A THC-induced in vitro blood-brain barrier injury model, construction method and its application

Abstract

The present invention discloses a THC-induced in vitro blood-brain barrier injury model, a construction method and its application, including: preparing a THC solution; evaluating the toxic effect of THC on hCMEC / D3 and determining the dosing dose and dosing time; constructing a co-culture BBB model; evaluating comprehensive indicators of the blood-brain barrier after the action of THC. The present invention constructs a co-culture BBB model of hCMEC / D3 cells and U-87MG cells, clarifies the detailed construction process and evaluation indicators, improves the problem that THC is difficult to be directly administered by water-soluble dissolution, comprehensively evaluates the pathological change process of THC-induced BBB injury, and provides a reference for the precise use of THC. The present invention provides a more real and accurate THC-induced BBB injury model, and this model provides a more effective tool for studying the influence of THC on the central nervous system and its potential neurotoxicity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a THC-induced in vitro blood-brain barrier injury model, a construction method, an evaluation index and its application. Background Art

[0002] Neuroscience is a research field related to the structure, function and diseases of the nervous system, involving a wide range of studies from the molecular level to the whole biological behavior. The blood-brain barrier (BBB) is an important part of the cerebrovascular system, which maintains the stability of the internal microenvironment of the brain by restricting the free passage of substances. In neuroscience research, it is crucial to understand the function of the BBB and study its injury mechanism. The injury of the BBB is related to various drug addictions and their associated nervous system diseases. Therefore, researchers are committed to developing various in vitro BBB injury models to simulate these disease states and study their specific mechanisms of action.

[0003] In biomedical research, in vitro models are important tools, especially those for studying the BBB. Traditional in vitro BBB models include cell line and tissue slice models. Although certain progress has been made in simulating the structure and function of the BBB, these models still face limitations, such as difficulties in simulating the real dynamic blood-brain barrier interaction and the lack of specific disease models. Currently, the research trend is to develop in vitro BBB models that can better simulate the function of the BBB, which can accurately simulate the microenvironment of the BBB, cell-cell interactions and the mechanism of substance passage. Specifically, it includes the development of models using techniques such as multi-dimensional cell culture systems and microfluidic technology.

[0004] Δ 9 Delta-9-tetrahydrocannabinol (THC) is one of the main active ingredients in the cannabis plant, mediating the addiction of cannabis. Its mechanism of action in the nervous system has been widely studied. THC can affect neurons and glial cells through various pathways, but currently, the effect of THC on the BBB is still uncertain, and the specific mechanism involved is still unclear. Therefore, constructing a model of THC-induced BBB injury helps to deeply understand the mechanism of action of THC on the BBB and its potential neurotoxicity.

[0005] Currently, the development of cannabis-based drugs is also ongoing. THC exerts its neurological effects by first passing through the BBB. The lipophilic properties of THC and the cannabinoid receptor type 1 (CB1R) on brain microvascular endothelial cells also constitute the structural basis for THC to damage the BBB. Therefore, it is particularly important to develop a model that can rapidly evaluate the ability of drugs to cross the BBB and their potential toxicity. The development of a THC-induced BBB damage model can provide a more realistic platform for researchers to evaluate neurotoxicity. Summary of the Invention

[0006] The technical problems to be solved by the present invention include:

[0007] (1) To clarify the toxic effects of THC on human brain microvascular endothelial cells (hCMEC / D3);

[0008] (2) To construct a multi-cell co-culture system, introduce astrocytes and co-culture them with hCMEC / D3 cells to construct a more comprehensive and dynamic model;

[0009] (3) To determine the THC administration method and conditions;

[0010] (4) To comprehensively evaluate the effects of THC on the structure and function of the BBB.

[0011] The technical solution of the present invention is as follows:

[0012] A method for constructing a THC-induced in vitro blood-brain barrier damage model, comprising the following steps:

[0013] (1) Preparation of the THC solution: Dissolve THC in DMSO, and then dilute it with DMSO, PBS, and basal medium respectively according to the steps described in the present invention. Finally, add it to the transwell chamber.

[0014] (2) Evaluating the toxic effects of THC on hCMEC / D3 and determining the dosing regimen: After treating hCMEC / D3 cells with different concentrations of THC (e.g., 0, 1, 10, 20, 40 μM) for different times (e.g., 0, 6, 12, 24, 48 h)), observe the cell morphological changes caused by the action of THC through an inverted microscope, evaluate the effects of THC on the proliferation activity and toxic effects of hCMEC / D3 by CCK8, and evaluate the direct effects of THC on the tight junctions between hCMEC / D3 cells by Western Blot. Finally, clarify the dosing regimen of THC, including the dosing dose (the concentration of THC) and the dosing time (the time of acting on hCMEC / D3 cells).

[0015] (3) Construction of a co-culture BBB model: A model of co-culture of endothelial cells and astrocytes is constructed in vitro, with increased cell-cell interaction, more realistically simulating the structural and functional characteristics of the BBB.

[0016] (4) Evaluation of comprehensive indicators of the blood-brain barrier after THC treatment: Use a molecular marker (NaF) to observe its diffusion between the upper and lower chambers to evaluate the permeability of the BBB model to molecules; measure the transmembrane resistance between the upper and lower chambers in the BBB model to evaluate the structural integrity and functional state of the BBB; detect the expression of tight junction proteins (such as Claudin5, Occludin, JAMA, etc.) on the cell surface in the blood-brain barrier model to evaluate the formation of cell-cell junctions and barriers; by detecting inflammation factor-related indicators (such as IL-1β, IL-6, IL-10) and oxidative stress-related indicators (such as ROS, MDA, GPX, CAT), the physiological state of the BBB can be comprehensively judged.

[0017] The present invention provides a more realistic and accurate THC-induced BBB injury model, which provides a more effective tool for studying the effects of THC on the central nervous system and its potential neurotoxicity.

[0018] Preferably, in the process of dissolving THC in the present invention, multiple methods such as stepwise dilution, ultrasonic oscillation, and water bath are adopted.

[0019] More preferably, the present invention selects the action concentration of THC to be 10 μM and the action time to be 12 h.

[0020] More preferably, the endothelial cells selected in the present invention are human cerebral microvascular endothelial cells (hCMEC / D3 cells), and the astrocytes selected are human cerebral astrocytes (U-87MG cells).

[0021] More preferably, in the method for constructing the co-culture model provided by the present invention, the seeding density of hCMEC / D3 cells is 6×10 5 cells / cm 2 , and the seeding density of U-87MG cells is 5×10 4 cells / cm 2 .

[0022] More preferably, the cells are replaced with fresh medium multiple times as needed during the culture period and washed with PBS.

[0023] More preferably, the Transwell chamber is produced by Corning, a 24-well plate specification is selected, and the chamber is coated with a polycarbonate filter membrane with a pore size of 0.4 μm.

[0024] More preferably, when constructing the co-culture BBB model, the present invention selects a working solution of rat tail collagen type I at 0.012 mg / mL to coat the Transwell chamber.

[0025] More preferably, the present invention inoculates U-87MG cells on the bottom layer of the Transwell chamber instead of the bottom layer of the 24-well plate, enabling them to interact better with hCMEC / D3 cells.

[0026] More preferably, the present invention selects to co-culture the cells for 3 days to construct a stable and complete BBB model.

[0027] More preferably, the method for constructing the co-culture model provided by the present invention, in addition to detecting the barrier characteristics and transport functions of the constructed model, also includes the evaluation of the physiological and pathological states of the model, including inflammatory response and oxidative stress response.

[0028] The beneficial effects of the present invention include:

[0029] (1) The present invention constructs a co-culture BBB model of hCMEC / D3 cells and U-87MG cells, and clarifies the detailed construction process and evaluation indicators;

[0030] (2) It improves the problem that THC is difficult to be directly administered by water-soluble dissolution;

[0031] (3) It comprehensively evaluates the pathological change process of THC-induced BBB injury, providing a reference for the precise use of THC. Description of the Drawings

[0032] Figure 1 : The effects of THC on the activity of hCMEC / D3 cells and the expression level of tight junction proteins.

[0033] Figure 2 : Schematic diagram of the construction process of the BBB co-culture model.

[0034] Figure 3 : Growth curve of hCMEC / D3 cells, represented by TEER value.

[0035] Figure 4 : Functional evaluation of the BBB, including the expression of tight junction proteins, detection of cell permeability, and detection of transcellular electrical resistance.

[0036] Figure 5 : Physiological state evaluation of the BBB, including the detection of inflammatory factors and oxidative stress factors. Detailed Embodiments

[0037] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. In the present invention, the cells, reagents, and materials involved can all be obtained through regular commercial purchases or by conventional technical means in this field.

[0038] Example 1: Preparation of THC Solution

[0039] Dissolve THC in DMSO, and then dilute it according to the following steps and add it to the transwell chamber as needed:

[0040] (a) 3.145 mg THC powder + 1 mL DMSO → 10 mM THC

[0041] 7.86 mg THC powder + 1 mL DMSO → 25 mM THC;

[0042] (b) 100 μL 10 mM THC + 900 μL PBS → sonicate for 5 min → water bath at 60 °C for 10 min → 1 mM THC

[0043] 100 μL 25 mM THC + 900 μL PBS → sonicate for 5 min → water bath at 60 °C for 10 min → 2.5 mM THC;

[0044] (c) 1 μL 1 mM THC + 999 μL basal medium → 1 μM THC

[0045] 5 μL 1 mM THC + 995 μL basal medium → 5 μM THC

[0046] 10 μL 1 mM THC + 990 μL basal medium → 10 μM THC

[0047] 8 μL 2.5 mM THC + 994 μL basal medium → 20 μM THC

[0048] 16 μL 2.5 mM THC + 992 μL basal medium → 40 μM THC

[0049] 32 μL 2.5 mM THC + 992 μL basal medium → 80 μM THC

[0050] 64 μL 2.5 mM THC + 992 μL basal medium → 160 μM THC.

[0051] The basal medium should be preheated to 37 °C in advance.

[0052] The THC solution prepared in step (a) can be stored stably for a long time; the THC solutions prepared in steps (b) and (c) must be prepared and used immediately and cannot be stored for a long time.

[0053] Example 2: Evaluate the toxic effect of THC on hCMEC / D3 and determine the dosing regimen

[0054] The steps of the toxic effect experiment include:

[0055] (1) Cell culture: The brain microvascular endothelial cells hCMEC / D3 cells are cultured in the complete medium for hCMEC / D3 cells. The complete medium for hCMEC / D3 cells is prepared by mixing 5% fetal bovine serum, 1% endothelial cell growth factor, 1% double antibody, and 93% ECM medium. The cells are cultured in a constant temperature cell incubator at 5% CO2 and 37°C.

[0056] (2) Cell seeding: Select cells in the logarithmic growth phase and perform seeding of hCMEC / D3 cells: A cell suspension of hCMEC / D3 cells with a density of 3×10 6 cells / cm 2 is added to a 6-well plate with a medium volume of 400 μL / well, and then 1 mL / well of the complete medium is added. The plate lid is covered and placed in the cell incubator for culture for subsequent experiments. In a 96-well plate, 100 μL of the hCMEC / D3 cell suspension (cell density of 4×10 5 cells / cm 2 ) is added. After culturing in the cell incubator for 24 h, it is used for subsequent experiments.

[0057] (3) When the cell growth confluence in the 6-well plate reaches 80%, the intracellular medium is aspirated, and the cells are washed three times with 500 μL / well of PBS. Then, the basal medium containing different concentrations of THC prepared in Example 1 (0, 1, 10, 20, 40, 80, 160 μM; where the THC concentration of 0 μM represents the control group) is used to act on hCMEC / D3 cells for different times (0, 3, 6, 12, 24, 48 h; where the time of 0 h represents the control group), and then subsequent experiments are carried out.

[0058] (4) Observe the cell morphology after the action of THC through an inverted microscope. It can be seen that the cells in the Vehicle group have plump and bright cell bodies, good refractive properties, smooth cell membranes and clear boundaries, and the cell morphology is good; while for the cells in the THC group, with the continuous increase of the drug concentration and the continuous extension of the action time, the cell bodies shrink and become round, and some cytoplasm shows vacuolar-like structures. With the increase of the action concentration and the extension of the action time, some cells even detach from the wall and float and die.

[0059] (5) Evaluate the effect of THC on the proliferation activity and toxicity of hCMEC / D3 cells by CCK8. First, add the basal medium containing different concentrations of THC (0, 1, 10, 20, 40, 80, 160 μM) prepared in Example 1 to the 96-well plate in step (2) of Example 2. After incubating the 96-well plate in the incubator for an appropriate time (3, 6, 12, 24, 48 h), add 10 μL of CCK-8 solution to each well (pay attention not to generate bubbles in the wells). Then continue to incubate the 96-well plate in the incubator for 2 h. Finally, measure the absorbance at 450 nm using a microplate reader. The proliferation activity of the cells is represented by the cell survival rate, and its specific calculation method is as follows:

[0060] Cell survival rate = [(As - Ab) / (Ac - Ab)] × 100%

[0061] As: Absorbance of the experimental well (medium containing cells, CCK-8 reagent, THC)

[0062] Ac: Absorbance of the control well (medium containing cells, CCK-8 reagent, without THC)

[0063] Ab: Absorbance of the blank well (medium without cells and THC, CCK-8 reagent)

[0064] It was found through the experimental results that after treating the cells with 40 μM THC for 24 h, the cell activity changed significantly, and THC had a toxic effect on the cells (as shown in Figure 1 A and B).

[0065] (6) Collect the cells after THC treatment in step (3) of Example 2, extract proteins, perform Western Blot, and detect the expression of Claudin5 and Occludin to evaluate the direct effect of THC on the tight junctions between hCMEC / D3 cells. The results showed that THC caused a concentration-dependent decrease (as shown in Figure 1 C) and a time-dependent decrease (as shown in Figure 1 D) in the concentration of tight junction proteins (Claudin5, Occludin) between hCMEC / D3 cells, and significant changes occurred at 10 μM and 12 h.

[0066] (7) Confirm the administration concentration and time of THC based on the above results: When performing THC administration in the follow-up, we will use 10 μM of THC for 12 h to explore the effect of THC on the co-cultured BBB model.

[0067] Example 3: Construction of a co-cultured BBB model

[0068] The regulatory role of BBB is mainly achieved by astrocytes communicating with endothelial cells, thereby regulating the function and role of BBB. We constructed a co-culture model of endothelial cells and astrocytes in vitro to better simulate the structural function of BBB. The steps are as follows (as Figure 2 shown):

[0069] (1) Cell culture: Brain microvascular endothelial cells were cultured in hCMEC / D3 cell complete medium. hCMEC / D3 cell complete medium was prepared by mixing 5% fetal bovine serum, 1% endothelial cell growth factor, 1% double antibody, and 93% ECM medium. Astrocyte U-87MG cells were cultured in U-87MG cell complete medium. U-87MG cell complete medium was prepared by mixing 8% fetal bovine serum, 1% double antibody, and 91% DMEM medium. Both types of cells were cultured in a constant temperature cell incubator at 5% CO2 and 37°C.

[0070] (2) Preparation of rat tail collagen type I working solution: Prepare a 0.006 mol / L acetic acid working solution with sterile acetic acid and sterilized ddH2O, filter it with a 0.22 μm microporous filter membrane, and store it for later use. Dilute the rat tail collagen type I stock solution with the acetic acid working solution to a 0.012 mg / mL rat tail collagen type I working solution.

[0071] (3) Coating: Add 200 μL of rat tail collagen type I working solution to the upper layer of the Transwell cell culture insert, place the Transwell cell culture plate in the ultra-clean bench overnight, and store it for later use.

[0072] (4) Cell seeding: Select cells in the logarithmic growth phase for cell seeding: ① Invert the coated 24-well Transwell insert in a 6-well plate, add a U-87MG cell suspension with a cell density of 5×10 4 cells / cm 2 to the bottom of the Transwell insert at a medium volume of 200 μL / well, cover the plate lid, and place it in the cell culture incubator for 4 h to allow the cells to adhere and grow. ② After the U-87MG cells have adhered, take out the 6-well plate from the incubator, place the Transwell insert upright in its original 24-well plate, and add the prepared U-87MG cell complete medium to the lower chamber. ③ Add an hCMEC / D3 cell suspension with a cell density of 6×10 5 cells / cm 2 to the upper chamber of the Transwell insert at a medium volume of 200 μL / well. ④ Make the liquid levels of the media inside and outside the Transwell insert flush, cover the plate lid, and place it in the cell culture incubator for culture.

[0073] (5)Model construction completed: Every day, the transendothelial electrical resistance (TEER) between the upper and lower chambers of the Transwell chamber was recorded using an epithelial volt-ohmmeter. The specific steps were as follows: First, the electrode plates of the transendothelial electrical resistance meter were soaked in alcohol for 10 minutes to disinfect the electrode plates; then soaked in ECM medium for 10 minutes to balance the electrode plates; the electrode plates were connected to the transendothelial electrical resistance meter, the long ends of the electrode plates were inserted into the lower chamber of the Transwell chamber, and the short ends were inserted into the upper chamber of the Transwell chamber, and the electrode plates were kept vertical and stationary, and the resistance value on the resistance meter was recorded; the insertion position of the electrode plates was changed, and the resistance values at different positions of the same chamber were measured again in the same way. The resistance values at at least 3 different positions of the same chamber were measured, and then the average value of the three resistance values was taken as the transendothelial electrical resistance value of this chamber. The resistance values of the blank group and the experimental group were measured in the same way, and the TEER value was calculated according to the following formula.

[0074] TEER (Ω·cm 2 ) = (resistance of the target chamber - resistance of the blank chamber) × chamber area

[0075] It was found that the cells formed the highest TEER value on the third day and remained stable for a period of time thereafter (as Figure 3 shown). Therefore, 3 days after culturing was selected as the time point when the co-culture BBB model was successfully constructed, and subsequent experiments were carried out thereafter.

[0076] Example 4: Evaluation of blood-brain barrier indicators after THC treatment

[0077] This example includes the following steps:

[0078] (1) 10 μM THC prepared in Example 1 was added to the successfully constructed co-culture BBB model in Example 3, and subsequent experiments were carried out after incubation for 12 h.

[0079] (2) TEER measurement: According to the method in step (5) of Example 3, the TEER value between the upper and lower chambers of the Transwell chamber after THC incubation was detected to evaluate the structural integrity and functional state of the BBB. A higher TEER value usually indicates a better barrier function. It was found that THC could reduce the TEER value of the co-culture BBB model (as Figure 4 shown in C).

[0080] (3) Permeability marker determination: Use a molecular marker (NaF) to observe its diffusion between the upper and lower chambers to evaluate the permeability of the blood-brain barrier to the molecule. Weigh an appropriate amount of NaF, dissolve it in HBSS solution and make up the volume to 1 mg / mL to obtain a NaF stock solution. Then measure an appropriate amount of the stock solution, dilute it with HBSS solution to obtain a 20 μg / mL NaF standard solution, and dilute it in the same way to obtain NaF standard curve solutions with concentrations of 2.5, 1, 0.5, 0.1, 0.01 and 0.001 μg / mL.

[0081] After the TEER measurement is completed, discard the culture media in the upper and lower chambers of the Transwell chamber, add HBSS solution with the same volume as the culture media to both the upper and lower chambers, and place it in the incubator for 30 min; discard the HBSS solution, add 400 μL of NaF working solution (20 μg / mL) to the upper chamber, add 1.5 mL of blank HBSS solution to the lower chamber, and incubate it in the incubator for 1 h. Then take out 100 μL of the transport solution from the lower chamber and place it in a light-shielded 96-well plate, and measure the fluorescence intensity of NaF in the taken-out solution on an enzyme-linked immunosorbent assay (ELISA) reader (excitation wavelength is 485 nm, emission wavelength is 528 nm). At the same time, add a series of different concentrations of NaF standard curve solutions prepared above to the 96-well plate at 100 μL / well for measurement. According to the concentration and fluorescence intensity of the NaF standard curve solution, calculate the standard curve with fluorescence intensity as the abscissa (i.e., X value) and the concentration of the NaF standard curve solution as the ordinate (i.e., Y value), and then calculate the NaF content in the test well according to the fluorescence intensity of the test well and the standard curve, so as to characterize the NaF content in the lower chamber.

[0082] According to the results, THC can cause an increase in the NaF content in the lower chamber, indicating that THC increases the permeability of the co-cultured BBB model (as Figure 4 shown in B).

[0083] (3) Tight junction protein expression: Collect the hCMEC / D3 cells in the upper chamber after the action of THC in step (2) of Example 4, extract proteins, and perform Western Blot experiments to detect the expression of Claudin5, Occludin, and JAMA to evaluate the effect of THC on the tight junctions between hCMEC / D3 cells in the co-cultured BBB model. The results show that the action of THC significantly reduces the expression of tight junction proteins (as Figure 4 shown in A).

[0084] (4) BBB status assessment: To comprehensively evaluate the physiological and pathological status of the co-cultured BBB model, the present invention detected inflammatory cytokines, including IL-1β, IL-6, and IL-10, using an ELISA kit to characterize the inflammatory response. The results showed that the expression levels of the pro-inflammatory cytokines IL-1β and IL-6 increased after THC treatment, while the expression level of the anti-inflammatory cytokine IL-10 decreased after THC treatment (as shown in Figure 5 E-G), indicating that THC activated the inflammatory response in the co-cultured BBB model. The present invention also detected the expression of some oxidative stress molecules and antioxidant enzymes using a kit, and the results showed that the expression levels of ROS and MDA increased after THC treatment, while the expression levels of GPX and CAT decreased after THC treatment (as shown in Figure 5 A-D), indicating that THC also caused disorders in oxidative stress in the co-cultured BBB model. By comprehensively analyzing these inflammatory factors and oxidative stress indicators, it was found that THC treatment disrupted the normal physiological state of the co-cultured BBB model and shifted it to a pathological state, which may be the basis for blood-brain barrier function impairment.

[0085] In summary, the present invention constructed a method for damaging the co-cultured BBB model by THC, clarified the detailed construction process and evaluation indicators, and comprehensively elaborated and characterized the changes in the physiological and pathological processes of THC-induced BBB damage; at the same time, it improved the problem that THC is difficult to dissolve in water-soluble solutions due to its lipophilicity, providing a reference for the subsequent construction of a THC administration model. This comprehensive evaluation method not only helps to reveal the process of THC-induced changes in BBB function but also can evaluate its response ability under pathological conditions. This is of great significance for studying the role of THC in neurological diseases and developing targeted treatment strategies.

[0086] Unless otherwise specified, the terms in the present invention are interpreted by common general knowledge, including:

[0087] The term "BBB" refers to the Blood-Brain Barrier (BBB).

[0088] The term "THC" refers to Δ 9 -tetrahydrocannabinol (Tetrahydrocannabinol, THC), also known as: dronabinol, drobinone, Δ 1 -tetrahydrocannabinol, etc.;

[0089] The term "transwell chamber" refers to a permeable cell culture chamber that can perform experiments on the permeability, barrier function, and substance transport function of cell layers.

[0090] The term "PBS" refers to phosphate buffered saline, which is mainly used for cell washing and dilution.

[0091] The term "HBSS" refers to Hanks' Balanced Salt Solution, a buffer solution commonly used in cell culture. It contains various salts and nutrients to help maintain the physiological state of cells and does not contain phenol red.

[0092] The term "DMEM" refers to a widely used basal medium.

[0093] The term "ECM" refers to endothelial cell medium, which is used for the optimal growth of normal human microvascular endothelial cells in vitro.

[0094] The term "DMSO" refers to dimethyl sulfoxide.

[0095] The term "TEER" refers to trans-epithelial electrical resistance, which can help evaluate the integrity of the barrier and its ability to block external substances. A higher TEER value usually indicates a stronger barrier function of the cell layer; while a lower TEER value may imply impaired integrity of the barrier.

[0096] The term "NaF" refers to sodium fluorescein, a commonly used fluorescent dye.

[0097] The term "IL-1β" refers to interleukin-1β, an important inflammatory mediator that participates in regulating immune responses and inflammatory processes.

[0098] The term "IL-6" refers to interleukin-6, an important inflammatory mediator that participates in regulating immune responses and inflammatory processes.

[0099] The term "IL-10" refers to interleukin-10, an important inflammatory mediator that participates in regulating immune responses and inflammatory processes.

[0100] The term "ROS" refers to reactive oxygen species, a class of oxygen-containing chemical substances that include oxidants and free radicals, such as superoxide anion, hydrogen peroxide, and hydroxyl radical.

[0101] The term "MDA" refers to malondialdehyde, a marker of lipid peroxidation that can reflect the degree of oxidative stress in the body.

[0102] The term "GPX" refers to glutathione peroxidase, a class of important antioxidant enzymes that are mainly responsible for reducing and neutralizing organic peroxides in the body, thereby protecting cells from oxidative damage.

[0103] The term "CAT" refers to catalase, which is mainly responsible for decomposing hydrogen peroxide into water and oxygen, and can prevent hydrogen peroxide from causing oxidative damage to cells.

[0104] The term "Western Blot" refers to protein blotting, which is a common experimental technique in the biological field for detecting and analyzing proteins. It can quantitatively and qualitatively analyze proteins, and identify the presence, size, and expression level of specific proteins.

[0105] The term "ELISA" refers to enzyme-linked immunosorbent assay, which is a common experimental method in the biological field for detecting and quantifying antigens or antibodies in samples.

Claims

1. A Δ 9 -tetrahydrocannabinol-induced in vitro blood-brain barrier injury model construction method, characterized in that, The steps include the following: S1, Configure Δ 9 -tetrahydrocannabinol solution, and add Δ 9 -tetrahydrocannabinol is dissolved in DMSO, diluted with DMSO, PBS, and basal medium respectively, and finally added to the transwell chamber; the dilution is carried out by stepwise dilution combined with ultrasonic oscillation and water bath method; S2, Evaluate Δ 9 - The toxic effects of tetrahydrocannabinol on hCMEC / D3 and determine the dosage and administration time, including: After treating hCMEC / D3 cells with Δ-tetrahydrocannabinol at a concentration of 1 - 40 μM for 6 - 48 h, the morphological changes of the cells caused by the action of Δ-tetrahydrocannabinol were observed through an inverted microscope, and the effect of Δ-tetrahydrocannabinol on the proliferation activity of hCMEC / D3 and its toxic effect were evaluated by CCK8, and the direct effect of Δ-tetrahydrocannabinol on the tight junctions between hCMEC / D3 cells was determined, as well as the administration concentration and action time of Δ-tetrahydrocannabinol; 9 9 9 9 9 ​​​​​ S3. Construct a co-culture blood-brain barrier model by constructing a model of co-culture of endothelial cells and astrocytes in vitro; the seeding density of the hCMEC / D3 cells is 6×10 5 cells / cm 2 , and the seeding density of the U-87 MG cells is 5×10 4 cells / cm 2 . Seed the U-87 MG cells on the bottom layer of the Transwell chamber to allow them to interact with the hCMEC / D3 cells; S4, Δ 9 - Evaluation of comprehensive indicators of blood-brain barrier after the action of tetrahydrocannabinol.

2. The method for constructing a Δ 9 -tetrahydrocannabinol-induced in vitro blood-brain barrier injury model, characterized in that The step S4 includes: Using the molecular marker NaF to observe its diffusion between the upper and lower chambers to evaluate the permeability of the blood-brain barrier model to molecules; measuring the transmembrane resistance between the upper and lower chambers in the blood-brain barrier model to evaluate the structural integrity and functional state of the blood-brain barrier; detecting the expression of tight junction proteins on the cell surface in the blood-brain barrier model to evaluate the formation of cell-cell junctions and barriers; and comprehensively judging the physiological state of the blood-brain barrier by detecting inflammation factor-related indicators and oxidative stress-related indicators.

3. The Δ according to claim 1 9 - A method for constructing an in vitro blood-brain barrier injury model induced by tetrahydrocannabinol, characterized in that In the step S2: The said Δ 9 The administration concentration of Δ-9-tetrahydrocannabinol is 10 μM, and the action time is 12 h.

4. A Δ-tetrahydrocannabinol-induced in vitro blood-brain barrier injury model constructed by the method according to any one of claims 1-3 9 -tetrahydrocannabinol-induced in vitro blood-brain barrier injury model 9 -tetrahydrocannabinol-induced in vitro blood-brain barrier injury model.

5. A Δ according to claim 4 9 -tetrahydrocannabinol-induced in vitro blood-brain barrier injury model in the study of Δ 9 -tetrahydrocannabinol on the central nervous system and its potential neurotoxicity.

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