Application of recombinant collagen in preparation of preparation for increasing expression quantity of skin fibroblast proliferation related genes

Abstract

The invention provides application of recombinant collagen in preparation of a preparation for increasing the expression quantity of skin fibroblast proliferation related genes. The amino acid sequence of the recombinant collagen is shown as SEQ ID NO: 1. The expression condition of a series of genes capable of enhancing skin fibroblast proliferation is detected in a transcriptomics mode, and it is proved that the recombinant collagen can promote expression of the skin fibroblast proliferation genes. The collagen can be used for development and application of various preparation products, and endows the preparation products with the efficacy and effect of promoting skin fibroblast proliferation.

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 proliferation of skin fibroblasts. Background Art

[0002] Collagen is a family of proteins. At least 30 coding genes for collagen chains have been discovered, which can form more than 16 types of collagen molecules. According to their structure, they can be divided into fibrous collagen, basement membrane collagen, microfibrillar collagen, anchoring collagen, hexagonal reticular collagen, non-fibrous collagen, transmembrane collagen, etc.

[0003] According to their distribution and functional characteristics in the body, collagen can be divided into interstitial collagen, basement membrane collagen and pericellular collagen. Interstitial collagen molecules account for the vast majority of collagen in the whole body, including type I, II and III collagen molecules. Collagen has good biocompatibility, biodegradability and biological activity, so it is widely used in food, medicine, tissue engineering, cosmetics and other fields.

[0004] Fibroblasts (existing in the dermis) are the main cell components of loose connective tissue. Fibroblasts synthesize and secrete collagen and elastin, generate collagen fibers, reticular fibers and elastic fibers, and synthesize and secrete matrix components such as glycosaminoglycans and glycoproteins. Collagen peptides have biological activities, such as repairing skin damage, antioxidant activity, antihypertensive activity, and fat-reducing activity.

[0005] At present, collagen is mainly prepared and separated from the tissues of animals such as fish, pigs, and cattle, which may pose a risk of virus transmission. In addition, animal collagen has different sources, and its quality is difficult to control, and the preparation process is complicated. Therefore, providing a recombinant collagen produced by genetic engineering technology has important application value. Summary of the invention

[0006] 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 proliferation of skin fibroblasts. The present invention detects the expression of a series of genes that can enhance the proliferation of skin fibroblasts by transcriptomics, and proves that the recombinant collagen SEQ ID NO: 1 can promote the expression of skin fibroblast proliferation genes. The collagen can be used for the development and application of a variety of cosmetic products, giving cosmetic products the efficacy and effect of promoting the proliferation of skin fibroblasts. At the same time, this series of genes can also be used as a reference for future evaluation of the ability of other raw materials or cosmetics to enhance the proliferation of skin fibroblasts.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] 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 the proliferation of skin fibroblasts, wherein the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:1.

[0009] Preferably, the skin fibroblast proliferation-related genes include: any one or a combination of at least two of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA.

[0010] The present invention detects the expression of a series of genes that can enhance the proliferation of skin fibroblasts by transcriptomics, and the results show that the recombinant collagen can promote the expression of skin fibroblast proliferation genes. The collagen can be used for the development and application of a variety of cosmetic products, giving the cosmetic products the efficacy and effect of promoting the proliferation of skin fibroblasts.

[0011] Preferably, the skin fibroblast proliferation-related genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 and VEGFA.

[0012] Preferably, the preparation comprises a medicine or a cosmetic.

[0013] 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, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL or 0.6 mg / mL.

[0014] Preferably, the cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

[0015] The present invention also provides an expression promoter for increasing the expression of genes related to skin fibroblast proliferation. The expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes that shown in SEQ ID NO:1.

[0016] In the present invention, after the recombinant collagen is administered, the relative expression level of the skin fibroblast proliferation-related gene is 1.821.

[0017] In the present invention, a relative expression promotion of 1.19 is considered to be promotion. In bioinformatics analysis, a log2foldchange 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.

[0018] The present invention provides a biomarker for evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin fibroblasts. The biomarker is a gene related to the proliferation of skin fibroblasts, and the gene includes any one of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA, or a combination of at least two of them.

[0019] In a second aspect, the present invention provides the use of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to enhance the proliferation of skin fibroblasts, wherein the biomarker is a gene related to the proliferation of skin fibroblasts, and the gene includes: any one of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA, or a combination of at least two of them.

[0020] Preferably, the genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 and VEGFA.

[0021] Preferably, the product comprises a primer set, a reagent or a detection model.

[0022] Preferably, the reagents include qPCR detection reagents.

[0023] In a third aspect, the present invention provides a primer combination for detecting the expression level of a biomarker, the primer combination comprising:

[0024] The upstream primer for amplifying BMP2 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:6;

[0025] The upstream primer for amplifying CCL2 is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8;

[0026] The upstream primer for amplifying CCN1 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:10;

[0027] The upstream primer for amplifying CCND1 is shown in SEQ ID NO: 11, and the downstream primer is shown in SEQ ID NO: 12;

[0028] The upstream primer for amplifying EDN1 is shown in SEQ ID NO: 13, and the downstream primer is shown in SEQ ID NO: 14;

[0029] The upstream primer for amplifying EDN2 is shown in SEQ ID NO: 15, and the downstream primer is shown in SEQ ID NO: 16;

[0030] The upstream primer for amplifying FGF1 is shown in SEQ ID NO: 17, and the downstream primer is shown in SEQ ID NO: 18;

[0031] The upstream primer for amplifying FOXC2 is shown in SEQ ID NO: 19, and the downstream primer is shown in SEQ ID NO: 20;

[0032] The upstream primer for amplifying FOXF1 is shown in SEQ ID NO:21, and the downstream primer is shown in SEQ ID NO:22;

[0033] The upstream primer for amplifying HGF is shown in SEQ ID NO:23, and the downstream primer is shown in SEQ ID NO:24;

[0034] The upstream primer for amplifying KLF4 is shown in SEQ ID NO:25, and the downstream primer is shown in SEQ ID NO:26;

[0035] The upstream primer for amplifying LIF is shown in SEQ ID NO:27, and the downstream primer is shown in SEQ ID NO:28;

[0036] The upstream primer for amplifying MYC is shown in SEQ ID NO:29, and the downstream primer is shown in SEQ ID NO:30;

[0037] The upstream primer for amplifying NGF is shown in SEQ ID NO:31, and the downstream primer is shown in SEQ ID NO:32;

[0038] The upstream primer for amplifying NOG is shown in SEQ ID NO:33, and the downstream primer is shown in SEQ ID NO:34;

[0039] The upstream primer for amplifying PDGFA is shown in SEQ ID NO:35, and the downstream primer is shown in SEQ ID NO:36; the upstream primer for amplifying PDGFB is shown in SEQ ID NO:37, and the downstream primer is shown in SEQ ID NO:38; the upstream primer for amplifying RUNX2 is shown in SEQ ID NO:39, and the downstream primer is shown in SEQ ID NO:40; the upstream primer for amplifying SERPINE1 is shown in SEQ ID NO:41, and the downstream primer is shown in SEQ ID NO:42; the upstream primer for amplifying SFRP1 is shown in SEQ ID NO:43, and the downstream primer is shown in SEQ ID NO:44;

[0040] The upstream primer for amplifying SMAD3 is shown in SEQ ID NO:45, and the downstream primer is shown in SEQ ID NO:46; the upstream primer for amplifying SNAI1 is shown in SEQ ID NO:47, and the downstream primer is shown in SEQ ID NO:48;

[0041] The upstream primer for amplifying STAT1 is shown in SEQ ID NO:49, and the downstream primer is shown in SEQ ID NO:50;

[0042] The upstream primer for amplifying STAT3 is shown in SEQ ID NO:51, and the downstream primer is shown in SEQ ID NO:52;

[0043] The upstream primer for amplifying TGFB1 is shown in SEQ ID NO:53, and the downstream primer is shown in SEQ ID NO:54;

[0044] The upstream primer for amplifying TGFB2 is shown in SEQ ID NO:55, and the downstream primer is shown in SEQ ID NO:56;

[0045] The upstream primer for amplifying VEGFA is shown in SEQ ID NO: 57, and the downstream primer is shown in SEQ ID NO: 58. The primers are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049] 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.

[0050] 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.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention screens out a group of genes that promote fibroblast proliferation through transcriptomics, which can be used as detection targets for promoting fibroblast proliferation in other cosmetic raw materials or finished products in the future.

[0053] The present invention develops a new collagen protein, which exhibits a better function on fibroblast proliferation than the existing collagen protein, and can be added to skin care products as a raw material in the future to achieve better skin care effects.

[0054] The present invention designs and summarizes a series of primers of genes that promote the proliferation of fibroblasts, thereby facilitating the rapid and accurate detection of related genes in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Heat maps of the experimental and control groups.

[0056] Figure 2 The figure shows the result of fibroblast proliferation experiment at 0 hour in the control group.

[0057] Figure 3 This is the result of 24-hour fibroblast proliferation experiment in the control group.

[0058] Figure 4 These are the results of the fibroblast proliferation experiment in the experimental group at 0.5 mg / mL and 0 hours.

[0059] Figure 5 These are the results of the fibroblast proliferation experiment in the experimental group at 0.5 mg / mL and 24 hours.

[0060] Figure 6 These are the results of the fibroblast proliferation experiment in the experimental group at 0.2 mg / mL and 0 hours.

[0061] Figure 7 These are the results of the fibroblast proliferation experiment in the experimental group at 0.2 mg / mL and 24 hours.

[0062] Figure 8These are the results of the fibroblast proliferation experiment in the experimental group at 0.8 mg / mL and 0 hours.

[0063] Fig. 9 These are the results of the fibroblast proliferation experiment in the experimental group at 0.8 mg / mL and 24 hours. DETAILED DESCRIPTION

[0064] 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.

[0065] 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.

[0066] Example 1

[0067] In this example, an expression promoter for increasing the expression of genes related to the proliferation of skin fibroblasts 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.

[0068] 1. Fermentation process

[0069] First, the strain was activated, including the following steps: streaking activation on a YPD medium plate, and culturing at 30°C for 72 hours. Then, the seed solution was prepared, including the following steps: selecting a single colony with good growth in a clean bench, inoculating it into a triangular flask with YPD medium, and culturing it for 30 hours in a shaking incubator at 30°C and 250rpm. Then, the fermenter was inoculated, and the seed solution was transferred to a fermenter containing 40g / L glycerol and 4.35mL / L BSM medium. The fermentation process control is shown in Table 2.

[0070] Table 2

[0071]

[0072] The experimental samples and equipment include: a ceramic membrane with a pore size of 0.45 μm, a Tofflon hydrophobic chromatography column Phevl Purose6FF; and PrePack50 / 200.

[0073] 2. Purification process

[0074] (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 3.

[0075] Table 3

[0076] 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

[0077] (2) Preparation of experimental materials and buffer solution:

[0078] Distilled water: 5L;

[0079] Equilibration buffer: 20 mM PB, 1 M ammonium sulfate, pH 7, 5 L;

[0080] Elution buffer: 20 mM PB, pH 7, 5 L;

[0081] Wash buffer: 1M NaOH, 1 L;

[0082] Note: The above buffers must be filtered through a 0.45μm filter membrane.

[0083] (3) The purification experimental steps are as follows:

[0084] 1) The column was flushed with equilibration buffer at a linear flow rate of 90 cm / h for 3 CV (column volume) until the pH, UV, and Cond (conductivity) detection baselines were stable and consistent with the equilibration buffer.

[0085] 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.

[0086] 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.

[0087] 4) The elution buffer and the equilibrium buffer were eluted at a rate of 90 cm / h (30 mL / min) at a ratio of 6:4 to elute the target protein and collect the eluted fractions.

[0088] 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.

[0089] 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.

[0090] 7) Wash with 3CV of 20% ethanol to prevent microbial growth.

[0091] Example 2

[0092] This example uses transcriptomic sequencing to study the effect of the recombinant collagen in Example 1 on the proliferation of skin fibroblasts.

[0093] The recombinant collagen was prepared into a solution with a concentration of 0.5 mg / mL and added to the cell culture medium of BJ cells (human skin fibroblasts) that were growing to about 70% of the bottom area of ​​the culture dish at a final concentration of 0.1%. 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.

[0094] 1. Analysis plan

[0095] 1.1 Species name: Homo sapiens; Source: NCBI; Reference genome version: GCF_000001405.40_GRCh38.p14.

[0096] 1.2 Difference Grouping Settings

[0097] One of the important analysis points of transcriptome sequencing is the comparison of expression differences. The control and treatment conditions in the pairwise comparison are shown in Table 4 below.

[0098] Table 4

[0099] serial number Comparison deal with 1 BJ_untreated (BJ cells are not treated) BJ_treated (BJ cells have been treated)

[0100] 2. Experimental process

[0101] 2.1 mRNA library construction process

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 4) The ligation product is amplified by PCR using specific primers.

[0106] 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.

[0107] 6) Sequencing on a machine.

[0108] 4. Data analysis and results

[0109] 4.1 Data Analysis Process

[0110] 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 clean 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.

[0111] 4.2 The obtained transcriptomic sequencing results were retained with a p value > 0.05. The FPKM values ​​and log2 fold change values ​​of genes related to skin fibroblast proliferation in the experimental group and the control group were extracted.

[0112] 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 5. The heat map was obtained by heat map analysis of the Kidio Bioinformatics Cloud tool, as shown in Figure 1 .

[0113] Table 5

[0114]

[0115]

[0116] As can be seen from Table 5, the final calculated log2 fold change was about 0.777, which means that gene expression was promoted by about 1.713 times.

[0117] This example detects the expression of a series of genes related to skin fibroblast proliferation by transcriptomics, proving that recombinant collagen can enhance the proliferation ability of skin fibroblasts.

[0118] Example 3

[0119] 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 skin fibroblast proliferation-related genes. The experimental steps include:

[0120] 1. Cell sample culture

[0121] 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.

[0122] 2. Extraction and purification of total RNA in cells

[0123] (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.

[0124] (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.

[0125] (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.

[0126] (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.

[0127] (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.

[0128] 3. RNA concentration determination

[0129] (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.

[0130] (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.

[0131] (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.

[0132] (4) According to the formula OD260 × 0.04 × dilution factor, calculate the RNA solution concentration in μg / μL. Dilute with DEPC-water to adjust the RNA concentration to 1 μg / μL.

[0133] 4. Reverse transcription

[0134] (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.

[0135] (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.

[0136] 5. Fluorescence quantitative PCR

[0137] (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.

[0138] (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.

[0139] (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.

[0140] (4) After the PCR process is completed, the software automatically generates the CT value for each sample.

[0141] 6. Quantitative Analysis of Results

[0142] (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.

[0143] (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.

[0144] (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.

[0145] The experimental results are shown in Table 6 below.

[0146] Table 6

[0147]

[0148]

[0149] As shown in Table 6, by treating cells with different concentrations of collagen and then measuring gene expression, the concentration range of 0.2mg / mL-0.8mg / mL can ensure that the expression of all target genes is promoted. Among them, 0.5mg / mL is the concentration with the highest promotion rate.

[0150] Example 4

[0151] This example uses the recombinant collagen obtained in Example 1 to conduct a fibroblast proliferation experiment to verify the effects of different concentrations of recombinant collagen on fibroblast proliferation. The experimental steps include:

[0152] (1) Digest the BJ fibroblasts that have entered the exponential culture phase with trypsin for about 20 seconds, add DMEM medium to terminate the digestion, and resuspend for later use.

[0153] (2) Place a 2-well cell scratch plug in the center of each well of a six-well cell culture dish, and slowly drip the resuspended BJ cells into the well. After culturing for 24 hours until the cells are completely attached to the wall, remove the scratch plug and add 2 mL of DMEM culture medium to each well. Add 1% sterile water to the control well. Add 1% of 0.2 mg / mL, 0.5 mg / mL, and 0.8 mg / mL recombinant collagen solutions to the experimental wells, respectively. Place in a cell culture incubator and culture for 24 hours. Repeat the experiment twice for each group.

[0154] (3) Observe and calculate the cell scratch recovery. Figure 2-9 The scratch width (unit / micrometer) statistics are shown in Table 7.

[0155] Table 7

[0156]

[0157] The calculation method of the recovery rate in Table 7 is: recovery rate = (0 hour average - 24 hour average) / 0 hour average.

[0158] From the results in Table 7, it can be seen that recombinant collagen has a significant effect on promoting the proliferation of fibroblasts and can restore the scratches of fibroblasts more quickly.

[0159] Example 5

[0160] 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 proliferation of skin fibroblasts.

[0161] 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.

[0162] The experimental steps include:

[0163] 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.

[0164] 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 8.

[0165] Table 8

[0166] Gene name Collagen 1 Collagen 2 Collagen 3 Recombinant collagen BMP2 1.0562734 1.000666701 1.066354872 1.3534789 CCL2 1.630 2.923312155 1.186 3.239 CCN1 1.658 2.097748796 1.294 2.149 CCND1 1.138 1.406657597 0.990 1.579 EDN1 1.103 1.483990484 1.590 1.610 EDN2 3.304 2.960170724 2.053 4.899 FGF1 1.194 1.1461919 1.046 1.629 FOXC2 1.060 1.048093457 1.182 1.305 FOXF1 1.244 1.275773295 1.002 1.531 HGF 1.031 0.98381206 1.107 1.319 KLF4 1.642 1.762915839 1.502 2.012 LIF 1.560 1.739070287 1.503 1.837 MYC 1.327 1.243265916 1.615 1.738 NGF 1.141 1.163790948 1.255 1.399 NOG 2.321 1.572934147 1.517 2.757 PDGFA 1.295 1.060638894 1.131 1.498 PDGFB 1.189 1.394875664 1.058 1.738 RUNX2 1.223 1.005002722 1.415 1.489 SERPINE1 1.063 0.997182978 1.226 1.398 SFRP1 1.607 1.098984148 1.328 1.769 SMAD3 1.301 1.238970938 1.232 1.448 SNAI1 1.220 1.281740576 1.083 1.397 STAT1 1.266 1.162384917 1.240 1.837 STAT3 1.255 1.36856676 1.686 1.740 TGFB1 1.316 1.118575767 1.435 1.578 TGFB2 1.004 1.157160274 1.146 1.236 VEGFA 1.436 1.437608877 1.291 1.659 Mean 1.392 1.412225438 1.303 1.813

[0167] From the qPCR results in the table, it can be seen that the recombinant collagen of the present invention can upregulate 27 skin fibroblast proliferation-related genes, while the other collagens 1, 2, and 3 cannot upregulate all of the above 27 genes, and their upregulation effects are not as good as the recombinant collagen of the present invention. It can be seen that the collagen of the present invention has a better effect of promoting fibroblast proliferation, which is mutually confirmed by the conclusion in the aforementioned Example 3, and the recombinant collagen of the present invention has a significant effect of promoting fibroblast proliferation.

[0168] Application Example 1

[0169] 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.

[0170] Application 1, preparation of facial cream, formulated according to the following ratio: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 5% liquid paraffin, 4% glycerol, 3% propylene glycol, 3% ethylhexyl palmitate, 3% petrolatum, 2% cetearyl ether-21, 2% dimethicone, 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 (65.2%).

[0171] Application 2, preparation of emulsion, formulated according to the following ratio: 0.1% recombinant collagen, 5% glycerin, 5% hydrogenated polyisobutene, 4% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% dimethicone, 1.5% ceteareth-21, 1.2% ceteareth-2, 0.5% tocopherol, 0.2% methylparaben, 0.1% triethanolamine, 0.1% ethylparaben, 0.1% carbomer, the balance is water (74.2%).

[0172] 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%).

[0173] In summary, the present invention detects the expression of a series of genes related to skin fibroblast proliferation by transcriptomics, proving that recombinant collagen can promote the expression of genes related to skin fibroblast proliferation. The collagen can be used for the development and application of a variety of cosmetic products, giving cosmetic products the efficacy and effect of promoting skin fibroblast proliferation. At the same time, the series of genes can also be used as a reference for future evaluation of the ability of other raw materials or cosmetics to promote skin fibroblast proliferation.

[0174] 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 proliferation of skin fibroblasts, 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 fibroblast proliferation-related genes include: any one of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA, or a combination of at least two of them.

3. The use according to claim 1 or 2, characterized in that: The skin fibroblast proliferation-related genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 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 enhance the proliferation of skin fibroblasts, characterized in that: The biomarker is a gene related to skin fibroblast proliferation, and the gene includes: any one of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 or VEGFA, or a combination of at least two of them.

8. The use according to claim 7, characterized in that: The genes include: a combination of BMP2, CCL2, CCN1, CCND1, EDN1, EDN2, FGF1, FOXC2, FOXF1, HGF, KLF4, LIF, MYC, NGF, NOG, PDGFA, PDGFB, RUNX2, SERPINE1, SFRP1, SMAD3, SNAI1, STAT1, STAT3, TGFB1, TGFB2 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 BMP2 is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:6; The upstream primer for amplifying CCL2 is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; The upstream primer for amplifying CCN1 is shown in SEQ ID NO:9, and the downstream primer is shown in SEQ ID NO:10; The upstream primer for amplifying CCND1 is shown in SEQ ID NO: 11, and the downstream primer is shown in SEQ ID NO: 12; The upstream primer for amplifying EDN1 is shown in SEQ ID NO: 13, and the downstream primer is shown in SEQ ID NO: 14; The upstream primer for amplifying EDN2 is shown in SEQ ID NO: 15, and the downstream primer is shown in SEQ ID NO: 16; The upstream primer for amplifying FGF1 is shown in SEQ ID NO: 17, and the downstream primer is shown in SEQ ID NO: 18; The upstream primer for amplifying FOXC2 is shown in SEQ ID NO: 19, and the downstream primer is shown in SEQ ID NO: 20; The upstream primer for amplifying FOXF1 is shown in SEQ ID NO:21, and the downstream primer is shown in SEQ ID NO:22; The upstream primer for amplifying HGF is shown in SEQ ID NO:23, and the downstream primer is shown in SEQ ID NO:24; The upstream primer for amplifying KLF4 is shown in SEQ ID NO:25, and the downstream primer is shown in SEQ ID NO:26; The upstream primer for amplifying LIF is shown in SEQ ID NO:27, and the downstream primer is shown in SEQ ID NO:28; The upstream primer for amplifying MYC is shown in SEQ ID NO:29, and the downstream primer is shown in SEQ ID NO:30; The upstream primer for amplifying NGF is shown in SEQ ID NO:31, and the downstream primer is shown in SEQ ID NO:32; The upstream primer for amplifying NOG is shown in SEQ ID NO:33, and the downstream primer is shown in SEQ ID NO:34; The upstream primer for amplifying PDGFA is shown in SEQ ID NO:35, and the downstream primer is shown in SEQ ID NO:36; The upstream primer for amplifying PDGFB is shown in SEQ ID NO:37, and the downstream primer is shown in SEQ ID NO:38; The upstream primer for amplifying RUNX2 is shown in SEQ ID NO:39, and the downstream primer is shown in SEQ ID NO:40; The upstream primer for amplifying SERPINE1 is shown in SEQ ID NO:41, and the downstream primer is shown in SEQ ID NO:42; The upstream primer for amplifying SFRP1 is shown in SEQ ID NO:43, and the downstream primer is shown in SEQ ID NO:44; The upstream primer for amplifying SMAD3 is shown in SEQ ID NO:45, and the downstream primer is shown in SEQ ID NO:46; The upstream primer for amplifying SNAI1 is shown in SEQ ID NO:47, and the downstream primer is shown in SEQ ID NO:48; The upstream primer for amplifying STAT1 is shown in SEQ ID NO:49, and the downstream primer is shown in SEQ ID NO:50; The upstream primer for amplifying STAT3 is shown in SEQ ID NO:51, and the downstream primer is shown in SEQ ID NO:52; The upstream primer for amplifying TGFB1 is shown in SEQ ID NO:53, and the downstream primer is shown in SEQ ID NO:54; The upstream primer for amplifying TGFB2 is shown in SEQ ID NO:55, and the downstream primer is shown in SEQ ID NO:56; The upstream primer for amplifying VEGFA is shown as SEQ ID NO:57, and the downstream primer is shown as SEQ ID NO:58.

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