Method for establishing blood-brain barrier organ aging model and method for intervening blood-brain barrier organ aging model

Abstract

The invention relates to a method for establishing a blood-brain barrier organ aging model and an intervention method for the model, and the method comprises the following steps: S1, model preparation: preparing cryopreserved brain microvascular endothelial cells, resuscitating the cryopreserved brain microvascular endothelial cells, preparing a culture bottle containing a culture medium for later use, preparing a culture chip and an inducer, and treating the culture chip and the inducer for later use; s2, cell culture: taking the recovered brain microvascular endothelial cells in S1, inoculating the brain microvascular endothelial cells in a culture bottle, culturing and carrying out cell passage to obtain brain microvascular endothelial cells in a logarithmic phase; s3, constructing a blood-brain barrier, inoculating the brain microvascular endothelial cells in the logarithmic phase obtained in the S2 to a chip, and culturing to obtain a blood-brain barrier model; and S4, constructing an aging model, taking the blood-brain barrier model in S3, and performing injury treatment to obtain the blood-brain barrier organ aging model. A D-galactose drug group is used for successfully inducing the aging injury of the blood-brain barrier organ model, and a reliable in-vitro blood-brain barrier organ aging injury model is provided.

Description

Technical fields:

[0001] This invention relates to the field of cell biology, and in particular to a method for establishing an organoid aging model of the blood-brain barrier and an intervention method for the model. Background Technology

[0002] With the increasing aging of the global population, neurodegenerative diseases have become a growing public health problem. Neurodegenerative diseases are a broad category of diseases caused by the degeneration of neurons, including Alzheimer's disease (AD) and Parkinson's disease (PD). Studies have indicated that aging is a major risk factor for most neurodegenerative diseases, and senescent cells may affect the function of surrounding cells by secreting related factors.

[0003] The blood-brain barrier (BBB) ​​is a highly selective barrier between brain tissue and the peripheral circulation. Composed of brain capillary endothelial cells, pericytes, and astrocyte terminale, it plays a crucial role in maintaining the homeostasis of the central nervous system. With age, the structure and function of the BBB change, leading to a series of age-related changes such as increased permeability and elevated oxidative stress levels, which in turn affect the normal function of the nervous system. For example, in Alzheimer's disease patients, BBB disruption manifests as reduced expression of tight junction proteins and degeneration of endothelial cells and pericytes. Therefore, constructing reliable organoid models of the blood-brain barrier and their aging models is of great significance for in-depth research into the physiological and pathological mechanisms of the blood-brain barrier and for developing therapeutic strategies for neurodegenerative diseases. In view of this, this application proposes a method for establishing a blood-brain barrier organoid aging damage model and an intervention method for this model. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention discloses a method for establishing a blood-brain barrier organoid aging model, characterized by comprising the following steps:

[0005] S1, Model preparation: Prepare frozen brain microvascular endothelial cells and revive them; prepare prepared culture medium and culture flasks for later use; prepare culture chips and inducing agents after treatment for later use.

[0006] S2, cell culture: take the revived brain microvascular endothelial cells from S1, inoculate them in a culture flask, add culture medium, culture and passage the cells to obtain brain microvascular endothelial cells in the logarithmic growth phase.

[0007] S3, construct the blood-brain barrier. Take the brain microvascular endothelial cells in the logarithmic growth phase obtained in S2 and the cell barrier model culture chip in S1. Seed the brain microvascular endothelial cells onto the chip, and then supplement the cell culture medium to the middle and left and right chambers. After culturing for a period of time, the blood-brain barrier organoid model is obtained.

[0008] S4. Construct an aging model. Take the blood-brain barrier organoid model and the inducer from S3. Replace the culture medium in the blood-brain barrier organoid model with the inducer. After the damage treatment is completed, the blood-brain barrier organoid aging model is obtained.

[0009] Specifically, the culture medium in the S2 culture flask is an endothelial cell culture medium containing 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin / streptomycin.

[0010] Specifically, the cell barrier model culture chip in S3 is a barrier function organoid chip, and the brain microvascular endothelial cells are seeded onto the lower surface of the membrane of the middle chamber of the chip.

[0011] Specifically, in step S3, before inoculating brain microvascular endothelial cells onto the cell barrier model culture chip, rat tail collagen needs to be added to pre-coat the lower culture chamber of the chip.

[0012] Specifically, in step S3, the density of brain microvascular endothelial cells seeded on the cell barrier model culture chip is 2.5 × 10⁻⁶. 6 The cell count was 15 μL, and the cell suspension was added at a rate of 10 cells / mL.

[0013] Specifically, in S3, the culture medium for the chip is added to the upper surface of the membrane of the chip's intermediate chamber, i.e., the liquid storage chamber of the chip's intermediate chamber, and the amount of culture medium added to the liquid storage chamber is 100 μL.

[0014] Specifically, the inducing agent in S4 is a mixture of the treatment agent and the culture medium, and the treatment agent is a D-galactose drug group.

[0015] Furthermore, an intervention method using the aforementioned blood-brain barrier organoid aging injury model includes the following operations:

[0016] The blood-brain barrier organoid aging model was constructed in the order of S1 to S4. In step S4, a mixture of inducer and intervention agent was added to the obtained blood-brain barrier organoid model. After incubation with the intervention agent, the blood-brain barrier organoid aging injury intervention model was obtained.

[0017] Specifically, the intervention agent is a nervonic acid solution.

[0018] Advantages and effects

[0019] This invention successfully induced aging damage in a blood-brain barrier organoid model by using D-galactose, providing a reliable in vitro model for studying the aging mechanism of BBB and the treatment of related diseases, and contributing to a deeper understanding of the pathophysiological processes of neurodegenerative diseases. Attached Figure Description

[0020] Figure 1 The present invention refers to brain microvascular endothelial cells in a conventional culture flask;

[0021] Figure 2 The brain microvascular endothelial cells in the barrier function organoid chip of the present invention;

[0022] Figure 3 Images showing the β-galactosidase staining results of Example 1 and the control group of the present invention;

[0023] Figure 4 Images showing the cell viability and mortality staining results of Example 1 and the control group of the present invention;

[0024] Figure 5 Images showing the ROS detection results of Example 1 and the control group of the present invention;

[0025] Figure 6 This is a statistical graph showing the expression changes of the target gene in Experiment Example 4 of this invention;

[0026] Figure 7 This is a statistical graph showing the cell viability values ​​in a comparative experiment of the intervention method of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but is not limited to the contents of the specification.

[0028] This invention relates to a method for establishing a blood-brain barrier organoid aging model, which includes the following steps.

[0029] S1, Model preparation: Prepare frozen brain microvascular endothelial cells and revive them; prepare culture medium and culture flasks containing the prepared medium; prepare culture chips and inducing agents after treatment for later use.

[0030] Specifically, cryopreserved brain microvascular endothelial cells are used. After being removed from the cryopreservation environment, the cells are thawed and then used for subsequent culture. The culture flasks are filled with endothelial cell culture medium containing 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin / streptomycin. The cell barrier model culture chip is the M1 barrier chip manufactured by DaXiang Technology. This cell barrier model culture chip has three chambers: a left chamber, a middle chamber, and a right chamber. Each chamber has its own liquid filling well, and the left and right chambers are connected to the middle chamber through channels. The middle chamber has a barrier membrane structure that divides the middle chamber into upper and lower chambers, both of which can be seeded with cells for cell culture.

[0031] S2, cell culture: take the revived brain microvascular endothelial cells from S1, seed them in a culture flask, add culture medium, culture and passage the cells to obtain brain microvascular endothelial cells in the logarithmic growth phase.

[0032] Specifically, the resuscitated brain microvascular endothelial cells from step S1 are seeded into culture flasks, and 5-10 mL of culture medium is added. The culture flasks are then placed in a cell culture incubator at 37°C and 5% CO2. The culture medium is changed every two days during the culture process, and the growth status is as follows. Figure 1 As shown. When the cells reach 80%-90% confluence, they are passaged, and brain microvascular endothelial cells in the logarithmic growth phase are used for subsequent blood-brain barrier construction.

[0033] S3, constructing the blood-brain barrier: Logarithmically growing brain microvascular endothelial cells obtained in S2 and the cell barrier model culture chip from S1 were used. Culture medium was added to the cell barrier model culture chip, and the brain microvascular endothelial cells were seeded onto the chip for culture. After seeding, the chip was inverted in an incubator and cultured for 3 hours to allow the cells to fully adhere and grow on the lower side of the barrier membrane. Subsequently, the chip was placed upright and culture medium was added. After a period of culture, the following results were obtained: Figure 2 The blood-brain barrier organoid model shown;

[0034] Specifically, the logarithmic growth phase brain microvascular endothelial cells obtained in step S2 are seeded onto an M1 chip. The seeding site is the lower layer of the barrier membrane in the middle chamber of the M1 chip. Before seeding, rat tail collagen is added to pre-coat the lower chamber of the chip's barrier membrane. The cells are then cultured on a shaker at 37°C for 3 hours to facilitate cell adhesion and growth on the chip. The brain microvascular endothelial cells are then seeded at a rate of 2-3 × 10⁻⁶ cells / cm². 6 pcs / cm 3Cells were seeded at a density below the barrier membrane in the middle chamber of the microfluidic M1 chip. Excessive density weakened the damage treatment effect of subsequent inducing agents, while insufficient density prevented the formation of the blood-brain barrier. 15 μL of cell suspension was then added, followed by incubation. During culture, the M1 cell barrier model chip was incubated upside down in an incubator for 3 hours to allow cell adhesion before adding more culture medium. Incubation shorter than 3 hours prevented cells from adhering properly to the barrier membrane structure, resulting in excessive adhesion to the bottom of the chamber and affecting barrier formation. A total of 200 μL of culture medium was added to the left and right chambers (which are connected), and 100 μL to the middle chamber. The chips were then cultured on a shaker at 30°C and 1 r / min. After 2 days of culture, a blood-brain barrier organoid model was obtained. The culture medium was then changed, and a blood-brain barrier organoid aging and damage model was constructed.

[0035] S4. Construct an aging model. Take the blood-brain barrier organoid model and the inducer from S3. Replace the culture medium in the blood-brain barrier organoid model with the inducer. After the damage treatment is completed, the blood-brain barrier organoid aging model is obtained.

[0036] Specifically, the cultured blood-brain barrier organoid model is taken, and the culture medium on the cell barrier model culture chip is replaced with an inducer. The inducer is a mixture of the treatment agent and the culture medium. The culture medium is brain microvascular endothelial cell culture medium, and the treatment agent is a D-galactose drug group. The D-galactose drug group used in this invention is a solid powder of D-galactose, which is dissolved in the culture medium to form an inducer. The concentration of D-galactose in the mixture is 45-60 mg / mL. After 2 days of treatment, the blood-brain barrier organoid aging model is constructed.

[0037] Example 1

[0038] Perform steps S1 to S4 of the above-described method for establishing an organoid aging and injury model of the blood-brain barrier, wherein the seeding density of brain microvascular endothelial cells in step B is 2.5 × 10⁻⁶. 6 pcs / cm 3 In step S4, the inducing agent used is a mixture of D-galactose at a concentration of 50 mg / mL and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0039] Example 2

[0040] This embodiment 2 uses essentially the same method as embodiment 1, except that the seeding density of brain microvascular endothelial cells in step S2 is 2 × 10⁻⁶. 6 pcs / cm 3 In step S4, the inducing agent used is a mixture of D-galactose at a concentration of 45 mg / mL and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0041] Example 3

[0042] This embodiment 3 uses essentially the same method as embodiment 1, except that the seeding density of brain microvascular endothelial cells in step S2 is 3 × 10⁻⁶. 6 pcs / cm 3 In step S4, the inducing agent used is a mixture of D-galactose at a concentration of 60 mg / mL and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0043] Comparative Example 1

[0044] This comparative example is operated in basically the same way as Example 1, except that the inducing agent used in step S4 is a mixture of D-galactose at a concentration of 25 mg / mL and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0045] Comparative Example 2

[0046] This comparative example is operated in basically the same way as Example 1, except that the inducing agent used in step S4 is a mixture of D-galactose at a concentration of 80 mg / mL and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0047] Comparative Example 3

[0048] This comparative example uses essentially the same method as Example 1, except that the seeding density of brain microvascular endothelial cells in step S2 is 1.5 × 10⁻⁶. 6 pcs / cm 3 After damage treatment, a blood-brain barrier organoid aging damage model was obtained.

[0049] Comparative Example 4

[0050] This comparative example uses essentially the same method as Example 1, except that the seeding density of brain microvascular endothelial cells in step S2 is 4 × 10⁻⁶. 6 pcs / cm 3 After processing, an organoid aging and injury model of the blood-brain barrier was obtained;

[0051] Comparative Example 5

[0052] This comparative example is operated in basically the same way as Example 1, except that the treatment agent used in step S4 is a mixture of glucose solution with a concentration of 35 mmol / L (5.4 mg / mL) and culture medium. After treatment, a blood-brain barrier organoid aging injury model is obtained.

[0053] During the culture process, it was observed that Comparative Example 3 did not form a blood-brain barrier within the same culture time as other examples and comparative examples, and no subsequent damage treatment was performed.

[0054] The blood-brain barrier organoid aging injury models obtained in Examples 1-3 and Comparative Examples 1, 2, 4, and 5 above, along with an untreated blood-brain barrier organoid model as a control group, were used to detect the following injury manifestations:

[0055] Experimental Example 1

[0056] Transendothelial Electrical Resistance (TEER) was measured using an IBACTEER resistance meter. After the blood-brain barrier organoids matured, the original solution in the chip was removed, and 200 μL and 100 μL of PBS solution were added to the left and middle reservoirs of each chip unit, respectively. The detection electrodes were then inserted into the left and middle reservoirs of the chip for TEER measurement. The formula is TEER (Ω·cm). 2 = (Measured hole resistance value - Blank hole resistance value) × Film area (cm²) 2 The more significant the damage effect, the lower the TEER value.

[0057] See Table 1 for specific test results.

[0058] Table 1

[0059]

[0060] The resistance of blood-brain barrier organoids was measured using an IBAC TEER resistance meter. The results showed that the TEER value of the control group was (22.07±0.21) Ω·cm. 2 The TEER value of the model group obtained after two days of treatment in Example 1 was significantly reduced to (19.46±0.98) Ω·cm. 2 This indicates that D-galactose treatment can disrupt the integrity of the blood-brain barrier.

[0061] Excessive D-galactose concentration (Comparative Example 2) severely damages the blood-brain barrier, making it insufficient to form a barrier structure. The electrical resistance value becomes extremely low, failing to meet application requirements and ultimately preventing the formation of an aging-related injury model. In contrast, excessively low D-galactose concentration (Comparative Example 1) produces very weak damage, causing almost no harm and also failing to establish an aging-related injury model. Excessive cell concentration dilutes the damaging effect of D-galactose, resulting in very weak damage.

[0062] Experimental Example 2

[0063] Detection of Papp value of fluorescent yellow

[0064] Prepare a 5 mg / mL stock solution of fluorescein using DMSO. Dilute the fluorescein stock solution to a 25 μg / mL working solution using PBS. Remove the culture medium from the test unit and rinse once with PBS. Add 200 μL of fluorescein working solution to the left well of the test unit and 100 μL of PBS to the middle chamber. Place the test chip back in the incubator on a shaker for dynamic incubation for 1–2 hours. After incubation, collect the supernatant from the middle chamber into a 96-well plate for fluorescence detection at excitation / emission wavelengths of 428 / 536 nm.

[0065] See Table 2 for specific test results.

[0066] Table 2

[0067]

[0068] The Papp value test results for the fluorescein permeability showed that the drug permeability of the model group obtained after 2 days of treatment in Example 1 was significantly higher than that of the control group, at (1.08±0.02)×10⁻⁶. -4 cm / s and (4.39±0.01)×10 -5 cm / s, indicating that D-galactose treatment increased the permeability of the blood-brain barrier, consistent with the decrease in TEER resistance.

[0069] Experimental Example 3

[0070] Example 1, which showed good modeling effect for aging damage, and the control group were compared to the intracellular β-galactosidase activity, live / dead cell levels, and intracellular LDH and ROS levels.

[0071] The detection method is as follows:

[0072] β-Galactosidase staining: Prepare the staining working solution, remove the original culture medium, rinse once with PBS, and fix with fixative at room temperature for 15 min. Then wash twice with PBS, 200 μL each time. Use a pipette to slowly rinse during washing to prevent cell detachment. Add 15 μL of staining solution to each well of the left and right chambers of the cell barrier model culture chip to cover the cell growth area. Add a small amount of staining solution (10 μL) to the middle chamber to prevent drying. Then incubate overnight in a carbon dioxide-free incubator at 37°C. During the process, a sealing film and outer PBS are required to prevent evaporation. After that, the staining results can be observed using the observation device. If crystals are encountered during observation, rinse with 70% ethanol solution to remove the staining solution. Add 30 μL of PBS and store in a 4°C refrigerator for up to one week for observation.

[0073] Live / dead cell staining: Prepare staining working solution, remove the original culture medium, add 15 μL of staining solution to the left and right chambers of the cell barrier model culture chip, and do not add staining solution to the middle chamber. Then observe the staining situation with a fluorescence microscope.

[0074] ROS detection: Using a ROS kit, dilute the probe to a ratio of 2000:1 (diluent:probe = 2000:1) to prepare the staining solution. Remove the original culture medium from the chip, do not rinse with PBS, and add 200 μL of staining solution to the left and right chambers of the chip under light-protected conditions. Add 30 μL of staining solution to the middle chamber to prevent drying. Incubate in the dark for 20 min, then remove the staining solution and rinse twice with 200 μL of PBS each time. Rinse slowly to prevent cell detachment. When observing under a fluorescence microscope, first irradiate with strong green fluorescence for 30-60 s. After observing the fluorescence excitation phenomenon, adjust the brightness and take a picture to obtain the staining image.

[0075] The final cell staining results are as follows: Figure 3 , Figure 4 and Figure 5 As shown.

[0076] Figure 3 The β-galactosidase staining results showed (A in the figure is the control group, B is the model group) that the β-galactosidase activity in the cells of the model group constructed in Example 1 was significantly increased, and the blue staining area was significantly more than that in the control group.

[0077] Figure 4 The results of live / dead cell staining showed that the green fluorescence intensity of the control group was greater than that of the model group, while the red fluorescence intensity was lower than that of the model group, indicating that more cells died in the model group.

[0078] Figure 5 The ROS detection results showed (A in the figure is the control group, B is the model group) that the intracellular ROS level in the model group was significantly increased. The green fluorescence intensity of the model group in Example 1 was higher than that in the control group, indicating that D-galactose treatment induced intracellular oxidative stress response.

[0079] Test Example 4

[0080] For Example 1, which showed good modeling performance, the damage model was subjected to the following PCR experiment: Cells in the chip were digested with trypsin and incubated for 5 minutes until cell detachment. Serum-containing culture medium was added to inactivate trypsin. The cell solution was transferred to an RNase-free centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The cell pellet was collected. Total RNA was extracted from the cells using the TRNzol method, reverse transcribed into cDNA, and then amplified by PCR. The reaction program was: 94℃ pre-denaturation for 30 seconds, 94℃ for 5 seconds, 60℃ for 30 seconds, for a total of 40 cycles. Primer sequences are shown in Table 3. GAPDH was used as an internal control, and 2...-ΔΔCt Calculate the change in target gene expression relative to the control group.

[0081] Table 3 Target gene and primer design

[0082]

[0083] like Figure 6 The bar graph PCR results shown indicate that, compared to the control group, the mRNA expression levels of tight junction proteins Occuldin and ZO-1 were downregulated in the injury model group (p<0.001), while the mRNA expression of heme oxygenase HO-1, antioxidant transcription factor Nrf2, and aging-related protein p53 were significantly upregulated (p<0.001). This suggests that the tight junction function of the blood-brain barrier organoids induced by D-galactose was affected, resulting in aging and oxidative stress damage within the cells, leading to increased expression of stress response protein genes. As can be seen from the data in the figure, the blood-brain barrier organoid aging injury model was successfully established using the model establishment method of this invention.

[0084] Further analysis of the expression of the target genes revealed that the D-galactose-induced aging mechanism of the blood-brain barrier may be related to oxidative stress. Oxidative stress disrupts the redox balance of cells, leading to cellular dysfunction and structural damage, which in turn affects the expression and function of tight junction proteins, increasing blood-brain barrier permeability. Downregulation of tight junction proteins ZO-1 and occludin further confirmed the destructive effect of oxidative stress on the blood-brain barrier structure. In addition, upregulation of HO-1 and Nrf2 mRNA expression indicated that cells initiated antioxidant defense mechanisms to cope with oxidative stress. HO-1 is an antioxidant enzyme, and its product has antioxidant and anti-inflammatory effects; Nrf... 2 is a major regulator of HO-1, enhancing cellular antioxidant capacity by activating the expression of antioxidant genes. However, this upregulation did not completely counteract the damage caused by D-galactose, suggesting that the cellular antioxidant defense capacity may be limited. The upregulation of p53 may be related to the cellular stress response to damage, indicating that the cells are under stress and may even initiate apoptosis. p53 is activated under oxidative stress, regulating the expression of downstream genes, leading to cell cycle arrest or apoptosis. In summary, D-galactose induces oxidative stress, disrupts the expression and function of tight junction proteins, and ultimately achieves the modeling method of this invention, which can construct an organoid aging damage model of hemorrhage-brain barrier.

[0085] Based on the ability to construct an organoid aging and injury model of the blood-brain barrier, this invention also intervenes in the blood-brain barrier. Specifically, during the establishment of the blood-brain barrier organoid aging and injury model, in step S4, when treating the blood-brain barrier with a mixture of D-galactose and culture medium, nervonic acid (NA) solution is simultaneously added to the mixture as an intervention solution to intervene in the aging and injury model. In the groups where the intervention effect is detected, this invention also detects the intervention effect of different concentrations of nervonic acid, including 1 μmol / L nervonic acid, 50 μmol / L nervonic acid, and 100 μmol / L nervonic acid. The final aging and injury model is then subjected to CCK-8 cell viability testing. The degree of blood-brain barrier damage in the aging and injury model is determined by the cell viability value. Simultaneously, the uninterrupted blood-brain barrier organoid aging and injury model obtained by this method is also subjected to CCK-8 cell viability testing. The test results are shown in Table 4.

[0086] Table 4

[0087]

[0088]

[0089] The results showed that, after the same treatment time, the cell viability of the blood-brain barrier injury model treated with nervonic acid was higher than that of the untreated aging injury model. Furthermore, the experimental procedures described in Examples 1 to 4 were performed on the nervonic acid-treated blood-brain barrier organoid aging injury model. The results showed that the blood-brain barrier was also in a state of aging injury after nervonic acid intervention; that is, the duration of the aging injury state in the blood-brain barrier organoid model was prolonged before the cells lost activity. This injury intervention method allows for a longer period of time for subsequent research (such as injury mechanisms, various cellular indicator levels caused by injury), observation, and recording using the blood-brain barrier organoid aging injury model of this application. Moreover, the survival time of cells in the aging injury model after injury intervention was longer than that of the untreated aging injury model, as confirmed by CCK-8 cell viability testing.

[0090] Regarding the operational methods of injury intervention, the researchers also conducted different intervention methods. One method was to add 50 mg / mL D-galactose solution to S4 for 24 hours based on Example 1, and then add nervonic acid solution for intervention.

[0091] Another approach is to add nervonic acid solution before step D treatment, based on Example 1, and then perform S4 operation 24 hours after the injury intervention to establish a blood-brain barrier organoid aging injury model.

[0092] After detecting CCK-8 cell viability using the two intervention methods described above, the results were compared with those of the present invention, which involved treatment with D-galactose solution followed by intervention with nervonic acid solution. Figure 7 As shown in the statistical chart Figure 7 In the mean square brackets: 'a' indicates a difference from the control group, 'ab' indicates a difference from both the control group and the G1 group, P < 0.05;

[0093] CON: Control group, cultured in ordinary culture medium for 48 hours;

[0094] G1: First treat with 50 mg / mL D-galactose for 24 h, then switch to normal culture medium and continue culturing for another 24 h;

[0095] G2: First, treat with 50 mg / mL D-galactose for 24 h, then switch to 50 μM NA for 24 h.

[0096] G3: First, use 50 μM NA for 24 h, then switch to 50 mg / mL D-galactose for 24 h.

[0097] G4: First, treat with a mixture of 50 mg / mL D-galactose and 50 μM NA for 24 h, then culture in normal medium for 24 h.

[0098] As can be seen from the cell viability test values ​​in the statistical graph, the damage intervention effect of the present invention is the best, with the model in an ideal state of aging and damage, and a relatively long cell survival time.

[0099] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all embodiments here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for establishing a blood-brain barrier organoid aging model, characterized by comprising the following steps: It comprises the following steps ​ S1, model preparation, prepare cryopreserved brain microvascular endothelial cells and recover them, prepare culture bottles containing prepared culture medium for standby, prepare culture chips and induce agents after treatment for standby; S2, cell culture, take the recovered brain microvascular endothelial cells in S1, inoculate them in culture bottles for culture and cell passage, and obtain brain microvascular endothelial cells in the logarithmic growth phase; S3, construction of blood-brain barrier, take the logarithmic growth phase brain microvascular endothelial cells obtained in S2 and the cell barrier model culture chip in S1, inoculate the brain microvascular endothelial cells on the chip for culture, supplement the culture medium to the chip, and after a period of culture, obtain a blood-brain barrier organoid model; S4, construction of aging model, take the blood-brain barrier organoid model in S3 and the inducing agent, replace the culture medium in the blood-brain barrier organoid model with the inducing agent, and after the damage treatment is completed, obtain a blood-brain barrier organoid aging model.

2. The method of claim 1, wherein the blood brain barrier organoid aging model is established by: The culture medium in the S2 culture bottle is an endothelial cell culture medium containing 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin / streptomycin.

3. The method for establishing a blood-brain barrier organoid aging model according to claim 1, characterized in that: The cell barrier model culture chip in S3 is a barrier function organoid chip, and the brain microvascular endothelial cells are inoculated under the barrier membrane.

4. The method for establishing a blood-brain barrier organoid aging model according to claim 1, characterized in that: Before inoculating the brain microvascular endothelial cells on the cell barrier model culture chip in S3, mouse tail collagen needs to be added to coat the lower culture chamber of the chip in advance.

5. The method for establishing a blood-brain barrier organoid aging model according to claim 1, characterized in that: The density of the brain microvascular endothelial cells seeded on the cell barrier model culture chip in S3 is 2-3 x 10 6 cells / mL, and the added amount of the cell suspension is 15 μL.

6. The method of claim 1, wherein the blood brain barrier organoid aging model is established by: In S3, the culture medium supplemented to the cell barrier model culture chip is: 200 μL of culture medium is added to the left and right chambers of the chip, and 100 μL of culture medium is added to the middle chamber.

7. The method of claim 1, wherein the blood brain barrier organoid aging model is established by: The inducing agent in S4 is a mixture of a treatment agent and a culture medium, and the treatment agent is a D-galactose drug group.

8. An intervention method using the senescence damage model according to claims 1 to 7, characterized by: It comprises the following operations According to the order of S1 to S4, a blood-brain barrier organoid aging model is constructed, a mixture of an inducing agent and an intervention agent is added to the obtained blood-brain barrier organoid model in step S4, and after incubation with the intervention agent, a blood-brain barrier organoid aging damage intervention model is obtained.

9. The intervention method for the senescence-impaired model according to claim 8, characterized by: The intervention agent is a nervonic acid solution.

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