Use of recombinant collagen in preparation of a preparation for improving expression of skin barrier function-related genes

Abstract

The application provides application of recombinant collagen in preparation of a preparation for improving expression of skin barrier function related genes, and an amino acid sequence of the recombinant collagen comprises SEQ ID NO: 45. The application detects expression of a series of skin barrier function related genes in a transcriptomic mode, and proves that the recombinant collagen provided by the application can promote expression of the skin barrier function related genes. The collagen can be used for development and application of various cosmetic products, and endows the cosmetic products with the efficacy and effect of maintaining skin barrier function. Meanwhile, the series of skin barrier function related genes screened can be used as a reference for evaluating effects of other raw materials or cosmetics on enhancement of skin barrier function.

Description

TECHNICAL FIELD

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

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

[0003] Keratinocytes undergo a series of changes via the basal layer, the prickle cell layer, and the granular cell layer, and finally become the outermost layer of the epidermis, the stratum corneum; the stratum corneum plays an important role in protecting skin homeostasis. During the migration of epidermal cells from the basal layer to the stratum corneum, the lipid content also changes, and in the granular layer, lipids are contained in the intercellular lamellar bodies, and when the cells move to the stratum corneum, the lipids are squeezed into the intercellular space and form a lipid bilayer structure with the keratinocytes. The hydrophobic lipids outside the keratinocytes prevent water loss and regulate the movement of electrolytes in the stratum corneum.

[0004] Skin, as the first barrier of the body, can protect the body from external factors. Excessive cleaning, ultraviolet radiation, etc. can cause damage to the skin barrier. Improper cleansing products can cause excessive cleaning of the skin surface, thereby damaging the skin barrier; long-term exposure of the skin to ultraviolet light can also cause damage to the skin barrier. Recombinant collagen can quickly respond to skin damage, initiate barrier repair mechanisms, and strengthen barrier function.

[0005] CN202311119152.9 discloses a recombinant type III humanized collagen dressing mask for damaged skin barrier and a preparation method thereof. It contains recombinant type III humanized collagen, which can inhibit and reduce the release of IL-6 factor and TRPV1 itch regulatory factor during the inflammatory response process, and has the effect of alleviating and improving dry skin.

[0006] CN202410106634.9 discloses a skin barrier repair composition and its application, a repair cosmetic and a preparation method thereof. The skin barrier repair composition contains recombinant type III humanized collagen, recombinant humanized fibronectin, and silk core protein composition. The combination of the above three proteins takes the advantages of each other and improves the repair effect of the composition on the skin barrier and function.

[0007] Many compositions containing recombinant humanized collagen, cosmetics, and their effects on skin barrier repair are disclosed in the prior art, but there is no research on the expression of genes related to the enhancement of skin barrier function by recombinant humanized collagen, and the effect of recombinant humanized collagen on the expression of skin barrier function related genes is not disclosed, so it is necessary to study the gene expression of collagen and skin barrier repair, and provide a new type of collagen that can enhance the expression of skin barrier function related genes. SUMMARY

[0008] In view of the deficiencies in the prior art, the purpose of the present application is to provide the use of recombinant collagen in the preparation of a preparation for increasing the expression amount of skin barrier function related genes. The present application detects a series of genes related to the enhancement of skin barrier function by transcriptomics, and proves that the recombinant collagen of the present application can promote the expression of genes related to the skin barrier function.

[0009] To achieve the purpose of the present application, the following technical solutions are adopted:

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

[0011] Preferably, the genes include any one or a combination of at least two 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 or TP63.

[0012] ALOX gene family: involved in the oxidation of lipids in the stratum corneum, regulates the generation and metabolism of skin barrier lipids, thereby maintaining the integrity of the stratum corneum. It includes: ALOX12B, ALOXE3, ALOX15.

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

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

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

[0016] CRYAB gene: As a molecular chaperone, it protects skin cells from stress damage and maintains barrier integrity.

[0017] ELOVL4 gene: Encodes a fatty acid elongase responsible for the synthesis of long-chain fatty acids, involved in the formation of stratum corneum lipids, ensuring the normal functioning of the skin barrier.

[0018] GJB4 gene: Involved in intercellular communication, supporting the health of the skin barrier.

[0019] GRHL3 gene: Plays a role in skin development and wound healing, helping the formation of the epidermal barrier.

[0020] KRT6 gene: Involved in the differentiation of keratinocytes, ensuring the health of the stratum corneum, thus maintaining the barrier function of the skin.

[0021] LAMA5 gene: Encodes laminin alpha 5 chain, plays an important role in the basement membrane, maintaining the integrity of the dermal-epidermal junction.

[0022] LIPN gene: Involved in lipid metabolism, helping to maintain skin hydration and barrier function.

[0023] NTRK1 gene: Involved in cell signaling, affecting the growth of the skin barrier and wound healing.

[0024] PKP1 gene: Involved in the formation of intercellular adhesion complexes, enhancing the skin barrier function.

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

[0026] RNF31 gene: Involved in cell signal transduction, affecting the growth and repair of skin cells.

[0027] SPINK1 gene: Regulates protease activity in the epidermal layer of the skin, protecting the skin barrier.

[0028] STX17 gene: Regulates intercellular vesicle transport in the skin, ensuring the exchange of substances between keratinocytes, supporting barrier function.

[0029] TCHH gene: Encodes hair transparent protein, involved in keratinocyte differentiation, ensuring the maintenance of barrier function.

[0030] TGM1 gene: Encodes type I transglutaminase, catalyzes cross-linking reactions between proteins during the terminal differentiation of keratinocytes, forming the barrier structure of the stratum corneum.

[0031] TP63 gene: Regulates the proliferation and differentiation of skin stem cells, supporting skin barrier regeneration.

[0032] Preferably, the preparation increases the expression 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.

[0033] Preferably, the preparation is 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, etc.

[0035] Preferably, the cosmetic includes a cream, a lotion, a gel, a cosmetic water, an essence, a mask, an eye cream, an aerosol cleansing foam, a spray, a shower gel or a facial cleanser.

[0036] The present application also provides an expression promoter for increasing the expression of skin barrier function-related genes, wherein the expression promoter is a recombinant collagen, and the amino acid sequence of the recombinant collagen comprises SEQ ID NO: 45.

[0037] In the present application, after the recombinant collagen is administered, the relative expression of the skin barrier function-related genes is increased.

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

[0039] The present application also provides a recombinant collagen for improving skin barrier function, wherein the amino acid sequence of the recombinant collagen comprises SEQ ID NO: 45.

[0040] The present application also provides a biomarker for evaluating the effect of a cosmetic or raw material thereof on increasing skin barrier function, the biomarker being 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 application provides use of a product for detecting expression level of a biomarker in evaluating the effect of a cosmetic or raw material thereof on increasing skin barrier function, the biomarker being 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 a detection model for detecting expression level of a biomarker, the PCR detection kit comprising a primer combination for amplifying 22 skin barrier function-related genes.

[0044] In a third aspect, the present application provides a primer combination for detecting expression level of a biomarker, the biomarker being 22 skin barrier function-related genes.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0064] Table 1

[0065]

[0066]

[0067] The primer combination in the present application has the following beneficial effects: the present application designs a series of primers related to skin barrier function genes, which can be used for rapid and accurate detection of the expression levels of these genes in the future, and further evaluate the promotion or inhibition of skin barrier function of a certain to-be-tested product.

[0068] The numerical range described in the present application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and the present application does not exhaustively list the specific point values included in the range for the sake of brevity and simplicity.

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

[0070] (1) The present application detects the expression of a series of genes related to the enhancement of skin barrier function by transcriptomics, and proves that the recombinant collagen protein of the present application can promote the expression of genes related to skin barrier function. The collagen protein can be used for the development and application of various cosmetic products, and endows the cosmetic products with 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 evaluating other raw materials or cosmetics to enhance skin barrier function in the future.

[0071] (2) The application develops a new recombinant collagen which can significantly improve the expression of skin barrier function related genes compared with other collagens, and the expression ability of related genes of a series of concentrations of recombinant collagen is tested, and the highest concentration of recombinant collagen with the highest promotion rate is selected, which can be used as a reference for future design of cosmetic raw material formula.

[0072] (3) The application designs a series of primers related to skin barrier function genes, and detects and verifies through experiments. It can be used for rapid and accurate detection of the expression level of these genes in the future, and then evaluate the promotion or inhibition of the skin barrier function of a certain to-be-tested product. BRIEF DESCRIPTION OF DRAWINGS

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

[0074] The technical solutions of the application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the application and should not be regarded as specific limitations on the application.

[0075] If a specific technology or condition is not specified in the embodiments, it is performed according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0076] Example 1

[0077] 1. Culture of Hacat cells

[0078] (1) Take the Hacat human immortalized epidermal cells (hereinafter referred to as Hacat cells) stored at -80℃, quickly thaw in a 37℃ water bath. The frozen cells are directly transferred to a culture dish with 10mL DEME medium (containing 10% fetal bovine serum and 1% double antibody). Incubate at 37℃ in a cell incubator with 5% carbon dioxide for 24 hours to allow the cells to grow adherently.

[0079] (2) After the recovery of Hacat cells, the culture medium is removed, and 2mL of 37℃ preheated PBS is used to gently wash the cell surface, and the process is repeated twice.

[0080] (3) Add 1mL of 37℃ preheated trypsin, gently shake the culture medium to evenly distribute the trypsin in the culture dish, and put it in the incubator for 2 minutes. After taking it out, immediately add 37℃ preheated DEME medium to terminate digestion, and gently blow the adherent Hacat cells with a pipette gun. Take a part and count using a cell counting plate.

[0081] (4) The cells blown down were placed in a 15 mL centrifuge tube, and the centrifuge was set to 500 RPM for 5 minutes. After centrifugation, the cells were resuspended with 1 mL of DEME medium, and 5 x 10 5

[0082] 2. Recombinant collagen co-cultured with Hacat cells

[0083] (1) The recombinant collagen was prepared into multiple 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) The Hacat cells in the six-well plate were aspirated, and 200 μL of preheated PBS was added to each well to gently wash the cell surface. Then, 2 mL of DEME medium was added to each well, and the recombinant collagen test solution at each concentration was added to each test well at a proportion of 0.1%. An equal volume of sterile water was added to the control wells, and two replicates were performed for each test concentration. The recombinant collagen and Hacat cells were placed in a cell incubator for co-culture for 24 hours.

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

[0086] GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0087] 3. Lysis of Hacat cells

[0088] (1) The Hacat cells co-cultured in the six-well culture dish were removed from the incubator, the medium was aspirated, and 200 μL of preheated PBS was added to each well to gently wash the cell surface. Then, the PBS was aspirated and 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 the lysate. Divide into 1.5 mL centrifuge tubes, label, and use for subsequent analysis.

[0090] Example 2

[0091] Transcriptome sequencing was performed on the cell lysate in Example 1.

[0092] 1. Analysis scheme

[0093] 1.1 Species name: Homo sapiens; source: NCBI; reference genome version: GCF_000001405.40 GRCh38.p14.

[0094] 1.2 Differential grouping settings

[0095] One of the important analysis points of transcriptome sequencing is the differential comparison of expression levels. In pairwise comparison, the control and treatment are set as shown in Table 2.

[0096] Table 2

[0097] Control Treatment HaCat_untreated (HaCat cells untreated) HaCat_treated (Hacat cells treated)

[0098] 2. Experimental procedure

[0099] (1) mRNA library construction procedure

[0100] 1) total RNA is treated with mRNA enrichment or rRNA removal method. mRNA enrichment: mRNA with polyA tail is enriched with OligodT magnetic beads; rRNA removal: rRNA is hybridized with DNA probe, DNA / RNA hybrid chain is selectively digested by RNaseH, DNA probe is digested by DNaseI, and the desired RNA is obtained after purification.

[0101] 2) The obtained RNA is fragmented with fragmentation buffer, and random N6 primers are used for reverse transcription, and then cDNA double strands are synthesized to form double-stranded DNA.

[0102] 3) The synthesized double-stranded DNA is end-repaired and 5' phosphorylated, and a 3' end is formed with a "A" overhanging sticky end, and then a 3' end with a "T" overhanging bubble-shaped adapter is connected.

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

[0104] 5) The PCR product is heat denatured into single-stranded DNA, 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 machine.

[0106] 3) Filtering of sequencing data

[0107] The raw data of sequencing contains reads with low quality, adapter contamination and too much N content. These reads should be removed before data analysis to ensure the reliability of the results. The analysis methods of data filtering include:

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

[0109] 2) Remove reads with more than 1% N content.

[0110] 3) Remove reads with low quality (we define reads with more than 40% bases with quality value less than 15 as low quality reads).

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

[0112] 4) Reference genome alignment

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

[0114] 5) Reference gene alignment

[0115] Use Bowtie2 to align Clean Reads to the reference gene sequence, and then use RSEM to calculate the expression level of genes and transcripts.

[0116] 6) Gene annotation

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

[0118] (2) Use diamond to align mRNA to NR, KOG, KEGG, uniprot, COG, TF (Animal Transcription Factor Database is AnimalTFDB3, Plant Transcription Factor Database is PlantTFdb5), PRGDB database; based on uniprot (including sprot and trembl) alignment information to obtain GO annotation information.

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

[0120] 7) Gene quantification

[0121] Clean reads were aligned to the genome sequence using Bowtie2, and then RSEM was used to calculate the gene expression level of each sample. RSEM is a software package for RNA-seq reads to calculate gene and transcript subtype expression.

[0122] 8. Differential expression analysis

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

[0124] According to the GO annotation results and official classification, the differential 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 differential genes were classified into biological pathways, and enrichment analysis was performed using the phyper function in R software.

[0126] 9. Results analysis

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

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

[0129] Table 3

[0130]

[0131] From Table 3, it can be seen that the Hacat cells treated with recombinant collagen have a mean Log2 fold change of 1.072 between the experimental group and the control group, i.e., the difference multiple is 2.102, which is higher than the promotion standard of 1.19. It is proved that recombinant collagen can promote the expression of skin barrier function related genes.

[0132] In this example, a series of skin barrier function related gene expression situations were detected by transcriptomics, which proved that recombinant collagen can enhance skin barrier function.

[0133] Example 3

[0134] This embodiment uses recombinant collagen for experiment, to verify the promoting effect of different concentrations of recombinant collagen on the expression of a series of skin barrier function related genes. The experimental steps include:

[0135] 1. Cell sample culture

[0136] Collagen was prepared into 0.1 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 1.5 mg / mL concentration of test solution. The above test solution was added to the culture medium of the BJ cells growing on the substrate to about 70% of the area of the culture dish at a final concentration of 0.1%, and cultured for 24 hours.

[0137] 2. Total RNA extraction and purification

[0138] (1) The cultured cells were removed from the culture medium, washed twice with pre-cooled PBS, 1 mL of TRIzol reagent was added, and the collected centrifuge tube was shaken at room temperature for 5 minutes.

[0139] (2) 200 μL of chloroform was added to the centrifuge tube, the centrifuge tube was shaken vigorously for 15 seconds, and after standing for 10 minutes, it was centrifuged at 4°C at 12000g for 15 minutes.

[0140] (3) After centrifugation, the liquid was layered, and the RNA was in the upper colorless transparent aqueous phase layer with a volume of about 600 μL. After transferring to a new centrifuge tube, 500 μL of isopropanol was added, mixed slowly by inverting several times, and after standing for 15 minutes, it was centrifuged at 4°C at 12000g for 10 minutes.

[0141] (4) After centrifugation, the RNA precipitate was at the bottom of the centrifuge tube, and a gelatinous precipitate was visible when facing the light source. After removing the supernatant, 1 mL of 75% ethanol was added to resuspend and wash the RNA, and it was centrifuged at 4°C at 7500g for 5 minutes.

[0142] (5) The ethanol supernatant was removed, the RNA was dried for 5 minutes, and 20 μL of DEPC-water was added 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 a total volume of 100 μL. Add the diluted RNA solution to the 96-well UV ultraviolet version.

[0145] (2) Place the 96-well version in the full-wavelength enzyme marker, select the area to be tested, and set the program as follows: shake for 10 seconds, measure the absorbance at 260 nm, measure the absorbance at 280 nm, and read the values.

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

[0147] (4) According to the formula OD 260 × 0.04 × dilution factor, the RNA solution concentration is calculated, which is μg / μL. Adjust the RNA concentration to 1 μg / μL with DEPC-water.

[0148] 4. Reverse transcription

[0149] (1) Use the reverse transcription kit (Novozyme one-step reverse transcription kit R333) to prepare the reverse transcription system in a 200 μL PCR tube, which contains: a) RNA solution with a concentration of 1 μg / μL, 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, which is mixed uniformly with a pipette and centrifuged for 30 seconds with a palm centrifuge.

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

[0151] 5. Fluorescent quantitative PCR

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

[0153] (2) Each sample needs three repeated holes for each molecule to be tested, and a blank control without adding cDNA is set. In addition to the molecules to be tested, actin beta (ACTB) is also needed as a reference molecule.

[0154] (3) Place the eight-tube PCR tube in the fluorescent quantitative PCR instrument, and set the reaction program as follows: a) 95℃ for 2 minutes; b) 95℃ for 5 seconds; c) 60℃ for 10 seconds; d) constant temperature preservation at 20℃. The steps b and c are repeated for 45 cycles.

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

[0156] 6. Quantitative analysis of results

[0157] (1) Average the CT values of each sample in triplicate, calculate the difference between the CT value of the molecule to be tested and the CT value of the reference molecule (ACTB), namely 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, namely ΔΔCT.

[0159] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the index, namely 2 -△△CT , of each concentration of the parallel group -△△CT , 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 as shown in Table 4.

[0160] Table 4

[0161]

[0162] From Table 4, it can be seen that 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 recombinant collagen protein for increasing the expression of skin barrier function related genes.

[0163] Example 4

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

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

[0166] SEQ ID NO: 46:

[0167] LPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGASPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVRGAIGSKGESGVDGLMGPAGPKGQPGDPGPQGPPGLDGKPGRE

[0168] SEQ ID NO: 47:

[0169] GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP

[0170] SEQ ID NO: 48:

[0171] GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT

[0172] The experimental procedure included:

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

[0174] 2. The subsequent RNA extraction, reverse transcription, and qPCR experiment were operated according to the operation of Example 3. The experimental results are shown in Table 5.

[0175] Table 5

[0176]

[0177]

[0178] From Table 5, it can be seen that the recombinant collagen of the present application can up-regulate 21 kinds of skin fibroblast proliferation related genes, while other collagen 1, collagen 2, collagen 3 cannot up-regulate all the 21 kinds of genes, and the up-regulation effect is not as good as the recombinant collagen of the present application. It can be seen that the collagen of the present application has better effect of improving skin barrier function, which is mutually confirmed with the conclusion in the foregoing Example 3, and the recombinant collagen of the present application has a significant improvement effect on improving skin barrier function.

[0179] Application Example 1

[0180] The recombinant collagen in the present application can be used as an active ingredient for the preparation of skin conditioners and cosmetics. For example, it can be a cream, emulsion, jelly, cosmetic water, serum, mask, eye cream, aerosol cleaning foam, spray, shower gel or facial cleanser.

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

[0182] Application 2, preparation of emulsion, prepared 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% cetostearyl alcohol ether-21, 1.2% cetostearyl alcohol ether-2, 0.5% tocopherol, 0.2% nipagin methyl ester, 0.1% triethanolamine, 0.1% nipagin ethyl ester, 0.1% carbomer, the rest is water (74.2%).

[0183] Application 3, preparation of cosmetic water, prepared according to the following ratio: 0.1% recombinant collagen, 6% glycerin, 2% propylene glycol, 0.2% diazime imidazole alkyl urea, the rest is water (91.7%).

[0184] In summary, the present application detects the expression of a series of skin barrier function related genes by transcriptomics, and proves that recombinant collagen can promote the expression of skin barrier function related genes. The collagen can be used for the development and application of various cosmetic products, and endows the cosmetic products with increased skin barrier function. At the same time, the series of genes can also be used as a reference for evaluating the effect of other raw materials or cosmetics on promoting skin barrier function in the future.

[0185] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. Use of a recombinant collagen for the preparation of a cosmetic product for improving the barrier function of the skin, characterized in that, The amino acid sequence of the recombinant collagen is shown as SEQ ID NO:

45.

2. The use according to claim 1 in the preparation of a cosmetic product for improving the barrier function of the skin, characterized in that, The cosmetic increases the expression amount 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.

3. The use according to claim 1 in the preparation of a cosmetic product for improving the barrier function of the skin, characterized in that, The concentration of the recombinant collagen in the cosmetic is 0.9-1.1 mg / mL.

4. The use according to claim 1 in the preparation of a cosmetic product for improving the barrier function of the skin, characterized in that, The cosmetic includes a face cream, a lotion, a gel, a cosmetic water, an essence, a mask, an eye cream, an aerosol cleaning foam, a spray, a shower gel, or a facial cleanser.

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