CN110628866A - Skin light irritation evaluation method based on double-cell level - Google Patents
Skin light irritation evaluation method based on double-cell level Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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Abstract
The invention discloses a skin light irritation evaluation method based on a double-cell level, which comprises the following steps of: (1) obtaining in vitro normal human skin keratinocytes and fibroblasts, and performing cell plating; (2) taking a cell plate, removing the upper culture solution, respectively adding a series of test substances with different concentrations, placing under UV for irradiation, and recording as UV +, placing in a dark place for the same time, and recording as UV-; (3) after exposure, the test substance was tested for cellular activity at different concentrations and IC was calculated50Value, obtaining keratinocyte and fibroblast IC50Value (UV +) and IC50Value (UV-); (4) calculating a ratio of light stimuli, and classifying the test substance into a non-skin light stimulus, a suspected skin light stimulus and a skin light stimulus according to the ratio. The method has the advantages of high speed, high flux, good repeatability, high correlation with human and the like, can quickly evaluate various tested substances, and greatly reduces the use of experimental animals.
Description
Technical Field
The invention belongs to the technical field of non-animal testing methods, and particularly relates to a skin light irritation evaluation method based on a double-cell level.
Background
Certain compounds, called light-sensitive substances, transform from a stable state to an excited state under the action of light (mainly ultraviolet light), and thus have a harmful effect on cell tissues. The skin covers the surface of the human body and is the outermost barrier between the human body and the external environment. However, the skin is an organ directly contacted with ultraviolet rays, and drugs, cosmetics and the like applied on the surface of the skin can cause light irritation, and usually shows itching, pain, red swelling, bright red papules and blisters on the irradiated part, and even serious injuries such as skin tissue necrosis and the like.
In daily contact with chemical substances, such as asphalt, coal tar, fuel and the like, in cosmetics and spices, such as bergamot essential oil, lemon oil, sandalwood oil and the like, in plants, such as lemon, amaranth, caraway and the like, in traditional Chinese medicines, such as fructus psoraleae, radix angelicae, divaricate saposhnikovia root and the like, in medicines, such as sulfanilamide, chlorpromazine, salicylate and estrogen in oral contraceptives, all are light-sensitive substances, and can cause photoreactivity of skin and even whole body.
The existing skin phototoxicity evaluation method is a guinea pig animal experiment, the skin of the guinea pig is irradiated after being smeared with a test object, and the erythema edema of the skin after irradiation is observed for evaluation and prediction. But the defects of poor subjectivity/repeatability, species difference, long period, high cost, low flux, pain and death of animals caused by using living animals and the like are gradually eliminated by modern toxicology. At present, the test method using in vitro technology as the core is gradually accepted and approved by the regulatory authorities in many countries and regions. The 3T3 phototoxicity test based on the cell level is accepted by a multi-party supervision organization and is widely applied to phototoxicity evaluation, but the test has a high false positive rate, adopts cells derived from mice, does not specifically aim at local skin photostimulation, and still has certain limitation. There is currently no recognized in vitro test method specifically for skin light irritation.
Human skin is composed mainly of the stratum corneum of the epidermis and dermal fibroblasts, and studies have shown that UVA radiation can reach the dermis, which is primarily the stratum corneum and dermis, while UVB is primarily the epidermis, and not the dermis. In addition, the isolated keratinocytes and fibroblasts of the skin in vitro can be cultured in vitro and generate corresponding biological functions, and the isolated keratinocytes and fibroblasts are widely applied to various skin-related safety and efficacy evaluations.
Disclosure of Invention
The invention aims to provide a method for evaluating the skin light irritation based on a double-cell level, which evaluates whether a substance to be tested has the skin light irritation by analyzing the existence of two skin-related cells, namely keratinocytes and fibroblasts and measuring the survival condition of the cells under the ultraviolet irradiation, has the characteristics of quickness, simplicity, convenience, high flux, high correlation with human and the like, can quickly evaluate various tested substances, and greatly reduces the use of experimental animals.
The above object of the present invention can be achieved by the following technical solutions: a skin light irritation evaluation method based on a double-cell level comprises the following steps:
(1) obtaining isolated normal human skin keratinocytes and fibroblasts, and respectively performing cell plating after culturing and identifying by adopting a conventional culture solution;
(2) taking the cells in the step (1), plating, removing the culture solution on the upper part, keeping the keratinocytes and the fibroblasts at the bottom, respectively adding a series of test substances with different concentrations, pretreating, placing under UV for irradiation, and recording as UV +, placing in a dark place for the same time and recording as UV-;
(3) after the exposure is finished, re-culturing is carried out, then the cell activity of the tested substance at different concentrations is tested, and the IC is calculated50Value, i.e. concentration which causes half of the cell inhibition, obtaining keratinocyte and fibroblast IC50Value (UV +) and IC50Value (UV-);
(4) calculating the ratio of light stimulus RPV to IC50(UV-)/IC50(UV +), according to which ratio the test substances are distinguished as non-skin light irritants, suspected skin light irritants and skin light irritants, as follows:
in the above method for evaluating skin light irritation based on a two-cell level:
optionally, the ex vivo normal human skin keratinocytes and fibroblasts in step (1) are derived from surgically isolated skin tissue.
In the invention, because the cells are derived from normal human skin, the experimental result can truly reflect the photodamage effect of two main cells (fibroblasts and keratinocytes) in normal human skin tissues on the tested substances, and the method can also be used as a supplement of the existing animal phototoxicity experiment and cell phototoxicity experiment.
Optionally, when the cells are plated in step (1): subjecting keratinocyte cell and fibroblast cell to treatment with (0.5-2) × 105Inoculating to 96-well plate at density of one/mL, culturing in incubator overnight for 18-22h under 37 + -0.5 deg.C and (5 + -1)% CO2Concentration, saturation humidity, two 96-well plates identical for each cell were prepared.
Optionally, one or more of the test chemical, surfactant, cosmetic perfume, plant extract, Chinese medicine extract and western medicine in step (2).
Further, the chemical substance in the step (2) is chlorpromazine hydrochloride (CPZ) and the like, and the surfactant is sodium dodecyl sulfate (SLS) and the like.
Optionally, the series of different concentrations of the test substance in step (2) is set in a gradient, and each test substance is set at 6-12 concentrations.
Alternatively, the series of different concentrations of the test substance in step (2) may be obtained by diluting with a buffer solution, which may be Hank's Balanced Salt Solution (HBSS), phosphate buffer solution containing calcium and magnesium ions (DPBS), Phosphate Buffer Solution (PBS), or the like.
Optionally, the pretreatment in the step (2) is to return to the incubator for further culture for 0.5-6 h.
Optionally, when the irradiation is carried out under UV in the step (2), the irradiation dose of the fibroblasts is UVA2-12J/cm2(ii) a The keratinocyte irradiation dose is UVA2-12J/cm2Or UVB100-1000mJ/cm2。
The method of the invention utilizes two human skin cells to be simultaneously exposed to a tested substance and UV irradiation, measures the cell activity by analyzing whether the UV irradiation exists or not, reflects the damage degree of the tested substance to the cells under the condition of existence of the UV irradiation, and predicts whether the tested substance is a skin light irritant or not.
Optionally, when re-culturing in the step (3), cleaning the cell layer for 1-3 times, adding the culture solution again, culturing in an incubator for 20 ± 2 hours, and measuring the cell activity of the test object under different concentrations.
Alternatively, in the step (3), when the cell activities of the test substances at different concentrations are measured, the relative cell activities at different concentrations are calculated by using the MTT, XTT, neutral Red or CCK-8 method.
Compared with the prior art, the invention has the following advantages:
(1) the method of the invention uses the human skin tissue, and the separated cells can maintain the corresponding biological function and have scientificity and human body relativity;
(2) the method of the invention tests through two main cells of the skin, avoids the limitation of a single cell, and approaches to the actual exposure condition;
(3) the method established by the invention can be directly used for evaluating the skin light irritation evaluation of the drugs and raw materials applied to the skin surface or skin contact product components;
(4) the method has the advantages of high flux, repeatability, high correlation with human, multi-index evaluation and the like, can quickly evaluate various tested objects, and greatly reduces the use of experimental animals.
Drawings
FIG. 1 is a morphological observation of cells in example 1, wherein A keratinocytes, B fibroblasts;
FIG. 2 is the result of the CPZ test of example 1, wherein A keratinocytes, B fibroblasts;
FIG. 3 is the SLS test results of example 1, wherein A keratinocytes, B fibroblasts;
FIG. 4 shows the results of the test sample of example 2, wherein A keratinocytes and B fibroblasts are present.
Detailed Description
Example 1
The method for evaluating the skin light irritation based on the double-cell level provided by the embodiment comprises the following steps of:
1. preparation of the experiment
1.1 sterile skin specimens of Normal humans (about 4 cm)2)；
1.2 culture solution:
fibroblast cell: DMEM medium containing 10% fetal bovine serum;
keratinocyte cell: Keratinocyte-SFM serum-free culture solution;
1.3 other reagents: 0.25% pancreatin-EDTA, HBSS, PBS buffer, 75% sterile alcohol, Dispases II, MTT solution, DMSO, chlorpromazine hydrochloride (CPZ), Sodium Lauryl Sulfate (SLS);
1.4 culture conditions: temperature 37. + -. 1 ℃ and 5% CO2Concentration, saturated relative humidity;
2 test procedure
2.1 cell isolation and in vitro culture
2.1.1 skin treatment: thoroughly washing fresh foreskin of infant cut by surgery with 0.01mol/L PBS buffer solution containing 100U/mL penicillin and 100ug/mL streptomycin for 2-3 times; then removing subcutaneous fat and connective tissue under aseptic conditions; cutting the cleaned foreskin into pieces of about 2.0mm x 3.0 mm; spreading the skin, soaking the epidermis facing upwards in 0.25% lyase (Dispases II) to separate epidermis and dermis, and digesting at 37 deg.C for 2 + -0.5 hr or at 4 deg.C overnight for 12-18 hr; separation: taking the separated skin pieces out of lyase (Dispases II), gently separating epidermis and dermis by using tweezers,
2.1.2 keratinocyte isolation: collecting epidermis, repeatedly washing in PBS containing double antibody for 2-3 times, and thoroughly cutting tissue with sterilized ophthalmic surgical scissors; cutting epidermis, placing in 0.25% pancreatin-EDTA, digesting at 37 deg.C for 15min, and blowing for several times to assist cell dissociation. After digestion, cells were collected by centrifugation and repeated 2 times. Filtration was performed with a 200 mesh filter and counted. After being resuspended, the cells are placed in a culture flask and then are placed in an incubator for culture. It takes 3-5 days to grow well.
2.1.3 fibroblast isolation: collecting dermis, repeatedly washing in PBS containing double antibody for 2-3 times, and thoroughly cutting dermal tissue with sterilized ophthalmic surgical scissors; placing the cut dermal tissue into a culture bottle, uniformly spreading, adding a small amount of culture medium, removing tissue fragments after the fibroblasts climb out, and adding a DMEM culture medium containing 10% newborn calf serum for culture. Passage was performed once 3 days after cell stabilization.
2.1.4 keratinocyte identification: morphological observations, staining of keratin
2.1.5 fibroblast identification: morphological observations, vimentin staining
2.2 test substance preparation
SLS was formulated with HBSS at a concentration of 100mg/mL, diluted with HBSS to 10mg/mL before use as the highest concentration tested, with 7 concentrations diluted 3.16-fold in sequence.
After dissolution of CPZ in ethanol, 0.1mg/mL of CPZ was diluted with HBSS before use as the highest concentration tested, and 7 concentrations were sequentially diluted.
2.3 cell plating
Cells were plated at 0.8 x 105The cells were inoculated in 96-well plates at a density of 100. mu.L/well and incubated in an incubator for 18-22 h. For each cell, 2 96-well plates were prepared, each 96-well plate was seeded in the middle 60 wells, and the peripheral wells were filled with PBS.
2.4 pretreatment of the test substance
The 96-well plate was removed, the stock culture (the keratinocytes and fibroblasts at the bottom were retained), and after diluting the test substance at 8 concentrations with HBSS, 100 μ L per well was added to the plate, while setting the control group (containing no test substance) and the solvent control group. Returning to the incubator for pretreatment for 1.5 h.
2.5 light Exposure
After the pretreatment, 1 culture plate of keratinocytes and fibroblasts was randomly removed and exposed to UV irradiation (UV +), with the fibroblast irradiation dose UVA 3J/cm2And the keratinocyte irradiation dose UVB is 500mJ/cm2The other plate was left in the dark (UV-) for the same time.
After irradiation, the cell layer was washed with DPBS 2 times, and fresh culture medium was added again for 20 + -1 h in the incubator. After the specified culture time is reached, the relative cell activities at each concentration are calculated by a method for measuring cell activities such as MTT/XTT/neutral red/CCK-8 and the like, based on 100% of the control group. In this example, an MTT method was used.
2.6 measurement of cell Activity
The cell status was observed, MTT solution (5mg/mL) was added to each well, and the incubator was left for 3 hours. The culture plate was removed, the MTT-containing culture solution was removed, 150. mu.L of DMSO was added to each well, formazan was dissolved by shaking at room temperature in the dark for 15min, and the absorbance value was measured at a wavelength of 570 nm. The cell activity was calculated at each concentration with the cell activity of the control group as 100%, and the results were expressed as mean ± standard deviation.
MTT is called 3- (4,5) -dimethylthiohia azo (-z-y1) -3, 5-di-phenylyttrazolumro amide in English, and has a chemical name of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide, a trade name: thiazole blue, a yellow dye, is a commonly used method for detecting cell survival and growth by MTT colorimetry.
IC calculation by relative cellular Activity Using non-Linear regression50Value, i.e. the concentration which causes half of the cell inhibition, obtaining the IC of keratinocytes and fibroblasts50(UV + and UV-), for a total of 4 ICs50The value is obtained. By calculating the ratio of photo-stimulation (RPV) to IC50UV+/IC50UV-. Each test is repeated at least 2 times, and the judgment can be made when a consistent prediction result is obtained.
Other conditions may be predicted as suspected skin light irritants, usually requiring a third test or prediction in conjunction with other types of tests/data, which the present invention has not done for the time being.
3 results
3.1: keratinocyte cell: under the optical microscope, the keratinocyte is observed to be polygonal or irregular, such as a 'paving stone' shape, and the keratinocyte is of typical epithelial-like characteristics, has high nuclear plasma proportion, compact cell arrangement and clear outline and good refractivity. It can be morphologically determined as keratinocytes. (see FIG. 1). The cells are identified to be positive for keratin by an immunocytochemistry method.
Fibroblast cell: under a light microscope, fibroblast cell bodies are fusiform or triangular, have 2-3 protrusions with different lengths, are oval, are positioned in the middle of the cell bodies, and are closely arranged after the cells are converged and run in a typical vortex shape and a radial shape (see figure 1). And identifying that the cells are Vimentin positive by adopting an immunocytochemistry method.
3.2 cytotoxic results
The results of the CPZ test are shown in table 1 and fig. 2.
TABLE 1 CPZ test results
The SLS test results are shown in fig. 3 and table 2.
TABLE 2 SLS test results
3.3 conclusion
According to the results in table 1 and table 2, the following predictions are made: CPZ is a skin light irritant and SLS is a non-skin light irritant.
Example 2
The method for evaluating the skin light irritation based on the double-cell level provided by the embodiment comprises the following steps of:
1. preparation of the experiment
1.1 human Primary skin keratinocytes and human Primary skin fibroblasts
1.2 culture solution: fibroblast cell: DMEM medium containing 10% fetal bovine serum
Keratinocyte cell: Keratinocyte-SFM serum-free culture solution
1.3 other reagents: 0.25% pancreatin-EDTA, HBSS, PBS buffer, 75% sterile alcohol, MTT solution, DMSO, sample 1 (some plant leaf extract).
1.4 culture conditions: temperature ofDegree of 37 +/-1 ℃ and 5% CO2Concentration, saturated relative humidity
2 test procedure
2.1 test substance preparation
The test article (sample 1) was formulated with HBSS at a concentration of 200mg/mL, diluted to 100mg/mL with HBSS prior to use, and subsequently diluted in a 10-fold gradient to set a total of 9 concentrations tested.
2.2 cell plating
Cells were plated at 0.8 x 105The cells were inoculated in 96-well plates at a density of 100. mu.L/well and incubated in an incubator for 18-22 h. For each cell, 2 96-well plates were prepared, each 96-well plate was seeded in the middle 60 wells, and the peripheral wells were filled with PBS.
2.3 pretreatment of the test substance
The 96-well plate was removed, the original culture solution was removed, and after the test substance was diluted to 9 concentrations in HBSS, 100. mu.L per well was added to the plate, while the culture solution control group and the solvent control group were set. Returning to the incubator for pretreatment for 1.5 h.
2.4 light Exposure
After the pretreatment, 1 culture plate of keratinocytes and fibroblasts was randomly removed and exposed to UV irradiation (UV +), with the fibroblast irradiation dose UVA 3J/cm2And the keratinocyte irradiation dose UVB is 500mJ/cm2The other plate was left in the dark (UV-) for the same time.
After irradiation, the cell layer was washed with DPBS 2 times, and fresh culture medium was added again for 20 + -1 h in the incubator.
2.5 measurement of cellular Activity
The cell status was observed, MTT solution (5mg/mL) was added to each well, and the incubator was left for 3 hours. The culture plate was removed, the MTT-containing culture solution was removed, 150. mu.L of DMSO (dimethyl sulfoxide) was added to each well, formazan was dissolved by shaking at room temperature in the dark for 15min, and the absorbance value was measured at a wavelength of 570 nm. The cell activity was calculated at each concentration with the cell activity of the control group as 100%, and the results were expressed as mean ± standard deviation.
2.6 prediction of results
Calculating the ratio of light stimulus RPV to IC50(UV-)/IC50(UV+)
3 results
3.1 cytotoxic results
The cytotoxicity results are shown in table 3 and fig. 4.
Table 3 sample 1 test results
3.2 conclusion
And predicting according to the result: sample 1 was a non-skin light irritant.
Example 3
The method for evaluating the skin light irritation based on the double-cell level provided by the embodiment comprises the following steps of:
1. preparation of the experiment
1.1 human Primary skin keratinocytes and human Primary skin fibroblasts
1.2 culture solution: fibroblast cell: DMEM medium containing 10% fetal bovine serum
Keratinocyte cell: Keratinocyte-SFM serum-free culture solution
1.3 other reagents: 0.25% pancreatin-EDTA, HBSS, PBS buffer, 75% sterile alcohol, neutral Red, anhydrous alcohol, glacial acetic acid, sample 2 (certain herb extract)
1.4 culture conditions: temperature 37. + -. 1 ℃ and 5% CO2Concentration, saturated relative humidity
2 test procedure
2.1 test substance preparation
The test substance (sample 2) was formulated with HBSS at a concentration of 200mg/mL, diluted with HBSS to 100mg/mL prior to use, and subsequently diluted in a 10-fold gradient to set a total of 9 concentrations tested.
2.2 cell plating
Cells were plated at 0.8 x 105The cells were inoculated in 96-well plates at a density of 100. mu.L/well and incubated in an incubator for 18-22 h. Preparing 2 96-well plates for each cell, inoculating 60 wells in the middle of each 96-well plate, and inoculating four wells to each 96-well plateThe PBS was filled.
2.3 pretreatment of the test substance
The 96-well plate was removed, the original culture solution was removed, and after the test substance was diluted to 9 concentrations in HBSS, 100. mu.L per well was added to the plate, while a culture solution control group (containing no test substance) and a solvent control group were set. Returning to the incubator for pretreatment for 1.5 h.
2.4 light Exposure
After the pretreatment, 1 culture plate of keratinocytes and fibroblasts was randomly removed and exposed to UV irradiation (UV +), with the fibroblast irradiation dose being UVA 4J/cm2And keratinocyte irradiation dose UVB 700mJ/cm2The other plate was left in the dark (UV-) for the same time.
After irradiation, the cell layer was washed with DPBS 2 times, and fresh culture medium was added again for 20 + -1 h in the incubator.
2.5 measurement of cellular Activity
The state of the cells was observed, the culture medium was removed from the wells, a neutral red solution (50. mu.l/mL) was added to each well, and the incubator was left for 3 hours. The plate was removed, the wells were washed 1 time with buffer, 200. mu.L of desorption solution (absolute ethanol: water: glacial acetic acid 49:50:1) was added to each well, the mixture was shaken at room temperature in the dark for 25 to 30min, neutral red was dissolved, and the absorbance value was measured at a wavelength of 540 nm. The cell activity was calculated at each concentration with the cell activity of the control group as 100%, and the results were expressed as mean ± standard deviation.
2.6 prediction of results
Calculating the ratio of light stimulus RPV to IC50(UV-)/IC50(UV+)
3 results
3.1 cytotoxicity results: the cytotoxicity results are shown in table 4.
Table 4 sample 1 test results
3.2 conclusion
And predicting according to the result: sample 2 was a suspected skin light irritant.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention. The present invention is only illustrative of some of the chemical and surfactant samples. However, other non-single component systems mentioned in the present invention, such as daily exposure to chemicals, cosmetics, perfumes, plants, plant extracts, herbs, herbal extracts, etc., as well as drugs and oral contraceptives, etc., can also be evaluated for skin photostimulation by the method of the present invention, and in addition, when measuring the cell activity of the test substance at different concentrations, the relative cell activity at different concentrations can be calculated by methods such as XTT or CCK-8, besides the MTT method and the neutrophilic erythropoiesis method, which are not listed herein.
Claims (10)
1. A skin light irritation evaluation method based on a double-cell level is characterized by comprising the following steps:
(1) obtaining isolated normal human skin keratinocytes and fibroblasts, and respectively performing cell plating after culturing and identifying by adopting a conventional culture solution;
(2) taking the cells in the step (1), plating, removing the culture solution on the upper part, keeping the keratinocytes and the fibroblasts at the bottom, respectively adding a series of test substances with different concentrations, pretreating, placing under UV for irradiation, and recording as UV +, placing in a dark place for the same time and recording as UV-;
(3) after the exposure is finished, re-culturing is carried out, then the cell activity of the tested substance at different concentrations is tested, and the IC is calculated50Value, i.e. concentration which causes half of the cell inhibition, obtaining keratinocyte and fibroblast IC50Value (UV +) and IC50Value (UV-);
(4) calculating the ratio of light stimulus RPV to IC50(UV-)/IC50(UV +) according to the ratioThe values, which distinguish the test subjects as non-skin light irritants, suspected skin light irritants, and skin light irritants, are as follows:
2. the method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: the ex vivo normal human skin keratinocytes and fibroblasts in step (1) are derived from surgically isolated skin tissue.
3. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: when the cells are plated in the step (1): subjecting keratinocyte cell and fibroblast cell to treatment with (0.5-2) × 105Inoculating to 96-well plate at density of one/mL, culturing in incubator overnight for 18-22h under 37 + -0.5 deg.C and (5 + -1)% CO2Concentration, saturation humidity, two 96-well plates identical for each cell were prepared.
4. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: the test substance in the step (2) comprises one or more of chemical substances, surfactants, cosmetic perfumes, plants, plant extracts, traditional Chinese medicines, traditional Chinese medicine extracts and western medicines.
5. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: in the step (2), the series of test objects with different concentrations are arranged in a gradient way, and each test object is provided with 6-12 concentrations.
6. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: the series of test substances with different concentrations in the step (2) are obtained by diluting with a buffer solution, wherein the buffer solution is Hank's Balanced Salt Solution (HBSS), phosphate buffer solution (DPBS) containing calcium and magnesium ions or Phosphate Buffer Solution (PBS).
7. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: and (3) returning the pretreatment in the step (2) to an incubator for continuous culture for 0.5-6 h.
8. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: when the fibroblast is irradiated under UV in the step (2), the irradiation dose of the fibroblast is UVA2-12J/cm2(ii) a The keratinocyte irradiation dose is UVA2-12J/cm2Or UVB100-1000mJ/cm2。
9. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: and (4) when re-culturing in the step (3), cleaning the cell layer for 1-3 times, adding the culture solution again, culturing for 20 +/-2 hours in the incubator, and measuring the cell activity of the tested object under different concentrations.
10. The method for evaluating skin light irritation based on a two-cell level according to claim 1, wherein: when the cell activity of the test substance is measured at different concentrations in the step (3), the relative cell activity at different concentrations is calculated by using a method of MTT, XTT, neutral red or CCK-8.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0497399A1 (en) * | 1991-01-28 | 1992-08-05 | The Procter & Gamble Company | Process for evaluating skin irritancy |
| US20100075360A1 (en) * | 2008-09-22 | 2010-03-25 | Muhammed Majeed | Uv based cell viability as an indicator of sun protection factor and methods of measurement thereof |
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|---|---|---|---|---|
| EP0497399A1 (en) * | 1991-01-28 | 1992-08-05 | The Procter & Gamble Company | Process for evaluating skin irritancy |
| US20100075360A1 (en) * | 2008-09-22 | 2010-03-25 | Muhammed Majeed | Uv based cell viability as an indicator of sun protection factor and methods of measurement thereof |
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| ALENA RAJNOCHOVÁ SVOBODOVÁ等: "Human keratinocyte cell line as a suitable alternative model for in vitro phototoxicity testing", 《AN BRAS DERMATOL》 * |
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