阅读说明：本技术 一种基于双细胞水平的皮肤光刺激性评价方法 (Skin light irritation evaluation method based on double-cell level ) 是由 黄健聪 程树军 冯鉴鸿 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种基于双细胞水平的皮肤光刺激性评价方法,包括以下步骤：(1)获取离体正常人皮肤角质细胞和成纤维细胞,进行细胞铺板；(2)取细胞铺板,去除上部培养液,分别加入一系列不同浓度的受试物,置于UV下进行辐照,记为UV+,在暗处放置相同时间,记为UV-；(3)暴露结束后,测试受试物在不同浓度下的细胞活性,计算IC<Sub>50</Sub>值,获取角质细胞和成纤维细胞IC<Sub>50</Sub>值(UV+)和IC<Sub>50</Sub>值(UV-)；(4)计算光刺激比值,根据该比值,将受试物分为非皮肤光刺激物、可疑皮肤光刺激物和皮肤光刺激物。该法具有快速、通量高、可重复性良好、与人相关性高等优点,可对多种受试物进行快速评价,大大减少了实验动物的使用。(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 calculated 50 Value, obtaining keratinocyte and fibroblast IC 50 Value (UV +) and IC 50 Value (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.)

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.

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