Application of recombinant collagen in preparation of preparation for improving expression quantity of related genes of skin barrier function

Abstract

The invention provides application of recombinant collagen in preparation of a preparation for improving the expression quantity of genes related to a skin barrier function. The amino acid sequence of the recombinant collagen is shown as SEQ ID NO: 45. The expression conditions of a series of related genes capable of enhancing the skin barrier function are detected in a transcriptomics mode, and it is proved that the recombinant collagen provided by the invention can promote the expression of the related genes capable of enhancing the skin barrier function. The collagen can be used for development and application of various cosmetic products, and the cosmetic products are endowed with the efficacy and effect of maintaining the skin barrier function. Meanwhile, a series of screened genes related to the skin barrier function can also be used as references for evaluating the effect of enhancing the skin barrier function by other raw materials or cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of research on the efficacy of cosmetic raw materials, and specifically relates to the application of recombinant collagen in the preparation of a preparation for improving the expression of genes related to skin barrier function. Background Art

[0002] The structural basis of the skin barrier includes the stratum corneum formed by surface keratinocytes, lipids between keratinocytes, and natural protective factors. Its main function is to prevent the loss of water and electrolytes in the body, prevent invasion by the external environment, and maintain homeostasis of the body.

[0003] Keratinocytes undergo a series of changes, passing through the basal layer, spinous cell layer, and granular cell layer, and finally become the stratum corneum, the outermost layer of the epidermis; the stratum corneum plays an important role in protecting the homeostasis of the skin. During the migration of epidermal cells from the basal layer to the stratum corneum, the fat content in them also changes continuously. In the granular layer, lipids are contained in the intercellular lamellar bodies. When the cells move to the stratum corneum, lipids are squeezed into the intercellular spaces and form lipid bimolecular structures with keratinocytes. The hydrophobic lipids outside the stratum corneum cells prevent water from losing outward and regulate the movement of electrolytes in the stratum corneum.

[0004] As the first barrier of the body, the skin can protect the body from external factors. Excessive cleaning, ultraviolet radiation, etc. can cause damage to the skin barrier. Inappropriate cleansing products will cause the oil on the surface of the skin to be over-cleaned, thereby damaging the skin barrier; long-term exposure of the skin to ultraviolet rays will also cause damage to the skin barrier. Recombinant collagen can quickly respond to skin damage, activate the barrier repair mechanism, and strengthen the barrier function.

[0005] CN202311119152.9 discloses a recombinant type III humanized collagen dressing mask for damaged skin barrier and its preparation method, which contains recombinant type III humanized collagen, which can inhibit and reduce the release of IL-6 factor and TRPV1 itch regulating factor in the inflammatory response process, and play a role in alleviating and improving dry skin.

[0006] CN202410106634.9 discloses a skin barrier repair composition and its application, repair cosmetics and its preparation method. The skin barrier repair composition contains recombinant humanized type III collagen, recombinant humanized fibronectin and silk fibroin composition. The combination of the above three proteins complements each other and jointly improves the repair effect of the composition on the skin barrier and function.

[0007] The prior art discloses many compositions and cosmetics containing recombinant humanized collagen, as well as their role in skin barrier repair. However, there is no research on the effect of recombinant humanized collagen on the expression of genes related to skin barrier function enhancement, nor is there any disclosure of the effect of recombinant humanized collagen on the expression of genes related to skin barrier function. Therefore, it is necessary to study the gene expression of collagen and skin barrier repair, and to provide a new type of collagen that enhances the expression of genes related to skin barrier function. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an application of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to skin barrier function. The present invention detects the expression of a series of genes related to enhancing skin barrier function by transcriptomics, proving that the recombinant collagen of the present invention can promote the expression of genes related to skin barrier function.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides the use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to skin barrier function, wherein the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:45.

[0011] Preferably, the gene includes any one or a combination of at least two of the following 22 skin barrier function-related genes, including: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 or TP63.

[0012] ALOX gene family: participates in the oxidation of lipids in the stratum corneum, regulates the production and metabolism of skin barrier lipids, and thus maintains the integrity of the stratum corneum. Includes: ALOX12B, ALOXE3, ALOX15.

[0013] AP1S3 gene: regulates the immune response and balance in the skin to ensure the integrity of the barrier function.

[0014] CDH1 gene: encodes E-Cadherin, a cell adhesion molecule that maintains tight junctions between keratinocytes and ensures the structural stability of the skin barrier.

[0015] CHST8 gene: Participates in the glycosylation process and regulates the synthesis of skin barrier glycoproteins.

[0016] CRYAB genes: Act as molecular chaperones, protecting skin cells from stress damage and maintaining barrier integrity.

[0017] ELOVL4 gene: encodes fatty acid elongase, which is responsible for the synthesis of long-chain fatty acids, participates in the formation of stratum corneum lipids, and ensures the normal function of the skin barrier.

[0018] GJB4 gene: Involved in cell-to-cell communication and supports the health of the skin barrier.

[0019] GRHL3 gene: plays a role in skin development and wound healing, helping to form the epidermal barrier.

[0020] KRT6 gene: Participates in the differentiation of keratinocytes, ensures the health of the stratum corneum, and thus maintains the barrier function of the skin.

[0021] LAMA5 gene: encodes the laminin α5 chain, which plays an important role in the basement membrane and maintains the integrity of the dermal-epidermal junction.

[0022] LIPN gene: Participates in lipid metabolism and helps maintain skin hydration and barrier function.

[0023] NTRK1 gene: involved in cell signal transduction, affecting the growth of skin barrier and wound healing.

[0024] PKP1 gene: Participates in the formation of intercellular adhesion complex and enhances skin barrier function.

[0025] PRR7 gene: plays a role in skin cell proliferation and survival, supporting barrier function.

[0026] RNF31 gene: Participates in cell signal transduction and affects the growth and repair of skin cells.

[0027] SPINK1 gene: regulates the activity of proteases in the epidermis and protects the skin barrier.

[0028] STX17 gene: regulates intercellular vesicle transport in the skin, ensures the exchange of substances between stratum corneum cells, and supports barrier function.

[0029] TCHH gene: encodes hair transparent protein, participates in keratinocyte differentiation, and ensures the maintenance of barrier function.

[0030] TGM1 gene: encodes type I transglutaminase, which catalyzes the cross-linking reaction between proteins during the terminal differentiation of keratinocytes to form the barrier structure of the stratum corneum.

[0031] TP63 gene: regulates the proliferation and differentiation of skin stem cells and supports skin barrier regeneration.

[0032] Preferably, the preparation increases the expression levels of the following 22 skin barrier function-related genes: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 and TP63.

[0033] Preferably, the preparation comprises a medicine or a cosmetic.

[0034] Preferably, the concentration of the recombinant collagen in the preparation is 0.9-1.1 mg / mL, for example, it can be 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.05 mg / mL or 1.1 mg / mL.

[0035] Preferably, the cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

[0036] The present invention also provides an expression promoter for increasing the expression amount of genes related to skin barrier function, wherein the expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:45.

[0037] In the present invention, after the recombinant collagen is applied, the relative expression level of the skin barrier function-related gene is increased.

[0038] In the present invention, a relative expression promotion of 1.19 is considered to be promotion. In bioinformatics analysis, a log2fold change value of 0.25 is usually used as a threshold for screening differentially expressed genes, which is converted into a relative expression multiple of about 1.19.

[0039] The present invention also provides a recombinant collagen protein for improving skin barrier function, wherein the amino acid sequence of the recombinant collagen protein includes that shown in SEQ ID NO:45.

[0040] The present invention also provides a biomarker for evaluating the effect of cosmetics or their raw materials on increasing skin barrier function, wherein the biomarker is any one or a combination of at least two of 22 skin barrier function-related genes, including: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 or TP63.

[0041] In a second aspect, the present invention provides an application of a product for detecting the expression level of a biomarker in evaluating the effect of cosmetics or their raw materials on increasing skin barrier function, wherein the biomarker is any one or a combination of at least two of 22 skin barrier function-related genes, including: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 or TP63.

[0042] Preferably, the biomarker is a combination of 22 skin barrier function-related genes: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 and TP63.

[0043] Preferably, the product is a PCR detection kit or detection model for detecting the expression level of biomarkers, and the PCR detection kit includes a primer combination for amplifying 22 skin barrier function-related genes.

[0044] In a third aspect, the present invention provides a primer combination for detecting the expression level of biomarkers, wherein the biomarkers are 22 skin barrier function-related genes;

[0045] The upstream primer for amplifying ALOX12B is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 23;

[0046] The upstream primer for amplifying ALOX15 is shown in SEQ ID NO:2, and the downstream primer is shown in SEQ ID NO:24;

[0047] The upstream primer for amplifying ALOXE3 is shown in SEQ ID NO:3, and the downstream primer is shown in SEQ ID NO:25x;

[0048] The upstream primer for amplifying AP1S3 is shown in SEQ ID NO:4, and the downstream primer is shown in SEQ ID NO:26;

[0049] The upstream primer for amplifying CDH1 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:27;

[0050] The upstream primer for amplifying CHST8 is shown in SEQ ID NO:6, and the downstream primer is shown in SEQ ID NO:28;

[0051] The upstream primer for amplifying CRYAB is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:29;

[0052] The upstream primer for amplifying ELOVL4 is shown in SEQ ID NO:8, and the downstream primer is shown in SEQ ID NO:30;

[0053] The upstream primer for amplifying GJB4 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:31;

[0054] The upstream primer for amplifying GRHL3 is shown in SEQ ID NO: 10, and the downstream primer is shown in SEQ ID NO: 32;

[0055] The upstream primer for amplifying KRT6B is shown in SEQ ID NO:11, and the downstream primer is shown in SEQ ID NO:33;

[0056] The upstream primer for amplifying LAMA5 is shown in SEQ ID NO: 12, and the downstream primer is shown in SEQ ID NO: 34;

[0057] The upstream primer for amplifying LIPN is shown in SEQ ID NO:13, and the downstream primer is shown in SEQ ID NO:35;

[0058] The upstream primer for amplifying NTRK1 is shown in SEQ ID NO: 14, and the downstream primer is shown in SEQ ID NO: 36;

[0059] The upstream primer for amplifying PKP1 is shown in SEQ ID NO: 15, and the downstream primer is shown in SEQ ID NO: 37;

[0060] The upstream primer for amplifying PRR7 is shown in SEQ ID NO: 16, and the downstream primer is shown in SEQ ID NO: 38;

[0061] The upstream primer for amplifying RNF31-1 is shown in SEQ ID NO: 17, and the downstream primer is shown in SEQ ID NO: 39;

[0062] The upstream primer for amplifying SPINK1 is shown in SEQ ID NO: 18, and the downstream primer is shown in SEQ ID NO: 40;

[0063] The upstream primer for amplifying STX17 is shown in SEQ ID NO: 19, and the downstream primer is shown in SEQ ID NO: 41; the upstream primer for amplifying TCHH is shown in SEQ ID NO: 20, and the downstream primer is shown in SEQ ID NO: 42; the upstream primer for amplifying TGM1 is shown in SEQ ID NO: 21, and the downstream primer is shown in SEQ ID NO: 43; the upstream primer for amplifying TP63 is shown in SEQ ID NO: 22, and the downstream primer is shown in SEQ ID NO: 44. The primers are shown in Table 1.

[0064] Table 1

[0065]

[0066]

[0067] Beneficial effects of the primer combination of the present invention: The present invention designs a series of primers related to skin barrier function genes, which can be used to quickly and accurately detect the expression levels of these genes in the future, and then evaluate the promoting or inhibiting effect of a certain test product on skin barrier function.

[0068] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] (1) The present invention detects the expression of a series of genes related to enhancing skin barrier function by transcriptomics, proving that the recombinant collagen of the present invention can promote the expression of genes related to skin barrier function. The collagen can be used for the development and application of a variety of cosmetic products, giving cosmetic products the efficacy and effect of maintaining skin barrier function. At the same time, the series of genes can also be used as a reference for future evaluation of other raw materials or cosmetics to enhance skin barrier function.

[0071] (2) The present invention has developed a new recombinant collagen that can significantly enhance the expression of genes related to skin barrier function compared with other collagens. By setting a series of concentrations of recombinant collagen to test the expression ability of related genes, the recombinant collagen with the highest promotion rate is selected, which can be used as a reference for designing formulas for cosmetic raw materials in the future.

[0072] (3) The present invention designs a series of primers related to skin barrier function genes and verifies them through experiments. It can be used to quickly and accurately detect the expression levels of these genes in the future, and then evaluate the promoting or inhibiting effect of a certain test product on skin barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 is a heat map analysis of genes that maintain skin barrier function. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0075] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0076] Example 1

[0077] 1. Culture of Hacat cells

[0078] (1) Take the Hacat human immortalized epidermal cells (hereinafter referred to as Hacat cells) frozen at -80°C and thaw quickly in a 37°C water bath. Transfer the frozen cells directly to a culture dish with 10 mL of DEME medium (containing 10% fetal bovine serum and 1% double antibody). Resuscitate in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours to allow the cells to adhere to the wall and grow.

[0079] (2) After the Hacat cells have recovered, remove the culture medium and gently rinse the cell surface with 2 mL of PBS preheated at 37°C. Repeat twice.

[0080] (3) Add 1 mL of 37°C preheated trypsin, gently shake the culture medium to evenly distribute the trypsin in the culture dish, and place it in the incubator for digestion for 2 minutes. After taking it out, immediately add 37°C preheated DEME culture medium to terminate the digestion, and gently blow down the attached Hacat cells with a pipette, and take a portion to count using a cell counting plate.

[0081] (4) Place the cells in a 15 mL centrifuge tube and centrifuge at 500 RPM for 5 minutes. Resuspend the cells in 1 mL of DEME medium and inoculate 5 × 10 cells per well. 5 The cells were seeded into six-well plates with 2 mL of culture medium per well and placed in an incubator for 24 hours.

[0082] 2. Co-culture of recombinant collagen and Hacat cells

[0083] (1) Recombinant collagen was prepared into a plurality of test solutions at concentrations of 0.1 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 1.5 mg / mL.

[0084] (2) Remove the Hacat cells from the six-well plate and add 200 μL of preheated PBS to each well to gently rinse the cell surface. Then add 2 mL of DEME medium to each well. Add each concentration of recombinant collagen to each well at a ratio of 0.1%. Add an equal volume of sterile water to the wells of the control group. Repeat twice for all concentrations to be tested. Place the recombinant collagen and Hacat cells in a cell culture incubator and culture for 24 hours.

[0085] The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:45.

[0086] GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGA PGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0087] 3. Lysis of Hacat cells

[0088] (1) The six-well culture dish of Hacat cells after co-culture was taken out of the incubator, the culture medium was removed by aspiration, and 200 μL of preheated PBS was added to each well to gently rinse the cell surface, and then the PBS was removed by aspiration. This was repeated twice.

[0089] (2) Add 200 μL of TRIZOL reagent to each well to be tested, and use a pipette to blow the bottom of the culture dish to completely dissolve the cells in each well in the lysis solution. Dispense into 1.5 mL centrifuge tubes and mark them for subsequent analysis.

[0090] Example 2

[0091] The cell lysate in Example 1 was subjected to transcriptome sequencing.

[0092] 1. Analysis plan

[0093] 1.1 Species name: Homo sapiens; Source: NCBI; Reference genome version: GCF_000001405.40_GRCh38.p14.

[0094] 1.2 Difference Grouping Settings

[0095] One of the important analysis points of transcriptome sequencing is the comparison of expression levels. The control and treatment conditions in the pairwise comparison are shown in Table 2 below.

[0096] Table 2

[0097] Comparison deal with HaCat_utreated (HaCat cells untreated) HaCat_treated (Hacat cells treated)

[0098] 2. Experimental process

[0099] (1) mRNA library construction process

[0100] 1) Total RNA is processed by mRNA enrichment or rRNA removal. mRNA enrichment: mRNA with polyA tail is enriched by magnetic beads with OligodT; rRNA removal: rRNA is hybridized with DNA probe, RNaseH selectively digests DNA / RNA hybrid chains, and then DNaseI is used to digest the DNA probe. The desired RNA is obtained after purification.

[0101] 2) The obtained RNA was fragmented using a shearing buffer, reverse transcribed using a random N6 primer, and then the second strand of cDNA was synthesized to form double-stranded DNA.

[0102] 3) The ends of the synthesized double-stranded DNA are filled in and the 5' end is phosphorylated, forming a sticky end with a protruding "A" at the 3' end, and then a bubble-shaped linker with a protruding "T" at the 3' end is connected.

[0103] 4) The ligation product is amplified by PCR using specific primers.

[0104] 5) The PCR product is heat denatured into a single strand, and then a bridge primer is used to circularize the single-stranded DNA to obtain a single-stranded circular DNA library.

[0105] 6) Sequencing on a machine.

[0106] 3. Sequencing data filtering

[0107] The raw sequencing data contains reads with low quality, contaminated adapters, and high unknown base N content. These reads need to be removed before data analysis to ensure the reliability of the results. The analysis methods for data filtering include:

[0108] 1) Removal of reads containing adapters (adapter contamination).

[0109] 2) Remove reads with unknown base N content greater than 1%.

[0110] 3) Remove low-quality reads (we define low-quality reads as those in which the ratio of bases with a quality value lower than 15 to the total number of bases in the read is greater than 40%).

[0111] The filtered “Clean Reads” are saved in FASTQ format.

[0112] 4. Reference genome alignment

[0113] After obtaining Clean Reads, HISAT is used to align Clean Reads to the reference genome sequence.

[0114] 5. Reference gene comparison

[0115] Bowtie2 was used to align clean reads to the reference gene sequences, and RSEM was then used to calculate the expression levels of genes and transcripts.

[0116] 6. Gene Annotation

[0117] (1) Use blastn to align mRNA to the NT database.

[0118] (2) Diamond was used to align mRNA to NR, KOG, KEGG, uniprot, COG, TF (the animal transcription factor database is AnimalTFDB3, the plant transcription factor database is PlantTFdb5), and PRGDB databases; GO annotation information was obtained based on the uniprot (including sprot and trembl) alignment information.

[0119] (3) Use infernal to align lncRNA / mRNA to Rfam; use diamond to align lncRNA to miRBase (miRNA prerequisite sequence).

[0120] 7. Gene quantification

[0121] Bowtie2 was used to align clean reads to genomic sequences, and then RSEM was used to calculate gene expression levels for each sample. RSEM is a software package for RNA-seq reads to calculate gene and transcript isoform expression levels.

[0122] 8. Differential Expression Analysis

[0123] According to the results of differential gene detection, hierarchical cluster analysis was performed using the pheatmap function in R software.

[0124] According to the GO annotation results and official classification, the differentially expressed genes were functionally classified, and enrichment analysis was performed using the phyper function in R software.

[0125] According to the KEGG annotation results and official classification, the differentially expressed genes were classified into biological pathways, and the phyper function in R software was used for enrichment analysis.

[0126] 9. Results Analysis

[0127] The obtained transcriptomic sequencing results retained data with a p value > 0.05. The Log2 fold change values ​​of genes related to maintaining the homeostasis of skin extracellular matrix in the experimental group and the control group were extracted.

[0128] The FPKM values ​​of the above genes in the experimental group and the control group were integrated into the same table (Table 3). The heat map was obtained by heat map analysis of the Kidio Bioinformatics Cloud tool, as shown in Figure 1 shown.

[0129] Table 3

[0130]

[0131] As shown in Table 3, after the Hacat cells were treated with recombinant collagen, the mean Log2fold change of the above gene experimental group compared with the control group was calculated to be 1.072, that is, the difference fold was 2.102, which was 1.19 higher than the promotion standard. This proves that recombinant collagen promotes the expression of genes related to skin barrier function.

[0132] This example detects the expression of a series of genes related to skin barrier function by transcriptomics, proving that recombinant collagen can enhance skin barrier function.

[0133] Example 3

[0134] This example uses recombinant collagen to conduct experiments to verify the effects of different concentrations of recombinant collagen on the expression of a series of genes related to skin barrier function. The experimental steps include:

[0135] 1. Cell sample culture

[0136] Collagen was prepared into test solutions with concentrations of 0.1 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, respectively. The test solutions were added to the culture medium of BJ cells that were growing to about 70% of the bottom area of ​​the culture dish at a final concentration of 0.1%, and cultured for 24 hours.

[0137] 2. Extraction and purification of total RNA in cells

[0138] (1) Remove the culture medium from the cultured cells, wash twice with pre-cooled PBS, add 1 mL of TRIzol reagent, pipette and collect into a centrifuge tube, and leave at room temperature for 5 minutes.

[0139] (2) Add 200 μL of chloroform to the centrifuge tube, shake the tube vigorously for 15 seconds, let it stand for 10 minutes, and then centrifuge it at 12,000 g for 15 minutes at 4°C.

[0140] (3) After centrifugation, the liquid is separated into layers. RNA is present in the upper colorless and transparent aqueous layer with a volume of about 600 μL. After transferring to a new centrifuge tube, 500 μL of isopropanol is added and the tube is slowly inverted several times to mix. After standing for 15 minutes, centrifuge at 12,000 g for 10 minutes at 4°C.

[0141] (4) After centrifugation, the RNA precipitates at the bottom of the centrifuge tube. When facing the light source, it can be seen as a gelatinous precipitate. After aspirating the supernatant, add 1 mL of 75% ethanol to resuspend and wash the RNA, and centrifuge at 7500g for 5 minutes at 4°C.

[0142] (5) Remove the ethanol supernatant, let stand for 5 minutes to dry the RNA, and add 20 μL of DEPC-water to dissolve the RNA.

[0143] 3. RNA concentration determination

[0144] (1) Take 1 μL of the extracted RNA and add it to 99 μL of DEPC-water to make the total volume 100 μL. Add the diluted RNA solution to a 96-well UV plate.

[0145] (2) Place the 96-well plate in a full-wavelength microplate reader, select the area to be measured, and set the program as follows: oscillate for 10 seconds, measure the absorbance at 260 nm, measure the absorbance at 280 nm, and read the value.

[0146] (3) Calculation of OD 260 / OD 280Ratio: A ratio between 1.8 and 2.0 indicates that the quality of the extracted RNA meets subsequent needs.

[0147] (4) According to the formula OD 260 × 0.04 × dilution factor, calculate the RNA solution concentration in μg / μL. Dilute with DEPC-water to adjust the RNA concentration to 1 μg / μL.

[0148] 4. Reverse transcription

[0149] (1) Using a reverse transcription kit (Novozyme's one-step reverse transcription kit R333), prepare a reverse transcription system in a 200 μL PCR tube, including: a) 1 μg / μL RNA solution, 1 μL; b) 5×qRT SuperMix, 4 μL; c) premixed enzyme solution, 1 μL; d) DEPC-water, 14 μL. The final volume of the reverse transcription system is 20 μL. Use a pipette to gently pipette and mix well. Centrifuge briefly for 30 seconds in a handheld centrifuge.

[0150] (2) Place the reverse transcription system PCR tube into the PCR instrument and set the reaction program as follows: a) incubate at 50°C for 15 minutes; b) instant inactivate at 85°C for 10 seconds. The mRNA in the system is reverse transcribed into cDNA.

[0151] 5. Fluorescence quantitative PCR

[0152] (1) Using the reagents in the SYBR Green fluorescent quantitative kit, prepare the reaction system in an eight-link PCR tube, including: a) 2×SYBR Green PCR premix, volume 10μL; b) forward and reverse primers, volume 1.5μL each; c) cDNA solution with a concentration of 1μg / μL, volume 1μL; d) sterilized first-grade water, volume 6μL. The final volume of the PCR system is 20μL, and it is briefly centrifuged for 30 seconds using a handheld centrifuge.

[0153] (2) Each sample needs three replicate wells for each molecule to be tested, and a blank control without adding cDNA is set up; in addition to the molecule to be tested, actin β (ACTB) is also required as a reference molecule.

[0154] (3) Place the eight-link PCR tube into a fluorescent quantitative PCR instrument and set the reaction program: a) 95°C for 2 minutes; b) 95°C for 5 seconds; c) 60°C for 10 seconds; d) 20°C constant temperature storage. Steps b and c are repeated for 45 cycles.

[0155] (4) After the PCR process is completed, the software automatically generates the CT value for each sample.

[0156] 6. Quantitative analysis of results

[0157] (1) The CT values ​​of three replicates of each sample were averaged, and the difference between the CT value of the test molecule and the CT value of the reference molecule (ACTB) was calculated, i.e., ΔCT.

[0158] (2) Calculate the difference between the ΔCT value of the molecule to be tested in the cells treated with the raw material and the ΔCT value of the molecule to be tested in the untreated cells, i.e., ΔΔCT.

[0159] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the exponent, that is, 2 -△△CT , 2 parallel groups of each concentration -△△CT The average value was taken as the quantitative result of the molecule to be tested in each sample to achieve quantitative detection at the gene level. The experimental results are shown in Table 4 below.

[0160] Table 4

[0161]

[0162] As can be seen from Table 4, by treating Hacat cells with different concentrations of collagen and measuring the relative expression of related genes, it can be found that only when the concentration is 1.0 mg / mL, all genes are promoted and the average promotion rate is the highest. That is, 1.0 mg / mL is the most suitable concentration of the recombinant collagen of the present invention to improve the expression of genes related to skin barrier function.

[0163] Example 4

[0164] This example simultaneously uses recombinant collagen (SEQ ID NO: 45) and collagen 1, collagen 2, and collagen 3 to test their effects on promoting the expression of a series of genes related to skin barrier function.

[0165] The amino acid sequence of collagen 1 is shown in SEQ ID NO:46; the amino acid sequence of collagen 2 is shown in SEQ ID NO:47; and the amino acid sequence of collagen 3 is shown in SEQ ID NO:48.

[0166] SEQ ID NO:46:

[0167] LPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGASPGEPG YMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVRGAIGSKGESGVDGLMGPAGPKGQPGDPGPQGPPGLDGKPGRE

[0168] SEQ ID NO:47:

[0169] GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP

[0170] SEQ ID NO:48:

[0171] GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT

[0172] The experimental steps include:

[0173] 1. Recombinant collagen (SEQ ID NO: 45) and collagen 1, collagen 2, and collagen 3 were prepared into 1.0 mg / mL test solution, and added into the culture medium of Hacat cells that were growing to about 70% of the bottom area of ​​the culture dish at a final concentration of 0.1%, and cultured for 24 hours.

[0174] 2. The subsequent RNA extraction, reverse transcription, and qPCR experimental operations were the same as in Example 3. The experimental results are shown in Table 5.

[0175] Table 5

[0176]

[0177]

[0178] As can be seen from Table 5, the recombinant collagen of the present invention can upregulate 21 skin fibroblast proliferation-related genes, while the other collagens 1, 2, and 3 cannot upregulate all of the above 21 genes, and their upregulation effects are not as good as the recombinant collagen of the present invention. It can be seen that the collagen of the present invention has a better effect of improving the skin barrier function, which is mutually confirmed by the conclusion in the aforementioned Example 3. The recombinant collagen of the present invention has a significant effect on improving the skin barrier function.

[0179] Application Example 1

[0180] The recombinant collagen of the present invention can be used as an active ingredient in the preparation of skin conditioners and cosmetics, such as creams, lotions, gels, toners, essences, masks, eye creams, aerosol cleansing foams, sprays, shower gels or facial cleansers.

[0181] Application 1, preparation of facial cream, formulated according to the following ratio: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 4% liquid paraffin, 3% glycerol, 5% propylene glycol, 3% ethylhexyl palmitate, 3% petrolatum, 2% ceteareth-21, 1.5% dimethicone, 1.5% cetearyl alcohol, 1.5% cetearyl ether-2, 1.5% glyceryl monostearate, 0.2% methylparaben, 0.2% carbomer, 0.2% triethanolamine, 0.1% ethylparaben, the balance is water (65.2%).

[0182] Application 2, preparation of emulsion, formulated according to the following ratio: 0.1% recombinant collagen, 4.5% glycerin, 6% hydrogenated polyisobutene, 3% ethylhexyl palmitate, 3% propylene glycol, 3.5% caprylic / capric triglyceride, 2% dimethicone, 1.5% ceteareth-21, 1.2% ceteareth-2, 0.5% tocopherol, 0.2% methylparaben, 0.1% triethanolamine, 0.1% ethylparaben, 0.1% carbomer, the balance is water (74.2%).

[0183] Application 3, preparation of lotion, according to the following ratio: 0.1% recombinant collagen, 6% glycerol, 2% propylene glycol, 0.2% diazoimidazole urea, the balance is water (91.7%).

[0184] In summary, the present invention detects the expression of a series of genes related to skin barrier function by transcriptomics, proving that recombinant collagen can promote the expression of genes related to skin barrier function. The collagen can be used for the development and application of a variety of cosmetic products, giving cosmetic products increased skin barrier function. At the same time, the series of genes can also be used as a reference for future evaluation of the effects of other raw materials or cosmetics in promoting skin barrier function.

[0185] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. The use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

45.

2. The use of claim 1 in preparing a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The genes include any one or a combination of at least two of the following 22 skin barrier function-related genes, including: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 or TP63.

3. The use of claim 1 or 2 in preparing a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The preparation increases the expression levels of the following 22 skin barrier function-related genes: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 and TP63.

4. The use of any one of claims 1 to 3 in preparing a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The preparation includes a medicine or a cosmetic.

5. The use of claim 4 in preparing a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The concentration of the recombinant collagen in the preparation is 0.9-1.1 mg / mL.

6. The use of claim 4 or 5 in preparing a preparation for increasing the expression of genes related to skin barrier function, characterized in that: The cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

7. Use of a product for detecting the expression level of a biomarker in evaluating the effect of cosmetics or their raw materials on increasing skin barrier function, characterized in that: The biomarker is any one or a combination of at least two of 22 skin barrier function-related genes, including: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 or TP63.

8. Use of the product for detecting the expression level of biomarkers according to claim 7 in evaluating the effect of cosmetics or their raw materials on increasing skin barrier function, characterized in that: The biomarkers are a combination of 22 genes related to skin barrier function: ALOX12B, ALOX15, ALOXE3, AP1S3, CDH1, CHST8, CRYAB, ELOVL4, GJB4, GRHL3, KRT6B, LAMA5, LIPN, NTRK1, PKP1, PRR7, RNF31-1, SPINK1, STX17, TCHH, TGM1 and TP63.

9. Use of the product for detecting the expression level of biomarkers according to claim 7 or 8 in evaluating the effect of cosmetics or their raw materials on increasing skin barrier function, characterized in that: The product is a PCR detection kit or detection model for detecting the expression level of biomarkers, and the PCR detection kit includes a primer combination for amplifying 22 skin barrier function-related genes.

10. A primer combination for detecting the expression level of a biomarker, characterized in that: The biomarkers are 22 genes related to skin barrier function; The upstream primer for amplifying ALOX12B is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 23; The upstream primer for amplifying ALOX15 is shown in SEQ ID NO:2, and the downstream primer is shown in SEQ ID NO:24; The upstream primer for amplifying ALOXE3 is shown in SEQ ID NO:3, and the downstream primer is shown in SEQ ID NO:25x; The upstream primer for amplifying AP1S3 is shown in SEQ ID NO:4, and the downstream primer is shown in SEQ ID NO:26; The upstream primer for amplifying CDH1 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:27; The upstream primer for amplifying CHST8 is shown in SEQ ID NO:6, and the downstream primer is shown in SEQ ID NO:28; The upstream primer for amplifying CRYAB is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:29; The upstream primer for amplifying ELOVL4 is shown in SEQ ID NO:8, and the downstream primer is shown in SEQ ID NO:30; The upstream primer for amplifying GJB4 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:31; The upstream primer for amplifying GRHL3 is shown in SEQ ID NO: 10, and the downstream primer is shown in SEQ ID NO: 32; The upstream primer for amplifying KRT6B is shown in SEQ ID NO:11, and the downstream primer is shown in SEQ ID NO:33; The upstream primer for amplifying LAMA5 is shown in SEQ ID NO: 12, and the downstream primer is shown in SEQ ID NO: 34; The upstream primer for amplifying LIPN is shown in SEQ ID NO:13, and the downstream primer is shown in SEQ ID NO:35; The upstream primer for amplifying NTRK1 is shown in SEQ ID NO: 14, and the downstream primer is shown in SEQ ID NO: 36; The upstream primer for amplifying PKP1 is shown in SEQ ID NO: 15, and the downstream primer is shown in SEQ ID NO: 37; The upstream primer for amplifying PRR7 is shown in SEQ ID NO: 16, and the downstream primer is shown in SEQ ID NO: 38; The upstream primer for amplifying RNF31-1 is shown in SEQ ID NO: 17, and the downstream primer is shown in SEQ ID NO: 39; The upstream primer for amplifying SPINK1 is shown in SEQ ID NO: 18, and the downstream primer is shown in SEQ ID NO: 40; The upstream primer for amplifying STX17 is shown in SEQ ID NO: 19, and the downstream primer is shown in SEQ ID NO: 41; The upstream primer for amplifying TCHH is shown in SEQ ID NO:20, and the downstream primer is shown in SEQ ID NO:42; The upstream primer for amplifying TGM1 is shown in SEQ ID NO:21, and the downstream primer is shown in SEQ ID NO:43; The upstream primer for amplifying TP63 is shown in SEQ ID NO:22, and the downstream primer is shown in SEQ ID NO:44.

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