Application of recombinant collagen in preparation of preparation for increasing expression quantity of skin extracellular matrix homeostasis related genes

Abstract

The invention provides application of recombinant collagen in preparation of a preparation for increasing the expression quantity of skin extracellular matrix homeostasis related genes. The amino acid sequence of the recombinant collagen is shown as SEQ ID NO: 1. The invention also provides a biomarker, wherein the biomarker is any one or a combination of at least two of skin extracellular matrix homeostasis related genes including CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA.

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 increasing the expression of genes related to the homeostasis of skin extracellular matrix. Background Art

[0002] Collagen is a biological macromolecule and the main component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25-30% of the total protein, and even as high as 80% or more in some organisms.

[0003] There are many types of collagen, the most common types are type I, type II, type III, type V and type XI. Collagen has good biocompatibility, biodegradability and biological activity, so it is widely used in food, medicine, tissue engineering, cosmetics and other fields.

[0004] Collagen plays a very important supporting role in the skin, maintaining the firmness and elasticity of the skin, especially type I collagen, which is the most abundant collagen in the skin, accounting for about 90% of the total collagen content. However, as we age, the amount of collagen synthesis in the human body gradually decreases, while the rate of collagen loss remains the same or even faster, so the total content of natural collagen decreases.

[0005] Collagen has excellent biocompatibility and bioactivity, can effectively promote wound healing, and plays an important role in the repair of skin, bones, tendons and other tissues. Collagen can regulate inflammatory responses, affect cell growth, differentiation, apoptosis and other processes, thereby maintaining tissue homeostasis.

[0006] Existing research and literature disclose many experimental methods for detecting the effect of collagen on maintaining tissue homeostasis, most of which are based on macroscopic detection indicators such as cell morphology, elasticity, skin regeneration, and skin repair. In addition to macroscopic detection indicators, we can also use skin extracellular matrix homeostasis-related genes as detection indicators to evaluate the regulatory effect of collagen or other effective raw materials on skin homeostasis. However, there is a lack of relevant biomarkers at present. Therefore, the development of new collagen and biomarkers related to skin extracellular matrix homeostasis has important application value. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide the use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to the homeostasis of skin extracellular matrix. The present invention screened a series of genes related to the homeostasis of skin extracellular matrix by transcriptomics, and proved that the recombinant collagen in the present invention can promote the expression of genes related to the homeostasis of skin extracellular matrix, which can be used for the development and application of a variety of cosmetic products with the effect of promoting the homeostasis of skin extracellular matrix.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] 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 extracellular matrix homeostasis, wherein the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:1.

[0010] Preferably, the skin extracellular matrix homeostasis-related genes include any one or a combination of at least two of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA.

[0011] The present invention detects the expression of a series of genes related to the homeostasis of skin extracellular matrix by transcriptomics, proving that the recombinant collagen can promote the expression of genes related to the homeostasis of skin extracellular matrix. It can be used for the development and application of a variety of cosmetic products with the efficacy of promoting the homeostasis of skin extracellular matrix. At the same time, the above series of genes can also be used as a reference for evaluating the ability of other raw materials or cosmetics to maintain the homeostasis of skin extracellular matrix in the future.

[0012] Preferably, the genes related to skin extracellular matrix homeostasis include: a combination of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 and VEGFA.

[0013] Preferably, the preparation comprises a medicine or a cosmetic.

[0014] Preferably, the concentration of the recombinant collagen in the preparation is 0.4-0.6 mg / mL, for example, it can be 0.4 mg / mL, 0.5 mg / mL or 0.6 mg / mL.

[0015] Preferably, the cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

[0016] The present invention also provides an expression promoter for increasing the expression of genes related to the homeostasis of skin extracellular matrix. The expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:1.

[0017] In a preferred embodiment of the present invention, after the recombinant collagen is administered, the average relative expression level of the skin extracellular matrix homeostasis-related genes is 2.005.

[0018] 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.

[0019] The present invention provides a biomarker for evaluating the ability of cosmetics or their raw materials to maintain the homeostasis of skin extracellular matrix. The biomarker is a gene related to the homeostasis of skin extracellular matrix. The gene includes any one of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA, or a combination of at least two of them.

[0020] Preferably, the genes include: CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 and VEGFA.

[0021] In a second aspect, the present invention provides an application of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to maintain homeostasis of skin extracellular matrix, wherein the biomarker is a gene related to homeostasis of skin extracellular matrix, and the gene includes any one of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA, or a combination of at least two thereof.

[0022] Preferably, the genes include: CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 and VEGFA.

[0023] Preferably, the product comprises a primer set, a reagent or a detection model.

[0024] Preferably, the reagents include qPCR detection reagents.

[0025] In a third aspect, the present invention provides a primer combination for detecting the expression level of a biomarker, the primer combination comprising:

[0026] The upstream primer for amplifying CD44 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:6;

[0027] The upstream primer for amplifying COL16A1 is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; the upstream primer for amplifying COL23A1 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:10; the upstream primer for amplifying COL27A1 is shown in SEQ ID NO:11, and the downstream primer is shown in SEQ ID NO:12; the upstream primer for amplifying COL4A1 is shown in SEQ ID NO:13, and the downstream primer is shown in SEQ ID NO:14; the upstream primer for amplifying COL6A3 is shown in SEQ ID NO:15, and the downstream primer is shown in SEQ ID NO:16; the upstream primer for amplifying COL6A6 is shown in SEQ ID NO:17, and the downstream primer is shown in SEQ ID NO:18; the upstream primer for amplifying COL7A1 is shown in SEQ ID NO:19, and the downstream primer is shown in SEQ ID NO:20; the upstream primer for amplifying DPT is shown in SEQ ID NO:21, and the downstream primer is shown in SEQ ID NO:22;

[0028] The upstream primer for amplifying ELN is shown in SEQ ID NO:23, and the downstream primer is shown in SEQ ID NO:24;

[0029] The upstream primer for amplifying HAS2 is shown in SEQ ID NO:25, and the downstream primer is shown in SEQ ID NO:26;

[0030] The upstream primer for amplifying HAS3 is shown in SEQ ID NO:27, and the downstream primer is shown in SEQ ID NO:28;

[0031] The upstream primer for amplifying IGFBP5 is shown in SEQ ID NO:29, and the downstream primer is shown in SEQ ID NO:30; the upstream primer for amplifying LAMB3 is shown in SEQ ID NO:31, and the downstream primer is shown in SEQ ID NO:32; the upstream primer for amplifying LAMB4 is shown in SEQ ID NO:33, and the downstream primer is shown in SEQ ID NO:34; the upstream primer for amplifying LAMC2 is shown in SEQ ID NO:35, and the downstream primer is shown in SEQ ID NO:36; the upstream primer for amplifying SLIT2 is shown in SEQ ID NO:37, and the downstream primer is shown in SEQ ID NO:38;

[0032] The upstream primer for amplifying LUM is shown in SEQ ID NO:39, and the downstream primer is shown in SEQ ID NO:40;

[0033] The upstream primer for amplifying P4HA3 is shown in SEQ ID NO:41, and the downstream primer is shown in SEQ ID NO:42;

[0034] The upstream primer for amplifying PDGFA is shown in SEQ ID NO:43, and the downstream primer is shown in SEQ ID NO:44; the upstream primer for amplifying PTX3 is shown in SEQ ID NO:45, and the downstream primer is shown in SEQ ID NO:46;

[0035] The upstream primer for amplifying SERPINE1 is shown in SEQ ID NO:47, and the downstream primer is shown in SEQ ID NO:48; the upstream primer for amplifying SPP1 is shown in SEQ ID NO:49, and the downstream primer is shown in SEQ ID NO:50;

[0036] The upstream primer for amplifying TGFB2 is shown in SEQ ID NO:51, and the downstream primer is shown in SEQ ID NO:52;

[0037] The upstream primer for amplifying TGFBR2 is shown in SEQ ID NO:53, and the downstream primer is shown in SEQ ID NO:54; the upstream primer for amplifying VEGFA is shown in SEQ ID NO:55, and the downstream primer is shown in SEQ ID NO:56. The sequences of the primer combinations are shown in Table 1:

[0038] Table 1

[0039]

[0040]

[0041] The present invention can be used to quickly verify the ability of new raw materials or products to promote fibroblast proliferation by screening a series of gene primers, and ensure the accuracy of experimental results.

[0042] 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.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention detects the expression of a series of genes related to the homeostasis of extracellular matrix of skin cells by transcriptomics, and proves that the recombinant collagen can promote the expression of genes related to the homeostasis of extracellular matrix of skin cells. It can be used for the development and application of a variety of cosmetic products with the effect of promoting the homeostasis of extracellular matrix of skin cells. At the same time, the above series of genes can also be used as a reference for evaluating the ability of other raw materials or cosmetics to maintain the homeostasis of extracellular matrix of skin cells in the future. The present invention summarizes a series of primers of genes that promote the homeostasis of extracellular matrix by design, so as to facilitate the rapid and accurate detection of the expression ability of related genes in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a heat map of transcriptomic analysis. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] Example 1

[0049] In this embodiment, an expression promoter for increasing the expression of genes related to skin extracellular matrix homeostasis is prepared. The expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:1.

[0050] GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGA PGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0051] 1. Fermentation process

[0052] (1) Strain activation: Streak out the strain on a YPD medium plate and culture at 30°C for 72 hours.

[0053] (2) Seed solution preparation: single colonies with good growth were selected in a clean bench, inoculated into a conical flask containing YPD medium, and cultured in a shaking incubator at 30°C and 250 rpm for 30 hours.

[0054] (3) Fermentation tank inoculation: transfer the seed solution to a fermentation tank containing 40 g / L glycerol and 4.35 mL / L BSM medium. The fermentation stage is controlled according to the following conditions:

[0055] 1): Initial glycerol consumption stage: the temperature is maintained at 30°C, the pH is maintained at 5.5 with ammonia water, the air flow is 2vvm, and the dissolved oxygen is stirred and maintained at 20%-40%. After the dissolved oxygen value soars, enter the additional glycerol supplementation stage.

[0056] 2): Glycerol addition stage: Keep the temperature, pH, and air flow constant, and add 80-100 mL of 50% glycerol. Observe the change rate of DO to control the flow rate, and monitor OD at all times 600 . Waiting for OD 600 When the cell count reaches 250, stop adding glycerol and starve the cell for about an hour.

[0057] 3): Methanol induction stage: Control the temperature to 25℃, pH to 6, add 30-50mL of pure methanol at a flow rate of 35mL / h. Similarly, observe the change of DO to determine the flow rate. Finally, based on the non-accumulation of methanol, the flow rate stabilizes at about 10mL / L / h. Fermentation ends after 96-120 hours of pure methanol induction.

[0058] Experimental samples and equipment: ceramic membrane with a pore size of 0.45 μm, Dongfulong hydrophobic chromatography column Phevl Purose6FF; PrePack50 / 200.

[0059] 2. Purification process

[0060] (1) The obtained fermentation broth was first filtered using a 0.45 μm ceramic membrane and then subjected to a hydrophobic chromatography purification process. The specifications of the sample and the chromatography column are shown in Table 2.

[0061] Table 2

[0062] sample Recombinant collagen fermentation broth filler Phenyl Purose 6FF Column Packing Specifications PrePack 50×200 Sample volume 2CV Experimental flow rate 20mL / min Detection wavelength 220nm

[0063] (2) Preparation of experimental materials and buffer solution:

[0064] Distilled water: 5L;

[0065] Equilibration buffer: 20 mM PB, 1 M ammonium sulfate, pH 7, 5 L;

[0066] Elution buffer: 20 mM PB, pH 7, 5 L;

[0067] Wash buffer: 1M NaOH, 1 L;

[0068] Note: The above buffers must be filtered through a 0.45μm filter membrane.

[0069] (3) The purification experimental steps are as follows:

[0070] 1) Flush the column with equilibration buffer at a linear flow rate of 90 cm / h for 3 CV until the pH, UV, and Cond (conductivity) detection baselines are stable and consistent with the equilibration buffer.

[0071] 2) Load the sample and equilibration buffer at a ratio of 1:1 at a rate of 60 cm / h (20 mL / min), collect the flow-through components during the loading process and detect the purity and content of the target protein.

[0072] 3) After loading, flush with equilibrium buffer at a speed of 90 cm / h (30 mL / min) for 2CV to wash out unbound materials until the UV detection value drops to the initial value.

[0073] 4) The elution buffer and the equilibrium buffer were eluted at a rate of 90 cm / h (30 mL / min) in a ratio of 6:4 to elute the target protein and collect the eluted fractions.

[0074] 5) Wash the column with at least 2 column volumes of 1 M NaOH at a rate of 60 cm / h (20 mL / min) to remove residual contaminants such as lipids, endotoxins, nucleic acids, etc. in the packed column.

[0075] 6) Wash the column with 5-10 CV of distilled water at a rate of 60 cm / h (20 mL / min) until the pH of the effluent is neutral and the Cond (conductivity) is close to 0 to remove 1 M NaOH.

[0076] 7) Wash with 3CV of 20% ethanol to prevent microbial growth.

[0077] Example 2

[0078] This example uses transcriptomic sequencing to study the effect of the recombinant collagen in Example 1 on skin homeostasis.

[0079] The recombinant collagen was prepared into a solution with a concentration of 1 mg / mL and added to the cell culture medium of BJ cells (human skin fibroblasts) growing on the wall. After 24 hours of culture, the cells were lysed with Trizon and the cell lysate was sent to BGI for transcriptome sequencing. The analysis scheme is shown below.

[0080] 1. Analysis plan

[0081] 1.1 Species name: Homo sapiens; Source: NCBI; Reference genome version: GCF_000001405.40_GRCh38.p14.

[0082] 1.2 Difference Grouping Settings

[0083] 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 3 below.

[0084] Table 3

[0085] serial number Comparison deal with 1 BJ_control group (untreated) BJ_experimental group (treated)

[0086] 2. Experimental process

[0087] 2.1 mRNA library construction process

[0088] 1) Treat total RNA using mRNA enrichment or rRNA removal methods; mRNA enrichment: use magnetic beads with OligodT to enrich mRNA with polyA tails; rRNA removal: use DNA probes to hybridize rRNA, RNaseH selectively digests the DNA / RNA hybrid chain, and then use DNaseI to digest the DNA probe, and then obtain the desired RNA after purification.

[0089] 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.

[0090] 3) The ends of the synthesized double-stranded DNA are filled in and phosphorylated at the 5' end, forming a sticky end with a protruding "A" at the 3' end, and then connected to a bubble-shaped linker with a protruding "T" at the 3' end.

[0091] 4) The ligation product is amplified by PCR using specific primers.

[0092] 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.

[0093] 6) Sequencing on a machine.

[0094] 4. Data analysis and results

[0095] 4.1 Data Analysis Process

[0096] The original data obtained by sequencing is called raw reads. First, we filter out reads with low quality, connector contamination, and high unknown base N content. The filtered data is called clean reads. Then we align the cleanl reads to the reference genome, and then perform new transcript prediction, SNP&InDel and differential splicing gene detection. After obtaining the new transcript, we add the new transcript with protein coding potential to the reference gene sequence to form a complete reference sequence, and then calculate the gene expression level. Finally, for multiple samples, we detect differentially expressed genes between different samples according to needs, and perform in-depth clustering analysis and functional enrichment analysis on the differentially expressed genes.

[0097] 4.2 The transcriptomic sequencing results obtained were retained with a p value > 0.05. The FPKM values ​​and 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. The selected genes are as follows:

[0098] CD44: a cell surface receptor that binds to hyaluronic acid and other ECM components, regulating cell-ECM interactions and tissue stability.

[0099] COL gene family: Encodes the main structural protein in the skin: collagen. The fibers composed of collagen expressed by various COL gene subtypes form a mesh structure in the ECM to maintain the firmness of the skin.

[0100] DPT: This gene encodes a glycoprotein that builds support and regulates the role of collagen in the ECM of the skin and is involved in the structure and repair of the skin.

[0101] ELN: encodes elastin, an important component of skin ECM and plays an important role in maintaining homeostasis.

[0102] HAS gene family: Encodes hyaluronan synthase, catalyzing the synthesis of hyaluronan. Hyaluronan is an important glycosaminoglycan in the skin that maintains moisture, elasticity and volume of the skin and is essential for ECM homeostasis.

[0103] IGFBP5: can directly stimulate fibroblasts to synthesize key components of ECM, including collagen and elastin, thereby promoting the formation and maintenance of ECM and improving the structure and elasticity of the skin;

[0104] LAM gene family: encodes laminins, which anchor skin cells to the ECM and interact with other proteins in the ECM, affecting their stability and degradation.

[0105] SLIT2: encodes slipopolysaccharide type 2, which plays a role in regulating cell migration and ECM tissue remodeling, affecting ECM structure and cell behavior.

[0106] LUM: encodes proteoglycans in the ECM, regulates collagen fiber formation and tissue repair, and helps maintain the homeostasis of skin and connective tissue.

[0107] P4HA3: The enzyme encoded by this gene is mainly responsible for hydroxylating proline residues in collagen to 4-hydroxyproline. This modification is a key step in the stabilization and normalization of collagen molecules. It plays an important role in the homeostasis of ECM.

[0108] PDGF: plays an important role in cell proliferation and angiogenesis, and promotes ECM synthesis and tissue repair by regulating the activity of fibroblasts.

[0109] PTX3: This gene can bind to various proteins in the ECM and participate in the remodeling process of the ECM. During wound healing or tissue repair, the expression of PTX3 increases, helping to regulate the structure and function of the newly formed ECM.

[0110] SERPINE1: This gene participates in the regulation of ECM degradation balance by inhibiting the activity of plasminogen and is related to skin healing and tissue remodeling.

[0111] SPP1: SPP is involved in the interaction between cells and ECM and plays an important role in the health of skin tissue by regulating the integration of ECM components and tissue stability.

[0112] TGFB2, TGFB2R: These genes and receptors play an important role in tissue repair and fibrosis. They regulate the balance of ECM synthesis and degradation and participate in wound healing and tissue remodeling.

[0113] VEGFA: The growth factor encoded by this gene not only promotes angiogenesis, but also promotes skin damage repair and tissue regeneration by regulating the composition and metabolism of ECM.

[0114] 4.3 The FPKM values ​​of the above genes in the experimental group and the control group were integrated into the same table, see Table 4. The heat map was obtained by heat map analysis of the Kidio Bioinformatics Cloud tool, as shown in Figure 1 .

[0115] Table 4

[0116]

[0117]

[0118] As can be seen from Table 4, the final calculated log2 fold change was about 0.75, which means the relative expression was about 1.68437.

[0119] This example detects the expression of a series of genes related to the homeostasis of skin extracellular matrix by transcriptomics, proving that recombinant collagen can promote the expression of genes related to the homeostasis of skin extracellular matrix.

[0120] Example 3

[0121] This example uses the recombinant collagen obtained in Example 1 to conduct experiments to verify the effects of different concentrations of recombinant collagen on the expression of a series of genes related to the homeostasis of extracellular matrix in skin cells. The experimental steps include:

[0122] 1. Cell sample culture

[0123] Collagen was prepared into test solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1.0 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.

[0124] 2. Extraction and purification of total RNA in cells

[0125] (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.

[0126] (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.

[0127] (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.

[0128] (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.

[0129] (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.

[0130] 3. RNA concentration determination

[0131] (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.

[0132] (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.

[0133] (3) Calculation of OD 260 / OD 280 Ratio: A ratio between 1.8 and 2.0 indicates that the quality of the extracted RNA meets subsequent needs.

[0134] (4) According to the formula OD 260 × 0.04 × dilution factor to calculate the RNA solution concentration in μg / μL; dilute with DEPC-water to adjust the RNA concentration to 1 μg / μL.

[0135] 4. Reverse transcription

[0136] (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.

[0137] (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.

[0138] 5. Fluorescence quantitative PCR

[0139] (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.

[0140] (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.

[0141] (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.

[0142] (4) After the PCR process is completed, the software automatically generates the CT value for each sample.

[0143] 6. Quantitative analysis of results

[0144] (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.

[0145] (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.

[0146] (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 is taken as the quantitative result of the molecule to be tested in each sample to achieve quantitative detection at the gene level.

[0147] The experimental results are shown in Table 5.

[0148] Table 5

[0149]

[0150]

[0151] As can be seen from Table 5, compared with other concentrations, 0.5 mg / mL of recombinant collagen can ensure the promotion of all genes related to skin extracellular matrix homeostasis, and the average promotion rate is the highest.

[0152] Example 4

[0153] This example uses the recombinant collagen obtained in Example 1 as well as collagen 1, collagen 2, and collagen 3 to simultaneously test their effects on promoting the expression of a series of genes related to the homeostasis of skin extracellular matrix.

[0154] The amino acid sequence of collagen 1 is shown in SEQ ID NO:2; the amino acid sequence of collagen 2 is shown in SEQ ID NO:3; and the amino acid sequence of collagen 3 is shown in SEQ ID NO:4.

[0155] SEQ ID NO:2:

[0156] LPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGASPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVRGAIGSKGESGVDGLMGPAGPKGQPGDPGPQGPPGLDGKPGRE。

[0157] SEQ ID NO:3:

[0158] GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP。

[0159] SEQ ID NO:4:

[0160] GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGP PGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPP GPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT.

[0161] The experimental steps include:

[0162] 1. Take the recombinant collagen obtained in Example 1 and collagen 1, collagen 2, and collagen 3 respectively, prepare them into 0.5 mg / mL test solution, add them into the culture medium of BJ cells that are growing on the wall to about 70% of the bottom area of ​​the culture dish at a final concentration of 0.1%, and culture for 24 hours.

[0163] 2. The subsequent RNA extraction, reverse transcription, and qPCR experimental operations were the same as those described in Example 3. The experimental results are shown in Table 6.

[0164] Table 6

[0165] Gene Recombinant collagen Collagen 1 Collagen 2 Collagen 3 CD44 1.209 1.143 1.088 1.180 COL16A1 1.314 1.185 1.045 1.202 COL23A1 4.919 3.075 2.277 3.938 COL27A1 1.561 1.241 1.073 1.422 COL4A1 1.317 1.119 0.892 1.126 COL6A3 1.389 1.338 1.249 1.263 COL6A6 1.623 0.944 1.368 1.081 COL7A1 1.300 1.021 0.915 1.098 DPT 6.058 1.857 5.288 4.099 ELN 1.305 0.921 1.185 1.289 HAS2 2.620 1.316 1.274 1.016 HAS3 1.964 1.355 1.231 1.806 IGFBP5 1.196 0.954 1.009 1.151 LAMB3 1.340 0.993 1.098 1.303 LAMB4 1.781 1.368 1.043 1.500 LAMC2 1.622 1.554 1.126 1.388 SLIT2 1.244 1.030 0.896 1.076 LUM 2.350 1.138 1.508 2.057 P4HA3 1.446 1.183 1.266 1.400 PDGFA 1.350 0.978 1.245 1.332 PTX3 5.787 1.124 4.288 4.522 SERPINE1 1.350 1.134 1.249 1.177 SPP1 1.703 1.547 1.244 1.403 TGFB2 1.571 1.147 1.337 1.522 TGFBR2 1.269 1.065 0.903 1.203 VEGFA 1.550 0.940 1.439 1.141 average value 2.005 1.257 1.482 1.642

[0166] It can be seen from Table 6 that, compared with other collagens, the recombinant collagen of the present invention can ensure the promotion of all genes related to the homeostasis of the skin extracellular matrix, and the average promotion rate is the highest.

[0167] Application Example 1

[0168] 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.

[0169] Application 1, preparation of facial cream, formulated according to the following ratio: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 5.5% liquid paraffin, 4% glycerol, 3.5% propylene glycol, 3% ethylhexyl palmitate, 4% petrolatum, 2% cetearyl ether-21, 2% polydimethylsiloxane, 2% 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 (63.2%).

[0170] Application 2, preparation of emulsion, formulated according to the following ratio: 0.1% recombinant collagen, 4% glycerin, 5.5% hydrogenated polyisobutene, 4.5% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% dimethicone, 1.5% ceteareth-21, 1.0% ceteareth-2, 0.5% tocopherol, 0.2% methylparaben, 0.1% triethanolamine, 0.1% ethylparaben, 0.1% carbomer, the balance is water (74.4%).

[0171] Application 3, preparation of lotion, prepared according to the following ratio: 0.1% recombinant collagen, 5% glycerol, 3% propylene glycol, 0.2% diazoimidazole urea, and the balance is water (91.7%).

[0172] In summary, the present invention detects the expression of a series of genes related to the homeostasis of skin extracellular matrix by transcriptomics, and proves that recombinant collagen can promote the expression of genes related to the homeostasis of skin extracellular matrix. The collagen can be used for the development and application of various cosmetic products, giving cosmetic products the efficacy and effect of promoting the homeostasis of skin extracellular matrix. At the same time, the series of genes can also be used as a reference for evaluating the ability of other raw materials or cosmetics to maintain the homeostasis of skin extracellular matrix in the future.

[0173] 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 the homeostasis of skin extracellular matrix, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The skin extracellular matrix homeostasis-related genes include: any one of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA, or a combination of at least two thereof.

3. The use according to claim 1 or 2, characterized in that: The skin extracellular matrix homeostasis-related genes include: a combination of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 and VEGFA.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation includes a medicine or a cosmetic.

5. The use according to any one of claims 1 to 4, characterized in that: The concentration of recombinant collagen in the preparation is 0.4-0.6 mg / mL.

6. The use according to claim 4 or 5, 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 ability of cosmetics or their raw materials to maintain the homeostasis of the extracellular matrix of the skin, characterized in that: The biomarker is a gene related to skin extracellular matrix homeostasis, and the gene includes: any one of CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 or VEGFA, or a combination of at least two thereof.

8. The use according to claim 7, characterized in that: The genes include: CD44, COL16A1, COL23A1, COL27A1, COL4A1, COL6A3, COL6A6, COL7A1, DPT, ELN, HAS2, HAS3, IGFBP5, LAMB3, LAMB4, LAMC2, SLIT2, LUM, P4HA3, PDGFA, PTX3, SERPINE1, SPP1, TGFB2, TGFBR2 and VEGFA.

9. The use according to claim 7 or 8, characterized in that: The products include primer sets, reagents or detection models.

10. A primer combination for detecting the expression level of a biomarker, characterized in that: The primer combination comprises: The upstream primer for amplifying CD44 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:6; The upstream primer for amplifying COL16A1 is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; The upstream primer for amplifying COL23A1 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:10; The upstream primer for amplifying COL27A1 is shown in SEQ ID NO: 11, and the downstream primer is shown in SEQ ID NO: 12; The upstream primer for amplifying COL4A1 is shown in SEQ ID NO: 13, and the downstream primer is shown in SEQ ID NO: 14; The upstream primer for amplifying COL6A3 is shown in SEQ ID NO: 15, and the downstream primer is shown in SEQ ID NO: 16; The upstream primer for amplifying COL6A6 is shown in SEQ ID NO: 17, and the downstream primer is shown in SEQ ID NO: 18; The upstream primer for amplifying COL7A1 is shown in SEQ ID NO: 19, and the downstream primer is shown in SEQ ID NO: 20; The upstream primer for amplifying DPT is shown in SEQ ID NO:21, and the downstream primer is shown in SEQ ID NO:22; The upstream primer for amplifying ELN is shown in SEQ ID NO:23, and the downstream primer is shown in SEQ ID NO:24; The upstream primer for amplifying HAS2 is shown in SEQ ID NO:25, and the downstream primer is shown in SEQ ID NO:26; The upstream primer for amplifying HAS3 is shown in SEQ ID NO:27, and the downstream primer is shown in SEQ ID NO:28; The upstream primer for amplifying IGFBP5 is shown in SEQ ID NO:29, and the downstream primer is shown in SEQ ID NO:30; The upstream primer for amplifying LAMB3 is shown in SEQ ID NO:31, and the downstream primer is shown in SEQ ID NO:32; The upstream primer for amplifying LAMB4 is shown in SEQ ID NO:33, and the downstream primer is shown in SEQ ID NO:34; The upstream primer for amplifying LAMC2 is shown in SEQ ID NO:35, and the downstream primer is shown in SEQ ID NO:36; The upstream primer for amplifying SLIT2 is shown in SEQ ID NO:37, and the downstream primer is shown in SEQ ID NO:38; The upstream primer for amplifying LUM is shown in SEQ ID NO:39, and the downstream primer is shown in SEQ ID NO:40; The upstream primer for amplifying P4HA3 is shown in SEQ ID NO:41, and the downstream primer is shown in SEQ ID NO:42; The upstream primer for amplifying PDGFA is shown in SEQ ID NO:43, and the downstream primer is shown in SEQ ID NO:44; The upstream primer for amplifying PTX3 is shown in SEQ ID NO:45, and the downstream primer is shown in SEQ ID NO:46; The upstream primer for amplifying SERPINE1 is shown in SEQ ID NO:47, and the downstream primer is shown in SEQ ID NO:48; The upstream primer for amplifying SPP1 is shown in SEQ ID NO:49, and the downstream primer is shown in SEQ ID NO:50; The upstream primer for amplifying TGFB2 is shown in SEQ ID NO:51, and the downstream primer is shown in SEQ ID NO:52; The upstream primer for amplifying TGFBR2 is shown in SEQ ID NO:53, and the downstream primer is shown in SEQ ID NO:54; The upstream primer for amplifying VEGFA is shown as SEQ ID NO:55, and the downstream primer is shown as SEQ ID NO:56.

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