Construction method and application of Alzheimer's disease cell model

Abstract

The invention discloses a construction method and application of an Alzheimer's disease cell model. Specifically, a beta-amyloid protein oligomer (A beta oligomer) is used for stimulating highly differentiated rat adrenal pheochromocytoma cells (PC-12 cells) so as to simulate physiological changes of neurons in brains of patients with the Alzheimer's disease and construct the cell model of the Alzheimer's disease. The Alzheimer's disease cell model constructed by the method can simulate the physiological status of neurons in the brain of a patient with Alzheimer's disease in vitro more truly and reliably, and an important experimental material is provided for studying the pathogenesis of Alzheimer's disease and screening drugs for preventing or treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro cell model construction. More specifically, it relates to a method for constructing an Alzheimer's disease cell model and its application. Background Art

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and its main features include cognitive impairment and behavioral damage. Currently, the prevalence and mortality of Alzheimer's disease are increasing year by year. However, the pathogenesis of Alzheimer's disease is not yet clear, so there are no effective prevention and treatment means.

[0003] β-amyloid protein (Aβ) is a metabolite of neurons, and Aβ plaques formed by the aggregation of Aβ in the brains of Alzheimer's disease patients are considered to be one of the most important physiological markers of Alzheimer's disease. Currently, the academic community generally believes that a series of physiological changes caused by the abnormal increase of Aβ ultimately lead to the abnormal death of neurons, which is an important mechanism for the onset of Alzheimer's disease.

[0004] In order to study the pathogenesis of Alzheimer's disease, a reliable cell model that can reflect the changes of neurons in the brains of Alzheimer's disease patients needs to be provided. Currently, the main method for constructing a cell model for studying Alzheimer's disease is to directly induce nerve cells with β-amyloid oligomers (Aβ oligomers). Specifically, nerve cells need to be cultured in a medium containing Aβ oligomers so that they can exhibit corresponding characteristics similar to the changes of neurons in the brains of Alzheimer's disease patients under the stimulation of Aβ oligomers, such as oxidative stress caused by excessive reactive oxygen species.

[0005] However, research has confirmed that in the currently used method for constructing an Alzheimer's disease cell model, the preparation process of the medium containing Aβ oligomers will cause a decrease in the content of nutrients necessary for cell growth, such as serum and non-essential amino acids in the medium. As a result, the physiological changes of the constructed cell model cannot be completely attributed to the influence of Aβ oligomers. Therefore, there is a large difference between the constructed Alzheimer's disease cell model and the real environment of neurons in the patient's brain, resulting in the constructed cell model being unable to truly and reliably reflect the physiological changes of neurons in the brains of Alzheimer's disease patients, thus limiting the research on the pathogenesis of Alzheimer's disease, the screening of preventive or therapeutic drugs, etc.

[0006] Therefore, there is an urgent need to provide a cell model that can truly and reliably reflect the changes of neurons in the brains of Alzheimer's disease patients for the study of the pathogenesis of Alzheimer's disease and the screening of drugs for preventing or treating Alzheimer's disease. Summary of the Invention

[0007] In view of the above technical problems, an object of the present invention is to provide a new method for constructing an Alzheimer's disease cell model. The cell model constructed by this construction method can more truly and accurately simulate the physiological processes occurring in neurons in the brains of Alzheimer's disease patients.

[0008] Another object of the present invention is to provide the application of the constructed Alzheimer's disease cell model in studying the pathogenesis of Alzheimer's disease.

[0009] Still another object of the present invention is to provide the application of the constructed Alzheimer's disease cell model in screening drugs for preventing or treating Alzheimer's disease.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] In the first aspect, the present invention provides a method for constructing an Alzheimer's disease cell model, which includes stimulating rat pheochromocytoma cells (PC-12) with β-amyloid oligomers (Aβ oligomers) to simulate the physiological changes of neurons in the brains of Alzheimer's disease patients, and constructing an Alzheimer's disease cell model.

[0012] The construction method specifically includes:

[0013] (1) Prepare a culture medium containing β-amyloid oligomers, and the components of the culture medium include: β-amyloid oligomers, high-glucose DMEM, fetal bovine serum, non-essential amino acids, and penicillin-streptomycin;

[0014] (2) Expand the culture of rat pheochromocytoma cells until the density reaches 80%-90%;

[0015] (3) Discard the culture medium in step (2), replace it with the culture medium containing β-amyloid oligomers in step (1), and continue the culture to obtain an Alzheimer's disease cell model.

[0016] It should be noted that steps (1) and (2) do not have a sequential order. When completing the present invention, the culture medium containing β-amyloid oligomers can be prepared first, or the rat pheochromocytoma cells can be expanded in culture first, as long as the work contents of steps (1) and (2) are completed when performing step (3).

[0017] In addition, the expansion culture of rat pheochromocytoma cells can be carried out using a culture medium known in the prior art.

[0018] Further, in step (1), the volume ratio of each component in the culture medium containing β-amyloid oligomers is β-amyloid oligomer solution: high-glucose DMEM: fetal bovine serum: non-essential amino acids: penicillin-streptomycin = 1: (4.54 - 18.61): (0.52 - 2.12): (0.05 - 0.21): (0.05 - 0.21), and the concentration of β-amyloid oligomers in the culture medium containing β-amyloid oligomers is 5 - 15 μM.

[0019] Further, in step (3), the culture temperature is 37 °C and the culture time is 24 - 48 h.

[0020] Further, the Aβ oligomers of the present invention can be obtained by commercial purchase or by aggregating and synthesizing β-amyloid peptide (Aβ peptide) using known methods. For a clear understanding of the present invention, a method for preparing Aβ oligomers is provided herein, specifically including:

[0021] Dissolve Aβ peptide in hexafluoroisopropanol, let it stand and react for a period of time, then add high-glucose DMEM, and let it stand and react for another period of time, centrifuge at low temperature and collect the supernatant, and after bubbling with nitrogen, oscillate at a constant temperature to obtain β-amyloid oligomers.

[0022] Further, the mass-volume ratio of the Aβ peptide to hexafluoroisopropanol is 1 mg: (300 - 500) μL.

[0023] Further, the volume ratio of hexafluoroisopropanol to high-glucose DMEM is 1: (8 - 10).

[0024] Further, the temperature for the standing reaction is 20 - 25 °C; the reaction time is 15 - 20 min.

[0025] Further, the low-temperature centrifugation temperature is 4 °C; the centrifugal force is 14000 × G; the centrifugation time is 10 - 15 min.

[0026] Further, the nitrogen bubbling time is 15 - 20 min.

[0027] Further, the constant-temperature oscillation temperature is 20 - 24 °C; the oscillation rate is 450 - 550 rpm; the oscillation time is 22 - 26 h.

[0028] In the second aspect, the present invention also provides an Alzheimer's disease cell model constructed by using the above construction method.

[0029] In the third aspect, the present invention also provides the application of the above Alzheimer's disease cell model in studying the pathogenesis of Alzheimer's disease and the application of the above Alzheimer's disease cell model in screening drugs for preventing or treating Alzheimer's disease.

[0030] The beneficial effects of the present invention are as follows:

[0031] The method for constructing an Alzheimer's disease cell model provided by the present invention solves the problem that there are significant differences between the Alzheimer's disease cell model and the real environment of neurons in the patient's brain by improving the types and dosage ratios of the components in the culture medium containing Aβ oligomers, enabling the physiological changes of the constructed cell model to be completely attributed to the influence of Aβ oligomers and being able to more realistically simulate the physiological state of neurons in the brain of Alzheimer's disease patients in vitro. Therefore, the Alzheimer's disease cell model provided by the present invention can more realistically and reliably simulate the physiological processes occurring in neurons in the brain during the onset of Alzheimer's disease patients, providing an important experimental model for studying the pathogenesis of Alzheimer's disease and screening drugs for preventing or treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following further details the specific embodiments of the present invention in conjunction with the drawings.

[0033] Figure 1 Schematic diagram showing the comparison of cell viability of the control cell model 1, control cell model 2, and normally growing cells constructed in Example 2.

[0034] Figure 2 Schematic diagram showing the comparison of cell viability of the Alzheimer's disease cell model and normally growing cells constructed in Example 3.

[0035] Figure 3 Schematic diagram showing the apoptosis and necrosis of the Alzheimer's disease cell model constructed in Example 4.

[0036] Figure 4 Schematic diagram showing the apoptosis and necrosis of normally growing cells in Example 4.

[0037] Figure 5 Schematic diagram showing the comparison of BCECF-AM staining results of the Alzheimer's disease cell model and normally growing cells constructed in Example 5, n > 20.

[0038] Figure 6 Schematic diagram showing the comparison of Fluo-4AM staining results of the Alzheimer's disease cell model and normally growing cells constructed in Example 5, n > 20.

[0039] Figure 7 Schematic diagram showing the comparison of DCFH-DA staining results of the Alzheimer's disease cell model and normally growing cells constructed in Example 5, n > 20. DETAILED DESCRIPTION OF THE INVENTION

[0040] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0041] In the following embodiments, the main test materials are:

[0042] β-amyloid peptide (Aβ peptide) was purchased from GL Biochem (Shanghai) Ltd.;

[0043] DMEM is a culture medium containing various amino acids and glucose, and was developed on the basis of MEM medium; compared with MEM, the amounts of various components were increased, and it is also divided into high-glucose type (higher than 4500 mg / L) and low-glucose type (lower than 1000 mg / L). The high-glucose type DMEM used in the present invention was purchased from Thermo Fisher Scientific;

[0044] Fetal bovine serum was purchased from Vicente Biotechnology (Nanjing) Co., Ltd.;

[0045] Non-essential amino acids were purchased from Thermo Fisher Scientific, and included glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The concentration of the non-essential amino acids was 8-12 mM;

[0046] Penicillin-streptomycin was purchased from Thermo Fisher Scientific. The concentration of penicillin in the penicillin-streptomycin was 9,000-11,000 U / mL, and the concentration of streptomycin was 9,000-11,000 μg / mL;

[0047] Highly differentiated rat adrenal pheochromocytoma cells (PC-12) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0048] Example 1 Construction of an Alzheimer's disease cell model

[0049] It includes the following steps:

[0050] (1) Synthesis of Aβ oligomers

[0051] Add Aβ peptide (0.25 mg) to 100 μL of hexafluoroisopropanol, and let it stand and react at 25 °C for 20 min. Then, add 900 μL of high-glucose type DMEM, and let it stand and react at 25 °C for 20 min. Centrifuge at 4 °C and 14,000×G for 15 min, and collect the supernatant. Bubble the liquid with nitrogen for 20 min, and keep it at a constant temperature and oscillate at 22 °C and 500 rpm for 24 h to obtain an Aβ oligomer solution, and the concentration of the Aβ oligomer solution is 61.54 μM.

[0052] (2) Preparation of a culture medium containing Aβ oligomers

[0053] Mix the Aβ oligomer solution with high-glucose DMEM, fetal bovine serum, non-essential amino acids, and penicillin-streptomycin at a volume ratio of 1:4.6:0.46:0.046:0.046 to obtain a culture medium containing Aβ oligomers, where the concentration of Aβ oligomers is 10 μM.

[0054] (3) Cultivation of PC-12 cells

[0055] Take cells from PC-12 cells in the logarithmic growth phase for digestion and collection. Count the PC-12 cells and adjust the cell density to 5×10 4 / mL. Inoculate the adjusted PC-12 cells into a 60-mm cell culture dish, add 5 mL of the culture medium, which is high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Transfer the inoculated PC-12 cells to a constant-temperature cell culture incubator at 37 °C and 5% CO2 for cultivation.

[0056] (4) Stimulation of PC-12 cells with the culture medium containing Aβ oligomers

[0057] When the PC-12 cells inoculated in the 60-mm cell culture dish are cultured to a density of 80 - 90%, discard the original culture medium and replace it with 5 mL of the culture medium containing Aβ oligomers at the concentration of step (2), and continue to culture for 48 h to obtain an Alzheimer's disease cell model.

[0058] Example 2 Verification of the reliability of the Alzheimer's disease cell model in simulating the neuronal state in the patient's brain

[0059] To prove that the Alzheimer's disease cell model constructed by the present invention can more realistically and reliably simulate the physiological state of neurons in the brain of Alzheimer's disease patients in vitro, it is necessary to prove that the physiological changes of cells in the constructed cell model can be completely attributed to the cytotoxic effect of Aβ oligomers.

[0060] Following the construction method of the Alzheimer's disease cell model, a control cell model was constructed, that is, following the same method as the construction process of the Alzheimer's disease cell model, a control culture medium without Aβ oligomers was prepared, and the cells were transferred to the control culture medium for cultivation. The cell viability of the normally growing cell model and the control cell model was evaluated by the MTT method and compared.

[0061] (1) Normally growing cell model

[0062] Inoculate PC-12 cells into a 96-well cell culture plate (5×10 cells per well 3In each well of a 96-well cell culture plate inoculated with 5×10⁴ cells, 1 mL of medium was added. The medium was high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. The inoculated PC-12 cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO₂ and cultured until the density reached 80 - 90%.

[0063] (2) Control group cell model 1

[0064] Following the method for constructing an Alzheimer's disease cell model commonly used in the current academic community, control medium 1 was prepared: After allowing hexafluoroisopropanol to stand for 20 min, high-glucose DMEM was added (the volume ratio of hexafluoroisopropanol to high-glucose DMEM was 1:9). After standing and mixing at 25 °C for 20 min, centrifugation was carried out at 4 °C, 14000×G for 15 min to collect the supernatant. After bubbling with nitrogen for 20 min, it was oscillated at a constant temperature of 22 °C at 500 rpm for 24 h to obtain a solution without Aβ oligomers.

[0065] The solution without Aβ oligomers was directly added to the medium for normally growing cells (high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin) to obtain control medium 1.

[0066] PC-12 cells were inoculated into a 96-well cell culture plate (5×10⁴ cells per well), and 1 mL of medium was added to each well. The medium was high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Then, the inoculated PC-12 cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO₂ for culture. 3 After the cells were cultured until the density reached 80 - 90%, the original medium was discarded and replaced with 1 mL of control medium 1. The cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO₂ and continued to be cultured for 48 h.

[0067] After the cells were cultured until the density reached 80 - 90%, the original medium was discarded and replaced with 1 mL of control medium 1. The cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO₂ and continued to be cultured for 48 h.

[0068] (3) Control group cell model 2

[0069] Following the method for constructing an Alzheimer's disease cell model in Example 1 of the present invention, control medium 2 was prepared: After allowing hexafluoroisopropanol to stand for 20 min, high-glucose DMEM was added (the volume ratio of hexafluoroisopropanol to high-glucose DMEM was 1:9). After standing and mixing at 25 °C for 20 min, centrifugation was carried out at 4 °C, 14000×G for 15 min to collect the supernatant. After bubbling with nitrogen for 20 min, it was oscillated at a constant temperature of 22 °C at 500 rpm for 24 h to obtain a solution without Aβ oligomers.

[0070] Different from (2), instead of directly mixing the solution without Aβ oligomers with the culture medium used for normally growing cells, a control group culture medium 2 was obtained by mixing the solution without Aβ oligomers with high-glucose DMEM, fetal bovine serum, non-essential amino acids, and penicillin-streptomycin in a ratio of 1:4.6:0.46:0.046:0.046.

[0071] PC-12 cells were seeded into a 96-well cell culture plate (5×10 3 cells per well), and 1 mL of culture medium was added to each well. The culture medium was high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. The seeded PC-12 cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO2 for culture.

[0072] After the cells were cultured to a density of 80 - 90%, the original culture medium was discarded and replaced with 1 mL of the control group culture medium 2, and the cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO2 for continued culture for 48 h.

[0073] (4) Evaluation of cell viability by MTT method

[0074] The original culture medium of the cell models in (1)-(3) was discarded, gently washed 3 times with PBS, and replaced with 100 μL of culture medium containing MTT, which included additionally supplemented 1 mg / mL MTT, 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. The cells were transferred to a constant-temperature cell incubator at 37 °C and 5% CO2 for continued culture.

[0075] After 4 h of culture, the original culture medium was discarded and replaced with 100 μL of DMSO. The absorbance at 490 nm was measured on an enzyme-linked immunosorbent assay detector. Within a certain range, the cell viability value showed a linear relationship with the absorbance value at 490 nm.

[0076] The cell viabilities of the normally growing cell model, the control group cell model 1, and the control group cell model 2 were compared, and the results were as Figure 1 shown. From Figure 1It can be seen that, compared with the normally growing cell model, the cell viability of the control cell model 1 constructed by imitating the construction method of the Alzheimer's disease cell model commonly used in the current academic community has decreased significantly. This decrease is attributed to the preparation method of the control medium 1, which directly mixes the solution without Aβ oligomers with the medium used for normally growing cells, without considering the problems such as the decrease in the content of nutrients necessary for cell growth, such as serum and non-essential amino acids, caused by the mixing process. Therefore, the Alzheimer's disease cell model commonly used in the current academic community does not take this influence into account, resulting in the physiological changes of cells in the cell model not being completely attributed to Aβ oligomers, but also to the influence of the decrease in the content of nutrients necessary for cell growth. Therefore, it cannot truly simulate the physiological state of neurons in the brains of Alzheimer's disease patients.

[0077] However, by imitating the construction method of the Alzheimer's disease cell model in Example 1 of the present invention, the cell viability of the control cell model 2 constructed remains at 100%, showing no significant difference from the normally growing cell model. This indicates that the construction method of the Alzheimer's disease cell model provided by the present invention solves the above problems. The physiological changes of cells in the Alzheimer's disease cell model constructed by the method provided by the present invention can be completely attributed to the influence of Aβ oligomers. Therefore, it can more truly simulate the physiological state of neurons in the brains of Alzheimer's disease patients in vitro.

[0078] Example 3 MTT experiment to detect the cell viability of the constructed Alzheimer's disease cell model

[0079] The cell viability of the constructed Alzheimer's disease cell model (the model construction refers to Example 1) was evaluated by the MTT method.

[0080] PC-12 cells were inoculated into a 96-well cell culture plate (5×10 3 cells per well), and 1 mL of medium was added to each well. The medium was high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. The inoculated PC-12 cells were transferred to a constant-temperature cell culture incubator at 37°C and 5% CO2 for culture.

[0081] After the cells were cultured to a density of 80-90%, the original medium was discarded and replaced with 1 mL of medium containing Aβ oligomers at a concentration of 10 μM. The cells were transferred to a constant-temperature cell culture incubator at 37°C and 5% CO2 for continued culture.

[0082] After culturing for 24 or 48 h, discard the original medium, gently wash 3 times with PBS, and replace it with 100 μL of medium containing MTT, which includes an additional supplement of 1 mg / mL MTT, 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Transfer the cells to a constant temperature cell incubator at 37 °C and 5% CO2 for continued culture.

[0083] After culturing for 4 h, discard the original medium and replace it with 100 μL of DMSO. Measure the absorbance at 490 nm on an enzyme-linked immunosorbent assay detector. Within a certain range, the cell viability value shows a linear relationship with the absorbance value at 490 nm.

[0084] Compare the cell viability of the Alzheimer's disease cell model with that of normally growing cells. The results are as Figure 2 shown. It can be seen from Figure 2 that the cell viability of the Alzheimer's disease cell model is reduced by more than 75% compared with normal cells, and the cell viability is significantly reduced. Moreover, compared with the Alzheimer's disease cell model constructed for 24 h, the cell viability of the Alzheimer's disease cell model constructed for 48 h further decreases. The significant decrease in cell viability indicates to a certain extent that the present invention has successfully constructed an Alzheimer's disease cell model.

[0085] Example 4 Flow cytometry experiment to detect apoptosis and necrosis of the constructed Alzheimer's disease cell model

[0086] Detect the apoptosis and necrosis of the constructed Alzheimer's disease cell model (model construction refers to Example 1) by flow cytometry experiment.

[0087] Adjust the cell density of PC-12 cells to 5×10 4 / mL, inoculate into a 60 mm cell culture dish, and add 5 mL of medium. The medium is high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Transfer the inoculated PC-12 cells to a constant temperature cell incubator at 37 °C and 5% CO2 for culture.

[0088] After the cells are cultured to a density of 80-90%, discard the original medium and replace it with 5 mL of medium containing Aβ oligomers at a concentration of 10 μM. Transfer the cells to a constant temperature cell incubator at 37 °C and 5% CO2 for continued culture.

[0089] After culturing for 24 h, discard the original medium, gently wash 3 times with PBS, and replace it with 1 mL of 0.25% trypsin for digestion.

[0090] After digestion for 2 min, add 4 mL of high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Centrifuge the mixture at 4°C and 300×G for 5 min, and collect the precipitate. Discard the original medium, gently wash 3 times with PBS, centrifuge the mixture at 4°C and 300×G for 5 min, and collect the precipitate.

[0091] Dilute 1 mL of PBS containing 5 μL of Annexin V-FITC and 5 μL of PI and add it to the precipitate. On the flow cytometer, use a 488 nm laser for excitation to measure the cell counting gate and distinguish live cells, apoptotic cells, and necrotic cells. At least 5000 cells in each sample are counted and analyzed from within the gate.

[0092] Compare the apoptosis and necrosis of the Alzheimer's disease cell model with those of normally growing cells. The results are as Figure 3 and Figure 4 shown. Figure 3 and Figure 4 In, the Q1 region represents the proportion of necrotic cells, the Q2 region represents the proportion of cells in the late apoptotic stage, the Q3 region represents the proportion of cells in the early apoptotic stage, and the Q4 region represents the proportion of normally growing cells.

[0093] From Figure 3 and Figure 4 it can be seen that the proportions of necrotic cells, cells in the early apoptotic stage, and cells in the late apoptotic stage in the Alzheimer's disease cell model all increase significantly, and the proportion of normally growing cells decreases significantly. This result to a certain extent indicates that the present invention has successfully constructed an Alzheimer's disease cell model.

[0094] Example 5 Application of Alzheimer's Disease Cell Model in Studying the Pathogenesis of Alzheimer's Disease

[0095] Detect the changes in the concentrations of H + , Ca 2+ and ROS in the constructed Alzheimer's disease cell model (model construction refers to Example 1) by fluorescence dye staining method to study the pathogenesis of Alzheimer's disease.

[0096] Adjust the cell density of PC-12 cells to 5×10 4Inoculate at [X] cells / mL into a 29-mm glass-bottom cell culture dish, and add 1 mL of medium. The medium is high-glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin. Transfer the inoculated PC-12 cells to a constant-temperature cell incubator at 37 °C and 5% CO₂ for culture. After the cells are cultured to a density of 80 - 90%, discard the original medium and replace it with 1 mL of medium containing Aβ oligomers at a concentration of 10 μM. Then transfer the cells to a constant-temperature cell incubator at 37 °C and 5% CO₂ for continued culture.

[0097] After culturing for 0, 3, 6, 9, 12, and 24 h respectively, discard the original medium and replace it with medium containing 2 μM BCECF AM, 1 μM Fluo-4 AM, and 2 μM DCFH-DA respectively. Then transfer the cells to a constant-temperature cell incubator at 37 °C and 5% CO₂ for continued culture.

[0098] After culturing for 15 min, discard the original medium, gently wash the cells 3 times with PBS, and replace it with 1 mL of medium.

[0099] Image the cells under a laser confocal microscope, excite with a 488-nm laser, and collect fluorescence images at 500 - 550 nm. The results are as Figure 5 , Figure 6 and Figure 7 shown. As can be seen from Figure 5 , compared with the normally growing cells, the fluorescence intensity of BCECF AM in the Alzheimer's disease cell model decreases significantly with the increase in the stimulation time of the medium containing Aβ oligomers, corresponding to a significant increase in the concentration of H + with the increase in the stimulation time. As can be seen from Figure 6 and Figure 7 , compared with the normally growing cells, the fluorescence intensities of Fluo-4 AM and DCFH-DA in the Alzheimer's disease cell model first increase significantly and then decrease with the increase in the stimulation time of the medium containing Aβ oligomers, corresponding to a significant increase and then decrease in the concentrations of Ca 2+ and ROS respectively. This result shows that the Alzheimer's disease cell model constructed in the present invention can be used for the detection of H + , Ca 2+ and ROS, etc., and can be used to study the pathogenesis of Alzheimer's disease.

[0100] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for constructing a cell model of Alzheimer's disease, characterized in that, Including stimulating rat pheochromocytoma cells with β-amyloid oligomers to simulate the physiological changes of neurons in the brains of Alzheimer's disease patients, and constructing an Alzheimer's disease cell model.

2. The construction method according to claim 1, characterized in that Specifically including: (1) Preparing a culture medium containing β-amyloid oligomers, and the components of the culture medium include: β-amyloid oligomers, high-glucose DMEM, fetal bovine serum, non-essential amino acids, and penicillin-streptomycin; (2) Expanding the culture of rat pheochromocytoma cells until the density reaches 80%-90%; (3) Discarding the culture medium in step (2), replacing it with the culture medium containing β-amyloid oligomers in step (1), and continuing the culture to obtain an Alzheimer's disease cell model.

3. The construction method according to claim 2, characterized in that In step (1), the volume ratio of each component in the culture medium containing β-amyloid oligomers is β-amyloid oligomer solution: high-glucose DMEM: fetal bovine serum: non-essential amino acids: penicillin-streptomycin = 1: (4.54 - 18.61): (0.52 - 2.12): (0.05 - 0.21): (0.05 - 0.21), and the concentration of β-amyloid oligomers in the culture medium containing β-amyloid oligomers is 5 - 15 μM.

4. The construction method according to claim 2, characterized in that, In step (3), the culture temperature is 37 °C, and the culture time is 24 - 48 h.

5. The construction method according to claim 2, characterized in that The β-amyloid oligomers are prepared by the following method: dissolving β-amyloid peptide in hexafluoroisopropanol, standing for a reaction for a period of time, then adding high-glucose DMEM, standing for a reaction for a period of time again, centrifuging at low temperature and collecting the supernatant, bubbling with nitrogen, and then oscillating at a constant temperature to obtain β-amyloid oligomers.

6. The construction method according to claim 5, characterized in that The mass-volume ratio of the β-amyloid peptide to hexafluoroisopropanol is 1 mg: (300 - 500) μL; Preferably, the volume ratio of hexafluoroisopropanol to high-glucose DMEM is 1: (8 - 10).

7. The construction method according to claim 5, characterized in that The temperature of the standing reaction is 20 - 25 °C; the reaction time is 15 - 20 min; Preferably, the low-temperature centrifugation temperature is 4 °C; the centrifugal force is 14000×G; the centrifugation time is 10 - 15 min; Preferably, the constant-temperature oscillation temperature is 20 - 24 °C; the oscillation rate is 450 - 550 rpm; the oscillation time is 22 - 26 h.

8. An Alzheimer's disease cell model constructed by the construction method according to any one of claims 1 - 7.

9. Use of the Alzheimer's disease cell model according to claim 8 in studying the pathogenesis of Alzheimer's disease.

10. Use of the Alzheimer's disease cell model according to claim 8 in screening drugs for preventing or treating Alzheimer's disease.

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