Immune cell-based raw material screening model and its application in screening raw materials for sensitive skin

Abstract

The present invention provides a raw material screening model based on immune cells and its use in screening sensitive skin raw materials, belonging to the field of skin biology technology. The raw material screening model is a mast cell RBL-2H3 cell line and an epidermal model Episkin TM The present invention innovatively co-cultivates immune cells with a 3D epidermal model to simulate sensitive skin caused by a sensitive immune system in a real environment, thereby addressing the lack of an effective model for screening sensitive skin in the market. The present invention can evaluate whether the test substance is suitable for sensitive skin from an immunological perspective. The method of the present invention can replace living animal and human skin and be directly used to screen chemicals, cosmetics, pharmaceuticals, etc. for sensitive skin.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin biology, and in particular relates to an immune cell-based raw material screening model and its application in screening sensitive skin raw materials. Background Art

[0002] Sensitive skin (SS) refers specifically to a highly reactive state of the skin under physiological or pathological conditions, mainly occurring on the face. Clinically, the skin is prone to subjective symptoms such as burning, stinging, itching, and tightness when stimulated by physical, chemical, or mental factors, with or without objective signs such as erythema, scaling, and capillary dilation.

[0003] The main characteristic of sensitive skin is a series of unpleasant subjective sensations (such as stinging, burning, pain, itching, tingling, etc.) induced by usually harmless endogenous or exogenous factors, emphasizing subjectivity and responsiveness. Sensitive skin may affect the skin all over the body, but it is particularly common on the face. The appearance of the affected skin may be normal or accompanied by objective symptoms such as erythema. Although the definition points out that the symptoms of sensitive skin cannot be fully explained by the lesions of other skin diseases, many skin diseases may be accompanied by symptoms of sensitive skin, and the pathogenesis is somewhat related. Sensitive skin can be considered as a skin manifestation of inflammatory skin diseases or systemic diseases.

[0004] There are many reasons for sensitive skin, including race, gender, skin type, and the physiological health of the skin. For example, generally speaking, women are more sensitive to skin irritation than men. This may be because women's skin has a higher pH value and a poorer buffering capacity for irritation. Reed et al. conducted research based on the 6-type skin classification method and believed that skin barrier function is related to skin type, but not to race or gender. Current research believes that the occurrence of sensitive skin is a complex process involving the skin barrier, neurovascular system, and immune inflammation. Under the interaction of internal and external factors, the skin barrier function is damaged, causing an increase in sensory nerve afferent signals, resulting in increased skin reactivity to external stimuli and triggering a skin immune inflammatory response.

[0005] Numerous in vitro models, such as 2D / 3D cell culture, reconstructed human epidermis, reconstructed full-thickness human skin, ex vivo skin biopsies, and skin chips, have been proposed for screening active ingredients in cosmetics. Due to restrictions on animal use, the use of animal models for verifying the safety and efficacy of personal care products is avoided. Single-cell lineage-based systems lack intercellular communication with other cell types, and the lack of a multilayered epidermis in 2D culture systems limits the testing of skin barrier function. In vitro reconstructed epidermis, composed of human keratinocytes, is a three-dimensional reconstructed model with a structure and function similar to native human skin and is widely used in dermatological research and irritation assessment. It combines the physiological properties of skin, including its barrier function, three-dimensional structure, and immune response, in a single system. However, it is impossible to generate a true immune response in epidermal models. Ex vivo skin biopsies can reveal the essential components of skin, but the variability and limitations of skin donors are major issues and limitations. Consequently, few established models of sensitive skin exist. Summary of the Invention

[0006] In view of the above shortcomings, the present invention provides a raw material screening model based on immune cells and its application in screening sensitive muscle raw materials. The raw material screening model is a mast cell RBL-2H3 cell line and an epidermal model Episkin TM The present invention innovatively co-cultivates immune cells with a 3D epidermal model to simulate sensitive skin caused by a sensitive immune system in a real environment, thereby addressing the lack of an effective model for screening sensitive skin in the market. The present invention can evaluate whether the test substance is suitable for sensitive skin from an immunological perspective. The method of the present invention can replace living animal and human skin and be directly used to screen chemicals, cosmetics, pharmaceuticals, etc. for sensitive skin.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a raw material screening model based on immune cells, wherein the raw material screening model is a co-culture model of mast cells and epidermal models.

[0009] Specifically, the mast cells include primary isolated mast cells or mast cell lines.

[0010] More specifically, the mast cells include but are not limited to RBL-2H3 cell line, Ku812 cell line, P815 cell line, and LAD2 cell line.

[0011] Preferably, the mast cells are RBL-2H3 cell line.

[0012] Specifically, the epidermal model includes a self-built epidermal model or a purchased commercial epidermal model.

[0013] More specifically, the epidermal model includes but is not limited to Altskin or Episkin.

[0014] Preferably, the epidermal model is Episkin TM .

[0015] Specifically, the ratio of the number of mast cells to the surface area of ​​the epidermal model is 1x10 5 -1x10 7 cell:1-1.5cm 2 .

[0016] Preferably, the ratio of the number of mast cells to the surface area of ​​the epidermal model is 1x10 6 cell:1-1.07cm 2 Epidermal model.

[0017] Preferably, the co-culture method is an indirect contact culture based on an insert-type cell culture vessel.

[0018] On the other hand, the present invention provides a method for preparing the above-mentioned raw material screening model, wherein the preparation method

[0019] The method comprises co-culturing mast cells and the epidermal model for 1-2 hours.

[0020] Preferably, the mast cells are RBL-2H3 cell line; the epidermal model is Episkin TM .

[0021] Specifically, the mast cell culture method is as follows: RBL-2H3 cells are cultured at a rate of 1×10 5 -1x10 6 The cells / well were inoculated into 12 wells containing culture medium and cultured in an incubator for 18-24 hours.

[0022] Preferably, the RBL-2H3 cells are grown at 4×10 5 cells / well were seeded in 12 wells containing culture medium.

[0023] Preferably, the culture medium is DMEM culture medium containing 10% fetal bovine serum.

[0024] Specifically, the culture method of the epidermal model is: TM Transfer to

[0025] The culture well plate containing culture medium was placed in an incubator for overnight culture.

[0026] Preferably, the maintenance culture medium is a serum-free culture medium provided with the epidermal model.

[0027] Preferably, the preparation method comprises: forming an epidermal model (area 1.07 cm 2 ) and mast cells (1x10 6 cell) for 1 h to obtain the culture medium of the co-culture system.

[0028] Specifically, the co-culture conditions are a temperature of 36-38° C., a humidity of 85%-95%, and a CO 2 of 4%-6%.

[0029] Preferably, the co-cultivation culture conditions are a temperature of 37° C., a humidity of 90%, and a CO 2 concentration of 5%.

[0030] In another aspect, the present invention provides an application of the above-mentioned raw material screening model in screening raw materials suitable for sensitive skin.

[0031] In another aspect, the present invention provides a method for screening raw materials suitable for sensitive skin, wherein the method comprises using the above-mentioned raw material screening method. Specifically, the screening method comprises the following steps:

[0032] S1. Add the sample to be tested into the raw material screening model and culture it;

[0033] S2, remove the sample solution to be tested, add mast cell activator, and incubate;

[0034] S3, replace with fresh culture medium and continue culturing;

[0035] S4. The culture medium was centrifuged and the supernatant was collected for detection of sensitive muscle inflammatory factors;

[0036] S5. Take the epidermal model and perform pathological examination.

[0037] Preferably, the test sample is added to the culture medium in the bottom chamber in step S1.

[0038] The time is 10-15h.

[0039] Further preferably, the test sample in step S1 is added to the culture medium in the bottom chamber, and the culture time is 12 hours.

[0040] Preferably, the mast cell activator in step S2 is C48 / 80.

[0041] Further preferably, the concentration of the mast cell activator in step S2 is 5-15 μg / mL, and the incubation time is 1-3 h.

[0042] Still more preferably, the concentration of the mast cell activator in step S2 is 10 μg / mL, and the incubation time is 2 h.

[0043] Preferably, the culture duration of the continued culture in step S3 is 24-36 hours.

[0044] Further preferably, the culturing time of the continued culturing in step S3 is 24 hours.

[0045] Preferably, the sensitive muscle inflammatory factor detection in step S4 includes detecting factors related to red blood vessels, itching, redness, edema, and nervous tension.

[0046] Further preferably, the detection of sensitive muscle inflammatory factors in step S4 includes but is not limited to the detection of VEGF, histamine, sICAM-1, TNF-α, and Neurotensin.

[0047] Preferably, the pathological test in step S5 includes but is not limited to epidermal thickness detection, epidermal hyperplasia detection, and epidermal differentiation detection.

[0048] Preferably, the sensitive muscle inflammatory factor detection described in step S4 or the pathological detection laboratory group described in step S5 has statistical differences with the control group, and the detection parameters are fully mutually verified, indicating that the raw material has the possible effect of promoting or inhibiting sensitive muscle.

[0049] The beneficial effects of the present invention are:

[0050] (1) The present invention provides an in vitro model based on the co-culture of immune cells with a 3D epidermal model. This method can be widely used to screen ingredients suitable for sensitive skin. The model is simple to operate and provides intuitive results, making it an ideal model for predicting whether an ingredient is suitable for sensitive skin.

[0051] (2) The present invention uses mast cells co-cultured with an epidermal model (Episkin) and uses an ELISA kit to measure the inflammatory factor TNF-α, the redness-related factors VEGF and sICAM-1, and the itch-sensitization-related factor histamine to screen whether the raw materials are suitable for sensitive skin. At the same time, tissue sections of the epidermal model are taken to observe whether the test substance causes structural abnormalities in the epidermal model. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the co-culture model.

[0053] Figure 2Comparison of TNF-α differences among different groups; # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0054] Figure 3 Comparison of VEGF differences among different groups; # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0055] Figure 4 Comparison of sICAM-1 differences among different groups; # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0056] Figure 5 Comparison of histamine differences among different groups; # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0057] Figure 6 The results of HE staining of the epidermal model are shown (microscope 400X). DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] 1. The experimental materials used in the present invention are shown in Table 1:

[0060] Table 1 Experimental materials

[0061] name factory Item No. RBL-2H3 cell line Cell Bank of Chinese Academy of Sciences SCSP-518 <![CDATA[Episkin TM and its accompanying serum-free medium]]> Shanghai Sianfuno Biotechnology Co., Ltd. <![CDATA[Episkin TM ]]> Compound C48 / 80 CAYMAN CHEMICAL 22173 Human TNF-α ELISA kit Xinbosheng Bio EHC103a.96 Human VEGF ELISA kit Xinbosheng Bio EHC108.96 Human sICAM-1(CD54)ELISA kit Xinbosheng Bio EHC109.96

[0062] 2. Data Analysis

[0063] Data were expressed as mean ± standard deviation and analyzed using SPSS. One-way analysis of variance (ANOVA) and least significant difference (LSD) test were used to analyze the differences between groups; P < 0.05 was considered statistically significant.

[0064] Example 1: Raw material screening model based on immune cells

[0065] 1. Experimental conditions

[0066] (1) Incubator conditions: temperature 37±1°C, humidity 90±5%, CO2 5±1%.

[0067] (2) Culture medium: DMEM medium containing 10% fetal bovine serum.

[0068] 2. Experimental process:

[0069] (1) Mast cell preparation: RBL-2H3 cell line was routinely cultured and the cells were plated at 4x10 5 The cells / well were inoculated into 12 wells containing culture medium and cultured in an incubator for 24 h.

[0070] (2) Preparation of epidermal model: Prepare the epidermal model Episkin TM The cells were transferred to a culture plate containing maintenance culture medium (serum-free culture medium provided with the epidermal model) and cultured in an incubator overnight.

[0071] (3) Co-culture: The epidermal model (area 1.07 cm) prepared in step (2) was placed in the 2 ) and step (1) were cultured for 24 h and then mast cells (1x10 6 cell) for 1 h to obtain the culture medium of the co-culture system.

[0072] (4) Drug exposure: After removing the culture medium, add the test drugs to the co-culture system according to the grouping in Table 2 and incubate in the incubator for 12 h;

[0073] Table 2 Experimental groups

[0074]

[0075] (5) Mast cell activation: Aspirate the drug solution to be tested, and add 2 mL of culture medium containing 10 μg / mL C48 / 80 to each group except the negative control group, place in an incubator, and allow to act for 2 h.

[0076] (6) Post-incubation: Replace with fresh culture medium and culture in an incubator for 24 hours.

[0077] (7) Collecting culture medium: Centrifuge at 10,000°C for 10 min to collect the supernatant, store at -80°C, and use ELISA kits to measure TNF-α, VEGF, sICAM-1, and histamine according to the instructions.

[0078] (8) Remove the epidermal tissue from the epidermal model using a punch and transfer it to a 2 mL EP centrifuge tube. Add 2 mL of neutral fixative (neutral formaldehyde) to each tube and fix it at room temperature for at least 24 h. After routine embedding, sectioning, and H&E staining, observe the morphology under a microscope.

[0079] 3. Experimental results

[0080] 3.1 TNF-α, VEGF, and sICAM-1 expression levels

[0081] The results of the determination of the contents and relative contents of TNF-α, VEGF, sICAM-1 and histamine in different groups are shown in Tables 3-6 and Figure 2-Figure 5 shown.

[0082] Table 3 Comparison of TNF-α levels in different groups (Mean±SD)

[0083]

[0084]

[0085] Note: In Table 3, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0086] Table 4 Comparison of VEGF differences among different groups (Mean±SD)

[0087] Group VEGF content (pg / mL) Relative content (%) Negative control group (NC) 13.77±2.11* 50.10±7.66* Model control group (M) 27.50±2.73# 100.00±9.93# Positive control group (PC) 15.85±1.78* 57.65±6.47* Sample group (TA) 16.83±1.53* 61.23±5.56*

[0088] Note: In Table 4, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0089] Table 5 Comparison of sICAM-1 levels in different groups (Mean±SD)

[0090] Group sICAM-1 content (pg / mL) Relative content (%) Negative control group (NC) 1.42±0.07* 45.39±2.23* Model control group (M) 3.13±0.23# 100.00±7.32# Positive control group (PC) 1.92±0.17* 61.26±5.34* Sample group (TA) 2.08±0.16* 66.33±5.12*

[0091] Note: In Table 5, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0092] Table 6 Comparison of histamine levels in different groups (Mean±SD)

[0093] Group Histamine content (ng / mL) Relative content (%) Negative control group (NC) 0.26±0.01* 32.05±0.87* Model control group (M) 0.82±0.09# 100.00±8.84# Positive control group (PC) 0.47±0.01* 57.26±1.26* Sample group (TA) 0.55±0.08* 67.19±8.11*

[0094] Note: In Table 6, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0095] The results showed that the relative levels of TNF-α, VEGF, sICAM-1, and histamine in the model control group (M) were significantly higher than those in the negative control group (NC) (p < 0.05), indicating successful model establishment. The relative levels of TNF-α, VEGF, sICAM-1, and histamine in the positive control group were significantly lower than those in the model control group (p < 0.05). At 100% test concentration, the relative levels of TNF-α, VEGF, sICAM-1, and histamine in the "Yugan Composition" sample were significantly lower than those in the model control group, with statistically significant differences (p < 0.05).

[0096] 3.2 Histological HE staining results of epidermal model

[0097] Figure 6 It was found that compared with the negative control group (NC), the model control group (M) had a reduced number of viable cell layers (reduced epidermal model thickness), indicating that the inflammatory response triggered by C48 / 80 on mast cells inhibited the epidermal model. Compared with the model control group (M), the positive control group (PC) and the sample group (healing composition) had clear boundaries in the four-layer structure of the model, compact cell arrangement, and an increased number of viable cell layers, indicating that the positive control and the sample (healing composition) had an effect on ameliorating the inflammatory stimulation caused by C48 / 80.

[0098] In summary, the immune cell-based raw material screening model provided by the present invention can be effectively used to screen raw materials suitable for sensitive skin.

[0099] Example 2 Immune cell-based raw material screening model for raw material screening

[0100] 1. Experimental conditions

[0101] (1) Incubator conditions: temperature 37±1°C, humidity 90±5%, CO2 5±1%.

[0102] (2) Culture medium: DMEM medium containing 10% fetal bovine serum.

[0103] 2. Experimental process:

[0104] (1) Mast cell preparation: RBL-2H3 cell line was routinely cultured and the cells were plated at 4x10 5 The cells / well were inoculated into 12 wells containing culture medium and cultured in an incubator for 24 h.

[0105] (2) Preparation of epidermal model: Prepare the epidermal model Episkin TM The cells were transferred to a culture plate containing maintenance culture medium (serum-free culture medium provided with the epidermal model) and cultured in an incubator overnight.

[0106] (3) Co-culture: The epidermal model (area 1.07 cm) prepared in step (2) was placed in the 2 ) and step (1) were cultured for 24 h and then mast cells (1x10 6 cell) for 1 h to obtain the culture medium of the co-culture system.

[0107] (4) Drug exposure: After removing the culture medium, add the test drugs to the co-culture system according to the grouping in Table 7 and incubate in the incubator for 12 h;

[0108] Table 7 Experimental groups

[0109]

[0110]

[0111] (5) Mast cell activation: Aspirate the drug solution to be tested, and add 2 mL of culture medium containing 10 μg / mL C48 / 80 to each group except the negative control group, place in an incubator, and allow to act for 2 h.

[0112] (6) Post-incubation: Replace with fresh culture medium and culture in an incubator for 24 hours.

[0113] (7) Collecting culture medium: Centrifuge at 10,000°C for 10 min to collect the supernatant, store at -80°C, and use ELISA kits to measure TNF-α, VEGF, sICAM-1, and histamine according to the instructions.

[0114] 3. Experimental results

[0115] The determination results of the contents and relative contents of TNF-α, VEGF, sICAM-1 and histamine in different groups are shown in Tables 8 to 11.

[0116] Table 8 Comparison of TNF-α levels in different groups (Mean±SD)

[0117] Group TNF-α content (pg / mL) Relative content (%) Negative control group (NC) 35.54±4.72* 38.95±5.17* Model control group (M) 91.25±7.83# 100.00±8.58# Positive control group (PC) 52.6±4.27* 57.70±4.68* Sample set 1 (TA1) 69.04±3.23* 75.66±3.54* Sample set 2 (TA2) 74.51±2.81* 81.66±3.07* Sample set 3 (TA3) 83.74±2.97 91.77±3.25

[0118] Note: In Table 8, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0119] Table 9 Comparison of VEGF differences among different groups (Mean±SD)

[0120] Group VEGF content (pg / mL) Relative content (%) Negative control group (NC) 17.80±1.82* 51.99±5.32* Model control group (M) 34.24±1.74# 100.00±5.07# Positive control group (PC) 25.84±1.36* 75.46±3.98* Sample set 1 (TA1) 25.50±1.98* 74.48±5.80* Sample set 2 (TA2) 26.07±1.79* 76.12±5.23* Sample set 3 (TA3) 26.69±1.97* 77.94±5.76*

[0121] Note: In Table 9, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0122] Table 10 Comparison of sICAM-1 levels in different groups (Mean±SD)

[0123] Group sICAM-1 content (pg / mL) Relative content (%) Negative control group (NC) 1.17±0.12* 53.44±5.53* Model control group (M) 2.19±0.11# 100.00±5.23# Positive control group (PC) 1.23±0.05* 56.38±2.30* Sample set 1 (TA1) 1.15±0.04* 52.73±2.00* Sample set 2 (TA2) 1.27±0.02* 58.04±1.03* Sample set 3 (TA3) 1.29±0.03* 58.93±1.59*

[0124] Note: In Table 10, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0125] Table 11 Comparison of histamine levels in different groups (Mean±SD)

[0126] Group Histamine content (ng / mL) Relative content (%) Negative control group (NC) 1.17±0.04* 54.52±2.06* Model control group (M) 2.15±0.04# 100.00±1.68# Positive control group (PC) 1.35±0.06* 62.67±2.60* Sample set 1 (TA1) 1.29±0.05* 59.90±2.46* Sample set 2 (TA2) 1.38±0.09* 63.97±4.21* Sample set 3 (TA3) 1.44±0.02* 66.92±1.01*

[0127] Note: In Table 11, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0128] While 0.1% carboxymethyl chitosan performed well in both redness and itching-related indicators, it showed no significant inhibitory effect on TNF-α. However, 0.5% biosaccharide gum-2 and 0.0005% ceramide performed well in all four indicators. In summary, the immune cell-based raw material screening model provided by this invention can be effectively used to screen raw materials suitable for sensitive skin and can distinguish their specific mechanisms of action.

[0129] Comparative Example 1

[0130] The difference between Comparative Example 1 and Example 1 is only that the experimental process is different.

[0131] (1) Mast cell preparation: RBL-2H3 cell line was routinely cultured and the cells were plated at 4x10 5 The cells / well were inoculated into 12 wells containing culture medium and cultured in an incubator for 24 h.

[0132] (2) After culturing mast cells (1x10 6 cell) for 1 h to obtain the culture fluid.

[0133] (3) Drug exposure: After removing the culture medium, add the drugs to be tested into the co-culture system according to the groups in Table 2 and incubate in the incubator for 12 h;

[0134] (4) Mast cell activation: The drug solution to be tested was aspirated, and 2 mL of culture medium containing 10 μg / mL C48 / 80 was added to each group except the negative control group. The cells were placed in an incubator and allowed to act for 2 h.

[0135] (5) Post-incubation: Replace with fresh culture medium and culture in an incubator for 24 hours.

[0136] (6) Collecting culture medium: The supernatant was collected by centrifugation at 10,000°C for 10 min and stored at -80°C. The TNF-α expression level was determined using an ELISA kit according to the instructions. The results are shown in Table 12.

[0137] Table 12 Comparative Example 1 Comparison of TNF-α differences in different groups (Mean ± SD)

[0138] Group TNF-α content (pg / mL) Relative content (%) Negative control group (NC) 73.21±2.63* 43.50±1.57* Model control group (M) 168.32±4.34# 100.00±2.28# Positive control group (PC) 108.37±6.12* 64.38±3.65* Sample group (TA) 143.59±4.87* 85.31±2.90*

[0139] Note: In Table 12, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0140] In Comparative Example 1, the differences between the model control group, positive control group, and sample group and the negative control group were statistically significant. Furthermore, the differences between the model control group, positive control group, and sample group were statistically significant. Although Comparative Example 1 can be used to screen suitable raw materials for sensitive skin, the effect is not as good as Example 1.

[0141] Comparative Example 2

[0142] The difference between Comparative Example 2 and Example 1 is only that the experimental process is different.

[0143] (1) Preparation of epidermal model: TM The cells were transferred to a culture plate containing maintenance culture medium (serum-free culture medium provided with the epidermal model) and cultured in an incubator overnight.

[0144] (2) Co-culture: The epidermal model prepared in step (1) (area 1.07 cm 2 ) Culture for 1 hour to obtain culture medium.

[0145] (3) Drug exposure: After removing the culture medium, add the drugs to be tested into the co-culture system according to the groups in Table 2 and incubate in the incubator for 12 h;

[0146] (4) Cell activation: The drug solution to be tested was aspirated, and 2 mL of culture medium containing 10 μg / mL C48 / 80 was added to each group except the negative control group, and the cells were placed in an incubator for 2 h.

[0147] (5) Post-incubation: Replace with fresh culture medium and culture in an incubator for 24 hours.

[0148] (6) Collecting the culture medium: The supernatant was collected by centrifugation at 10,000°C for 10 min and stored at -80°C. The TNF-α expression level was determined using an ELISA kit according to the instructions. The results are shown in Table 13.

[0149] Table 13 Comparative Example 2 Comparison of TNF-α differences in different groups (Mean ± SD)

[0150] Group TNF-α content (pg / mL) Relative content (%) Negative control group (NC) 41.19±3.91* 79.69±7.57* Model control group (M) 51.67±3.24# 100.00±6.27# Positive control group (PC) 46.08±3.74* 89.15±7.24* Sample group (TA) 50.20±2.95# 97.12±5.71#

[0151] Note: In Table 13, # indicates that the difference is statistically significant compared with the negative control group (p<0.05); * indicates that the difference is statistically significant compared with the model control group (p<0.05).

[0152] In Comparative Example 2, the model control group, positive control group, and sample group showed statistically significant differences compared with the negative control group. The differences between the model control group and the positive control group were statistically significant, but the differences between the model control group and the sample group were not statistically significant, indicating that the screening model provided in Comparative Example 2 cannot be used for screening raw materials suitable for sensitive skin.

[0153] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A method for screening raw materials suitable for sensitive skin, characterized in that: The screening method comprises the following steps: S1. Add the sample to be tested to the raw material screening model and culture it; the raw material screening model is a co-culture model of mast cells and epidermal model; the mast cells are RBL-2H3 cell line, and the epidermal model is Episkin; the culture method is indirect contact culture based on an insert cell culture apparatus, the sample to be tested is added to the bottom chamber culture medium, and the culture time is 10-15 hours; S2. Remove the test sample solution, add a mast cell activator, and incubate; the mast cell activator is C48 / 80 at a concentration of 10 μg / mL, and the incubation time is 2 hours; the test sample includes a culture medium containing 0.1% carboxymethyl chitosan, a culture medium containing 0.5% biosaccharide gum-2, and a culture medium containing 0.0005% ceramide; S3, replace with fresh culture medium and continue culturing; S4. The culture medium is centrifuged and the supernatant is collected for detection of sensitive muscle inflammatory factors; the inflammatory factors are composed of VEGF, histamine, sICAM-1, and TNF-α; S5. Take the epidermal model and perform pathological examination.

2. The screening method according to claim 1, wherein The culture duration of the continued culture in step S3 is 24-36 hours.

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