Construction method and application of intestinal wall cell barrier model

Abstract

The invention relates to the field of biomedical engineering, and discloses a construction method and application of an intestinal wall cell barrier model. The method comprises the following steps: performing intestinal wall cell culture on a metasurface plasmon resonance chip of which the surface is subjected to bionic treatment in a culture medium to obtain a chip plate attached to the wall of intestinal wall cells, and monitoring the forming process of a barrier layer of the intestinal wall cells in real time by detecting the refractive index change of the surface of the metasurface plasmon resonance chip. The intestinal wall cell barrier model obtained by the construction method can realize unmarked real-time continuous monitoring of the formation process of the intestinal wall cell barrier layer and dynamic observation of physiological changes of cells, and the method has high sensitivity and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to biomedical engineering, in particular to a method for constructing an intestinal wall cell barrier model and its application. BACKGROUND

[0002] Intestinal wall cell barriers play a crucial role in maintaining intestinal homeostasis and preventing pathogen invasion. However, traditional cell barrier models have significant limitations in real-time monitoring of cell barrier layer formation processes. These models usually rely on labeling techniques such as fluorescent labeling or radioactive labeling, which can provide some information but also have some drawbacks, such as the label may interfere with the normal physiological function of the cells, the labeling process is complex and costly. In addition, the lack of sensitivity and spatial resolution in existing technology limits its application in complex biological systems.

[0003] To overcome these challenges, Surface Plasmon Resonance (SPR) technology is introduced as a powerful tool for label-free, real-time monitoring of biomolecular interactions. SPR technology detects the resonance changes of surface plasmon waves on the surface of a metal thin film to monitor the interactions between biomolecules. However, traditional SPR systems still have limitations in sensitivity, making it difficult to dynamically observe the physiological changes of cells, which limits their application in cell barrier dynamics research. SUMMARY

[0004] The purpose of the present application is to overcome the problem of low sensitivity of existing cell barrier models and the difficulty of dynamically observing cell behavior, and to provide a method for constructing an intestinal wall cell barrier model and its application. The intestinal wall cell barrier model obtained by the construction method can achieve label-free real-time continuous monitoring of the formation process of the intestinal wall cell barrier layer, dynamically observe the physiological changes of the cells, and the method has high sensitivity and stability.

[0005] To achieve the above-mentioned purpose, the first aspect of the present application provides a method for constructing an intestinal wall cell barrier model, which comprises the following steps: culturing intestinal wall cells on a surface biomimetic treated super surface plasmon resonance chip in a culture medium to obtain an intestinal wall cell adherent chip plate, and detecting the refractive index change on the surface of the super surface plasmon resonance chip to monitor the formation process of the barrier layer of the intestinal wall cells in real time.

[0006] Preferably, the surface plasmon resonance chip comprises a surface plasmon resonance chip plate and a MetaSPR sensor chip integrated on the surface plasmon resonance chip plate; the surface plasmon resonance chip plate is provided with a plurality of chip micropores, and the MetaSPR sensor chip comprises a substrate, a plurality of nanopores stamped on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores, the metal film comprising a transition metal film layer and a noble metal film layer arranged from inside to outside.

[0007] Preferably, the substrate is made of PET material.

[0008] Preferably, the transition metal film layer is a titanium film layer with a thickness of 5-20 nm, and the noble metal film layer comprises a silver film layer with a thickness of 40-100 nm and / or a gold film layer with a thickness of 5-60 nm.

[0009] Preferably, the noble metal film layer comprises a silver film layer and a gold film layer arranged from inside to outside.

[0010] Preferably, the nanopores are frustum-shaped, and the diameter of the large opening is 150-250 nm and the depth is 400-600 nm, and the spacing between two adjacent chip micropores is 200-500 nm.

[0011] Preferably, the construction method uses a WeSPR multifunctional molecular detector with the surface plasmon resonance biochip to detect the refractive index change of the surface of the surface plasmon resonance biochip in real time.

[0012] Preferably, the process of the biomimetic treatment comprises: after sterilization, washing and drying of the surface plasmon resonance biochip, adding a cell adhesion agent into the nanopores to form a film, and then removing the cell adhesion agent and washing.

[0013] Preferably, the cell adhesion agent is at least one selected from the group consisting of polylysine, polyethyleneimine and type IV collagen, and is preferably polyethyleneimine.

[0014] Preferably, the cell adhesion agent in a single nanopore is added in the form of a solution with a concentration of 5-1000 μg / mL and an addition amount of 50-200 μL.

[0015] Preferably, the film forming conditions comprise at least a temperature of 35-40 ℃ and a time of 1-3 h.

[0016] Preferably, the culture medium is at least one selected from the group consisting of DMEM medium, RPMI 1640 medium and Ham's F-12 medium.

[0017] Preferably, the culture medium further contains a culture medium additive, which is fetal bovine serum and / or penicillin-streptomycin.

[0018] Preferably, the culture medium additive is fetal bovine serum and penicillin-streptomycin.

[0019] Preferably, based on the mass of the culture medium, the mass of the fetal bovine serum is 5-20 wt%, and the mass of the penicillin-streptomycin is 1-10 wt%.

[0020] Preferably, the amount of culture medium added in a single microwell of the chip is 50-200 μL.

[0021] Preferably, the conditions for culturing the intestinal wall cells include at least the following: 10,000 to 30,000 cells seeded in a single microwell of the chip, a temperature of 35 to 40°C, a CO2 concentration of 4 to 6%, and a humidity of 80 to 100%.

[0022] Preferably, the method further includes: detecting color changes of the cell-attached chip plate to form a real-time change imaging platform.

[0023] Preferably, the color of the chip plate to which the cells adhere is detected using a scanning electron microscope.

[0024] The second aspect of this invention provides the application of the intestinal wall cell barrier model obtained by the construction method described in the first aspect above in evaluating alcohol-induced intestinal wall cell damage.

[0025] The third aspect of this invention provides the application of the intestinal wall cell barrier model obtained by the construction method described in the first aspect above in evaluating the protective or repairing effects of drugs or probiotics on intestinal wall cell damage.

[0026] Preferably, the intestinal wall cell damage is alcohol-induced intestinal wall cell damage.

[0027] Preferably, the drug is dihydroquercetin; the probiotic is selected from at least one of Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus curvularis, Lactobacillus pentosus, Bifidobacterium breve, Lactobacillus gasseri, Lactobacillus casei subsp. casei, and Lactobacillus casei subsp. casei.

[0028] The beneficial effects of the present invention through the above technical solution are as follows: The intestinal wall cell barrier model construction method provided by this invention employs MetaSPR technology. It dynamically monitors the formation process of the intestinal wall cell barrier layer in real time by detecting changes in the refractive index of a metasurface plasmon resonance chip. This enables label-free, real-time, and continuous monitoring of the intestinal wall cell barrier layer formation process, allowing dynamic observation of physiological changes in cells. This method exhibits high sensitivity and stability. Furthermore, the intestinal wall cell barrier model provided by this invention can simulate a real physiological environment, allowing cells to maintain normal physiological functions within the model, thus improving the reliability and applicability of the model. Attached Figure Description

[0029] Figure 1 This is a flowchart of a model of intestinal wall cell barrier monitoring cell behavior; Figure 2 This is a graph showing the effects of different adherent agents on Caco-2 cells cultured on a MetaSPR chip for 72 hours in Example 1. Figure 3 This is a color change diagram of the MetaSPR chip under a microscope in Example 2; Figure 4 This is the full absorption spectrum of the MetaSPR chip for glycerol aqueous solution in Example 2; Figure 5 This is the result of the cell-killing effect of different concentrations of alcohol in Example 3; Figure 6 This is a comparison of the detection results of the intestinal wall cell barrier model in Example 3 with the detection results of the traditional CCK8 method; Figure 7 This is the result of the cell-killing effect of different concentrations of alcohol in Example 4; Figure 8 This is the result of the cell repair effect of different concentrations of dihydroquercetin in Example 4. Detailed Implementation

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In a first aspect, the present invention provides a method for constructing an intestinal wall cell barrier model, the method comprising the following steps: culturing intestinal wall cells in a culture medium using a metasurface plasmon resonance chip with a biomimetic surface treatment to obtain a chip plate in which intestinal wall cells adhere; and monitoring the formation process of the barrier layer of the intestinal wall cells in real time by detecting the change in refractive index on the surface of the metasurface plasmon resonance chip.

[0032] During their research, the inventors of this invention discovered that by employing MetaSPR technology to monitor the formation process of the intestinal wall cell barrier layer in real time by detecting changes in the refractive index of a metasurface plasmon resonance chip, label-free, real-time, and continuous monitoring of the intestinal wall cell barrier layer formation process can be achieved, allowing for dynamic observation of physiological changes in cells. This method exhibits high sensitivity and stability. Furthermore, the intestinal wall cell barrier model provided by this invention can simulate a real physiological environment, enabling cells to maintain normal physiological functions within the model, thus improving the model's reliability and applicability.

[0033] In this invention, intestinal wall cells refer to various cells that make up the intestinal wall, preferably Caco-2 cells, which are derived from human colon adenocarcinoma cells. When cultured in vitro, they can spontaneously differentiate into cells with intestinal epithelial cell characteristics, forming a monolayer cell structure similar to small intestinal epithelium, possessing typical intestinal epithelial features such as microvilli, and can simulate the intestinal barrier function.

[0034] According to the present invention, preferably, the metasurface plasmon resonance chip includes a metasurface plasmon resonance chip board and a MetaSPR (nano-metasurface plasmon resonance detection) sensor chip integrated on the metasurface plasmon resonance chip board; the metasurface plasmon resonance chip board is provided with a plurality of chip micropores, and the MetaSPR sensor chip includes a substrate, a plurality of nanopores imprinted on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores, the metal film including a transition metal film layer and a noble metal film layer disposed from the inside to the outside. The inventors have found that, under this preferred embodiment, it is beneficial to improve the sensitivity and stability of the SPR signal of the chip, enabling more accurate detection of minute changes in cells, thereby improving the sensitivity and stability of the intestinal wall cell barrier model.

[0035] According to the present invention, in order to further improve the sensitivity and stability of the intestinal wall cell barrier model, preferably, the substrate is made of PET material.

[0036] According to the present invention, in order to further improve the sensitivity and stability of the intestinal wall cell barrier model, preferably, the transition metal film layer is a titanium film layer with a thickness of 5-20 nm, and the noble metal film layer includes a silver film layer with a thickness of 40-100 nm and / or a gold film layer with a thickness of 5-60 nm. The transition metal film layer and the noble metal film layer can be coated on the surface of the nanopore by vapor deposition.

[0037] According to the present invention, in order to further improve the sensitivity and stability of the intestinal wall cell barrier model, preferably, the noble metal membrane layer includes a silver membrane layer and a gold membrane layer arranged from the inside to the outside.

[0038] According to the present invention, in order to further improve the sensitivity and stability of the intestinal wall cell barrier model, preferably, the nanopore is in the shape of a frustum cone, and the diameter of its large opening is 150-250 nm, the depth is 400-600 nm, and the spacing between two adjacent micropores of the chip is 200-500 nm.

[0039] It should be noted that when using the metasurface plasmon resonance biochip, double-sided adhesive is used to integrate the chip with a bottomless 96-well plate to create a 96-throughput microplate. According to the present invention, preferably, this construction method employs a WeSPR multifunctional molecular detector with the metasurface plasmon resonance biochip to detect changes in the refractive index of the metasurface plasmon resonance biochip surface in real time. For example, after assembling the metasurface plasmon resonance biochip into the aforementioned microwell chip, it is placed in a WeSPR™ Cell Analyzer SP real-time label-free cell analyzer to detect changes in the refractive index of the metasurface plasmon resonance biochip. Different adhesion states of intestinal wall cells result in different refractive indices of light passing through the metasurface plasmon resonance biochip, leading to different signal response values. The WeSPR™ Cell Analyzer SP real-time label-free cell analyzer can form a real-time response platform, enabling monitoring of the dynamic processes of intestinal wall cells at different stages, demonstrating the complete process from cell seeding to repair. A flowchart of the intestinal wall cell barrier model monitoring cell behavior is shown below. Figure 1 .

[0040] According to the present invention, preferably, the biomimetic treatment process includes: sterilizing, washing, and drying the metasurface plasmon resonance biochip, adding an adhesion agent into the nanopores for coating, and then removing the adhesion agent and washing. The inventors have found that, under this preferred embodiment, it is beneficial for the culture and fixation of intestinal wall cells on the MetaSPR chip surface, promoting the normal growth of intestinal wall cells and the formation of barrier function, thereby improving the sensitivity and stability of the intestinal wall cell barrier model.

[0041] In this invention, the sterilization process can be carried out by ultraviolet irradiation for 20-40 minutes; the washing process can be carried out by washing with ultrapure water 2-3 times.

[0042] According to the present invention, preferably, the adhesion agent is selected from at least one of poly-L-lysine (PLL), polyethyleneimine (PEI), and IV collagen, and more preferably polyethyleneimine. The inventors have found that, under this preferred embodiment, it is beneficial to improve the adhesion effect of intestinal wall cells on the chip plate, promote the normal growth of intestinal wall cells and the formation of barrier function, thereby improving the sensitivity and stability of the intestinal wall cell barrier model.

[0043] According to the present invention, preferably, the wall-adhering agent is added to each nanopore in the form of a solution with a concentration of 5-1000 μg / mL and the amount added is 50-200 μL. The concentration of the wall-adhering agent refers to the total concentration of all wall-adhering agent components in the solution.

[0044] According to the present invention, preferably, the coating conditions include at least: a temperature of 35-40°C, specifically 35°C, 37°C, 40°C, or any value between the two aforementioned values; and a time of 1-3 hours, specifically 1 hour, 2 hours, 3 hours, or any value between the two aforementioned values. The inventors have found that, under this preferred embodiment, it is beneficial for the adhesion agent to form a coating on the metasurface ion resonance biochip, thereby improving the adhesion effect of intestinal wall cells.

[0045] According to the present invention, preferably, the culture medium is selected from at least one of DMEM complete medium, RPMI 1640 medium, and Ham's F-12 medium. The inventors have found that this preferred embodiment is beneficial for promoting the culture of intestinal wall cells and the formation of the cell barrier, thereby improving the sensitivity and stability of the intestinal wall cell barrier model.

[0046] According to the present invention, preferably, the culture medium further contains a culture medium additive, which is fetal bovine serum (FBS) and / or penicillin-streptomycin (DUB). The inventors have found that, under this preferred embodiment, it is beneficial to improve the adhesion of intestinal wall cells, promote the formation of the intestinal wall cell barrier, and thus improve the sensitivity and stability of the intestinal wall cell barrier model.

[0047] According to the present invention, in order to further improve the adhesion effect of intestinal wall cells and the sensitivity and stability of the model, preferably, the culture medium additive is fetal bovine serum and penicillin-streptomycin.

[0048] According to the present invention, preferably, based on the mass of the culture medium, the mass of the fetal bovine serum is 5-20 wt%, specifically 5 wt%, 10 wt%, 15 wt%, 20 wt%, or any value between the two aforementioned values; the mass of the penicillin-streptomycin is 1-10 wt%, specifically 1 wt%, 5 wt%, 10 wt%, or any value between the two aforementioned values.

[0049] According to the present invention, in order to further improve the adhesion effect of intestinal wall cells and the sensitivity and stability of the model, preferably, the amount of culture medium added in a single microwell of the chip is 50-200 μL, specifically 100 μL, 150 μL, 200 μL, or any value between the two aforementioned values.

[0050] According to the present invention, in order to further improve the adhesion effect of intestinal wall cells and the sensitivity and stability of the model, preferably, the intestinal wall cell culture conditions include at least the following: the number of cells seeded in a single microwell of the chip is 10,000 to 30,000, specifically 10,000, 20,000, 30,000, or any value between the two aforementioned values; the temperature is 35-40 ℃, specifically 35 ℃, 37 ℃, 40 ℃, or any value between the two aforementioned values; the CO2 concentration is 4-6%, specifically 4%, 5%, 6%, or any value between the two aforementioned values, wherein the CO2 concentration of 4-6% means that the volume of CO2 in the air is 4-6%; and the humidity is 80-100%, specifically 80%, 90%, 100%, or any value between the two aforementioned values.

[0051] According to the present invention, preferably, the method further includes: detecting color changes in the cell-attached chip plate to form a real-time change imaging platform. The metasurface plasmon resonance biochip has high sensitivity to changes in refractive index. When the concentration of the solution on the chip surface is different, the refractive index of light is different, resulting in different observed chip colors. This enables the formation of a real-time change imaging platform, which is beneficial for real-time monitoring of the dynamic behavior of cells and further improves the sensitivity and reliability of the intestinal wall cell barrier model.

[0052] According to the present invention, in order to further improve the sensitivity and reliability of the intestinal wall cell barrier model, preferably, scanning electron microscopy (SEM) is used to detect the color of the chip plate on which the cells adhere.

[0053] According to a particularly preferred embodiment of the present invention, a method for constructing an intestinal wall cell barrier model is provided. The method includes the following steps: culturing intestinal wall cells in a culture medium using a metasurface plasmon resonance chip with a biomimetic surface treatment to obtain a chip plate with intestinal wall cells adhering to the chip; detecting the refractive index change of the surface of the metasurface plasmon resonance chip using a WeSPR multifunctional molecular detector with a metasurface plasmon resonance biochip; detecting the color of the chip plate with cell adhering to the chip plate using a scanning electron microscope; and monitoring the formation process of the barrier layer of intestinal wall cells in real time. The metasurface plasma resonance chip includes a metasurface plasma resonance chip board and a MetaSPR sensor chip integrated on the metasurface plasma resonance chip board. The metasurface plasma resonance chip board has multiple chip micropores. The MetaSPR sensor chip includes a substrate, multiple nanopores imprinted on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores. The metal film includes a titanium film layer with a thickness of 5-20 nm, a silver film layer with a thickness of 40-100 nm, and a gold film layer with a thickness of 5-60 nm arranged from the inside to the outside. The nanopores are frustoconical in shape, with a diameter of 150-250 nm and a depth of 400-600 nm for the largest opening. The spacing between two adjacent chip micropores is 200-500 nm. The substrate is made of PET material. The biomimetic treatment process includes: sterilizing, washing, and drying the metasurface plasmon resonance biochip; adding an adhesion agent into the nanopores and coating the chip at 35-40 ℃ for 1-3 hours; removing the adhesion agent and washing; the adhesion agent is polyethyleneimine, and the adhesion agent is added to each nanopore in the form of a solution with a concentration of 5-1000 μg / mL, with an addition volume of 50-200 μL; the culture medium is selected from at least one of DMEM medium, RPMI 1640 medium, and Ham's F-12 medium, and the culture medium also contains 5-20 wt% fetal bovine serum and 1-10 wt% penicillin-streptomycin, with the addition volume of the culture medium in each micropore of the chip being 50-200 μL; the intestinal wall cell culture conditions include at least the following: the number of cells seeded in each micropore of the chip is 10,000-30,000, the temperature is 35-40 ℃, the CO2 concentration is 4-6%, and the humidity is 80-100%.

[0054] The intestinal cell barrier model constructed through the above-described preferred embodiments enables label-free, real-time, and continuous monitoring of the intestinal cell barrier layer formation process, allowing dynamic observation of cellular physiological changes. This method exhibits high sensitivity and stability. Furthermore, this intestinal cell barrier model can simulate a real physiological environment, allowing cells to maintain normal physiological functions within the model, thus improving the model's reliability and applicability.

[0055] Secondly, the present invention provides the application of the intestinal wall cell barrier model obtained by the construction method described in the first aspect above in evaluating alcohol-induced intestinal wall cell damage.

[0056] In this invention, the intestinal wall cell barrier model obtained by the above construction method can simulate the real physiological environment, such as the physiological changes of intestinal wall cells under alcohol induction. By evaluating the effect of alcohol on cell barrier function, it lays the foundation for research on the protection or repair of intestinal wall cells by drugs or probiotics, and has significant market prospects.

[0057] Thirdly, the present invention provides the application of the intestinal wall cell barrier model obtained by the construction method described in the first aspect above in evaluating the protective or repairing effects of drugs or probiotics on intestinal wall cell damage.

[0058] In this invention, the intestinal wall cell barrier model obtained by the above construction method can simulate the real physiological environment. When intestinal wall cells are damaged, the constructed intestinal wall cell barrier model can evaluate the protective or repairing effects of drugs or probiotics on intestinal wall cell damage, providing an efficient and accurate platform for drug or probiotic screening. It can quickly assess the impact of drugs or probiotics on cell barrier function, accelerate the drug development process, and has current market prospects.

[0059] According to the present invention, intestinal wall cell damage can be drug and toxin (e.g., alcohol) induced intestinal wall cell damage, ischemia-hypoxia induced intestinal wall cell damage, infectious intestinal wall cell damage, etc., preferably, the intestinal wall cell damage is alcohol induced intestinal wall cell damage.

[0060] According to the present invention, the drug or probiotic refers to a drug or probiotic that can protect or repair damaged intestinal wall cells. Preferably, the drug is dihydroquercetin (DHQ); the probiotic is selected from at least one of Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus curvatureis, Lactobacillus pentosus, Bifidobacterium breve, Lactobacillus gasseri, Lactobacillus casei subsp. casei, and Lactobacillus casei subsp. casei.

[0061] The present invention will be described in detail below through embodiments.

[0062] Unless otherwise specified, the experimental methods and equipment described in the following embodiments are conventional methods and equipment. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0063] Caco-2 cells were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.; DMEM complete culture medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product model KGL1206-500; poly-L-lysine (PLL) was purchased from Shanghai Beyotime Biotechnology Co., Ltd., product model ST508; polyethyleneimine (PEI) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; type IV collagen was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; fetal bovine serum was purchased from Gibco, product model 430167; penicillin-streptomycin was purchased from Gibco, product model 15140122.

[0064] Example 1: Establishment of an intestinal wall cell barrier model 1.1 Chip Fabrication The MetaSPR sensor chip includes a substrate (PET material), multiple nanopores imprinted on the substrate, and a metal film covering the surface of the nanopores. The metal film includes a transition metal film layer and a noble metal film layer arranged from the inside to the outside. The nanopores are frustoconical in shape with a diameter of 200 nm and a depth of 500 nm for their large openings. The spacing between two adjacent nanopores is 400 nm. The transition metal film layer includes a titanium film layer with a thickness of 15 nm, and the noble metal film layer includes a silver film layer with a thickness of 70 nm and a gold film layer with a thickness of 20 nm arranged from the inside to the outside. The specific fabrication process is as follows: the surface of the PET substrate is pre-imprinted with a matrix of nanopores, and then the metal film is deposited on the substrate.

[0065] The MetaSPR sensor chip was cut into 13 cm × 8.5 cm segments and attached to a 96-well metasurface plasmon resonance chip board. The chip board has chip micropores corresponding to the nanopores. Each chip micropore has a height of 500 nm, an upper circle radius of 200 nm at the opening, and a bottom circle diameter of 100 nm, to serve as the MetaSPR chip.

[0066] The nanostructure of the MetaSPR chip was examined using a scanning electron microscope (SEM), demonstrating its structural integrity and consistency.

[0067] 1.2 Screening of conditions for culturing Caco-2 cells using MetaSPR microarray (1) Pretreatment of Caco-2 cells: Sterilize the clean bench with UV light for 30 min. First, place DMEM complete medium (DMEM medium pre-added with 10wt% FBS and 1wt% antibiotics), PBS, and trypsin in a water bath at 37℃ for 30 min. Take Caco-2 cells from the incubator to the clean bench and pour the medium into the waste container. Add 5 mL of PBS and gently rotate it around the bottom of the culture dish to wash away the residual medium. Repeat twice. Add 2 mL of trypsin and place Caco-2 cells in the incubator to digest for 2 min. Add 4 mL of DMEM complete medium to stop the digestion. After the Caco-2 cells are evenly pipetted, add them to a centrifuge tube and centrifuge at 200 G for 7 min. Take out the centrifuge tube, discard the supernatant, and add 2 mL of fresh DMEM complete medium to resuspend the cells. (2) Sterilize the MetaSPR chip with ultraviolet light for 30 min. After sterilization, rinse each chip microwell twice with 150 μL of ultrapure water. After rinsing, shake off the ultrapure water on the chip board surface. (3) The MetaSPR chip was treated with 100 μg / mL PLL, 100 μg / mL PEI, 100 μg / mL collagen IV and ultrapure water respectively, and applied to the chip plate at a volume of 50 μL / well; after incubation at 37 ℃ for 2 hours, the pre-treated chip was taken out of the incubator, the treatment solution was discarded, and the chip was washed twice with ultrapure water. (4) The Caco-2 cells obtained in step (1) were seeded into pre-coated plates at standard densities (150 μL of culture medium per well, with cell counts of 0, 5000, 10000, and 20000 cells per well). Under different substrate conditions, the dynamics of cell adhesion and proliferation were continuously monitored over 72 hours using the WeSPR™ Cell Analyzer automated detection instrument, with a scan interval of 15 minutes.

[0068] See the screening results Figure 2 It can be seen that among the four groups of chips—PLL, PEI, IV collagen, and untreated—the chip treated with PEI showed the best cell growth when seeded at a density of 2 w cells / well.

[0069] 1.3 MetaSPR microarray culture of Caco-2 cells Caco-2 cells were seeded on the surface of the MetaSPR chip and pretreated with 10 μg / mL PEI. 20,000 cells were seeded per well and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (150 µL DMEM per well) and maintained at 37°C, 5% CO2 and 100% humidity.

[0070] 1.4 Real-time monitoring The formation process of the cell barrier layer is monitored in real time using SPR technology. The WeSPR™ cell analyzer monitors the dynamics of cell attachment and proliferation every 15 minutes. Different cell adhesion states cause different changes in refractive index. By detecting these changes in the refractive index of the cell layer, the dynamic changes of cells are reflected, recording the process of cell growth and barrier function establishment. Simultaneously, the MetaSPR chip is observed under a microscope. Different solution concentrations on the chip surface cause different refractive indices of light, resulting in different observed chip colors, forming a real-time imaging platform.

[0071] Example 2 Model Validation 2.1 By comparing the detection results of traditional cell barrier models and the model provided in this invention, the accuracy and reliability of the model in this invention are verified. CCK-8 and crystal violet were used as detection reagents to detect the formation process of the intestinal wall cell barrier layer. To assess the cytotoxicity of different concentrations of ethanol to cells, Caco-2 cells were seeded in 96-well cell culture plates (15,000 cells per well, 100 μL of DMEM medium). After a 12-hour cell attachment and proliferation phase, cells were treated with 10%, 5%, 2.5%, 1.25%, 0.63%, and 0.31% ethanol for 12 hours, respectively. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 °C and 5% CO2 for 2 hours. The absorbance at 450 nm was measured using a microplate reader. To assess the cytotoxicity of various alcoholic beverages to cells, cells were subjected to attachment and proliferation as described above. Then, different types of alcohol were mixed with DMEM medium at a 1:1 ratio and added to 96-well plates, and incubated for another 12 hours. Then, CCK-8 reagent was added according to the previously described protocol, and absorbance at 450 nm was measured to quantify cell viability. To evaluate the repair effect of dihydroquercetin on ethanol-induced intestinal barrier damage, cells were first attached and proliferated as described above. Subsequently, cells in the wells were treated with 2.5% ethanol for 12 hours to induce cell damage. After ethanol treatment, the culture medium was aspirated, and dihydroquercetin was supplemented at concentrations of 50 μmol, 40 μmol, 30 μmol, 20 μmol, 10 μmol, and 5 μmol, respectively. After a 24-hour recovery period, CCK-8 reagent was added according to the established method, and absorbance was measured at 450 nm to assess cell viability.

[0072] To assess the cytotoxicity of different concentrations of ethanol to cells, Caco-2 cells were seeded into 6-well plates at a density of 2,000 cells per well and 2 mL of culture medium per well. After cell formation, the cells were treated with 10%, 5%, 2.5%, 1.25%, 0.63%, and 0.31% ethanol for 12 hours, respectively. After treatment, the supernatant was removed, and the cells were washed three times with PBS. Then, the cells were fixed with 1.5 mL of 4% paraformaldehyde for 15 minutes, followed by three washes with PBS. The cells were stained with crystal violet solution for 15 minutes, and then thoroughly washed with deionized water to remove residual dye. The dried samples were recorded using imaging techniques. To assess the cytotoxicity of various alcoholic beverages to cells, cell seeding was performed according to the established experimental protocol. After clonal growth, different types of alcohol were mixed with DMEM medium at a 1:1 ratio and added to the wells. After 12 hours of incubation, the cells were fixed and stained according to the above method to quantitatively analyze clonal viability. To evaluate the repair effect of dihydroquercetin on intestinal cell barrier damage, cells were treated with 2.5% ethanol for 12 hours after colony formation to induce cell damage. After ethanol exposure, the culture medium was aspirated, and fresh culture medium containing dihydroquercetin at concentrations of 50 μmol, 40 μmol, 30 μmol, 20 μmol, 10 μmol, and 5 μmol were added, respectively. After a 24-hour recovery period, the cells were fixed and stained according to the above method to assess their morphological recovery.

[0073] Parallel validation using CCK-8 and crystal violet staining showed a high degree of consistency with MetaSPR data, with Pearson correlation coefficients of 0.9437 (CCK-8) and 0.9068 (crystal violet), confirming methodological consistency. Furthermore, traditional endpoint assays require cell fixation / staining (inherent cytotoxicity), while the intestinal wall cell barrier model constructed in this study allows for non-destructive real-time tracking over a longer period while maintaining cell viability. This comparison highlights the practical advantage of the MetaSPR system in preserving native cell physiology during dynamic therapeutic assessment.

[0074] 2.2 Different concentrations of glycerol-water were added to the surface of the MetaSPR chip, and the color changes on the chip surface were observed using a scanning electron microscope to verify the real-time monitoring capability of the MetaSPR chip. Glycerol aqueous solutions of different concentrations (0 wt%, 1 wt%, 3 wt%, 5 wt%, 7 wt%, 9 wt%, and 11 wt%) were prepared. 50 μL of each glycerol solution was added to each well of the MetaSPR chip. Absorption spectra were measured in the 500-700 nm wavelength range using a microplate reader in full-wavelength scanning mode. The color change of the MetaSPR chip under a microscope is shown in the figure. Figure 3The full absorption spectrum of the glycerol aqueous solution is shown below. Figure 4 .

[0075] from Figure 3 It can be seen that the MetaSPR chip changes with the concentration of added glycerol water, indicating that the chip is very sensitive to light scattering. Figure 4 As can be seen, the spectrum of the chip gradually redshifts with different RI values, indicating that the MetaSPR chip can monitor refractive index changes in real time and has high sensitivity and reliability.

[0076] Example 3: Evaluation of alcohol-induced intestinal barrier damage 3.1 Alcohol Treatment: Caco-2 cells were cultured on the surface of a chip (the same MetaSPR chip prepared in Example 1.1) (following the steps in Example 1.3). After monitoring their growth for 12 h, different brands of baijiu (ethanol concentration 38-52% v / v) and beer (ethanol concentration 2.5-4.7% v / v) were added, diluted with DMEM complete medium at a volume ratio of 1:1, and changes in cell state were observed in real time. Specific baijiu and beer included: Maopu buckwheat wine, Luzhou Laojiao, Lvdou Daqu, Guojiao 1573, Mingniang tea wine, Niulanshan Erguotou, Budweiser, Snow Beer, Heineken, and China Jinjiu. The control group was set as follows: ultrapure water diluted with DMEM complete medium at a volume ratio of 1:1.

[0077] 3.2 Real-time monitoring: SPR technology was used to monitor changes in cells after drug treatment in real time. The cell-killing effect of alcohol was reflected by detecting changes in the refractive index of the cell layer. The results are shown in [Figure number missing]. Figure 5 .

[0078] 3.3 Data Analysis: The differences between different treatment groups and the control group were analyzed to assess the effects of different types of alcohol on intestinal wall cells. The practicality and accuracy of the model were verified by comparing it with the traditional CCK8 method. Results are shown in [Figure number missing]. Figure 6 .

[0079] 3.4 Results: Compared with the control group, all types of alcoholic beverages showed a significant killing effect on intestinal wall cells, with Guojiao 1573 showing the most significant effect, which was significantly greater than that of Niulanshan and Luzhou Laojiao.

[0080] Example 4: Evaluation of the damaging effects of alcohol on intestinal wall cells and the repairing effects of dihydroquercetin on intestinal wall cell damage. 4.1 Cell Treatment: Following the method in Example 3.1, intestinal wall cells were killed using different concentrations of ethanol. Caco-2 cells were cultured on a MetaSPR chip, with 100 μL of culture medium per well and a seeding density of 20,000 cells per well. Cell kinetics were assessed in real time by scanning every 15 minutes using a cell analyzer: after a 12-hour incubation period when the cells were in the logarithmic growth phase, different concentrations (0 wt%, 0.3 wt%, 0.6 wt%, 1.25 wt%, 2.5 wt%, 5 wt%, and 10 wt%) of ethanol solution were added; continuous monitoring was then performed to assess the extent of ethanol-induced intestinal barrier damage; real-time cell changes were simultaneously captured using an imaging system; subsequently, the data were processed by analyzing changes in R channel values, and these data were used to generate real-time growth curves to illustrate changes in SPR readings as the cell life cycle progressed.

[0081] Caco-2 cells were cultured on a 96-well MetaSPR chip, with 100 μL of medium per well and a standard density of 20,000 cells per well. Real-time cell kinetics were observed using a cell analyzer, with scans every 15 minutes. After 12 hours of culture, during which the cells were in the logarithmic growth phase, 2.5% (wt) ethanol solution was introduced, and cell status was continuously monitored for another 12 hours. After ethanol-induced intestinal barrier disruption, the treatment medium was replaced with fresh medium containing different concentrations of DHQ (0, 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM). Real-time monitoring was then performed for 24 hours to quantitatively evaluate the compound's restorative effect on ethanol-induced intestinal barrier dysfunction.

[0082] 4.2 Real-time monitoring: SPR technology was used to monitor cell changes after drug treatment in real time. Changes in the refractive index of the cell layer were detected to reflect the cell-killing effect of alcohol and the cell-repairing effect of the drug. Results of the cell-killing effect of different concentrations of alcohol are shown below. Figure 7 The results of the cell repair effects of different concentrations of dihydroquercetin (DHQ) are shown in the figure. Figure 8 .

[0083] 4.3 Results: 10% alcohol can completely kill cells, while alcohol concentrations below 0.6% have no cell-killing effect. 20-50 μM dihydroquercetin has a repairing effect on damaged intestinal wall cells, while dihydroquercetin below 10 μM has no repairing effect on cells damaged to varying degrees.

[0084] Example 5 5.1 Probiotic Treatment: Different types of probiotics (Lactobacillus rhamnosus LRa05, Bifidobacterium lactis BLa08, Lactobacillus plantarum 6, Lactobacillus plantarum 445, Lactobacillus curvularis 884, Lactobacillus pentosus 1388, Bifidobacterium breve BBr60, Lactobacillus gasseri LG08, Lactobacillus casei subsp. casei 84, and Lactobacillus casei subsp. casei 1320, among which Lactobacillus rhamnosus LRa05, Bifidobacterium lactis BLa08, Bifidobacterium breve BBr60, and Lactobacillus gasseri LG08 were from Microcare Probiotics (Suzhou) Co., Ltd., and the remaining probiotics were from the LICME research group strain preservation bank of Nanjing Normal University) were set up with different concentration gradients of probiotic treatment groups and control groups.

[0085] 5.2 Real-time monitoring: SPR technology is used to monitor changes in cell barrier function after probiotic treatment in real time. The changes in the refractive index of the cell layer are detected to reflect the protective or repairing effect of probiotics on the cell barrier.

[0086] 5.3 Data Analysis: The differences between different probiotic treatment groups and the control group were analyzed to screen out probiotics that have a significant protective or repairing effect on the intestinal wall cell barrier function.

[0087] 5.4 Expected Results: A certain concentration of probiotics can protect intestinal wall cells and prevent cell damage. Compared to the control group, a higher concentration of alcohol is required to have a killing effect on cells. Furthermore, probiotics have a repairing effect on cells already damaged by alcohol.

[0088] Comparative Example Real-time monitoring of Caco-2 cells was performed using a conventional SPR chip (purchased from Liangzhun (Shanghai) Medical Devices Co., Ltd., product model G10002). The steps in Examples 1.3 and 1.4 were used to monitor the formation of the intestinal wall cell barrier, and the steps in Example 4 were used to evaluate the damaging effects of alcohol on intestinal wall cells and the repairing effects of dihydroquercetin on intestinal wall cell damage. The results showed that the conventional chip had low sensitivity during detection, and Caco-2 cells could not effectively adhere to the intestinal wall. Ultimately, the relative response values ​​and sensitivity of each experimental group were insufficient to demonstrate the damaging effects of alcohol on intestinal wall cells and the repairing effects of dihydroquercetin on intestinal wall cell damage.

[0089] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for constructing an intestinal wall cell barrier model, characterized in that, The method includes the following steps: culturing intestinal wall cells in a culture medium using a metasurface plasmon resonance chip with a biomimetic surface treatment to obtain a chip plate with intestinal wall cells adhering to the chip; and monitoring the formation process of the barrier layer of the intestinal wall cells in real time by detecting the change in refractive index on the surface of the metasurface plasmon resonance chip.

2. The construction method according to claim 1, characterized in that, The metasurface plasma resonance chip includes a metasurface plasma resonance chip board and a MetaSPR sensor chip integrated on the metasurface plasma resonance chip board; the metasurface plasma resonance chip board is provided with a plurality of chip micropores, and the MetaSPR sensor chip includes a substrate, a plurality of nanopores imprinted on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores, the metal film including a transition metal film layer and a noble metal film layer disposed from the inside to the outside; Preferably, the substrate is made of PET material; Preferably, the transition metal film is a titanium film with a thickness of 5-20 nm, and the noble metal film includes a silver film with a thickness of 40-100 nm and / or a gold film with a thickness of 5-60 nm. Preferably, the noble metal film layer includes a silver film layer and a gold film layer disposed from the inside out; Preferably, the nanopores are frustoconical in shape, with a diameter of 150-250 nm and a depth of 400-600 nm for the large opening, and a spacing of 200-500 nm between two adjacent micropores.

3. The construction method according to claim 2, characterized in that, The construction method uses a WeSPR multifunctional molecular detector with the aforementioned metasurface plasmon resonance biochip to detect the refractive index change on the surface of the metasurface plasmon resonance biochip in real time.

4. The construction method according to claim 2, characterized in that, The biomimetic treatment process includes: sterilizing, washing, and drying the metasurface plasmon resonance biochip, adding an adhesion agent to the nanopores for coating, and then removing the adhesion agent and washing.

5. The construction method according to claim 4, characterized in that, The adhesion agent is selected from at least one of polylysine, polyethyleneimine, and IV collagen, preferably polyethyleneimine; Preferably, the adhesion agent is added to each nanopore in the form of a solution with a concentration of 5-1000 μg / mL and the amount added is 50-200 μL; Preferably, the coating conditions include at least the following: a temperature of 35-40 ℃ and a time of 1-3 h.

6. The construction method according to claim 2, characterized in that, The culture medium is selected from at least one of DMEM medium, RPMI 1640 medium and Ham's F-12 medium; Preferably, the culture medium further contains a culture medium additive, which is fetal bovine serum and / or penicillin-streptomycin; Preferably, the culture medium additive is fetal bovine serum and penicillin-streptomycin; Preferably, based on the mass of the culture medium, the fetal bovine serum is 5-20 wt%, and the penicillin-streptomycin is 1-10 wt%. Preferably, the amount of culture medium added in a single microwell of the chip is 50-200 μL.

7. The construction method according to claim 2, characterized in that, The conditions for intestinal wall cell culture include at least the following: 10,000 to 30,000 cells seeded in a single microwell of the chip, a temperature of 35 to 40 °C, a CO2 concentration of 4 to 6%, and a humidity of 80 to 100%.

8. The construction method according to any one of claims 1 to 7, characterized in that, The method also includes: detecting color changes in the chip plate to which the cells adhere, forming a real-time change imaging platform; Preferably, the color of the chip plate to which the cells adhere is detected using a scanning electron microscope.

9. The application of the intestinal wall cell barrier model obtained by the construction method according to any one of claims 1 to 8 in evaluating alcohol-induced intestinal wall cell damage.

10. The application of the intestinal wall cell barrier model obtained by the construction method according to any one of claims 1 to 8 in evaluating the protective or repairing effects of drugs or probiotics on intestinal wall cell barrier damage; Preferably, the intestinal wall cell damage is alcohol-induced intestinal wall cell damage; Preferably, the drug is dihydroquercetin; the probiotic is selected from at least one of Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus curvularis, Lactobacillus pentosus, Bifidobacterium breve, Lactobacillus gasseri, Lactobacillus casei subsp. casei, and Lactobacillus casei subsp. casei.

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