Cell chip for evaluating skin discomfort caused by product and application of cell chip

Abstract

The invention discloses a cell chip for evaluating skin discomfort caused by a product and application of the cell chip. The cell chip comprises a detection hole of an overexpression cell containing a sensory receptor gene, the overexpression cells of the sensory receptor gene comprise various overexpression cells of a TRPs receptor family, a histamine receptor family, a protease activated receptor family and a prostaglandin receptor family. According to the invention, a plurality of key sensory receptors participating in skin discomfort regulation are adopted as comprehensive evaluation indexes, and a stable cell strain constructed in an overexpression mode is adopted, so that the skin sensory receptors with high expression quantity under abnormal sensitive perception of skin can be simulated; then, the change multiple of the in-vitro intracellular calcium ion fluorescence signal intensity is used for calculating and relating to the evaluation of clinical skin discomfort through a specific score, so that the evaluation of an in-vitro cell chip can be used as a prediction model for the evaluation of the clinical skin discomfort; and an innovative and accurate in-vitro cytology method capable of replacing clinic is provided for evaluating the skin discomfort.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an in vitro cell evaluation chip for evaluating skin discomfort caused by a product and an application thereof. Background Art

[0002] The somatosensory nervous system of mammals can detect a variety of sensations, including touch, pressure, cold / heat, pain, and itch. Therefore, the somatosensory nervous system can quickly detect various potentially harmful stimuli and produce protective behavioral responses. The skin is one of the main sources of sensory input to the somatosensory nervous system, and is the initial induction and response to various external stimuli (such as mechanical, cold / heat, chemical, light, etc.). When the skin is stimulated by external stimuli, various receptors in the skin will be activated and transmit the stimulation signals to the brain through nerve fibers. These signals are interpreted and integrated in the brain, allowing us to perceive various external stimuli and make corresponding responses and adaptations. According to the different types of stimuli to which these receptors respond and the different sensory information transmitted, these receptors can be divided into pain receptors, itch receptors, touch receptors, cold / heat receptors, pressure receptors, and olfactory receptors. These receptors have different distribution densities and types in different areas of the skin, allowing the human body to make fine perceptions and responses to different types of stimuli.

[0003] Skin discomfort is a complex concept involving a variety of internal and external factors, but it usually refers to a series of unpleasant perceptions that occur when the skin is stimulated by physical, chemical or mental factors, such as tightness, tingling, burning, itching, heat pain, pain, etc. In 2020, a survey on the symptoms of sensitive skin in Chinese women showed that 78.9% of the people experienced itching and 65.5% experienced tingling.

[0004] At present, the evaluation of skin discomfort mainly adopts clinical evaluation. The advantage of clinical evaluation is that it is closest to the real effect, but its disadvantages are as follows: 1) It cannot be evaluated by instruments, and the evaluation is based on the subjective perception of volunteers, which varies greatly; 2) At least three testers are required, one of whom is a clinical doctor; 3) At least 30 effective volunteers are required, and it is difficult to recruit volunteers; 4) Clinical evaluation has great safety risks, and volunteers may damage their skin after participating, which requires a certain amount of time to recover; 5) It cannot be carried out in a high-throughput manner, and generally only one sample can be tested at a time, which is not suitable for screening or testing of cosmetic raw materials; 6) The testing cost is relatively expensive, and the cost of one sample may be more than RMB 10,000; 7) The clinical evaluation has large errors, and different parts of the same volunteer vary greatly, and the same part of the same volunteer has large differences in evaluation at different times.

[0005] The advantages of conventional in vitro cell evaluation methods are as follows: 1) High-throughput screening is possible; 2) The detection cycle is short. Generally, 7 steps are required from the determination to the data results, and skilled operation may take 5 working days; 3) Fewer testers are required: generally, one sample requires one professional tester; 4) The test cost is low; generally, the test cost of 10 samples is about RMB 10,000; 5) The error is minimal; 6) The test results are stable. The same treatment method and different personnel test the results. However, the current in vitro cell evaluation methods still have the following disadvantages: 1) The correlation with clinical evaluation results is relatively low; 2) Due to the low expression of skin discomfort-related receptors in conventional cells, the skin discomfort state cannot be simulated and cannot be used for skin perception evaluation. 3) Skin discomfort perception involves multiple skin receptor regulatory pathways, and a single evaluation method is difficult to truly evaluate its normal physiological function.

[0006] With the development of cell and molecular biology technology, in-depth research on symptoms and mechanisms, high-content cell screening and high-throughput composite screening, potential risk substances, active substances and their targets can be identified from a large number of compound libraries in a short time. Cell chip technology is a high-tech technology involving multiple cross-fields. It realizes high-throughput screening and functional research of biological cells by culturing, processing and analyzing cells on microchips. Cell chip technology has the advantages of high throughput, parallelism and automation, which makes it have broad application prospects in drug research, ultra-high throughput screening platforms, pharmacogenomics, drug toxicology research, drug analysis resistance research, personalized medicine and treatment research. There are reports on the use of cell chips in basic research related to skin. However, there are no reports on the use of cell chips to evaluate the skin discomfort caused by products. Summary of the invention

[0007] In view of this, an object of the present invention is to provide a cell chip for evaluating skin discomfort caused by a product and its application. The method of evaluating skin discomfort caused by a product using the cell chip enables the cell chip to conveniently, quickly, accurately and sensitively evaluate skin discomfort caused by a product.

[0008] Another object of the present invention is to provide the use of the above cell chip in evaluating the skin discomfort caused by cosmetics, toiletries and health products.

[0009] Skin perception refers to the process by which the skin responds to various external stimuli through its complex system of nerves and cells. This process involves two main pathways:

[0010] (I) Initial perception of the skin: When external stimuli act directly on the keratinocytes of the skin, the keratinocytes release a variety of bioactive molecules, such as cytokines, prostaglandins, and chemokines, in response to these stimuli. This process is called the initial perception of the skin. This response is non-neural and mainly involves the function of the skin barrier and the activation of the immune response.

[0011] (II) Neural perception of the skin: External stimuli can also directly act on the nerve fibers in the skin. The nerve fibers will respond to these stimuli and transmit them to the brain through nerve signals for decoding, producing corresponding perceptions, such as pain, temperature, touch, etc. This process is called neural perception of the skin. The nerve fibers in the skin can be divided into many types, each with a specific range of perception and response characteristics, allowing us to perceive a rich variety of sensory information.

[0012] Normal skin perception function is conducive to the adaptation of organisms to the environment and protection from harm. Highly sensitive abnormal skin sensation brings unpleasant sensations (skin discomfort) to organisms and reduces the quality of life. In the present invention, the host cell selection of the cell chip technology that causes skin discomfort through the initial perception of the skin is HaCaT cells (human immortalized keratinocytes) that overexpress sensory receptor genes. In the present invention, the host cell selection of the cell chip technology that causes skin discomfort through the neural perception of the skin is SH-SY5Y cells (human neuroblastoma cell line) that overexpress sensory receptor genes.

[0013] The occurrence of skin discomfort is the result of the interaction of different receptors. The skin discomfort in the present invention is mainly pain, itching, and heat, supplemented by smell, pressure, cold, and touch. The sensory receptor design of the cell chip technology for detecting products that cause skin discomfort is based on this.

[0014] According to structural classification, the heat / pain / itch receptors are mainly ion channel receptors and G protein coupled receptors, and other types of receptors are supplementary. In the present invention, the sensory receptor genes for detecting skin discomfort caused by the product are selected from ion channel receptors and G protein coupled receptors.

[0015] The occurrence of skin discomfort is the result of a complex regulatory response activated by various external stimuli. It is a complex, mixed unpleasant sensation involving the activation and interaction of pain, itch, heat / cold, smell, pressure, and touch receptors.

[0016] The sensory receptor genes involved in the regulation of skin pain mainly come from the TRPs (Transient receptor potential, TRPs) receptor family, endothelin receptors family, cannabinoid receptors family, galanin receptors family, GABA receptors, neurokinin receptors (Neurokinin receptors, NKRs) family, opioid receptors (Opioid receptors), acid-sensing ion channel receptors (Acid-sensing ion channels, ASICs) family, adrenergic receptors (Adrenergic receptors, ARs), Mrgpr receptors (Mas-related G-protein-coupled receptors) family, CGRP receptors (Calcitonin gene-related peptide receptors) family, serotonin receptor family (5-HTreceptors, HTRs), prostaglandin receptors (Prostaglandin receptors, PTGs), cytokine receptors, and Toll-like receptors (TLRs).

[0017] The sensory receptor genes involved in the regulation of skin itch mainly come from the TRPs (Transient receptor potential, TRPs) receptor family, neurokinin receptors (Neurokinin receptors, NKRs), platelet-activating factor receptors (Platelet-activating factor receptors PAF-Rs), lysophosphatidic acid receptors (Lysophosphatidic acid receptors, LPA receptor) family, protease-activated receptor family (Protease-activated receptors, PARs), histamine receptors (Histamine receptors, HRs) family, serotonin receptor family (HTRs), Mrgpr receptor (Mas-related G-protein-coupled receptors) family, TSLP receptor (Tcell activating and proliferation-inducing molecule receptor, TSLPR), cytokine receptors (Cytokine Receptors), Toll-like receptors (TLRs), and leukotriene receptors (Leukotriene receptors, LTs).

[0018] The sensory receptor genes involved in the regulation of skin touch mainly come from the PIEZO family and adhesion G-protein-coupled receptors (aGPCRs).

[0019] The sensory receptor genes involved in the regulation of skin cold / heat mainly come from the TRPs (Transient receptor potential, TRPs) receptor family and the serotonin receptor family (HTRs).

[0020] The sensory receptor genes involved in the regulation of skin pressure sensation mainly come from the serotonin receptor family (HTRs), neurokinin receptors (NKRs), adrenergic receptors (ARs), endothelin receptors family, serotonin receptor family (HTRs), and galanin receptors (Galaninreceptors) family.

[0021] The sensory receptor genes involved in olfactory regulation mainly come from olfactory receptors (OR).

[0022] The objective of the present invention is achieved through the following technical solutions:

[0023] In a first aspect, the present invention provides an application of a sensory receptor gene overexpressing cell in the preparation of a product for evaluating whether the product causes skin discomfort, wherein the sensory receptor gene overexpressing cell includes a TRPs receptor family overexpressing cell, a histamine receptor family overexpressing cell, a protease activated receptor family overexpressing cell and a prostaglandin receptor family overexpressing cell.

[0024] As a preferred embodiment, the TRPs receptor family includes at least one of TRPV1, TRPV2, TRPV3, TRPV4, TRPC1, TRPC4, TRPM8, and TRPA1;

[0025] The histamine receptor family includes at least one of HRH1, HRH2, and HRH4;

[0026] The protease activated receptors include at least one of PAR-2 ​​and PAR-4;

[0027] The prostaglandin receptor family includes at least one of EP1 and EP4.

[0028] As a further preferred embodiment, the TRPs receptor family includes TRPV1, TRPV3, and TRPV4; the histamine receptor family includes HRH1; the protease-activated receptor includes PAR-2; and the prostate receptor family includes EP1.

[0029] As a further preferred embodiment, the sensory receptor gene overexpressing cells include TRPV1 overexpressing cells, TRPV3 overexpressing cells, TRPV4 overexpressing cells, HRH1 overexpressing cells, PAR-2 ​​overexpressing cells and EP1 overexpressing cells.

[0030] As a preferred embodiment, the sensory receptor gene overexpressing cells further include at least one of endothelin receptor family overexpressing cells, acid-sensing ion channel receptor family overexpressing cells, adrenergic receptor family overexpressing cells, and Mrgpr receptor family overexpressing cells;

[0031] The endothelin receptor family includes at least one of EDNRA and EDNRB;

[0032] The acid-sensing ion channel receptor family includes ASIC3;

[0033] The adrenergic receptor family includes at least one of ADRA1A, ADRA1B, and ADRA1D;

[0034] The Mrgpr receptor family includes at least one of MRGPRX1, MRGPRX4, MRGPRF, MRGPRF, and MRGPRA3.

[0035] As a further preferred embodiment, the endothelin receptor family includes EDNRA; the acid-sensing ion channel receptor family includes ASIC3; the adrenergic receptor family includes ADRA1A; and the Mrgpr receptor family includes MRGPRX1.

[0036] As a further preferred embodiment, the sensory receptor gene overexpression cells include TRPV1 overexpression cells, TRPV3 overexpression cells, TRPV4 overexpression cells, HRH1 overexpression cells, PAR-2 ​​overexpression cells and EP1 overexpression cells, and also include EDNRA overexpression cells, ASIC3 overexpression cells, ADRA1A overexpression cells, MRGPRX1 overexpression cells at least one. Most preferably, the sensory receptor gene overexpression cells include TRPV1 overexpression cells, TRPV3 overexpression cells, TRPV4 overexpression cells, HRH1 overexpression cells, PAR-2 ​​overexpression cells and EP1 overexpression cells, EDNRA overexpression cells, ASIC3 overexpression cells, ADRA1A overexpression cells, MRGPRX1 overexpression cells.

[0037] As a preferred embodiment, the sensory receptor gene overexpressing cell is a HaCaT cell or SH-SY5Y cell overexpressing the sensory receptor gene.

[0038] As a preferred embodiment, the sensory receptor gene overexpression cells include TRPV1 overexpression HaCaT cells, TRPV3 overexpression HaCaT cells, TRPV4 overexpression HaCaT cells, HRH1 overexpression HaCaT cells, PAR-2 ​​overexpression HaCaT cells and EP1 overexpression HaCaT cells, or TRPV1 overexpression SH-SY5Y cells, TRPV3 overexpression SH-SY5Y cells, TRPV4 overexpression SH-SY5Y cells, HRH1 overexpression SH-SY5Y cells, PAR-2 ​​overexpression HaCaT cells and EP1 overexpression HaCaT cells. R-2 overexpression SH-SY5Y cells and EP1 overexpression SH-SY5Y cells; also include at least one of EDNRA overexpression HaCaT cells, ASIC3 overexpression HaCaT cells, ADRA1A overexpression HaCaT cells, MRGPRX1 overexpression HaCaT cells, or at least one of EDNRA overexpression SH-SY5Y cells, ASIC3 overexpression SH-SY5Y cells, ADRA1A overexpression SH-SY5Y cells, MRGPRX1 overexpression SH-SY5Y cells.

[0039] In a second aspect, the present invention provides a cell chip for evaluating skin discomfort caused by a product, the cell chip comprising detection wells containing overexpressed cells of sensory receptor genes;

[0040] The sensory receptor genes include overexpression cells of the TRPs receptor family, overexpression cells of the histamine receptor family, overexpression cells of the protease activated receptor family, and overexpression cells of the prostaglandin receptor family;

[0041] The TRPs receptor family includes at least one of TRPV1, TRPV2, TRPV3, TRPV4, TRPC1, TRPC4, TRPM8, and TRPA1;

[0042] The histamine receptor family includes at least one of HRH1, HRH2, and HRH4;

[0043] The protease activated receptors include at least one of PAR-2 ​​and PAR-4;

[0044] The prostaglandin receptor family includes at least one of EP1 and EP4.

[0045] As a preferred embodiment, the sensory receptor gene overexpressing cells further include at least one of endothelin receptor family overexpressing cells, acid-sensing ion channel receptor family overexpressing cells, adrenergic receptor family overexpressing cells, and Mrgpr receptor family overexpressing cells;

[0046] The endothelin receptor family includes at least one of EDNRA and EDNRB;

[0047] The acid-sensing ion channel receptor family includes ASIC3;

[0048] The adrenergic receptor family includes at least one of ADRA1A, ADRA1B, and ADRA1D;

[0049] The Mrgpr receptor family includes at least one of MRGPRX1, MRGPRX4, MRGPRF, MRGPRF, and MRGPRA3.

[0050] As a preferred embodiment, the sensory receptor gene overexpressing cell is a HaCaT cell or SH-SY5Y cell overexpressing the sensory receptor gene.

[0051] As a preferred embodiment, the number of detection wells containing cells overexpressing each sensory receptor gene is 3-5.

[0052] In a third aspect, the present invention also provides a method for non-disease diagnosis purposes for evaluating skin discomfort caused by a product, comprising the following steps:

[0053] S1. Cell culture: adding culture medium to the detection wells containing overexpressed cells of each sensory receptor gene in the cell chip for culture;

[0054] S2, staining: discard the culture medium, wash with buffer, and then add a staining solution containing a calcium ion fluorescent probe to stain the overexpressing cells of each sensory receptor gene;

[0055] S3. After staining, wash with buffer solution, add buffer solution again, prepare the sample to be tested, the instrument automatically adds the sample to be tested, and performs real-time detection;

[0056] S4. Process the collected data, calculate the change multiple of the fluorescence intensity of the calcium ion signal of each detection well, then convert the change multiple of the fluorescence intensity of the calcium ion signal into a skin discomfort score, and then calculate the comprehensive score of skin discomfort. Finally, determine the level of skin discomfort caused by the product to be tested according to the comprehensive score of skin discomfort.

[0057] As a preferred solution, in step S4, the calculation formula for the change multiple of the fluorescence intensity of the calcium ion signal is shown in the following formula (1):

[0058] RFU fold change =(RFU 待测产品组 -RFU 阴性对照组 ) / RFU 阴性对照组 (1)

[0059] Among them, RFU fold change RFU is the change factor of the fluorescence intensity of calcium ion signal; 待测产品组 RFU is the fluorescence intensity value of the calcium ion signal of the product to be tested. 阴性对照组 is the fluorescence intensity value of calcium ion signal after adding 1×HHBS buffer.

[0060] As a preferred solution, in step S4, the conversion standard of the skin discomfort score is as follows:

[0061] RFU fold change <3, DS = 0;

[0062] 3≦RFU fold change ≦10, DS=1;

[0063] 10 <RFU fold change ≦20, DS=2;

[0064] 20 <RFU fold change ≦30, DS=3;

[0065] 30 < RFU fold change When it is ≤ 40, DS = 4;

[0066] RFU fold change When it is > 40, DS = 5;

[0067] Among them, DS is the skin discomfort score.

[0068] As an optimal solution, in step S4, the calculation formula of the comprehensive score of the skin discomfort is shown in the following formula (2):

[0069] TS = ∑i(DSi) (2)

[0070] Among them, TS is the comprehensive score of skin discomfort; i is the number of sensory receptor genes detected; DSi is the average value of the skin discomfort scores of each replicate well of the sensory receptor gene;

[0071] In step S4, the grading standard of the skin discomfort is as follows:

[0072] When 0 ≤ TS ≤ 1, the skin discomfort level is: no skin discomfort;

[0073] When 1 < TS ≤ 2, the skin discomfort level is: mild skin discomfort;

[0074] When TS > 2, the skin discomfort level is: moderate / severe skin discomfort.

[0075] As an optimal solution, in step S1, in each detection well, the overexpressed cells containing each sensory receptor gene are cells in the logarithmic growth phase, and the seeding density is 3.5w / well to 0.6w / well; the number of detection wells of the chip can be 384 or 96; the culture time is 18 - 20h.

[0076] As an optimal solution, in step S2, the volume of the staining solution containing the calcium ion fluorescent probe added is 30 - 100 μL; when the number of wells of the chip is 96, 100 μL is added; when the number of wells is 384, 30 μL is added. The calcium ion fluorescent probe is Cal 520-AM.

[0077] As an optimal solution, in steps S2 and S3, the buffer solution is 1×HHBS buffer solution; the staining time is 55 - 65 min.

[0078] As an optimal solution, in step S3, the test sample needs to meet the following conditions:

[0079] a) Be uniformly dissolved in an aqueous solution;

[0080] b) Do not change the cell morphology and have no cytotoxicity;

[0081] c) No spontaneous luminescence, no binding with the selected calcium ion fluorescent probe.

[0082] As a preferred embodiment, when the sample to be tested can directly induce skin discomfort through the skin initial perception pathway, HaCaT cells that overexpress sensory receptor genes are used, such as histamine, capsaicin, lactic acid, etc.; when the sample to be tested can directly induce skin discomfort through the skin nerve perception pathway, SH-SY5Y cells that overexpress sensory receptor genes are used, such as adrenaline, histamine, lactic acid, etc.

[0083] As a preferred solution, in step S3, the sample to be tested is mixed with 1×HHBS buffer to form a 2× working solution and then added.

[0084] As a preferred solution, in step S3, the volume of the buffer added is 20-50 μL; if the number of wells of the chip is 96, 50 μL is added; if the number of wells is 384, 20 μL is added.

[0085] As a preferred solution, in step S3, the instrument is a calcium flow detection instrument, such as FlexStation 3 (Molecular Devices) or FLIPR Tetra system (Molecular Devices). The added volume of the sample to be tested is the same as the volume of the buffer.

[0086] Different receptors have different signal pathways after activation and induce different physiological functions. The results of the cell chip for detecting skin discomfort caused by the product in the present invention are based on the calcium ion (Ca) after target activation. 2+ ) signal change multiples are scored, and then the scores of each sensory receptor gene are summed up to obtain the final score, so that the scoring results of skin discomfort caused by evaluation samples of different concentrations can be obtained.

[0087] The cell chip for detecting skin discomfort caused by the product of the present invention was used to detect different concentrations of capsaicin, histamine, sodium dodecyl sulfate (SLS), and DL-lactic acid. The results showed that 10,000 times the cell detection concentration was consistent with the clinical skin discomfort score results. When the chip was used to calculate and score a single sensory receptor separately, some evaluation results had great errors; the more sensory receptors selected, the more relevant the cell evaluation results were to the clinical evaluation results. When applied to the detection of specific products, the cells in the cell chip technology that can directly induce skin discomfort through the initial skin perception pathway preferentially select HaCaT cells that overexpress sensory receptor genes. When applied to the detection of specific products, the cells in the cell chip technology that can directly induce skin discomfort through the skin nerve perception pathway preferentially select SH-SY5Y cells that overexpress sensory receptor genes.

[0088] It can be seen from the above technical solutions that the cell chip for detecting skin discomfort caused by products of the present invention contains key receptors for skin discomfort. Through specific scoring calculation results, it can provide a reference for products in improving skin discomfort, raw material screening and individualized skin product testing.

[0089] Compared with the prior art, the present invention has the following beneficial effects:

[0090] 1. Compared with the current single perceptual evaluation, the present invention summarizes the skin discomfort as a whole, and finds the key sensory receptors involved in the regulation of skin discomfort, and selects multiple sensory receptor genes from the TRPs receptor family, histamine receptor family, protease-activated receptor family, prostaglandin receptor family, endothelin receptor family, acid-sensitive ion channel receptor family, adrenergic receptor family and Mrgpr receptor family as comprehensive evaluation indicators, thereby obtaining a more accurate method and product for evaluating skin discomfort caused by products.

[0091] 2. The stable cell lines constructed by the overexpression method of the present invention can simulate the skin sensory receptors with high expression under abnormal skin sensitivity. These cells are made into cell chips, and the change multiple of the intensity of the intracellular calcium ion fluorescence signal in vitro is used to calculate the correlation with the evaluation of clinical skin discomfort through specific scoring, so that the evaluation of the in vitro cell chip can be used as a prediction model for the evaluation of clinical skin discomfort. In this way, the evaluation of skin discomfort for cosmetics, skin care products, skin external application products and other products is carried out, and an innovative, accurate and clinical alternative in vitro cytology method is provided for the evaluation of skin discomfort.

[0092] 3. The cell chip technology provided by the present invention has a strong correlation with the clinical evaluation results, with fewer false positive results, and compared with the clinical evaluation method, it has the advantages of lower testing cost, high repeatability, short testing cycle and high screening throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0094] Figure 1 This is an example of the gene expression identification results of the overexpression stable cell lines constructed with HaCaT cells and SH-SY5Y cells in Example 1;

[0095] Figure 2The identification results of the overexpressed gene functions of the overexpression stable cell lines constructed with HaCaT cells and SH-SY5Y cells in Example 1 are given as examples; wherein, Capsaicin activation can identify the TRPV1 gene function, Histamine activation can identify the HRH1 gene function, 2-Aminoethyl diphenylborinate activation can identify the TRPV3 gene function, TRPV4agonist-1free base activation can identify the TRPV4 gene function, Endothelin 1 activation can identify the EDNRA gene function, SLIGRL-NH2 activation can identify the PAR-2 ​​gene function, Phenylephrine hydrochloride activation can identify the ADRA1A gene function, 0.004% DL-lactic acid + GMQ activation can identify the ASIC3 gene function, MRGPRX1agonist 1 activation can identify the MRGPRX1 gene function, and PGE2 activation can identify the EP1 gene function;

[0096] Figure 3 The results of the clinical evaluation of skin surface changes in Example 4 are exemplified. DETAILED DESCRIPTION

[0097] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0098] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0099] It should be understood that, except in any operating examples, or otherwise indicated, all numbers representing the amount of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary according to the desired performance to be obtained by the present invention. At least, it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0100] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0101] In the applicant's previous research, a cytological method for evaluating the discomfort of sensitive skin was disclosed (see patent document CN112626163 A). By using the TRPV1 high-expression HaCaT cell line to simulate sensitive skin, it was used for large-scale in vitro screening to verify the efficacy of plant extracts and various active ingredients. However, this method only evaluates the results of a single sensory receptor, and only uses "comparing the fluorescence value of the experimental group containing the irritant with the fluorescence value of the blank group without the irritant, and the difference in fluorescence value is greater than 30, then it is irritating" as the evaluation standard. In actual application, it can only make a preliminary assessment of whether it is irritating, and it is impossible to make a more accurate assessment of the severity of the irritation. When evaluating samples of different concentrations, the error of the evaluation results is large. Based on this, further research of the present invention was carried out.

[0102] Keratinocytes are the main cell type in the epidermis, and they constitute the majority of the epidermis. Keratinocytes play a key role in the initial perception process of the skin, especially in terms of perception and response to external stimuli. The present invention constructs a stable HaCaT (human immortalized keratinocyte) cell strain or a stable SH-SY5Y cell strain overexpressing through the skin receptor gene that plays a key role in the discomfort process of the skin, and prepares these stable overexpression cell strains into cell chips to simulate the abnormally sensitive perception of the skin and the evaluation of skin discomfort. At the same time, the correlation between the in vitro cell chip and clinical skin discomfort is established by known substances that can cause skin discomfort. The results of the in vitro cell chip evaluation can be improved by this method to be closer to the clinical evaluation results, and an objective basis is provided for the evaluation of skin discomfort by external skin application substances such as cosmetics.

[0103] The overexpression stable cell line provided in the present invention is provided by our laboratory, and the materials and reagents used in other cell chip technical solutions can be purchased from the market.

[0104] The overexpression cell lines used in the following examples were constructed and identified by our laboratory. The construction method is a conventional method and is not particularly limited in the present invention.

[0105] The complete culture medium used in the following examples is: containing 10% fetal bovine serum, 0.1% penicillin and streptomycin mixed solution, and the remainder is DMEM basic culture medium.

[0106] Example 1

[0107] This embodiment provides a method for constructing an overexpression stable cell line, and the specific steps are as follows:

[0108] 1) Selection of target genes: Check the full-length CDS (coding sequences, CDS) sequences of key sensory receptor genes in the initial skin perception process (TRPV1 (geneID:AY131289.1), TRPV3 (geneID:NM_001258205.2), TRPV4 (gene ID:NM_021625.5), HRH1 (gene ID:NM_000861.3), PAR2 (gene ID:NM_005242.6), ASIC3 (gene ID:NM_020321.3), MRGPRX1 (gene ID:NM_147199.4), EP1 (gene ID:NM_000955.3), EDNRA (gene ID:NM_001957.4), ADRA1A (gene ID:NM_033303.4));

[0109] 2) Construction of recombinant lentiviral plasmids: Sequences containing two restriction sites (XhoIⅠ / EcoRI) were obtained by synthesis, and cloned into PHY008 plasmids to obtain PHY008-TRPV1, PHY008-TRPV3, PHY008-TRPV4, PHY008-HRH1, PHY008-EDNRA, PHY008-PAR2, PHY008-ASIC3, PHY008-EDNRA, PHY008-ADRA1A, PHY008-EP1, and PHY008-MRGPRX1 recombinant plasmids. This process can be completed by a third party. In the present invention, the recombinant plasmid was completed by Shanghai Mosen Biotechnology.

[0110] 3) Construction of overexpression cell lines: The recombinant lentiviral plasmid containing the target gene and the lentiviral packaging system (psPAX2 and pMD2.G) were combined by Lipofectamine TM 3000 were co-transfected into 293T cells, and the virus supernatant was collected after continued culture for 48 hours. A universal virus concentration kit was used to concentrate the lentiviral particles. The concentrated lentiviral supernatant was directly added to HaCaT cells and SH-SY5Y cells for infection; after 72 hours, 1 μg / mL puromycin was used for screening to select cells that successfully integrated the lentiviral vector and expressed the target gene. In the present invention, the overexpression cell lines were constructed according to the above method, and the instruments or reagents used are shown in Table 1.

[0111] Table 1

[0112] Instrument / reagent name brand Model / Article No. Biological Safety Cabinets Thermo Fisher 1379 Inverted microscope Leica DMi1 <![CDATA[CO 2 Incubator]]> Thermo Fisher 371 Fluorescence microscopy Leica DMi8 96-well clear cell culture plate Corning 3599 6-well cell culture plate corning 3516 100mm cell culture dish corning 430167 T175 culture flask corning - DMEM high glucose medium Source Bio L110KJ Trypsin-EDTA (0.25%), with phenol red Gibco 25200072 Fetal bovine serum (FBS) Ausbian WS500T Penicillin-Streptomycin (PS) Blue Sky ST488 HaCaT cells North BNCC337693 SH-SY5Y cells North BNCC100158 293T cells North BNCC353535 <![CDATA[Lipofectamine TM 3000 Transfection Reagent]]> Invitrogen L3000008 Opti-MEM Gibco 31985062 Puromycin Blue Sky ST551-50mg Polybrene (Hexadimethrine Bromide) Blue Sky C0351-1ml Lentiviral packaging system Shanghai Mosen Biotechnology / Universal Virus Concentration Kit Blue Sky C2901S

[0113] 4) Identification: Detect the expression level of the target gene in cells by qRT-PCR; detect the changes in intracellular calcium ion fluorescence signals by FlexStation3 instrument to detect the function of overexpression stable cell lines. Verify the successful construction of stable cell lines from two aspects: expression and function. Figure 1 and Figure 2 As shown. The results show that this embodiment successfully constructed TRPV1 overexpressing cells (HaCaT-TRPV1-OE), TRPV3 overexpressing cells (HaCaT-TRPV3-OE), TRPV4 overexpressing cells (HaCaT-TRPV4-OE), HRH1 overexpressing cells (HaCaT-HRH1-OE), PAR2 overexpressing cells (HaCaT-PAR2-OE), ASIC3 overexpressing cells (HaCaT-ASIC3-OE), MRGPRX1 overexpressing cells (HaCaT-MRGPRX1-OE), EP1 overexpressing cells (HaCaT-EP1-OE), EDNRA overexpressing cells (HaCaT-EDNRA-OE) and ADRA1A overexpressing cells (HaCaT-ADRA1A-OE) based on HaCaT cells; based on SH -SY5Y cells were TRPV1 overexpressing cells (SH-SY5Y-TRPV1-OE), TRPV3 overexpressing cells (SH-SY5Y-TRPV3-OE), TRPV4 overexpressing cells (SH-SY5Y-TRPV4-OE), HRH1 overexpressing cells (SH-SY5Y-HRH1-OE), PAR2 overexpressing cells (SH-SY5Y-PAR2-OE), ASIC3 overexpressing cells (SH-SY5Y-ASIC3-OE), MRGPRX1 overexpressing cells (SH-SY5Y-MRGPRX1-OE), EP1 overexpressing cells (SH-SY5Y-EP1-OE), EDNRA overexpressing cells (SH-SY5Y-EDNRA-OE), and ADRA1A overexpressing cells (SH-SY5Y-ADRA1A-OE).

[0114] Example 2

[0115] This embodiment provides an evaluation of skin discomfort caused by different concentrations of several substances in each overexpression cell based on HaCaT cells. The products to be tested for skin discomfort evaluation include histamine, capsaicin, sodium dodecyl sulfate (SLS), DL-lactic acid (80%), and levofloxacin. Histamine, capsaicin, sodium dodecyl sulfate (SLS), and DL-lactic acid (80%) were all purchased from the market, and levofloxacin was provided by Shanghai Jiakai Biological Co., Ltd. The specific operation process is as follows:

[0116] 1) Cell plating: HaCaT-TRPV1-OE, HaCaT-TRPV3-OE, HaCaT-TRPV4-OE, HaCaT-HRH1-OE, HaCaT-PAR2-OE, HaCaT-ASIC3-OE, HaCaT-MRGPRX1-OE, HaCaT-EP1-OE, HaCaT-EDNRA-OE and HaCaT-ADRA1A-OE, overexpressing cells containing sensory receptor genes, were seeded into cell chips (96-well plates) at 3.5 w / well;

[0117] 2) On the second day, discard the old culture medium, use 1×HHBS buffer, wash gently twice, add 100 μL 1×HHBS to each well, add 25 μL Cal 520-AM staining solution, and stain for 60 minutes;

[0118] 3) Preparation of working solution of the product to be tested: Prepare 2× working solution of the product to be tested using 1× HHBS buffer, as shown in Table 2-Table 6 below:

[0119] Table 2

[0120]

[0121] Table 3

[0122]

[0123] Table 4

[0124]

[0125] Table 5

[0126]

[0127] Table 6

[0128]

[0129] 4) Experimental groups: blank control group of the test product: working solution of the test product, without overexpressed cells; negative control group: containing overexpressed cells + 1×HHBS buffer; test product group: containing overexpressed cells + working solution of the test product. Solvent control group: 1×HHBS buffer, without overexpressed cells.

[0130] 5) After staining, use 1×HHBS buffer to wash twice, add 50μL of 1×HHBS buffer to each well, and then use the calcium flow detection instrument to use the FlexStation 3 automatic sample loading module to automatically add 50μL of the working solution of the product to be tested to the cell culture plate, and perform real-time rapid kinetic detection to collect data for the product group to be tested; automatically add 50μL of 1×HHBS buffer to the cell culture plate to collect data for the negative control group. The fluorescence intensity value of the product group to be tested must first be deducted from the fluorescence intensity value of the blank control group of the product to be tested, and the fluorescence intensity value of the negative control group must be deducted from the fluorescence intensity value of the solvent control group. Then, based on the deduction, the change multiple of the fluorescence intensity of the calcium ion signal (RFU fold change ), the calculation formula is as follows:

[0131] RFU fold change =(RFU 待测产品组 -RFU 阴性对照组 ) / RFU 阴性对照组

[0132] Among them, RFU fold change RFU is the change factor of the fluorescence intensity of calcium ion signal; 待测产品组 RFU is the fluorescence intensity value of the calcium ion signal of the product to be tested. 阴性对照组 is the fluorescence intensity value of calcium ion signal after adding 1×HHBS buffer.

[0133] RFU received fold change The results are shown in Tables 7 to 11 below (results from three replicate wells for each cell):

[0134] Table 7

[0135]

[0136] Table 8

[0137]

[0138]

[0139] Table 9

[0140]

[0141]

[0142] Table 10

[0143]

[0144] Table 11

[0145]

[0146] 6) RFU according to the conversion standard fold change The DS score is converted into a DS score. The conversion standard is shown in Table 12. The obtained DS score is then substituted into the following formula to calculate the comprehensive score (TS) of the skin discomfort caused by the tested product:

[0147] TS=Σi(DSi)

[0148] Where i is the total number of sensory receptor genes, and DSi is the average of the skin discomfort scores of sensory receptor genes in each complex well.

[0149] Table 12

[0150] <![CDATA[RFU fold change ]]> DS score / points <![CDATA[RFU fold change <3]]> 0 <![CDATA[3≦RFU fold change ≦10]]> 1 <![CDATA[10<RFU fold change ≦20]]> 2 <![CDATA[20<RFU fold change ≦30]]> 3 <![CDATA[30<RFU fold change ≦40]]> 4 <![CDATA[RFU fold change >40]]> 5

[0151] Then, based on the results of TS, the skin discomfort caused by the tested products is graded, and the classification criteria are shown in Table 13.

[0152] Table 13

[0153] TS Range Skin discomfort level 0≦TS≦1 No skin discomfort (none) 1<TS≦2 Causes mild skin discomfort (mild) TS>2 Causes moderate / severe skin discomfort (severe / moderate)

[0154] The DS score, TS score, skin discomfort level of each product to be tested and the clinical evaluation predicted concentration corresponding to each concentration are shown in Tables 14 to 23. The cell evaluation concentration represents the concentration used in the cell chip evaluation, and the clinical evaluation predicted concentration is the concentration predicted to be used clinically by amplifying the cell evaluation concentration by 10,000 times and the level classification of skin discomfort caused by the predicted concentration. Among them, combination one is the result obtained using 10 overexpression cells (HaCaT-TRPV1-OE, HaCaT-TRPV3-OE, HaCaT-TRPV4-OE, HaCaT-HRH1-OE, HaCaT-PAR2-OE, HaCaT-ASIC3-OE, HaCaT-MRGPRX1-OE, HaCaT-EP1-OE, HaCaT-EDNRA-OE and HaCaT-ADRA1A-OE); combination two is the result obtained using 6 overexpression cells (HaCaT-TRPV1-OE, HaCaT-TRPV3-OE, HaCaT-TRPV4-OE, HaCaT-HRH1-OE, HaCaT-PAR2-OE, HaCaT-EP1-OE).

[0155] Table 14

[0156]

[0157]

[0158] Table 15

[0159]

[0160] Table 16

[0161]

[0162]

[0163] Table 17

[0164]

[0165]

[0166] Table 18

[0167]

[0168] Table 19

[0169]

[0170]

[0171] Table 20

[0172]

[0173]

[0174] Table 21

[0175]

[0176] Table 22

[0177]

[0178] Table 23

[0179]

[0180]

[0181] Example 3

[0182] This example provides an evaluation of skin discomfort caused by different concentrations of several substances in each overexpression cell based on SH-SY5Y cells. The products to be tested for skin discomfort evaluation include histamine, capsaicin, DL-lactic acid (80%), and levofloxacin. Histamine, capsaicin, and DL-lactic acid (80%) were purchased from the market, and levofloxacin was provided by Shanghai Jiakai Biological Co., Ltd. The specific operation process is as follows:

[0183] 1) Cell plating: Overexpression cells containing sensory receptor genes SH-SY5Y-TRPV1-OE, SH-SY5Y-TRPV3-OE, SH-SY5Y-TRPV4-OE, SH-SY5Y-HRH1-OE, SH-SY5Y-PAR2-OE, SH-SY5Y-ASIC3-OE, SH-SY5Y-MRGPRX1-OE, SH-SY5Y-EP1-OE, SH-SY5Y-EDNRA-OE and SH-SY5Y-ADRA1A-OE were seeded in 96-well black cell culture plates at 3.5 w / well;

[0184] The step 2) is the same as step 2) in Example 2.

[0185] 3) Preparation of working solution of the product to be tested: The product to be tested is prepared into 2× working solution using 1×HHBS buffer, wherein the preparation of capsaicin, histamine, SLS, and levofloxacin is the same as step 3) in Example 2, and the preparation of DL-lactic acid is as shown in Table 24:

[0186] Table 24

[0187]

[0188] The steps 4)-5) are the same as the steps 4)-5) in Example 2.

[0189] RFU received fold change The results are shown in Tables 25-29 below (results from three replicate wells for each cell):

[0190] Table 25

[0191]

[0192]

[0193] Table 26

[0194]

[0195]

[0196] Table 27

[0197]

[0198] Table 28 Table 29

[0199]

[0200]

[0201] 6) is the same as step 6) in Example 2. The DS score, TS score, skin discomfort level results of each product to be tested and the clinical evaluation predicted concentration corresponding to each concentration are shown in Tables 30 to 39 below. Among them, combination 1 is to use 10 overexpression cells (SH-SY5Y-TRPV1-OE, SH-SY5Y-TRPV3-OE, SH-SY5Y-TRPV4-OE, SH-SY5Y-HRH1-OE, SH-SY5Y-PAR2-OE, SH-SY5Y-ASIC3-OE, SH-SY5Y-MRGPRX1-OE, SH-SY5Y-EP1-OE, SH-SY5Y-EDNRA -OE and SH-SY5Y-ADRA1A-OE); combination 2 is the results obtained using 7 overexpression cells (SH-SY5Y-TRPV1-OE, SH-SY5Y-TRPV3-OE, SH-SY5Y-HRH1-OE, SH-SY5Y-EDNRA-OE, SH-SY5Y-ASIC3-OE, SH-SY5Y-ADRA1A-OE, SH-SY5Y-EP1-OE).

[0202] Table 30

[0203]

[0204]

[0205] Table 31

[0206]

[0207]

[0208] Table 32

[0209]

[0210] Table 33

[0211]

[0212] Table 34

[0213]

[0214]

[0215] Table 35

[0216]

[0217]

[0218] Table 36

[0219]

[0220] Table 37

[0221]

[0222]

[0223] Table 38

[0224]

[0225]

[0226] Table 39

[0227]

[0228]

[0229] Example 4

[0230] This example provides clinical evaluation results of skin discomfort caused by several products to be tested. The products to be tested for skin discomfort evaluation include histamine, capsaicin, sodium lauryl sulfate (SLS), DL-lactic acid (80%), and levofloxacin. Histamine, capsaicin, sodium lauryl sulfate (SLS), and DL-lactic acid (80%) were all purchased from the market, and levofloxacin was provided by Shanghai Jiakai Biological Co., Ltd. The specific operation process is as follows:

[0231] 1) The experimental method was approved by the Ethics Committee and the volunteers signed the informed consent.

[0232] 2) The age range of volunteers is 18 to 65 years old. The clinical evaluation is conducted according to the standard process. Within a certain period of time after applying the product to be tested, the subjects are required to fill in the skin discomfort evaluation form.

[0233] The classification standards for the clinical evaluation of skin discomfort levels are shown in Table 40 below:

[0234] Table 40

[0235]

[0236] 3) The evaluation information and evaluation results of the product to be tested are shown in Table 41 below:

[0237] Table 41

[0238]

[0239] Take photos of abnormalities on the skin surface. Figure 3As shown, 200μg / mL capsaicin, 1.7mg / mL histamine, 20mg / mL histamine, and 50mg / mL SLS all caused erythema and / or wheal, accompanied by obvious skin discomfort. 5% DL-lactic acid and 10% DL-lactic acid both caused obvious skin discomfort, but no abnormality was found on the skin surface. 1μg / mL capsaicin, 0.01mg / mL histamine, 10% Yuefuning (a commercially available product of Shanghai Jiakai Biological Co., Ltd.), 0.1mg / mL SLS, and 0.1% DL-lactic acid did not cause skin discomfort. This result is basically consistent with the prediction results of Examples 2 and 3 using cell chips, and is also basically consistent with the concentrations reported in the literature that cause skin discomfort.

[0240] The results of this example show that the evaluation results of skin discomfort obtained by the methods of Examples 2 and 3 are correlated with the clinical evaluation results and can be used as a prediction model for clinical evaluation.

[0241] It should be noted that the cell chip and method of the present invention are not limited to the evaluation of skin discomfort of the test samples in the embodiments, but can also be used to evaluate whether other test samples cause skin discomfort, which will not be listed one by one in the present invention.

[0242] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be pointed out that the above embodiments are only used to illustrate the present invention, and are not used to limit the protection scope of the present invention. For those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a sensory receptor gene overexpressing cell in the preparation of a product for evaluating skin discomfort caused by a product, characterized in that: The sensory receptor gene overexpressing cells include TRPs receptor family overexpressing cells, histamine receptor family overexpressing cells, protease activated receptor family overexpressing cells and prostaglandin receptor family overexpressing cells.

2. The use according to claim 1, characterized in that: The TRPs receptor family includes at least one of TRPV1, TRPV2, TRPV3, TRPV4, TRPC1, TRPC4, TRPM8, and TRPA1; The histamine receptor family includes at least one of HRH1, HRH2, and HRH4; The protease activated receptors include at least one of PAR-2 ​​and PAR-4; The prostaglandin receptor family includes at least one of EP1 and EP4.

3. The use according to claim 1, characterized in that: The sensory receptor gene overexpressing cells also include at least one of endothelin receptor family overexpressing cells, acid-sensing ion channel receptor family overexpressing cells, adrenergic receptor family overexpressing cells, and Mrgpr receptor family overexpressing cells; The endothelin receptor family includes at least one of EDNRA and EDNRB; The acid-sensing ion channel receptor family includes ASIC3; The adrenergic receptor family includes at least one of ADRA1A, ADRA1B, and ADRA1D; The Mrgpr receptor family includes at least one of MRGPRX1, MRGPRX4, MRGPRF, MRGPRF, and MRGPRA3.

4. A cell chip for evaluating skin discomfort caused by a product, characterized in that: The cell chip includes detection wells containing overexpressed cells of sensory receptor genes; The sensory receptor genes include overexpression cells of the TRPs receptor family, overexpression cells of the histamine receptor family, overexpression cells of the protease activated receptor family, and overexpression cells of the prostaglandin receptor family; The TRPs receptor family includes at least one of TRPV1, TRPV2, TRPV3, TRPV4, TRPC1, TRPC4, TRPM8, and TRPA1; The histamine receptor family includes at least one of HRH1, HRH2, and HRH4; The protease activated receptors include at least one of PAR-2 ​​and PAR-4; The prostaglandin receptor family includes at least one of EP1 and EP4.

5. The cell chip for evaluating skin discomfort caused by a product according to claim 4, characterized in that: The sensory receptor gene overexpressing cells also include at least one of endothelin receptor family overexpressing cells, acid-sensing ion channel receptor family overexpressing cells, adrenergic receptor family overexpressing cells, and Mrgpr receptor family overexpressing cells; The endothelin receptor family includes at least one of EDNRA and EDNRB; The acid-sensing ion channel receptor family includes ASIC3; The adrenergic receptor family includes at least one of ADRA1A, ADRA1B, and ADRA1D; The Mrgpr receptor family includes at least one of MRGPRX1, MRGPRX4, MRGPRF, MRGPRF, and MRGPRA3.

6. The cell chip for evaluating skin discomfort caused by a product according to any one of claims 4-5, characterized in that: The sensory receptor gene overexpressing cell is a HaCaT cell or SH-SY5Y cell overexpressing the sensory receptor gene.

7. A method for evaluating skin discomfort caused by a product for non-disease diagnosis purposes, characterized in that: The following steps are involved: S1. Cell culture: Add culture medium to the detection wells of the overexpressing cells containing each sensory receptor gene in the cell chip according to any one of claims 4-6 for culture; S2. Staining: Discard the culture medium, wash with buffer, and then add a staining solution containing a calcium ion fluorescent probe to stain the overexpressing cells of each sensory receptor gene; S3. After the staining is completed, wash with buffer, then add buffer, prepare the test sample, and the instrument automatically adds the test sample and performs real-time detection; S4. Process the collected data, calculate the change multiple of the calcium ion signal fluorescence intensity of each detection well, then convert the change multiple of the calcium ion signal fluorescence intensity into a skin discomfort score, and then calculate the comprehensive score of skin discomfort. Finally, determine the level of skin discomfort caused by the test product based on the comprehensive score of skin discomfort.

8. The method for evaluating skin discomfort caused by a product according to claim 7, characterized in that: In step S4, the calculation formula for the change multiple of the calcium ion signal fluorescence intensity is shown as formula (1) below: RFU fold change =(RFU 待测产品组 -RFU 阴性对照组 ) / RFU 阴性对照缚 (1) Among them, RFU f old change RFU is the change factor of the fluorescence intensity of calcium ion signal; 待测产品组 RFU is the fluorescence intensity value of the calcium ion signal of the product to be tested. 阴性对照组 is the fluorescence intensity value of calcium ion signal after adding 1×HHBS buffer.

9. The method for evaluating skin discomfort caused by a product according to claim 7, characterized in that: In step S4, the conversion standard for the skin discomfort score is as follows: RFU f old change <3, DS = 0; 3≦RFU f old change ≦10, DS=1; 10 <RFU f old change ≦20, DS=2; 20 <RFU f old change ≦30, DS=3; 30 <RFU f old change ≦40, DS=4; RFU f old change >40, DS=5; where DS is the skin discomfort score.

10. The method for evaluating skin discomfort caused by a product according to claim 7, characterized in that: In step S4, the calculation formula for the comprehensive score of skin discomfort is shown as formula (2) below: TS=∑ i (DS i ) (2) where TS is the comprehensive score of skin discomfort; i is the number of sensory receptor genes detected; DSi is the average value of the skin discomfort scores of the sensory receptor genes in each replicate well; In step S4, the grading standard for skin discomfort is as follows: When 0 ≦ TS ≦ 1, the skin discomfort level is: no skin discomfort; When 1 < TS ≦ 2, the skin discomfort level is: mild skin discomfort; When TS > 2, the skin discomfort level is: moderate / severe skin discomfort.

Images