Smoke disease brain microvascular endothelial cell model based on RNF213 mutation and application of smoke disease brain microvascular endothelial cell model
By directed differentiation of iPSCs in patients with smoke disease carrying RNF213p.R4810K mutation, a brain microvascular endothelial cell model was constructed, which solved the problem of lack of reliable models in existing studies, and achieved efficient disease simulation and new drug screening.
Patent Information
- Application Number
- CN202510182884.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-30
AI Technical Summary
The lack of reliable cerebral microvascular endothelial cell models in existing smoke disease studies has led to poor repetition and inefficiency in experiments, and limited understanding of the impact of RNF213p.R4810K mutation.
By directed differentiation of iPSCs in patients with smoke disease carrying RNF213p.R4810K mutation, a brain microvascular endothelial cell model can be constructed, which can be used to simulate the pathological processes triggered by mutations in specific genes of the disease.
The construction of a stable and reliable brain microvascular endothelial cell model is achieved, which can truly simulate the cytopathological characteristics of smoke disease, and improve the depth of research and the efficiency of new drug screening.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and particularly relates to a Moyamoya disease cerebral microvascular endothelial cell model based on the mutation of ring finger protein 213 ( RNF213 ), and its application. Background Art
[0002] Moyamoya disease is a chronic cerebrovascular disease, characterized by progressive stenosis or occlusion at the terminal ends of bilateral internal carotid arteries and / or the origins of the anterior cerebral artery and middle cerebral artery, accompanied by the formation of abnormal vascular networks in the skull base region. This disease shows obvious familial aggregation, and genetic factors play an important role in its pathogenesis.
[0003] RNF213 The gene is the first Moyamoya disease susceptibility gene identified by Japanese scholars. The RNF213 protein contains multiple domains, and most of the mutations in patients are located in the C-terminal region, including domains such as Back, RING, Shell, ZF2, Core, and CTD; among them, the p.R4810K mutation is located in the Core domain. Research shows that RNF213 the p.R4810K mutation is significantly associated with the risk of Moyamoya disease in different populations: the occurrence rate of this mutation is about 90% in Japanese patients, 79% in Korean patients, and about 31% in Chinese patients. At the same time, RNF213 the p.R4810K mutation of the gene is closely related to the clinical phenotypes, preoperative imaging features, postoperative collateral circulation compensation, and clinical prognosis of Moyamoya disease patients.
[0004] In current research, the understanding of how RNF213 the p.R4810K mutation affects the function of cerebral microvascular endothelial cells is still limited, confined to ordinary endothelial cells or not focusing on the p.R4810K variant. In addition, most existing research methods rely on patient tissues or primary endothelial cells with limited amplification ability, resulting in poor experimental repeatability and low efficiency. The development of iPSC technology provides the possibility to simulate disease-specific gene mutations, but the establishment of a cerebral microvascular endothelial model for the RNF213 p.R4810K mutation of Moyamoya disease is still in the exploratory stage. There is currently no relevant report on successfully constructing a cerebral microvascular endothelial cell model using iPSCs from patients with the RNF213 p.R4810K mutation. Summary of the Invention
[0005] The present invention aims to solve the problem of the lack of a reliable cerebral microvascular endothelial cell model in the existing research on Moyamoya disease, and provides a Moyamoya disease cerebral microvascular endothelial model based on RNF213 the p.R4810K mutation, which is used to deeply clarify the disease mechanism and conduct new drug screening and drug evaluation.
[0006] In a first aspect, the present invention claims protection for a method for constructing brain microvascular endothelial cells as a moyamoya disease model.
[0007] The method for constructing brain microvascular endothelial cells as a moyamoya disease model claimed by the present invention may include the following steps: (A1) Reprogram CD34 RNF213 cells in isolated PBMCs from moyamoya disease patients carrying the + p.R4810K mutation to obtain induced pluripotent stem cells (iPSCs); (A2) Directly induce the differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells, and the obtained brain microvascular endothelial cells are the brain microvascular endothelial cells that can be used as a moyamoya disease model.
[0008] Further, in step (A1), the CD34 + cells can be obtained by the following method: Isolate PBMCs from the peripheral blood of moyamoya disease patients carrying the RNF213 p.R4810K mutation, and then inoculate them into a CD34 + cell expansion medium for culture to promote the proliferation of CD34 + cells; the CD34 + cell expansion medium is StemSpan SFEMII.
[0009] Further, in step (A1), the reprogramming is to co-introduce the OCT4 gene, SOX2 gene, KLF4 gene, and c-MYC gene into the CD34 + cells and culture until the induced pluripotent stem cells are obtained.
[0010] Even further, in step (A1), OCT4 gene, SOX2 gene, KLF4 gene, and c-MYC gene are introduced into the CD34 + cells in the form of a recombinant vector; the recombinant vector is 4 recombinant plasmids obtained by cloning the OCT4 gene, SOX2 gene, KLF4 gene, and c- MYC gene into the pCXWB-EBNA1 vector respectively. Specifically, the recombinant vector is obtained by cloning the OCT4 gene, SOX2 gene, KLF4 gene, and c-MYCFour recombinant plasmids obtained by separately replacing small fragments between two EcoR I sites in the pCXWB-EBNA1 vector with genes.
[0011] Furthermore, in step (A1), OCT4 gene, SOX2 gene, KLF4 gene and c-MYC gene were co-introduced into the CD34 + cells, and then cultured according to the following method: On the 1st day after transfection, the treated CD34 + cells were transferred to a culture plate pre-coated with laminin and cultured with mTeSR™1 medium, and the medium was changed daily until induced pluripotent stem cells (iPSCs) were obtained. Among them, iPSC-like cells appeared approximately 2 - 3 days, and colony formation occurred around 1 week.
[0012] In an embodiment of the present invention, in step (A2), the method for directing the differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells is as follows: (a1) Before induction of differentiation, the induced pluripotent stem cells were seeded on a culture plate pre-coated with Matrigel, and then 10 μM Y-27632 was added to the culture system of the induced pluripotent stem cells and cultured for 3 days; (a2) The induced pluripotent stem cells treated in (a1) were cultured in DeSR1 medium supplemented with 6 μM CHIR99021 for 24 hours; The DeSR1 medium consists of MEM non-essential amino acid solution (100X), GlutaMAX, β-mercaptoethanol and basal medium 1; in the DeSR1 medium, the final concentration of the MEM non-essential amino acid solution (100X) is 1% by volume, the final concentration of GlutaMAX is 0.5% by volume, the final concentration of β-mercaptoethanol is 0.1 mM, and the balance is the basal medium 1; Furthermore, the basal medium 1 can be DMEM / F12 medium.
[0013] (a3) The medium of the induced pluripotent stem cells treated in (a2) was changed to DeSR2 medium and cultured for 5 days, and the fresh DeSR2 medium was changed every day during this period; The DeSR2 medium is obtained by adding B27 to the DeSR1 medium; the final concentration of B27 in the DeSR2 medium is 1% by volume.
[0014] (a4) Replace the culture medium of the induced pluripotent stem cells after being processed in (a3) with hECSR1 medium and culture for 2 days; The hECSR1 medium consists of basic fibroblast growth factor (bFGF), retinoic acid (RA), B27 and basal medium 2; in the hECSR1 medium, the final concentration of the basic fibroblast growth factor (bFGF) is 20 ng / mL, the final concentration of the retinoic acid (RA) is 10 μM, the final concentration of β-mercaptoethanol is 0.1 mM, and the balance is the basal medium 2; Further, the basal medium 2 can be human endothelial SFM medium.
[0015] (a5) Transfer the induced pluripotent stem cells after being processed in (a4) to a culture plate coated with Matrigel, and on the second day, replace the culture medium with hECSR2 medium. After culturing for 1 day, the induced pluripotent stem cells are directed to differentiate into brain microvascular endothelial cells; The hECSR2 medium is the medium obtained by removing the fibroblast growth factor (bFGF) and retinoic acid (RA) from the hECSR1 medium.
[0016] In the second aspect, the present invention claims to protect the brain microvascular endothelial cells prepared by the method described in the first aspect above.
[0017] In the third aspect, the present invention claims to protect the application of the brain microvascular endothelial cells prepared by the method described in the first aspect above in being used as or preparing a moyamoya disease model.
[0018] In the fourth aspect, the present invention claims to protect the application of the method described in the first aspect above or the brain microvascular endothelial cells described in the second aspect above in any of the following: (B1) Drug screening and / or drug evaluation for treating and / or preventing moyamoya disease; (B2) Studying the pathogenesis of moyamoya disease; (B3) Evaluating gene therapy strategies for moyamoya disease; (B4) Preparing products for drug screening and / or drug evaluation for treating and / or preventing moyamoya disease; (B5) Preparing products for studying the pathogenesis of moyamoya disease; (B6) Preparing products for evaluating gene therapy strategies for moyamoya disease.
[0019] In the third aspect and the fourth aspect above, the moyamoya disease may be moyamoya disease carrying RNF213 the p.R4810K mutation.
[0020] Fifth aspect, the present invention claims a method for preparing brain microvascular endothelial cells with reduced tube formation ability.
[0021] The method for preparing brain microvascular endothelial cells with reduced tube formation ability claimed by the present invention may include the steps described in the first aspect above; in step (A2), the brain microvascular endothelial cells differentiated from the induced pluripotent stem cells are the brain microvascular endothelial cells with reduced tube formation ability.
[0022] Sixth aspect, the present invention claims the use of the brain microvascular endothelial cells prepared by the method described in the fifth aspect above in screening drugs capable of enhancing the tube formation ability of brain microvascular endothelial cells.
[0023] In the above related aspects, the RNF213 p.R4810K mutation means that the 4810th amino acid of the RNF213 protein is mutated from R to K. The amino acid sequence of the unmutated RNF213 protein is as shown in NCBI Reference Sequence: NP_001243000.2 (see NCBI database, Update: PRI 12-NOV-2024). The amino acid sequence of the RNF213 protein with p.R4810K mutation is the sequence obtained by mutating the 4810th amino acid of the amino acid sequence of the unmutated RNF213 protein from R to K. The 160-15783rd positions of NCBI Reference Sequence: NM_001256071.3 (see NCBI database, Update: PRI 12-NOV-2024) are the coding gene sequence of the unmutated RNF213 protein, and the coding gene sequence of the RNF213 protein with p.R4810K mutation is the sequence obtained by mutating the 14429th nucleotide of the coding gene sequence of the unmutated RNF213 protein from G to A.
[0024] In the above related aspects, the moyamoya disease model is a cell model of moyamoya disease.
[0025] The present invention obtains a cell model similar to the phenotype and function of brain microvascular endothelial cells by directed differentiation of iPSCs derived from moyamoya disease patients, thereby precisely simulating RNF213 the related pathological processes caused by the mutation at the cellular level. This model can be used to clarify the genetic and molecular mechanisms of moyamoya disease, and on this basis, realize new drug screening, high-throughput drug evaluation and the development of personalized treatment strategies, with potential clinical translation value and broad application prospects.
[0026] Advantages of the present invention: 1. High stability: By using iPSC technology, unlimited amplification can be achieved, enabling the continuous acquisition of highly pure and phenotypically stable brain microvascular endothelial cells, providing a long-term and reliable cell source for research and applications.
[0027] 2. High disease relevance: Brain microvascular endothelial cells carrying RNF213 gene mutations showed significantly reduced angiogenesis ability in tube formation assays, truly simulating and reproducing the characteristic cellular pathological features of moyamoya disease.
[0028] 3. Wide application range: This model can be used to deeply analyze the molecular mechanism of moyamoya disease, assist in the research and screening of new drugs, and evaluate gene therapy strategies, providing important references for clinical translation and the design of personalized treatment plans. Brief Description of the Drawings
[0029] Figure 1 For Sanger sequencing. A: RNF213 There is no mutation in p.R4810K; B: RNF213 p.R4810K mutation.
[0030] Figure 2 For immunofluorescence staining of iPSC pluripotent stem cell specific markers. A: TRA-1-60 (S); B: Sox2; C: SSEA4; D: OCT-4A.
[0031] Figure 3 For RNF213 Karyotype analysis of iPSCs from moyamoya disease patients with p.R4810K mutation, showing no abnormal chromosome number (A) and no abnormal chromosome structure (B).
[0032] Figure 4 For immunofluorescence staining of brain microvascular endothelial cell specific markers differentiated from iPSCs. A: CD31; B: ZO-1; C: VE-Cadherin; D: Claudin-5.
[0033] Figure 5 For healthy individuals (without RNF213 p.R4810K mutation), RNF213 moyamoya disease patients without p.R4810K mutation, and RNF213 6-hour tube formation images (A) and tube formation statistics (B) of brain microvascular endothelial cells differentiated from iPSCs of moyamoya disease patients with p.R4810K mutation. In the figure, HC represents healthy individuals; MMD represents RNF213 moyamoya disease patients without p.R4810K mutation; MMD + p.R4810K represents RNF213 moyamoya disease patients with p.R4810K mutation. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Detailed implementation manners
[0034] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.
[0035] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0036] In the following embodiments, RNF213 The p.R4810K mutation means that the 4810th amino acid of the RNF213 protein is mutated from R to K. The amino acid sequence of the unmutated RNF213 protein is as shown in NCBI Reference Sequence: NP_001243000.2 (see NCBI database, Update: PRI 12-NOV-2024). The amino acid sequence of the RNF213 protein with the p.R4810K mutation is the sequence obtained by mutating the 4810th amino acid of the amino acid sequence of the unmutated RNF213 protein from R to K. The 160th to 15783rd positions of NCBI Reference Sequence: NM_001256071.3 (see NCBI database, Update: PRI 12-NOV-2024) are the coding gene sequence of the unmutated RNF213 protein, and the coding gene sequence of the RNF213 protein with the p.R4810K mutation is the sequence obtained by mutating the 14429th nucleotide of the coding gene sequence of the unmutated RNF213 protein from G to A.
[0037] Example 1. Construction and identification of a RNF213 cerebral microvascular endothelial cell model of moyamoya disease based on mutation 1. Sample preparation Collect 5 - 10 mL of peripheral venous blood samples from healthy donors and patients diagnosed with moyamoya disease. It is required that the donors have no history of other serious diseases. Use aseptic operation during collection and add anticoagulant (EDTA) to prevent blood coagulation. Subsequently, slowly layer the collected whole blood on the upper layer of the pre-prepared Ficoll-Paque density gradient solution, keeping the interface clear and avoiding agitation. Then centrifuge at 500×g for 20 minutes (room temperature, no braking) to isolate peripheral blood mononuclear cells (PBMCs). After centrifugation, carefully transfer the PBMC layer at the interface to a new centrifuge tube and centrifuge at 500×g for 5 minutes to precipitate the cells. Finally, resuspend the precipitated PBMCs with cell cryopreservation solution, first perform gradient cooling preservation at -80°C for 24 hours, and then transfer to liquid nitrogen for long-term storage.
[0038] 2. Sanger Sequencing When collecting peripheral venous blood samples in Step 1, 1 mL of whole blood is separately reserved for each subject. Use DNeasy Blood and Tissue Kits to extract DNA from this 1 mL of whole blood, and use Sanger sequencing technology to detect RNF213 the p.R4810K mutation status. After detection, select the following samples for subsequent experimental studies: samples from healthy controls without RNF213 the p.R4810K mutation, samples from moyamoya disease patients without RNF213 the p.R4810K mutation, and samples from moyamoya disease patients with RNF213 the p.R4810K mutation. The Sanger sequencing results are shown in Figure 1 , where Figure 1 A in RNF213 is the result of no p.R4810K mutation, Figure 1 and B in RNF213 is the result of p.R4810K mutation.
[0039] 3. Construction of iPSC Resuscitate the selected healthy control PBMCs without RNF213 the p.R4810K mutation, moyamoya disease patient PBMCs without RNF213 the p.R4810K mutation, and moyamoya disease patient PBMCs carrying RNF213 the p.R4810K mutation, and inoculate them into the specific CD34 + cell expansion medium StemSpan SFEM Ⅱ (STEMCELL; 09605), and culture under the constant temperature culture conditions of 37°C and 5% CO 2 . During the culture period, change the medium every 2 days, and at the same time observe the cell morphology using an inverted microscope, and appropriately control the seeding density to promote CD34 +For the effective proliferation of cells, after culturing for 6 - 9 days, CD34 + cells reached the peak, and the cells could be electroporated without purification.
[0040] Select the episomal vector pCXWB - EBNA1 (Addgene, #37624), and replace the small fragment between the two EcoRⅠ restriction sites of the pCXWB - EBNA1 vector with the OCT4 gene (positions 63 - 1145 of NCBI ReferenceSequence: NM_002701.6, see NCBI database, Update: PRI 09 - JUL - 2024), SOX2 gene (positions 437 - 1390 of NCBI Reference Sequence: NM_003106.4, see NCBI database, Update: PRI 12 - NOV - 2024), KLF4 gene (positions 598 - 2037 of NCBI Reference Sequence: NM_004235.6, see NCBI database, Update: PRI 24 - OCT - 2024) and c-MYC gene (positions 364 - 1728 of NCBI ReferenceSequence: NM_002467.6, see NCBI database, Update: PRI 11 - DEC - 2024) respectively to obtain 4 recombinant plasmids. According to the different foreign genes, the 4 recombinant plasmids are named pCXWB - OCT4, pCXWB - SOX2, pCXWB - KLF4, and pCXWB - c - MYC respectively. Use the Qiagen Maxi Kit to obtain endotoxin - free high - purity plasmids to ensure high purity and remove endotoxins and other impurities. Subsequently, use the Lonza Nucleofector 2B system to electroporate CD34 + cells, and co - efficiently introduce the above 4 recombinant plasmids into the cells (during transfection, each plasmid is transfected according to 1μg / 5×10 6 cell number), so as to achieve the stable expression of related reprogramming factors.
[0041] On the 1st day after transfection, transfer the treated cells to a culture plate pre - coated with laminin, and culture them using mTeSR™1 medium (STEMCELL; 85850), changing the medium daily. During the culture process, observe the cells under a microscope. After about 14 days, select clones with regular morphology and clear edges from the formed iPSC clones, pick them, and transfer them to a new culture plate to continue culturing in mTeSR™1 medium to ensure the homogeneity and pluripotency of the iPSC cell population.
[0042] The obtained iPSCs were subjected to immunofluorescent staining with antibodies against pluripotent stem cell-specific markers TRA-1-60 (S) (CST; 4746T), Sox2 (CST; 23064T), SSEA4 (CST; 4755T), and OCT-4A (CST; 2890T) to confirm the pluripotency of the iPSCs. Figure 2 For RNF213 Staining results of iPSCs derived from a patient with p.R4810K mutant moyamoya disease, showing positive staining of pluripotent stem cell-specific markers in the constructed iPSCs. Meanwhile, karyotype analysis of the iPSCs was performed, and the results showed no abnormalities in the chromosome number and structure of the constructed iPSC line ( Figure 3 ). The above results indicate that the karyotype of the constructed iPSCs did not change during the reprogramming process.
[0043] 4. Disease model validation (1) Differentiation of iPSCs into brain microvascular endothelial cells Before differentiation, iPSCs were dissociated using Accutase and seeded at a density of 25,000 to 50,000 cells per square centimeter on a culture plate pre-coated with Matrigel. The culture medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632, and incubated at 37 °C and 5% CO 2Amplify and culture for 3 days under the conditions. Differentiation was initiated on day 0 by adding 6 μM CHIR99021 to DeSR1 medium. The composition of DeSR1 medium is as follows: DMEM / F12 (Gibco; 11320033) as the basal medium, MEM non-essential amino acid solution (100X) (Gibco; 11140050) at a volume percentage of 1%, GlutaMAX™ supplement (Gibco; 35050061) at a volume percentage of 0.5%, and 0.1 mM β-mercaptoethanol (Solarbio; M8211) (each concentration is the final concentration in the medium). After 24 hours, the medium was changed to DeSR2 medium, that is, adding B27 (Gibco; 17504044) at a final concentration of 1% volume percentage on the basis of DeSR1 medium, and the medium was changed daily for the next 5 days. On day 6, the medium was changed to hECSR1 medium. The composition of hECSR1 medium is as follows: supplement 20 ng / ml basic fibroblast growth factor (bFGF), 10 μM retinoic acid (RA), and 1% volume percentage of B27 (Gibco; 17504044) (each concentration is the final concentration in the medium) in human endothelial SFM medium (Gibco; 11111044). After culturing in hECSR1 medium for 2 days, the cells on day 8 were digested with Accutase and seeded at a density of 1,000,000 cells per square centimeter on a 48-well tissue culture plate coated with 100 μg / ml Matrigel. On day 9, the medium was changed to hECSR2 medium, that is, hECSR1 medium without RA and bFGF, to ensure that the cells formed a confluent monolayer and the differentiation of brain microvascular endothelial cells was completed.
[0044] (2) Confirmation of the differentiation of brain microvascular endothelial cells The endothelial cells differentiated in step (1) were subjected to immunofluorescence staining with antibodies against endothelial-specific markers CD31 (CST; 3528T), ZO-1 (CST; 15652T), VE-Cadherin (CST; 2500T), and Claudin-5 (CST; 49564T) to verify the endothelial phenotype and confirm the successful differentiation. Figure 4 Shown are from RNF213 The immunofluorescence staining results of brain microvascular endothelial cells differentiated from iPSCs of p.R4810K mutant moyamoya disease patients. The results indicate that the differentiated cells are positive for endothelial-specific marker staining.
[0045] (3) Detection of angiogenesis ability Luminal formation assay: The cerebral microvascular endothelial cells obtained by the above differentiation were suspended in Matrigel matrix and cultured at 37 °C for 6 hours. The formation of tubular structures in the matrix was recorded by microscopic observation to evaluate the angiogenesis ability. The experiment was set with 3 replicates, and 2 sub-wells were set for each group in each replicate. ImageJ software was used to measure the node count of tube formation.
[0046] The experimental results showed that: RNF213 The cerebral microvascular endothelial cells differentiated from iPSCs of Moyamoya disease patients with p.R4810K mutation showed significantly weaker tube formation ability during this process, which was significantly lower than that of RNF213 healthy controls without RNF213 p.R4810K mutation and cerebral microvascular endothelial cells differentiated from iPSCs of Moyamoya disease patients without Figure 5 p.R4810K mutation ( RNF213 ). This result indicates dysfunction of cerebral microvascular endothelial cells differentiated from Moyamoya disease patients, and
[0047] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made with conventional techniques known in the art that are outside the scope disclosed in this application.
Claims
1. A method for constructing brain microvascular endothelial cells as a moyamoya disease model, comprising the following steps: (A1) For those carrying RNF213 CD34 in ex vivo PBMCs of patients with moyamoya disease with p.R4810K mutation + The cells are reprogrammed to obtain induced pluripotent stem cells; (A2) Directly inducing the differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells, and the obtained brain microvascular endothelial cells are brain microvascular endothelial cells that can be used as a moyamoya disease model.
2. The method according to claim 1, characterized in that: In step (A1), the reprogramming is to OCT4 Gene, SOX2 Gene, KLF4 Genes and c-MYC Gene co-introduction into the CD34 + cells, and cultured until the induced pluripotent stem cells are obtained.
3. The method according to claim 2, characterized in that: In step (A1), OCT4 Gene, SOX2 Gene, KLF4 Genes and c-MYC The gene is introduced into the CD34 + The recombinant vector is OCT4 Gene, SOX2 Gene, KLF4 Genes and c-MYC The four recombinant plasmids were obtained by cloning the genes into the pCXWB-EBNA1 vector.
4. The method according to any one of claims 1 to 3, characterized in that: In step (A2), the induced pluripotent stem cells are directed to differentiate into brain microvascular endothelial cells according to a method comprising the following steps: (a1) before inducing differentiation, adding 10 μM Y-27632 to the culture system of the induced pluripotent stem cells and culturing for 3 days; (a2) culturing the induced pluripotent stem cells treated in (a1) in DeSR1 medium supplemented with 6 μM CHIR99021 for 24 hours; The DeSR1 culture medium is composed of MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol and basic culture medium 1; in the DeSR1 culture medium, the final concentration of the MEM non-essential amino acid solution is 1% by volume, the final concentration of the GlutaMAX is 0.5% by volume, the final concentration of the β-mercaptoethanol is 0.1 mM, and the remainder is the basic culture medium 1; Further, the basic culture medium 1 is DMEM / F-12 culture medium; (a3) replacing the culture medium of the induced pluripotent stem cells treated in (a2) with DeSR2 culture medium, and culturing for 5 days, during which time the DeSR2 culture medium is replaced with fresh one every day; The DeSR2 medium is obtained by adding B27 to the DeSR1 medium; the final concentration of B27 in the DeSR2 medium is 1% by volume; (a4) replacing the culture medium of the induced pluripotent stem cells treated in (a3) with hECSR1 culture medium and culturing for 2 days; The hECSR1 culture medium is composed of basic fibroblast growth factor, retinoic acid, B27 and basic culture medium 2; in the hECSR1 culture medium, the final concentration of the basic fibroblast growth factor is 20 ng / mL, the final concentration of the retinoic acid is 10 μM, the final concentration of the β-mercaptoethanol is 0.1 mM, and the remainder is the basic culture medium 2; Further, the basic culture medium 2 is human endothelial SFM culture medium; (a5) transferring the induced pluripotent stem cells treated in (a4) to a culture plate coated with matrix gel, replacing the culture medium with hECSR2 culture medium on the second day, and achieving directed induction and differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells after one day of culture; The hECSR2 culture medium is a culture medium obtained by removing fibroblast growth factor and retinoic acid from the hECSR1 culture medium.
5. Brain microvascular endothelial cells prepared by the method according to any one of claims 1 to 4.
6. Use of the moyamoya disease cerebral microvascular endothelial cells according to claim 5 in forming or preparing a moyamoya disease model.
7. Use of the method according to any one of claims 1 to 4 or the brain microvascular endothelial cells according to claim 5 in any of the following: (B1) Drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B2) Study the pathogenesis of moyamoya disease; (B3) Evaluate gene therapy strategies for moyamoya disease; (B4) Preparation of products for drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B5) Preparation of products for studying the pathogenesis of moyamoya disease; (B6) Preparing products for evaluating gene therapy strategies for moyamoya disease.
8. The use according to claim 6 or 7, characterized in that: The moyamoya disease is a carrier of RNF213 Moyamoya disease with the p.R4810K mutation.
9. A method for preparing brain microvascular endothelial cells with reduced tube-forming ability, comprising the steps described in any one of claims 1 to 4; in step (A2), the brain microvascular endothelial cells differentiated by directed induction of the induced pluripotent stem cells are the brain microvascular endothelial cells with reduced tube-forming ability.
10. Use of brain microvascular endothelial cells prepared by the method of claim 9 in screening drugs that can enhance the tube-forming ability of brain microvascular endothelial cells.