Model construction method for testing toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells and application

Abstract

The invention relates to a model construction method and application for testing toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells. The method comprises the following steps: uniformly mixing primary arterial smooth muscle cells and a BD-Matrigel matrigel solution by adopting a convection type double-channel microchip, filling a cell culture channel with the mixture, filling a single-cell suspension of the primary arterial endothelial cells, constructing a primary arterial smooth muscle cell-endothelial cell in-vitro layered co-culture model, and after the model is intervened by environmental pollutants, establishing a primary arterial smooth muscle cell-endothelial cell in-vitro layered co-culture model. Collecting liquid in the diffusion pool and detecting the secretion level of the liquid; and observing the state and dynamic change of the gap junction channel formed by the smooth muscle cell-endothelial cell and the expression condition of the gap junction protein by using a laser confocal microscope. The construction of the model provides a new target for occurrence and development of arterial vascular remodeling and functional lesions caused by environmental exposure of harmful substances and drug prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical research technology, and particularly relates to the construction of an arterial disease research model, specifically a model construction method and application for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells. Background Art

[0002] Epidemiological surveys show that the incidence of diseases such as atherosclerosis, pulmonary hypertension, and right heart failure caused by arterial vascular disease has been increasing year by year, and environmental pollutants are one of the common risk factors. The pathogenesis of arterial vascular disease is complex, and its most basic pathological manifestation is the disorder of communication between endothelial cells and smooth muscle cells. The communication between endothelial cells and smooth muscle cells is divided into two types: autocrine and paracrine, and cell junctions. The disorder of communication is mainly manifested in changes in secretion levels and changes in the state of gap junction channels. Therefore, studying the toxic effects of environmental pollutants on primary arterial smooth muscle cells-endothelial cells is of great significance for preventing and treating various diseases caused by arterial vascular disease and providing new therapeutic targets.

[0003] Existing studies on gap junction channels between endothelial cells and smooth muscle cells mostly select one of the cells to study the structure and expression changes of gap junction proteins on its membrane, and there are few studies on gap junctions formed by co-culture of endothelial cells and smooth muscle cells. Studies on gap junctions formed by co-culture have been reported by simulating the tissue anatomical structure of blood vessels by inoculating endothelial cells and smooth muscle cells on both sides of the transwell chamber. Although this model based on the structure of the transwell chamber simulates vascular tissue in structure, the two cells can only communicate by producing a small amount of gap junction proteins through the transwell membrane pores, and it is difficult to obtain gap junction proteins and observe their morphology.

[0004] At present, the microfluidics field has proposed a variety of channel microchips as carriers for constructing a co-culture model of vascular smooth muscle cells and endothelial cells for the study of hemodynamic effects. For example, the invention patent with the announcement number CN114149923B discloses "a method for constructing a single-channel microchip model for co-culture of endothelial cells and smooth muscle cells". This method uses arterial blood vessels as prototypes to establish a single-channel microchip model for layered culture of endothelial cells and smooth muscle cells, which is used to dynamically monitor the cell morphology and functional changes of endothelial cells and smooth muscle cells under the same flow stimulation. Although this model solves the problem of direct physical contact between the two cells in the same microchannel, it does not propose a detection method for the gap junction channels between endothelial cells and smooth muscle cells. The confounding effect caused by the fluid shear force generated by its fluid input method will affect the study of the stimulation of a single environmental pollutant. Summary of the invention

[0005] The purpose of the present invention is to solve the problems in the prior art that endothelial cells and smooth muscle cells cannot be in direct physical contact, the gap junction channels between the two cells cannot be directly observed, and there are confounding factors in the research on environmental pollutant intervention. A model construction method and application for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is provided.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, comprising the following steps:

[0008] A. Cultivation of primary arterial mouse smooth muscle cells to obtain single-cell suspension of smooth muscle cells.

[0009] B. Cultivation of primary arterial mouse endothelial cells to obtain endothelial cell single cell suspension.

[0010] C. Construction of an in vitro layered co-culture model of primary arterial smooth muscle cells and endothelial cells.

[0011] a. Preparation of hardware system: The hardware system includes liquid input system, cell culture system and liquid recovery pool.

[0012] The liquid infusion system includes a mechanical flow controller to which a syringe is mounted.

[0013] The cell culture system includes a bottom plate, a first cover plate, a second cover plate and a third cover plate, wherein the bottom plate is spliced ​​and combined with the first cover plate, the second cover plate and the third cover plate to form different convection dual-channel microchips; the bottom plate and the first cover plate are spliced ​​and combined to form a first convection dual-channel microchip, wherein a first culture channel is formed inside the first convection dual-channel microchip, a first liquid input port and a first liquid output port communicating with the first culture channel are provided on the first cover plate, the first liquid input port is used to be connected to a syringe through a liquid input tube, and the first liquid output port is used to be connected to a liquid recovery pool through a liquid output tube; the bottom plate and the second cover plate are spliced ​​and combined to form a second convection dual-channel microchip, wherein a second culture channel is formed inside the second convection dual-channel microchip, and a second liquid input port and a second liquid output port communicating with the second culture channel are provided on the second cover plate The second liquid input port is used to connect to the syringe through the liquid input tube, and the second liquid output port is used to connect to the liquid recovery pool through the liquid output tube; the bottom plate and the third cover plate are spliced ​​and combined to form a third convection dual-channel microchip, and a third culture channel is formed inside the third convection dual-channel microchip. The third cover plate is provided with a third liquid input port and a third liquid output port that are communicated with the third culture channel, the third liquid input port is used to connect to the syringe through the liquid input tube, and the third liquid output port is used to connect to the liquid recovery pool through the liquid output tube; the third cover plate is provided with a diffusion pool, and the diffusion pool and the third culture channel are separated by a semipermeable membrane. The bottom plate and the third cover plate are combined to form an intervention channel without mixed dynamic factors. When an environmental pollutant solution is added to the diffusion pool, the pollutant molecules can pass through the semipermeable membrane without generating fluid shear force.

[0014] b. Soak and wipe all components of the cell culture system with 75% alcohol, then irradiate with ultraviolet light for 30 minutes for disinfection.

[0015] c. Prepare smooth muscle cell-BD-Matrixgel solution from smooth muscle cell single cell suspension;

[0016] d. Assemble the bottom plate and the first cover plate into a first convection dual-channel microchip, connect the first liquid input port on the first cover plate to the syringe through the liquid input tube, and connect the first liquid output port on the first cover plate to the liquid recovery pool through the liquid output tube; load the smooth muscle cell-BD-Matrixgel matrix gel solution into the syringe, turn on the mechanical flow controller, and inject the smooth muscle cell-BD-Matrixgel matrix gel solution into the first culture channel of the first convection dual-channel microchip at a speed of 2-10μl / min. After 30 minutes, the smooth muscle cell-BD-Matrixgel matrix gel solution in the first culture channel solidifies to form a flexible and flat smooth muscle cell culture layer.

[0017] The first cover plate is removed from the base plate, and the second cover plate is spliced ​​onto the base plate to form a second convection dual-channel microchip. The second liquid input port on the second cover plate is connected to the syringe through the liquid input tube, and the second liquid output port on the second cover plate is connected to the liquid recovery pool through the liquid output tube; the endothelial cell single-cell suspension is loaded into the syringe, and the mechanical flow controller is turned on to inject the endothelial cell single-cell suspension into the second culture channel of the second convection dual-channel microchip at a rate of 1-2 μl / min. After 12 hours, the endothelial cells in the endothelial cell single-cell suspension adhere to the smooth muscle cell culture layer, and the in vitro layered co-culture model of primary arterial smooth muscle cells-endothelial cells is completed.

[0018] D. Construction of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells.

[0019] On the second day, the residual culture medium liquid in the second culture channel is drained by pushing the airflow generated by the syringe; the second cover plate is removed from the base plate, and the third cover plate is spliced ​​on the base plate to form a third convection dual-channel microchip, the third liquid input port on the third cover plate is connected to the syringe through a liquid input tube, and the third liquid output port on the third cover plate is connected to a liquid recovery pool through a liquid output tube; the syringe is filled with culture medium containing 20% ​​fetal bovine serum, the mechanical flow controller is turned on, and the culture medium containing 20% ​​fetal bovine serum is poured into the third culture channel of the third convection dual-channel microchip at a rate of 2-10 μL / min until the culture medium containing 20% ​​fetal bovine serum fills the entire third culture channel; the pre-prepared environmental toxicant solution is added to the diffusion pool on the third cover plate for intervention. After the intervention is completed, the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is completed.

[0020] As a preferred technical solution, after the construction of the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model is completed, it is identified, specifically: the residual culture medium liquid in the second culture channel is drained by pushing the airflow generated by the syringe; the sterile PBS liquid is loaded into the syringe, the mechanical flow controller is turned on, and the sterile PBS liquid is poured into the second culture channel at a rate of 5 μl / min to complete the flushing of the second culture channel; after the flushing is completed, the Dil-Ac-LDL dye solution is loaded into the syringe, the mechanical flow controller is turned on, and the Dil-Ac-LDL dye solution is poured into the second culture channel at a rate of 5 μl / min until the Dil -Ac-LDL dye solution filled the entire second culture channel, and then placed in a cell culture incubator for incubation for 2 hours; after the incubation was completed, the residual Dil-Ac-LDL dye solution in the second culture channel was drained by pushing the airflow generated by the syringe; sterile PBS liquid was filled into the syringe, and the mechanical flow controller was turned on to inject sterile PBS liquid into the second culture channel at a rate of 5μl / min to complete the flushing of the second culture channel; through a laser confocal microscope, under the conditions of excitation wave 495nm and emission wave 565nm, the model was successfully constructed if the red fluorescent markers were arranged neatly and regularly; if the red fluorescence was scattered sporadically, the model construction failed.

[0021] As a preferred technical solution, the output end of the mechanical flow controller is connected to the piston of the syringe to control the flow rate of the liquid in the syringe; the capacity of the syringe is 1ml or 5ml.

[0022] As a preferred technical solution, in the first convection dual-channel microchip, the first culture channel has a height of 30 μm, a length of 6000 μm, and a width of 30 μm, and the first culture channel is used to culture smooth muscle cells; in the second convection dual-channel microchip, the second culture channel has a height of 60 μm, a length of 6000 μm, and a width of 30 μm, and the second culture channel is used to culture endothelial cells; in the third convection dual-channel microchip, the third culture channel has a height of 60 μm, a length of 6000 μm, and a width of 30 μm, and the third culture channel is used to infuse a culture medium containing 20% ​​fetal bovine serum or other liquids required for model applications; in the third convection dual-channel microchip, the diffusion pool adopts a cubic pool body with a length of 4000 μm, a width of 30 μm, and a height of 60 μm.

[0023] As a preferred technical solution, the material of the diffusion cell (13) is PDMS; the material of the semipermeable membrane is PET, and its pore size is 0.4 μm.

[0024] As a preferred technical solution, primary arterial mouse smooth muscle cells and primary arterial mouse endothelial cells are both derived from secondary and tertiary arteries of mice.

[0025] As a preferred technical solution, the method for preparing the smooth muscle cell-BD-Matrixgel matrix gel solution is as follows: take a single cell suspension of smooth muscle cells and make it into a suspension with a density greater than 1×10 7 To the single-cell suspension of 100 μg / mL, add 20 μl / ml of BD-Matrixgel and shake gently to mix.

[0026] Furthermore, the present invention also provides the above-mentioned model application for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, and the application mainly includes the following two aspects:

[0027] The first application is: after the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed, the liquid in the diffusion pool is collected, and the levels of inflammatory factors such as IL-6 and TNF-α are detected using ELISA kits to reflect whether the communication between smooth muscle cells and endothelial cells is normal, which is helpful to study the effect of environmental pollutants on the information exchange between smooth muscle cells and endothelial cells.

[0028] The second application is to use Fluo-4 / AM and Rhod-4 / AM as fluorescent dyes to pre-stain endothelial cells and smooth muscle cells respectively, and then use laser confocal microscopy to observe the Ca2+ in smooth muscle cells and endothelial cells before and after the intervention of environmental pollutants in this model. 2+ The dynamic distribution of , specifically includes the following steps:

[0029] S1. Basically the same as step A in the above-mentioned model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the cultured smooth muscle cells are washed 3 times with PBS, 1 ml of Rhod-4 / AM dye solution is added, and the cells are placed in a cell culture incubator for 30 minutes, the dye solution is discarded, and the cells are washed 3 times with PBS to obtain a pre-stained smooth muscle cell single cell suspension;

[0030] S2. Basically the same as step B in the above-mentioned model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the cultured endothelial cells are washed 3 times with PBS, 1 ml of Fluo-4 / AM dye solution is added, and the cells are placed in a cell culture incubator for 30 minutes, the dye solution is discarded, and the cells are washed 3 times with PBS to obtain a pre-stained endothelial cell single cell suspension;

[0031] S3. is basically the same as step C in the above-mentioned method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the smooth muscle cell single cell suspension used is replaced by a pre-stained smooth muscle cell single cell suspension, and the endothelial cell single cell suspension used is replaced by a pre-stained endothelial cell single cell suspension;

[0032] S4. This is basically the same as step D in the above-mentioned method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that after the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed, a laser confocal microscope is used to observe the Ca2+ in smooth muscle cells and endothelial cells under the conditions of an excitation wave of 494 nm and an emission wave of 550 nm. 2+ The dynamic distribution of .

[0033] In the present invention, the gap junctions formed by the smooth muscle cells and the endothelial cells are formed at the junction of the smooth muscle-BD-Matrixgel layer and the endothelial cells. The smooth muscle cells and endothelial cells are pre-stained with red and green Ca 2+ Fluorescent probes, after environmental pollutants were intervened, the dynamic changes of gap junction channels were observed by laser confocal microscopy, namely, the Ca 2+ Fluorescent probes can observe the state of gap junction channels, and after staining, the expression of gap junction proteins can be directly observed, which is helpful to study the role of gap junction channels in the occurrence and development of arterial remodeling caused by environmental exposure to harmful substances, and provide new targets for drug prevention and treatment of various diseases caused by arterial vascular lesions.

[0034] The model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cell-endothelial cells described in the present invention solves the problem that gap junctions between vascular endothelial cells and smooth muscle cells are produced less and cannot be directly observed, eliminates the influence of confounding factors in the intervention of environmental pollutants, and realizes the dynamic detection of the state of gap junction channels, which is helpful to study the role of gap junction channels in the occurrence and development of diseases related to arterial remodeling caused by environmental exposure to harmful substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments of the present invention are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 It is a schematic diagram of the sagittal plane of the model used for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to the present invention.

[0037] Figure 2 Schematic diagram of the components constituting the convection dual-channel microchip of the present invention.

[0038] Figure 3 It is a schematic diagram of the structure of the first convection dual-channel microchip in the present invention in a sagittal plane.

[0039] Figure 4 It is a schematic diagram of the structure of the second convection dual-channel microchip of the present invention in a sagittal plane.

[0040] Figure 5 It is a schematic diagram of the structure of the third convection dual-channel microchip of the present invention in a sagittal plane.

[0041] Figure 6 The Ca2+-β-catenin-type model used in the present invention to test the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is shown in FIG. 2+ Schematic diagram of fluorescent probe tracing.

[0042] In the figure: 1-mechanical flow controller, 2-syringe, 3-liquid input tube, 4-convection dual-channel microchip, 5-liquid output tube, 6-liquid recovery pool, 7-diffusion pool, 8-bottom plate, 9-first cover plate, 10-second cover plate, 11-third cover plate, 12-first convection dual-channel microchip, 13-first culture channel, 14-first liquid input port, 15-first liquid output port, 16-second convection dual-channel microchip, 17-second culture channel, 18-second liquid input port, 19-second liquid output port, 20-third convection dual-channel microchip, 21-third culture channel, 22-third liquid input port, 23-third liquid output port, 24-semipermeable membrane. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0044] The present invention provides a model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, comprising the following steps:

[0045] A. Cultivation of primary arterial mouse smooth muscle cells to obtain a single cell suspension of smooth muscle cells; wherein, primary arterial mouse smooth muscle cells are derived from the secondary and tertiary arteries of mice.

[0046] B. Cultivation of primary arterial mouse endothelial cells to obtain endothelial cell single cell suspension; wherein, primary arterial mouse endothelial cells are from the secondary and tertiary arteries of mice.

[0047] C. Construction of an in vitro layered co-culture model of primary arterial smooth muscle cells and endothelial cells.

[0048] a. Preparation of hardware system: The hardware system includes liquid input system, cell culture system and liquid recovery pool 6.

[0049] The liquid input system includes a mechanical flow controller 1, which is equipped with a syringe 2; the output end of the mechanical flow controller 1 is connected to the piston of the syringe 2 to control the flow rate of the liquid in the syringe 2; the capacity of the syringe 2 is 1ml or 5ml.

[0050] The cell culture system includes a bottom plate 8, a first cover plate 9, a second cover plate 10 and a third cover plate 11. The bottom plate 8 is spliced ​​and combined with the first cover plate 9, the second cover plate 10 and the third cover plate 11 to form different convection dual-channel microchips 4. The bottom plate 8 and the first cover plate 9 are spliced ​​and combined to form a first convection dual-channel microchip 12. A first culture channel 13 is formed inside the first convection dual-channel microchip 12. The first cover plate 9 is provided with a first liquid input port 14 and a first liquid output port 15 which are communicated with the first culture channel 13. The first liquid input port 14 is used to connect to the syringe 2 through the liquid input tube 3, and the first liquid output port 15 is used to connect to the liquid recovery pool 6 through the liquid output tube 5; wherein the first culture channel 13 has a height of 30 μm, a length of 6000 μm and a width of 30 μm, and is used to culture smooth muscle cells. The bottom plate 8 and the second cover plate 10 are spliced ​​and combined to form a second convection dual-channel microchip 16. A second culture channel 17 is formed inside the second convection dual-channel microchip 16. The second cover plate 10 is provided with a second liquid input port 18 and a second liquid output port 19 which are communicated with the second culture channel 17. The second liquid input port 18 is used to connect to the syringe 2 through the liquid input tube 3, and the second liquid output port 19 is used to connect to the liquid recovery pool 6 through the liquid output tube 5. The second culture channel 17 has a height of 60 μm, a length of 6000 μm, and a width of 30 μm. The second culture channel 17 is used to culture endothelial cells. The bottom plate 8 and the third cover plate 11 are spliced ​​and combined to form a third convection dual-channel microchip 20. A third culture channel 21 is formed inside the third convection dual-channel microchip 20. The third cover plate 11 is provided with a third liquid input port 22 and a third liquid output port 23 which are communicated with the third culture channel 21. The third liquid input port 22 is used to connect with the syringe 2 through the liquid input tube 3, and the third liquid output port 23 is used to connect with the liquid recovery pool 6 through the liquid output tube 5. A diffusion pool 7 is provided on the third cover plate 11. The diffusion pool 7 and the third culture channel 21 are separated by a semipermeable membrane 24. The third culture channel 21 has a height of 60 μm, a length of 6000 μm, and a width of 30 μm. The third culture channel 21 is used to infuse a culture medium containing 20% ​​fetal bovine serum or other liquids required for model application. The diffusion pool 7 adopts a cubic pool body with a length of 4000 μm, a width of 30 μm, and a height of 60 μm. The semipermeable membrane 24 is made of PET and has a pore size of 0.4 μm.

[0051] b. Soak and wipe the components of the cell culture system with 75% alcohol, and then irradiate with ultraviolet light for 30 minutes for disinfection;

[0052] c. Prepare smooth muscle cell-BD-Matrixgel matrix gel solution from smooth muscle cell single cell suspension. The specific method is as follows: take smooth muscle cell single cell suspension and make it into a solution with a density greater than 1×10 7 To the single-cell suspension of 100 μg / mL, add 20 μl / ml of BD-Matrixgel and shake gently to mix.

[0053] d. Assemble the bottom plate 8 and the first cover plate 9 to form a first convection dual-channel microchip 12, connect the first liquid input port 14 on the first cover plate 9 to the syringe 2 through the liquid input tube 3, and connect the first liquid output port 15 on the first cover plate 9 to the liquid recovery pool 6 through the liquid output tube 5; load the smooth muscle cell-BD-Matrixgel matrix gel solution into the syringe 2, turn on the mechanical flow controller 1, and inject the smooth muscle cell-BD-Matrixgel matrix gel solution into the first culture channel 13 of the first convection dual-channel microchip 12 at a speed of 2-10 μl / min. After 30 minutes, the smooth muscle cell-BD-Matrixgel matrix gel solution in the first culture channel 13 solidifies to form a flexible and flat smooth muscle cell culture layer;

[0054] The first cover plate 9 is disassembled from the bottom plate 8, and the second cover plate 10 is spliced ​​onto the bottom plate 8 to form a second convection dual-channel microchip 16. The second liquid input port 18 on the second cover plate 10 is connected to the syringe 2 through the liquid input tube 3, and the second liquid output port 19 on the second cover plate 10 is connected to the liquid recovery pool 6 through the liquid output tube 5; the endothelial cell single-cell suspension is loaded into the syringe 2, and the mechanical flow controller 1 is turned on to inject the endothelial cell single-cell suspension into the second culture channel 17 of the second convection dual-channel microchip 16 at a speed of 1-2 μl / min. After 12 hours, the endothelial cells in the endothelial cell single-cell suspension adhere to the smooth muscle cell culture layer, and the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model is completed;

[0055] D. Construct a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells;

[0056] On the second day, the residual culture medium liquid in the second culture channel 17 is drained by pushing the airflow generated by the syringe 2; the second cover plate 10 is disassembled from the bottom plate 8, and the third cover plate 11 is spliced ​​on the bottom plate 8 to form a third convection dual-channel microchip 20, the third liquid input port 22 on the third cover plate 11 is connected to the syringe 2 through the liquid input tube 3, and the third liquid output port 23 on the third cover plate 11 is connected to the liquid recovery pool 6 through the liquid output tube 5; the syringe 2 is filled with a culture medium containing 20% ​​fetal bovine serum, the mechanical flow controller 1 is turned on, and the culture medium containing 20% ​​fetal bovine serum is poured into the third culture channel 21 of the third convection dual-channel microchip 20 at a speed of 2-10 μL / min until the culture medium containing 20% ​​fetal bovine serum fills the entire third culture channel 21; the pre-prepared environmental toxicant solution is added to the diffusion pool 7 on the third cover plate 11 for intervention. After the intervention is completed, the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is completed.

[0057] Furthermore, after the construction of the in vitro layered co-culture model of primary arterial smooth muscle cells-endothelial cells was completed, and before the construction of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, the constructed in vitro layered co-culture model of primary arterial smooth muscle cells-endothelial cells was identified, specifically as follows: on the second day, the residual culture medium liquid in the second culture channel 17 was drained by pushing the airflow generated by the syringe 2; sterile PBS liquid was filled into the syringe 2, and the mechanical flow controller 1 was turned on to inject sterile PBS liquid into the second culture channel 17 at a rate of 5μl / min to complete the flushing of the second culture channel 17; after the flushing was completed, Dil-Ac-LDL dye solution was filled into the syringe 2, and the mechanical flow controller 1 was turned on to inject sterile PBS liquid into the second culture channel 17 at a rate of 5μl / min to complete the flushing of the second culture channel 17. The second culture channel 17 was filled with Dil-Ac-LDL dye solution until the Dil-Ac-LDL dye solution filled the entire second culture channel 17, and then placed in a cell culture incubator for incubation for 2 hours; after the incubation was completed, the residual Dil-Ac-LDL dye solution in the second culture channel 17 was drained by pushing the airflow generated by the syringe 2; sterile PBS liquid was filled into the syringe 2, and the mechanical flow controller 1 was turned on to inject sterile PBS liquid into the second culture channel 17 at a rate of 5 μl / min to complete the flushing of the second culture channel 17; laser confocal microscopy was used to observe under the conditions of excitation wave 495nm and emission wave 565nm, and it was found that the red fluorescent markers were arranged neatly and regularly, indicating that the in vitro layered co-culture model of primary arterial smooth muscle cells-endothelial cells was successfully constructed.

[0058] The above-mentioned model application for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells includes the following two aspects:

[0059] The first application is: after the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed, the liquid in the diffusion cell 7 is collected and the levels of inflammatory factors such as IL-6 and TNF-α are detected using ELISA kits.

[0060] The second application is to use Fluo-4 / AM and Rhod-4 / AM as fluorescent dyes to pre-stain endothelial cells and smooth muscle cells respectively, and then use laser confocal microscopy to observe the Ca2+ in smooth muscle cells and endothelial cells before and after the intervention of environmental pollutants in this model. 2+ The dynamic distribution of , specifically includes the following steps:

[0061] S1. Basically the same as step A in the above-mentioned model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the cultured smooth muscle cells are washed 3 times with PBS, 1 ml of Rhod-4 / AM dye solution is added, and the cells are placed in a cell culture incubator for 30 minutes, the dye solution is discarded, and the cells are washed 3 times with PBS to obtain a pre-stained smooth muscle cell single cell suspension;

[0062] S2. Basically the same as step B in the above-mentioned model construction method for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the cultured endothelial cells are washed 3 times with PBS, 1 ml of Fluo-4 / AM dye solution is added, and the cells are placed in a cell culture incubator for 30 minutes, the dye solution is discarded, and the cells are washed 3 times with PBS to obtain a pre-stained endothelial cell single cell suspension;

[0063] S3. is basically the same as step C in the above-mentioned method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that the smooth muscle cell single cell suspension used is replaced by a pre-stained smooth muscle cell single cell suspension, and the endothelial cell single cell suspension used is replaced by a pre-stained endothelial cell single cell suspension;

[0064] S4. This is basically the same as step D in the above-mentioned method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, except that after the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed, a laser confocal microscope is used to observe the Ca2+ in smooth muscle cells and endothelial cells under the conditions of an excitation wave of 494 nm and an emission wave of 550 nm. 2+ The dynamic distribution of .

[0065] The method of the present invention first cultures primary mouse arterial smooth muscle cells and endothelial cells, and then uses a homemade convection dual-channel microchip to co-culture the two cells in vitro, thereby establishing a model for testing the toxicity of environmental pollutants to primary mouse arterial smooth muscle cells-endothelial cells. The toxic effects of environmental pollutants on the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model are mainly manifested in the disorder of cell communication between endothelial cells and smooth muscle cells, which is specifically manifested as increased inflammation levels and abnormal gap junction channel switching. Before and after environmental pollutants intervene in the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model, the liquid in the diffusion cell 7 is collected and the levels of inflammatory factors such as IL-6 and TNF-α are detected. Ca 2+ It is a signal that has been clearly identified for information transmission through gap junction channels, so it was selected as an indicator of the switch state of gap junction channels between smooth muscle cells and endothelial cells. Rhod-4 / AM and Fluo-4 / AM were used as fluorescent dyes to pre-stain smooth muscle cells and endothelial cells respectively, and laser confocal microscopy was used to observe Ca2+ before and after environmental pollutants intervened in this model. 2+ The dynamic distribution of the gap junction proteins was investigated; and their structural characteristics were observed after staining.

[0066] The technical solution of the present invention is further described below in conjunction with several specific embodiments:

[0067] Example 1: Construction and identification of an in vitro co-culture model of primary mouse arterial smooth muscle cells and endothelial cells (taking the pulmonary artery as an example)

[0068] A. Culture of mouse primary pulmonary artery smooth muscle cells

[0069] Experimental animals: 5-7 week-old C57BL / 6J mice were purchased from the Experimental Animal Center of Shanxi Medical University.

[0070] The mice were anesthetized intraperitoneally with sodium pentobarbital, and the chest was opened quickly under sterile conditions; pre-cooled PBS was perfused along the apex until the mouse lungs turned white; agarose iron powder solution (agarose: iron powder: M199 culture medium = 1:1:200) was perfused along the same position until the mouse lungs turned gray-purple; the heart and lung tissues were removed and rinsed with pre-cooled PBS 3-5 times until the surface blood coagulation was cleaned; each lung lobe was cut with scissors and placed in a new 35mm culture dish, 1ml of pre-cooled PBS was added, and the lung tissue was cut into 1mm pieces with a blade. 3small pieces; transfer the tissue block into a centrifuge tube with a Pasteur pipette, place the centrifuge tube on a magnetic separator, wait for the iron-containing vascular tissue to be adsorbed on the side of the centrifuge tube near the magnet, wash it 3 times with pre-cooled PBS, and then suck out the residual PBS liquid in the tube; add collagenase solution (0.002g / ml), blow and wash the tissue block and resuspend it, move it to a new 60mm culture dish, and place it in a 37°C, 5% CO2 incubator for culture; after 1h, take out the culture dish, transfer the tissue block in the dish into a centrifuge tube, and repeatedly aspirate 4-5 times with a 20mL syringe to further resuspend it; place the centrifuge tube on a magnetic separator, wait for the iron-containing tissue block to be adsorbed on the tube wall, wash the collagenase solution in the tube, wash the tissue 3 times with M199 culture medium, and wash out the residual collagenase solution in the tube; add 3mL of M199 culture medium containing serum, resuspend the tissue block and transfer it to a 60mm culture dish, and place it in a CO2 incubator (37°C, 5% CO2) for culture overnight. On the next day, remove the culture dish, transfer the tissue blocks in the dish into a centrifuge tube and place it on a magnetic separator. After aspirating the culture medium, wash three times with M199 medium, add 3 mL of M199 medium containing serum, resuspend the tissue blocks and transfer them into a 60 mm culture dish, and place them in a CO2 incubator (37°C, 5% CO2) for culture.

[0071] After the cells adhered, fresh culture medium was replaced every 2-3 days until the cells were fully grown and passaged.

[0072] B. Cultivation of primary mouse pulmonary artery endothelial cells

[0073] Experimental animals: 5-7 week-old C57BL / 6J mice were purchased from the Experimental Animal Center of Shanxi Medical University.

[0074] The mice were anesthetized intraperitoneally with sodium pentobarbital, and the chest was opened quickly under sterile conditions to remove the heart and lung tissues. Under a stereoscopic microscope, the heart and five lung lobes were fixed with acupuncture needles, and the mouse pulmonary artery was isolated. The isolated pulmonary artery tissue was placed in a pre-cooled PBS solution, washed with PBS three times, and the residual liquid was discarded. The lung tissue was cut into 1 mm pieces with a blade. 3 The chopped tissue pieces were transferred to a new culture dish and digested with 0.005 g / ml collagenase; after 20 min, the tissue in the dish was transferred to a centrifuge tube, centrifuged at 1700 rpm for 3 min, the supernatant was discarded, 0.7 mL of EGM-2 medium containing serum was added, and the dish was cultured in a CO2 incubator (37°C, 5% CO2); after 6 h, the non-adherent tissue in the dish was discarded, and 3 mL of EGM-2 medium containing serum was added for culture.

[0075] After the cells adhered, fresh culture medium was replaced every 2-3 days until the cells were fully grown and passaged.

[0076] C. Construction of an in vitro co-culture model of mouse primary pulmonary artery smooth muscle cells and endothelial cells

[0077] This embodiment proposes a new carrier - a convection dual-channel microchip. All components of the convection dual-channel microchip are prepared according to the design drawing, and PDMS is selected as the material to obtain the components of the convection dual-channel microchip.

[0078] All components of the convection dual-channel microchip were wiped with sterile gauze soaked in 75% alcohol and sterilized by ultraviolet irradiation for 30 min.

[0079] Preparation of smooth muscle cell-BD-Matrixgel matrix gel solution: Culture primary smooth muscle cells to a density greater than 1×10 7 To the single-cell suspension of 100 μl / mL, add 20 μl / mL of BD-Matrixgel and shake gently to mix.

[0080] The piston of the syringe 2 is connected to the output end of the mechanical flow controller 1, and the liquid outlet of the syringe 2 is connected to the liquid input tube 3. The bottom plate 8 and the first cover plate 9 are spliced ​​and assembled into a first convection dual-channel microchip 12, the first liquid input port 14 is connected to the liquid input tube 3, the first liquid output port 15 is connected to the liquid output tube 5, and the other end of the liquid output tube 5 is connected to the liquid recovery pool 6 to form a passage. The smooth muscle cell-BD-Matrixgel matrix gel solution is loaded into the syringe 2 of 1mL, the mechanical flow controller 1 is turned on, and the smooth muscle cell-BD-Matrixgel matrix gel solution is poured into the first culture channel 13 of the first convection dual-channel microchip 12 at a speed of 2-10μL / min. After waiting for 30min, the smooth muscle cell-BD-Matrixgel matrix gel solution solidifies to form a flexible and flat smooth muscle cell culture layer.

[0081] Remove the first cover plate 9, and combine the second cover plate 10 with the bottom plate 8 to form a second convection dual-channel microchip 16. Connect the piston of the syringe 2 to the output end of the mechanical flow controller 1, connect the liquid outlet of the syringe 2 to one end of the liquid input tube 3, connect the second liquid input port 18 on the second cover plate 10 to the other end of the liquid input tube 3, connect the second liquid output port 19 to the liquid output tube 5, and connect the other end of the liquid output tube 5 to the liquid recovery pool 6 to form a passage. Load the 1ml endothelial cell single cell suspension into the syringe 2, turn on the mechanical flow controller 1, and pour the endothelial cell single cell suspension into the second culture channel 17 of the second convection dual-channel microchip 16 at a speed of 1-2μL / min, and wait for the endothelial cells to adhere to the wall; after 12h, the endothelial cells in the endothelial cell single cell suspension adhere to the smooth muscle cell culture layer, and the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model is completed.

[0082] D. Identification of primary arterial smooth muscle cell-endothelial cell layered co-culture model in vitro

[0083] Preparation of Dil-Ac-LDL stock solution: Take 50 μg of Fluo-4 / AM and add 54.6 μl of DMSO to prepare a 1 mM stock solution.

[0084] Preparation of Dil-Ac-LDL working solution: Take 4 μl of Fluo-4 / AM stock solution, add 1 ml of EGM-2 medium, mix well and set aside.

[0085] On the second day, push the piston of syringe 2 to drain the residual culture medium in the second culture channel 17; add 500 μl of sterile PBS to the 1 mL syringe 2 and perfuse at a rate of 5 μl / min until there is no PBS liquid in the second culture channel 17; add 1 ml of Dil-Ac-LDL dye solution to the 1 mL syringe 2 and perfuse at a rate of 5 μl / min until the dye solution is discharged from the liquid output tube 5. Place in a cell culture incubator and incubate for 30 min.

[0086] Push the piston of syringe 2 to drain the Fluo-4 / AM dye solution in the second culture channel 17 , and add 500 μl of sterile PBS into the 1 mL syringe 2 and perfuse at a speed of 5 μl / min until there is no PBS liquid in the second culture channel 17 .

[0087] Add 1 ml of EGM-2 culture medium into the 1 ml syringe 2, and infuse at a rate of 5 μl / min until EGM-2 culture medium is discharged from the liquid output tube 5. After waiting for 30 minutes, observe through a laser confocal microscope under the conditions of excitation wave 495 nm and emission wave 565 nm. If the red fluorescent markers are arranged neatly and regularly, the model is successfully constructed; if the red fluorescence is scattered sporadically, the model construction fails.

[0088] Example 2: Construction of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells

[0089] An in vitro co-culture model of mouse primary pulmonary artery smooth muscle cells and endothelial cells was constructed. The specific method is shown in Example 1. On the second day, the piston of the syringe 2 was pushed to drain the residual culture medium liquid in the second culture channel 17.

[0090] Remove the second cover plate 10, combine the third cover plate 11 and the bottom plate 8 to form a third convection dual-channel microchip 20, fill the 1mL syringe 2 with a culture medium containing 20% ​​fetal bovine serum, connect the piston of the syringe 2 to the output end of the mechanical flow controller 1, connect the liquid outlet of the syringe 2 to one end of the liquid input tube 3, connect the third liquid input port 22 on the third cover plate 11 to the other end of the liquid input tube 3, connect the third liquid output port 23 to the liquid output tube 5, and connect the other end of the liquid output tube 5 to the liquid recovery pool 6 to form a passage. Open the mechanical flow controller 1, and fill the third culture channel 21 of the third convection dual-channel microchip 20 with a culture medium containing 20% ​​fetal bovine serum at a speed of 2-10 μL / min until the liquid fills the entire third culture channel 21. Open the diffusion pool 7, and add the pre-prepared environmental toxicant solution to the diffusion pool 7 for intervention.

[0091] After the intervention, the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells was established. The fluid in the diffusion cell 7 was collected to detect the levels of IL-6, TNF-α, etc. in the secretion.

[0092] Example 3: Observation of Ca before and after environmental pollutant intervention 2+ Distribution in smooth muscle cells and endothelial cells

[0093] Preparation of Fluo-4 / AM stock solution (1 mM): Take 50 μg of Fluo-4 / AM and add 54.6 μl of DMSO to prepare a 1 mM stock solution.

[0094] Preparation of Fluo-4 / AM working solution: Take 4 μl of Fluo-4 / AM mother solution, add 1 ml of EGM-2 medium, mix well and set aside.

[0095] Preparation of Rhod-4 / AM stock solution (4 mM): Take 50 μg of Rhod-4 / AM, add 12.3 μl of DMSO, heat at 45°C for 30 min, and prepare a 4 mM stock solution.

[0096] Preparation of Rhod-4 / AM working solution: Take 1 μl of Rhod-4 / AM stock solution, add 1 mL of EGM-2 medium, mix well and set aside.

[0097] A. Pre-staining of primary mouse arterial smooth muscle cells

[0098] The cultured primary mouse arterial smooth muscle cells were washed three times with PBS, 1 mL of Rhod-4 / AM dye solution was added, and the cells were placed in a cell culture incubator for 30 min, the dye solution was discarded, and the cells were washed three times with PBS; a smooth muscle cell-BD-Matrixgel matrix gel solution was prepared, as shown in Example 1 for the specific method.

[0099] B. Pre-staining of primary mouse arterial endothelial cells

[0100] The cultured primary mouse arterial endothelial cells were washed three times with PBS, 1 ml of Fluo-4 / AM staining solution was added, and the cells were placed in a cell culture incubator for incubation for 30 min. The staining solution was discarded, and the cells were washed three times with PBS to obtain a pre-stained endothelial cell single cell suspension.

[0101] C. Construction of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells

[0102] The method is basically the same as Example 2, except that the smooth muscle cell single cell suspension used is replaced by a pre-stained smooth muscle cell single cell suspension, and the endothelial cell single cell suspension used is replaced by a pre-stained endothelial cell single cell suspension.

[0103] D. After observing the environmental toxicants intervention, Ca 2+ Distribution in smooth muscle cells and endothelial cells.

[0104] After the intervention is completed, push the piston of syringe 2 to drain the residual culture medium in the third culture channel 21. Add 500 μl of sterile PBS to the 1 mL syringe 2 and perfuse at a rate of 5 μl / min until there is no PBS liquid in the third culture channel 21. Add 1 ml of Fluo-4 / AM dye solution to the 1 mL syringe 2 and perfuse at a rate of 5 μl / min until the dye solution is discharged from the liquid output tube 5, and place it in the cell culture incubator for 30 min.

[0105] Push the piston of syringe 2 to drain the Fluo-4 / AM dye solution in the third culture channel 21 , and add 500 μl of sterile PBS into the 1 mL syringe and perfuse at a speed of 5 μl / min until there is no PBS liquid in the third culture channel 21 .

[0106] Add 1 ml of EGM-2 medium into the 1 ml syringe 2 and infuse at a rate of 5 μl / min until medium is discharged from the liquid output tube 5; wait for 30 minutes and observe the Ca2+ in the smooth muscle cells and endothelial cells under the conditions of excitation wave 494 nm and emission wave 550 nm using a laser confocal microscope. 2+ The distribution of .

[0107] Example 4: Immunofluorescence staining to observe gap junctions after environmental pollutant intervention

[0108] Construct an environmental pollutant intervention model, see Example 3.

[0109] The third cover plate 11 was opened, and the matrix gel in the channel was gently removed with a knife and tweezers, and the sample was placed on a freezing embedding tray, embedded with an ice-cold embedding medium (OTC), and sliced; primary antibody against Factin (cytoskeleton protein) and secondary antibody labeled with Alexfluo-594 were added to the slices.

[0110] Laser confocal microscopy was used to observe the expression of Cx43 protein.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells, characterized in that: The following steps are involved: A. Cultivation of primary arterial mouse smooth muscle cells to obtain single-cell suspension of smooth muscle cells; B. Cultivation of primary arterial mouse endothelial cells to obtain endothelial cell single cell suspension; C. Establishment of an in vitro layered co-culture model of primary arterial smooth muscle cells and endothelial cells; a. Preparation of hardware system: The hardware system includes liquid input system, cell culture system and liquid recovery pool (6); The liquid input system comprises a mechanical flow controller (1) which is equipped with a syringe (2); the cell culture system comprises a bottom plate (8), a first cover plate (9), a second cover plate (10) and a third cover plate (11); the bottom plate (8) is spliced ​​and combined with the first cover plate (9), the second cover plate (10) and the third cover plate (11) to form different convection-type dual-channel microchips (4); the bottom plate (8) is spliced ​​and combined with the first cover plate (9) to form a first convection-type dual-channel microchip (12); a first convection-type dual-channel microchip (12) is formed inside The first cover plate (9) is provided with a first liquid input port (14) and a first liquid output port (15) which are in communication with the first culture channel (13); the first liquid input port (14) is used to connect to the syringe (2) via the liquid input tube (3); the first liquid output port (15) is used to connect to the liquid recovery pool (6) via the liquid output tube (5); the bottom plate (8) and the second cover plate (10) are spliced ​​and combined to form a second convection dual-channel microchip (16); the second convection dual-channel microchip (16) has a The second culture channel (17) is provided with a second liquid input port (18) and a second liquid output port (19) which are in communication with the second culture channel (17); the second liquid input port (18) is used to be connected to the syringe (2) via the liquid input tube (3); the second liquid output port (19) is used to be connected to the liquid recovery pool (6) via the liquid output tube (5); the bottom plate (8) and the third cover plate (11) are spliced ​​and combined to form a third convection dual-channel microchip (20); the third convection dual-channel microchip (20) has an inner A third culture channel (21) is formed, and a third liquid input port (22) and a third liquid output port (23) which are in communication with the third culture channel (21) are provided on the third cover plate (11); the third liquid input port (22) is used to be connected to the syringe (2) via a liquid input tube (3), and the third liquid output port (23) is used to be connected to a liquid recovery pool (6) via a liquid output tube (5); a diffusion pool (7) is provided on the third cover plate (11), and the diffusion pool (7) and the third culture channel (21) are separated by a semipermeable membrane (24); b. Soak and wipe the components of the cell culture system with 75% alcohol, and then irradiate with ultraviolet light for 30 minutes for disinfection; c. Prepare smooth muscle cell-BD-Matrixgel matrix gel solution from smooth muscle cell single cell suspension; d. Assemble the bottom plate (8) and the first cover plate (9) to form a first convection dual-channel microchip (12), connect the first liquid input port (14) on the first cover plate (9) to the syringe (2) through the liquid input tube (3), and connect the first liquid output port (15) on the first cover plate (9) to the liquid recovery pool (6) through the liquid output tube (5); load the smooth muscle cell-BD-Matrixgel matrix gel solution into the syringe (2), turn on the mechanical flow controller (1), and inject the smooth muscle cell-BD-Matrixgel matrix gel solution into the first culture channel (13) of the first convection dual-channel microchip (12) at a rate of 2-10 μl / min. After 30 minutes, the smooth muscle cell-BD-Matrixgel matrix gel solution in the first culture channel (13) solidifies to form a flexible and flat smooth muscle cell culture layer; The first cover plate (9) is removed from the bottom plate (8), and the second cover plate (10) is spliced ​​onto the bottom plate (8) to form a second convection dual-channel microchip (16). The second liquid input port (18) on the second cover plate (10) is connected to the syringe (2) through the liquid input tube (3), and the second liquid output port (19) on the second cover plate (10) is connected to the liquid recovery pool (6) through the liquid output tube (5); the endothelial cell single cell suspension is loaded into the syringe (2), the mechanical flow controller (1) is turned on, and the endothelial cell single cell suspension is injected into the second culture channel (17) of the second convection dual-channel microchip (16) at a rate of 1-2 μl / min. After 12 hours, the endothelial cells in the endothelial cell single cell suspension adhere to the smooth muscle cell culture layer, and the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model is constructed; To establish a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells; On the second day, the residual culture medium liquid in the second culture channel (17) is drained by pushing the airflow generated by the syringe (2); the second cover plate (10) is removed from the base plate (8), and the third cover plate (11) is spliced ​​onto the base plate (8) to form a third convection dual-channel microchip (20); the third liquid input port (22) on the third cover plate (11) is connected to the syringe (2) through the liquid input tube (3), and the third liquid output port (23) on the third cover plate (11) is connected to the liquid recovery pool (6) through the liquid output tube (5); the culture medium containing 20% ​​fetal bovine serum is filled into the syringe (2), and the mechanical flow controller (1) is turned on to adjust the flow rate at a rate of 2-10 A culture medium containing 20% ​​fetal bovine serum is poured into the third culture channel (21) of the third convection dual-channel microchip (20) at a rate of μL / min until the culture medium containing 20% ​​fetal bovine serum fills the entire third culture channel (21); a pre-prepared environmental toxicant solution is added to the diffusion pool (7) on the third cover plate (11) for intervention. After the intervention is completed, a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed.

2. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: After the construction of the primary arterial smooth muscle cell-endothelial cell in vitro layered co-culture model was completed, it was identified, specifically: the residual culture medium liquid in the second culture channel (17) was drained by pushing the airflow generated by the syringe (2); sterile PBS liquid was filled into the syringe (2), and the mechanical flow controller (1) was turned on to inject the sterile PBS liquid into the second culture channel (17) at a rate of 5 μl / min to complete the flushing of the second culture channel (17); after the flushing was completed, Dil-Ac-LDL dye solution was filled into the syringe (2), and the mechanical flow controller (1) was turned on to inject the Dil-Ac-LDL dye solution into the second culture channel (17) at a rate of 5 μl / min until the Dil-Ac-LDL dye solution filled the entire second culture channel (17), and then the cell was placed in a cell culture incubator and incubated for 2 hours. h; after the incubation is completed, the residual Dil-Ac-LDL dye in the second culture channel (17) is drained out by pushing the airflow generated by the syringe (2); sterile PBS liquid is filled into the syringe (2), and the mechanical flow controller (1) is turned on to inject sterile PBS liquid into the second culture channel (17) at a rate of 5 μl / min to complete the flushing of the second culture channel (17); observation is carried out under the conditions of excitation wave 495 nm and emission wave 565 nm by laser confocal microscopy. If the red fluorescent markers are arranged neatly and regularly, the model construction is successful; if the red fluorescent markers are scattered, the model construction fails.

3. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: The output end of the mechanical flow controller (1) is connected to the piston of the syringe (2) to control the flow rate of the liquid in the syringe (2); the capacity of the syringe (2) is 1 ml or 5 ml.

4. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: In the first convection dual-channel microchip (12), the first culture channel (13) has a height of 30 μm, a length of 6000 μm and a width of 30 μm, and the first culture channel (13) is used to culture smooth muscle cells; in the second convection dual-channel microchip (16), the second culture channel (17) has a height of 60 μm, a length of 6000 μm and a width of 30 μm, and the second culture channel (17) is used to culture endothelial cells; in the third convection dual-channel microchip (20), the third culture channel (21) has a height of 60 μm, a length of 6000 μm and a width of 30 μm, and the third culture channel (21) is used to perfuse a culture medium containing 20% ​​fetal bovine serum or other liquids required for model applications; in the third convection dual-channel microchip (20), the diffusion pool (7) uses a cubic pool body with a length of 4000 μm, a width of 30 μm and a height of 60 μm.

5. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: The material of the diffusion cell (7) is PDMS; the material of the semipermeable membrane (24) is PET, and its pore size is 0.4 μm.

6. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: Primary arterial mouse smooth muscle cells and primary arterial mouse endothelial cells are derived from secondary and tertiary arteries of mice.

7. The method for constructing a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells according to claim 1, characterized in that: The method for preparing smooth muscle cell-BD-Matrixgel matrix gel solution is as follows: take a single cell suspension of smooth muscle cells and make it into a suspension with a density greater than 1×10 7 To the single-cell suspension of 100 μg / mL, add 20 μl / ml of BD-Matrixgel and shake gently to mix.

8. The use of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells as claimed in claim 1, characterized in that: After the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells was constructed, the liquid in the diffusion cell (7) was collected and the levels of IL-6 and TNF-α inflammatory factors were detected using ELISA kits.

9. The use of a model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells as claimed in claim 1, characterized in that: The steps include: S1. The method is substantially the same as step A in claim 1, except that the cultured smooth muscle cells are washed three times with PBS, 1 ml of Rhod-4 / AM stain is added, the cells are placed in a cell culture incubator and incubated for 30 min, the stain is discarded, and the cells are washed three times with PBS to obtain a pre-stained smooth muscle cell single cell suspension; S2. The method is substantially the same as step B in claim 1, except that the cultured endothelial cells are washed three times with PBS, 1 ml of Fluo-4 / AM stain is added, the cells are placed in a cell culture incubator and incubated for 30 min, the stain is discarded, and the cells are washed three times with PBS to obtain a pre-stained endothelial cell single cell suspension; S3. The method of claim 1, wherein step C is substantially the same as step C, except that the single cell suspension of smooth muscle cells is replaced by a pre-stained single cell suspension of smooth muscle cells, and the single cell suspension of endothelial cells is replaced by a pre-stained single cell suspension of endothelial cells; S4. Basically the same as step D in claim 1, except that after the model for testing the toxicity of environmental pollutants to primary arterial smooth muscle cells-endothelial cells is constructed, a laser confocal microscope is used to observe the Ca2+ in smooth muscle cells and endothelial cells under the conditions of an excitation wave of 494 nm and an emission wave of 550 nm. 2+ The dynamic distribution of .

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