Use of recombinant collagen in the preparation of a preparation for increasing the expression of genes associated with the extracellular matrix homeostasis of the skin

Abstract

The application provides application of recombinant collagen in preparation of a preparation for increasing expression of a skin extracellular matrix homeostasis related gene, and an amino acid sequence of the recombinant collagen comprises SEQ ID NO: 1. The application also provides a biomarker, and 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 application belongs to the field of cosmetic raw material efficacy research, and particularly relates to application of recombinant collagen in preparation of a preparation for increasing expression amount of a skin extracellular matrix homeostasis related gene. BACKGROUND

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

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

[0004] Collagen plays a very important supporting role in the skin and can maintain the tightness and elasticity of the skin. In particular, type I collagen is the most abundant collagen in the skin, accounting for about 90% of the total collagen content. With age, the synthesis of collagen in the human body gradually decreases, but the loss of collagen remains constant or even faster, and the total content of natural collagen becomes less and less.

[0005] Collagen has excellent biocompatibility and biological activity, and can effectively promote wound healing and has an important role in the repair of skin, bone and tendon tissues. Collagen can regulate inflammatory response and affect cell growth, differentiation, apoptosis and other processes, thereby maintaining tissue homeostasis.

[0006] Many experimental methods for detecting the effect of collagen on maintaining tissue homeostasis are disclosed in existing research and literature, and most of them are based on macroscopic detection indexes such as cell morphology, elasticity, skin regeneration and skin repair. In addition to macroscopic detection indexes, skin extracellular matrix homeostasis related genes can also be used as detection indexes to evaluate the regulatory effect of collagen or other functional raw materials on skin homeostasis. However, there is currently a lack of related biomarkers, and therefore, the development of new collagen and skin extracellular matrix homeostasis related biomarkers has important application value. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application aims to provide the use of recombinant collagen in the preparation of a preparation for increasing the expression amount of skin extracellular matrix homeostasis-related genes.

[0008] To achieve the object of the present application, the present application adopts the following technical solutions:

[0009] 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 extracellular matrix homeostasis-related genes, wherein the amino acid sequence of the recombinant collagen comprises SEQ ID NO: 1.

[0010] Preferably, the skin extracellular matrix homeostasis-related genes comprise 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, and VEGFA.

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

[0012] Preferably, the skin extracellular matrix homeostasis-related genes comprise 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 drug 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, etc.

[0015] Preferably, the cosmetic product includes: a cream, a lotion, a jelly, a cosmetic water, an essence, a mask, an eye cream, an aerosol cleansing foam, a spray, a shower gel or a facial cleanser.

[0016] The present application also provides an expression promoter for increasing the expression amount of a skin extracellular matrix homeostasis-related gene, the expression promoter being a recombinant collagen, the amino acid sequence of the recombinant collagen comprising SEQ ID NO: 1.

[0017] In a preferred embodiment of the present application, the average relative expression amount of the skin extracellular matrix homeostasis-related gene after administration of the recombinant collagen is 2.005.

[0018] In the present application, a relative expression amount promotion amount of 1.19 is considered to be promotion. In bioinformatics analysis, a log2 fold change value of 0.25 is usually used as a set threshold for screening differentially expressed genes, which is converted to a relative expression amount multiple of about 1.19.

[0019] The present application provides a biomarker for evaluating the ability of a cosmetic product or its raw material to maintain skin extracellular matrix homeostasis, the biomarker being a skin extracellular matrix homeostasis-related gene, the gene comprising any one of 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 and VEGFA.

[0020] Preferably, the gene comprises 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 application provides use of a product for detecting expression level of a biomarker, which is a skin extracellular matrix homeostasis related gene, including 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, in evaluating ability of a cosmetic or a raw material thereof in maintaining skin extracellular matrix homeostasis.

[0022] Preferably, the gene includes 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 includes a primer combination, a reagent or a detection model.

[0024] Preferably, the reagent includes a qPCR detection reagent.

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

[0026] an upstream primer for amplifying CD44 as shown in SEQ ID NO: 5 and a downstream primer as shown in SEQ ID NO: 6;

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

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

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

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

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

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

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

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

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

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

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

[0038] Table 1

[0039]

[0040]

[0041] The present application can be quickly used for qPCR experiment verification of the promotion ability of new raw materials or products on fibroblast proliferation by screening primers of a series of genes, and the accuracy of the experimental results is ensured.

[0042] The numerical range described in the present application not only includes the point values listed above, but also includes any point values between the above numerical ranges that are not listed, and the present application does not enumerate the specific point values included in the range for the sake of brevity and simplicity.

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

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

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

[0046] The technical solutions of the present application will be further illustrated by the specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations on the present application.

[0047] Unless otherwise specified in the examples, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0048] Example 1

[0049] In this embodiment, an expression promoter for increasing the expression amount of skin extracellular matrix homeostasis-related genes is prepared, and the expression promoter is a recombinant collagen, and the amino acid sequence of the recombinant collagen comprises SEQ ID NO: 1.

[0050] GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0051] 1. Fermentation process

[0052] (1) Strain activation: streak activation on YPD medium plate, 30℃ culture for 72 hours.

[0053] (2) Seed liquid preparation: select single colony with good growth in the clean bench, inoculate into YPD medium in a triangular flask, cultivate for 30 hours at 30°C and 250 rpm.

[0054] (3) Inoculation of the fermenter, transfer the seed liquid to the fermenter containing 40 g / L glycerol and 4.35 mL / L BSM medium. Control the fermentation stage as follows:

[0055] 1) Initial glycerol consumption stage: maintain the temperature at 30°C, maintain the pH at 5.5 with ammonia water, maintain the air flow at 2 vvm, and maintain the dissolved oxygen stirring correlation at 20%-40%. After the dissolved oxygen value rises, enter the additional glycerol supplement stage.

[0056] 2) Glycerol supplement stage: maintain the temperature, pH, and air flow unchanged, supplement 80-100 mL of 50% glycerol. Control the flow rate by observing the change rate of DO, and detect the OD at all times. 600 When the OD 600 increases to 250, stop supplementing glycerol and starve for about one hour.

[0057] 3) Methanol induction stage: control the temperature at 25°C, the pH at 6, and input 30-50 mL of pure methanol at a flow rate of 35 mL / h. Similarly, observe the DO change to determine the flow rate, and finally stabilize the flow rate at about 10 mL / L / h based on the non-accumulation of methanol. After 96-120 hours of pure methanol induction, the fermentation is complete.

[0058] Experimental samples and equipment: ceramic membrane with a pore size of 0.45 μm, hydrophobic chromatography column Phevl Purose 6FF; PrePack 50 / 200.

[0059] 2. Purification process

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

[0061] Table 2

[0062] Sample Recombinant collagen fermentation broth Filler Phenyl Purose 6FF Column packing specification PrePack 50x200 Sample loading amount 2 CV Experimental flow rate 20 mL / min Detection wavelength 220 nm

[0063] (2) Experimental material preparation and buffer preparation:

[0064] Distilled water: 5 L;

[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] Washing buffer: 1 M NaOH, 1 L;

[0068] Note: The above buffers must be filtered through 0.45 pm membrane.

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

[0070] 1) The chromatography column was flushed with 3 CV of equilibration buffer at a linear flow rate of 90 cm / h until the pH, UV, and Cond (conductivity) detection baseline was stable and consistent with the equilibration buffer.

[0071] 2) The sample and equilibration buffer were loaded at a ratio of 1:1 at a speed of 60 cm / h (20 mL / min), and the flow-through components during loading were collected and detected for the purity and content of the target protein.

[0072] 3) After the loading was completed, the column was flushed with 2 CV of equilibration buffer at a speed of 90 cm / h (30 mL / min) to wash out the unbound materials until the UV detection value decreased to the initial value.

[0073] 4) The elution buffer and equilibration buffer were eluted at a ratio of 6:4 at a speed of 90 cm / h (30 mL / min) to elute the target protein, and the elution components were collected.

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

[0075] 6) The chromatography column was washed with 5-10 CV of distilled water at a speed of 60 cm / h (20 mL / min) to flush the effluent to neutral pH and Cond (conductivity) close to 0 to remove 1 M NaOH.

[0076] 7) 3 CV of 20% ethanol was used for preservation to prevent microbial growth.

[0077] Example 2

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

[0079] The recombinant collagen was prepared into a solution with a concentration of 1 mg / mL, added to the cell culture medium of BJ cells (human skin fibroblasts) growing adherently, and cultured for 24 hours. Then, the cells were lysed with Trizon, and the cell lysate was sent to Huada Gene for transcriptome sequencing. The analysis scheme is as follows.

[0080] 1. Analysis scheme

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

[0082] 1.2 Differential group setting

[0083] One of the important analysis points of transcriptome sequencing is the differential comparison of expression amount. The setting of control and treatment in pairwise comparison is shown in Table 3.

[0084] Table 3

[0085] Number Control Treatment 1 BJ_untreated group BJ_treated group

[0086] 2, Experimental process

[0087] 2.1 mRNA library construction flow

[0088] 1) Total RNA is treated by mRNA enrichment method or rRNA removal method; mRNA enrichment: mRNA with polyA tail is enriched by magnetic beads with OligodT; 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.

[0089] 2) The obtained RNA is fragmented by fragmentation buffer, and then reverse transcribed by random N6 primer, and double-stranded DNA is formed by synthesizing cDNA second strand.

[0090] 3) The synthesized double-stranded DNA is end-repaired and 5'-phosphorylated, and a 3'-end sticky end with a protruding "A" is formed, and then a bubble-shaped adapter with a protruding "T" at the 3'-end is connected.

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

[0092] 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.

[0093] 6) Sequencing.

[0094] 4, Data analysis and results

[0095] 4.1 Data analysis flow

[0096] The raw data obtained by sequencing is called raw reads. First, we filter out low-quality, adapter contamination, and too many unknown bases N content reads, and the filtered data is called clean reads, then align the clean reads to the reference genome, and then perform new transcript prediction, SNP & InDel and differential splicing gene detection. After obtaining the new transcripts, we add the new transcripts 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, according to the needs, detect the differentially expressed genes between different samples, and do in-depth cluster analysis and functional enrichment analysis of the differentially expressed genes, etc.

[0097] 4.2 The transcriptomic sequencing results obtained are retained for data with p-value > 0.05. The FPKM values and log2 fold change values of the genes related to maintaining skin extracellular matrix homeostasis in the experimental group and the control group are extracted. The following genes are selected:

[0098] CD44: a cell surface receptor that can bind to hyaluronic acid and other ECM components, regulating cell-ECM interaction 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 network structure in the ECM, maintaining the tightness of the skin.

[0100] DPT: The glycoprotein encoded by this gene constructs support and regulates the role of collagen in the skin ECM, involving the structure and repair of the skin.

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

[0102] HAS gene family: encodes hyaluronic acid synthase, which catalyzes the synthesis of hyaluronic acid. Hyaluronic acid is an important glycosaminoglycan in the skin, which can maintain the moisture, elasticity and volume of the skin, and is crucial 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, improving the structure and elasticity of the skin;

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

[0105] SLIT2: Encodes Slit guidance molecule 2, plays a role in regulating cell migration and ECM tissue remodeling, affecting ECM structure and cell behavior.

[0106] LUM: Encodes Lumican, a proteoglycan in the ECM, regulates collagen fiber formation and tissue repair, helps maintain skin and connective tissue homeostasis.

[0107] P4HA3: The gene encodes an enzyme responsible for hydroxylation of proline residues in collagen to 4-hydroxyproline. This modification is a key step in collagen molecule stabilization and functional normalization. Plays an important role in ECM homeostasis.

[0108] PDGF: Plays an important role in cell proliferation and angiogenesis, while promoting ECM synthesis and tissue repair by regulating fibroblast activity.

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

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

[0111] SPP1: SPP is involved in cell-ECM interaction and plays an important role in skin tissue health by regulating ECM component integration and tissue stability.

[0112] TGFB2, TGFB2R: The gene and receptor play an important role in tissue repair and fibrogenesis. They regulate the balance of ECM synthesis and degradation, involved in wound healing and tissue remodeling.

[0113] VEGFA: The gene encodes a growth factor that not only promotes angiogenesis, but also promotes skin damage repair and tissue regeneration by regulating ECM components and metabolism.

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

[0115] Table 4

[0116]

[0117]

[0118] As shown in Table 4, the final calculated overall improvement of log2 fold change is approximately 0.75, which means the relative expression level is approximately 1.68437.

[0119] This embodiment uses transcriptomics to detect the expression of a series of genes related to skin extracellular matrix homeostasis, demonstrating that recombinant collagen can promote the expression of genes related to skin extracellular matrix homeostasis.

[0120] Example 3

[0121] This embodiment uses the recombinant collagen obtained in Example 1 to verify the promoting effect of different concentrations of recombinant collagen on the expression levels of a series of skin extracellular matrix homeostasis-related genes. 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 BJ cell culture medium that was adhering to the culture dish and 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 from cells

[0125] (1) Take the cultured cells, remove the culture medium, wash twice with pre-cooled PBS, add 1 mL of TRIzol reagent, pipette and collect into a centrifuge tube, and let stand at room temperature for 5 minutes.

[0126] (2) Add 200 μL of chloroform to the centrifuge tube, shake the centrifuge tube vigorously for 15 seconds, let it stand for 10 minutes, and then centrifuge at 12000g for 15 minutes at 4℃.

[0127] (3) After centrifugation, the liquid is separated into layers. The RNA is present in the upper colorless and transparent aqueous phase layer with a volume of about 600 μL. After transferring to a new centrifuge tube, add 500 μL of isopropanol, slowly invert the tube several times to mix, let stand for 15 minutes, and then centrifuge at 12000g for 10 minutes at 4℃.

[0128] (4) After centrifugation, the RNA precipitates at the bottom of the centrifuge tube and appears as a gel-like precipitate when facing the light source. After removing 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 a total volume of 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 to: shake for 10 seconds, measure the absorbance at 260 nm, measure the absorbance at 280 nm, and read the values.

[0133] (3) Calculate OD 260 / OD 280 The ratio, which is between 1.8 and 2.0, indicates that the quality of the extracted RNA meets the subsequent needs.

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

[0135] 4. Reverse transcription

[0136] (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, use a pipette to mix gently, and use a portable centrifuge for short centrifugation for 30 seconds.

[0137] (2) Place the reverse transcription system PCR tube in the RCR instrument, set the reaction program to: a) incubate at 50°C for 15 minutes; b) inactivate instantly at 85°C for 10 seconds. The mRNA in the system is reverse transcribed into cDNA.

[0138] 5. Fluorescent quantitative PCR

[0139] (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, use a portable centrifuge for short centrifugation for 30 seconds.

[0140] (2) Each sample requires three repeated holes for each molecule to be measured, and a blank control without cDNA is set; in addition to the molecules to be measured, actin beta (ACTB) is also needed as a reference molecule.

[0141] (3) Put the eight PCR tubes into the fluorescence quantitative PCR instrument, and set the reaction program: a) 95℃ for 2 minutes; b) 95℃ for 5 seconds; c) 60℃ for 10 seconds; d) 20℃ constant temperature storage. The steps b and c are repeated for 45 cycles.

[0142] (4) After the end of the PCR program, the software automatically generates the CT value of each sample.

[0143] 6. Quantitative analysis of results

[0144] (1) Take the average of the CT values of three repeats of each sample, and calculate the difference between the CT value of the tested molecule and the CT value of the reference molecule (ACTB), that is, ΔCT.

[0145] (2) Calculate the difference between the ΔCT value of the tested molecule in the cells treated with raw materials and the ΔCT value of the tested molecule in the untreated cells, that is, ΔΔCT.

[0146] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the exponent, that is, 2 -△△CT Take the average of 2 -△△CT of the parallel groups of each concentration as the quantitative result of the tested molecule in each sample, and realize quantitative detection at the gene level.

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

[0148] Table 5

[0149]

[0150]

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

[0152] Example 4

[0153] In this example, the recombinant collagen protein obtained in Example 1 and collagen proteins 1, 2 and 3 are used to test their promotion effects on the expression amounts of a series of skin extracellular matrix homeostasis related genes.

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

[0161] The experimental steps include:

[0162] 1. Take the recombinant collagen obtained in Example 1 and collagen 1, collagen 2 and collagen 3 respectively, and prepare 0.5 mg / mL of the test solution. Add 0.1% of the test solution into the culture medium of the BJ cells which are growing on the wall and occupy about 70% of the area of the culture dish, and culture for 24 hours.

[0163] 2. The subsequent RNA extraction, reverse transcription and qPCR experiment are operated according to the description 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] As shown in Table 6, compared with other collagens, the recombinant collagen of the present application can ensure the promotion of all the genes related to the skin extracellular matrix homeostasis, and has the highest average promotion rate.

[0167] Application Example 1

[0168] The recombinant collagen described in the present application can be used as an active substance for the preparation of skin conditioners and cosmetics. For example, it can be a cream, a lotion, a gel, a toner, an essence, a mask, an eye cream, an aerosol cleansing foam, a spray, a shower gel or a facial cleanser.

[0169] Application 1, preparation of a cream, formulated as follows: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 5.5% liquid paraffin, 4% glycerin, 3.5% propylene glycol, 3% ethylhexyl palmitate, 4% petrolatum, 2% cetostearyl alcohol ether-21, 2% dimethicone, 2% cetostearyl alcohol, 1.5% cetostearyl 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 water (63.2%).

[0170] Application 2, preparation of an emulsion, formulated as follows: 0.1% recombinant collagen, 4% glycerin, 5.5% hydrogenated polyisobutylene, 4.5% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% dimethicone, 1.5% cetostearyl alcohol ether-21, 1.0% cetostearyl alcohol ether-2, 0.5% tocopherol, 0.2% nipagin methyl ester, 0.1% triethanolamine, 0.1% nipagin ethyl ester, 0.1% carbomer, and the balance water (74.4%).

[0171] Application 3, preparation of a cosmetic water, formulated as follows: 0.1% recombinant collagen, 5% glycerin, 3% propylene glycol, 0.2% diazimidazolidinyl urea, and the balance water (91.7%).

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

[0173] The applicant states that the above description 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 those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. The application of recombinant collagen in the preparation of cosmetics that increase the expression levels of genes related to skin extracellular matrix homeostasis, characterized in that, The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:1; The skin extracellular matrix homeostasis-related genes are 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.

2. The application according to claim 1, characterized in that, The concentration of recombinant collagen in the cosmetic product is 0.4-0.6 mg / mL.

3. The application according to claim 1, characterized in that, The cosmetics include: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

Images