CN119257972B

CN119257972B - Application of recombinant collagen in the preparation of preparations for increasing the expression of genes related to proliferation of skin fibroblasts

Info

Publication number: CN119257972B
Application number: CN202411686478.4A
Authority: CN
Inventors: 孙怀庆; 郭朝万; 马博闻; 王宁; 蒲艳; 叶林
Original assignee: Guangdong Marubi Biological Technology Co Ltd
Current assignee: Guangdong Marubi Biological Technology Co Ltd
Priority date: 2024-11-22
Filing date: 2024-11-22
Publication date: 2025-10-10
Anticipated expiration: 2044-11-22
Also published as: CN119257972A

Abstract

The invention provides application of recombinant collagen in preparation of a preparation for increasing the expression level of genes related to skin fibroblast proliferation, wherein the amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1. The invention detects the expression condition of a series of genes capable of enhancing the proliferation of the skin fibroblasts by means of transcriptome, and proves that the recombinant collagen can promote the expression of the skin fibroblast proliferation genes. The collagen can be used for developing and applying various preparation products, and endowing the preparation products with the efficacy and effect of promoting the proliferation of skin fibroblasts.

Description

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

Technical Field

The invention belongs to the field of research on efficacy of cosmetic raw materials, and particularly relates to application of recombinant collagen in preparation of a preparation for increasing expression quantity of genes related to proliferation of skin fibroblasts.

Background

Collagen is a family of proteins, and at least 30 genes encoding collagen chains have been found to form more than 16 types of collagen molecules, which can be classified into fibrillar collagen, basement membrane collagen, microfibril collagen, anchored collagen, hexagonal reticulocyte collagen, non-fibrillar collagen, transmembrane collagen, and the like, depending on their structure.

Collagen can be classified into interstitial collagen, basement membrane collagen and extracellular collagen according to their distribution and functional characteristics in the body. The interstitial type collagen molecules account for the vast majority of the whole body collagen, including type I, II, III collagen molecules. Collagen has excellent biocompatibility, biodegradability and bioactivity, so that it has been widely used in the fields of foods, medicines, tissue engineering, cosmetics, etc.

Fibroblasts (present in the dermis) are the major cellular component of loose connective tissue, and they both synthesize and secrete collagen, elastin, collagen fibers, reticulate fibers, and elastin, and also matrix components such as glycosaminoglycans and glycoproteins. The collagen peptide has biological activity, such as repairing skin injury, antioxidant activity, antihypertensive activity, and fat reducing activity.

At present, collagen is mainly prepared and separated from tissues of animals such as fish, pigs, cattle and the like, and the risk of virus transmission possibly exists, and the quality of the animal collagen is difficult to control because of different sources, so that the preparation process is complex. Therefore, the recombinant collagen prepared based on the genetic engineering technology has important application value.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide the application of the recombinant collagen in preparing a preparation for increasing the expression quantity of genes related to the proliferation of skin fibroblasts. The invention detects the expression condition of a series of genes capable of enhancing the proliferation of the skin fibroblasts by means of transcriptome, and proves that the recombinant collagen SEQ ID NO. 1 can promote the expression of the proliferation genes of the skin fibroblasts, and the collagen can be used for developing and applying various cosmetic products, and endows the cosmetic products with the efficacy and effect of promoting the proliferation of the skin fibroblasts. Meanwhile, the series of genes can also be used as a reference for evaluating the proliferation capacity of other raw materials or cosmetics for enhancing the skin fibroblast.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect, the invention provides an application of recombinant collagen in preparing a preparation for increasing the expression level of genes related to skin fibroblast proliferation, wherein the amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1.

Preferably, the skin fibroblast proliferation-related gene comprises 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.

According to the invention, a series of expression situations of genes capable of enhancing the proliferation of the skin fibroblasts are detected by a transcriptome mode, and as a result, the recombinant collagen can promote the expression of the skin fibroblast proliferation genes. The collagen can be used for developing and applying various cosmetic products, and endowing the cosmetic products with the efficacy and effect of promoting the proliferation of skin fibroblasts.

Preferably, the skin fibroblast proliferation-related gene comprises 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.

Preferably, the formulation comprises a pharmaceutical or cosmetic product.

Preferably, the concentration of recombinant collagen in the formulation is 0.4-0.6mg/mL, which may be, for example, 0.4mg/mL, 0.45mg/mL, 0.5mg/mL, 0.55mg/mL, or 0.6mg/mL, etc.

Preferably, the cosmetic comprises face cream, emulsion, gel, toning lotion, essence, facial mask, eye cream, aerosol cleaning foam, spray, bath lotion or facial cleanser.

The invention also provides an expression promoter for improving the expression quantity of the skin fibroblast proliferation related gene, wherein the expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen comprises the amino acid sequence shown in SEQ ID NO. 1.

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

In the present invention, the relative expression promoting amount was regarded as promoting by 1.19. In bioinformatics analysis, a value of log2foldchange is usually 0.25 as a set threshold for screening for differentially expressed genes, which is converted to a relative expression level multiple of about 1.19.

The invention provides a biomarker for evaluating the capability of cosmetics or raw materials thereof to enhance skin fibroblast proliferation, wherein the biomarker is a skin fibroblast proliferation related gene, and the gene comprises 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.

In a second aspect, the invention provides the use of a product for detecting the expression level of a biomarker, which is a skin fibroblast proliferation-related gene comprising 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, in evaluating the ability of a cosmetic or a raw material thereof to enhance skin fibroblast proliferation.

Preferably, the genes comprise 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.

Preferably, the product comprises a primer combination, a reagent or a detection model.

Preferably, the reagent comprises a qPCR detection reagent.

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

The upstream primer for amplifying BMP2 is shown as SEQ ID NO. 5, and the downstream primer is shown as SEQ ID NO. 6;

The upstream primer of the amplified CCL2 is shown as SEQ ID NO. 7, and the downstream primer is shown as SEQ ID NO. 8;

The upstream primer for amplifying CCN1 is shown as SEQ ID NO. 9, and the downstream primer is shown as SEQ ID NO. 10;

The upstream primer of the amplified CCND1 is shown as SEQ ID NO. 11, and the downstream primer is shown as SEQ ID NO. 12;

The upstream primer of the amplified EDN1 is shown as SEQ ID NO. 13, and the downstream primer is shown as SEQ ID NO. 14;

The upstream primer of the amplified EDN2 is shown as SEQ ID NO. 15, and the downstream primer is shown as SEQ ID NO. 16;

The upstream primer of the amplified FGF1 is shown as SEQ ID NO. 17, and the downstream primer is shown as SEQ ID NO. 18;

the upstream primer of the amplified FOXC2 is shown as SEQ ID NO. 19, and the downstream primer is shown as SEQ ID NO. 20;

the upstream primer of the amplification FOXF1 is shown as SEQ ID NO. 21, and the downstream primer is shown as SEQ ID NO. 22;

The upstream primer of the amplified HGF is shown as SEQ ID NO. 23, and the downstream primer is shown as SEQ ID NO. 24;

The upstream primer for amplifying KLF4 is shown as SEQ ID NO. 25, and the downstream primer is shown as SEQ ID NO. 26;

the upstream primer for amplifying LIF is shown as SEQ ID NO. 27, and the downstream primer is shown as SEQ ID NO. 28;

The upstream primer for amplifying MYC is shown as SEQ ID NO. 29, and the downstream primer is shown as SEQ ID NO. 30;

The upstream primer for amplifying NGF is shown as SEQ ID NO. 31, and the downstream primer is shown as SEQ ID NO. 32;

The upstream primer for amplifying NOG is shown as SEQ ID NO. 33, and the downstream primer is shown as SEQ ID NO. 34;

The upstream primer of amplified PDGFA is shown as SEQ ID NO. 35, the downstream primer is shown as SEQ ID NO. 36, the upstream primer of amplified PDGFB is shown as SEQ ID NO. 37, the downstream primer is shown as SEQ ID NO. 38, the upstream primer of amplified RUNX2 is shown as SEQ ID NO. 39, the downstream primer is shown as SEQ ID NO. 40, the upstream primer of amplified SERPINE1 is shown as SEQ ID NO. 41, the downstream primer is shown as SEQ ID NO. 42, the upstream primer of amplified SFRP1 is shown as SEQ ID NO. 43, and the downstream primer is shown as SEQ ID NO. 44;

The upstream primer of the amplified SMAD3 is shown as SEQ ID NO. 45, the downstream primer is shown as SEQ ID NO. 46, the upstream primer of the amplified SNAI1 is shown as SEQ ID NO. 47, and the downstream primer is shown as SEQ ID NO. 48;

the upstream primer of the amplified STAT1 is shown as SEQ ID NO. 49, and the downstream primer is shown as SEQ ID NO. 50;

the upstream primer of the amplified STAT3 is shown as SEQ ID NO. 51, and the downstream primer is shown as SEQ ID NO. 52;

The upstream primer of the amplified TGFB1 is shown as SEQ ID NO. 53, and the downstream primer is shown as SEQ ID NO. 54;

The upstream primer of the amplified TGFB2 is shown as SEQ ID NO. 55, and the downstream primer is shown as 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. The primers are shown in Table 1.

TABLE 1

The invention can be rapidly used for qPCR experiments to verify the promotion capability of new raw materials or products on fibroblast proliferation by screening a series of primers of genes, and ensure the accuracy of experimental results.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens out a group of genes which play a role in promoting fibroblast proliferation through transcriptomics, and can be used for detecting targets of promoting fibroblast proliferation of other cosmetic raw materials or finished products in the future.

By developing a new collagen, the invention has better function of proliferation of fibroblasts than the existing collagen, and can be used as a raw material to be added with skin care products in the future to achieve better skin care effect.

The invention provides convenience for rapid and accurate detection of related genes in the future by designing and summarizing a series of primers of genes which play a role in promoting fibroblast proliferation.

Drawings

FIG. 1 is a heat map of the experimental and control groups.

FIG. 2 shows the results of a control group of 0 hour fibroblast proliferation assay.

FIG. 3 shows the results of a 24-hour fibroblast proliferation assay in the control group.

FIG. 4 shows the results of the 0.5mg/mL, 0 hour fibroblast proliferation test of the experimental group.

FIG. 5 shows the results of a fibroblast proliferation assay of 0.5mg/mL and 24 hours for the experimental group.

FIG. 6 shows the results of the 0.2mg/mL, 0-hour fibroblast proliferation test of the experimental group.

FIG. 7 shows the results of a fibroblast proliferation assay of 0.2mg/mL and 24 hours for the experimental group.

FIG. 8 shows the results of the 0.8mg/mL, 0-hour fibroblast proliferation test.

FIG. 9 shows the results of a fibroblast proliferation assay of 0.8mg/mL and 24 hours for the experimental group.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1

The expression promoter for increasing the expression level of the skin fibroblast proliferation-related gene is prepared in the embodiment, wherein the expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1.

1. Fermentation process

The strain activation was first performed, and the steps include streaking activation on YPD medium plates, and culturing at 30℃for 72 hours. Then preparing seed liquid, namely selecting single colony with good growth condition in an ultra clean bench, inoculating the single colony into a triangular flask with YPD culture medium, and culturing for 30 hours at the temperature of a shaking table of 30 ℃ and at the speed of 250 rpm. 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.

TABLE 2

Experimental samples and equipment included ceramic membranes with pore sizes of 0.45 μm, dongfu Long Shushui chromatography column Phevl Purose FF, prePack50/200.

2. Purification process

(1) The obtained fermentation broth was first filtered using a ceramic membrane of 0.45 μm and then subjected to a hydrophobic chromatography purification process. The sample and column specifications are shown in Table 3.

TABLE 3 Table 3

Sample of	Recombinant collagen fermentation broth
		Packing material	Phenyl Purose 6FF
Column mounting specification	PrePack 50×200
		Sample loading amount	2CV
Flow rate of experiment	20mL/min
		Detection wavelength	220nm

(2) Preparing experimental materials, and preparing a buffer solution:

distilled water 5L;

equilibration buffer 20mM PB,1M ammonium sulfate, pH7,5L;

Elution buffer 20mM PB, pH7,5L;

Washing buffer solution, 1M NaOH,1L;

Note that the above buffer solution was filtered with a 0.45 μm filter.

(3) The purification experimental procedure is as follows:

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

2) Sample and balance buffer are loaded at a speed of 60cm/h (20 mL/min) according to a ratio of 1:1, and flow-through components in the loading process are collected and the purity and content of target proteins are detected.

3) After the loading was completed, the sample was washed with an equilibration buffer at a rate of 90cm/h (30 mL/min) for 2CV to wash out unbound material until the UV detection value was reduced to the initial value.

4) Eluting the target protein by eluting the eluting buffer and the balance buffer at a speed of 90cm/h (30 mL/min) according to a ratio of 6:4, and collecting the eluting component.

5) The column is washed with at least 2 column volumes of 1m NaOH at a rate of 60cm/h (20 mL/min) to remove residual contaminants such as lipids, endotoxins, nucleic acids, etc. from the packed column.

6) The column was washed with 5-10CV of distilled water at a rate of 60cm/h (20 mL/min), rinsed until the effluent pH was neutral, and Cond (conductivity) was near 0 to remove 1M NaOH.

7) The stock was washed with 3CV of 20% ethanol to prevent microbial growth.

Example 2

This example uses transcriptomic sequencing to investigate the effect of recombinant collagen in example 1 on skin fibroblast proliferation.

Recombinant collagen was prepared as a solution at a concentration of 0.5mg/mL, added to a cell culture medium in which BJ cells (human skin fibroblasts) were growing on the wall to about 70% of the bottom area of the dish at a final concentration of 0.1%, cultured for 24 hours, lysed with Trizon, and the cell lysate was sent to the Huada genes for transcriptome sequencing, with the following analysis protocol.

1. Analytical protocol

1.1 Species name homosapiens, source NCBI, reference genome version GCF_000001405.40_GRCh38.p14.

1.2 Differential packet setup

One of the important analysis points of transcriptome sequencing is a differential comparison of expression levels, and the cases of setting controls and treatments in the pairwise comparison are shown in Table 4 below.

TABLE 4 Table 4

Numbering device	Control	Treatment of
			1	BJ_ untreated (BJ cell untreated)	BJ_treated (BJ cells treated)

2. Experimental procedure

2.1MRNA library construction flow

1) Treating total RNA by using mRNA enrichment method or rRNA removal method, enriching mRNA with OligodT magnetic beads and polyA tail, removing rRNA, using DNA probe to hybridize rRNA, using RNaseH to selectively digest DNA/RNA hybridized chain, using DNaseI to digest DNA probe, and purifying so as to obtain the invented required RNA.

2) The obtained RNA is fragmented by using a breaking buffer solution, and a random N6 primer is subjected to reverse transcription, so that cDNA two-strand is synthesized to form double-stranded DNA.

3) The synthesized double-stranded DNA is terminated by filling in and phosphorylating at the 5 end, the 3 'end forms a sticky end protruding from the A, and then a bubbling-shaped joint with a protruding T at the 3' end is connected.

4) The ligation products were PCR amplified by specific primers.

5) The PCR product is thermally denatured into single strands, and a segment of bridge primer is used for cyclizing the single-strand DNA to obtain a single-strand circular DNA library.

6) Sequencing on a machine.

4. Data analysis and results

4.1 Data analysis flow

The raw data from sequencing is called raw reads. First, we filtered reads of low quality, linker contamination, and too high an unknown base N content, the filtered data was called CLEAN READS (clean reads, i.e., filtered reads), then aligned CLEAN READS onto the reference genome, followed by new transcript prediction, SNP & InDel, and differential splicing gene detection. After obtaining new transcripts, we add new transcripts with protein coding potential to the reference gene sequence to form a complete reference sequence, and then calculate gene expression levels. Finally, the differentially expressed genes between the different samples are detected for a plurality of samples according to the requirements, and deep cluster analysis, functional enrichment analysis and the like are performed on the differentially expressed genes.

4.2 Transcriptomic sequencing results obtained, data with p-value >0.05 were retained. FPKM values and log2 fold change values of the skin fibroblast proliferation-related genes in the experimental group and the control group were extracted.

4.3 The FPKM values of the above genes of 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 kendion cloud tool, as in fig. 1.

TABLE 5

As can be seen from Table 5, the overall improvement in log2 fold change was calculated to be about 0.777, i.e., gene expression was promoted by about 1.713-fold.

In this example, a series of expression of genes related to skin fibroblast proliferation were detected by transcriptome, which demonstrates that recombinant collagen can enhance skin fibroblast proliferation.

Example 3

In this example, experiments were performed using the recombinant collagen obtained in example 1 to verify the effect of different concentrations of recombinant collagen on promoting the expression level of a series of genes related to skin fibroblast proliferation. The experimental steps include:

1. Cell sample culture

Collagen is prepared into a liquid to be tested with the concentration of 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 0.8mg/mL and 1.0mg/mL respectively, and the liquid to be tested is added into BJ cell culture medium which is growing on the wall to about 70 percent of the bottom area of a culture dish according to the final concentration of 0.1 percent, and is cultured for 24 hours.

2. Intracellular total RNA extraction and purification

(1) The cultured cells were removed by pipetting the medium, washed twice with pre-chilled PBS, 1mL of TRIzol reagent was added, and the cells were collected by pipetting into a centrifuge tube and allowed to stand at room temperature for 5 minutes.

(2) 200. Mu.L of chloroform was added to the tube, the tube was vigorously shaken for 15 seconds, and after 10 minutes of standing, 12000g was centrifuged at 4℃for 15 minutes.

(3) After centrifugation, the liquid was separated, RNA was present in the upper colorless transparent aqueous layer, the volume was about 600. Mu.L, and after transferring to a new centrifuge tube, 500. Mu.L of isopropyl alcohol was added, and the mixture was slowly turned upside down and mixed several times, and after standing for 15 minutes, 12000g was centrifuged at 4℃for 10 minutes.

(4) After centrifugation, RNA was pelleted at the bottom of the tube and seen as a gelatinous pellet facing the light source, after which the supernatant was aspirated, the RNA was washed by resuspension with 1mL of 75% ethanol and centrifuged at 7500g for 5 min at 4 ℃.

(5) The ethanol supernatant was removed by pipetting, left to stand for 5 minutes to dry the RNA, and 20. Mu.L of DEPC-water was added to dissolve the RNA.

3. RNA concentration determination

(1) The extracted RNA was taken at 1. Mu.L and added to 99. Mu.L of DEPC-water to make the total volume 100. Mu.L. The diluted RNA solution was added to a 96-well UV plate.

(2) The 96-well plate is put into a full-wavelength enzyme-labeled instrument, the region to be measured is selected, the program is that oscillation is carried out for 10 seconds, the light absorption value of 260nm is measured, the light absorption value of 280nm is measured, and the value is read.

(3) Calculating the OD ₂₆₀/OD₂₈₀ ratio, the ratio is between 1.8 and 2.0, which shows that the quality of the extracted RNA meets the subsequent requirement.

(4) RNA solution concentration in μg/μl was calculated according to the formula OD ₂₆₀ X0.04X dilution. RNA concentration was adjusted to 1. Mu.g/. Mu.L by dilution with DEPC-water.

4. Reverse transcription

(1) A reverse transcription system was prepared in a 200. Mu.L PCR tube using a reverse transcription kit (Novazal one-step reverse transcription kit R333) comprising a) 1. Mu.g/. Mu.L concentration of RNA solution in 1. Mu.L volume, b) 5 XqRT Supermix in 4. Mu.L volume, c) premix enzyme solution in 1. Mu.L volume, d) DEPC-water in 14. Mu.L volume. The final volume of the reverse transcription system was 20. Mu.L, and the mixture was gently stirred and mixed by a pipette, and centrifuged briefly in a palm centrifuge for 30 seconds.

(2) The reverse transcription system PCR tube was placed in an RCR apparatus and the reaction procedure was set to a) incubate at 50℃for 15 minutes and b) transiently inactivate at 85℃for 10 seconds. mRNA in the system was reverse transcribed into cDNA.

5. Fluorescent quantitative PCR

(1) A reaction system is prepared in an eight-link PCR tube by using reagents in a SYBR Green fluorescence quantitative kit, wherein the reaction system comprises a) 2×SYBR Green PCR premix solution with the volume of 10 μL, b) forward and reverse primers with the volume of 1.5 μL each, c) cDNA solution with the concentration of 1 μg/μL with the volume of 1 μL, d) sterilized primary water with the volume of 6 μL. The final volume of the PCR system was 20. Mu.L and centrifuged briefly for 30 seconds using a palm centrifuge.

(2) Three duplicate wells are needed for each test molecule of each sample, a blank control without cDNA is set, and actin beta (ACTB) is also needed as a reference molecule in addition to the test molecule.

(3) The eight-linked PCR tube is put into a fluorescent quantitative PCR instrument, and a reaction program is set, wherein the reaction program is a) 95 ℃ for 2 minutes, b) 95 ℃ for 5 seconds, C) 60 ℃ for 10 seconds, and d) 20 ℃ for constant-temperature storage. Wherein steps b and c are repeated for 45 cycles.

(4) After the PCR procedure is completed, the software automatically generates CT values for each sample.

6. Quantitative analysis of results

(1) The three repeated CT values of each sample are averaged, and the difference of the CT value of the molecule to be detected minus the CT value of the reference molecule (ACTB), i.e., deltaCT, is calculated.

(2) And (3) calculating the difference of delta CT value of the molecules to be detected in the cells after the raw material treatment minus delta CT value of the molecules to be detected in the untreated cells, namely delta CT.

(3) Calculating an index value with 2 as a base number and (-delta CT) as an index, namely 2 ^-△△CT, taking an average value of 2 ^-△△CT of a parallel group of each concentration as a quantitative result of molecules to be detected in each sample, and realizing quantitative detection of gene level.

The experimental results are shown in table 6 below.

TABLE 6

As is clear from Table 6, by detecting the gene expression level after treating cells with this collagen without using the concentration, the concentration range of 0.2mg/mL to 0.8mg/mL can ensure that the expression of all target genes is promoted. Wherein 0.5mg/mL is the concentration with the highest promotion rate.

Example 4

In this example, the recombinant collagen obtained in example 1 was used for fibroblast proliferation experiments, and the effect of different concentrations of recombinant collagen on fibroblast proliferation was verified. The experimental steps include:

(1) The BJ fibroblasts entering the index culture period were digested with pancreatin for about 20 seconds, and the digestion and resuspension were stopped by adding DMEM medium.

(2) 2 Wells of cell scratch inserts were placed in the center of each well of a six well cell culture dish, and slowly resuspended BJ cells were dropped into the wells. After incubation for 24 hours, after the cells were completely adherent, the scratch inserts were removed and 2mLDMEM medium was added to each well and 1% sterile water was added to the control wells. 1% of a recombinant collagen solution of 0.2mg/mL,0.5mg/mL and 0.8mg/mL was added to each experimental well. The cells were placed in a cell incubator for 24 hours and the experiment was repeated for each group.

(3) Cell scratch recovery was observed and calculated. The results are shown in FIGS. 2-9, and the scratch width (units/micron) statistics are shown in Table 7.

TABLE 7

The recovery rate in table 7 was calculated as recovery rate= (0 hour mean-24 hour mean)/0 hour mean.

From the results in Table 7, it is clear that the recombinant collagen has a remarkable effect of promoting fibroblast proliferation and can restore the scratches of the fibroblasts more quickly.

Example 5

In this example, the recombinant collagen obtained in example 1 was used as well as collagen 1, collagen 2 and collagen 3 to test the effect of promoting the expression level of a series of genes related to skin fibroblast proliferation.

The amino acid sequence of the collagen 1 is shown as SEQ ID NO. 2, the amino acid sequence of the collagen 2 is shown as SEQ ID NO. 3, and the amino acid sequence of the collagen 3 is shown as SEQ ID NO. 4.

The experimental steps include:

1. The recombinant collagen obtained in example 1, collagen 2 and collagen 3 were prepared as 0.5mg/mL of the solution to be tested, and added to BJ cell culture medium which was growing on the wall to about 70% of the bottom area of the culture dish at a final concentration of 0.1%, followed by culturing for 24 hours.

2. Subsequent RNA extraction, reverse transcription, qPCR experimental procedure was as described in example 3. The experimental results are shown in table 8.

TABLE 8

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 value of	1.392	1.412225438	1.303	1.813

From the qPCR results in the table, the recombinant collagen of the present invention can up-regulate 27 skin fibroblast proliferation related genes, while other collagen 1, collagen 2, collagen 3 could not up-regulate all of the 27 genes, and the up-regulation effect was inferior to that of 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 with the conclusion in the foregoing example 3, and the recombinant collagen of the present invention has a remarkable promoting effect on fibroblast proliferation.

Application example 1

The recombinant collagen protein can be used as an active substance for preparing skin conditioning agents and cosmetics. For example, it may be a face cream, an emulsion, a gel, a lotion, an essence, a mask, an eye cream, an aerosol cleansing foam, a spray, a shower gel or a facial cleanser.

The cream is prepared by using 0.1% of recombinant collagen, 7% of caprylic/capric triglyceride, 5% of liquid paraffin, 4% of glycerol, 3% of propylene glycol, 3% of ethylhexyl palmitate, 3% of vaseline, 2% of cetostearyl ether-21, 2% of polydimethylsiloxane, 2% of cetostearyl alcohol, 1.5% of cetostearyl ether-2, 1.5% of glyceryl monostearate, 0.2% of methyl paraben, 0.2% of carbomer, 0.2% of triethanolamine, 0.1% of ethyl paraben and the balance of water (65.2%).

The emulsion was prepared using 2, the emulsion was prepared from 0.1% recombinant collagen, 5% glycerol, 5% hydrogenated polyisobutene, 4% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic/capric triglyceride, 2% polydimethylsiloxane, 1.5% cetylstearyl ether-21, 1.2% cetylstearyl ether-2, 0.5% tocopherol, 0.2% methyl paraben, 0.1% triethanolamine, 0.1% ethyl paraben, 0.1% carbomer, the balance being water (74.2%).

The cosmetic water is prepared from (by weight) recombinant collagen 0.1%, glycerol 5%, propylene glycol 3%, diazo imidazolidinyl urea 0.2%, and water 91.7%.

In conclusion, the invention detects the expression condition of a series of genes related to the proliferation of the skin fibroblasts by a transcriptomic mode, and proves that the recombinant collagen can promote the expression of the genes related to the proliferation of the skin fibroblasts. The collagen can be used for developing and applying various cosmetic products, and endowing the cosmetic products with the efficacy and effect of promoting the proliferation of skin fibroblasts. Meanwhile, the series of genes can also be used as a reference for evaluating the proliferation capacity of other raw materials or cosmetics for promoting the skin fibroblast.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims

1. Use of recombinant collagen in preparing cosmetics, characterized in that the amino acid sequence of the recombinant collagen is as shown in SEQ ID NO: 1.

2. The use according to claim 1, characterized in that the cosmetic can increase the expression of genes related to skin fibroblast proliferation, and the skin fibroblast proliferation-related genes include: 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, any one or a combination of at least two.

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

4. The use according to claim 3, wherein the cosmetics include: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.