Construction method and application of atherosclerosis blood vessel model

Abstract

The invention discloses a construction method and application of an atherosclerosis blood vessel model, and the construction method comprises the following steps: (1) manufacturing a PDMS chip with a sample introduction channel, and then carrying out surface modification on the sample introduction channel to enable the sample introduction channel to be used for GelMA attachment; (2) mixing GelMA and SMCs, pouring the mixture into the surface-modified sample introduction channel, curing and crosslinking the mixture on the inner wall of the sample introduction channel to form a simulated artery blood vessel wall middle membrane layer, and annularly culturing GFP-HUVECs in the simulated artery blood vessel wall, so as to obtain an endodermis in tubular arrangement, and further obtain a simulated artery blood vessel endodermis; and (3) introducing a culture medium containing ox-LDL, TNF-alpha and IL-1beta into the simulated artery blood vessel obtained in the step (2), and culturing to obtain the atherosclerosis blood vessel model with the physiological artery structure. The atherosclerosis blood vessel model constructed by the method is very close to an in-vivo microenvironment, and atherosclerosis candidate drugs can be effectively screened and tested in vitro.

Description

Technical Field

[0001] The invention belongs to the technical field of disease model construction, and specifically relates to a method for constructing an atherosclerotic blood vessel model and an application thereof. Background Art

[0002] Cardiovascular disease has always been the disease category with the highest morbidity and mortality in the world. It is estimated that more than 17 million people die from cardiovascular disease each year worldwide. The World Health Organization predicts that by 2030, this number may rise to about 23 million. Atherosclerosis (AS) is the main pathological basis of cardiovascular diseases, including ischemic heart disease, coronary heart disease, acute myocardial infarction and cerebrovascular disease, posing a serious threat to human health. Therefore, in-depth research on the pathogenesis of AS, the development of drug treatments that can prevent the early occurrence of AS, and the establishment of new, accurate and efficient drug screening methods are of great significance for clinical treatment.

[0003] The causes and pathogenesis of AS are extremely complex, involving multiple aspects such as inflammatory response, immune system function, and lipid metabolism disorders. Its pathological characteristics include lipid accumulation, thickening and loss of elasticity of arterial walls, and increased inflammatory factors. In particular, the deposition of oxidized low-density lipoprotein (ox-LDL) in the arterial intima is considered to be the initiating factor in the development of AS. Endothelial cells (ECs) on the inner wall of blood vessels are the physical barrier between blood and vascular tissue. They are not only responsible for the exchange of substances between blood and tissue fluid, but also maintain the normal function of blood vessels by synthesizing or secreting a variety of bioactive substances, including regulating vascular tension, inflammatory response, blood pressure, and coagulation and anticoagulation balance. When ECs are stimulated by proinflammatory factors, hyperlipidemia, oxidative stress, etc., they will show functional disorders, leading to the destruction of vascular dynamic balance and may induce inflammatory response. ECs can also regulate the growth behavior and functional expression of smooth muscle cells (SMCs) and promote the repair of diseased vascular intimal tissue by synthesizing or secreting bioactive substances such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and interleukin-1 (IL-1). In the early stage of AS, mature SMCs in the vascular tunica media present a contractile phenotype and maintain low synthetic activity. However, when the local microenvironment of the blood vessel changes, SMCs will transform into a synthetic phenotype, enhance the ability of cell proliferation, migration, and matrix synthesis, and promote the formation of early AS plaques. In the late stage of AS, the apoptosis and decline of SMCs will lead to the thinning of the plaque fibrous cap, and their phenotype will be transformed into derived macrophages, which may promote inflammation, lead to the formation of necrotic cores, increase the vulnerability of plaques, and aggravate the condition of AS. Therefore, the complex factor expression, functional mechanism and phenotypic transformation of cells such as ECs and SMCs are important factors in the occurrence and development of AS vascular diseases. Effective use of drugs to control the development of AS is the key to solving cardiovascular diseases caused by AS.

[0004] Drug screening is the process of evaluating the biological activity, pharmacological effects and medicinal value of potential drug substances using appropriate methods. Constructing a drug screening model is an experimental method to prove that a substance has pharmacological activity. AS is a chronic inflammatory disease of the arterial wall. In the process of finding effective therapeutic drugs, commonly used animal screening models can intuitively reflect the therapeutic effects, adverse reactions and toxic side effects of drugs. However, due to the particularity of animal samples, the number of samples that can be screened is limited, and the screening process is complicated, resulting in low efficiency and high cost of animal model screening. Although the drug screening model at the cellular and molecular levels has high screening efficiency, it ignores the complex 3D structure between cells, the biochemical signal regulation feedback mode between cells and cells, and between cells and microenvironments during the AS pathological process, and cannot achieve effective simulation of inflammatory response, coagulation and anticoagulation balance, resulting in the inability to accurately screen out effective drugs. At present, the screening model based on the tissue and organ level can realize the co-culture of multiple cells of the same tissue, but the culture time is short and it is impossible to form tissue units with physiological functions such as vascular tissue structure and vascular lesion microenvironment. At the same time, for the complex pathogenesis of AS, these models cannot accurately simulate the pathological behavior characteristics of proinflammatory factors on tissue samples such as endothelial loss, foam cell formation, and plaque occurrence. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a method for constructing an atherosclerotic blood vessel model.

[0006] Another object of the present invention is to provide an application of the atherosclerotic vascular model constructed by the above construction method.

[0007] The technical solution of the present invention is as follows:

[0008] A method for constructing an atherosclerotic blood vessel model comprises the following steps:

[0009] (1) manufacturing a PDMS chip with an injection channel, and then performing surface modification on the injection channel to enable it to be used for GelMA attachment;

[0010] (2) GelMA and SMCs are mixed and perfused into the surface-modified injection channel, and the mixture is cured and cross-linked to the inner wall of the injection channel to form a simulated arterial vessel wall, and GFP-HUVECs are cultured in a ring shape in the simulated arterial vessel wall to obtain an endothelial layer arranged in a tubular shape, thereby obtaining a simulated arterial vessel;

[0011] (3) A culture medium containing ox-LDL, TNF-α and IL-1β is introduced into the simulated artery obtained in step (2) for culturing, thereby obtaining an atherosclerotic blood vessel model with a physiological arterial structure.

[0012] In a preferred embodiment of the present invention, the surface modification is sequentially treated with isocyanate ions and immersed in a polylysine solution.

[0013] Further preferably, the concentration of the polylysine solution is 0.8-1.2%; more preferably, the concentration of the polylysine solution is 1%.

[0014] In a preferred embodiment of the present invention, the degree of substitution of GelMA is 29-31%, and the concentration is 9-11%; more preferably, the degree of substitution of GelMA is 30%, and the concentration is 10%.

[0015] Further preferably, the density of the SMCs in the GelMA is 3.8-4.1×10 6 More preferably, the density of the SMCs in the GelMA is 4×10 6 Pieces / mL.

[0016] In a preferred embodiment of the present invention, the surface modification is sequentially treated with isocyanate ions and immersed in a polylysine solution with a concentration of 0.8-1.2%; the degree of substitution of the GelMA is 29-31% and the concentration is 9-11%; the density of the SMCs in the GelMA is 3.8-4.1×10 6 / mL; more preferably, the surface modification is sequentially treated with isocyanate ions and immersed in a 1% polylysine solution; the substitution degree of GelMA is 30% and the concentration is 10%; the density of SMCs in the GelMA is 4×10 6 Pieces / mL.

[0017] In a preferred embodiment of the present invention, the concentrations of ox-LDL, TNF-α and IL-1β in the culture medium are 48-52 μg / mL, 1.5-2.4 ng / mL and 1.5-2.4 ng / mL, respectively.

[0018] Further preferably, in step (3), the speed of introducing the culture medium is 18-22 μL / h (preferably 20 μL / h), and the culture time is 20-25 h.

[0019] The application of the atherosclerotic vascular model constructed by the above construction method in in vitro screening tests of atherosclerosis therapeutic drugs.

[0020] An in vitro screening test method for atherosclerosis therapeutic drugs uses the atherosclerosis blood vessel model constructed by the above construction method.

[0021] The beneficial effect of the present invention is that the atherosclerotic vascular model constructed by the present invention is very close to the in vivo microenvironment and can effectively screen and test candidate atherosclerotic drugs in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the PMMA template design diagram in Example 1 of the present invention.

[0023] Figure 2 Schematic diagram of the production process of the PDMS chip in Example 1 of the present invention.

[0024] Figure 3 Schematic diagram of the three-dimensional structure (A), a real picture (B) and a longitudinal section diagram of the injection channel (C) of the PDMS chip prepared in Example 1 of the present invention.

[0025] Figure 4 This is an electron microscope photograph of the perfusable channel in Example 2 of the present invention.

[0026] Figure 5 This is a biological characterization diagram of the healthy three-dimensional bionic vascular model and HUVECs and SMCs in Example 2 of the present invention.

[0027] Figure 6 It is a graph showing the experimental results of endothelial dysfunction in Example 4 of the present invention and the levels of ox-LDL, ROS, TG and CH in different model groups measured by ELISA.

[0028] Figure 7 This is the concentration response curve of rapamycin in Example 4 of the present invention in the 2D well plate model and the 3D perfusable blood vessel chip model. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0030] The experimental materials and reagents in the following examples are specifically as follows:

[0031]

[0032]

[0033] Example 1 Design and fabrication of microfluidic perfusion chip structure

[0034] (1) Design PMMA template: Figure 1 As shown, the design of the microchannel includes the specifications of the injection channel being 2 mm×2 mm×80 mm, the chip width being 20 mm, and the structure being (straight-shaped).

[0035] (2) Make PDMS chips, such as Figure 2 As shown, the specific steps include:

[0036] A. Substrate preparation: First, use an electronic analytical balance to ensure that the weighed Dow Corning PDMS prepolymer A glue and B glue are mixed in a mass ratio of 10:1, stir with a glass rod for 5 minutes, and pour into the PMMA mold after fully mixing; then put it into the PDMS degassing and curing machine, degas for 15 minutes, and cure for 60 minutes until it cools to room temperature; finally, remove the PDMS substrate from the PMMA template and wrap it with plastic wrap to prevent dust from falling.

[0037] B. Bonding of substrate and cover sheet: Prepare a 100μm thick PDMS cover sheet, use a chip cutter to cut the edges of the substrate and cover sheet flat, so that one chip corresponds to one cover sheet, and wrap the substrate and cover sheet with clean plastic wrap. Take out the substrate and cover sheet, put the substrate with the channel side facing up and put it into the quartz chamber of PLASMACLEANER together with the cover sheet, so that the sample is as close to the middle position as possible (the glow in the middle position is the strongest). Open the vacuum valve to evacuate for about 5 minutes, so that the vacuum degree in the chamber reaches below 400mtorr, then open the oxygen valve, put in pure oxygen for 5 seconds, add high voltage to 740V to make the oxygen in the vacuum chamber glow, bombard the surface of the PDMS cover sheet and substrate with oxygen plasma for 30 seconds, turn off the high voltage switch and the vacuum pump, turn the vent valve to balance the air pressure inside and outside the chamber, open the chamber door, and immediately take out the substrate and cover sheet for bonding.

[0038] C. External steel pipe connection: Prepare a steel pipe with an outer diameter of 2.2mm, a wall thickness of 0.2mm, a length of 15mm, and a material of 316 stainless steel. Apply 5mm long high-transparency UV shadowless glue evenly on the outer layer, and then insert it into the hollow channel of the bonded chip from both sides. Then use a UV lamp to irradiate the front and back sides of the connection for 30s each to fix and seal the steel pipe and the PDMS chip. Finally, Figure 3 The PDMS chip shown was sterilized by high-pressure steam until ready for use.

[0039] (3) PDMS chip surface modification: The contact angle of the scaffold material GelMA on polydimethylsiloxane (PDMS) was measured using a contact angle meter. It was observed that the water droplet showed a higher contact angle, indicating that it has strong hydrophobicity, which is not conducive to the adhesion of the scaffold material GelMA. The above PDMS chip was then placed in PLASMACLEANER for oxygen plasma treatment and soaked in 1% polylysine for 1 minute, which reduced the contact angle, improved the lipophilicity of the PDMS surface, and enhanced its adhesion to other materials; in addition, adding other polymers (1% polylysine) to the PDMS chip can also change its surface properties and enhance its mechanical or chemical properties.

[0040] Example 2 Characterization of 3D co-culture of SMCs and GFP-HUVECs on PDMS chip

[0041] (1) Characterization of the physical and chemical properties of the scaffold material GelMA: Gelatin and methacrylic anhydride were used as raw materials. GelMA with 30%, 60%, and 90% substitution degree was prepared by adjusting the feed ratio of gelatin and methacrylic anhydride. GelMA with different substitution degrees was prepared into different concentrations such as 6%, 8%, 10%, and 12%, and its rheological properties were measured. The photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was added to different GelMA solutions to explore the effect of GelMA with different substitution degrees and concentrations on the growth of SMCs. The compressive strength and tensile properties of the GelMA scaffold were tested using a micro-universal material testing machine to provide a real ECM growth microenvironment for SMCs. Based on the above analysis of the results, GelMA with 30% substitution and 10% concentration was finally selected for co-culture of cells.

[0042] (2) Characterization of the perfusionability of PDMS chips: Use polymer hydrogel GelMA mixed with SMCs as the arterial vascular wall, and culture ECs in a ring inside the vascular wall to obtain a tubularly arranged endothelial layer, thereby constructing a hollow vascular structure with a perfusable channel. First, prepare a steel needle with a diameter of 1 mm and a length of 100 mm, coat the surface with a small amount of lubricating oil, and insert it into the middle channel of the PDMS chip. Taking advantage of the thermosensitivity and photocuring properties of GelMA, when it is in liquid form at 37°C, it is sent into the PDMS chip with the steel needle inserted. Then, after irradiating with a portable curing light source (420 nm) for 11 seconds each on the front and back, GelMA solidifies into a gel state, and then slowly pulls out the steel needle from one side, forming a cylindrical perfusable channel in the middle (such as Figure 4 As shown). Then, an external pump was connected through a PVC tube, and the culture medium was introduced at a flow rate of 200 μL / h, 100 μL / h, 50 μL / h, and 20 μL / h for 24 hours to observe the swelling of GelMA and the perfusion of the channel. After ECs were introduced and cultured for 6 hours, the culture medium was introduced at a flow rate of 20 μL / h to observe the adhesion of endothelial cells and the swelling of GelMA after 1 day, 2 days, and 3 days.

[0043] (3) Cultivation of SMCs and GFP-HUVECs in GelMA hydrogels: Qtracker TM 655 labeled SMCs were 4×10 6The cells were mixed at a density of 100 / mL in a GelMA solution containing 0.5% (w / v) LAP. A steel needle with a diameter of 1 mm and a length of 100 mm was prepared. After the surface was treated with lubricating oil, it was inserted into the middle channel of the PDMS chip that had been soaked in 1% polylysine for 1 min. Then the GelMA containing SMCs was passed into the hollow channel of the chip and laid on the inner surface of the channel without affecting its perfusionability. Blue light (420 nm) was irradiated for 11 seconds each in both the positive and negative directions to evenly wrap the SMCs inside the cross-linked GelMA hydrogel. After curing, the steel needle in the middle was pulled out to form a hollow perfusable channel in the middle. Ordinary culture medium was passed to flush away the lubricating oil in the channel, and then GFP-HUVECs were inoculated at a rate of 4×10 6 The cells were mixed with a cell density of 100 μg / mL in the culture medium of GFP-HUVECs, passed into the hollow channel, and evenly laid on the inner surface of the polymerized GelMA. After being placed in a saturated humidity, 37°C, 5% CO2 incubator and statically cultured for 6 hours, they were arranged in a tubular shape to obtain simulated arterial blood vessels. Finally, the culture medium of GFP-HUVECs and SMCs was mixed in a 1:1 ratio and moved into a syringe, connected to an external pump, and passed into the above-mentioned simulated arterial blood vessels at a flow rate of 20 μL / h. The culture medium of GFP-HUVECs was a mixture of F12 and ECM in a 1:1 ratio, and then 10% (w / v) FBS and 1% (w / v) double antibodies were added. The culture medium of SMCs was a mixture of 1640 and SMCM in a 1:1 ratio, and then 10% (w / v) FBS and 1% (w / v) double antibodies were added. The cell growth and vascularization of GFP-HUVECs were observed under a fluorescence microscope. The growth and distribution of SMCs in GelMA hydrogels at different times, as well as the distribution of SMCs and GFP-HUVECs in the hollow perfusable model were observed using a confocal laser scanning microscopy (CLSM). Figure 5 AC). It was observed that a small amount of GFP-HUVECs were distributed in the hollow channel at 6 hours. After 24 hours, ECs were obviously proliferated and the connections between cells increased significantly. GelMA with good biocompatibility provided good conditions for GFP-HUVECs to adhere to and proliferate on its surface. At the same time, it was observed that the SMCs wrapped in it also showed good proliferation and ductility after 24 hours, showing signs of early angiogenesis (such as Figure 5DI). The results showed that GelMA could well enclose HUVECs and SMCs in the perfusable hollow artery model without affecting cell growth, and achieved the relative spatial distribution of GFP-HUVECs and SMCs, simulating the annular hollow structure of the bionic arterial wall. The specific protein expression of HUVECs and SMCs was observed by high-resolution fluorescence images, further confirming the cell activity and cell function in the perfusable bionic artery. The fluorescence (yellow) image of α-smooth muscle actin (α-SMA) of SMCs showed ( Figure 5 J), influenced by the physical and chemical properties of the three-dimensional scaffold GelMA, SMCs formed a self-organized tissue-like structure in GelMA after 24 hours of culture, and began to stretch and differentiate to form intercellular protrusions. At the same time, the VE-Cad fluorescence (purple) of GFP-HUVECs also showed a highly organized network ( Figure 5 K), which is due to the presence of GelMA hydrogel remodeling the ECM. In addition, the obvious increase in the purple fluorescence level of VE-Cad and the tubular growth indicate that the tissue vascularization of HUVECs is also enhanced with the extension of culture time.

[0044] Example 3 Construction of Pathological Blood Vessel Chip

[0045] (1) Simulation of high-fat conditions: Red fluorescent-labeled ox-LDL (Dil-ox-LDL, 10-50 μg / mL) was added to the culture medium and passed into the circulating vascular chip. After 1, 3, and 5 days, the cells were compared with the normal blank group to detect the phagocytosis of ox-LDL, the expression of von Willebrand factor (vWF) protein, the content of NO, proteoglycan and collagen type II in the culture medium, and the PCR and ELISA were used to detect the gene and protein levels of TNF-α, IL-1β, IL-6, COX-2, CXCR-4, and MMPs (such as MMP-3 / 9 / 13) to optimize the concentration of ox-LDL.

[0046] (2) Simulation of inflammatory environment: Inflammatory factors IL-1β (1-5 ng / mL) and TNF-α (1-5 ng / mL) were added to the culture medium and passed into the circulating vascular chip. After 1, 3, and 5 days, the contents of NO, proteoglycan, and collagen type II in the culture medium were detected compared with those in the normal blank group. PCR and ELISA were used to detect the gene and protein levels of TNF-α, IL-1β, IL-6, COX-2, CXCR-4, and MMPs (such as MMP-3 / 9 / 13) to optimize the concentration of inflammatory factors.

[0047] (3) Establishment of AS disease model induced by high fat and inflammatory factors: Preliminary concentration exploration showed that ox-LDL (50 μg / mL), TNF-α (2 ng / mL) and IL-1β (2 ng / mL) could simulate the early pathological conditions of atherosclerosis. They were added to the culture medium at the above concentrations and 2 mL was filled with a syringe for later use. The entire PDMS chip was placed in a sterile incubator with saturated humidity, 37°C, and 5% CO2. It was connected to a syringe containing culture medium (containing the above concentration of stimulating factors) through a PVC tube and an adapter. Then, it was placed on a syringe pump and the flow rate was adjusted to 20 μL / h to pass through the simulated artery. After 24 hours, the specific protein expression of ECs, the migration of SMCs, and the distribution of the two cells were observed by cell immunofluorescence staining. The content of TG, CH, ROS, and ox-LDL was detected by enzyme-linked immunosorbent assay (ELISA) to verify the successful construction of the AS vascular disease model.

[0048] Example 4 Pathological blood vessel chip for drug screening

[0049] 1. Experimental Methods

[0050] (1) Immunofluorescence staining of cell-specific proteins

[0051] Fixation: After the incubation, wash the samples gently with PBS for 3 times, fix with 4% paraformaldehyde for 1 hour, and rinse with PBS for 3 times, 5 minutes each time. Punching: Add 1mL of 0.1% Triton X-100 (solvent is PBS) to each group of samples at room temperature for 20 minutes, and rinse with PBS for 3 times, 5 minutes each time. Blocking: Add 1mL of 5% BSA (10mg / mL, solvent is PBS) to each well, incubate on a shaker at room temperature for 1 hour, and rinse with PBS for 3 times. Primary antibody incubation: Dilute VE-cad and α-SMA primary antibodies according to the dosage at a dilution ratio of 1:200 (solvent is PBS), mix well and use, add 500μL to each well, fill the outer circle of the well plate with PBS, seal with plastic wrap, place in a moisturizing box, and incubate at 4℃ overnight (12-14h). Rinse with PBS 3 times after completion. Secondary antibody incubation in the dark: VE-cad goat anti-rabbit IgG (H+L)-CoraLite59 secondary antibody and α-SMA goat anti-rabbit IgG H&L (Alexa 488) The secondary antibody is diluted at a ratio of 1:200 (the solvent is PBS) according to the dosage, mixed and ready to use, add 500μL to each well, fill the gap of the well plate with PBS, seal it with plastic wrap and tin foil, incubate it on a shaker at room temperature for 1h, and rinse it with PBS 5 times. Cell nucleus staining: add 500μLDA I stain (1μg / mL, the solvent is 1% BSA) to each well, treat it at room temperature for 10min, and wash it with PBS 3 times. The sample is soaked in a small amount of PBS solution and photographed as soon as possible. If it needs to be placed for a long time, the sample can be soaked in a small amount of secondary antibody solution for preservation, or anti-fluorescence quenching agent can be used for sealing. Finally, CLSM is used to observe and photograph at 488nm and 655nm channels, and the control group is used for parameter setting and calibration. Subsequently, the batch of samples is uniformly photographed using the predetermined uniform parameters. Finally, the image is quantitatively analyzed for fluorescence area using Image J.

[0052] (2) Detection of the content of pathological model-related molecules

[0053] Ox-LDL (50 μg / mL), TNF-α (2 ng / mL), and IL-1β (2 ng / mL) were added to the culture medium and passed into the simulated vascular model for 24 hours, and the supernatant after circulation was collected. Then the cells in the model were taken out and treated with an ultrasonic crusher, and the supernatant was centrifuged and mixed with the supernatant taken after circulation. Specific antibodies were used to bind to the molecules to be detected, and then the antibody-antigen binding events were detected by enzyme-labeled secondary antibodies. The relative contents of ROS, TG, CH, and ox-LDL were detected by ELISA. At the same time, a 2D flat plate culture group and a 3D healthy vascular model group were set up as controls to verify the successful construction of the AS vascular disease model. The specific steps are as follows: first add the sample: set 10 wells with 5 concentration points for the standard, set parallel wells for each concentration, add 50μL of standard of different concentrations, set 1 well for the blank well and add 50μL of distilled water (no sample and enzyme-labeled reagent are added to the blank control well, and the rest of the steps are the same), add 40μL of sample diluent to the well of the sample to be tested on the enzyme-labeled coated plate, and then add 10μL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Incubation: Seal the plate with a sealing film and incubate at 37℃ for 30min. Liquid preparation: dilute the 20-fold concentrated washing solution with 20-fold distilled water for use. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat this 5 times, and pat dry. Add enzyme: add 50μL of enzyme-labeled reagent to each well, except for the blank well. Then incubate and wash, and the specific operation is the same as above. Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color development at 37℃ in the dark for 15min. Termination: Add 50μL of stop solution to each well to terminate the reaction (the blue color turns yellow immediately). Measurement: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15min after adding the stop solution. Finally, use GraphPad Prism for statistical analysis and drawing.

[0054] (3) Cell viability and drug toxicity testing

[0055] Rapamycin (RAP) was used as a model drug. This example observed the cell growth of RAP in different models. Two types of models were set up: 2D flat plate culture and 3D perfusable vascular model (model successfully constructed using Example 3 of the present invention). The 3D perfusable model was divided into a healthy group and a pathological group. RPM with concentrations of 100, 80, 60, 40, 30, 20, and 10 μM was introduced into the simulated arterial blood vessels of the above-mentioned PDMS chip and cultured for 24 hours (the culture medium was the culture medium of GFP-HUVECs and the culture medium of SMCs in Example 2 mixed in a 1:1 ratio). The CCK-8 method was used to observe the activity of the cells and the effect of the drug on the cell proliferation activity. The cell survival rate (%) was calculated according to the following formula:

[0056] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0057] As: absorbance of the experimental well (culture medium containing cells, CCK-8, and drug to be tested)

[0058] Ac: Absorbance of control well (culture medium containing cells, CCK-8, no test drug)

[0059] Ab: absorbance of blank well (medium without cells and drug to be tested, CCK-8)

[0060] And draw the cell survival rate curve to determine the drug concentration with 50% inhibition rate (IC50). The above analysis set the cells cultured on 2D plates as the control group, including three groups of ECs, SMCs and 1:1 co-culture of ECs and SMCs, and compared them with the drug evaluation results obtained using the AS disease vascular model to improve the efficiency of drug screening.

[0061] 2. Experimental Results

[0062] (1) Cell-specific protein expression

[0063] VE-cadherin is an adhesion molecule expressed in vascular endothelial cells, which is mainly responsible for maintaining the integrity of the endothelial barrier and regulating the adhesion strength between cells. In this experiment, we used immunofluorescence staining to compare the expression level of VE-cad in endothelial cells in the vascular chip before and after treatment with ox-LDL (50μg / mL), TNF-α (2ng / mL) and IL-1β (2ng / mL), and calculated the area and average fluorescence intensity of the two groups of cells by software statistics (such as Figure 6AB). The results showed that after inflammatory factor treatment, the cell area and mean fluorescence intensity of VE-cad in the model group were significantly reduced. This suggests that inflammatory factors may weaken the adhesion strength between endothelial cells by downregulating the expression of VE-cad or changing its distribution, thereby destroying the integrity of the endothelial barrier. The loss of endothelial barrier function may lead to increased permeability of the vascular wall, allowing more inflammatory cells and harmful substances to enter the arterial wall, thereby aggravating the lesions of atherosclerosis.

[0064] (2) Quantitative analysis of related molecules and proteins in pathological models

[0065] To further verify the biological efficacy of the 3D AS biomimetic vascular model, the levels of ox-LDL, ROS, human triglyceride (TG), and human cholesterol (CH) were measured and compared with the HUVECs and SMCs co-culture model on a 2D plate and the 3D normal vascular model group without the addition of inflammatory factors. It is worth noting that enzyme-linked immunosorbent assay (ELISA) provides a highly sensitive and specific detection for analyzing protein levels, allowing accurate quantification of target proteins in samples. Figure 6 As shown in CD, compared with the 2D flat co-culture model group, ox-LDL retention and ROS production were higher in the 3DAS model after 24 hours of incubation with inflammatory factors. The results showed that the increase in ox-LDL retention and ROS production in the 3D cultured cell group was attributed to the penetration and diffusion of cytokines and the mechanical stimulation generated when the cells were co-cultured with GelMA, and excessive ROS would cause cellular oxidative damage and dysfunction, further accelerating the development of AS. In addition, in the constructed 3D AS pathological model, TG, a risk factor for cardiovascular disease, increased significantly after 24 hours of inflammatory factor stimulation (P<0.00001), indicating that it promotes the occurrence of AS to a certain extent ( Figure 6 E). At the same time, CH increased significantly in the AS pathological model group (P = 0.0004). On the one hand, it shows that excessive CH deposited in the bionic vascular model accelerated the formation of the core part of the atherosclerotic plaque. On the other hand, CH crystals can activate NLRP3 inflammasomes, leading to the release of inflammatory factors (such as IL-1β), aggravating local inflammatory reactions and promoting the development of AS ( Figure 6 F).

[0066] (3) Models for drug screening

[0067] In the preclinical drug development process, the purpose of using organ-on-a-chip models for drug screening is to evaluate the safety, efficacy and potential side effects of drugs under earlier and more realistic physiological conditions. This innovative model combines microfluidics and cell culture technology to more accurately simulate the microenvironment of human organs and tissues, providing more relevant and predictive results than traditional 2D cell culture or animal models. According to the CCK-8 cytotoxicity results, the absorbance (OD value) and the best fit half-maximum inhibitory concentration (IC50) of the anti-inflammatory drug RAP in each group were analyzed, and the concentration-absorbance curve and dose-response curve ( Figure 7 AD). The results showed that the IC50 values ​​of 2D cultured HUVECs, SMCs and 1:1 co-culture were 22.29, 27.71 and 32.69 μM, respectively. In the 3D co-cultured SMCs and HUVECs biomimetic arterial wall model, the IC50 values ​​of the healthy group without inflammatory factors and the pathological group with inflammatory factors were 140.2 μM and 55.36 μM, respectively. The IC50 value of the drug in the 3D culture model is higher than that in the 2D culture model. On the one hand, the scaffold material GelMA limits the diffusion and penetration of the drug to a certain extent. On the other hand, the 3D structure formed by GelMA is closer to the in vivo tissue. The behavior and response of cells in the 3D environment are different from those in the 2D environment. Cells may enhance their tolerance to drugs by regulating gene expression and cell signaling pathways, thereby requiring higher drug concentrations to inhibit cell activity, showing higher survival rates and stronger drug resistance. In addition, in a 3D environment, the interactions between cells and surrounding cells and matrix are more complex, and these interactions can also affect cell growth and response to drugs. The above results indicate that these factors make the bionic AS pathological vascular model constructed by 3D co-culture in this example closer to the in vivo microenvironment and can be used as a representative drug testing platform.

[0068] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for constructing an atherosclerotic blood vessel model, characterized in that: The steps include: (1) manufacturing a PDMS chip with an injection channel, and then performing surface modification on the injection channel to enable it to be used for GelMA attachment; (2) GelMA and SMCs are mixed and perfused into the surface-modified injection channel, and the mixture is cured and cross-linked to the inner wall of the injection channel to form a simulated arterial wall, and GFP-HUVECs are cultured in a ring shape in the simulated arterial wall to obtain an endothelial layer arranged in a tubular shape, thereby obtaining a simulated arterial vessel; (3) introducing a culture medium containing ox-LDL, TNF-α and IL-1β into the simulated artery obtained in step (2) for culturing.

2. The construction method according to claim 1, characterized in that: The surface modification is carried out by sequentially treating with isocyanate ions and soaking in a polylysine solution.

3. The construction method according to claim 2, characterized in that: The concentration of the polylysine solution is 0.8-1.2%.

4. The construction method according to claim 1, characterized in that: The substitution degree of the GelMA is 29-31%, and the concentration is 9-11%.

5. The construction method according to claim 4, characterized in that: The density of the SMCs in the GelMA was 3.8-4.1×10 6 Pieces / mL.

6. The construction method according to claim 1, characterized in that: The surface modification is sequentially treated with isocyanate ions and immersed in a polylysine solution with a concentration of 0.8-1.2%; the substitution degree of the GelMA is 29-31% and the concentration is 9-11%; the density of the SMCs in the GelMA is 3.8-4.1×10 6 Pieces / mL.

7. The construction method according to any one of claims 1 to 6, characterized in that: The concentrations of ox-LDL, TNF-α and IL-1β in the culture medium were 48-52 μg / mL, 1.5-2.4 ng / mL and 1.5-2.4 ng / mL, respectively.

8. The construction method according to claim 7, characterized in that: In the step (3), the culture medium is introduced at a rate of 18-22 μL / h, and the culture time is 20-25 h.

9. Use of the atherosclerotic vascular model constructed by the construction method according to any one of claims 1 to 8 in in vitro screening tests of therapeutic drugs for atherosclerosis.

10. An in vitro screening test method for treating atherosclerosis, characterized in that: An atherosclerotic vascular model constructed by the construction method described in any one of claims 1 to 8.

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