THP-1 complete cell culture medium containing 10~30 ng / mL Aβ1-40 oligomers.

[0013] In some embodiments of the present invention, the human bone marrow mesenchymal stem cells include at least one of primary human bone marrow mesenchymal stem cells and immortalized cell lines. Preferably, the human bone marrow mesenchymal stem cells are immortalized human bone marrow mesenchymal stem cells (hBMSCs).

[0014] In step three, the human microglia are HMC3 cells, preferably HMC3 cells in the logarithmic growth phase; the culture time is 12-24 h, and the culture temperature is 30-37 ℃. In some embodiments of the present invention, the culture time is 24 h, and the culture temperature is 37 ℃. In some embodiments of the present invention, the human microglia include at least one of different primary human microglia and immortalized cell lines. In other embodiments of the present invention, the seeding density of the human microglia is 6-15 × 10⁻⁶ cells / year. 4 / cm 2 .

[0015] In some embodiments of the present invention, in step three, the human microglia are cultured using HMC3 complete cell culture medium. The HMC3 complete cell culture medium is MEM medium containing 10% v / v premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 100 mg / mL amphotericin B.

[0016] In step four, the human neuroblastoma cells are SKNMC cells or SHSY5Y cells, preferably human neuroblastoma cells in the logarithmic growth phase. The culture is carried out in a low-absorption plate for 12-24 hours at a temperature of 30-37°C. In some embodiments of the invention, the culture time is 24 hours and the culture temperature is 37°C. In other embodiments of the invention, the low-absorption plate includes at least one of 6-well, 12-well, 24-well, and 96-well plates, preferably a 24-well plate. In some embodiments of the invention, the human neuroblastoma cells SKNMC are cultured using SKNMC complete cell culture medium. The SKNMC complete cell culture medium is MEM medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 100 mg / mL amphotericin B. The human neuroblastoma cells SHSY5Y were cultured using SHSY5Y complete cell culture medium, which contained 10% v / v premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 100 mg / mL amphotericin B in DMEM medium. In other embodiments of the invention, the seeding density of the human neuroblastoma cells was 6–15 × 10⁶ cells / mL. 4 / cm 2 .

[0017] In step five, the conditioned medium contains 40% to 70% v / v HMC3 cell complete culture medium; that is, the conditioned medium is a combination of the cell supernatant and the HMC3 cell complete culture medium. In some embodiments of the present invention, the conditioned medium contains 40% v / v cell supernatant and 60% v / v HMC3 cell complete culture medium.

[0018] In step five, human microglia are seeded in the upper chamber, and human neuroblastoma cells are seeded in the lower chamber for co-culture. In some embodiments of the present invention, the culture system in the upper chamber is a conditioned medium containing human microglia, and the culture system in the lower chamber is the human neuroblastoma cell culture medium obtained in step four.

[0019] In some embodiments of the present invention, the relative immunofluorescence intensity of Aβ1-42 in the Alzheimer's disease "bone-brain" axis 3D cell model prepared by the present invention is significantly upregulated.

[0020] Secondly, the present invention provides a 3D cell model of the Alzheimer's disease "bone-brain" axis constructed by the construction method described in the first aspect.

[0021] Thirdly, the present invention provides the application of the 3D cell model of the Alzheimer's disease "bone-brain" axis described in the second aspect in the treatment of Alzheimer's disease drugs, screening Alzheimer's disease drug targets and / or studying the immune molecular mechanisms of Alzheimer's disease.

[0022] Beneficial effects: This invention is based on the pathogenesis theory of "kidney deficiency leading to toxin accumulation - toxin lurking in the bone marrow - damage to the brain and marrow." It utilizes Transwell chambers to co-culture human peripheral blood mononuclear cells and bone marrow mesenchymal stem cells to prepare conditioned medium, and then uses this medium to intervene in a co-culture system of human microglia and neurons, thereby simulating the in vitro interaction between peripheral immunity, bone marrow, and the central nervous system. The model construction method of this invention is simple, highly reproducible, and has long-term stability, effectively filling the gap in research models of "peripheral-bone marrow-central" interaction, and providing a reliable in vitro model and technical support for the study of Alzheimer's disease pathogenesis and high-throughput drug screening and evaluation. Attached Figure Description

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0024] Figure 1 This is a schematic diagram of the 3D model process for cell co-culture of the present invention.

[0025] Figure 2The effects of adding phorbol ester (PMA) to Tranwell chambers for induction intervention and the effect of induction time on THP-1 cells are shown in Figure A, where A represents the effect of adding PMA, and B represents microscopic images of THP-1 cells after 24 h, 48 h, and 72 h of intervention with 100 ng / mL PMA.

[0026] Figure 3 Immunofluorescence images of SKNMC cells and relative fluorescence intensity results of Aβ1-42 obtained from conditioned media with different ratios.

[0027] Figure 4 The image shows the Western blot results of SHSY5Y cells obtained from conditioned media with different ratios.

[0028] Figure 5 Immunofluorescence images of 3D cultured SKNMC cells. Red represents Aβ1-42, and blue represents neurons. Detailed Implementation

[0029] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0030] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, the materials and reagents used in these examples are commercially available. Unless otherwise specified, the solvent for all solutions used in the following examples is sterile PBS buffer, with a concentration of 0.01 M and a pH of 7.4.

[0031] Example 1 Cell Culture The monocytic leukemia cell line (THP-1), immortalized human bone marrow mesenchymal stem cells (hBMSC), and human neuroblastoma cells (SKNMC) described in this invention were all purchased from a biotechnology company; the human microglia (HMC3) and human neuroblastoma cells (SHSY5Y) described were all purchased from the Cell Bank of the Chinese Academy of Sciences.

[0032] The specific methods for preparing the culture medium and drug stock solution used in this invention are as follows: Human peripheral blood mononuclear cell (THP-1) complete culture medium: RPMI Medium 1640 medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 100 mg / mL amphotericin B. Human bone marrow mesenchymal stem cell (hBMSC) complete culture medium: MEM-α medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 100 mg / mL amphotericin B. Human microglia (HMC3) complete culture medium: MEM medium containing 10% v / v premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 100 mg / mL amphotericin B; Human neuroblastoma cells (SKNMC) complete culture medium: MEM medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 100 mg / mL amphotericin B; Human neuroblastoma cells (SHSY5Y) complete culture medium: DMEM medium containing 10% v / v Australian premium fetal bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 100 mg / mL amphotericin B.

[0033] Preparation of PMA stock solution: Weigh 1 mg PMA, dissolve it in 1 mL DMSO solution to obtain a 1 mg / mL PMA stock solution, filter sterilize using a 0.22 μm filter, dispense into containers, and store at -20°C. The working concentration of PMA is 100 ng / mL. Preparation of Aβ1-40 oligomer stock solution: Weigh 1 mg of Aβ1-40 monomer and dissolve it in 1 mL of 25 mM sodium hydroxide solution. Centrifuge at 2000 rpm for 4 min at 4°C using a 0.22 μm centrifuge tube filter to remove large aggregates, thus preparing a 1 mg / mL Aβ1-40 stock solution. Dilute the Aβ1-40 stock solution with PBS, aliquot, and store at -20°C. The working concentration of Aβ1-40 is 20 ng / mL.

[0034] The specific cell culture methods of the present invention are as follows: THP-1 cells were cultured in THP-1 complete cell culture medium at 37 °C in a 5% CO2 cell culture incubator. When the cell suspension turned yellow and reached 80% confluence, the cell medium was changed and the cells were passaged. Cells in the logarithmic growth phase were selected for experiments. hBMSCs were cultured using complete hBMSC cell culture medium; HMC3 cells were cultured using complete HMC3 cell culture medium; SKNMC cells were cultured using complete SKNMC cell culture medium; and SHSY5Y cells were cultured using complete SHSY5Y cell culture medium. All cells were cultured in a 37 °C, 5% CO2 cell culture incubator. When the cells reached 90% confluence with the bottom of the dish, they were trypsinized and passaged. Cells in the logarithmic growth phase were selected for experiments.

[0035] Example 2: Construction of a 3D model for co-culturing THP-1-hBMSC+HMC3-SKNMC or THP-1-hBMSC+HMC3-SHSY5Y cells The cell culture methods for each cell in the logarithmic growth phase described in this embodiment are given in Example 1. A schematic diagram of the construction of the 3D cell co-culture model (taking THP-1-hBMSC+HMC3-SKNMC as an example) is shown below. Figure 1 As shown, the specific method is described in the following steps: Step 1: Take THP-1 cells in the logarithmic growth phase, centrifuge at 600-800 rpm for 5 min, discard the cell supernatant, resuspend the cells in THP-1 complete cell culture medium containing 100 ng / mL PMA, and seed them into the upper Transwell chambers of a 6-well plate (the permeable membrane of the chamber has a pore size of 0.4 μm) at a seeding density of 40 × 10⁶ cells / well. 4 / mL, placed in a 37 °C, 5% CO2 cell culture incubator for 24 h, THP-1 cells transformed from a suspension state into adherent macrophages, such as Figure 2 As shown in A. In this step, the cell culture time was optimized, as follows: Figure 2 As shown in B, under the microscope at 48 h and 72 h, an increase in dead cells (from adherent state back to suspension state) was observed, and the differentiated macrophages did not produce a significant proliferation effect. Therefore, the intervention time was selected as 24 h for subsequent experiments.

[0036] Step 2: Take hBMSCs in the logarithmic growth phase, digest with trypsin, centrifuge at 1000 rpm for 5 min, discard the cell supernatant, resuspend the cells in complete hBMSC culture medium, and seed hBMSCs into the lower layer of a 6-well plate at a density of 4-8 × 10⁶ cells / well, using 2 mL of the liquid medium per well. 4 / cm 2 (The specific inoculation density in this embodiment is 8×10) 4 / cm 2The cell supernatant of macrophages cultured in the upper chamber of step one was replaced with THP-1 complete cell culture medium containing 20 ng / mL Aβ1-40. The membrane of the upper chamber was placed in the lower chamber of the hBMSC complete cell culture and co-cultured at 37 °C in a 5% CO2 cell culture incubator for 24 h. After co-culturing, the hBMSC cell supernatant from the lower chamber of the 6-well plate was taken and filtered through a 0.22 μm filter to obtain hBMSC cell supernatant. The excess hBMSC cell supernatant was frozen and stored at -80 °C for later use.

[0037] Step 3: Take HMC3 cells in the logarithmic growth phase, digest with trypsin, centrifuge at 1000 rpm for 5 min, discard the cell supernatant, resuspend the cells in HMC3 complete cell culture medium, and seed them into the upper Transwell chambers (0.4 μm pore size) of a 24-well plate at a density of 6–15 × 10⁶ cells / well, using 200 μL of liquid per upper chamber. 4 / cm 2 (The inoculation density in this embodiment is specifically 10×10) 4 / cm 2 Place in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0038] Step 4: Take human neuroblastoma cells (SKNMC or SHSY5Y) in the logarithmic growth phase, digest with trypsin, centrifuge at 1000 rpm for 5 min, discard the cell supernatant, resuspend the cells in the corresponding complete human neuroblastoma cell culture medium, and seed them into the lower layer of a 24-well low-absorption plate at a density of 600 μL per lower chamber, with a seeding density of 6~15 × 10⁶ cells / well. 4 / cm 2 (The inoculation density in this embodiment is specifically 10×10) 4 / cm 2 Place in a 37 °C, 5% CO2 cell culture incubator for 24 h.

[0039] Step 5: The hBMSC cell supernatant from Step 2 was used to prepare HMC3 conditioned medium with different volume ratios (3:7, 4:6, 5:5, 6:4, 7:3) by mixing hBMSC cell supernatant with HMC3 complete cell culture medium. The hBMSC cell supernatant was then replaced with the conditioned medium in the above-mentioned different ratios and placed in the upper chamber as the model group. The HMC3 cell complete culture medium containing 40% hBMSC supernatant (with a volume ratio of 4:6) was used as the control group. Both the hBMSC cell supernatant and the lower layer of human neuroblastoma cells obtained in Step 4 were co-cultured at 37 °C in a 5% CO2 cell incubator for 24 h, ultimately obtaining the "bone-brain" axis 3D cell model.

[0040] The transformation of THP-1 cells into macrophages induced by PMA in step one was observed using an inverted white field microscope, and the results are as follows: Figure 2 As shown, THP-1 cells changed from a suspended state to an adherent state, and the cell morphology changed from round to fibrous spindle-shaped cells.

[0041] This embodiment uses immunofluorescence microscopy to observe the immunofluorescence expression of Aβ1-42 in human neuroblastoma cells. The specific method is as follows: Taking SKNMC as an example, after step five, the chamber was removed, and SKNMC cells were washed three times with PBS. They were then fixed with 4% paraformaldehyde for 10 min, and 0.5% Triton was added to permeabilize the membrane for 15 min. Immunofluorescence blocking buffer was added to each well for 1 h. Aβ1-42 antibody was diluted 1:250 with immunostaining primary antibody dilution buffer, and 50 μL was added to each well. The wells were wrapped in aluminum foil to protect from light and incubated overnight at 4°C. After 1 h of warming at room temperature, the corresponding fluorescent secondary antibody was diluted 1:250 with immunostaining secondary antibody dilution buffer, and 50 μL was added to each well. The wells were incubated at room temperature in the dark for 1 h, and then 50 μL of DAPI staining solution was added and incubated at room temperature in the dark for 5 min. The wells were then mounted with anti-fluorescence quencher, and images were acquired under a fluorescence microscope. ImageJ software was used for image analysis. The results are as follows: Figure 3 As shown in Table 1, compared with the control group, the relative fluorescence intensity of Aβ1-42 in the model group (especially the 40% group, i.e., the volume ratio of hBMSC supernatant to HMC3 cell complete culture medium was 4:6) was significantly increased (P<0.01), indicating that the culture medium after Aβ1-40 intervention of peripheral macrophage THP-1 affecting human bone marrow mesenchymal stem cells (hBMSCs) can promote the generation of Aβ1-42 in SKNMC cells in the HMC3-SKNMC cell co-culture system.

[0042] Table 1. Relative fluorescence intensity of Aβ1-42 in the control group and the model group.

[0043] In addition, this embodiment also used Western blotting to detect the expression levels of β-amyloid-beta precursor protein (APP), β-secretase 1 (BACE1), and tumor necrosis factor (TNF-α) in human neuroblastoma cells. Specifically, taking SHSY5Y cells as an example, after step five, the chamber was removed, and SHSY5Y cells were washed three times with PBS. Total cell protein was extracted using RIPA lysis buffer. The cells were then subjected to SDS-PAGE wet electrophoresis, transferred to a membrane, incubated with primary antibody overnight, incubated with secondary antibody, washed, and then photographed using a chemiluminescent gel imaging system. The absorbance of the bands was analyzed using ImageJ software. The results are as follows: Figure 4 As shown in Table 2. Compared with the control group, the expression levels of APP and TNF-α in the model group were positively correlated with the proportion of conditioned medium (p<0.05); while the expression level of BACE1 showed a trend of first increasing and then decreasing with the increase of the proportion, reaching a peak at 40% conditioned medium (i.e., the volume ratio of hBMSC supernatant to HMC3 cell complete medium was 4:6).

[0044] Table 2. Expression levels of relevant proteins in the model group and the control group.

[0045] The results showed that the co-culture model of THP-1-hBMSC+HMC3-SKNMC cells could increase the production of Aβ1-42, and the co-culture model of THP-1-hBMSC+HMC3-SHSY5Y cells could increase the expression levels of APP, BACE1, and TNF-α proteins. Furthermore, based on immunofluorescence and Western blotting results, a 40% concentration of conditioned medium was most suitable for establishing a "bone-brain axis" cell model to study amyloid deposition. According to Western blotting results, if studying neuroinflammatory responses related to elevated inflammatory markers such as tumor necrosis factor, the concentration of conditioned medium could be controlled at 50%–70%. In conclusion, a 3D cell model of the "bone-brain axis" pathogenesis of Alzheimer's disease, characterized by "kidney deficiency leading to toxin accumulation – toxin lurking in the bone marrow – damage to the brain," was successfully constructed.

[0046] Fluorescence microscopy was used to observe the formation of SKNMC 3D cell spheroids. Red represents Aβ1-42 cells surrounding the cell, and blue represents neurons. Figure 5 As shown, the SKNMC neuron (blue) grows upright, forming a spherical structure with an Aβ1-42 (red) surrounding it.

[0047] This invention provides a 3D cell model of the "bone-brain" axis in Alzheimer's disease, its construction method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a 3D cell model of the "bone-brain" axis in Alzheimer's disease, characterized in that, Includes the following steps: Step 1: Induce human peripheral blood mononuclear cells to differentiate into macrophages; Step 2: Co-culture human bone marrow mesenchymal stem cells and macrophages obtained in Step 1, filter, and obtain cell supernatant; Step 3: Culture human microglia; Step 4: Culture human neuroblastoma cells; Step 5: Discard the culture supernatant of human microglia obtained in Step 3, replace it with conditioned medium, and co-culture it with human neuroblastoma cells obtained in Step 4. Collect the human neuroblastoma cells to obtain the Alzheimer's disease "bone-brain" axis 3D cell model. The conditioned medium is a culture medium containing the cell supernatant obtained in Step 2.

2. The construction method according to claim 1, characterized in that, In step one, the human peripheral blood mononuclear cells are THP-1 cells; the induction of differentiation is performed by inducing differentiation with 50-200 ng / mL phorbol ester for 12-24 h.

3. The construction method according to claim 1, characterized in that, In steps two and five, a Transwell system is used for co-culture, which includes an upper chamber and a lower chamber. The bottom of the upper chamber is a permeable membrane with a pore size of 0.4 μm; The co-culture process involves a culture time of 12-24 hours and a culture temperature of 30-37 ℃.

4. The construction method according to claim 3, characterized in that, In step two, the human bone marrow mesenchymal stem cells are seeded in the lower chamber, and the macrophages are seeded in the upper chamber for co-culture. The culture medium for the macrophages contained 10-30 ng / mL Aβ1-40 oligomers; The aforementioned filtration process involves filtering the human bone marrow mesenchymal stem cell culture system seeded in the lower chamber, with a filter membrane pore size of 0.2~0.3 μm.

5. The construction method according to claim 1, characterized in that, In step three, the human microglia are HMC3 cells, and the culture time is 12-24 h, and the culture temperature is 30-37 ℃.

6. The construction method according to claim 1, characterized in that, In step four, the human neuroblastoma cells are SKNMC cells or SHSY5Y cells, and the culture is carried out in a low-absorption plate for 12-24 hours at a temperature of 30-37°C.

7. The construction method according to claim 1, characterized in that, In step five, the conditioned medium contains 40% to 70% v / v cell supernatant.

8. The construction method according to claim 3, characterized in that, In step five, human microglia are seeded into the upper chamber and human neuroblastoma cells are seeded into the lower chamber for co-culture.

9. The Alzheimer's disease "bone-brain" axis 3D cell model constructed by the construction method according to any one of claims 1 to 8.

10. The application of the Alzheimer's disease "bone-brain" axis 3D cell model as described in claim 9 in screening Alzheimer's disease drug targets and / or studying the immune molecular mechanisms of Alzheimer's disease.

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