Intestinal cell model as well as preparation method and application thereof

Abstract

The invention provides an intestinal cell model as well as a preparation method and application thereof. According to the method, a Caco-2 / THP-1 cell co-culture system is constructed to simulate the dynamic interaction between epithelial cells and immune cells in human intestinal tracts, and finally comprehensive evaluation on digestion efficiency, absorption efficiency, bioavailability, metabolites, inflammatory reaction influence and the like of drugs, nutrient substances (such as milk powder) and other components is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an intestinal cell model, its preparation method, and its application. Background Technology

[0002] Currently, commonly used in vitro intestinal models mainly rely on single cell line culture, such as the human colon adenocarcinoma Caco-2 cell model. Caco-2 cells are widely used to simulate the transepithelial transport of drugs or nutrients because they can form a monolayer structure similar to the small intestinal epithelium and possess microvilli and tight junction proteins.

[0003] However, the Caco-2 cell model has the following limitations: (1) Single cell composition: The Caco-2 cell model is composed only of epithelial cells and lacks key physiological components such as intestinal mucus layer and immune cells (such as macrophages and dendritic cells), which cannot simulate the complex intercellular interactions of the intestine; (2) Incomplete metabolic enzyme system: Some metabolic enzymes (such as cytochrome P450 enzyme system) in Caco-2 cells have low expression levels, resulting in incomplete research on the metabolism of nutrients or drugs; (3) Insufficient simulation of inflammatory response: Existing models are difficult to reproduce the pathophysiological processes such as impaired epithelial barrier function and impaired absorption and transport in intestinal inflammatory states (such as inflammatory bowel disease).

[0004] To overcome these shortcomings, some studies have attempted to construct models that more closely resemble the in vivo environment by co-culturing other cells (such as fibroblasts and endothelial cells) or adding mucus layer components. However, such methods still suffer from problems such as incomplete intercellular signal transduction mechanisms, high model complexity, and high costs, making it difficult to achieve standardized applications. Summary of the Invention

[0005] The purpose of this invention is to provide an intestinal cell model, its preparation method, and its application. By constructing a Caco-2 / THP-1 cell co-culture system (i.e., an intestinal cell model), the dynamic interaction between epithelial cells and immune cells in the human intestine is simulated, ultimately enabling a comprehensive evaluation of the digestion efficiency, absorption efficiency, bioavailability, metabolites, and inflammatory response effects of drugs, nutrients (such as milk powder), and other components.

[0006] This invention provides a method for preparing an intestinal cell model, comprising the following steps:

[0007] Caco-2 cells were seeded into the upper chamber of a Transwell culture plate for the first culture, and then THP-1 cells were seeded into the lower chamber of the Transwell culture plate for the second culture, thus obtaining an intestinal cell model.

[0008] In the preparation method described above, the culture medium for Caco-2 cells in the first culture is EMEM medium.

[0009] In the preparation method described above, the seeding amount of Caco-2 cells in the first culture is 0.5-1.5 × 10⁻⁶. 5 per mL.

[0010] In the preparation method described above, the endpoint of the first culture is a transmembrane resistance value of a Caco-2 cell layer composed of Caco-2 cells > 500 Ω·cm. 2 .

[0011] In the preparation method described above, in the second culture, the culture medium for THP-1 cells is obtained by mixing EMEM medium and RPMI-1640 medium at a volume ratio of 2-4:1.

[0012] In the preparation method described above, the seeding density of THP-1 cells in the second culture is 0.5-1.5 × 10⁻⁶. 5 per mL.

[0013] In the preparation method described above, the second culture time is 3-5 days.

[0014] In the preparation method described above, the volume of the culture medium in the upper compartment of the Transwell culture plate is 0.5-1.5 mL; and / or,

[0015] The culture medium volume in the lower compartment of the Transwell culture plate is 0.5-1.5 mL.

[0016] This invention provides an intestinal cell model, which is prepared by the above-described preparation method.

[0017] This invention provides an intestinal cell model prepared by the above-described preparation method, or the application of the above-described intestinal cell model in evaluating the digestion, absorption, or bioavailability of drugs, nutrients, and other components.

[0018] This invention provides an intestinal cell model in which Caco-2 cells simulate intestinal epithelium in the upper chamber and THP-1 cells simulate intestinal mucosal immune cells in the lower chamber through Transwell non-contact co-culture. The model promotes cross-barrier signal transmission between the two cells through soluble factors in the culture medium, accurately reproducing the bidirectional interaction process in the human intestinal epithelial cells that involves sensing external components, secreting signaling molecules, regulating immune cell activation, secreting factors by immune cells, and counter-regulating epithelial cell function. This enables an integrated assessment of absorption, metabolism, and immunity. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a two-dimensional intestinal cell model in one embodiment of the present invention;

[0020] Figure 2 This is a graph showing the transmembrane resistance values ​​of the Caco-2 cell layer in one embodiment of the present invention;

[0021] Figure 3 This is a graph showing the protein count results of the digestion and absorption groups in one embodiment of the present invention;

[0022] Figure 4 This is a differential protein volcano diagram of the digestive and absorptive groups in one embodiment of the present invention;

[0023] Figure 5 This is a graph showing the peptide count results for the digestion and absorption groups in one embodiment of the present invention;

[0024] Figure 6 This is a differential peptide volcano diagram of the digestive and absorptive groups in one embodiment of the present invention;

[0025] Figure 7 This is a diagram showing the free fatty acid content of different milk powders in a two-dimensional intestinal cell model in one embodiment of the present invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] To develop a simple, low-cost intestinal cell model that more closely resembles the intestinal environment, the first aspect of this invention provides a method for preparing an intestinal cell model, comprising the following steps:

[0028] Caco-2 cells were seeded into the upper chamber of a Transwell culture plate for the first culture, and then THP-1 cells were seeded into the lower chamber of the Transwell culture plate for the second culture, thus obtaining an intestinal cell model.

[0029] Caco-2 cells, derived from human colon adenocarcinoma cells, possess the same microvilli structure and tight junctions as small intestinal epithelial cells. They contain enzymes associated with the brush border epithelium of the small intestine and can reflect information related to the absorption, metabolism, and transport of drugs, nutrients, and other components through the small intestinal mucosa at the cellular level. They are commonly used as models to study intestinal absorption and transport mechanisms. THP-1 cells, on the other hand, are a human macrophage line isolated from the peripheral blood of a boy with acute monocytic leukemia. Under local stimulation, they can differentiate into mature macrophages and play a central role in cell-mediated immune and inflammatory responses. They are widely used in studies of monocyte and macrophage-related mechanisms and signaling pathways, such as inflammatory responses and immune regulation. Therefore, this invention constructs a non-contact co-culture system including Caco-2 and THP-1 cells to simulate the dynamic interaction between epithelial cells and immune cells in the human intestine, ultimately enabling a comprehensive evaluation of the digestibility, absorption efficiency, bioavailability, metabolites, and inflammatory effects of drugs, nutrients (such as milk powder), and other components.

[0030] Specifically, the present invention has the following benefits:

[0031] (1) Restore the bidirectional interaction mechanism between intestinal epithelium and immunity: The core function of the human gut depends on the dynamic collaboration between intestinal epithelial cells and intestinal mucosal immune cells. However, in existing technologies, models using Caco-2 cells alone can only simulate the intestinal epithelial barrier and absorption function, failing to reflect the regulation of absorption and metabolism by immune cells. For example, inflammatory mediators and enzymes released by inflammatory cells such as THP-1 cells can affect the tight junctions of the intestinal mucosa. Tight junctions are important connection structures between intestinal epithelial cells, preventing harmful substances from entering the bloodstream and maintaining selective permeability of the intestinal mucosa. For instance, TNF-α can induce downregulation of the expression of genes encoding tight junction proteins (such as occludin and claudin) or alter their protein structure, increasing intestinal mucosal permeability. Simultaneously, cytokines released by inflammatory cells can regulate the expression and function of some transporters. For example, IL-1β can reduce the expression and activity of glucose transporters (such as SGLT-1 and GLUT-2) in intestinal epithelial cells, decreasing glucose absorption. Inflammation may also affect the distribution of transporters, causing them to migrate from the apical membrane of intestinal mucosal cells to the basolateral membrane or be internalized, leading to malabsorption. Furthermore, models using THP-1 cells alone can only study immune responses, lacking the barrier metabolic basis of epithelial cells. This invention utilizes Transwell non-contact co-culture, where Caco-2 cells simulate intestinal epithelium in the upper chamber and THP-1 cells simulate intestinal mucosal immune cells in the lower chamber. This facilitates cross-barrier signal transmission between the two cells through soluble factors (such as cytokines and metabolites) in the culture medium. It precisely recreates the bidirectional interaction process in the human intestine where epithelial cells sense external components, secrete signaling molecules, regulate immune cell activation, secrete immune cells secrete factors, and then counter-regulate epithelial cell function. Therefore, the physiological relevance of the intestinal cell model of this invention is significantly better than that of the single-cell model.

[0032] (2) Integrated assessment of absorption, metabolism and immune response: In the existing technology, if it is necessary to study absorption, metabolism and immune response at the same time, Caco-2 model and THP-1 model need to be constructed separately for independent experiments. This is not only cumbersome, but also cannot eliminate the interference of variables such as culture environment and reagent batch in the two independent experiments, resulting in insufficient correlation and reliability of experimental results. However, the Caco-2 / THP-1 cell co-culture system constructed in this invention can simultaneously achieve the following in the same system: (1) the absorption efficiency, transport mechanism, metabolite analysis and metabolic enzyme activity of drugs, nutrients (such as milk powder) and other components through the intestinal epithelium; (2) the effects of drugs, nutrients (such as milk powder) and other components on the activation state of intestinal immune cells, secretion of inflammatory factors, and regulation of inflammatory mediators; (3) the feedback of the immune response caused by drugs, nutrients (such as milk powder) and other components on the absorption / metabolism function of epithelial cells, realizing the integrated assessment of absorption-metabolism-immunity, and greatly improving the accuracy and correlation of data.

[0033] (3) Simple operation process and low cost: The preparation method of the present invention only requires routine cell culture operation. The Transwell culture plate used is a common consumable in cell biology laboratories. No special equipment or complex process is required. Ordinary R&D laboratories can achieve this. The technical threshold is low and the reproducibility is high.

[0034] (4) Compared with 3D intestinal organoid models or animal models, this invention uses two commercial cell lines that can be stably passaged, are inexpensive, and have a short culture cycle, which can significantly reduce experimental costs and time costs. They are suitable for high-throughput screening (such as simultaneously evaluating the effects of multiple food components or drugs) and have the potential for industrial application.

[0035] In summary, the Caco-2 / THP-1 cell co-culture system (i.e., intestinal cell model) constructed in this invention can better simulate the intestinal environment, comprehensively considering the two key factors of absorption and physiological activity. It can be used to study the interaction between intestinal epithelial cells and immune cells in processes such as inflammatory response, drug absorption and metabolism, and is expected to provide more accurate, reliable and comprehensive research methods for drug development, assessment of the bioavailability of nutrients and other fields.

[0036] In the preparation of the intestinal cell model of the present invention, Caco-2 cells and THP-1 cells can be routinely cultured in advance to obtain Caco-2 cells and THP-1 cells in the logarithmic growth phase.

[0037] The Caco-2 cell culture method can be as follows: Caco-2 cells are seeded in EMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin, and cultured in a constant temperature incubator at 37°C, 5% CO2, and 90% relative humidity. Fresh EMEM medium is replaced every other day. When the confluence of Caco-2 cells reaches about 80% under a microscope, Caco-2 cells are digested with 0.25% trypsin solution and passaged at a ratio of 1:3.

[0038] The THP-1 cell culture method is as follows: THP-1 cells are seeded in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a constant temperature incubator at 37°C, 5% CO2 and 90% relative humidity. The medium is replaced with fresh RPMI-1640 medium every 2-3 days. THP-1 cells are collected by centrifugation and passaged at a ratio of 1:3.

[0039] Next, Caco-2 cells in the logarithmic growth phase can be seeded into the upper chamber of a Transwell culture plate for the first culture, and then THP-1 cells in the logarithmic growth phase can be seeded into the lower chamber of a Transwell culture plate for the second culture, thus obtaining the Caco-2 / THP-1 cell co-culture system, i.e., the intestinal cell model.

[0040] In the above technical solution, the culture medium for Caco-2 cells in the first culture is EMEM medium. EMEM medium (Eagle's Minimum Essential Medium) is developed from Eagle's Basic Medium (BEM) and is the most basic and widely used animal cell culture medium. It contains only 12 essential amino acids, glutamine, and 8 vitamins. Its simple composition makes it mainly used for the culture of adherent cells.

[0041] Furthermore, fetal bovine serum and penicillin-streptomycin antibiotics can be added to the EMEM medium to promote better growth of Caco-2 cells. The concentration of fetal bovine serum can be 15-25% (v / v), and the concentration of penicillin-streptomycin antibiotics can be 0.5-1.5% (v / v).

[0042] In the above technical solution, during the first culture, the inoculum size of Caco-2 cells is 0.5-1.5 × 10⁻⁶. 5 per mL.

[0043] Because Caco-2 cells need to grow to 100% confluence on the filter membrane surface of the upper compartment of the Transwell to form a tight junction structure similar to intestinal epithelium, thereby mimicking the barrier function of the intestinal mucosa, limiting the inoculum size ensures that Caco-2 cells rapidly fuse to form a dense monolayer, constructing an effective physical barrier. If the inoculum size is less than 0.5 × 10⁻⁶ cells / mL... 5 A seeding density of 1.5 × 10⁶ cells / mL will result in excessively low cell density, requiring a longer time to fuse, and after fusion, gaps will remain in the monolayer of cells, resulting in incomplete tight junctions and weakened barrier function. If the seeding density exceeds 1.5 × 10⁶ cells / mL... 5 A cell density of 10 cells / mL can lead to excessively high cell density, limited growth space, and cell stacking and overproliferation. This can cause some cells to undergo apoptosis due to hypoxia and nutrient deficiency, or disrupt tight junction structures. In addition, overproliferated cells may secrete excessive metabolic waste, alter the local culture environment pH, inhibit the expression of brush border enzymes and transporters, and reduce the effectiveness of cell metabolism and absorption.

[0044] In the above technical solution, the endpoint of the first culture is a transmembrane resistance value of the Caco-2 cell layer composed of Caco-2 cells > 500 Ω·cm.2 .

[0045] In the intestinal cell model of this invention, the transmembrane resistance (TEER) of the Caco-2 cell layer is limited to >500 Ω·cm. 2 The purpose is to quantify the tight junction integrity of the Caco-2 cell layer, ensuring that the intestinal cell model can accurately simulate the physical barrier properties of the human intestinal mucosa, and to provide a reliable functional basis for subsequent integrated assessment of absorption, metabolism and immunity.

[0046] In some embodiments, Caco-2 cells in the logarithmic growth phase can be taken and the Caco-2 cell ratio adjusted to 0.5-1.5 × 10⁻⁶ cells using EMEM medium containing 15-25% fetal bovine serum and 0.5-1.5% penicillin-streptomycin. 5 Caco-2 cells / mL were seeded onto Transwell culture plates and cultured for 18-25 days. The transmembrane electrical resistance (TEER) of the Caco-2 cell layer was measured using a resistance meter. When TEER > 500 Ω·cm 2 This indicates that the Caco-2 cells have fully differentiated and can be cultured a second time (i.e., co-culture).

[0047] When co-culturing Caco-2 and THP-1 cells, the culture medium needs to be adjusted to meet the growth requirements of both Caco-2 and THP-1 cells. This co-culture system integrates the absorption and transport carriers, metabolic enzyme systems, and physiologically active factors and enzymes of both cell types. Furthermore, the two cell lines can exchange extracellular secretions across the membrane using Transwell plates. While absorption is a primary consideration for bioavailability, ensuring the cell's biological activity in vivo is the most crucial factor. Therefore, a cell model that reflects both absorption and physiological activity is urgently needed for more comprehensive and in-depth research on bioavailability.

[0048] By adjusting the culture medium to achieve the co-growth of two cell types, more stable and reliable experimental conditions are provided for the research. A suitable culture medium can provide the nutrients, growth factors, and appropriate environmental conditions required for cell growth. Simultaneously, the interaction of the absorption and transport carriers and metabolic enzyme systems of the two cell types in the co-culture system helps to provide a more comprehensive understanding of the metabolic processes of the analyte in vivo. The presence of physiologically active factors and enzyme systems provides important indicators and evidence for studying the bioactivity of the analyte.

[0049] Specifically, in the second culture (i.e. co-culture), the culture medium for THP-1 cells was obtained by mixing EMEM medium and RPMI-1640 medium at a volume ratio of 2-4:1.

[0050] RPMI 1640 is a commonly used cell culture medium, originally developed by Moore et al. in 1966 at the Roswell Park Cancer Institute in the United States, hence its name. It contains reduced glutathione and high concentrations of vitamins, offering rich nutrients and unique effects, and is widely used for the in vitro culture of various mammalian cells. Furthermore, fetal bovine serum and penicillin-streptomycin antibiotics can be added to RPMI 1640 medium to promote better growth of THP-1 or Caco-2 cells. The concentration of fetal bovine serum can be 5-10% (v / v), and the concentration of penicillin-streptomycin antibiotics can be 0.5-1.5% (v / v).

[0051] At this point, the upper compartment of the Transwell containing Caco-2 cells still uses EMEM medium, which is adapted to the epithelial differentiation and barrier function of Caco-2 cells. EMEM medium is a classic dedicated medium for Caco-2 cells, and its core advantages are: it contains a high concentration of glucose, meeting the high metabolic needs of Caco-2 cells and supporting their proliferation, fusion, and differentiation into mature intestinal epithelial cells; the ratio of nutrients (such as amino acids and vitamins) matches the physiological metabolic characteristics of intestinal epithelial cells, which can induce Caco-2 cells to highly express brush border enzymes (such as alkaline phosphatase and sucrase) and absorption transport carriers (such as amino acid transporters), ensuring the integrity of its intestinal epithelial absorption and metabolic functions.

[0052] The culture medium in the lower compartment of the Transwell containing THP-1 cells is a mixed medium obtained by mixing EMEM and RPMI-1640 media at a volume ratio of 2-4:1, which is suitable for the immune activity of THP-1 cells. The classic culture medium for THP-1 cells is RPMI-1640, but when used alone, its nutrient composition cannot meet the needs of transmembrane signaling interaction with Caco-2 cells in the co-culture system; while the high glucose concentration and high osmotic pressure of EMEM medium alone can lead to abnormal activation or increased apoptosis rate of THP-1 cells. The mixed culture medium of the present invention achieves dual adaptation through optimization of the 2-4:1 ratio: it can retain the high nutrient density of EMEM medium to provide sufficient energy for THP-1 cells (adapting to the increased nutrient consumption caused by Caco-2 cell metabolism in co-culture); and it can introduce the core components of RPMI-1640 medium to maintain the normal morphology and immune function of THP-1 cells. If the mixing ratio is <2:1 (i.e., the proportion of RPMI-1640 is too high), the nutrient concentration will be insufficient, and the accumulation of Caco-2 cell metabolites will be too rapid, resulting in the suppression of THP-1 cell proliferation and a decrease in immune activity. If the mixing ratio is >4:1 (i.e., the proportion of EMEM is too high), high glucose and high osmotic pressure will induce premature differentiation or activation of THP-1 cells, leading to false positive inflammatory responses and interfering with the assessment of the immunomodulatory effects of the target components.

[0053] Furthermore, the Caco-2 cells were cultured in pure EMEM (high-nutrient) medium, while the underlying THP-1 cells were cultured in a mixed medium (medium-nutrient). This design creates a nutrient concentration gradient that precisely mimics the physiological gradient between the lumen side (high-nutrient, derived from digested food) and the lamina propria side (medium-nutrient, dependent on blood supply) in the human intestine. This gradient not only drives the nutrient transport function of Caco-2 cells but also stimulates THP-1 cells to maintain the physiological state of mucosal immune cells, further enhancing the physiological relevance of the model.

[0054] Preferably, the mixed culture medium can be obtained by mixing EMEM medium and RPMI-1640 medium in a volume ratio of 3:1, which can further enhance the physiological relevance of the model.

[0055] In the above technical solution, during the second culture, the seeding density of THP-1 cells is 0.5-1.5 × 10⁻⁶. 5 The concentration range ensures that THP-1 cells are evenly distributed in the lower chamber, preventing insufficient immune signal transmission due to excessively low cell density, and avoiding excessive cell accumulation, nutrient competition, and apoptosis due to excessively high density. This ensures the stability of immune cell function and provides a reliable basis for subsequent detection of immune-related indicators.

[0056] In the above technical solution, the second culture time is 3-5 days.

[0057] The second culture time is set at 3-5 days. Sufficient co-culture time can promote the establishment of a stable interaction between Caco-2 and THP-1 cells, simulate the long-term physiological state of the intestinal barrier, and avoid insufficient intercellular signaling due to short culture time, which would fail to truly reflect the long-term effects of functional components.

[0058] In some embodiments, after Caco-2 cells have fully differentiated, THP-1 cells in the logarithmic growth phase can be taken and the THP-1 cell ratio adjusted to 0.5-1.5 × 10⁻⁶ cells using a mixed culture medium (EMEM medium containing 15-25% fetal bovine serum and 0.5-1.5% penicillin-streptomycin and RPMI-1640 medium containing 5-15% fetal bovine serum and 0.5-1.5% penicillin-streptomycin at a volume ratio of 2-4:1). 5 Cells / mL were seeded into the lower chamber of a Transwell culture plate. Subsequently, the Transwell culture plate was placed in a constant temperature incubator at 37°C, 5% CO2, and 90% relative humidity for Caco-2 / THP-1 co-culture for 3-5 days to obtain a two-dimensional intestinal cell model.

[0059] In the above technical solution, the culture medium volume in the upper compartment of the Transwell culture plate is 0.5-1.5 mL; the culture medium volume in the lower compartment of the Transwell culture plate is also 0.5-1.5 mL. That is, Caco-2 cells in the logarithmic growth phase can be taken and adjusted to a Caco-2 cell ratio of 0.5-1.5 × 10⁻⁶ cells using 0.5-1.5 mL of EMEM medium. 5 THP-1 cells / mL can be seeded into the chamber of a Transwell culture plate; alternatively, THP-1 cells in logarithmic growth phase can be taken and adjusted to a THP-1 cell ratio of 0.5-1.5 × 10⁶ cells using 0.5-1.5 mL of mixed culture medium (a mixture of EMEM and RPMI-1640 medium at a volume ratio of 2-4:1). 5 Cells / mL were seeded into the lower chamber of a Transwell culture plate to further precisely control the construction of the intestinal cell model and make it closely resemble the real intestinal microenvironment.

[0060] Based on the above research, the second aspect of the present invention provides an intestinal cell model, which is prepared by the above preparation method.

[0061] The intestinal cell model of the present invention can be used as an in vitro intestinal simulation tool, solving the pain points of traditional intestinal models such as single function, insufficient physiological simulation or high cost, and is widely applicable to the needs of multiple scenarios such as food nutrition research and development, drug development, and intestinal disease research.

[0062] A third aspect of the present invention provides an intestinal cell model prepared by the above-described preparation method, or the application of the above-described intestinal cell model in evaluating the digestion, absorption, or bioavailability of drugs, nutrients, and other components.

[0063] For example, the traditional Caco-2 cell model can only assess the intestinal absorption efficiency of proteins, probiotics, and functional components (such as lactoferrin and oligosaccharides), but cannot assess their regulatory effect on intestinal immunity; while the intestinal cell model prepared by the above-mentioned preparation method of the present invention or the above-mentioned intestinal cell model can simultaneously analyze: (1) the absorption efficiency of nutrients; ② the immunomodulatory effect of nutrients on THP-1 cells; (3) the changes in the integrity of the intestinal epithelial barrier after immunomodulation; thus providing a direct basis for the evaluation of the dual efficacy of food in terms of nutritional absorption and physiological activity, and assisting in the research and development of functional foods and the verification of efficacy claims.

[0064] The intestinal cell model prepared by the above-mentioned preparation method of the present invention or the above-mentioned intestinal cell model can also simultaneously evaluate: (1) the intestinal absorption rate and bioavailability of the drug; (2) the activation of intestinal immune cells by the drug (such as whether it triggers an inflammatory response); (3) the changes in drug absorption and metabolism under inflammatory conditions (such as the effect of increased intestinal permeability caused by inflammation on drug absorption), providing more comprehensive experimental data for drug dosage form design, dosage optimization and safety assessment, reducing reliance on animal experiments and accelerating the research and development process.

[0065] Furthermore, the core pathological mechanisms of inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease) are closely related to epithelial barrier damage and immune disorders. The intestinal cell model prepared by the above-described preparation method of this invention, or the intestinal cell model described above, can simulate the abnormal interaction between intestinal epithelium and immune cells under inflammatory conditions, and can be used to study disease pathogenesis, screen potential therapeutic drugs, and provide a reliable in vitro model tool for biomedical research and development.

[0066] In one embodiment, the intestinal cell model prepared by the above-described method, or the intestinal cell model described above combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) and proteomics analysis, can be used to detect the absorption of milk powder. The results showed that after absorption in the intestinal cell model, milk powder triggered differential expression of numerous intestinal cell-related proteins, with predominantly upregulated expression. This suggests that the nutrients in the milk powder may be effectively absorbed by intestinal epithelial cells and exert their biological effects by regulating intracellular protein expression. Furthermore, by combining the intestinal cell model with the functional annotation of differentially regulated proteins, the functions of upregulated or downregulated proteins can be inferred, thereby analyzing the specific functions and mechanisms of action of the milk powder. These results demonstrate that the intestinal cell model of the present invention can realistically simulate the absorption process of milk powder in the intestinal tract and accurately capture the proteomics changes during this process. This verifies the reliability and practicality of the model in food nutrition evaluation and functional component screening, providing an efficient in vitro evaluation tool for subsequent research on the nutritional mechanisms and functional optimization of milk powder and similar dairy products.

[0067] In another embodiment, the intestinal cell model prepared by the above-described method, or the intestinal cell model combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) and based on peptidomics analysis, can be used to detect the absorption of milk powder. The results showed that commercially available milk powder, after absorption in the intestinal cell model, yielded multiple peptides. Differentially regulated peptides accounted for 24.4% of the total identified peptides, and the ratio of upregulated to downregulated peptides was balanced. This indicates that the absorption of milk powder peptides in intestinal epithelial cells is selective; some peptides are efficiently absorbed (upregulated) due to structural adaptation to intestinal transport proteins, while others may be further degraded or not absorbed in the intestine (downregulated), clarifying the intestinal absorption pattern of milk powder peptides. Furthermore, the sequence analysis of differentially regulated peptides using the intestinal cell model can infer the functions of upregulated and downregulated differentially regulated peptides, providing a direct basis for the screening and identification of functional peptides in milk powder. This can guide the research and development of high-value functional milk powders, demonstrating reliability in the evaluation of functional peptides in dairy products and providing an efficient tool for the in vitro screening of functional components in the field of food science.

[0068] The technical solution of this application will be further explained below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or obtained through public channels.

[0069] Example 1: Construction of a two-dimensional intestinal cell model

[0070] The Caco-2 cell culture method is as follows: Caco-2 cells are seeded in EMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin, and cultured in a constant temperature incubator at 37°C, 5% CO2, and 90% relative humidity. Fresh EMEM medium is replaced every other day. When the confluence of Caco-2 cells reaches about 80% under a microscope, Caco-2 cells are digested with 0.25% trypsin solution and passaged at a ratio of 1:3.

[0071] The THP-1 cell culture method is as follows: THP-1 cells are seeded in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a constant temperature incubator at 37℃, 5% CO2 and 90% relative humidity. The medium is replaced with fresh RPMI-1640 medium every 2-3 days. THP-1 cells are collected by centrifugation and passaged at a ratio of 1:3.

[0072] The two-dimensional intestinal cell model was constructed as follows: Caco-2 cells in the logarithmic growth phase were harvested and the cell ratio was adjusted to 1×10⁻⁵ cells using 0.5 mL of EMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. 5 Caco-2 cells / mL were seeded into the chamber of a 12-well Transwell plate and cultured for 21 days. The transmembrane electrical resistance (TEER) of the Caco-2 cell layer was measured using a resistance meter. When TEER > 500 Ω·cm 2 This indicates that the Caco-2 cells have fully differentiated. After the Caco-2 cells have fully differentiated, THP-1 cells in the logarithmic growth phase were taken and the THP-1 cell ratio was adjusted to 1.5 × 10⁻⁶ cells using 1.5 mL of mixed culture medium (EMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin and RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a volume ratio of 3:1). 5 Cells / mL were seeded into the lower chamber of a 12-well Transwell plate. The Transwell plate was then co-cultured with Caco-2 / THP-1 at 37°C, 5% CO2, and 90% relative humidity for 4 days to obtain a two-dimensional intestinal cell model, as shown in the schematic diagram. Figure 1 .

[0073] Example 2: Detection of TEER

[0074] Starting from the seeding of Caco-2 cells into the chambers of 12-well Transwell culture plates in Example 1, the TEER of Caco-2 cells was measured daily using a resistance meter. The results are shown below. Figure 2 .

[0075] Figure 2 The results showed that the monolayer of Caco-2 cells formed by single-cell culture had a relatively stable TEER. After 21 days of Caco-2 cell culture, the TEER increased to over 600, indicating that the Caco-2 cell layer was successfully formed. Subsequently, Caco-2 cells and THP-1 cells were co-cultured. The co-culture of Caco-2 / THP-1 cells led to a decrease in the TEER of the Caco-2 cell layer, indicating that the co-culture system regulated the functional state of the tight junctions between Caco-2 cells through the interaction of epithelial cells and immune cells, resulting in changes in the permeability of the intestinal epithelial barrier. This further illustrates that the two-dimensional intestinal cell model constructed in this invention is closer to the functional state of the in vivo intestinal physiological barrier.

[0076] Example 3: Analysis of the effect of milk powder in a two-dimensional intestinal cell model based on proteomics

[0077] This embodiment uses liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect the proteome of commercially available milk powder after in vitro simulated gastrointestinal digestion, and the proteome of commercially available milk powder after in vitro simulated gastrointestinal digestion and subsequent absorption simulated by a two-dimensional intestinal cell model. The results are as follows: Figure 3 and Figure 4 As shown, the specific steps are as follows: (1) Prepare the gastrointestinal simulation solution:

[0078] Commercially available adult gastrointestinal mimicry solutions include saliva mimicry, gastric juice mimicry, and intestinal juice mimicry, the composition of which is shown in Table 1. Commercially available infant gastrointestinal mimicry solutions include gastric juice mimicry and intestinal juice mimicry, the composition of which is shown in Table 2. Adult and infant gastrointestinal mimicry solutions were prepared according to the instructions and Tables 1 and 2. The pH of the gastrointestinal mimicry solutions was adjusted to physiologically relevant values ​​using 5M NaOH and 5M HCl solutions. Furthermore, given that Ca... 2+ Precipitation is prone to occur in electrolyte stock solutions, so CaCl2 is added separately to the simulation solution before the digestion and absorption experiment.

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] (2) Digestion experiment in the adult group:

[0084] A. Oral digestion: The saliva simulation solution in the adult gastrointestinal simulation solution was preheated at 37°C. 2 mL of commercially available adult milk powder solution (5 mg / mL) was mixed with the saliva simulation solution at a volume ratio of 1:1. The pH was adjusted to 7.0, and the mixture was stirred at 37°C and 50 rpm for 2 min to obtain the oral digestive solution.

[0085] B. Gastric digestion: Preheat the gastric juice simulation solution in the adult gastrointestinal simulation solution at 37°C. Take 2 mL of the above oral digestive solution and mix it with the gastric juice simulation solution (excluding CaCl2 solution and other components) at a volume ratio of 1:1. Adjust the pH to 2.0, add CaCl2 solution and pepsin solution, adjust the pH to 2.0 again, and mix and digest at 37°C and 300 rpm for 2 h to obtain gastric digestive solution.

[0086] C. Intestinal digestion: Preheat the intestinal fluid simulation solution in the adult gastrointestinal simulation solution at 37°C and adjust the pH to 7.0. Take 4 mL of the above gastric digestion solution and mix it with the intestinal fluid simulation solution (excluding CaCl2 solution and other components) at a volume ratio of 1:1. Then add 0.032 g of bile and place it at 37°C and stir at 300 rpm. After the bile is completely dissolved, add CaCl2 solution and pancreatic enzyme solution, adjust the pH to 7.0 again, and mix and digest at 37°C and 300 rpm for 2 h to obtain the intestinal digestion solution.

[0087] The intestinal digestive fluid was quickly placed in a boiling water bath (100℃) and heated for 10 minutes, then immediately transferred to a refrigerator at -80℃ for storage to obtain the adult digestive group.

[0088] (3) Infant digestion experiment:

[0089] A. Gastric digestion: Add 5 mL of 2 mg / mL commercially available infant formula solution to a 50 mL beaker, then add 2.4 mL of simulated gastric juice solution and 0.24 mL of pepsin solution. Adjust the pH to 4.4 with HCl, and add an appropriate amount of distilled water until the total volume of the system is 8 mL. Place the beaker in a magnetic stirrer at 37℃ and stir at 180 rpm for 2 h to obtain gastric digestive juice.

[0090] B. Intestinal digestion: Add 11.2 mL of intestinal fluid simulation solution (excluding CaCl2 solution), 0.24 mL of CaCl2 solution, 2.4 g of bile and 4 mL of pancreatic enzyme solution to the above gastric digestion fluid. Then add HCl to adjust the pH to 6.5. Add an appropriate amount of distilled water until the total volume of the system is 24 mL. Place the mixture in a magnetic stirrer at 37℃ (180 r / min) and react for 2 h to obtain intestinal digestion fluid.

[0091] The intestinal digestive fluid was quickly placed in a boiling water bath (100℃) and heated for 10 minutes, then immediately transferred to a refrigerator at -80℃ for storage to obtain the infant digestive group.

[0092] (4) Absorption experiments in adult and infant groups:

[0093] The above-mentioned adult digestive samples or infant digestive samples were thawed at 4°C; after thawing, they were filtered through a 0.22 μm sterile filter membrane to obtain the samples to be absorbed.

[0094] Discard the culture medium from the upper and lower chambers of the Transwell culture plate used in the two-dimensional intestinal cell model of Example 1. Add 0.5 mL of preheated DuPont phosphate-buffered saline (DPBS) to the upper chamber and 1.5 mL to the lower chamber, respectively. Gently shake the Transwell culture plate, discard the buffer, and repeat the washing process three times. Add 0.5 mL of the sample to be absorbed to the upper chamber of the Transwell culture plate, and add 1.5 mL of serum-free EMEM culture medium to the lower chamber. Incubate the Transwell culture plate in a constant temperature incubator for 6 h. After incubation, collect all the culture medium from the lower chamber of the Transwell culture plate and store it at -80℃ to obtain the adult absorption group and the infant absorption group.

[0095] in, Figure 3 This is a graph showing the protein count results for the adult digestive and absorption groups. Figure 4 This is a volcano plot of differentially expressed proteins between the adult digestive and absorption groups. It can be seen that a total of 1131 total proteins were identified in both groups in this embodiment. Differentially expressed proteins were screened based on a fold change (FC) > 1.5 and P < 0.05. A total of 746 differentially expressed proteins were found between the adult digestive and absorption groups, divided into 702 upregulated proteins and 44 downregulated proteins.

[0096] The above results indicate that: (1) After being absorbed in the two-dimensional intestinal cell model, milk powder caused a large number of differentially expressed intestinal cell-related proteins (differential proteins accounted for 65.9% of the total identified proteins), and the expression was mainly upregulated (upregulated proteins accounted for 94.1% of the differentially expressed proteins), suggesting that the nutrients in milk powder (such as whey protein, casein hydrolysate, bioactive peptides, etc.) may be effectively absorbed by intestinal epithelial cells and exert biological effects by regulating intracellular protein expression; (2) The two-dimensional intestinal cell model combined with the functional annotation of differentially expressed proteins can infer the related functions of upregulated or downregulated proteins, and then analyze the specific functions and mechanisms of action of milk powder; (3) It suggests that the Caco-2 / THP-1 two-dimensional intestinal cell model constructed in Example 1 can realistically simulate the absorption process of milk powder in the intestine in vivo, and can accurately capture the proteomics changes in the process, verifying the reliability and practicality of the model in food nutrition evaluation and functional component screening, and providing an efficient in vitro evaluation tool for subsequent nutritional mechanism research and functional optimization of milk powder and similar dairy products.

[0097] Example 4: Analysis of the effect of milk powder in a two-dimensional intestinal cell model based on peptidomics

[0098] This embodiment uses liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect the polypeptide composition of commercially available milk powder after in vitro simulated gastrointestinal digestion, and also after in vitro simulated gastrointestinal digestion and subsequent absorption using a two-dimensional intestinal cell model. The results are as follows: Figure 5 and Figure 6 As shown, the specific steps can be found in Example 3. Wherein, Figure 5 This is a graph showing the peptide count results for the adult digestive and absorption groups. Figure 6 This is a volcano plot of differentially expressed peptides between the adult digestive and absorption groups. It can be seen that a total of 197 peptides were identified in both groups in this embodiment. Differentially expressed peptides were screened using FC>1.5 and P<0.1 as criteria. A total of 48 differentially expressed peptides were found between the adult digestive and absorption groups, divided into 27 upregulated peptides and 21 downregulated peptides.

[0099] The above results indicate that: (1) when milk powder is digested in a simulated gastrointestinal fluid and absorbed in a two-dimensional intestinal cell model, 197 peptides are produced, suggesting that the two-dimensional intestinal cell model can simulate the absorption process of milk powder protein after hydrolysis in the intestine; (2) differentially expressed peptides account for 24.4% of the total identified peptides, and the ratio of upregulated to downregulated peptides is balanced, indicating that the absorption process of milk powder peptides in intestinal epithelial cells is selective. Some peptides are efficiently absorbed (upregulated) due to their structural adaptation to intestinal transport proteins, while some peptides may be further degraded or not absorbed in the intestine (downregulated), clarifying the intestinal absorption pattern of milk powder peptides; (3) the two-dimensional intestinal cell model combined with the sequence analysis of differentially expressed peptides, It can be inferred that the functions of upregulating and downregulating differential peptides are directly based on the screening and identification of functional peptides in milk powder, and can guide the research and development of high-value functional milk powder; (4) Compared with traditional in vitro digestion models (which only focus on peptide generation and cannot simulate the absorption process), the Caco-2 / THP-1 two-dimensional intestinal cell model constructed in this invention can simultaneously simulate the epithelial absorption of milk powder and the interaction between epithelial and immune cells. Combined with peptide omics technology, it can clarify the dynamic changes of milk powder peptides during the absorption process, verifying the specificity and reliability of this two-dimensional intestinal cell model in the evaluation of functional peptides in dairy products, and providing an efficient tool for the in vitro screening of functional components in the field of food science.

[0100] Example 5: Study on intestinal absorption of different milk powders based on a two-dimensional intestinal cell model

[0101] This embodiment uses a BCA reagent kit to detect the protein content of commercially available infant formula after in vitro simulated gastrointestinal digestion, and the protein content of commercially available infant formula after in vitro simulated gastrointestinal digestion and subsequent absorption using a two-dimensional intestinal cell model. The results are as follows: Figure 7 As shown (A and B represent different brands of commercially available infant formula), the specific steps can be found in Example 3. Figure 7 This chart shows the protein content results for the infant digestion group, infant absorption group, adult digestion group, and adult absorption group. Comparing the adult digestion and absorption groups reveals that both brands of commercially available infant formula (A and B) have high protein content after in vitro simulated gastrointestinal digestion. However, the amount of protein that can be absorbed by the two-dimensional intestinal cell model is significantly reduced, indicating that the protein composition of commercially available infant formulas A and B can be further optimized to improve the absorption and utilization rate of protein in the adult intestine. Comparing the infant digestion and absorption groups also reveals that both brands of commercially available infant formula (A and B) have high protein content after in vitro simulated gastrointestinal digestion. However, the amount of protein that can be absorbed by the two-dimensional intestinal cell model is reduced, indicating that the protein composition of commercially available infant formulas A and B can be further optimized to improve the absorption and utilization rate of protein in the infant intestine.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an intestinal cell model, characterized in that, Includes the following steps: Caco-2 cells were seeded into the upper chamber of a Transwell culture plate for the first culture, and then THP-1 cells were seeded into the lower chamber of the Transwell culture plate for the second culture, thus obtaining the intestinal cell model.

2. The preparation method according to claim 1, characterized in that, In the first culture, the culture medium for the Caco-2 cells is EMEM medium.

3. The preparation method according to claim 1 or 2, characterized in that, In the first culture, the seeding density of the Caco-2 cells was 0.5-1.5 × 10⁻⁶. 5 per mL.

4. The preparation method according to any one of claims 1-3, characterized in that, The endpoint of the first culture is a transmembrane resistance value of >500 Ω·cm for the Caco-2 cell layer composed of the Caco-2 cells. 2 .

5. The preparation method according to any one of claims 1-4, characterized in that, In the second culture, the culture medium for the THP-1 cells is obtained by mixing EMEM medium and RPMI-1640 medium at a volume ratio of 2-4:

1.

6. The preparation method according to any one of claims 1-5, characterized in that, In the second culture, the seeding density of the THP-1 cells was 0.5-1.5 × 10⁻⁶. 5 per mL.

7. The preparation method according to any one of claims 1-6, characterized in that, The second culture period is 3-5 days.

8. The preparation method according to any one of claims 1-7, characterized in that, The culture medium volume in the upper compartment of the Transwell culture plate is 0.5-1.5 mL; and / or, The culture medium volume in the lower compartment of the Transwell culture plate is 0.5-1.5 mL.

9. An intestinal cell model, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of an intestinal cell model prepared by the preparation method according to any one of claims 1-8 or the intestinal cell model according to claim 9 in evaluating the digestion, absorption or bioavailability of drugs, nutrients and other components.

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