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

Abstract

The invention discloses a THC-induced in-vitro blood-brain barrier injury model as well as a construction method and application thereof. The construction method comprises the following steps: preparing a THC solution; the toxic effect of THC on hCMEC / D3 is evaluated, and the administration dosage and administration time are determined; constructing a co-culture BBB model; and evaluating comprehensive indexes of the blood-brain barrier after the THC acts. According to the invention, the hCMEC / D3 cell and U-87MG cell co-culture BBB model is constructed, a detailed construction process and evaluation indexes are defined, the problem that THC is difficult to directly carry out water-soluble dissolution administration is improved, the pathological change process of THC-induced BBB injury is comprehensively evaluated, and a reference is provided for THC precise medication. The invention provides a more real and accurate THC-induced BBB injury model, and the model provides a more effective tool for researching the influence of THC on the central nervous system and the potential neurotoxicity of THC.

Description

Technical Field

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

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

[0003] In biomedical research, in vitro models are important tools, especially in vitro models for studying the BBB. Traditional in vitro BBB models include cell lines and tissue slice models, which have made some progress in simulating the structure and function of the BBB, but still face limitations, such as difficulty in simulating the real dynamic blood-brain barrier interaction and lack of specific disease models. The current research trend tends to develop in vitro BBB models that can better simulate the function of the BBB. These models can accurately simulate the microenvironment, cell-cell interactions and substance passage mechanisms of the BBB, including the development of models using multidimensional cell culture systems, microfluidics and other technologies.

[0004] Δ 9 -Tetrahydrocannabinol (THC) is one of the main active ingredients in cannabis plants, mediating the addictiveness of cannabis, and its mechanism of action in the nervous system has been widely studied. THC can affect neurons and glial cells through various pathways, but the effects of THC on the BBB are still uncertain, and the specific mechanisms involved are still unclear. Therefore, constructing a model of THC-induced BBB damage will help to gain a deeper understanding of the mechanism of action of THC on the BBB and its potential neurotoxicity.

[0005] Currently, the development of cannabis drugs is also ongoing. THC must first pass through the BBB to exert its neurological effects. The lipid-soluble properties of THC and the type 1 cannabinoid receptor (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 quickly evaluate the ability of drugs to pass through the BBB and their potential toxicity. The development of a THC-induced BBB damage model can provide researchers with a more realistic neurotoxicity assessment platform. 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) construct a multicellular co-culture system, introduce astrocytes and co-culture them with hCMEC / D3 cells to build a more comprehensive and dynamic model;

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

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

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

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

[0013] (1) Preparation of THC solution: THC was dissolved in DMSO, and then diluted with DMSO, PBS, and basal culture medium according to the steps of the present invention, and finally added to the transwell chamber.

[0014] (2) Evaluate the toxic effect of THC on hCMEC / D3 and determine the dosing regimen: After using different concentrations of THC (e.g., 0, 1, 10, 20, 40 μM) to act on hCMEC / D3 cells for different periods of time (e.g., 0, 6, 12, 24, 48 h), observe the cell morphological changes caused by THC using an inverted microscope, evaluate the effect of THC on the proliferation activity and toxicity of hCMEC / D3 using CCK8, and evaluate the direct effect of THC on the tight junctions between hCMEC / D3 cells using Western Blot. Finally, determine the dosing regimen of THC, including the dosage (THC concentration) and the dosing time (the time of acting on hCMEC / D3 cells).

[0015] (3) Construction of a co-culture BBB model: A co-culture model of endothelial cells and astrocytes was constructed in vitro, and the interaction between cells was increased to more realistically simulate the structure and function of the BBB.

[0016] (4) Evaluation of comprehensive indicators of the blood-brain barrier after the action of THC: Use molecular markers (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 status 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 its intercellular connection and barrier formation; by detecting inflammatory 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 damage model, which provides a more effective tool for studying the effects of THC on the central nervous system and its potential neurotoxicity.

[0018] Preferably, the present invention adopts multiple methods such as gradual dilution, ultrasonic vibration, water bath, etc. in the process of dissolving THC.

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

[0020] More preferably, the selected endothelial cells of the present invention are human brain microvascular endothelial cells (hCMEC / D3 cells), and the selected astrocytes are human brain 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 Pieces / cm 2 The seeding density of U-87MG cells was 5×10 4 Pieces / cm 2 .

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

[0023] More preferably, the Transwell chamber is produced by Corning Incorporated, with a 24-well plate specification, and the coated chamber is 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 0.012 mg / mL rat tail collagen type I working solution to coat the Transwell chamber.

[0025] More preferably, in the present invention, U-87MG cells are seeded on the bottom layer of the Transwell chamber rather than the bottom layer of the 24-well plate, so that they can better interact with hCMEC / D3 cells.

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

[0027] More preferably, the method for constructing a co-culture model provided by the present invention, in addition to detecting the barrier properties and transport function of the constructed model, also includes an assessment of the physiological and pathological state 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 BBB model of co-culture 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 directly dissolve in water for administration;

[0031] (3) The pathological changes of THC-induced BBB damage were comprehensively evaluated, providing a reference for the precise use of THC. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0035] Figure 4 :BBB functional evaluation, including tight junction protein expression, cell permeability detection, and cell transmembrane resistance detection.

[0036] Figure 5 :Assessment of BBB physiological status, including detection of inflammatory factors and oxidative stress factors. DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to the accompanying drawings and examples. In the present invention, the cells, reagents and materials involved can be purchased from the market or obtained by conventional techniques in the art.

[0038] Example 1: Preparation of THC solution

[0039] Dissolve THC in DMSO and dilute as follows and add 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 → ultrasonic vibration for 5 min → 60 °C water bath for 10 min → 1 mM THC

[0043] 100μL 25mM THC + 900μL PBS → ultrasonic vibration for 5min → 60℃ water bath for 10min → 2.5mM THC;

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

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

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

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

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

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

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

[0051] The basal culture 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 solution prepared in steps (b) and (c) must be prepared and used immediately and cannot be stored for a long time.

[0053] Example 2: Evaluating the toxic effects of THC on hCMEC / D3 and determining the dosing regimen

[0054] The steps of toxicity experiment include:

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

[0056] (2) Cell inoculation: Select cells in the logarithmic growth phase and inoculate hCMEC / D3 cells: the cell density is 3×10 6 Pieces / cm 2 hCMEC / D3 cell suspension was added to a 6-well plate at a volume of 400 μL / well, and then 1 mL / well of complete culture medium was added. The plate was covered and placed in a cell culture incubator for subsequent experiments. 100 μL of hCMEC / D3 cell suspension (cell density was 4×10 5 Pieces / cm 2 ). Place the cells in a cell culture incubator and culture for 24 hours before use in subsequent experiments.

[0057] (3) When the cell growth confluence of the 6-well plate reached 80%, the intracellular culture medium was aspirated and the cells were washed three times with 500 μL / well PBS. Then, the basal culture medium containing different concentrations of THC prepared in Example 1 (0, 1, 10, 20, 40, 80, 160 μM; wherein the THC concentration of 0 μM represents the comparative example) was added to the hCMEC / D3 cells for different time periods (0, 3, 6, 12, 24, 48 h; wherein the time of 0 h represents the comparative example) and subsequent experiments were performed.

[0058] (4) The cell morphology after THC treatment was observed by inverted microscope. It can be seen that the cell bodies of the Vehicle group were plump and translucent, with good refractive index, smooth cell membrane, clear boundaries, and good cell morphology; while in the THC group, as the drug concentration continued to increase and the action time continued to prolong, the cell bodies shrank and became round, and some cytoplasms showed vacuolar structures. As the action concentration increased and the action time prolonged, some cells even detached from the wall and floated, and died.

[0059] (5) The effect of THC on the proliferation activity and toxicity of hCMEC / D3 was evaluated by CCK8. First, the basal medium containing different concentrations of THC (0, 1, 10, 20, 40, 80, 160 μM) prepared in Example 1 was added to the 96-well plate in step (2) of Example 2. After the 96-well plate was incubated in an incubator for an appropriate time (3, 6, 12, 24, 48 h), 10 uCCK-8 solution was added to each well (be careful not to generate bubbles in the well). Then the 96-well plate was continued to be incubated in the incubator for 2 h. Finally, the absorbance at 450 nm was measured with an ELISA instrument. The proliferation activity of the cells is expressed by the cell survival rate, and the specific calculation method is as follows:

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

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

[0062] Ac: Absorbance of control well (culture medium containing cells, CCK-8 reagent, no THC)

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

[0064] The experimental results show that after 24 hours of exposure to 40 μM THC, the cell activity changes significantly, and THC has a toxic effect on the cells (such as Figure 1 A and B).

[0065] (6) The cells after THC treatment in step (3) of Example 2 were collected, proteins were extracted, and Western Blot was performed to 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 in the tight junction proteins (Claudin5, Occludin) between hCMEC / D3 cells (e.g. Figure 1 C) and time-dependent decreases (as shown in Figure 1 D) and significant changes occurred at 10 μM and 12 h.

[0066] (7) The above results confirmed the concentration and time of THC administration: In the subsequent THC administration, we will use 10 μM THC for 12 h to explore the effect of THC on the co-culture BBB model.

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

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

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

[0070] (2) Prepare rat tail collagen type I working solution: Prepare 0.006 mol / L acetic acid working solution with sterile acetic acid and sterilized ddH2O, filter with a 0.22 μm microporous filter membrane and set aside. 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 chamber, and place the Transwell cell culture plate in a clean bench overnight for later use.

[0072] (4) Cell inoculation: Select cells in the logarithmic growth phase for cell inoculation: ① Place the coated 24-well Transwell chamber upside down in a 6-well plate and inoculate the cells at a density of 5×10 4 Pieces / cm 2 ②After the U-87MG cells have attached to the wall, take out the 6-well plate from the incubator, place the Transwell chamber upright in its original 24-well plate, and add the prepared U-87MG cell complete culture medium to the lower chamber. ③The cell density is 6×10 5 Pieces / cm 2 ④ Add the hCMEC / D3 cell suspension to the upper chamber of the Transwell chamber at a volume of 200 μL / well. ④ Add the culture medium level inside and outside the Transwell chamber to the same level, cover the plate, and place it in a cell culture incubator for culture.

[0073] (5) Model construction completed: The transendothelial electrical resistance (TEER) between the upper and lower chambers of the Transwell chamber was recorded every day using a transendothelial electrical resistance meter. The specific steps are as follows: First, soak the electrode of the transmembrane resistance meter in alcohol for 10 minutes to disinfect the electrode; then soak it in ECM culture medium for 10 minutes to balance the electrode; connect the electrode to the transmembrane resistance meter, insert the long end of the electrode into the lower chamber of the Transwell chamber, and the short end into the upper chamber of the Transwell chamber, and keep the electrode in a vertical and static state, and record the resistance value on the resistance meter; change the position where the electrode is inserted, and re-measure the resistance value at different positions of the same chamber in the same way. Measure the resistance value of at least 3 different positions in the same chamber, and then take the average of the three resistance values, which is the transmembrane 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 ) = (target cell resistance - blank cell resistance) × cell area

[0075] The results showed that the cells formed the highest TEER value on the third day and then remained stable for a period of time (such as Figure 3 Therefore, the time point after 3 days of culture was selected as the time point when the co-culture BBB model was successfully constructed, and subsequent experiments were performed after that.

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

[0077] This embodiment includes the following steps:

[0078] (1) The 10 μM THC prepared in Example 1 was added to the co-culture BBB model successfully constructed in Example 3, and subsequent experiments were performed 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 incubated with THC was measured to evaluate the structural integrity and functional status of the BBB. A higher TEER value generally indicates a better barrier function. The results showed that THC can reduce the TEER value of the co-cultured BBB model (e.g. Figure 4 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 molecules. Weigh an appropriate amount of NaF, add HBSS solution to dissolve, and dilute to 1 mg / mL to obtain a NaF stock solution. Then measure an appropriate amount of the stock solution and add HBSS solution to obtain a 20 μg / mL NaF standard solution. Dilute in the same way to obtain 2.5, 1, 0.5, 0.1, 0.01, and 0.001 μg / mL NaF standard curve solutions.

[0081] After the TEER determination is completed, the culture medium in the upper and lower chambers of the Transwell chamber is discarded, and an equal volume of HBSS solution as the culture medium is added to the upper and lower chambers, and the chambers are placed in an incubator for 30 minutes; the HBSS solution is discarded, 400 μL of NaF working solution (20 μg / mL) is added to the upper chamber, and 1.5 mL of blank HBSS solution is added to the lower chamber, and the chambers are placed in an incubator for incubation for 1 hour. Then 100 μL of the transport solution is taken out from the lower chamber into a light-shielded 96-well plate, and the fluorescence intensity of NaF in the taken out solution is measured on an ELISA reader (excitation wavelength is 485 nm, emission wavelength is 528 nm). At the same time, a series of NaF standard curve solutions of different concentrations configured above are also added to the 96-well plate at 100 μL / well for determination. According to the concentration and fluorescence intensity of the NaF standard curve solution, a standard curve with fluorescence intensity as the horizontal axis (i.e., X value) and NaF standard curve solution concentration as the vertical axis (i.e., Y value) is calculated, and then the NaF content in the well to be tested is calculated according to the fluorescence intensity of the well to be tested and the standard curve, thereby characterizing the NaF content in the lower chamber.

[0082] According to the results, THC caused an increase in the NaF content in the lower chamber, indicating that THC increased the permeability of the co-cultured BBB model (e.g. Figure 4 B).

[0083] (3) Expression of tight junction proteins: hCMEC / D3 cells in the upper chamber after THC treatment in step (2) of Example 4 were collected, proteins were extracted, and Western Blot experiments were performed to detect the expression of Claudin5, Occludin, and JAMA to evaluate the effect of THC on tight junctions between hCMEC / D3 cells in the co-cultured BBB model. The results showed that THC significantly reduced the expression of tight junction proteins (such as Figure 4 A).

[0084] (4) BBB status assessment: In order 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 pro-inflammatory cytokines IL-1β and IL-6 increased after THC treatment, while the expression level of anti-inflammatory cytokine IL-10 decreased after THC treatment (e.g. Figure 5 EG), 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 through a kit, and found that the expression of ROS and MDA increased after the action of THC, and the expression of GPX and CAT decreased after the action of THC (as shown in FIG. Figure 5 AD), indicating that THC also caused the disorder of oxidative stress in the co-culture BBB model. Comprehensive analysis of these inflammatory factors and oxidative stress indicators showed that THC treatment caused the normal physiological state of the co-culture BBB model to be destroyed and turned into a pathological state, which may be the basis for the damage of blood-brain barrier function.

[0085] In summary, the present invention constructs a method for THC-damaged co-cultured BBB model, clarifies the detailed construction process and evaluation indicators, comprehensively explains and characterizes the physiological and pathological changes of THC-induced BBB damage; at the same time, it improves the problem that THC is difficult to dissolve in water-soluble solutions due to its fat solubility, and provides a reference for the subsequent construction of THC administration models. This comprehensive evaluation method not only helps to reveal the process of changes in BBB function caused by THC, but also can evaluate its coping 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 defined according to common knowledge, including:

[0087] The term "BBB" refers to the blood-brain barrier (BBB).

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

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

[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 a variety of 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 culture medium.

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

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

[0095] The term "TEER" refers to transepithelial cell membrane electrical resistance, which can help assess the integrity of the barrier and its ability to block foreign substances. Higher TEER values ​​generally indicate that the barrier function of the cell layer is strong; while lower TEER values ​​may indicate that the integrity of the barrier is impaired.

[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 involved in regulating immune responses and inflammatory processes.

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

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

[0100] The term “ROS” refers to reactive oxygen species, a class of oxygen-containing chemical species that includes oxidants and free radicals, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals.

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

[0102] The term "GPX" refers to glutathione peroxidase, an important class of antioxidant enzymes that are primarily 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 primarily responsible for breaking down hydrogen peroxide into water and oxygen, thereby preventing 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 method for constructing a THC-induced in vitro blood-brain barrier injury model. It is characterized in that The steps include: S1, prepare THC solution; S2, evaluate the toxic effects of THC on hCMEC / D3 and determine the dosage and time of administration, including: After different concentrations of THC were applied to hCMEC / D3 cells for different periods of time, the cell morphological changes caused by THC were observed using an inverted microscope. The effects of THC on the proliferation activity and toxicity of hCMEC / D3 were evaluated by CCK8. The direct effects of THC on the tight junctions between hCMEC / D3 cells were evaluated by Western Blot to determine the administration concentration and action time of THC. S3, construction of co-culture BBB model; S4, Evaluation of comprehensive indicators of blood-brain barrier after THC action.

2. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 1, It is characterized in that The step S1 comprises: Dissolve THC in DMSO, then dilute it with DMSO, PBS, and basal culture medium according to certain steps, and finally add it to the transwell chamber.

3. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 1, It is characterized in that In step S2: The THC administration concentration is 1 to 40 μM; The action time is 6 to 48 hours.

4. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 1, It is characterized in that The step S3 comprises: Establishing a co-culture model of endothelial cells and astrocytes in vitro can increase cell-cell interactions and better simulate the structural function of the BBB.

5. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 1, It is characterized in that The step S4 comprises: The molecular marker NaF was used to observe its diffusion between the upper and lower chambers to evaluate the permeability of the BBB model to molecules. The transmembrane resistance between the upper and lower chambers in the BBB model was measured to evaluate the structural integrity and functional status of the BBB. The expression of tight junction proteins on the cell surface in the blood-brain barrier model was detected to evaluate its intercellular connection and barrier formation. The physiological status of the BBB was comprehensively judged by detecting indicators related to inflammatory factors and oxidative stress.

6. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 3, It is characterized in that In step S2: The administration concentration of THC is 10 μM, and the action time is 12 h.

7. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 4, Features: The endothelial cells are human brain microvascular endothelial cells hCMEC / D3 cells, and the astrocytes are human brain astrocytes U-87MG cells.

8. The method for constructing a THC-induced in vitro blood-brain barrier damage model according to claim 7, Features: The seeding density of hCMEC / D3 cells was 6×10 5 Pieces / cm 2 The seeding density of U-87MG cells was 5×10 4 Pieces / cm 2 .

9. A THC-induced in vitro blood-brain barrier injury model constructed according to the method for constructing a THC-induced in vitro blood-brain barrier injury model according to any one of claims 1 to 8.

10. Use of the THC-induced in vitro blood-brain barrier damage model according to claim 9 in studying the effects of THC on the central nervous system and its potential neurotoxicity.

Images