A cell model simulating sensitive skin, a construction method and application thereof

Abstract

The application discloses a cell model simulating sensitive skin, a construction method and application, and belongs to the technical field of biotechnology. In view of the defects of the single existing sensitive skin in-vitro model evaluation system and the absence of a sensitive gradient, the application provides two sensitive skin cell models, namely a double-layer model and a three-layer model. The double-layer model is composed of an epidermis simulation unit based on human keratinocytes and an immune simulation unit based on human mast cells, and is connected through a porous membrane to realize epidermis-immune synergistic simulation. The three-layer model is additionally provided with a true epidermis connection simulation unit based on human fibroblasts, and can simulate different sensitive skin by adjusting the thickness and density of the unit. The application also discloses a construction method of the corresponding model and application in in-vitro evaluation of the soothing effect of a test substance on sensitive skin or in-vitro screening of sensitive skin soothing active substances. The application makes up for the defects of traditional models, and can be widely applied to the fields of sensitive skin mechanism research, soothing active ingredient screening and skin care product efficacy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the construction of cell models, specifically to a cell model simulating sensitive muscle, its construction method, and its application. Background Technology

[0002] Sensitive skin, also known as sensitive skin syndrome (SSS), is a common skin problem with a high global prevalence. Its core clinical features include: subjective discomfort such as stinging, burning, itching, and tightness after exposure to routine non-sensitizing physical, chemical, or psychological stimuli, which may be accompanied by objective signs such as erythema, dryness, and desquamation. Currently, the clinical diagnosis of SSS relies heavily on patients' subjective symptom descriptions, and a unified, objective, and quantifiable assessment standard is still lacking.

[0003] The pathogenesis of sensitive skin is not fully understood. Current research generally suggests that the core mechanism driving the development of sensitive skin involves the interaction and synergistic amplification of three factors: hyperresponsiveness of skin sensory nerves, impaired barrier function, and abnormal immune activation. Transient receptor potential vanillic acid subtype 1 (TRPV1, capsaicin receptor) is considered to play a key role in signal transduction in sensitive skin, and it is expressed not only in sensory neurons but also functionally in keratinocytes. Mast cells, as key effector cells in skin immunity, undergo degranulation upon stimulation, releasing inflammatory mediators such as histamine and trypsinoids, which can directly or indirectly activate sensory nerve endings and further activate TRPV1, significantly exacerbating symptoms such as itching, stinging, and burning.

[0004] Current in vitro screening and evaluation methods for ingredients with soothing effects on sensitive skin are mostly established independently around three core pathways: neurosensitivity, skin barrier damage, and immune inflammatory response, without forming a unified and integrated evaluation system. In the evaluation of neurosensitivity, conventional techniques mainly rely on sensory neuron action potential detection, TRPV1 channel expression and fluorescence calcium imaging, and neurotransmitter release assays, which can only reflect changes in nerve signal transmission and cannot link the barrier and immune status. In the evaluation of skin barrier function, external stimuli such as SDS, H2O2, and UVB are usually used to induce keratinocyte damage individually, and the barrier repair effect is judged by detecting changes in the expression of genes or proteins such as tight junction proteins (e.g., ZO-1, Occludin), filaggrin, and lipofuscin. These methods only focus on the single dimension of the epidermal barrier and ignore the regulatory role of immune cells on the barrier. In the evaluation of immune inflammation, in vitro experiments such as mast cell degranulation rate, trypsin release, histamine content measurement, or macrophage NO release and inflammatory factor secretion are commonly used, which can only reflect the level of immune cell activation and cannot reflect the driving effect of barrier structure and nerve perception on immunity. In addition, some studies have used three-dimensional (3D) reconstructed artificial skin models for efficacy evaluation. Although the structure is closer to the real shape of human skin, it is still mainly based on single tissue simulation and has failed to achieve synergistic simulation of nerve-barrier-immunity. Furthermore, it has not established an adjustable sensitivity gradient and is difficult to fully reproduce the real pathological process of sensitive skin.

[0005] In order to overcome the many shortcomings of existing sensitive muscle models, this invention is proposed. Summary of the Invention

[0006] The first objective of this invention is to provide a cell model that simulates human sensitive skin, aiming to overcome the technical deficiency of existing in vitro models that can only simulate a single pathway of nerve sensitivity, barrier damage, or immune inflammation, and cannot achieve the synergistic simulation of nerve-barrier-immune linkage; the second objective is to provide a method for constructing the above-mentioned cell model; and the third objective is to provide the application of the above-mentioned cell model in in vitro evaluation of the soothing effect of test substances on sensitive skin or in vitro screening of active substances that soothe sensitive skin.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A cell model simulating human sensitive skin, the cell model comprising:

[0009] Epidermal simulation unit, the epidermal simulation unit comprising human keratinocytes and their culture medium;

[0010] An immune simulation unit, wherein the immune simulation unit comprises human mast cells and their culture medium;

[0011] The epidermal simulation unit is located above the immune simulation unit. The two are connected by a porous membrane that allows the culture medium and its soluble substances to pass through but prevents cells from passing through, thus forming an epidermal-immune bilayer sensitive muscle simulation system.

[0012] The culture medium of the epidermal simulation unit contains a TRPV1 receptor agonist, and the culture medium of the immune simulation unit contains a mast cell degranulation inducer.

[0013] In one specific embodiment, the human keratinocytes used are human immortalized keratinocytes, but other human keratinocytes may also be used.

[0014] In one specific embodiment, the TRPV1 receptor agonist used is capsaicin, a specific agonist of the TRPV1 receptor that can specifically activate the TRPV1 channel, triggering downstream nerve-sensitive signal transduction and inflammatory cascade responses. Other TRPV1 receptor agonists can also achieve the technical effects of this invention.

[0015] In one specific embodiment, the mast cell degranulation inducer used is C48 / 80. Those skilled in the art know that C48 / 80 is a mast cell-specific degranulation inducer that can efficiently activate human mast cells and promote their degranulation, releasing inflammatory mediators such as histamine and trypsin. Other mast cell degranulation inducers can also achieve the technical effects of this invention.

[0016] Furthermore, the cell model also includes:

[0017] The dermal-epidermal junction simulation unit is a cell layer constructed from human fibroblast culture.

[0018] The dermal-epidermal junction simulation unit is located between the epidermal simulation unit and the porous membrane, forming a three-layer sensitive muscle simulation system of epidermis-connection-immunity.

[0019] Furthermore, the cell layers of varying thicknesses and / or densities, together with the epidermal simulation unit and the immune simulation unit, constitute a three-layer sensitive skin simulation system that mimics skin with different levels of sensitivity. The thickness and / or density of the dermal-epidermal junction simulation unit is adjustable, thereby regulating the signal exchange intensity between the epidermal simulation unit and the immune simulation unit to achieve sensitive skin simulation with different levels of sensitivity.

[0020] Furthermore, the epidermal simulation unit is located in the upper chamber of the dual-chamber co-culture device, and the immune simulation unit is located in the lower chamber of the dual-chamber co-culture device.

[0021] Furthermore, the pore size of the porous membrane is 0.4 μm to 1.0 μm.

[0022] In one specific embodiment, the dual-chamber co-culture device uses a Transwell chamber. However, this is merely a readily available, standardized dual-chamber co-culture device, and considering its role in this invention, the Transwell chamber is not the only option. The porous membrane between the upper and lower chambers of the Transwell chamber is a porous permeable membrane with uniform pore size (0.4 μm). This membrane only allows the culture medium and dissolved signaling molecules, cytokines, inflammatory mediators, and small molecule metabolites to pass freely, enabling signal exchange and pathway linkage between the epidermal and immune mimicry units. Simultaneously, it effectively prevents transmembrane mixing between cells in the upper and lower chambers, ensuring the independent structure and clear boundaries of each functional unit, thereby stably maintaining the synergistic mimicry state of epidermal-immune or epidermal-connection-immune. A pore size range of 0.4 μm to 1.0 μm balances permeability efficiency and cell isolation (most cells have a diameter of 1–100 μm; see page 004 of *Biophysics*). Figure 1-1 Spatial Scales in Life: From Angles to Hundreds of Meters (Editor-in-Chief: Shi Yigong, Publication Date: 2025) adapts to the biomimetic requirements of this invention for the pathological microenvironment of sensitive muscle, and the membrane possesses good cell adhesion and biocompatibility, without affecting normal cell growth, factor secretion, and subsequent indicator detection. Those skilled in the art, based on common knowledge and the role of porous membranes in this invention, may also select porous membranes with other pore sizes.

[0023] Furthermore, the porous membrane is selected from either polycarbonate membranes or polyester membranes. In one specific embodiment, the dual-chamber co-culture device uses a Transwell chamber, and the porous membrane between the upper and lower chambers of the Transwell chamber is a polycarbonate membrane or a polyester membrane. However, those skilled in the art should know that any porous membrane material that is biocompatible, has a suitable pore size range, supports cell adhesion growth, allows the culture medium and soluble substances therein to pass through, but prevents cell passage, can be used in this invention and is within the scope of protection of this invention. In addition to polycarbonate membranes and polyester membranes, other membrane materials with equivalent functions, such as polyethylene terephthalate membranes, cellulose membranes, and collagen-modified membranes, can be used equivalently as long as they meet the requirements of pore size (e.g., 0.4 μm~1.0 μm), structural stability, and no cytotoxicity. Their core function is to realize signal transmission and spatial separation between the upper and lower units of the porous membrane, and does not affect the overall construction of the model and the sensitive muscle simulation effect of this invention.

[0024] A method for constructing the above-mentioned cell model (including an epidermal mimicry unit and an immune mimicry unit) includes the following steps:

[0025] Human keratinocytes and human mast cells were cultured separately. Human keratinocytes and their culture medium were placed above a porous membrane to form an epidermal simulation unit, and human mast cells and their culture medium were placed below the porous membrane to form an immune simulation unit. A TRPV1 receptor agonist was added to the culture medium above the porous membrane, and a mast cell degranulation inducer was added to the culture medium below the porous membrane to construct an epidermal-immune bilayer sensitive muscle simulation system.

[0026] A method for constructing the above-mentioned cell model (including an epidermal simulation unit, a dermal-epidermal junction simulation unit, and an immune simulation unit) includes the following steps:

[0027] Human keratinocytes, human mast cells, and human fibroblasts were cultured separately. Human fibroblasts were cultured above a porous membrane to form a dermal-epidermal junction simulation unit. Human keratinocytes and their culture medium were placed above the dermal-epidermal junction simulation unit to form an epidermal simulation unit. Human mast cells and their culture medium were placed below the porous membrane to form an immune simulation unit. A TRPV1 receptor agonist was added to the culture medium above the porous membrane, and a mast cell degranulation inducer was added to the culture medium below the porous membrane. This constructed a barrier-connection-immunity three-layer sensitive muscle simulation system.

[0028] The aforementioned cell models (including epidermal and immune mimicry units) are used in the in vitro evaluation of the soothing effects of test substances on sensitive skin or in the in vitro screening of active substances for soothing sensitive skin. This application, based on a biomimetic pathological environment synergistically stimulating the epidermal-immune pathway, can realistically recreate the chain reaction of nerve hypersensitivity, immune activation, and barrier damage after stimulation of sensitive skin at the cellular level. It allows for a comprehensive, objective, and quantifiable assessment of the soothing mechanism, intensity, and repair effects of test substances, and is particularly suitable for the rapid screening and efficacy verification of cosmetic raw materials, active ingredients, compound compositions, and semi-finished skincare products.

[0029] Furthermore, the application includes the following steps:

[0030] (1) Model preparation

[0031] Human keratinocytes were seeded on the upper part of a porous membrane and incubated until the cells adhered and reached a predetermined degree of confluence. The cells were then divided into two groups: the model group was cultured in medium containing a TRPV1 receptor agonist, and the test substance or candidate substance group was cultured in medium containing both a TRPV1 receptor agonist and the test substance or candidate substance. Human mast cells were seeded on the lower part of the porous membrane, and both the model group and the test substance or candidate substance group were cultured in medium containing a mast cell degranulation inducer.

[0032] (2) Model incubation

[0033] The prepared model was incubated under standard cell culture conditions for a preset time.

[0034] (3) Testing and evaluation

[0035] After incubation, cells from each group were collected, and the expression levels of indicators related to skin barrier, immune inflammation, nerve sensitivity, inflammatory mediators, and vascular response were detected. The soothing effect of the test substance on sensitive skin was evaluated based on the changes in the indicators, or sensitive skin soothing active substances were screened.

[0036] Furthermore, the skin barrier-related indicators are ZO-1 and Occludin, the immune inflammation-related indicators are TSLP, IL-4, and IL-13, the nerve sensitivity-related indicator is TRPV1, and the inflammatory mediator and vascular response-related indicators are PTGES2 and VEGF.

[0037] The aforementioned cell model (including an epidermal mimicry unit, a dermal-epidermal junction mimicry unit, and an immunomimicry unit) is used in in vitro evaluation of the soothing effects of test substances on sensitive skin or in in vitro screening of soothing active substances for sensitive skin. This three-layered model, based on bilayer epidermal-immunoassay co-culture, introduces an adjustable dermal-epidermal junction mimicry unit, highly replicating the actual tissue structure and signal transduction pathways of human skin. It can simulate different sensitivity gradients by adjusting DEJ thickness / density, thus meeting the efficacy evaluation needs for mild, moderate to severe sensitivity levels. This three-layered model can comprehensively, objectively, and quantify the soothing mechanism, intensity, and repair effects of test substances for sensitive skin of different sensitivity levels (mild, moderate, and severe), and is particularly suitable for rapid screening and efficacy verification of cosmetic raw materials, active ingredients, compound compositions, and semi-finished skincare products in multi-gradient sensitivity scenarios.

[0038] Furthermore, the application includes the following steps:

[0039] (1) Model preparation

[0040] Human fibroblasts were seeded on top of a porous membrane and incubated until adherence was achieved and the predetermined time was reached. Human keratinocytes were seeded on top of human fibroblasts and incubated until cell adherence was achieved and the predetermined degree of confluence was reached. The cells were then divided into groups: the model group was cultured in medium containing a TRPV1 receptor agonist, and the test substance or candidate substance group was cultured in medium containing both a TRPV1 receptor agonist and the test substance or candidate substance. Human mast cells were seeded below the porous membrane, and both the model group and the test substance or candidate substance group were cultured in medium containing a mast cell degranulation inducer.

[0041] (2) Model incubation

[0042] The prepared model was incubated under standard cell culture conditions for a preset time.

[0043] (3) Testing and evaluation

[0044] After incubation, cells from each group were collected, and the expression levels of indicators related to skin barrier, immune inflammation, nerve sensitivity, inflammatory mediators, and vascular response were detected. The soothing effect of the test substance on sensitive skin was evaluated based on the changes in the indicators, or sensitive skin soothing active substances were screened.

[0045] Furthermore, the skin barrier-related indicators are ZO-1 and Occludin, the immune inflammation-related indicators are TSLP, IL-4 and IL-13, the nerve sensitivity-related indicator is TRPV1, and the inflammatory mediator and vascular response-related indicators are PTGES2 and VEGF.

[0046] Beneficial effects:

[0047] 1. This invention employs a two-dimensional co-culture system of keratinocytes and mast cells, achieving signal exchange through a porous membrane. It can simultaneously simulate the core pathological features of barrier damage and abnormal immune activation in sensitive skin, overcoming the shortcomings of traditional in vitro models that only evaluate neural, barrier, or immune pathways and cannot accurately reflect the synergistic amplification effects between pathways. This makes in vitro evaluation more closely resemble the actual pathological process in humans. Furthermore, by introducing fibroblasts into the system to simulate the dermal-epidermal junction microenvironment, the thickness and density of the dermal-epidermal junction (DEJ) can be altered by controlling the culture days, regulating the signal exchange intensity between the epidermis and immune units. This allows for precise simulation of high, medium, and low-level sensitivity states, meeting the needs of mechanism research and efficacy evaluation for sensitive skin at different levels.

[0048] 2. This invention's model is based on two-dimensional cell culture, featuring a short construction cycle, simple operation, and low experimental cost. It eliminates the need for complex three-dimensional tissue culture equipment, enabling batch parallel experiments. It is suitable for high-throughput screening of soothing active ingredients and skincare raw materials, effectively improving R&D efficiency. Furthermore, this invention integrates multiple key indicators across multiple dimensions, including neurosensitivity, inflammatory mediators and vascular responses, immune inflammation, and the skin barrier, establishing a comprehensive sensitivity scoring system. This represents a shift from single-indicator detection to overall quantitative scoring, resulting in stable, repeatable, and highly standardized evaluation results. This addresses the current industry problem of a lack of unified, objective, and quantitative standards for in vitro evaluation of sensitive skin.

[0049] 3. The model of this invention is closer to the physiological environment of human skin, which greatly improves the reliability of in vitro prediction of clinical effects. It can be widely used in the study of sensitive skin mechanisms, screening of soothing active ingredients, evaluation of skin care product efficacy and assessment of raw material safety, and has good scientific research value and industrial application prospects. Attached Figure Description

[0050] Figure 1 A schematic diagram illustrating the construction and drug delivery of a bilayer sensitive muscle cell model;

[0051] Figure 2 A schematic diagram illustrating the construction and drug delivery of a three-layered sensitive muscle cell model;

[0052] Figure 3 HE staining results for DEJ simulation layers of different thicknesses (200x magnification, scale bar 100μm). Detailed Implementation

[0053] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0054] Example 1: Construction and Application of a Double-Layer Sensitive Muscle Cell Model

[0055] 1. Cell Culture

[0056] (1) Resuscitation and treatment of immortalized human keratinocytes

[0057] Immortalized human keratinocytes were rapidly thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium containing 10% FBS and 1% antibiotics. The cells were then transferred to cell culture dishes and cultured at 37°C in a 5% CO2 incubator until confluence. After confluence, the complete medium was discarded, the cells were washed twice with PBS, and trypsin solution containing 0.25% EDTA was added. Digestion was performed at 37°C in a 5% CO2 incubator for 7 min, followed by the addition of complete medium to terminate digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in DMEM complete medium, counted, and the cell density was adjusted for later use.

[0058] (2) Human mast cell resuscitation and treatment

[0059] Frozen human mast cells were rapidly thawed in a 37°C water bath, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in IMDM complete medium containing 10% FBS and 1% penicillin-dextrose antibody. The cells were then transferred to a cell culture flask and cultured until confluent. An appropriate amount of cell suspension was taken, centrifuged at 800 rpm for 5 min, the supernatant was discarded, the cells were resuspended in IMDM basal medium, and the cells were counted and the cell density was adjusted for later use.

[0060] 2. Experimental Grouping and Reagent Preparation

[0061] Two-chamber co-culture was performed using 12-well Transwell plates, divided into: a blank control group, a model group, a dexamethasone positive control group, and a soothing sample group. The blank control group was incubated in DMEM basal medium; the other groups were prepared with DMEM basal medium to form their respective drug delivery systems: the model group contained 50 μM / L capsaicin (a specific agonist of the TRPV1 receptor); the dexamethasone positive control group contained 50 μM / L capsaicin + 6 μg / mL dexamethasone; and the soothing sample group contained 50 μM / L capsaicin + the corresponding concentration of soothing sample (50 μg / mL α-bisabolol, 8 μg / mL asiaticoside, or 10 μg / mL tert-butylcyclohexanol).

[0062] 3. Model construction and drug administration (see schematic diagram) Figure 1 (As shown)

[0063] (1) Upper chamber lining and drug administration

[0064] Take the above-mentioned human immortalized keratinocyte suspension and evenly seed it into the upper chamber of Transwell, 0.5 mL per well, with a cell volume of 12-15 w / well. Incubate in a 37℃, 5% CO2 incubator until the cells adhere to the wall and the degree of confluence reaches 60%-70%. Discard the original culture medium in the upper chamber and add the corresponding culture medium according to the group, 0.5 mL per well.

[0065] (2) Lower chamber slab and induction

[0066] 1.5 mL of human mast cell suspension was added to the lower chamber of the blank control group; 1.5 mL of human mast cell suspension containing 10 μg / mL C48 / 80 (mast cell-specific degranulation inducer) was added to the lower chamber of the model group, dexamethasone positive drug group, and soothing sample group to induce mast cell degranulation and activate immune response.

[0067] The ratio of immortalized human keratinocytes in the upper chamber to human mast cells in the lower chamber was 2:1 to 3:1.

[0068] 4. Model incubation

[0069] The Transwell co-culture system, after model construction and drug administration, was incubated at 37°C and 5% CO2 for 12-24 hours to allow barrier damage and immune activation signals to fully interact and form a stable state of sensitive muscle simulation.

[0070] 5. Indicator Testing and Result Evaluation

[0071] After incubation, total RNA was collected from each group of cells. Using β-actin as an internal reference gene, RT-qPCR was used to detect the relative mRNA expression levels of several key indicators, including nerve sensitivity-related markers (TRPV1), inflammatory mediators and vascular response-related markers (PTGES2, VEGF), immune inflammatory markers (TSLP, IL-4, IL-13), and barrier function markers (ZO-1, Occludin). The fold increase in expression of each target gene was calculated using formula (Formula 1), and the CSS value was calculated by substituting the weights assigned to each indicator (Table 1) into the comprehensive sensitivity scoring formula (Formula 2). The CSS value for the model group was set to 100, and for the blank control group, it was 0. Lower CSS values ​​in other groups indicated better soothing effects of the corresponding components on sensitive skin.

[0072] Formula 1, Formula 2 and Table 1 are as follows.

[0073] Formula 1:

[0074]

[0075] in:

[0076] n: indicates the fold increase in the expression of the target gene;

[0077] C: Represents the CT value of the target gene in the blank control group / model group / dexamethasone positive drug group / soothing sample group;

[0078] C avg : Represents the average CT value of the internal reference gene in the blank control group / model group / dexamethasone positive drug group / soothing sample group;

[0079] C k : Represents the CT value of the target gene in the blank control group;

[0080] C kavg : Indicates the average CT value of the internal reference gene in the blank control group.

[0081] Formula 2:

[0082]

[0083] Among them: blank control group, model group, dexamethasone positive drug group, and soothing sample group.

[0084] CSS (Comprehensive Sensitivity Score): Represents the sensitivity index corresponding to a group;

[0085] W i : Indicates the weight corresponding to the i-th indicator;

[0086] V: represents the average fold increase in the expression of the target gene in the blank control group / model group / dexamethasone positive drug group / soothing sample group;

[0087] V k : Indicates the average fold increase in the expression of the target gene in the blank control group;

[0088] V m : Indicates the average fold increase in expression of the target gene in the model group.

[0089] Table 1 Weights of Each Indicator

[0090]

[0091] The test results and CSS values ​​for each group of indicators are shown in Tables 2, 3 (continued Table 2), and 4 (continued Table 2).

[0092] Table 2. Detection results and CSS values ​​for each group of indicators.

[0093]

[0094] Table 3 (Continued from Table 2) Detection results and CSS values ​​for each group of indicators

[0095]

[0096] Table 4 (Continued from Table 2) Detection results and CSS values ​​for each group of indicators

[0097]

[0098] Compared with the blank control group, the expression levels of nerve sensitivity-related indicators (TRPV1), inflammatory mediators and vascular response-related indicators (PTGES2, VEGF), and immune inflammation-related indicators (TSLP, IL-4, IL-13) were significantly increased in the model group cells, while the expression levels of skin barrier function-related indicators (ZO-1, Occludin) were significantly decreased. The overall expression trend was highly consistent with the pathophysiological changes of sensitive skin under external stimulation. Compared with the model group, the expression levels of nerve sensitivity-related indicators, inflammatory mediators and vascular response-related indicators, and immune inflammation-related indicators in the dexamethasone positive drug group and each soothing sample group cells were significantly reduced, while the expression levels of skin barrier function-related indicators were significantly increased, showing a clear soothing intervention effect. It is well known to those skilled in the art that dexamethasone is a commonly used anti-inflammatory glucocorticoid in clinical practice, which can broadly inhibit inflammatory responses and immune activation; α-bisabolol, asiaticoside, and tert-butylcyclohexanol are all active substances that have been proven to have soothing, anti-inflammatory, and barrier-protective effects on sensitive skin.

[0099] In summary, the bilayer sensitive muscle cell model constructed in this embodiment can effectively reproduce the synergistic changes in the neural-immune-barrier pathway of sensitive muscle under stimulation conditions. It can effectively distinguish whether the model is successfully established, the intervention effects of positive drugs and different soothing active substances, and can serve as a reliable model for evaluating the soothing efficacy of test substances in vitro and screening soothing active ingredients for sensitive muscle.

[0100] Example 2: Three-layer sensitive muscle cell model, construction and application examples

[0101] This embodiment was completed in the same batch of experiments as Embodiment 1, as detailed below.

[0102] 1. Cell Culture

[0103] (1) Resuscitation and treatment of immortalized human keratinocytes

[0104] Same as Example 1.

[0105] (2) Resuscitation and treatment of human fibroblasts

[0106] Frozen human fibroblasts were rapidly thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium containing 10% FBS and 1% antibiotics. The cells were then transferred to cell culture dishes and cultured at 37°C in a 5% CO2 incubator until confluence. After confluence, the complete medium was discarded, the cells were washed twice with PBS, and trypsin solution containing 0.25% EDTA was added. Digestion was performed at 37°C in a 5% CO2 incubator for 7 min, followed by the addition of complete medium to terminate digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in DMEM complete medium, counted, and the cell density was adjusted for later use.

[0107] (3) Human mast cell resuscitation and treatment

[0108] Same as Example 1.

[0109] 2. Experimental Grouping and Reagent Preparation

[0110] Two-chamber co-culture was performed using 12-well Transwell plates, divided into: a blank control group (same as in Example 1), a DEJ-free model group (same as the model group in Example 1), a thin DEJ model group, and a thick DEJ model group. Drug administration systems for each group were prepared using DMEM basal medium: the DEJ-free model group, the thin DEJ model group, and the thick DEJ model group all contained 50 μM / L capsaicin.

[0111] 3. Model construction and drug administration (see schematic diagram) Figure 2 (As shown)

[0112] (1) Upper chamber lining and drug administration

[0113] Human fibroblasts were seeded at a density of 20-25w / well in the upper chamber of a Transwell chamber, and DMEM complete medium containing 180-200mM ascorbic acid was added. The chambers were then incubated at 37°C in a 5% CO2 incubator with the medium changed every two days. The cells were cultured for 7 or 14 days according to the target sensitivity gradient to form DEJ-like layers of different thicknesses. Figure 3 (HE staining results for DEJ simulation layers of different thicknesses). Human immortalized keratinocyte suspension was added above the cultured fibroblast layer at a concentration of 12-15 weeks / well, 0.5 mL per well. Incubation continued until the human immortalized keratinocytes adhered and achieved 60-70% confluence. The original culture medium in the upper chamber was discarded, and the corresponding drug-treated culture medium was added to each well according to the group, 0.5 mL per well.

[0114] In this step, the operation of the blank control group and the DEJ-free model group (corresponding to the model group in Example 1) is the same as in Example 1; the DEJ thin model group and the DEJ thick model group are inoculated with cultured human fibroblasts in advance to ensure that the addition of human immortalized keratinocytes is synchronized with the blank control group and the DEJ-free model group.

[0115] (2) Lower chamber slab and induction

[0116] In the blank control group, 1.5 mL of human mast cell suspension was added to the lower chamber; in the DEJ-free model group, the DEJ thin model group, and the DEJ thick model group, 1.5 mL of human mast cell suspension containing 10 μg / mL C48 / 80 was added to the lower chamber to induce mast cell degranulation and activate the immune response.

[0117] The ratio of immortalized human keratinocytes in the upper chamber to human mast cells in the lower chamber was 2:1 to 3:1.

[0118] 4. Model incubation

[0119] The Transwell co-culture system, after model construction and drug administration, was incubated at 37°C and 5% CO2 for 12-24 hours to allow for full interaction of cell signals in each layer and to form a stable state of sensitive muscle simulation.

[0120] 5. Indicator Testing and Result Evaluation

[0121] Same as Example 1. The CSS value of the no-DEJ model group was set to 100, and the blank control group was 0. Different thicknesses of the DEJ simulation layer correspond to different CSS values. The thicker the DEJ simulation layer, the lower the CSS value, and the weaker the sensitivity of the simulated sensitive skin; conversely, the thinner the DEJ simulation layer, the higher the CSS value, and the stronger the sensitivity of the simulated sensitive skin.

[0122] The test results and CSS values ​​for each group of indicators are shown in Tables 5, 6 (continued from Table 5), and 7 (continued from Table 5).

[0123] Table 5. Detection results and CSS values ​​for each group of indicators.

[0124]

[0125] Table 6 (Continued from Table 5) Detection results and CSS values ​​for each group of indicators

[0126]

[0127] Table 7 (Continued from Table 5) Detection results and CSS values ​​for each group of indicators

[0128]

[0129] Compared with the blank control group, the expression levels of nerve sensitivity-related indicators (TRPV1), inflammatory mediators and vascular response-related indicators (PTGES2, VEGF), and immune inflammation-related indicators (TSLP, IL-4, IL-13) were significantly increased in the DEJ-free model group, while the expression levels of skin barrier function-related indicators (ZO-1, Occludin) were significantly decreased. The overall expression trend was highly consistent with the pathophysiological changes of sensitive skin under external stimulation. Compared with the DEJ-free model group, the expression levels of nerve sensitivity-related indicators, inflammatory mediators and vascular response-related indicators, and immune inflammation-related indicators were significantly decreased in the DEJ thin model group and the DEJ thick model group, while the expression levels of skin barrier function-related indicators were significantly increased, and the sensitivity was significantly reduced. The thicker the DEJ simulation layer, the lower the sensitivity of the sensitive skin model it represents.

[0130] In summary, the three-layer sensitive skin cell model constructed in this embodiment can flexibly adjust the thickness of the DEJ simulation layer by regulating the fibroblast culture time, thereby controlling the signal transmission intensity between the epidermal unit and the immune unit. It successfully constructs an in vitro simulation system of sensitive skin with different sensitivity gradients. The model structure is closer to the real physiological microenvironment of human skin and can effectively simulate skin states with different levels of sensitivity from mild to severe. It can provide a standardized and quantifiable experimental platform for screening effective ingredients, elucidating mechanisms of action, and evaluating soothing effects under different sensitivity levels.

[0131] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A cellular model simulating human sensitive skin, characterized in that, This cell model includes: Epidermal simulation unit, the epidermal simulation unit comprising human keratinocytes and their culture medium; An immune simulation unit, wherein the immune simulation unit comprises human mast cells and their culture medium; The epidermal simulation unit is located above the immune simulation unit. The two are connected by a porous membrane that allows the culture medium and its soluble substances to pass through but prevents cells from passing through, thus forming an epidermal-immune bilayer sensitive muscle simulation system. The culture medium of the epidermal simulation unit contains a TRPV1 receptor agonist, and the culture medium of the immune simulation unit contains a mast cell degranulation inducer.

2. The cell model according to claim 1, characterized in that, The cell model also includes: The dermal-epidermal junction simulation unit is a cell layer constructed from human fibroblast culture. The dermal-epidermal junction simulation unit is located between the epidermal simulation unit and the porous membrane, forming a three-layer sensitive muscle simulation system of epidermis-connection-immunity.

3. The cell model according to claim 2, characterized in that: The cell layers of different thicknesses and / or different densities, together with the epidermal simulation unit and the immune simulation unit, constitute a three-layer sensitive skin simulation system that simulates skin with different sensitivities.

4. The cell model according to any one of claims 1 to 3, characterized in that: The epidermal simulation unit is located in the upper chamber of the dual-chamber co-culture device, and the immune simulation unit is located in the lower chamber of the dual-chamber co-culture device.

5. The cell model according to any one of claims 1 to 3, characterized in that: The porous membrane has a pore size of 0.4 μm to 1.0 μm.

6. The cell model according to claim 5, characterized in that: The porous membrane is selected from either polycarbonate membrane or polyester membrane.

7. A method for constructing the cell model according to claim 1, characterized in that, Includes the following steps: Human keratinocytes and human mast cells were cultured separately. Human keratinocytes and their culture medium were placed above a porous membrane to form an epidermal simulation unit, and human mast cells and their culture medium were placed below the porous membrane to form an immune simulation unit. A TRPV1 receptor agonist was added to the culture medium above the porous membrane, and a mast cell degranulation inducer was added to the culture medium below the porous membrane to construct an epidermal-immune bilayer sensitive muscle simulation system.

8. A method for constructing the cell model according to claim 2, characterized in that, Includes the following steps: Human keratinocytes, human mast cells, and human fibroblasts were cultured separately. Human fibroblasts were cultured above a porous membrane to form a dermal-epidermal junction simulation unit. Human keratinocytes and their culture medium were placed above the dermal-epidermal junction simulation unit to form an epidermal simulation unit. Human mast cells and their culture medium were placed below the porous membrane to form an immune simulation unit. A TRPV1 receptor agonist was added to the culture medium above the porous membrane, and a mast cell degranulation inducer was added to the culture medium below the porous membrane. This constructed a barrier-connection-immunity three-layer sensitive muscle simulation system.

9. The application of the cell model described in claim 1 in in vitro evaluation of the soothing effect of the test substance on sensitive skin or in vitro screening of active substances for soothing sensitive skin.

10. The application according to claim 9, characterized in that, Includes the following steps: (1) Model preparation Human keratinocytes were seeded on the upper part of a porous membrane and incubated until the cells adhered and reached a predetermined degree of confluence. The cells were then divided into two groups: the model group was cultured in medium containing a TRPV1 receptor agonist, and the test substance or candidate substance group was cultured in medium containing both a TRPV1 receptor agonist and the test substance or candidate substance. Human mast cells were seeded on the lower part of the porous membrane, and both the model group and the test substance or candidate substance group were cultured in medium containing a mast cell degranulation inducer. (2) Model incubation The prepared model was incubated under standard cell culture conditions for a preset time. (3) Testing and evaluation After incubation, cells from each group were collected, and the expression levels of indicators related to skin barrier, immune inflammation, nerve sensitivity, inflammatory mediators, and vascular response were detected. The soothing effect of the test substance on sensitive skin was evaluated based on the changes in the indicators, or sensitive skin soothing active substances were screened.

11. The application according to claim 10, characterized in that: The skin barrier-related indicators were ZO-1 and Occludin; the immune inflammation-related indicators were TSLP, IL-4 and IL-13; the nerve sensitivity-related indicator was TRPV1; and the inflammatory mediator and vascular response-related indicators were PTGES2 and VEGF.

12. The use of the cell model according to claim 2 or 3 in in vitro evaluation of the soothing effect of the test substance on sensitive skin or in vitro screening of active substances for soothing sensitive skin.

13. The application according to claim 12, characterized in that, Includes the following steps: (1) Model preparation Human fibroblasts were seeded on top of a porous membrane and incubated until adherence was achieved and the predetermined time was reached. Human keratinocytes were seeded on top of human fibroblasts and incubated until cell adherence was achieved and the predetermined degree of confluence was reached. The cells were then divided into groups: the model group was cultured in medium containing a TRPV1 receptor agonist, and the test substance or candidate substance group was cultured in medium containing both a TRPV1 receptor agonist and the test substance or candidate substance. Human mast cells were seeded below the porous membrane, and both the model group and the test substance or candidate substance group were cultured in medium containing a mast cell degranulation inducer. (2) Model incubation The prepared model was incubated under standard cell culture conditions for a preset time. (3) Testing and evaluation After incubation, cells from each group were collected, and the expression levels of indicators related to skin barrier, immune inflammation, nerve sensitivity, inflammatory mediators, and vascular response were detected. The soothing effect of the test substance on sensitive skin was evaluated based on the changes in the indicators, or sensitive skin soothing active substances were screened.

14. The application according to claim 13, characterized in that: The skin barrier-related indicators were ZO-1 and Occludin; the immune inflammation-related indicators were TSLP, IL-4 and IL-13; the nerve sensitivity-related indicator was TRPV1; and the inflammatory mediator and vascular response-related indicators were PTGES2 and VEGF.

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