High-activity triple-helix mini collagen for skin light injury repair

Abstract

The invention belongs to the field of skin medicine (collagen biomimetic material technology), and particularly relates to a high-activity triple-helix mini collagen for skin light injury repair, the high-activity triple-helix mini collagen successfully simulates the structure and function of natural collagen, has no cytotoxicity and good water solubility, and can be used for repairing skin light injury. The high-activity triple-helix mini collagen can promote adhesion, migration and proliferation of fibroblasts and has a remarkable repairing effect on skin damaged by ultraviolet rays, meanwhile, the preparation method of the high-activity triple-helix mini collagen is simple and convenient, conditions are mild, and the high-activity triple-helix mini collagen has wide application prospects in the fields of tissue regeneration, skin medicine and the like.

Description

Technical Field

[0001] The present invention belongs to the field of skin medicine (collagen bionic material technology), and in particular relates to a highly active triple-helix mini collagen for repairing skin photodamage. Background Art

[0002] The skin is the largest organ in the human body, with a total area of ​​approximately 1.5-2.0m 2 , the total mass can account for 16% of the human body weight. As an important organ that is in direct contact with the outside world, the skin can protect the body from physical, chemical and biological harmful factors in the external environment. In addition, the skin plays an important role in maintaining the internal balance of the human body, regulating body temperature, transmitting sensations and participating in immune responses. The skin is easily damaged by ultraviolet rays in the sun when it is exposed to sunlight for a long time. Excessive ultraviolet radiation can cause varying degrees of photodamage, including edema, erythema, pigmentation, inflammation, DNA damage, photoaging and photocarcinogenesis.

[0003] Collagen is the main component of the extracellular matrix of the dermis of the skin, accounting for about 80-90% of the total extracellular matrix. Natural collagen has a unique triple helix structure and diverse biological functions. It can provide mechanical support for the skin and regulate cellular processes such as cell adhesion, migration, proliferation and differentiation. Ultraviolet radiation can cause abnormal degradation and cross-linking of collagen in the skin, resulting in decreased skin elasticity. As a bioactive polymer, collagen has excellent moisturizing, film-forming and regenerative properties, and can effectively repair damaged skin. Animal-derived collagen has a wide range of sources, good biocompatibility and high biological activity, and is widely used in the field of skin damage repair. However, animal-derived collagen has problems such as large batch differences, potential immunogenicity and pathogen transmission risks. It is difficult to achieve directional hydroxylation of proline in recombinant collagen prepared by genetic engineering.

[0004] Hydrolyzed collagen peptides are polypeptide fragments produced by further hydrolysis of collagen. They have a small molecular weight and low immunogenicity, and are gradually attracting attention in the field of skin damage repair. Hydrolyzed collagen peptides have good hygroscopic and moisturizing properties, can remove superoxide anions, hydroxyl radicals and inhibit lipid peroxidation, thereby inhibiting ultraviolet radiation-mediated skin photoaging. At present, hydrolyzed collagen peptides are mainly prepared by acid, alkali and enzyme hydrolysis. However, the hydrolyzed collagen peptides prepared by the above method have uncontrollable amino acid sequences and do not have the triple helix structure of collagen. They cannot mimic the structure and function of natural collagen, which affects their application in the field of dermatology.

[0005] The present invention provides a highly active triple-helical mini collagen, and for the first time applies the mini collagen to skin damage repair. The highly active triple-helical mini collagen is prepared by a solid phase synthesis method, has a controllable amino acid sequence and a complete triple-helical structure, including N1 and N2 domains containing repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa at both ends, and a biologically functional integrin binding site GFOGER between the N1 and N2 domains. The highly active triple-helical mini collagen can successfully simulate the structure and function of natural collagen, promote the adhesion, migration and proliferation of fibroblasts, accelerate the repair of acute ultraviolet light-damaged skin, and has broad application prospects in the field of cosmetics or medical beauty products. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide a highly active triple-helical mini collagen, which can successfully simulate the structure and function of natural collagen, promote the adhesion, migration and proliferation of fibroblasts, accelerate the repair of acute ultraviolet light-damaged skin, and has broad application prospects in the field of dermatology. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides a highly active triple-helical mini-collagen for repairing skin photodamage, wherein the polypeptide sequence of the highly active triple-helical mini-collagen is: N1-GFOGER-N2;

[0008] Wherein, the GFOGER is Gly-Phe-Hyp-Gly-Glu-Arg;

[0009] The N1 and N2 contain at least 3 repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa; the Xaa and Yaa are any different amino acids.

[0010] Preferably, the Xaa is Pro; and the Yaa is Hyp.

[0011] Preferably, N1 and N2 are (Gly-Pro-Hyp) n or (Gly-Pro-Pro) n , wherein n is any integer greater than or equal to 3.

[0012] Preferably, both N1 and N2 are (Gly-Pro-Hyp) n , wherein n is any integer greater than or equal to 3.

[0013] Preferably, n is 3, or 4, or 5.

[0014] Preferably, the polypeptide sequence of the highly active triple-helical mini-collagen is:

[0015] (Gly-Pro-Hyp)3GFOGER(Gly-Pro-Hyp)3, or

[0016] (Gly-Pro-Hyp)3GFOGER(Gly-Pro-Hyp)4, or

[0017] (Gly-Pro-Hyp)4GFOGER(Gly-Pro-Hyp)4, or

[0018] (Gly-Pro-Hyp)5GFOGER(Gly-Pro-Hyp)5.

[0019] Preferably, the polypeptide sequence of the highly active triple-helical mini collagen is: (Gly-Pro-Hyp)5GFOGER(Gly-Pro-Hyp)5.

[0020] In a second aspect, the present invention provides the use of the highly active triple-helix mini collagen described in the first aspect in the preparation of skin damage repair products.

[0021] Preferably, the skin damage is skin photodamage.

[0022] Preferably, the products for repairing skin photodamage include cosmetics, medical cosmetic products, and medicines.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention first provides a highly active triple-helical mini-collagen, comprising N1 and N2 domains containing repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa at both ends, and an integrin binding site GFOGER with biological function between the N1 and N2 domains;

[0025] (2) The highly active triple-helical mini-collagen described in the present invention can mimic the structure and function of natural collagen;

[0026] (3) The highly active triple-helical mini-collagen described in the present invention is non-cytotoxic and can promote the adhesion, migration and proliferation of fibroblasts;

[0027] (4) The highly active triple-helix mini collagen described in the present invention has a significant repairing effect on UV-damaged skin;

[0028] (5) The preparation method of the highly active triple-helix mini collagen described in the present invention is simple, the conditions are mild, and it has broad application prospects in the fields of tissue regeneration, skin medicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The circular dichroism spectrum of the collagen of the present invention; wherein au is respectively the CD full spectrum of MC-1, the thermal change curve of MC-1, the first derivative of the thermal change curve of MC-1, the CD full spectrum of MC-2, the thermal change curve of MC-2, the first derivative of the thermal change curve of MC-2, the CD full spectrum of MC-3, the thermal change curve of MC-3, the first derivative of the thermal change curve of MC-3, the CD full spectrum of MC-4, the thermal change curve of MC-4, the first derivative of the thermal change curve of MC-4, the CD full spectrum of SMP, the thermal change curve of SMP, the first derivative of the thermal change curve of SMP, the CD full spectrum of STP, the thermal change curve of STP, the first derivative of the thermal change curve of STP, the CD full spectrum of HCP, the thermal change curve of HCP, the first derivative of the thermal change curve of HCP;

[0030] Figure 2 The collagen described in the present invention is a cytotoxicity graph based on HFF-1 cells; wherein ah is the cytotoxicity graph of MC-1, MC-2, MC-3, MC-4, SMP, STP, HCP and Col, respectively;

[0031] Figure 3 The collagen described in the present invention is based on the cell adhesion map of HFF-1 cells; wherein aj is the cell adhesion map of PBS, MC-1, MC-2, MC-3, MC-4, BSA, SMP, STP, HCP and Col respectively;

[0032] Figure 4 The collagen prepared by the present invention is based on the cell proliferation diagram of HFF-1 cells;

[0033] Figure 5 Cell migration diagram of collagen prepared by the present invention based on HFF-1 cells;

[0034] Figure 6 Ultrasonic images of mouse skin tissues in the blank group, model group, and experimental group;

[0035] Figure 7 Density test results of mouse skin tissue in the blank group, model group, and experimental group;

[0036] Figure 8 Chroma detection results of mouse skin tissue in the blank group, model group, and experimental group;

[0037] Fig. 9 The moisture test results of mouse skin tissue in the blank group, model group, and experimental group;

[0038] Fig.10 The results of transepidermal water loss (TEWL) of mouse skin tissue in the blank group, model group, and experimental group;

[0039] Fig.11 HE staining results of mouse skin tissues in the blank group, model group, and experimental group;

[0040] Fig.12 Masson staining results of mouse skin tissue in the blank group, model group, and experimental group;

[0041] Fig.13 Results of collagen fiber content detection in the skin tissue of mice in the blank group, model group, and experimental group;

[0042] Fig.14 Detection results of hydroxyproline content in skin tissue of mice in the blank group, model group and experimental group. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to the following.

[0044] The sequences of the highly active triple-helical mini-collagen involved in the following examples are shown in Table 1 below, but are not limited to the sequences described in Table 1; the sequences of the comparative collagen polypeptides involved are shown in Table 2 below. Among them, the GPO sequence is the (Gly-Pro-Hyp) sequence described in this application; the GFOGER is (Gly-Phe-Hyp-Gly-Glu-Arg). And the highly active triple-helical mini-collagen sequences are all synthesized by solid phase synthesis, and the solid phase synthesis method is specifically as follows:

[0045] (1) Add 100 mg of Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0046] (2) removing the N-terminal Fmoc protecting group using a 20% piperidine / N,N-dimethylformamide (DMF) solution, and detecting complete removal of the protecting group using a colorimetric reaction;

[0047] (3) The amino acid (4 eq) with Fmoc protection at the N-terminus, HOBT (4 eq) and HBTU (4 eq) were dissolved in DMF and activated at low temperature for 20 min. DIEA (6 eq) was then added dropwise to the solution. The solution was mixed and added to the reactor for reaction for 3 h.

[0048] (4) After the reaction is completed, the reaction solution is extracted from the reactor, and the resin is washed three times with 5 mL of DMF and DCM, respectively. The color reaction is used to detect that the amino acid condensation is complete. The resin is treated with 20% piperidine / DMF solution three times for 5 min, 5 min and 15 min, respectively. The resin is washed three times with 5 mL of DMF and DCM, respectively, and the color reaction is used to detect that the protecting group is completely removed;

[0049] (5) Repeat steps (3) and (4) until the target peptide sequence is completed.

[0050] (6) Block the N-terminus with 25% acetic anhydride (DMF) for 25 minutes.

[0051] (7) Cleavage: At room temperature, use TFA (trifluoroacetic acid): triisopropylsilane: H2O = 95:2.5:2.5, shake the resin for 3 hours to complete the cleavage of the peptide. Precipitate with ice ether, centrifuge, and wash to obtain the crude product of mini collagen.

[0052] The crude mini collagen product was purified by reverse phase high performance liquid chromatography using a C18 chromatographic column and freeze-dried to obtain the target mini collagen shown in Table 1.

[0053] Table 1 Sequence list of highly active triple helical mini collagen

[0054]

[0055] Table 2 Sequence Listing of Collagen Peptides

[0056]

[0057] The collagen hydrolyzed peptides (HCP) involved in the following examples are from Hainan Yuanpeptide Biotechnology Co., Ltd., and collagen (Col) is from the Collagen Joint Research Center of Lanzhou University.

[0058] definition:

[0059] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the methods belong. Although any methods and compositions similar or equivalent to those described herein can also be used to achieve self-assembly of collagen polypeptides, they are not limited to the representative exemplary methods and compositions described in the following examples. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention and the appended claims.

[0060] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a mini-collagen" includes a plurality of such mini-collagens, reference to "the mini-collagen" is a reference to one or more mini-collagens and their equivalents known to those skilled in the art, and so on.

[0061] "Comprising" means "including but not limited to", and is not intended to exclude, for example, other components, integers, etc. In particular, in the statement "comprising at least 3 repeats of the Gly-Pro-Hyp sequence", it is explicitly included to include the listed contents (unless limiting terms such as "consisting of ..." are included), which means that other components, integers not listed in the steps are not intended to be excluded.

[0062] Where range values ​​are provided, it is understood that each intervening value between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the methods and compositions, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the methods and compositions.

[0063] It should be understood that, for the sake of clarity, certain features described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the methods and compositions described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0064] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and shown herein has individual components and features that may be readily separated or combined with the features of any other embodiment without departing from the scope or spirit of the present method. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0065] The term "mini collagen" refers to a highly active collagen biomimetic polypeptide with a triple helical structure and functional sequence.

[0066] The term "collagen" is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals. It has a trimer structure composed of three peptide chains, mainly including type I, type II, type III and type IV collagen.

[0067] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked to each other by peptide bonds or modified peptide bonds. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers that contain modified residues and non-naturally occurring amino acid polymers.

[0068] The term "thermal transition temperature" refers to the temperature at which half of the triple helical structure of the collagen polypeptide is converted into a random coil. The thermal transition temperature can be determined by known structural analysis methods such as circular dichroism (CD), but is not limited to these and can also be determined by other known methods. It is well known to those skilled in the art that collagen polypeptides with thermal transition temperatures all have a triple helical structure.

[0069] Example 1 Characterization of the properties of the mini collagen, collagen polypeptide, collagen hydrolyzed peptide and collagen described in this application

[0070] Circular dichroism (CD) spectra were obtained on a Chirascan CD spectrometer (Appliedphotophysics, England) equipped with a Peltier temperature controller. A 2 mg / mL aqueous solution of the peptide was prepared at pH 7.4. The sample was equilibrated at 4 °C for at least 24 h before measurement. A cuvette with a path length of 1 mm was used. The thermal curve was obtained by monitoring the change of the characteristic CD peak at a wavelength of 225 nm with temperature, ranging from 4 °C to 90 °C, with a heating rate of 0.5 °C / min. The first-order derivative of the thermal curve was taken, and the temperature corresponding to the extreme value was the thermal transition temperature (T m ).

[0071] The characterization results of mini collagen (GPO) 3GFOGER (GPO) 3 (MC-1) are as follows Figure 1 As shown in a, b, and c: The CD spectrum shows the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that mini collagen MC-1 has a triple helical structure. The results of the thermal curve and its first-order derivative show that MC-1 forms a stable triple helical structure with a thermal transition temperature of 37.9℃.

[0072] The characterization results of mini collagen (GPO) 3GFOGER (GPO) 4 (MC-2) are as follows Figure 1 As shown in d, e, and f: The CD spectrum shows the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that mini collagen MC-2 has a triple helical structure. The results of the thermal curve and its first-order derivative show that MC-2 forms a stable triple helical structure with a thermal transition temperature of 47.6℃.

[0073] The characterization results of mini collagen (GPO) 4GFOGER (GPO) 4 (MC-3) are as follows Figure 1As shown in g, h, and i: The CD spectrum shows the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that mini collagen MC-3 has a triple helical structure. The results of the thermal curve and its first-order derivative show that MC-3 forms a stable triple helical structure with a thermal transition temperature of 58.2℃.

[0074] The characterization results of mini collagen (GPO) 5GFOGER (GPO) 5 (MC-4) are as follows Figure 1 As shown in j, k, and l: The CD spectrum shows the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that the mini collagen MC-4 has a triple helical structure. The results of the thermal curve and its first-order derivative show that MC-4 forms a stable triple helical structure with a thermal transition temperature of 66.8℃.

[0075] The characterization results of collagen peptide GPOGFOGERGPO (SMP) are as follows Figure 1 As shown in m, n, and o: At 225 nm, the CD value of the collagen polypeptide GPOGFOGERGPO changes linearly with temperature, indicating that SMP has no triple helix structure.

[0076] The characterization results of collagen polypeptide (GPO) 8 (STP) are as follows Figure 1 As shown in p, q, and r: The full CD spectrum shows the highest absorption peak at 225nm and the lowest absorption peak at 198nm, indicating that the polypeptide STP has a triple helical structure. The results of the thermal curve and its first-order derivative show that STP forms a stable triple helical structure with a thermal transition temperature of 60.8℃.

[0077] The characterization results of hydrolyzed collagen polypeptide (HCP) are as follows Figure 1 As shown in s, t, and u: the CD spectrum has no highest absorption peak at 225 nm, and shows the lowest absorption peak at 198 nm. In addition, the CD value of collagen hydrolyzed peptide at 225 nm changes linearly with temperature, indicating that HCP has no triple helix structure.

[0078] Characterization of the properties of collagen (Col). The collagen Col used was from the Collagen Joint Research Center of Lanzhou University. The collagen Col has a stable triple helix structure and a thermal transition temperature of 42.7°C.

[0079] Example 2 Cell experiments on mini collagen, collagen polypeptides, collagen hydrolyzed peptides and collagen described in this application 1. Cytotoxicity experiment

[0080] The cytotoxicity of the mini collagen, collagen polypeptide, collagen hydrolyzed peptide and collagen HFF-1 cells described in Example 1 of the present application was determined using the CCK-8 method. HFF-1 cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) were cultured in a constant temperature incubator containing 5% CO2 (temperature set at 37°C). A volume of 100 μL of HFF-1 cell suspension (5×10 per well) was added to 3 The cells were inoculated into 96 microwells at a density of 10 cells / mL and incubated for 24 hours (37°C, 5% CO2) to ensure complete adhesion. Then, 100 μL of each sample solution of different concentrations (1 μg / mL, 10 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL and 1000 μg / mL) was added to the 96 microwell plates, and 100 μL of DMEM medium was added to the control wells, and the incubation was continued at 37°C for 24 hours. Then, 100 μL of 10% Cell Counting Kit-8 (CCK-8) was added to each well, and the cells were cultured in a cell culture incubator. The OD value at 450 nm was measured using a Tecan Infinite F200 / M200 multifunctional microplate reader (Tecan, Mannedor, Switzerland). The appropriate time was found, and the culture was stopped when the OD value of the well with only cells and no material reached about 1. The activity of HFF-1 cells was calculated as the average absorbance value of three measurements for each condition.

[0081] The results of cytotoxicity experiments were Figure 2 As shown: the mini collagen and collagen polypeptides, collagen hydrolyzed peptides and collagen described in Example 1 of the present application all have a cell survival rate of ~100% at different concentrations, indicating that the mini collagen described in the present application is non-cytotoxic.

[0082] 2. Cell Adhesion Assay

[0083] 24 microwell plates (untreated tissue) were coated with PBS, BSA, mini collagen, collagen peptides, collagen hydrolyzed peptides and collagen samples described in Example 1 of the present application. PBS and BSA were used as controls. Before adding HFF-1 cells, the coated wells were washed three times with PBS buffer (10 mM). Then 500 μL (1×10 5 The HFF-1 cell suspension (200 μg / mL, serum-free medium) was inoculated in a 24-well microplate and incubated at 37°C for 5 hours. Finally, the unattached HFF-1 cells were washed with 10 mM PBS buffer. The adhesion effect of HFF-1 cells was observed by inverted microscope.

[0084] The results of cell adhesion experiments were as follows Figure 3As shown: HFF-1 cells cultured on MC-1, MC-2, MC-3, MC-4, and Col samples spread into a spindle shape, HFF-1 cells cultured on the HCP surface showed an inconspicuous spindle shape, and some still maintained a spherical shape, and HFF-1 cells cultured on PBS, BSA, SMP, and STP samples maintained a spherical shape. This result shows that BSA, SMP, STP, and HCP have poor adhesion to HFF-1 cells, while the mini collagen described in this application is similar to natural collagen and exhibits good adhesion to HFF-1 cells.

[0085] 3. Cell Proliferation Assay

[0086] The ability of mini collagen to promote HFF-1 cell proliferation was detected by CCK-8 kit at 1, 3, 5 and 7 days. HFF-1 cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) were cultured in a constant temperature incubator containing 5% CO2 (temperature set at 37°C). A volume of 100 μL of HFF-1 cell suspension (5×10 cells per well) was added to 3 The cells were inoculated into 96 microwells at a density of 100 cells / mL and incubated for 24 hours (37°C, 5% CO2) to ensure complete adhesion. Then, 100 μL of the mini collagen MC-4 and collagen polypeptide, collagen hydrolyzed peptide and collagen sample solution described in Example 1 of the present application at a concentration of 0.1 mg / mL were added to the 96 microwell plates, and 100 μL of DMEM culture medium was added to the control wells, and the culture solution was replaced every two days. When the incubation was continued at 37°C for 1, 3, 5, and 7 days, 100 μL of 10% CCK-8 was added to each well, placed in a cell culture incubator, and the OD value at 450 nm was measured using a Tecan Infinite F200 / M200 multifunctional microplate reader. The activity of HFF-1 cells was calculated as the average absorbance value of three measurements for each condition.

[0087] The results of cell proliferation assays were Figure 4 As shown: when co-cultured with HFF-1 cells for 1 day and 3 days, the cell proliferation rates of MC-1, MC-2, MC-3, MC-4 and Col groups were slightly higher than those of other groups; on the 5th day, the cell proliferation rates of MC-1, MC-2, MC-3, and MC-4 groups were significantly higher than those of SMP and STP groups; on the 7th day, the cell proliferation rates of MC-1, MC-2, MC-3, and MC-4 were significantly higher than those of SMP, STP, and HCP groups. This result shows that the mini collagen described in this application has a good cell proliferation effect, and its cell proliferation promoting performance is significantly better than that of hydrolyzed collagen peptides.

[0088] 4. Cell Migration Assay

[0089] The ability of mini collagen to promote HFF-1 cell migration was determined by cell scratch method. Three horizontal lines were drawn on the bottom of 6-well plates treated with TC using a marker pen, with each horizontal line spaced 0.5-1 cm apart. HFF-1 cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) were cultured in a constant temperature incubator (temperature set at 37°C) containing 5% CO2. A volume of 2 mL of HFF-1 cell suspension (1×10 cells per well) was added to the plate. 6 The cells were inoculated into 6 microwells at a density of 10 cells / well and incubated for 24 hours (37°C, 5% CO2) to ensure complete adhesion. When the cells cover the bottom of the well plate, use a 10 μL gun tip to draw a vertical line along the ruler and perpendicular to the horizontal line at the bottom to form a cell scratch, and wash the well plate 3 times with PBS to remove the scratched cells. 2 mL of mini collagen MC-4 and collagen polypeptides, collagen hydrolyzed peptides and collagen sample solutions described in Example 1 of the present application were added to the 6-well plate, and the 6-well plate was placed in a cell culture incubator for culture. Then, the well plate was removed at 0h and 24h and the scratch area was photographed under an inverted microscope. The scratch area (S) was calculated using Image J, and the cell migration rate was calculated according to the following formula:

[0090]

[0091] Where S0 is the area of ​​cell scratch at 0 h, and St is the area of ​​cell scratch at 24 h.

[0092] The results of cell migration experiments were Figure 5 As shown in the figure, the cells in the blank group hardly migrated, with a migration rate of 19.09%; the migration rate of HFF-1 cells after 24 hours of culture with MC-4 reached 51.15%, which was significantly higher than 26.82% in the SMP group, 27.41% in the STP group, and 42.27% in the HCP group. The results show that the mini collagen described in the present application significantly promotes cell migration, and its effect is better than that of hydrolyzed collagen peptides.

[0093] Example 3 UV-damaged skin repair experiment

[0094] 1. UV-damaged skin modeling and treatment

[0095] Clean-grade male Kunming mice weighing 20±2g were selected, and the backs of the mice were shaved with a hair clipper, and then depilatory cream was used to remove hair, leaving the back epidermis completely bare. The depilatory area was approximately 2×4cm and was stable for 24h. The mice were randomly divided into three groups. The blank group did not receive any treatment. The back skin of the mice in the experimental group and the control group was irradiated with continuous wavelength ultraviolet lamps A (320-440nm) and B (280-320nm). The radiation was quantified using an ultraviolet irradiator to reach 200J / cm 2 This indicated that the acute UV damage model of mouse back skin was successfully established.

[0096] Deionized water was used to prepare the mini collagen MC-4, collagen polypeptide, collagen hydrolyzed peptide and collagen sample solutions described in Example 1 of the present application with a final concentration of 0.1 mg / mL, and 5 mL of the sample solutions were applied to the back skin of the mice, respectively, and reapplied every 24 hours. Among them, the mice in the experimental group were smeared with the sample solution of the example, the mice in the control group were smeared with normal saline, and the mice in the blank group were not irradiated with ultraviolet light and sample smeared.

[0097] 2. Ultrasound images of mouse skin tissue

[0098] Combo ultrasound imaging (ultrasound signal intensity white>yellow>red>blue>green>black) was used to detect the density and thickness of the skin in vivo and evaluate the ability of mini collagen to promote the repair of UV damage in mouse skin. Figure 6 As shown: On the first day after UV-damaged skin modeling, the epidermal density and thickness of the skin in each UV-damaged group increased, while the dermal density decreased. On the third day, the epidermal density and thickness of the skin in each experimental group were still high; the dermal density of the MC-4 and Col groups began to increase, and the Col group was slightly higher than the MC-4 group, while the dermal density of the SMP, STP, HCP and control groups did not increase significantly; on the fifth day, the epidermal thickness of the MC-4 and Col groups began to decrease, while the dermal density continued to increase, and the Col group was slightly higher than the MC-4 group, while the epidermal density and thickness of the SMP, STP, HCP and control groups were still high, and the dermal density began to increase, but was lower than the MC-4 and Col groups; on the seventh day, the epidermis and dermis of the MC-4 and Col groups returned to normal skin levels; the epidermal thickness of the SMP, STP, HCP and control groups decreased, and the dermal density increased, but none of them returned to normal skin levels. The above results show that the mini collagen described in the present application is similar to natural collagen and can accelerate the recovery of UV-damaged skin to normal density and thickness, and its effect is better than hydrolyzed collagen peptides.

[0099] 3. Density of Mouse Skin Tissue

[0100] Using the Combo ultrasound probe, the density of the mouse back skin was quantitatively detected in vivo to evaluate the ability of mini collagen to promote the restoration of skin density. Figure 7As shown: On the first day after skin UV damage repair, the skin density of MC-4, SMP, STP, HCP, Col and control group was significantly lower than that of the blank group, which decreased to 38.05±6.44, 35.68±4.08, 36.26±3.72, 34.92±3.02, 39.08±4.57 and 37.95±2.57, respectively, and there was no significant difference among the UV damage groups; on the third day, the skin density of MC-4 group increased to 54.26±9.36, and the skin density of Col group increased to 60.72±9.42, both of which were higher than 41.64±5.77 of SMP group, 43.31±5.46 of STP group, 44.70±10.02 of HCP group and 42.90±3.09 of control group. On the 5th day, the skin density of the MC-4 group and the Col group increased to 65.35±9.73 and 74.90±6.77, respectively, which were significantly different from the blank group, and higher than 55.13±7.55 in the SMP group, 54.35±4.94 in the STP group, and 58.59±5.38 in the HCP group. The skin density of each group was higher than 50.02±3.99 in the control group. On the 7th day, the skin density of the MC-4 and Col groups returned to the normal skin level, increasing to 84.77±7.87 and 89.10±4.14, respectively, which were higher than 59.93±6.14 in the SMP group, 56.03±10.04 in the STP group, and 66.75±5.98 in the HCP group. The skin density of the above groups was higher than 54.84±8.45 in the control group. The above results show that the mini collagen described in the present application is similar to natural collagen and helps to restore the density of UV-damaged skin, and its effect is better than that of hydrolyzed collagen peptides.

[0101] 4. Chromatography of Mouse Skin Tissue

[0102] The skin color probe of Combo was used to detect the color of the back skin of mice in vivo to evaluate the ability of mini collagen to reduce skin erythema. The erythema index of the skin can reflect the condition of skin capillaries and directly reflect the degree of inflammation. The lower the value, the better the repair effect.

[0103] The colorimetric results of mouse skin tissue are as follows Figure 8As shown in the figure: On the first day after skin UV damage repair, the skin chromaticity of each UV damage group increased and was significantly higher than that of the blank group. The skin chromaticity of the MC-4 group increased to 9.50±0.77, which was higher than 8.13±0.57 of the Col group, but significantly lower than 13.52±1.07 of the SMP group, 12.71±2.16 of the STP group, 12.49±0.60 of the HCP group, and 14.25±1.80 of the control group. On the third day, the skin chromaticity of each UV damage group continued to increase. The skin chromaticity of the MC-4 group increased to 13.76±1.90, which was higher than 11.08±1.04 of the Col group, but lower than 16.68±1.26 of the SMP group, 15.28±2.48 of the STP group, 14.56±1.72 of the HCP group, and 14.25±1.80 of the control group. On the 5th day, the skin chromaticity of the MC-4 group and the Col group decreased to 10.08±1.05 and 10.62±3.14, respectively, with no significant difference between the two groups, but significantly lower than 16.21±0.24 in the SMP group, 6.17±1.34 in the STP group, 15.19±0.94 in the HCP group, and 16.48±0.66 in the control group; on the 7th day, the skin chromaticity of the MC-4 group and the Col group returned to the normal skin level, decreasing to 6.05±0.47 and 5.32±0.54, respectively, significantly lower than 12.03±0.63 in the SMP group, 12.05±0.96 in the STP group, and 8.49±3.13 in the HCP group. The skin chromaticity of the above groups was lower than 16.27±0.70 in the control group. The above results show that the mini collagen described in the present application is similar to natural collagen and helps the skin to quickly return to normal color, and its effect is better than hydrolyzed collagen peptides.

[0104] 5. Moisture of Mouse Skin Tissue

[0105] The moisture probe of Combo was used to detect the moisture of the mouse back skin in vivo, and the ability of mini collagen to promote the rapid recovery of skin moisture to normal levels was evaluated. The higher the value obtained, the higher the moisture content of the skin stratum corneum.

[0106] The results of moisture test on mouse skin tissue are as follows Fig. 9As shown: On the first day of skin UV damage repair, the skin moisture of each experimental group was significantly higher than that of the blank group. The skin moisture of the MC-4 group increased to 541.75±121.29μS, and the skin moisture of the Col group increased to 520.40±71.89μS, which were significantly lower than 805.15±122.36μS of the SMP group, 846.73±109.27μS of the STP group, and 867.87±181.79μS of the HCP group. The skin moisture of the above groups was lower than that of the control group (1207.35±50.96μS). On the third day, the skin moisture of MC-4, SMP, STP, HCP, Col and the control group decreased to 149.61±8.27μS, 169.60±22.75μS, 168.48±6.63μS, 166.28±2.11μS, 169.94±17.89μS and 172.59±12. 60μS; on the 5th day, the skin moisture of the MC-4 group and the Col group began to increase, increasing to 177.76±15.56μS and 201.74±22.03μS, respectively, with no significant difference between the two groups, which were significantly higher than 43.06±7.71μS of the SMP group, 43.71±14.11μS of the STP group, 82.61±24.27μS of the HCP group, and 43.67±10.43μS of the control group; on the 7th day , the skin moisture of the MC-4 group and the Col group increased to the level of normal skin, increasing to 229.92±19.25μS and 236.35±13.71μS respectively, with no significant difference between the two, which was significantly higher than 144.62±12.27μS of the SMP group, 137.77±24.03μS of the STP group and 178.82±26.13μS of the HCP group, all higher than 68.67±14.57μS of the control group. The above results show that the mini collagen described in this application is similar to natural collagen, helps the skin moisture to quickly restore to normal moisture, and its effect is better than hydrolyzed collagen peptides.

[0107] 6. Transepidermal Water Loss (TEWL) of Mouse Skin Tissue

[0108] The Combo TEWL probe was used to detect the transepidermal water loss of the mouse back skin in vivo to evaluate the ability of mini collagen to promote the rapid recovery of skin TEWL to normal levels. TEWL refers to the loss of water in the dermis through epidermal evaporation. The higher the value, the more water loss in the skin and the worse the skin barrier function.

[0109] The results of transepidermal water loss test on mouse skin tissue are as follows Fig.10As shown in Figure 1, on the first day after skin UV damage repair, the TEWL of the skin in each experimental group was significantly higher than that in the blank group. The TEWL of the skin in the control group, MC-4, SMP, STP, HCP and Col groups increased to 65.17±3.21 g / m 2 *h, 60.85±5.36g / m 2 *h, 61.62±3.98g / m 2 *h, 65.87±1.76g / m 2 *h, 61.98±3.03g / m 2 *h and 58.46±7.57g / m 2 *h; On the 3rd day, the skin TEWL of the MC-4 group decreased to 53.19±2.348 g / m 2 *h, the skin TEWL of the Col group decreased to 49.88±5.81g / m 2 *h, there was no significant difference between the two groups, which was significantly lower than the 63.91±1.88g / m 2 *h, STP group 61.59±5.17g / m 2 *h, HCP group 60.71±5.38g / m 2 *h and 63.91±1.88g / m 2 *h; On the 5th day, the skin TEWL of the MC-4 group and the Col group continued to decrease, decreasing to 31.24±1.79 g / m 2 *h and 31.65±4.84g / m 2 *h, there was no significant difference between the two groups, both significantly lower than the 45.02±5.17g / m 2 *h, STP group 42.35±1.76g / m 2 *h, HCP group 39.44±1.17g / m 2 *h and 46.40±2.49g / m 2 *h; On the 7th day, the TEWL of the skin in the MC-4 group and the Col group returned to the normal skin level, decreasing to 20.42±1.99 g / m 2 *h and 20.39±1.00g / m 2 *h, there was no significant difference between the two groups, which was significantly lower than 32.76±2.04g / m 2 *h, STP group 30.18±5.44g / m 2 *27.88±3.38g / m in h and HCP groups 2 *h, which was lower than that of the control group (37.88±5.82g / m 2*h. The above results show that the mini collagen described in the present application is similar to natural collagen and helps the skin to quickly return to normal TEWL, and its effect is significantly better than that of hydrolyzed collagen peptides.

[0110] 7. HE staining of mouse skin tissue

[0111] HE staining was used to study the ability of mini collagen to promote the repair of acute UV-damaged skin at the tissue level. Fig.11 As shown: The back skin of mice in the blank group remained healthy throughout the experiment, with a thin epidermis, clear stratification of cells and orderly arrangement, a tight connection between the epidermis and dermis, a clear boundary between the two and a wavy junction, and continuous and dense fiber arrangement in the dermis, without inflammatory cell infiltration.

[0112] On the first day of skin UV damage repair, the epidermis of the back skin of mice in each UV damage group was abnormally thickened, and the thickness was uneven, and the boundary between the epidermis and dermis was blurred. The superficial dermis became lighter, with edema, inflammatory cell infiltration, and obvious inflammation. The fibers in the dermis were destroyed, the fibers were broken and curled, and the arrangement was disordered, and there was vascular dilation. On the third day, the epidermis of the MC-4 group and the Col group began to keratinize, and the connective tissue close to the epidermis protruded to the epidermis to form obvious dermal papillae. The dermis was edematous and accompanied by vascular dilation, the fibers in the dermis were curled and broken, and there was inflammatory cell infiltration. The epidermis of the SMP, STP, HCP and control groups began to keratinize, and the dermis was edematous and accompanied by vascular dilation. The fibers in the dermis were curled and broken, and there was inflammatory cell infiltration.

[0113] On the 5th day, the epidermis of the MC-4 and Col groups began to regenerate, the keratinized epidermis was not removed, and the dermal papilla near the epidermis was more obvious. The edema of the dermis basically subsided, the degree of vascular dilation and dermal fiber curling and breaking was slightly reduced compared with the 3rd day, and the infiltration of inflammatory cells decreased. The epidermis of the SMP, STP, HCP and control groups continued to keratinize, among which the connective tissue near the epidermis of the dermis in the SMP, STP and HCP groups protruded to the epidermis to form dermal papilla. The edema of the dermis in each group began to subside, the curling and breaking of the dermal fibers was slightly reduced, and there was inflammatory cell infiltration. On the 7th day, the keratinized epidermis of the MC-4 and Col groups was removed, and the new epidermis was slightly thicker than that of the normal group, and the cells were clearly layered and arranged neatly and orderly. The edema of the dermis completely faded, the fibers of the dermis were arranged in an orderly manner, and the vasodilation phenomenon and inflammatory response disappeared; the epidermis of SMP, STP, HCP and the control group continued to keratinize, and the edema and vasodilation of the dermis did not completely disappear. The degree of curling and breaking of the dermis fibers was reduced compared with the 5th day, the infiltration of inflammatory cells was reduced, and the skin morphology was still significantly different from that of the normal group. The above results show that the mini collagen described in this application has a good repair effect on acute ultraviolet light damaged skin, and its effect is significantly better than that of hydrolyzed collagen peptides.

[0114] 8. Masson staining of mouse skin tissue

[0115] Masson staining was used to study the ability of mini collagen to promote the repair and regeneration of collagen fibers in acute UV-damaged skin. Fig.12 As shown in the figure: The structure of the back skin of the blank group mice was normal during the whole experiment. The collagen fibers in the papillary layer were small, and the collagen fibers in the reticular layer were thick. They were tightly woven into a three-dimensional network and arranged in a wavy manner parallel to the epidermis. On the first day of skin UV damage repair, the collagen fibers in the dermis of the back skin of the mice in each UV damage group were broken and curled, some of them gathered into clumps, and the overall arrangement was disordered. Inflammatory cells infiltrated between the collagen fibers. On the third day, new collagen fibers began to be generated at the broken parts of the dermis collagen fibers in the MC-4 group and the Col group. The fibers were arranged in a disordered manner and some of them gathered into clumps. Inflammatory cells infiltrated between the collagen fibers. The collagen fibers in the dermis of the SMP, STP, HCP and control groups were still broken, the fibers were arranged in a disordered manner and some of them gathered into clumps. A large number of inflammatory cells infiltrated between the collagen fibers. The infiltration degree of the control group was the most serious.

[0116] On the 5th day, the collagen fibers in the dermis of the MC-4 group and the Col group continued to regenerate, the fiber arrangement began to become loose, some collagen fibers were disordered, and the number of inflammatory cells decreased; new collagen fibers began to be generated at the broken parts of the collagen fibers in the dermis of the SMP, STP, HCP and control groups, and the fiber regeneration speed was faster than that of the control group. The fibers were disordered and partially aggregated into clusters, and the number of inflammatory cells decreased. On the 7th day, the collagen fibers in the dermis of the MC-4 group and the Col group basically returned to normal, and skin inflammation disappeared; the collagen fibers in the dermis of the SMP, STP, HCP and control groups continued to increase, among which the fibers in the SMP, STP and HCP groups were loosely arranged, and some were still disordered, and the number of inflammatory cells decreased. The above results show that mini collagen can accelerate the repair and regeneration of collagen fibers in the dermis of acute UV-damaged skin, and its effect is better than that of hydrolyzed collagen peptides.

[0117] 9. Collagen fiber content in mouse skin tissue

[0118] The collagen fiber content in mouse skin tissue at different repair times was quantified by Image J software. The results of collagen fiber content in mouse skin tissue are shown in Fig.13 As shown: On the first day of skin UV damage repair, the collagen fiber content of the skin of MC-4, SMP, STP, HCP, Col and control groups was significantly lower than that of the blank group, decreasing to 13.49±1.03%, 13.33±0.83%, 13.64±2.10%, 13.14±1.70%, 13.37±1.03% and 13.81±0.75%, respectively, and there was no significant difference among the model groups; on the third day, the collagen fiber content of the skin of the MC-4 group increased to 19.16±2.05%, and the collagen fiber content of the skin of the Col group increased to 20.82±0.22%, both of which were higher than 16.70±0.21% of the SMP group, 16.73±1.05% of the STP group, 17.96±0.32% of the HCP group and 16.35±1.37% of the control group.

[0119] On the 5th day, the collagen fiber content of the skin of the MC-4 group and the Col group increased to 30.29±1.42% and 30.73±1.76%, respectively, which were significantly different from the collagen fiber content of normal skin, and significantly higher than 20.50±1.28% of the SMP group, 20.91±1.88% of the STP group, and 24.57±1.18% of the HCP group. The collagen fiber content of the above groups was higher than 19.06±1.21% of the control group. On the 7th day, the collagen fiber content of the skin of the MC-4 group and the Col group returned to the normal skin level, increasing to 35.93±0.91% and 36.47±1.97%, respectively, which were significantly higher than 26.13±2.27% of the SMP group, 26.55±1.72% of the STP group, and 29.25±1.01% of the HCP group. The collagen fiber content of the above groups was higher than 25.29±2.85% of the control group. The above results indicate that the mini collagen described in the present application can accelerate the expression of collagen fibers in UV-damaged skin, and its effect is better than that of hydrolyzed collagen peptides.

[0120] 10. Hydroxyproline content in mouse skin tissue

[0121] Take the back skin of the mouse and dry it in a 60℃ oven, then cut it into pieces, accurately weigh 20mg, add 2mL HCl (6mol / L) and mix well, place it in an 85℃ oven for 12h, take it out and cool it to room temperature, add an appropriate amount of NaOH solution (10mol / L) to adjust the pH to neutral. After filtering, discard the solid impurities, keep the supernatant, dilute it with water to 20.0mL, and get the sample test solution. Take 200μL of the sample test solution in a test tube, add deionized water to 1.0mL; add 2.0mL of chloramine T solution, shake well and place it at room temperature for 20min; add 2.0mL of color developer, mix well and quickly place the test tube in an 85℃ water bath to react for 10min; quickly cool to room temperature; use tube 1 as a blank control, measure the absorbance at a wavelength of 561nm, record the data, and calculate the hydroxyproline content according to the standard curve.

[0122] The experimental results are as follows Fig.14 As shown: On the 7th day, the hydroxyproline content in the skin of the MC-4 group and the Col group returned to the normal skin level, which was 28.88±3.16μg / mg and 31.24±2.13μg / mg, respectively. There was no significant difference between the two, and both were significantly higher than 18.54±0.65μg / mg in the SMP group, 18.33±1.72μg / mg in the STP group, and 20.76±1.32μg / mg in the HCP group. The hydroxyproline content in the skin of each group was higher than 16.17±0.66μg / mg in the control group. The experimental results show that the mini collagen described in this application is similar to natural collagen, can promote collagen regeneration, and its effect is significantly better than hydrolyzed collagen peptides.

[0123] In summary, the mini collagen described in the present invention (two end sequences N1 and N2 sequences, and the middle sequence GFOGER; the N1 and N2 sequences include repeated Gly-Pro-Hyp sequences and / or repeated Gly-Pro-Pro sequences, preferably, the N1 and N2 include at least 3 repeated Gly-Pro-Hyp sequences) can mimic the structure and function of natural collagen, effectively promote the adhesion, proliferation and migration of HFF-1 cells, accelerate the density, chromaticity, moisture and transepidermal water loss of UV-damaged skin to restore to a healthy skin state, and promote the restoration of the original structure and function of each layer of damaged skin, and its effect is significantly better than that of hydrolyzed collagen peptides.

[0124] The above description is only the details of individual exemplary implementation cases of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to specific preparation conditions during actual application, and is not intended to limit the present invention. Anything within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A highly active triple-helix mini collagen for repairing skin photodamage, characterized in that: The polypeptide sequence of the highly active triple-helical mini collagen is: N1-GFOGER-N2; Wherein, the GFOGER is Gly-Phe-Hyp-Gly-Glu-Arg; The N1 and N2 contain at least 3 repeated Gly-Xaa-Yaa and / or Gly-Xaa-Xaa; the Xaa and Yaa are any different amino acids.

2. The highly active triple-helix mini collagen according to claim 1, characterized in that: The Xaa is Pro; the Yaa is Hyp.

3. The highly active triple-helix mini collagen according to claim 2, characterized in that: The N1 and N2 are (Gly-Pro-Hyp) n or (Gly-Pro-Pro) n , wherein n is any integer greater than or equal to 3.

4. The highly active triple-helix mini collagen according to claim 3, characterized in that: Both N1 and N2 are (Gly-Pro-Hyp) n , wherein n is any integer greater than or equal to 3.

5. The highly active triple-helix mini collagen according to claim 4, characterized in that: The n is 3, 4 or 5.

6. The highly active triple-helix mini collagen according to claim 5, characterized in that: The polypeptide sequence of the highly active triple-helical mini collagen is: (Gly-Pro-Hyp)3GFOGER(Gly-Pro-Hyp)3, or (Gly-Pro-Hyp)3GFOGER(Gly-Pro-Hyp)4, or (Gly-Pro-Hyp)4GFOGER(Gly-Pro-Hyp)4, or (Gly-Pro-Hyp)5GFOGER(Gly-Pro-Hyp)5.

7. The highly active triple-helix mini collagen according to claim 6, characterized in that: The amino acid sequence of the highly active triple-helix mini collagen is: (Gly-Pro-Hyp)5GFOGER(Gly-Pro-Hyp)5.

8. Use of the highly active triple-helix mini collagen as described in any one of claims 1 to 7 in the preparation of skin damage repair products.

9. The use according to claim 8, characterized in that The skin damage is skin photodamage.

10. The use according to claim 9, characterized in that The products for repairing skin photodamage include cosmetics, medical beauty products, and medicines.

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