Marking factor for detecting skin sensitization and application thereof

Abstract

The invention discloses a marker factor for detecting skin sensitization and application of the marker factor. The marker factor comprises a DDIT4 gene and / or a CXCL8 gene. Whether skin sensitization exists or not is judged by detecting relative expression quantity changes of DDIT4 and CXCL8 genes after to-be-detected cosmetics, to-be-detected medical instruments or food related products and THP-1 cells are co-exposed, the method is a powerful supplement for skin sensitization detection through an h-CLAT method at present, and the method has the advantages of being rapid, sensitive, low in cost and the like, can meet the detection requirements of a large number of products and has a good application prospect. The sensitization risk of chemicals or medical instruments is preliminarily evaluated, and the requirements of industry development and supervision are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarker, and more specifically, the present invention relates to a biomarker and method for detecting skin sensitization. Background Art

[0002] Skin sensitization, also known as allergic contact dermatitis, is a delayed hypersensitivity reaction triggered by skin contact with sensitizing substances, and the clinical manifestations are local skin redness, swelling and itching. Due to its prevalence, persistence and impact on quality of life, skin allergy is recognized as an important occupational and environmental health problem, so it is very important to detect the skin sensitization of chemicals and cosmetic ingredients.

[0003] Skin sensitization is divided into two stages: induction and elicitation stages. Skin sensitization test is a requirement for chemical classification and labeling, and also an important part of toxicity evaluation. The classical skin sensitization detection methods are the local lymph node assay (LLNA) and the guinea pig maximization test (GPMT), both of which are animal evaluation methods. However, with the development of the "3R" principle and modern technology, the traditional animal test methods have gradually been unable to meet the needs of market development due to the relatively long cycle and large demand for animals. The in vitro alternative methods for skin sensitization mainly target different key events in the adverse outcome pathway, and the detection indicators are mostly the expression of the final product proteins, which makes these alternative methods have certain limitations, and the prediction accuracy may also be lower than that of animal experiments.

[0004] The human cell line activation test, namely the h-CLAT method (Human cell line activation test), simulates key event 3 (dendritic cell activation) of the AOP (Adverse outcome pathway) pathway during skin sensitization. After the sensitizer contacts the skin, dendritic cells differentiate and mature during the process of migrating to lymphoid organs, and up-regulate the expression of a series of cell surface molecules such as CD86 and CD54. In OECD 442E and the "Technical Specifications for Cosmetic Safety" in China, the h-CLAT method is to co-expose the in vitro cultured dendritic cell-like THP-1 with the test substance for 24 hours, then add dyes to perform fluorescence staining on CD54 and CD86, and use a flow cytometer to measure the expression of the two molecules CD86 and CD54. However, at present, most domestic research institutions or enterprises do not have high-end instruments such as flow cytometers, and the cost of flow antibodies is relatively high. The overall test operation is relatively complex, requiring high ability of the test personnel, and the test cycle is long. Therefore, it limits the practical use of this method. Summary of the Invention

[0005] Based on this, the object of the present invention is to provide a biomarker for detecting skin sensitization.

[0006] The technical solutions for achieving the above-mentioned invention objectives are as follows.

[0007] In the first aspect of the present invention, a marker factor for detecting skin sensitization is provided, which includes the DDIT4 gene and / or the CXCL8 gene.

[0008] In the second aspect of the present invention, the application of a reagent for detecting the expression level of a marker factor in the preparation of a product for detecting skin sensitization is provided, wherein the marker factor includes the DDIT4 gene and / or the CXCL8 gene.

[0009] In the third aspect of the present invention, a method for detecting skin sensitization is provided, including the following steps: after co-exposing a test substance to be detected with THP-1 cells, using the cDNA of the THP-1 cells as a template, and performing a qPCR reaction with the primers shown in SEQ ID NO:1 and SEQ ID NO:2, and / or SEQ ID NO:3 and SEQ ID NO:4.

[0010] Based on RNA-Seq sequencing, the inventors of the present invention performed differential gene expression analysis, target gene prediction, and enrichment analysis of target genes on all mRNAs of THP-1 cells co-exposed with sensitizers, screened 3 candidate marker factors for detecting skin sensitization, and further verified these 3 candidate marker factors by qRT-PCR, and determined that the DDIT4 gene and the CXCL8 gene can be used as marker factors for detecting skin sensitization.

[0011] The method for detecting skin sensitization of the present invention is to determine whether there is skin sensitization by detecting the change in the relative expression levels of the DDIT4 gene and the CXCL8 gene in THP-1 cells after co-exposing a test substance to be detected (including cosmetic raw materials or medical devices) with THP-1 cells. This detection method is consistent with the results of detecting the expression of CD86 and CD54 in THP-1 cells by the h-CLAT method at present. Therefore, the method of the present invention is a powerful supplement to the current h-CLAT method for detecting skin sensitization, and has the advantages of being fast, sensitive, and low-cost, can meet the detection needs of a large number of products, preliminarily evaluate the sensitization risk of chemicals, medical devices or food-related products, and meet the needs of industry development and supervision. Brief Description of the Drawings

[0012] Figure 1Quality detection results of total RNA of THP-1 cells in each allergen group in Example 1 of the present invention. Among them, lanes 1-3: blank control group; lanes 4-6: 2,4-dinitrochlorobenzene (DNCB) group; lanes 7-9: p-phenylenediamine (PPDA) group; lanes 10-12: nickel sulfate (NiSO4) group; lanes 13-15: 2-mercaptobenzothiazole group; lanes 16-18: limonene group; lanes 19-21: imidazolidinyl urea group.

[0013] Figure 2 Principal component analysis (PCA) results in Example 1 of the present invention. Among them, C: blank control group; D: DNCB group; M: imidazolidinyl urea group; N: limonene group; Ni: NiSO4 group; P: PPDA group; S: 2-mercaptobenzothiazole group.

[0014] Figure 3 mRNA heat map analysis results of different allergen groups and blank control group in Example 1 of the present invention.

[0015] Figure 4 Multi-group differential scatter plot in Example 1 of the present invention. Among them, C: blank control group; D: DNCB group; M: imidazolidinyl urea group; N: limonene group; Ni: NiSO4 group; P: PPDA group; S is 2-mercaptobenzothiazole group.

[0016] Figure 5 GO significant enrichment bubble chart in Example 1 of the present invention.

[0017] Figure 6 GO enrichment bar chart in Example 1 of the present invention.

[0018] Figure 7 KEGG significant enrichment bubble chart in Example 1 of the present invention.

[0019] Figure 8 KEGG significant enrichment bar chart in Example 1 of the present invention.

[0020] Figure 9 Relative expression levels of DDIT4 gene in each allergen group in Example 2 of the present invention; among them, compared with the blank control group, **P < 0.01.

[0021] Figure 10 Relative expression levels of CXCL8 gene in each allergen group in Example 2 of the present invention; among them, compared with the blank control group, **P < 0.01.

[0022] Figure 11 Relative expression levels of CXCL10 gene in each allergen group in Example 2 of the present invention; among them, compared with the blank control group, *P < 0.05, **P < 0.01.

[0023] Figure 12 The relative mRNA expression level of the DDIT4 gene after the co-exposure of cosmetic raw material 1 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0024] Figure 13 The relative mRNA expression level of the CXCL8 gene after the co-exposure of cosmetic raw material 1 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0025] Figure 14 The relative mRNA expression level of the DDIT4 gene after the co-exposure of cosmetic raw material 2 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0026] Figure 15 The relative mRNA expression level of the CXCL8 gene after the co-exposure of cosmetic raw material 2 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0027] Figure 16 The relative mRNA expression level of the DDIT4 gene after the co-exposure of cosmetic raw material 3 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0028] Figure 17 The relative mRNA expression level of the CXCL8 gene after the co-exposure of cosmetic raw material 3 and THP-1 cells in Example 3 of the present invention. Among them, the positive control was 3.00 μg / mL DNCB.

[0029] Figure 18 The detection result of CD54 after the co-exposure of cosmetic raw material 1 and THP-1 cells in Example 4 of the present invention.

[0030] Figure 19 The detection result of CD86 after the co-exposure of cosmetic raw material 1 and THP-1 cells in Example 4 of the present invention.

[0031] Figure 20 The detection result of CD54 after the co-exposure of cosmetic raw material 2 and THP-1 cells in Example 4 of the present invention.

[0032] Figure 21 The detection result of CD86 after the co-exposure of cosmetic raw material 2 and THP-1 cells in Example 4 of the present invention.

[0033] Figure 22This is the detection result of CD54 after the co - exposure of cosmetic raw material 3 and THP - 1 cells in Example 4 of the present invention.

[0034] Figure 23 This is the detection result of CD86 after the co - exposure of cosmetic raw material 3 and THP - 1 cells in Example 4 of the present invention.

[0035] Above Figures 18 to 23 , the solid line of the circular marker represents the relative cell survival rate; the solid line of the square marker represents the relative fluorescence intensity of CD54 or CD86; the black dashed line represents the fluorescence threshold for sensitization and non - sensitization; compared with the blank control group, **P < 0.01. Detailed implementation manners

[0036] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0038] The experimental methods without specific conditions noted in the following examples are generally in accordance with conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2013, or in accordance with the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0039] In some embodiments of the present invention, a biomarker for detecting skin sensitization is disclosed, which includes the DDIT4 gene and / or the CXCL8 gene.

[0040] In other embodiments of the present invention, the application of a reagent for detecting the expression level of the above - mentioned biomarker in the preparation of a product for detecting skin sensitization is disclosed, and the biomarker includes the DDIT4 gene and / or the CXCL8 gene.

[0041] In some embodiments, the expression level of the biomarker is detected by polymerase chain reaction technology, gene sequencing technology or protein immunoblotting technology.

[0042] In some of these embodiments, the polymerase chain reaction is qRT-PCR.

[0043] In some of these embodiments, the nucleotide sequences of the primers used in the qRT-PCR are as shown in SEQ ID NO:1 and SEQ ID NO:2, and / or, the nucleotide sequences of the primers used in the qRT-PCR are as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0044] In some of these embodiments, the product is a kit, a test strip or a chip.

[0045] In some of these embodiments, the kit includes a qRT-PCR kit, a Western blot detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, an immunohistochemical detection kit or an ELISA kit.

[0046] In some other embodiments of the present invention, a method for detecting skin sensitization is disclosed, comprising the following steps: after co-exposing a test substance to be tested with THP-1 cells, using the cDNA of the THP-1 cells as a template, performing a qPCR reaction with the primers shown in SEQ ID NO:1 and SEQ ID NO:2, and / or SEQ ID NO:3 and SEQ ID NO:4.

[0047] In some of these embodiments, the time of the co-exposure is 22h to 26h.

[0048] In some of these embodiments, when the test substance is a cosmetic, a medical device or a food-related product. When the test substance is a cosmetic or its raw material, a powdered medical device, a liquid medical device or a food-related product, it is formulated into a solution (the concentration matches its cytotoxicity result), and is mixed with the THP-1 cell suspension in equal proportion for 22h to 26h, and then a qRT-PCR reaction is carried out. When the test substance is a solid medical device, the solid medical device is extracted with a culture medium (such as RPMI 1640 medium) to obtain an extract, and then the extract is mixed with the THP-1 cell suspension in equal proportion for 22h to 26h, and then a qPCR reaction is carried out.

[0049] In some of these embodiments, it further includes a step of analyzing the result of the qPCR reaction: if the relative expression levels of the DDIT4 gene and / or the CXCL8 gene at the transcriptome level increase significantly, the test cosmetic, the test medical device or the food-related product has skin sensitization.

[0050] The THP-1 cells used in the following embodiments of the present invention were purchased from the National Institutes for Food and Drug Control. DNCB and PPDA were purchased from Aladdin; NiSO4, 2-mercaptobenzothiazole, imidazolidinyl urea, and limonene were purchased from Sigma, USA. RPMI 1640 medium and fetal bovine serum were purchased from Gibco, USA. TRIzol for total RNA extraction was purchased from Invitrogen, and PrimeScript TM RT MasterMix and TB Fast qPCR Mix were purchased from TaKaRa.

[0051] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0052] Example 1 Screening and detecting candidate biomarker factors for skin sensitization

[0053] In this example, genes with differential expression in dendritic cell-like THP-1 were screened by RNA-Seq sequencing and fluorescence quantitative PCR to obtain candidate biomarker factors that can be used to detect skin sensitization. The steps are as follows:

[0054] 1. Uniformly seed THP-1 cells at a quantity of 1×10 6 cells in a 24-well plate, add an equal volume of the test substance solution with twice the concentration, and incubate for 3 h; the test substance solution includes: 2 strong sensitizers (3.00 μg / mL DNCB, 24.00 μg / mL PPDA), 2 medium-strength sensitizers (40.00 μg / mL NiSO4, 200.00 μg / mL 2-mercaptobenzothiazole), and 2 weak sensitizers (500.00 μg / mL limonene, 40.00 μg / mL imidazolidinyl urea);

[0055] 2. Use the TRIzol method (collect THP-1 cells and cultures in a 1.5 mL centrifuge tube, centrifuge at 1000 rpm / min for 5 minutes, discard the supernatant, add PBS to resuspend the cells, and centrifuge once to obtain THP-1 cells; add TRIzol to lyse for 10 minutes, add 1 / 5 volume of chloroform, mix the liquid up and down, vortex vigorously for 15 minutes, centrifuge at 4°C and 12000 rpm / min for 15 minutes; carefully aspirate 400 μL of the supernatant and place the liquid in a new EP tube, add an equal volume of isopropanol, mix the liquid up and down, let stand for 15 minutes, centrifuge at 4°C and 12000 rpm / min for 15 minutes; after aspirating the supernatant, add 75% ethanol to wash 2 times, each time centrifuge at 4°C and 7500 rpm / min for 5 minutes. Open the lid and dry for 20 min) to extract the total RNA of THP-1 cells in different sensitizer groups; add 50 μL of enzyme-free water to dissolve the total RNA, and use an ultraviolet spectrophotometer to detect the purity and concentration of the total RNA.

[0056] 3. Hand over the total RNA samples to Guangzhou GeneDeeo Biotechnology Co., Ltd. for RNA quality detection and transcriptome sequencing. The results of the total RNA quality detection of THP-1 cells in different allergen groups are as Figure 1 shown. It can be seen from Figure 1 that the RNA quality detections of the cells corresponding to the blank control, 3.00 μg / mL DNCB, 24.00 μg / mL PPDA, 40.00 μg / mL NiSO4, 200.00 μg / mL 2-mercaptobenzothiazole, 40.00 μg / mL imidazolidinyl urea, and 500.00 μg / mL limonene groups are all qualified. Process the original sequencing data of each sample to obtain the mRNA expression profile, and conduct differential gene expression analysis, target gene prediction, and enrichment analysis of the target genes for all mRNAs. The specific steps are as follows:

[0057] (1) Based on the gene expression level information, use R (gmodels package) to perform principal component analysis (PCA, Principal Component Analysis) for comparison between intra-group and inter-group samples, and analyze the repeatability of intra-group samples and the difference degree of inter-group samples. The results of the principal component analysis are shown in Figure 2 . Except for one sample in the limonene group with a slightly larger deviation, the samples in the other groups have a relatively high similarity in gene expression patterns.

[0058] (2) Genes with similar expression patterns may have common functions or participate in common metabolic pathways and signaling pathways. Perform hierarchical clustering on the differential gene expression patterns, perform z-score processing on each gene, and use a heatmap to present the clustering results. Use a gradient color to intuitively display the expression levels of significantly different genes in each sample or group, so as to show the variation rules of genes in different samples or groups, and perform clustering analysis on samples and genes according to the gene expression levels. The heatmap analysis results of different allergens and the blank control are shown in Figure 3 .

[0059] (3) The screening criteria for differential mRNAs are that the expression level changes by more than 2 times and the P value is less than 0.05. Differential expression analysis was performed on all mRNAs. Taking the logarithm of the fold change between groups log2FC as the ordinate and the name of the comparison group as the abscissa, the multi-group differential scatter plot is shown in Figure 4The results showed that compared with the blank control group, there were 756 up-regulated genes and 2,338 down-regulated genes in the 3.00 μg / mL DNCB group; 102 up-regulated genes and 26 down-regulated genes in the 24.00 μg / mL PPDA group; 9 up-regulated genes and 2 down-regulated genes in the 40.00 μg / mL NiSO4 group; 284 up-regulated genes and 408 down-regulated genes in the 200.00 μg / mL 2-mercaptobenzothiazole group; 71 up-regulated genes and 440 down-regulated genes in the 40.00 μg / mL imidazolidinyl urea group; and 546 up-regulated genes and 1,323 down-regulated genes in the 500.00 μg / mL limonene group.

[0060] (4) Perform enrichment analysis of target genes for differentially expressed genes.

[0061] The results of GO enrichment analysis are as Figure 5 and Figure 6 shown. The target genes were mainly concentrated in pathways such as DNA binding in the regulatory region of RNA polymerase II, sequence-specific DNA binding in transcriptional regulatory sequences, DNA binding in regulatory regions, transcription of polymerase II promoters, macromolecule biosynthesis processes, metabolic processes of nucleobase-containing compounds, regulation of DNA and nucleic acid template transcription, and metabolic processes of cellulose aromatic compounds.

[0062] The results of KEGG enrichment analysis are as Figure 7 and Figure 8 shown. The target genes were mainly concentrated in pathways such as TGF-β signaling pathway, P53 signaling pathway, Toll-like receptor signaling pathway, JAK-STAT signaling pathway, ferroptosis, Notch signaling pathway, TNF signaling pathway, inflammation, NOD-like receptor signaling pathway, IL-17 signaling pathway, and interaction between factors and factor receptors.

[0063] Through the above steps, 3 significantly differentially expressed genes - DDIT4, CXCL8, and CXCL10 were finally screened out, which can be used as candidate biomarker factors for detecting skin sensitization.

[0064] Example 2 Determination of biomarker factors for detecting skin sensitization

[0065] In this example, qRT-PCR was used to verify the 3 candidate biomarker factors screened in Example 1, and it was determined that DDIT4 and CXCL8 can be used as biomarker factors for detecting skin sensitization. The steps are as follows:

[0066] 1. Design specific primers for the internal reference genes GAPDH, CXCL8, DDIT4, and CXCL10 genes, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primers are shown in Table 1.

[0067] Table 1

[0068] Gene Primer sequence GAPDH-F 5’-CCACTCCTCCACCTTTGAC-3’ (SEQ ID NO:5) GAPDH-R 5’-ACCCTGTTGCTGTAGCCA-3’ (SEQ ID NO:6) CXCL8-F 5’-TTTTGCCAAGGAGTGCTAAAGA-3’ (SEQ ID NO:1) CXCL8-R 5’-AACCCTCTGCACCCAGTTTTC-3’ (SEQ ID NO:2) DDIT4-F 5’-CGGAGGAAGACACGGCTTACC-3’ (SEQ ID NO:3) DDIT4-R 5’-GCTTACCAACTGGCTAGGCATCA-3’ (SEQ ID NO:4) CXCL10-F 5’-CCTGCTTCAAATATTTCCCT-3’ (SEQ ID NO:7) CXCL10-R 5’-CCTTCCTGTATGTGTTTGGA-3’ (SEQ ID NO:8)

[0069] 2. Extract the RNA of THP-1 cells treated with the sensitizer for 3 h (the sensitizer, its concentration, method, etc. are the same as in Example 1), and perform reverse transcription (the reaction system is: 2 μL of 5×PrimerScript RT MasterMix (Perfect Real Time), 5 μL of RNA (80 μg / μL), and add nuclease-free water to 10 μL; the reaction program is: 37°C for 15 min; 85°C for 5 sec; 4°C) to obtain cDNA.

[0070] 3. Add 30 μL of nuclease-free water to dilute the cDNA to obtain diluted cDNA; perform qPCR reaction. Among them, the amplification reaction system is shown in Table 2. The reaction conditions are: 95°C for 5 min; 95°C for 5 sec, 60°C for 30 sec, for 40 cycles. Melting conditions: 95°C for 1 min, 60°C for 15 sec, 98°C for 5 sec.

[0071] Table 2

[0072]

[0073]

[0074] The qPCR results are respectively as Figures 9 to 11 shown.

[0075] Compared with the blank control group, 3.00 μg / mL DNCB, 24.00 μg / mL PPDA, 40.00 μg / mL NiSO4, 200.00 μg / mL 2-mercaptobenzothiazole, 500.00 μg / mL limonene, and 40.00 μg / mL imidazolidinyl urea all caused an up-regulation of the relative expression level of the DDIT4 gene in THP-1 cells, and the difference was statistically significant (P<0.01)( Figure 9 ).

[0076] Compared with the blank control group, 3.00 μg / mL DNCB, 24.00 μg / mL PPDA, 40.00 μg / mL NiSO4, 200.00 μg / mL 2-mercaptobenzothiazole, 500.00 μg / mL limonene, and 40.00 μg / mL imidazolidinyl urea all caused an up-regulation of the relative expression level of the CXCL8 gene in THP-1 cells, and the difference was statistically significant (P<0.01)( Figure 10 ).

[0077] Compared with the blank control group, 24.00 μg / mL PPDA, 40.00 μg / mL NiSO4, 200.00 μg / mL 2-mercaptobenzothiazole, and 500.00 μg / mL limonene all caused an up-regulation of the relative expression level of the CXCL10 gene in THP-1 cells, with statistically significant differences (P<0.01). 3.00 μg / mL DNCB and 40.00 μg / mL imidazolidinyl urea caused a down-regulation of the relative expression level of the CXCL10 gene in THP-1 cells, with statistically significant differences (P<0.05). Figure 11 )

[0078] Therefore, the DDIT4 and CXCL8 genes can be used as marker factors for detecting skin sensitization.

[0079] Example 3 Detection of the sensitization of cosmetic raw materials using the marker factors of the present invention

[0080] Based on the fluorescence quantitative PCR method, this example detects whether cosmetic raw material 1, cosmetic raw material 2, and cosmetic raw material 3 have potential skin sensitization by detecting the relative expression levels of the marker factors DDIT4 and CXCL8 for detecting skin sensitization screened in Example 2. It includes the following steps:

[0081] 1. Cell seeding: Seed THP-1 cells at a density of 2×10 6 cells / mL, and pipette 500 μL of the cell suspension onto a 24-well plate.

[0082] 2. Co-exposure: Add 500 μL of each prepared sample working solution (including cosmetic raw material 1 at concentrations of 1.25 mg / mL, 2.50 mg / mL, and 5.00 mg / mL, cosmetic raw material 2 at concentrations of 0.25 mg / mL, 0.50 mg / mL, and 1.00 mg / mL, and cosmetic raw material 3 at concentrations of 1.25 mg / mL, 2.50 mg / mL, and 5.00 mg / mL) to the 24-well plate and co-expose for 24 h.

[0083] 3. Cell collection: After 24 h of co-exposure, transfer to a 1.5 mL centrifuge tube, wash the cells 2 times with staining buffer, and obtain the cells.

[0084] 4. Extract the RNA of THP-1 cells, reverse transcribe to obtain cDNA, and perform qPCR reactions, with the method the same as in Example 1.

[0085] The real-time fluorescence quantitative results are calculated using the 2 -△△CT method. Independent sample T-test analysis is used, and P<0.05 indicates that the difference is statistically significant. The detection results of the DDIT4 gene and CXCL8 gene in THP-1 cells after co-exposure with cosmetic raw material 1 are as shown in Figure 12 andFigure 13 As shown, the detection results of the DDIT4 gene and CXCL8 gene in THP-1 cells after co-exposure with cosmetic raw material 2 are as Figure 14 and Figure 15 shown. The detection results of the DDIT4 gene and CXCL8 gene in THP-1 cells after co-exposure with cosmetic raw material 3 are as Figure 16 and Figure 17 shown.

[0086] The results showed that there was no statistically significant difference in the relative expression levels of the DDIT4 and CXCL8 genes in THP-1 cells co-exposed with three doses of cosmetic raw material 1 and cosmetic raw material 2 (P>0.05). Therefore, cosmetic raw material 1 and cosmetic raw material 2 do not have potential skin sensitization. The relative expression levels of the DDIT4 and CXCL8 genes in THP-1 cells co-exposed with three doses of cosmetic raw material 3 were up-regulated, and the difference was statistically significant (P<0.05). Therefore, cosmetic raw material 3 has potential skin sensitization.

[0087] Example 4 Verification of the accuracy of the biomarker and detection method of the present invention

[0088] In this example, the h-CLAT method was used to detect the expression of CD86 and CD54 in THP-1 cells after co-exposure with cosmetic raw materials 1-3 to verify the accuracy of the method for detecting skin sensitization using the screened biomarkers of the present invention. The steps are as follows:

[0089] 1. Two weeks after the resuscitation of THP-1 cells, centrifuge at 300×g for 5 min, and add fresh complete medium to adjust the cell density to 1×10 5 cells / mL. Pipette 100 μL of the cell suspension and inoculate it on a 96-well plate. Six parallel wells are set for each treatment group, and 100 μL of the test substance solution (cosmetic raw material 1, cosmetic raw material 2, cosmetic raw material 3) with 2-fold volume of different concentrations (8 doses) are added respectively. After co-exposure in a cell culture incubator for 24 h, add CCK-8, mix well and culture, then detect the OD value of each well at 450 nm, calculate the relative cell viability of THP-1 cells, and calculate the cell 75% viability concentration value (75% cell viability, CV75).

[0090] 2. Centrifuge to obtain cells, and add fresh complete medium to adjust the cell density to 2×10 6cells / mL. Take 500 μL of the cell suspension and mix it with 500 μL of the working solution of cosmetic raw material 1 / cosmetic raw material 2 / cosmetic raw material 3, and inoculate it on a 24-well plate. Set up a blank control. After incubating the 24-well plate in a cell culture incubator for 24 h, transfer it to a 1.5 mL centrifuge tube, wash the cells 2 times with 1 mL of staining buffer (PBS solution with 0.1% fetal bovine serum), add a human Fc receptor blocker to block the Fc segment antibody of the cells (blocking concentration: 2.5 μg / 1×10 6 cells), and add 7-AAD staining solution (final concentration: 1 μg / mL) to stain the dead cells for 10 min. After washing the cells 2 times with the staining buffer, perform antibody staining.

[0091] 3. Divide each tube of cells into two equal parts. One part is used as the experimental group, add FITC-CD86 and PE-CD54, and add staining buffer to make the staining system up to 50 μL; the other part is used as the isotype control group, add FITC-IgG1 and PE-IgG1, and add staining buffer to make the staining system up to 50 μL. After adding the dyes to the two groups of cells, gently pipette several times to make the staining uniform, and stain at 4°C in the dark for 30 min. After the staining is completed, add the staining buffer to wash 2 times. Finally, resuspend the cells with 500 μL of staining buffer, transfer them to a flow tube for standby. Use a flow cytometer to measure the mean fluorescence intensity (Mean Fluorescence Intensity, MFI) of the FITC, PE, and 7-AAD dyes in each group of cells. Calculate the relative fluorescence intensity value (Relative Fluorescence Intensity, RFI) of the test substance.

[0092] The detection results of CD54 and CD86 in THP-1 cells after co-exposure with cosmetic raw material 1 are shown in Figure 18 and Figure 19 respectively. The detection results of CD54 and CD86 in THP-1 cells after co-exposure with cosmetic raw material 2 are shown in Figure 20 and Figure 21 respectively. The detection results of CD54 and CD86 in THP-1 cells after co-exposure with cosmetic raw material 3 are shown in Figure 22 and Figure 23 respectively. The results show that the RFI CD86 of 8 doses of cosmetic raw material 1 and cosmetic raw material 2 are all less than 150%, and the RFI CD54 are all less than 200%. According to the judgment rules of the h-CLAT method in the "Technical Specifications for Cosmetics Safety", cosmetic raw material 1 and cosmetic raw material 2 do not have potential skin sensitization. The RFI CD86 of 8 doses of cosmetic raw material 3 are all greater than 150%, and the RFI CD54 are all greater than 200%. According to the judgment rules of the h-CLAT method in the "Technical Specifications for Cosmetics Safety", cosmetic raw material 3 has potential skin sensitization.

[0093] The above results are consistent with the results of skin sensitization detection using the biomarker factors DDIT4 and CXCL8. Therefore, the biomarker factors DDIT4 and CXCL8 can be used as biomarker factors for detecting skin sensitization. By using fluorescence quantitative PCR to detect the changes in the DDIT4 and CXCL8 genes in THP-1 cells after co-exposure with cosmetic raw materials, it is possible to accurately determine whether the cosmetic raw materials have skin sensitization potential.

[0094] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0095] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A marker factor for detecting skin sensitization, characterized in that, It includes the DDIT4 gene and / or the CXCL8 gene.

2. Use of a reagent for detecting the expression level of a biomarker in the preparation of a product for detecting skin sensitization, characterized in that, The marker factor includes the DDIT4 gene and / or the CXCL8 gene.

3. The application according to claim 2, characterized in that, The expression level of the marker factor is detected by polymerase chain reaction technology, gene sequencing technology or Western blotting technology.

4. The application according to claim 3, characterized in that The polymerase chain reaction is qRT-PCR.

5. The application according to claim 4, wherein The primers used in the qRT-PCR are as shown in SEQ ID NO:1 and SEQ ID NO:2, and / or, the primers used in the qRT-PCR are as shown in SEQ ID NO:3 and SEQ ID NO:

4.

6. The application according to claim 2, wherein The product is a kit, a test strip or a chip.

7. The application according to claim 6, characterized in that, The kit includes a qRT-PCR kit, a Western blot detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, an immunohistochemical detection kit or an ELISA kit.

8. A method for detecting skin sensitization, characterized in that, It includes the following steps: After co-exposing the test substance to be tested with THP-1 cells, using the cDNA of the THP-1 cells as a template, qPCR reaction is carried out with the primers shown in SEQ ID NO:1 and SEQ ID NO:2, and / or SEQ ID NO:3 and SEQ ID NO:

4.

9. The method for detecting skin sensitization according to claim 8, wherein The test substance is a cosmetic, a medical device or a food; and / or, the time of the co-exposure is 22h to 26h.

10. The method for detecting skin sensitization according to claim 8 or 9, characterized in that, It also includes the step of analyzing the results of the qPCR reaction: if the relative expression levels of the DDIT4 gene and / or the CXCL8 gene increase significantly, the test substance to be tested has skin sensitization.

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