A microfluidic chip system and method for accurately simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress

Abstract

A microfluidic chip system and method for precisely simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress, belonging to the technical field of bio-microfluidic chips. According to the principles of fluid mechanics and microfluidic chip technology, a microfluidic chip and a circulation system are designed to precisely simulate the microenvironment of the synergistic effect of blood glucose fluctuations and pulsatile shear stress. The microfluidic chip is placed in a cell culture environment for long-term dynamic culture of endothelial cells. The endothelial cell monitoring device can observe the state of endothelial cells in real time and achieve multi-channel fluorescence microscopy imaging. The construction of the circulation system can save culture reagents and precisely simulate the synergistic effect of real blood glucose fluctuations and pulsatile shear stress in the body using PID feedback control. This system provides an experimental platform for analyzing and studying the law of pulsatile shear stress in the blood flow of diabetic patients after exercise to repair the endothelial cell damage caused by their blood glucose fluctuations, and the research results provide a scientific basis for delaying or even reversing the occurrence and development of atherosclerosis caused by diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-microfluidic chips. Based on the principles of hydrodynamics, mass transfer, and microfabrication technology, it specifically relates to a microfluidic chip system for accurately simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress. Background Art

[0002] Diabetes, as a common and frequently-occurring disease, is second only to cancer in terms of harm to the human body. China is the largest country with diabetes in the world. As of 2021, there are approximately 141 million diabetic patients aged 20 - 79 years. The terrifying aspect of diabetes lies in the various complications it may bring, among which the most fatal are cardiovascular and cerebrovascular complications, and atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular diseases. The damage of arterial endothelial cells is an important early event leading to atherosclerosis and abnormal vascular structure and function. Diabetic patients are more likely to induce endothelial dysfunction. Research has confirmed that fluctuating hyperglycemia causes more severe damage to umbilical vein endothelial cells than stable hyperglycemia. Endothelial cells are closely arranged on the inner surface of blood vessels and are constantly affected by blood flow shear stress. Many studies have shown that blood flow shear stress affects the structure and function of vascular endothelium by regulating the gene phenotype of endothelial cells, thereby influencing the development of atherosclerotic lesions. Based on this, constructing a microfluidic chip system that can accurately simulate the synergistic microenvironment of blood glucose fluctuations and pulsatile shear stress is crucial for studying how to improve endothelial cell function by regulating the synergistic microenvironment of blood glucose fluctuations and pulsatile shear stress, thereby delaying or even reversing the occurrence and development of atherosclerosis caused by diabetes.

[0003] In the past two decades, microfluidic chip technology has been continuously developed and widely applied in the fields of biology, medicine, chemistry, and environment. Due to characteristics such as miniaturized structure, microscale samples, precise flow control, and low processing cost, microfluidic chip technology provides key technology for constructing a microenvironment that can accurately simulate the synergistic effect of blood glucose fluctuations and pulsatile shear stress. In recent years, researchers have started to construct an in-vivo endothelial cell microenvironment system for diabetic patients based on microfluidic chip technology. However, the currently constructed microfluidic systems mostly focus on studying problems such as endothelial cell dysfunction in a hyperglycemic environment, and do not form a circulation loop. Long-term cell culture consumes a large amount of culture reagents, resulting in waste; for the construction of a blood glucose fluctuation environment, currently only a simple square wave signal can be generated, and it is impossible to accurately simulate the microenvironment of the combined action of blood glucose fluctuations and shear stress in which endothelial cells in diabetic patients are located. In view of this, there is an urgent need to develop a microfluidic chip system for accurately simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress. Summary of the Invention

[0004] The present invention aims to provide a microfluidic chip system that can accurately simulate the synergistic effect of blood glucose fluctuations and pulsatile shear stress. By connecting a programmable pulsatile pump, a programmable syringe pump, and a microfluidic chip, a microfluidic circulation loop is constructed. According to the principle of fluid mechanics, it can accurately simulate the microenvironment of the synergistic effect of blood glucose fluctuations and pulsatile shear stress, and is used to analyze and study the law and mechanism of pulsatile shear stress in repairing endothelial cell damage caused by blood glucose fluctuations.

[0005] The technical solution of the present invention is as follows:

[0006] A microfluidic chip system that accurately simulates the synergistic effect of blood glucose fluctuations and pulsatile shear stress, wherein the microfluidic chip system includes a microfluidic chip A, an endothelial cell monitoring device B, and a circulation system C;

[0007] The microfluidic chip A includes a microfluidic chip inlet 1-1, a cell culture chamber 1-2, and a microfluidic chip outlet 1-3, and is placed in the environment required for cell culture, and is used for long-term dynamic culture of endothelial cells;

[0008] The endothelial cell monitoring device B includes a computer 2-1 built by a multi-channel fluorescence microscope and a multi-channel fluorescence microscope 2-2, and is used for endothelial cell microscopy imaging and observation of multiple fluorescence channels;

[0009] The circulation system C is used to accurately construct in vivo shear stress waveforms and blood glucose waveforms, and includes a computer 3-1, a liquid storage tank 3-11, and a programmable pulsatile pump 3-2, a programmable syringe pump A 3-3, a programmable syringe pump B 3-4, a flow sensor, and a blood glucose sensor that are electrically connected to the computer 3-1; the circulation system C sets the required waveforms through programming by the computer 3-1. The programmable pulsatile pump 3-2 is used to generate shear stress waveforms; the programmable syringe pump A 3-3 is used to generate blood glucose waveforms; the programmable syringe pump B 3-4 is used to eliminate the remaining blood glucose waveforms in the loop and restore the blood glucose concentration in the loop to the same as the initial value; the flow sensor is used to monitor the flow rate change in real time; the blood glucose sensor is used to monitor the blood glucose change in real time; the liquid storage tank 3-11 is used to cooperate with the programmable syringe pump B 3-4 to eliminate the remaining blood glucose waveforms in the loop.

[0010] Further, the described circulation system C includes a computer 3-1, a T-shaped channel 3-10, a liquid storage tank 3-11, and a programmable pulsating pump 3-2, a programmable injection pump A 3-3, a programmable injection pump B 3-4, a flow sensor A 3-5, a flow sensor B 3-6, a flow sensor C 3-7, a blood glucose sensor A 3-8, and a blood glucose sensor B 3-9 that are electrically connected to the computer 3-1; normal blood glucose culture medium, high blood glucose culture medium, or sugar-free culture medium is respectively loaded in the programmable pulsating pump 3-2, the programmable injection pump A 3-3, and the programmable injection pump B 3-4; the programmable pulsating pump 3-2, the programmable injection pump A 3-3 are connected and converge to the T-shaped channel 3-10 through pipelines, a flow sensor A 3-5 is arranged between the programmable pulsating pump 3-2 and the T-shaped channel 3-10, a flow sensor B 3-6 is arranged between the programmable injection pump A 3-3 and the T-shaped channel 3-10, the converging pipe orifice of the T-shaped channel 3-10 is connected to the microfluidic chip inlet 1-1 through a pipeline, and a blood glucose sensor A 3-8 is arranged between the T-shaped channel 3-10 and the microfluidic chip inlet 1-1; the microfluidic chip outlet 1-3 is connected to the inlet of the liquid storage tank 3-11, and the programmable injection pump B 3-4 is also connected to the inlet of the liquid storage tank 3-11 through a pipeline, and a flow sensor C 3-7 is arranged between the programmable injection pump B 3-4 and the liquid storage tank 3-11; the outlet of the liquid storage tank 3-11 is connected to the programmable pulsating pump 3-2 through a pipeline, and a blood glucose sensor B 3-9 is arranged between the outlet of the liquid storage tank 3-11 and the programmable pulsating pump 3-2.

[0011] Further, the cell culture chamber 1-2 is a cubic flat structure, and the length L, width W, and height H of the cell culture chamber 1-2 satisfy: H << W and H << L. (The width W and length L are in millimeters, and the height H is in micrometers, so that the fluid in the entire cell culture chamber exhibits laminar flow characteristics.) Since the height of the cell culture chamber 1-2 is much smaller than the width and length, and the Reynolds number of the flow in the chamber is very low, satisfying the quasi-steady flow assumption, the shear stress formula in the cell culture chamber 1-2 can be simplified as:

[0012]

[0013] where μ is the fluid viscosity, and W and H are the width and height of the cell culture chamber. Therefore, the magnitude τ of the shear stress applied to the endothelial cells is proportional to the fluid flow rate Q through the cell culture chamber 1-2, and the magnitude of the shear stress can be quantitatively regulated by controlling the flow rate Q.

[0014] As Figure 3 shown, the output flow rate of the programmable pulsating pump 3-2 is Q1(t), the output flow rate of the programmable injection pump A 3-3 is Q2(t), and the flow rate of the cell culture chamber 1-2 is Q(t), satisfying fluid continuity:

[0015] Q(t) = Q1(t) + Q2(t) (2)

[0016] The programmable pulsatile pump 3-2 outputs a blood glucose concentration of C1, the programmable infusion pump A3-3 outputs a blood glucose concentration of C2, and the blood glucose concentration in the cell culture chamber 1-2 is C(t). According to the law of conservation of mass, we can obtain:

[0017] Q1(t)·C1 + Q2(t)·C2 = Q(t)C(t) (3)

[0018] Among them, C1 and C2 are constants, Q(t) is the flow waveform calculated from the shear stress waveform collected from the human body according to formula (1), that is, the target flow waveform, and C(t) is the blood glucose fluctuation waveform collected from the human body, that is, the target dynamic blood glucose waveform. That is, Q(t) and C(t) are known. According to formulas (1) and (3), we get:

[0019]

[0020] According to Q1(t)≈Q(t) and Q2(t) << Q1(t), we get:

[0021]

[0022] By regulating the output flow rate Q1(t) of the programmable pulsatile pump 3-2 and the output flow rate Q2(t) of the programmable infusion pump A3-3, a microenvironment that can accurately simulate the synergistic effect of shear stress and blood glucose fluctuation can be constructed.

[0023] Furthermore, the computer 3-1 sets the required target waveform as the flow waveform calculated from the pulsatile shear stress waveform in the real human environment collected. The pulsatile shear stress waveform includes the pulsatile shear stress waveform in the resting state, the pulsatile shear stress waveform corresponding to low-intensity exercise, the pulsatile shear stress waveform corresponding to moderate-intensity exercise, and the pulsatile shear stress waveform corresponding to high-intensity exercise; the blood glucose waveform includes the normal blood glucose waveform and the blood glucose fluctuation waveform of diabetic patients (as Figure 4 shown). By combining different pulsatile shear stress and blood glucose waveforms, the microenvironment of endothelial cells in the body of diabetic patients in the resting state and after different-intensity exercises can be accurately simulated.

[0024] Furthermore, the microfluidic chip A, the endothelial cell monitoring device B, and the circulatory system C are combined to construct the microfluidic chip system that accurately simulates the synergistic effect of blood glucose fluctuations and pulsatile shear stress. The microfluidic chip A can be obtained through microfabrication technology. The endothelial cell monitoring device B includes a computer and a multi-channel fluorescence microscope, and the whole set of devices is suitable for real-time monitoring of endothelial cells. The circulation loop C adds the microfluidic chip A and the endothelial cell monitoring device B into the loop, and uses a computer to control three programmable pumps. Together with five sensors, they respectively detect the flow rate and blood glucose concentration in real time and return the data to the computer, forming a feedback control to accurately simulate the synergistic effect of blood glucose fluctuations and pulsatile shear stress. When connecting all the devices, silicone tubes with corresponding inner diameters can be used, and the operation is simple. The whole set of devices can be easily built.

[0025] A method for accurately simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress by a microfluidic chip system for accurate simulation is as follows:

[0026] Step 1: After the microfluidic chip A that has been degassed and sterilized is coated with the required protein for a period of time, the endothelial cell suspension is perfused into the cell culture chamber 1-2 of the microfluidic chip A. It can be used for subsequent long-term dynamic culture only after the cells adhere and grow to a confluence of 50% - 80%.

[0027] Step 2: The collected in-vivo pulsatile shear stress waveform and blood glucose fluctuation waveform are used as the target pulsatile shear stress waveform and the target dynamic blood glucose waveform respectively. The target flow rate waveform is calculated, and then the computer 3-1 is used to set the output flow rate waveforms of the programmable pulsatile pump 3-2 loaded with normal blood glucose culture solution and the programmable injection pump A 3-3 loaded with hyperglycemic culture solution. The flow rate waveforms within one cycle are detected by the flow sensor A 3-5 and the flow sensor B 3-6 to see if they conform to the target flow rate waveform. If they do not conform to the target flow rate waveform, the detected data is returned to the computer 3-1 and adjusted through PID feedback control. After the output waveform conforms to the target flow rate waveform, the programmable pulsatile pump 3-2 and the programmable injection pump A 3-3 are connected in parallel with a T-shaped channel 4-1, and the blood glucose waveform within one cycle is detected by the blood glucose sensor A 3-8 to see if it conforms to the target dynamic blood glucose waveform. If it does not conform to the target dynamic blood glucose waveform, the detected data is returned and adjusted through PID feedback control.

[0028] Step 3: After the shear stress waveform and the blood glucose waveform are correct, the fluid flows through the microfluidic chip A to the reservoir 3-11. The solution in the reservoir 3-11 returns to the loop, and the blood glucose concentration in the solution is measured by the blood glucose sensor B 3-9. The programmable syringe B 3-4 loaded with sugar-free culture solution is controlled to inject a certain flow rate of solution into the reservoir 3-11 to make the solution concentration measured by the blood glucose sensor B 3-9 return to normal blood glucose, and the waveform of the flow sensor C 3-7 is recorded.

[0029] Step 4: After the entire loop flow is stable and error-free, place the microfluidic chip A containing endothelial cells in the circulation system C for culturing in a microenvironment with the synergistic action of shear stress and blood glucose fluctuations, for subsequent related cell biology and molecular biology research. Collect the shear stress waveforms corresponding to normal people during low-intensity exercise, moderate-intensity exercise, and high-intensity exercise, and repeat Steps 1 to 4 to obtain endothelial cell damage caused by regulating blood glucose fluctuations under different exercise intensity conditions. Using the results obtained from endothelial cells cultured with blood glucose fluctuations at rest as a control, further analyze whether the pulsatile shear stress corresponding to different exercise intensities can repair the endothelial cell damage caused by blood glucose fluctuations. Through subsequent related cell biology and molecular biology research, qualitatively and quantitatively analyze the results to reveal the law of pulsatile shear stress corresponding to different exercise intensities in repairing endothelial cell damage caused by blood glucose fluctuations.

[0030] Advantages of the present invention: The present invention can successfully achieve precise loading of pulsatile shear stress with different amplitudes and frequencies and real blood glucose fluctuation signals on endothelial cells cultured at the bottom of the microfluidic chip, and realize long-term dynamic culture of endothelial cells; the present invention adopts a circulating perfusion method, saving culture reagents; combined with a multi-channel fluorescence microscope imaging device, it can realize real-time monitoring of endothelial cells and simultaneous observation of multiple fluorescences; in vitro models can observe phenomena that cannot be observed in vivo, providing a scientific basis for revealing the law of pulsatile shear stress in repairing endothelial cell damage caused by blood glucose fluctuations and finding reasonable pulsatile blood flow shear stress intervention methods to delay or even reverse diabetic vascular endothelial cell dysfunction. Description of the Drawings

[0031] Figure 1 is a schematic structural diagram of the microfluidic chip A.

[0032] Figure 2 is the endothelial cell monitoring device B.

[0033] Figure 3 is the circulation system C.

[0034] Figure 4 is the pulsatile shear stress and dynamic blood glucose waveform diagram collected from the human body. (a) is the pulsatile shear stress waveform diagram, and (b) is the dynamic blood glucose waveform diagram.

[0035] Figure 5 is the schematic diagram of the generation principle of pulsatile shear stress and blood glucose waveforms.

[0036] In the figure: A is a microfluidic chip; B is an endothelial cell monitoring device; C is a circulatory system; 1-1 is the inlet of the microfluidic chip, 1-3 is the outlet of the microfluidic chip; 1-2 is the cell culture chamber; o, x, y, z are coordinate systems; L, W, H are the length, width, and height of the cell culture chamber respectively; 2-1 is the computer built by a multi-channel fluorescence microscope; 2-2 is a multi-channel fluorescence microscope; 3-1 is a computer; 3-2 is a programmable pulsatile pump; 3-3 is a programmable syringe pump A, 3-4 is a programmable syringe pump B; 3-5 is a flow sensor A, 3-6 is a flow sensor B, 3-7 is a flow sensor C; 3-8 is a blood glucose sensor A, 3-9 is a blood glucose sensor B; 3-10 is a T-shaped channel; 3-11 is a reservoir. Detailed implementation mode

[0037] The technical solution of the present invention will be further described below in conjunction with specific embodiments and drawings.

[0038] A microfluidic chip system that accurately simulates the synergistic effect of blood glucose fluctuations and pulsatile shear stress, as Figure 3 shown, the microfluidic chip system includes a microfluidic chip A, an endothelial cell monitoring device B, and a circulatory system C. The experiment using this device to repair endothelial cell damage caused by blood glucose fluctuations with pulsatile shear stress includes the following steps:

[0039] (1) First, the steps of designing and fabricating the microfluidic chip A are as follows:

[0040] Step 1: Both the inlet and outlet of the microfluidic chip A are rectangular channels with a length of 2 mm and a width of 1 mm. The cell culture chamber 1-2 is a rectangular chamber with a length of 10 mm and a width of 3 mm. All the channel and chamber structures on the chip are fabricated using a standardized microfabrication method with PDMS and permanently bonded and sealed with a clean cover glass to form a transparent glass-PDMS chip with good biocompatibility. The height of the chip is 150 μm.

[0041] Step 2: Place the fabricated microfluidic chip A in water to evacuate air bubbles, and after evacuating the air bubbles in the channels, place it in a high-temperature and high-pressure sterilizer for sterilization. The sterilized microfluidic chip A is placed in a laminar flow hood for standby.

[0042] (2) The steps of cell culture in the microfluidic chip A are as follows:

[0043] Step 3: Place the microfluidic chip A sterilized in Step 2 in a glass dish filled with normal culture medium, and use a 1 mL syringe to inject the Fibronection solution into the entire channel of the microfluidic chip A, and place it in a cell culture incubator for 4 hours.

[0044] Step 4: Inject the human umbilical vein endothelial cell suspension into microfluidic chip A using a syringe, and place it in a cell incubator containing 5% carbon dioxide at 37°C for culturing. When 50% of the cells have adhered, place it in the circulation loop for 48 hours of dynamic culturing.

[0045] (III) The steps for constructing and debugging the circulation loop are as follows:

[0046] Step 5: Collect the pulsatile shear stress waveform and normal blood glucose waveform in the resting state of normal people to determine the target pulsatile shear stress waveform and target dynamic blood glucose waveform (as Figure 4 shown). Take normal blood glucose and pulsatile shear stress in the resting state as specific examples. Calculate the required flow waveform according to the above principle formula, and set the output flow waveforms of the programmable pulsatile pump 3-2 loaded with 5.5 mmol / L normal blood glucose culture medium and the programmable syringe pump A 3-3 loaded with 5.5 mmol / L normal culture medium through the computer 3-1. Use the flow sensor A 3-5, flow sensor B 3-6, and blood glucose sensor A 3-8 to judge whether the waveform conforms to the target waveform; if it does not conform to the target waveform, the detected data is returned to the computer 3-1 and adjusted through PID feedback control. When the solution circulates to the blood glucose sensor B 3-9, record its concentration. The entire loop circulates for two minutes. After the waveforms of each sensor reach stability, the normal pulsatile shear stress and normal blood glucose circulation loop is constructed;

[0047] Step 6: Collect the waveform of blood glucose fluctuations in diabetic patients (as Figure 4 shown). According to the normal pulsatile shear stress and blood glucose fluctuation circulation loop constructed in Step 5, the difference is that the blood glucose concentration of the culture medium in the programmable syringe pump A 3-3 needs to be changed to 25 mmol / L, and 25 mmol / L of mannitol needs to be added to maintain the osmotic pressure balance inside and outside the cells; and before the loop circulation, the waveform of the blood glucose sensor B 3-9 needs to be eliminated using the programmable syringe pump B 3-4 loaded with sugar-free culture medium, and the flow rate of the flow sensor C 3-7 is recorded;

[0048] Step 7: After the normal pulsatile shear stress and normal blood glucose circulation loop and the normal pulsatile shear stress and blood glucose fluctuation are constructed, use this as a control group, and then change the shear stress conditions to repair the endothelial cell function, that is, collect the shear stress waveforms corresponding to normal people under low-intensity exercise, moderate-intensity exercise, and high-intensity exercise to construct a circulation loop of the synergistic effect of pulsatile shear stress and blood glucose fluctuation corresponding to different exercise intensities.

[0049] (IV) The experimental steps for repairing endothelial cell damage caused by blood glucose fluctuations with pulsatile shear stress corresponding to different exercise intensities are as follows:

[0050] Step 8: Place the microfluidic chip with 50% adherent endothelial cells in a circulation loop with normal pulsatile shear stress and normal blood glucose for 48 hours of culture as experimental control group 1; place the microfluidic chip with 50% adherent endothelial cells in a circulation loop with resting pulsatile shear stress and blood glucose fluctuations for 48 hours of culture as experimental control group 2; place the microfluidic chip with 50% adherent endothelial cells in a circulation loop with pulsatile shear stress corresponding to low-intensity exercise and blood glucose fluctuations for 48 hours of culture as repair group 1; place the microfluidic chip with 50% adherent endothelial cells in a circulation loop with pulsatile shear stress corresponding to moderate-intensity exercise and blood glucose fluctuations for 48 hours of culture as repair group 2; place the microfluidic chip with 50% adherent endothelial cells in a circulation loop with pulsatile shear stress corresponding to high-intensity exercise and blood glucose fluctuations for 48 hours of culture as repair group 3.

[0051] Step 9: Measure the morphological, cytoskeletal and related functional indices of the endothelial cells in the above five groups. For example, use Tubulin Tracker Deep Red, SiR700-Actin kits, and DiD to stain the microtubules, microfilaments and cell membranes of the endothelial cells in the above 5 groups respectively, and use a multi-channel fluorescence microscope 2-2 for photographing, observation and analysis. Load the fluorescence probe according to the instructions of the DAF-2DA nitric oxide detection probe kit, and use a flow cytometer to detect the NO content; load the fluorescence probe according to the instructions of the nitric oxide synthase detection probe kit, and use a flow cytometer to detect the eNOS content; detect the expression levels of p-eNOS Ser1177 and GRP78 proteins by Western Blot. Use an ELISA kit to detect the secretion levels of inflammatory factors such as IL-6, TNF-α and ICAM-1 and the content of advanced glycation end products AGEs. The damage caused by blood glucose fluctuations to endothelial cells can be obtained by comparing the cell morphology and protein expression of control groups 1 and 2; the repair effect of pulsatile shear stress on endothelial cell damage caused by blood glucose fluctuations can be obtained by comparing the cell morphology and protein expression of repair groups 1, 2 and 3 with control group 2. Among them, an increase in the production of eNOS and NO, an up-regulation of the expression level of p-eNOS Ser1177, a down-regulation of the expression level of GRP78 protein, a decrease in the secretion levels of inflammatory factors such as IL-6, TNF-α and ICAM-1, and a decrease in the AGEs content, etc. are regarded as the repair of endothelial cell damage caused by pulsatile shear stress to blood glucose fluctuations.

Claims

1. A microfluidic chip system that precisely simulates the synergistic effect of blood glucose fluctuations and pulsatile shear stress, characterized in that, The described microfluidic chip system includes a microfluidic chip (A), an endothelial cell monitoring device (B), and a circulatory system (C); The microfluidic chip (A) includes a microfluidic chip inlet (1-1), a cell culture chamber (1-2), and a microfluidic chip outlet (1-3), and is placed in an environment required for cell culture, and is used for long-term dynamic culture of endothelial cells; The endothelial cell monitoring device (B) includes a computer (2-1) built by a multi-channel fluorescence microscope and a multi-channel fluorescence microscope (2-2), and is used for endothelial cell microscope imaging and observation of multiple fluorescence channels; The circulatory system (C) is used to accurately construct in vivo shear stress waveforms and blood glucose waveforms, and includes a computer (3-1), a liquid storage tank (3-11), and a programmable pulsatile pump (3-2), a programmable injection pump A (3-3), a programmable injection pump B (3-4), a flow sensor, and a blood glucose sensor that are electrically connected to the computer (3-1); the circulatory system (C) sets the required waveforms through programming by the computer (3-1), the programmable pulsatile pump (3-2) is used to generate shear stress waveforms; the programmable injection pump A (3-3) is used to generate blood glucose waveforms; the programmable injection pump B (3-4) is used to eliminate the remaining blood glucose waveforms in the loop and restore the blood glucose concentration in the loop to the same as the initial value; the flow sensor is used to monitor the flow rate change in real time; the blood glucose sensor is used to monitor the blood glucose change in real time; the liquid storage tank (3-11) is used to cooperate with the programmable injection pump B (3-4) to eliminate the remaining blood glucose waveforms in the loop.

2. The microfluidic chip system for precisely simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress according to claim 1, wherein The described circulation system (C) includes a computer (3-1), a T-shaped channel (3-10), a liquid storage tank (3-11), and a programmable pulsating pump (3-2), a programmable injection pump A (3-3), a programmable injection pump B (3-4), a flow sensor A (3-5), a flow sensor B (3-6), a flow sensor C (3-7), a blood glucose sensor A (3-8), and a blood glucose sensor B (3-9) that are electrically connected to the computer (3-1); the programmable pulsating pump (3-2), the programmable injection pump A (3-3), and the programmable injection pump B (3-4) are respectively filled with normal blood glucose culture solution, high blood glucose culture solution, or sugar-free culture solution; the programmable pulsating pump (3-2), the programmable injection pump A (3-3) are connected to and converge with the T-shaped channel (3-10) through pipelines, a flow sensor A (3-5) is arranged between the programmable pulsating pump (3-2) and the T-shaped channel (3-10), a flow sensor B (3-6) is arranged between the programmable injection pump A (3-3) and the T-shaped channel (3-10), the converging nozzle of the T-shaped channel (3-10) is connected to the microfluidic chip inlet (1-1) through a pipeline, and a blood glucose sensor A (3-8) is arranged between the T-shaped channel (3-10) and the microfluidic chip inlet (1-1); the microfluidic chip outlet (1-3) is connected to the inlet of the liquid storage tank (3-11), the programmable injection pump B (3-4) is also connected to the inlet of the liquid storage tank (3-11) through a pipeline, and a flow sensor C (3-7) is arranged between the programmable injection pump B (3-4) and the liquid storage tank (3-11); the outlet of the liquid storage tank (3-11) is connected to the programmable pulsating pump (3-2) through a pipeline, and a blood glucose sensor B (3-9) is arranged between the outlet of the liquid storage tank (3-11) and the programmable pulsating pump (3-2).

3. A microfluidic chip system for precisely simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress according to claim 1, characterized in that, The described cell culture chamber (1-2) is a cubic flat structure, and the length L, width W, and height H of the cell culture chamber (1-2) satisfy: H << W and H << L; since the height of the cell culture chamber (1-2) is much smaller than the width and length, and the Reynolds number of the flow in the chamber is very low, satisfying the quasi-steady flow assumption, the shear stress formula in the cell culture chamber (1-2) can be simplified as: where μ is the fluid viscosity, and W and H are the width and height of the cell culture chamber; therefore, the magnitude τ of the shear stress applied to the endothelial cells is proportional to the fluid flow rate Q flowing through the cell culture chamber (1-2), and the magnitude of the shear stress can be quantitatively regulated by controlling the flow rate Q; The output flow rate of the programmable pulsating pump (3-2) is Q1(t), the output flow rate of the programmable injection pump A (3-3) is Q2(t), and the flow rate of the cell culture chamber (1-2) is Q(t), satisfying fluid continuity: Q(t) = Q1(t) + Q2(t) (2) The output blood glucose concentration of the programmable pulsating pump (3-2) is C1, the output blood glucose concentration of the programmable injection pump A (3-3) is C2, and the blood glucose concentration of the cell culture chamber (1-2) is C(t). According to the law of conservation of mass, we can obtain: Q1(t)·C1 + Q2(t)·C2 = Q(t)C(t) (3) Wherein, C1 and C2 are constants, Q(t) is the flow waveform calculated according to the shear stress waveform collected from the human body by formula (1), that is, the target flow waveform, and C(t) is the blood glucose fluctuation waveform collected from the human body, that is, the target dynamic blood glucose waveform, namely Q(t) and C(t) are known; according to formula (1) and (3), it is obtained that: According to Q1(t)≈Q(t) and Q2(t)<<Q1(t), it is obtained that: By regulating the output flow rate Q1(t) of the programmable pulsatile pump (3-2) and the output flow rate Q2(t) of the programmable syringe pump A (3-3), a microenvironment that can accurately simulate the synergistic effect of shear stress and blood glucose fluctuation can be constructed.

4. A microfluidic chip system for precisely simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress according to claim 1, 2 or 3, characterized in that, The computer (3-1) sets the required target waveform as the flow waveform calculated from the pulsatile shear stress waveform collected in the real human environment. The pulsatile shear stress waveform includes the pulsatile shear stress waveform at rest, the pulsatile shear stress waveform corresponding to low-intensity exercise, the pulsatile shear stress waveform corresponding to moderate-intensity exercise, and the pulsatile shear stress waveform corresponding to high-intensity exercise; the blood glucose waveform includes the normal blood glucose waveform and the blood glucose fluctuation waveform of diabetic patients; by combining different pulsatile shear stress and blood glucose waveforms, the microenvironment of endothelial cells in the body of diabetic patients at rest and after different intensities of exercise can be accurately simulated.

5. A microfluidic chip system for precisely simulating the synergistic effect of blood glucose fluctuations and pulsatile shear stress according to claim 1, 2 or 3, characterized in that The microfluidic chip (A), the endothelial cell monitoring device (B) and the circulatory system (C) are combined to construct the microfluidic chip system that accurately simulates the synergistic effect of blood glucose fluctuation and pulsatile shear stress; the microfluidic chip (A) can be obtained by microfabrication technology, and the endothelial cell monitoring device (B) includes a computer and a multi-channel fluorescence microscope. The whole set of devices is suitable for real-time monitoring of endothelial cells; the circulation loop (C) adds the microfluidic chip (A) and the endothelial cell monitoring device (B) into the loop, and uses a computer to regulate three programmable pumps, plus five sensors to respectively detect the flow rate and blood glucose concentration in real time and return to the computer, constituting a feedback control to accurately simulate the synergistic effect of blood glucose fluctuation and pulsatile shear stress.

6. A method for precise simulation using a microfluidic chip system that precisely simulates the synergistic effect of blood glucose fluctuations and pulsatile shear stress according to any one of claims 1-5, characterized in that The steps are as follows: Step 1: After the microfluidic chip (A) that has exhausted air bubbles and been sterilized is coated with the required protein for a period of time, the endothelial cell suspension is perfused into the cell culture chamber (1-2) of the microfluidic chip (A). It can be used for subsequent long-term dynamic culture only after the cells adhere and grow to a confluence of 50% - 80%. Step 2: Take the in-vivo pulsatile shear stress waveform and blood glucose fluctuation waveform collected as the target pulsatile shear stress waveform and the target dynamic blood glucose waveform respectively. Calculate the target flow waveform, and then use the computer (3-1) to set the output flow waveforms of the programmable pulsatile pump (3-2) loaded with normal blood glucose culture medium and the programmable syringe pump A (3-3) loaded with hyperglycemic culture medium. Detect whether the flow waveform within one cycle conforms to the target flow waveform through the flow sensor A (3-5) and the flow sensor B (3-6); if it does not conform to the target flow waveform, return the detected data to the computer (3-1) and adjust it through PID feedback control; after the output waveform conforms to the target flow waveform, parallel the programmable pulsatile pump (3-2) and the programmable syringe pump A (3-3) with a T-shaped channel (4-1), and use the blood glucose sensor A (3-8) to detect whether the blood glucose waveform within one cycle conforms to the target dynamic blood glucose waveform. If it does not conform to the target dynamic blood glucose waveform, return the detected data and adjust it through PID feedback control; Step 3: After the shear stress waveform and the blood glucose waveform are correct, flow through the microfluidic chip (A) to the reservoir (3-11). The solution in the reservoir (3-11) returns to the loop. Measure the blood glucose concentration in the solution with the blood glucose sensor B (3-9). Regulate the programmable syringe B (3-4) loaded with sugar-free culture medium to inject a certain flow of solution into the reservoir (3-11) so that the solution concentration measured by the blood glucose sensor B (3-9) returns to normal blood glucose, and record the waveform of the flow sensor C (3-7); Step 4: After the entire loop flows stably and correctly, place the microfluidic chip (A) containing endothelial cells in the circulatory system (C) for culturing under the microenvironment of the synergistic action of shear stress and blood glucose fluctuation for subsequent related cell biology and molecular biology research; collect the shear stress waveforms corresponding to normal people under low-intensity exercise, moderate-intensity exercise, and high-intensity exercise, and repeat Steps 1 to 4 to obtain endothelial cell damage caused by regulating blood glucose fluctuation under different exercise intensity conditions; Take the results obtained from endothelial cells cultured with blood glucose fluctuation in the resting state as a control, and then analyze whether the pulsatile shear stress corresponding to different exercise intensities can repair the endothelial cell damage caused by blood glucose fluctuation; through subsequent related cell biology and molecular biology research, qualitatively and quantitatively analyze the results to reveal the law of the pulsatile shear stress corresponding to different exercise intensities in repairing the endothelial cell damage caused by blood glucose fluctuation.

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