Establishment method of smog disease vascular endothelial cell model
The established vascular endothelial cell model of smoke disease was solved by culturing vascular endothelial cells in vitro and using heat-inactivated cerebrospinal fluid to stimulate culture medium for patients with hot-inactivated smog disease, which solved the problem that the existing model was limited to specific susceptible genes, and achieved effective research on the pathogenesis and drug screening of smoke disease.
Patent Information
- Application Number
- CN202510545363.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-08-08
AI Technical Summary
The existing vascular endothelial cell model of smoke disease is mainly limited to specific susceptibility genes, and it is impossible to comprehensively study multiple susceptibility genes, and the lack of stable animal models limits the in-depth research on the pathogenesis of smoke disease and drug treatment.
By culturing vascular endothelial cells in vitro and stimulating culture medium with heat-inactivated cerebrospinal fluid in patients with smoke disease, a vascular endothelial cell model that can reflect multiple susceptible genes was established.
This model successfully simulates the pathophysiological status of vascular endothelial cells in smoke disease in vitro, improving the reliability and efficiency of studying the pathogenesis of smoke disease and drug screening, which is low in cost and easy to construct.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of disease models, and in particular to a method for establishing a moyamoya disease vascular endothelial cell model and its application. Background Art
[0002] Moyamoya disease (MMD) is a chronic, progressive ischemic cerebrovascular disease of unknown etiology, characterized by chronic, progressive stenosis or occlusion of the distal ends of the bilateral internal carotid arteries and the development of an abnormal network of vessels at the base of the brain. This network of vessels resembles a puff of smoke on cerebral angiography, earning it the name "Moyamoya disease." The annual incidence of MMD in China is approximately 1.14 per 100,000 people, and is increasing annually. The annual incidence of cerebrovascular events (including stroke) caused by MMD in adults reaches 2.4-5.7%. Furthermore, MMD is also highly prevalent in children and young adults in my country, making it a significant cause of cerebrovascular accidents. Among ischemic strokes in children, MMD is the cause in up to 22%. Typical complications of MMD include hemiplegia, dysarthria, aphasia, cognitive impairment, and intracranial hemorrhage, leading to a high incidence of disability and even death.
[0003] Moyamoya disease (Moyamoya) is a progressive, chronic vascular stenosis characterized by progressive stenosis or occlusion of the intracranial internal carotid arteries (ICAs) and their proximal branches, followed by the formation of aberrant collateral vessels. The term "moyamoya" ("smoke puff" in Japanese) refers to the abnormal collateral vessels that appear adjacent to the stenotic vessels on cerebral angiography. Pathological specimens of Moyamoya disease patients show no arteriosclerotic or inflammatory changes leading to occlusion in the affected vessels. Instead, vascular occlusion results from a combination of endothelial cell proliferation and luminal thrombosis. The tunica media is typically thinned, and the elastic lamina is irregular. Moyamoya-associated collaterals are typically dilated perforating arteries, a combination of pre-existing and newly developed vessels. These collaterals show evidence of stress associated with increased flow, including disruption of the elastic lamina, thinning of the tunica media within the vessel wall, and the presence of microaneurysms. The mechanisms underlying these pathological changes remain unclear, and therefore, animal or cell models for mechanistic investigation are urgently needed for Moyamoya disease research.
[0004] Currently, research on the pathogenesis of moyamoya disease is primarily conducted in in vitro cell models. However, the currently commonly used vascular endothelial cell moyamoya disease model is constructed by overexpressing and knocking out specific genes. It is limited to a specific susceptibility gene and cannot target multiple possible susceptibility genes, limiting its value. Further exploration of the pathogenesis of moyamoya disease and screening of therapeutic drugs must be conducted in higher-level in vivo models. However, there is still no reliable and stable animal model for moyamoya disease, and modeling using the discovered susceptibility genes has failed.
[0005] Therefore, there is an urgent need for a stable cell model that can simply, stably and intuitively reflect the vascular pathological changes of moyamoya disease, especially the status of microvascular endothelial cells, to study the molecular mechanism of the pathogenesis and drug treatment of moyamoya disease. Summary of the Invention
[0006] The present invention aims to provide a method for establishing a moyamoya disease vascular endothelial cell model and its application.
[0007] In one aspect, the present invention provides a method for establishing a vascular endothelial cell model of moyamoya disease, the method comprising the following steps: (1) isolating vascular endothelial cells at a density of 1.25×10 5 -1.25×10 7 / ml and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 for at least 24 hours; (2) stimulating the vascular endothelial cells obtained in step (1) with cerebrospinal fluid stimulation medium; the cerebrospinal fluid stimulation medium contains 2.5% heat-inactivated cerebrospinal fluid from patients with moyamoya disease.
[0008] In one embodiment, the method comprises the following steps: (1) isolating vascular endothelial cells at a concentration of 1.25×10 6 / ml and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 for 24 hours; (2) stimulating the vascular endothelial cells obtained in step (1) with cerebrospinal fluid stimulation medium; the cerebrospinal fluid stimulation medium contains 2.5% heat-inactivated cerebrospinal fluid from patients with moyamoya disease.
[0009] In one embodiment, the temperature for thermal inactivation of cerebrospinal fluid of patients with moyamoya disease is 50 (±10)° C., and the inactivation time is not less than 20 minutes.
[0010] In one embodiment, the temperature for heat inactivation of cerebrospinal fluid of patients with moyamoya disease is 50° C., the inactivation time is 20 minutes, and the cerebrospinal fluid container is gently shaken at least every 5 minutes.
[0011] In one embodiment, the cerebrospinal fluid of a moyamoya disease patient is derived from a moyamoya disease patient aged 6-65 years, and the moyamoya disease patient does not suffer from hypertension, diabetes, hemolytic disease, immune disease, heart disease, or atherosclerotic disease.
[0012] In one embodiment, the seeding density of the vascular endothelial cells is 1.25×10 6 / ml.
[0013] In one embodiment, the cerebrospinal fluid stimulation medium is a complete HBMEC medium, which includes 1% P / S dual antibody and 1% endothelial cell growth supplement.
[0014] In one embodiment, the cerebrospinal fluid stimulation medium is a complete HBMEC medium, which includes 1% P / S dual antibody, 1% endothelial cell growth supplement and 2.5% heat-inactivated cerebrospinal fluid from patients with moyamoya disease.
[0015] In one embodiment, the endothelial cell growth supplement is EGM-2.
[0016] In one embodiment, the vascular endothelial cells are human brain microvascular endothelial cells (HBMECs).
[0017] In one embodiment, the culture medium is HBMEC complete culture medium, and the HBMEC complete culture medium is DMEM / F-12 (Cat. No. 11320033).
[0018] In one embodiment, the well plate inoculated with the vascular endothelial cells is a 6-well plate with a seeding density of 2.5×10 5 -2.5×10 7 / well, each well contains 2 ml of culture medium. Preferably, the seeding density is 2.5×10 6 / hole.
[0019] In one embodiment, the method comprises the steps of reviving, seeding and passaging a vascular endothelial cell line to obtain the vascular endothelial cells.
[0020] In one embodiment, the method comprises the steps of seeding and passage a vascular endothelial cell line to obtain the vascular endothelial cells.
[0021] In one embodiment, the method for inoculating the vascular endothelial cell line is as follows: 1.5 ml of the cell line is mixed with HBMEC complete medium in a 1:1 ratio, transferred to a 50 ml centrifuge tube, 6 ml of HBMEC complete medium is added to the centrifuge tube, and gently pipetted to mix; 800 rpm, 4 ° C, centrifuged for 5 minutes; discarded the supernatant in a sterile operating table, added 6 ml of HBMEC complete medium and evenly resuspended the cells; the cell resuspension is transferred to a cell culture flask and cultured in a constant temperature cell culture incubator at 37 ° C with 5% CO2.
[0022] In one embodiment, the method for passaging the vascular endothelial cell line is as follows: when the cells grow to a cell confluency of 90%, the culture medium is discarded; each bottle of cells is gently washed twice with 1-2 mL of PBS; the PBS is discarded; 2 mL of a digestion solution mixed with 0.25% trypsin and 0.02% EDTA is added, and the cells are gently shaken to allow the digestion solution to fully contact the cells; after observation under a microscope, when the cells become rounded, 2 mL of HBMEC complete culture medium is quickly added to terminate digestion, and the cells are gently pipetted; centrifugation is performed at 800 rpm and 4°C for 5 minutes; the supernatant is discarded; the cells are resuspended in HBMEC complete culture medium, placed in a culture flask, and after the cells are shaken to mix, the culture flask is transferred to a constant temperature cell culture incubator at 37°C and containing 5% CO2 for culture.
[0023] In one embodiment, the method further comprises the step of constructing a cell model using lentiviral transfection, adenoviral transfection or plasmid transfection methods.
[0024] On the other hand, the present invention provides a moyamoya disease vascular endothelial cell model, which is obtained by using the above-mentioned method for establishing a moyamoya disease vascular endothelial cell model.
[0025] In one embodiment, the proliferation capacity of vascular endothelial cells in the moyamoya disease vascular endothelial cell model is enhanced.
[0026] In one embodiment, the proliferation capacity of vascular endothelial cells stimulated by cerebrospinal fluid of patients with moyamoya disease is enhanced compared with vascular endothelial cells stimulated by cerebrospinal fluid of normal persons or compared with vascular endothelial cells without cerebrospinal fluid stimulation.
[0027] In one embodiment, the angiogenic capacity of the vascular endothelial cells in the moyamoya disease vascular endothelial cell model is enhanced.
[0028] In one embodiment, the angiogenic capacity of vascular endothelial cells stimulated by cerebrospinal fluid of a moyamoya disease patient is enhanced compared with vascular endothelial cells stimulated by cerebrospinal fluid of a normal person or compared with vascular endothelial cells without cerebrospinal fluid stimulation.
[0029] In one embodiment, the S phase ratio of vascular endothelial cells in the moyamoya disease vascular endothelial cell model is significantly increased.
[0030] In one embodiment, the S phase ratio of vascular endothelial cells stimulated by cerebrospinal fluid of patients with moyamoya disease is significantly increased compared with vascular endothelial cells stimulated by cerebrospinal fluid of normal persons or compared with vascular endothelial cells without cerebrospinal fluid stimulation.
[0031] In another aspect, the present invention provides the use of the above-mentioned moyamoya disease vascular endothelial cell model in studying the mechanism of moyamoya disease, or in screening or preparing drugs for moyamoya disease.
[0032] In another aspect, the present invention provides a method for screening a drug for moyamoya disease, comprising the step of contacting the drug with vascular endothelial cells in the above-mentioned moyamoya disease vascular endothelial cell model.
[0033] In another aspect, the present invention provides a method for studying the mechanism of moyamoya disease, comprising the step of conducting research using the above-mentioned moyamoya disease vascular endothelial cell model.
[0034] Beneficial effects
[0035] The moyamoya disease vascular endothelial cell model established by this invention effectively simulates the pathophysiological state of vascular endothelial cells during moyamoya disease in vitro, reflecting the phenotype of moyamoya disease vascular endothelial cells, and provides an important basis for exploring the pathogenesis of moyamoya disease and drug screening. Furthermore, the model construction has a high success rate, is simple, low-cost, and highly efficient. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 .EDU experiment was used to detect the cell proliferation ability of HBMEC after stimulation with cerebrospinal fluid.
[0037] Figure 2 .Tubule formation experimental results.
[0038] Figure 3 .Flow cytometer analysis results. DETAILED DESCRIPTION
[0039] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.
[0040] Example 1: Construction of a Moyamoya Disease HBMEC Cerebrospinal Fluid Stimulation Model
[0041] (1) Cerebrospinal fluid extraction from patients with moyamoya disease
[0042] 1. Collection of cerebrospinal fluid from patients with moyamoya disease: 2-5 ml of cerebrospinal fluid was collected from patients with moyamoya disease and healthy controls (controls) and stored at 4°C. Inclusion criteria for CSF donors from patients with moyamoya disease were: ① Patients diagnosed with moyamoya disease according to the 2021 revised guidelines for the diagnosis of moyamoya disease; ② Aged 6 to 65 years; ③ Excluding hypertension, diabetes, hemolytic diseases, immune diseases, heart disease, or atherosclerotic disease.
[0043] 2. Cerebrospinal fluid storage: Store the cerebrospinal fluid in a vacuum blood collection tube at -80℃ for future use.
[0044] (2) Culture of human brain microvascular endothelial cells
[0045] This step is the culture of human brain microvascular endothelial cells (HBMEC) before cerebrospinal fluid stimulation, which determines the growth state of HBMEC cells and plays a decisive role in the construction of the HBMEC cerebrospinal fluid stimulation model.
[0046] 1. HBMEC cell line recovery: The frozen human brain microvascular endothelial cell line (HBMECs, purchased from ScienCell) was taken out from the liquid nitrogen tank and thawed in a 37°C warm water bath.
[0047] 2. Plating HBMEC Cell Lines: Mix 1.5 ml of the thawed frozen cell line solution with HBMEC complete medium (DMEM / F-12, Cat. No. 11320033, GIBCO) at a 1:1 ratio. Add 6 ml of HBMEC complete medium to a centrifuge tube, mix thoroughly, and centrifuge at 800 rpm for 5 minutes at 4°C. Discard the supernatant and resuspend the cells in 6 ml of HBMEC complete medium. Then, aliquot the cell suspension and transfer it to T25 cell culture flasks. Each flask is filled to 5 ml with HBMEC complete medium. Incubate the flasks at 37°C, 5% CO2, with daily medium changes of 5 ml each time.
[0048] 3. Passaging HBMEC Cell Lines: Cells were passaged when they reached 90% confluency. After discarding the old culture medium, each flask of cells was washed one to two times with 1-2 mL of PBS buffer. The PBS was discarded and 2 mL of a mixture of 0.25% trypsin and 0.02% EDTA was added for digestion. After 30 seconds, cells were observed to have rounded under a microscope. After adding 2 mL of complete HBMEC medium, the cells were harvested and centrifuged at 800 rpm at 4°C for 5 minutes. The supernatant was discarded and the cells were resuspended in 15 mL of complete HBMEC medium. The cells were aliquoted into three T25 culture flasks, shaken well, and cultured in a 37°C, 5% CO2 incubator. The medium was changed daily, with 5 mL of complete HBMEC medium added each time.
[0049] (III) Cerebrospinal fluid stimulation of human brain microvascular endothelial cells
[0050] This step is the core step in constructing the HBMEC cerebrospinal fluid stimulation model.
[0051] 1. HBMEC inoculation: Digest the cells in the culture flask, centrifuge, resuspend in HBMEC complete medium, and inoculate into 6-well plates, with approximately 2.5×10 cells per well. 6The 6-well plate was placed in a 37°C, 5% CO2 constant temperature cell culture incubator and the medium was changed daily with 2 ml of HBMEC complete medium.
[0052] 2. Heat inactivation of patient cerebrospinal fluid: Take the cerebrospinal fluid of patients with moyamoya disease and healthy subjects (control) out of the -80℃ freezer, thaw in a 37℃ water bath, transfer the cerebrospinal fluid to a serum bottle, and heat inactivate it in a 50℃ water bath for 20 minutes. After inactivation, divide the bottle into 15ml centrifuge tubes and store at -20℃.
[0053] 3. HBMEC CSF stimulation: Thaw the heat-inactivated CSF in a 37°C water bath. After incubating HBMECs seeded in 6-well plates (divided into 2 groups, seeded in 2 6-well plates) for approximately 24 hours, discard the culture medium and gently wash with PBS 1-2 times. Replace the culture medium with 2 ml of HBMEC complete medium containing 1% P / S double antibody, 1% endothelial cell growth supplement (EGM-2), and 2.5% heat-inactivated CSF per well. Transfer the cells to a 37°C, 5% CO2 constant temperature cell culture incubator and culture the medium daily. The replacement volume is 2 ml per well of HBMEC complete medium containing 1% P / S double antibody, 1% endothelial cell growth supplement (EGM-2), and 2.5% heat-inactivated CSF. Observe the cell growth status every 8 hours. When the cell confluence reaches 90%, the HBMEC CSF stimulation model is established and can be used for related phenotypic experiments. Two groups of HBMECs were stimulated with two types of heat-inactivated cerebrospinal fluid (cerebrospinal fluid from patients with moyamoya disease and healthy subjects), respectively.
[0054] Example 2: EDU assay to detect HBMEC cell proliferation after cerebrospinal fluid stimulation
[0055] EdU (5-ethynyl-2'-deoxyuridine) is a thymidine analog. During cell proliferation, EdU is taken up into the cell nucleus and binds to deoxyribonucleic acid (DNA) during DNA synthesis, forming an EdU-DNA complex. This complex is recognized and bound by anti-Edu antibodies, forming an EdU-DNA-antibody complex. By measuring the signal intensity of this complex, cell proliferation can be determined.
[0056] In a 96-well plate, 1 × 10 4Each group of HBMEC cells in the HBMEC cerebrospinal fluid stimulation model constructed in Example 1 was inoculated at a density of 100 μg / well. After the cells were cultured overnight and returned to normal, the next step was carried out. Prepare 2× EdU working solution: dilute EdU (10mM) with HBMEC complete culture medium at 1:500 to obtain 2× EdU working solution (final concentration 20μM). Add equal volumes of 2× EdU working solution (20μM) preheated at 37°C to a 96-well plate so that the final concentration of EdU in the 96-well plate becomes 1× (final concentration 10μM), and continue to incubate the cells in the EdU working solution for 2 hours.
[0057] After EdU labeling, remove the culture medium and add 100 μL of 4% paraformaldehyde to each well for fixation at room temperature for 15 minutes. Remove the fixative and wash the cells three times with PBS for 5 minutes each. Remove the wash solution and add 100 μL of PBS containing 0.3% Triton X-100 to each well and incubate at room temperature for 15 minutes. Remove the permeabilization solution and wash the cells twice with PBS for 5 minutes each.
[0058] Prepare the Click Additive Solution: Dissolve one tube of Click Additive in 1.3 mL of deionized water and mix until completely dissolved. Prepare the Click Additive Solution (500 μL of reaction mixture per well of a six-well plate. For 12-, 24-, 48-, 96-, and 384-well plates, use 200 μL, 100 μL, 70 μL, 50 μL, and 20 μL of reaction mixture per well, respectively).
[0059]
[0060]
[0061] Add 50 μL of Click reaction solution to each well, gently shake the plate to ensure the reaction mixture evenly covers the sample, and incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash the plate three times with PBS buffer for 5 minutes each.
[0062] Nuclear staining: Dilute Hoechst 33342 with PBS at a ratio of 1:1000 to prepare Hoechst 33342 working solution. Add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes. Aspirate the 1× Hoechst 33342 solution and wash three times with PBS for 5 minutes each.
[0063] Observe under a microscope at 100 times magnification and take photos. Figure 1 As shown. Figure 1In the data, the control group represents HBMEC cells without CSF stimulation (blank control group), the HC group represents HBMEC cells stimulated with CSF from healthy individuals, and the MMD group represents HBMEC cells stimulated with CSF from patients with Moyamoya disease. Compared with the control group, the proliferation capacity of HBMEC cells in the HC group did not differ significantly. However, the proliferation capacity of HBMEC cells in the MMD group was significantly increased compared with the HC group. This indicates that the proliferation capacity of HBMEC cells in the constructed Moyamoya disease model stimulated with CSF from patients with Moyamoya disease is enhanced, which is consistent with the pathological state of intimal hyperplasia in patients with Moyamoya disease.
[0064] Example 3: Tubule formation experiment
[0065] The day before the experiment, Matrigel was taken out of the -20°C freezer and placed in a 4°C refrigerator to melt overnight. The pipette tip was placed in a -20°C refrigerator to pre-cool. It was taken out 30 minutes before the experiment and placed on ice. 50 μL of melted Matrigel was added to each well of a 96-well plate. Do not generate bubbles during the addition process. Shake gently until it is evenly spread. Let it stand in a 37°C incubator for 30 minutes to allow the Matrigel to fully solidify. The treated HBMEC cells were digested and centrifuged, resuspended in HBMEC complete medium, counted, and counted at 1.5×10 4 Add the cells to a 96-well plate at a density of 1000 cells / well, making sure not to touch the gel surface. Set up three replicates per well and culture in a 37°C incubator for 6 hours. After 2 hours of culture, closely observe the state of blood vessel formation. After 6 hours of culture, remove the 96-well plate and observe and photograph under a microscope at 200 times the magnification. The results are as follows: Figure 2 As shown in A. Figure 2 B is the number of branches formed by HBMEC cells in each group, Figure 2 C is the length of blood vessels formed by HBMEC cells in each group. Figure 2 In the study, the control group represents HBMEC cells not stimulated with cerebrospinal fluid (blank control group), the HC group represents HBMEC cells stimulated with cerebrospinal fluid from healthy individuals, and the MMD group represents HBMEC cells stimulated with cerebrospinal fluid from patients with moyamoya disease. Compared with the control group, there were no significant differences in the angiogenesis capacity, number, and length of blood vessel branches formed by HBMEC cells in the HC group. Compared with the HC group, the angiogenesis capacity, number, and length of blood vessel branches formed by HBMEC cells in the MMD group were significantly increased. Because moyamoya disease involves abnormal angiogenesis and intimal hyperplasia, this model demonstrates that the proliferation and angiogenesis capacity of MMD cells surpass those of HC cells, consistent with the pathological state of moyamoya disease.
[0066] Example 4: Flow cytometer detection of cell cycle
[0067] Immediately before use, prepare a staining solution using a 1:9 ratio of RNase A to PI working solution. Take cells from each group in the logarithmic growth phase and incubate them in HBMEC complete medium for 24 hours. Discard the medium and wash the cells twice with 0.5 mL of PBS. Discard the PBS and add 2 mL of 0.25% trypsin-free EDTA-free digestion solution. Then, add 2 mL of HBMEC complete medium to terminate the digestion. Gently pipette and collect the cells. Centrifuge at 2000 rpm, 4°C for 5 minutes, discard the supernatant, and collect and adjust the cell concentration to 1 × 10 6 / mL, take 1mL of single-cell suspension; after centrifugation of the prepared single-cell suspension, remove the supernatant, resuspend the cells in 500μL of 70% cold ethanol, fix, seal with sealing film, and fix at 4℃ overnight.
[0068] Centrifuge at 800 rpm for 15 minutes to collect fixed cells, wash twice with PBS; resuspend the cells in 0.4 mL of PBS and transfer to a tube and gently pipette to prevent cell breakage; add 500 μL of the previously prepared PI / RNase A staining solution and stain in an ice bath in the dark for 30 minutes; filter through a 300-mesh (pore size 40-50 μm) nylon mesh and analyze the results. Figure 3 As shown. Figure 3 In this study, the control group represents HBMEC cells not stimulated with cerebrospinal fluid (blank control group), the HC group represents HBMEC cells stimulated with cerebrospinal fluid from healthy individuals, and the MMD group represents HBMEC cells stimulated with cerebrospinal fluid from patients with moyamoya disease. Compared with the control group, the HBMEC cell cycle in the HC group showed no significant difference. However, the proportion of HBMEC cells in the S phase was significantly increased in the MMD group compared with the HC group. This increase in the S phase proportion in the MMD group indicates enhanced endothelial cell proliferation, consistent with the pathological mechanism of moyamoya disease.
[0069] The cell model of the present invention is constructed by stimulating HBMEC cells with heat-inactivated cerebrospinal fluid. Compared with heat-inactivated serum or peripheral blood stimulation, it can better reflect the impact of the intracranial microenvironment of moyamoya disease patients on HBMEC cells and the clinical phenotype of moyamoya disease.
[0070] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.
Claims
1. A method for establishing a moyamoya disease vascular endothelial cell model, characterized in that: The method comprises the following steps: (1) Vascular endothelial cells were cultured at a rate of 1.25×10 5 -1.25×10 7 / ml and cultured in a constant temperature cell culture incubator at 37 ° C with 5% CO2 for at least 24 hours; (2) stimulating the vascular endothelial cells obtained in step (1) with a cerebrospinal fluid stimulation medium; The cerebrospinal fluid stimulation culture medium contains 2.5% heat-inactivated cerebrospinal fluid from patients with moyamoya disease.
2. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 1, characterized in that: The temperature for thermal inactivation of cerebrospinal fluid of patients with moyamoya disease is 50 (±10)° C., and the inactivation time is not less than 20 minutes.
3. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 1, characterized in that: The cerebrospinal fluid of the moyamoya disease patient is derived from a moyamoya disease patient aged 6-65 years old, and the moyamoya disease patient does not suffer from hypertension, diabetes, hemolytic disease, immune disease, heart disease, or atherosclerotic disease.
4. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 1, characterized in that: The cerebrospinal fluid stimulation culture medium is an HBMEC complete culture medium comprising 1% P / S dual antibody, 1% endothelial cell growth supplement and 2.5% heat-inactivated cerebrospinal fluid from patients with moyamoya disease.
5. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 4, characterized in that: The HBMEC complete culture medium is DMEM / F-12.
6. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 1, characterized in that: The vascular endothelial cells are human brain microvascular endothelial cells (HBMECs).
7. The method for establishing a moyamoya disease vascular endothelial cell model according to claim 1, characterized in that: The method comprises the steps of inoculating and passage a vascular endothelial cell line to obtain the vascular endothelial cells.
8. A moyamoya disease vascular endothelial cell model, characterized in that: The moyamoya disease vascular endothelial cell model is obtained by using the method for establishing a moyamoya disease vascular endothelial cell model according to any one of claims 1 to 7.
9. Use of the moyamoya disease vascular endothelial cell model according to claim 8 in studying the mechanism of moyamoya disease, or in screening or preparing drugs for moyamoya disease. 10 . A method for screening a drug for moyamoya disease, comprising the step of contacting a drug with vascular endothelial cells in the moyamoya disease vascular endothelial cell model according to claim 8 .