A recombinant type IV humanized collagen and its application

Abstract

This invention belongs to the field of biomaterials, specifically relating to a recombinant type IV humanized collagen and its applications. This invention provides a recombinant type IV humanized collagen whose amino acid sequence comprises 10 repeating units directly linked together. Each repeating unit contains the amino acid sequence shown in SEQ ID NO. 1. The recombinant type IV humanized collagen used in this invention can achieve skin anti-wrinkle, firming, and repair effects.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a recombinant type IV humanized collagen and its applications. Background Technology

[0002] The basement membrane is an extremely thin sheet of extracellular matrix, approximately 100 nm thick, and is a specialized extracellular matrix located beneath epithelial cells, endothelial cells, peripheral nerve axons, adipocytes, and muscle cells. Type IV collagen is the main component maintaining the morphology of the basement membrane. In its N-terminal region, it forms tetramers cross-linked by disulfide bonds, while in its spherical form, it associates end-to-end. This bonding between the N-terminal and C-terminal regions constructs a two-dimensional network structure, constituting the main structure of the basement membrane. The type IV collagen network is essential for maintaining the basement membrane structure and provides a scaffold for other collagens and laminins.

[0003] In young skin, the basement membrane maintains homeostasis between the epidermis and dermis, ensuring hydration, suppleness, and elasticity. However, when exposed to external factors such as UV radiation, excessively dry air, or over-cleansing, or due to accelerated aging, the levels of type IV collagen and laminin, key components of the basement membrane, decrease, and they degrade and alter, disrupting the basement membrane's structure. This leads to reduced skin hydration and elasticity, abnormal keratin shedding, and loss of skin firmness and radiance, resulting in roughness, wrinkles, and other signs of aging. Therefore, type IV collagen is closely related to skin aging, and its reduction is a major factor contributing to wrinkles, dullness, loss of texture, and decreased elasticity.

[0004] Maintaining a healthy basement membrane structure is crucial for healthy skin. Timely repair and reconstruction of the basement membrane are essential for its health, providing timely basement membrane repair to the skin. For example, CN119925578A discloses recombinant type IV collagen (a truncated protein of human type IV collagen), peptides, and compositions using said recombinant type IV collagen and peptides as active ingredients, which possess activities for repairing the basement membrane and soothing redness. The recombinant type IV collagen and peptides have specific amino acid sequences, and when combined in a certain proportion, they exhibit synergistic effects in repairing the basement membrane and soothing redness. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] With age and the influence of external environmental factors such as UV radiation, skin exhibits signs of aging and sagging, leading to wrinkles. While existing technologies, such as those cited above, disclose products for repairing the skin's basal barrier, these studies are not yet comprehensive. Existing research on products has limitations, especially those applied to the skin's surface; their transdermal absorption and efficacy cannot be guaranteed. Furthermore, recombinant type IV collagen and peptides possess specific amino acid sequences, requiring further validation of their functionality and effectiveness. Currently, there is a significant market demand for products with good transdermal properties that achieve skin firming and anti-wrinkle repair effects.

[0007] During the in-depth study of the function of the recombinant type IV humanized collagen disclosed in ZL202311391711.1, it was unexpectedly discovered that it can achieve skin anti-wrinkle and firming repair and has good transdermal absorption performance.

[0008] Based on this, the purpose of the present invention is to provide the use of recombinant type IV humanized collagen with good transdermal absorption properties in skin anti-wrinkle and / or skin firming and / or skin repair.

[0009] Solution for solving the problem

[0010] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0011] [1]. A recombinant type IV humanized collagen; wherein the amino acid sequence of the recombinant type IV humanized collagen contains 10 repeating units, the repeating units are directly connected to each other, and the repeating units contain the amino acid sequence shown in SEQ ID NO.1.

[0012] [2]. The recombinant humanized type IV collagen according to [1], wherein the amino acid sequence of the recombinant humanized type IV collagen is as shown in SEQ ID NO.2.

[0013] [3]. Use of recombinant type IV humanized collagen as described in [1] or [2] in the preparation of products for skin anti-wrinkle and / or skin firming and / or skin repair.

[0014] [4]. According to the use described in [3], wherein the product for anti-wrinkle and / or skin firming and / or skin repair has transdermal absorption.

[0015] [5]. According to the use described in [3] or [4], wherein the skin anti-wrinkle and / or skin firming and / or skin repair includes any one or more of the following manifestations: improving the continuity of dermal-epidermal junction, increasing the thickness of the epidermal living cell layer, increasing the content of elastin, decreasing the content of matrix metalloproteinase 1, increasing the content of α-smooth muscle actin, increasing the content of epidermal morphogenetics, increasing the content of integrin interacting protein Kindlin-1, increasing the content of integrin β4, increasing the content of collagen fibers, and increasing the content of collagen.

[0016] [6]. According to the use described in [5], the increase in collagen content includes increasing the content of any one or more of Collagen I, Collagen III, Collagen V, Collagen VI, Collagen VII, Collagen XVII and Collagen XVIII.

[0017] [7]. Use according to any one of [3]-[6], wherein the product is selected from cosmetics, pharmaceuticals and medical devices;

[0018] Preferably, the product is a biological dressing, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material, a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, or a pharmaceutical excipient.

[0019] Preferably, the product comprises a pharmaceutically and / or cosmetically acceptable carrier;

[0020] Preferably, the product is a solid, liquid, or gel at room temperature;

[0021] Preferably, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation;

[0022] Preferably, the product is a product for topical application; more preferably, the topical application is a topical application to the skin; even more preferably, the topical application is a topical transdermal application to the facial skin.

[0023] [8]. Use according to any one of [3]-[7], wherein the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.3 ppm.

[0024] [9]. A product having the effects of anti-wrinkle and / or skin firming and / or skin repair, wherein the product comprises recombinant type IV humanized collagen; wherein the amino acid sequence of the recombinant type IV humanized collagen comprises 10 repeating units, the repeating units being directly connected to each other, and the repeating units comprising the amino acid sequence shown in SEQ ID NO.1;

[0025] Preferably, the amino acid sequence of the recombinant type IV humanized collagen is as shown in SEQ ID NO.2;

[0026] Preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.3 ppm;

[0027] Preferably, the product having anti-wrinkle and / or skin-firming and / or skin-repairing effects is transdermal absorbable.

[0028]

[10] . The product according to [9], wherein the product is selected from cosmetics, pharmaceuticals and medical devices;

[0029] Preferably, the product is a biological dressing, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material, a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, or a pharmaceutical excipient.

[0030] Preferably, the product comprises a pharmaceutically and / or cosmetically acceptable carrier;

[0031] Preferably, the product is a solid, liquid, or gel at room temperature and pressure;

[0032] Preferably, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation;

[0033] Preferably, the product is a product for topical application; more preferably, the topical application is a topical application to the skin; even more preferably, the topical application is a topical application to the facial skin.

[0034] The effects of the invention

[0035] This invention provides recombinant type IV humanized collagen and its use in the preparation of products for skin anti-wrinkle and / or skin firming and / or skin repair. The recombinant type IV humanized collagen used in this invention has excellent hydrophilicity and stability, its amino acid composition is 100% identical to the corresponding portions of the amino acid sequence of natural collagen, it does not cause immune rejection or allergic reactions when applied to the human body, and it has good permeability, allowing it to penetrate into the dermis layer of the skin. It can be widely used in the biopharmaceutical and cosmetic industries.

[0036] The recombinant type IV humanized collagen used in this invention exhibits excellent transdermal absorption properties, allowing it to cross the skin's epidermal barrier and act on deep skin tissues and cells. This greatly ensures the effectiveness of the recombinant type IV humanized collagen in anti-wrinkle, firming, and repairing effects on the skin during actual human use.

[0037] The recombinant type IV humanized collagen used in this invention can significantly repair the basement membrane and participate in the reconstruction of the basement membrane reticular structure. This is directly demonstrated by its ability to significantly alleviate the breakage of the dermal-epidermal junction structure and improve skin tissue morphology. Experimental data show that the recombinant type IV humanized collagen used in this invention can downregulate the content of matrix metalloproteinase 1 (MMP-1), and increase the content of α-smooth muscle actin, elastin, epidermal morphogenetics, integrin-interacting protein Kindlin-1, and integrin β4. It can also increase the content of various collagens, such as type I, type III, type V, type VI, type VII, type XVII, and type XVIII collagen, and increase the content of collagen fibers. The effect of recombinant type IV humanized collagen in increasing the expression levels of integrin-interacting protein Kindlin-1 and integrin β4 can strengthen the hemidesmosome junction between keratinocytes and the basement membrane, enhancing the mechanical anchoring force of the dermis. The enhancement of epidermal morphogenetic protein (EMP) expression levels by recombinant human type IV collagen can influence the integrity of the dermal-epidermal junction by regulating the secretion or assembly of basement membrane-related molecules (such as type IV collagen). The inhibition of matrix metalloproteinase 1 (MMP1) by recombinant human type IV collagen can slow down its degradation of type IV collagen in the basement membrane region, helping to maintain the structural integrity of the basement membrane. The promotion of type XVII collagen synthesis by recombinant human type IV collagen facilitates the connection between the basement membrane and keratinocytes, and also aids in the layered assembly of the basement membrane's compact and stratum lucidum. Simultaneously, the enhancement of other collagen and elastin levels by recombinant human type IV collagen can support the dermal structure and indirectly protect the basement membrane. Recombinant human type IV collagen can also promote myofibroblast differentiation, indirectly repairing the basement membrane and accelerating the skin repair process. Recombinant type IV humanized collagen further promotes the deposition of elastic fibers and type III collagen in the dermis, forming a stable mechanical support environment. This not only enables the regeneration and repair of the three-dimensional structure of the basement membrane, but also further achieves anti-wrinkle, firming and repair effects on the skin.

[0038] Furthermore, these experimental data are based on ex vivo skin tissue and 3D full-thickness skin models. This data was obtained from tests using an ex vivo skin model. Compared to existing cell detection models, ex vivo skin tissue testing more closely resembles the structure of real skin, possessing a complete stratum corneum and better simulating the skin barrier function, thus more realistically reflecting skin responses—something that single-type cell detection models cannot match. 3D full-thickness skin model. This invention can simulate the barrier function, cell layering, and intercellular interactions of human skin, while also possessing a functional stratum corneum, thus accurately mimicking the skin barrier. By utilizing these models in conjunction with molecular biology techniques, this invention enables more accurate and comprehensive efficacy evaluation. Attached Figure Description

[0039] Figure 1 Example 1: Multi-point Raman scanning spectrum of the entire skin layer.

[0040] Figure 2 Example 1: Total Intensity Statistics Chart.

[0041] Figure 3 Example 2: Transmission electron microscopy observation results of the continuity of the epidermal-dermal junction; where the arrows indicate the DEJ structure, and the NC group marks the damaged area.

[0042] Figure 4 Example 2: Tissue morphology test results.

[0043] Figure 5 Example 2: Bar chart showing the results of epidermal cell layer thickness detection.

[0044] Figure 6 Example 2: Elastin immunohistochemical detection results.

[0045] Figure 7 Example 2: Bar chart of average relative integrated optical density (IOD) of Elastin.

[0046] Figure 8 Example 2: MMP-1 immunohistochemical detection results.

[0047] Figure 9 Example 2: Bar chart of average relative integrated optical density (IOD) of MMP-1.

[0048] Figure 10 Example 2: α-SMA immunofluorescence detection results.

[0049] Figure 11 Example 2: Bar chart of average relative integrated optical density (IOD) of α-SMA.

[0050] Figure 12 Example 2: Syntaxin-2 immunofluorescence detection results.

[0051] Figure 13 Example 2: Bar chart of the average relative integrated optical density (IOD) of Syntaxin-2.

[0052] Figure 14Example 2: Results of Collagen XVII immunofluorescence assay.

[0053] Figure 15 Example 2: Histogram of relative integrated optical density (IOD) / area average of Collagen XVII.

[0054] Figure 16 Example 3: Tissue morphology test results.

[0055] Figure 17 Example 3: Bar chart of epidermal cell layer thickness detection results.

[0056] Figure 18 Example 3: Results of collagen fiber staining.

[0057] Figure 19 Example 3: Bar chart of the average relative area of ​​collagen fibers.

[0058] Figure 20 Example 3: Results of Collagen I immunofluorescence detection.

[0059] Figure 21 Example 3: Bar chart of average relative integrated optical density (IOD) of Collagen I.

[0060] Figure 22 Example 3: Kindlin-1 immunofluorescence detection results.

[0061] Figure 23 Example 3: Bar chart of average relative integrated optical density (IOD) of Kindlin-1.

[0062] Figure 24 Example 3: Results of integrin β4 immunofluorescence detection.

[0063] Figure 25 Example 3: Bar chart of relative integrated optical density (IOD) / area average of integrin β4.

[0064] Figure 26 Example 4: Results of Collagen III immunofluorescence detection; The images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen III. The stronger the green fluorescence, the higher the Collagen III content.

[0065] Figure 27 Example 4: Bar chart of average relative integrated optical density (IOD) of Collagen III.

[0066] Figure 28Example 4: Results of Collagen V immunofluorescence detection; The images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen V. The stronger the green fluorescence intensity, the higher the Collagen V content.

[0067] Figure 29 Example 4: Histogram of Collagen V relative integrated optical density (IOD) / area average.

[0068] Figure 30 Example 4: Results of Collagen VI immunofluorescence detection; The images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen VI. The stronger the green fluorescence, the higher the Collagen VI content.

[0069] Figure 31 Example 4: Colagen VI relative integrated optical density (IOD) / average area histogram.

[0070] Figure 32 Example 4: Results of Collagen VII immunofluorescence detection; The images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen VII. The stronger the green fluorescence, the higher the content of Collagen VII.

[0071] Figure 33 Example 4: Histogram of relative integrated optical density (IOD) / area average of Collagen VII.

[0072] Figure 34 Example 4: Results of Collagen XVIII immunofluorescence detection; The images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen XVIII. The stronger the green fluorescence, the higher the content of Collagen XVIII.

[0073] Figure 35 Example 4: Histogram of Collagen XVIII relative integrated optical density (IOD) / area average. Detailed Implementation

[0074] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0075] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0076] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0077] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0078] In this invention, the numerical range referred to as “value A to value B”, “value A to value B”, and “value A above / below” refers to the range that includes the endpoint values ​​A and B.

[0079] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0080] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0081] In this invention, all unit names used are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0082] In this invention, "normal temperature and pressure" refers to an ambient temperature of 25°C under standard atmospheric pressure.

[0083] In this invention, the term "identity" refers to the percentage of identical amino acids among two or more polypeptides. Sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions containing identical amino acid residues in the aligned polypeptides, comparing this to the number of positions containing different amino acid residues in the aligned polypeptides. Sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptide.

[0084] In this invention, amino acid addition can refer to adding 1, 2 or 3 or more amino acids at any position at the C-terminus, N-terminus or between the C-terminus and N-terminus of the amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0085] In this invention, amino acid substitution refers to the replacement of an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be conserved amino acid substitution, meaning that compared to the original amino acid sequence, several amino acids are replaced by amino acids with similar or related properties to form a peptide (conserved variant peptide). For example, these conserved variant peptides can be generated based on the following amino acid substitutions: substitution of Ala with Val, Leu, or Ile; substitution of Arg with Lys, Gln, Asn, or His; substitution of Asn with Gln, His, Lys, or Arg; substitution of Asp with Glu or Asn; substitution of Cys with Ser or Ala; substitution of Gln with Asn or Glu; substitution of Glu with Asp or Gln; substitution of Gly with Ala; substitution of His with Asn, Lys, Gln, or Arg; substitution of Cys with Leu, Met, Ala, Val, Phe, or leucine. Substitutions include: Ile substitutions; substitutions of Leu with Ile, Met, Ala, Val, Phe, or leucine; substitutions of Lys with Asn, Gln, or Arg; substitutions of Met with Ile, Leu, or Phe; substitutions of Phe with Leu, Val, Ile, Ala, or Tyr; substitutions of Pro with Ala; substitutions of Ser with Thr; substitutions of Thr with Ser or Val; substitutions of Trp with Phe or Tyr; substitutions of Tyr with Trp, Phe, Thr, or Ser; and substitutions of Val with Phe, Ala, Met, Ile, Leu, or leucine. Amino acid substitutions can also be non-conserved amino acid substitutions.

[0086] In this invention, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0087] In this invention, "hybridization" refers to the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without nonspecific binding to non-complementary sequences. The terms "medium stringent conditions," "medium-high stringent conditions," "high stringent conditions," or "very high stringent conditions" used in this invention describe the conditions for nucleic acid hybridization and washing. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0088] In this invention, the terms “application,” “giving,” and “treatment” when applied to animals, humans, experimental subjects, cells, tissues, or organs refer to the contact between exogenous drugs, therapeutic agents, diagnostic agents, medical devices, cosmetics, or compositions and animals, humans, experimental subjects, cells, tissues, or organs.

[0089] In this invention, the term "effective amount" includes an amount sufficient to improve or prevent physiological symptoms or conditions. The effective amount for a particular subject may vary depending on factors such as the symptoms to be improved, the subject's overall health, the route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0090] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0091] I. Recombinant Type IV Humanized Collagen

[0092] The recombinant type IV humanized collagen described in this invention is described in the patent application number 202311391711.1 entitled "Preparation method of biosynthetic human structural material type IV collagen", the contents of which are incorporated herein by reference.

[0093] In some embodiments, the amino acid sequence of the recombinant type IV humanized collagen comprises 10 repeating units directly linked together, and the repeating units comprise the amino acid sequence shown in SEQ ID NO.1.

[0094] SEQ ID NO. 1: GAKGDKGSKGEVGFPGLAGSPGIPGSKGEQ.

[0095] In some embodiments, the amino acid sequence of the recombinant type IV humanized collagen includes any one of the following (i)-(iv):

[0096] (i) A sequence as shown in SEQ ID NO.2;

[0097] (ii) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.2, and retaining the anti-wrinkle and / or skin-tightening and / or skin-repairing effects of the sequence shown in SEQ ID NO.2;

[0098] (iii) A sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO.2, and which retains the anti-wrinkle and / or skin firming and / or skin repair effects of the sequence shown in SEQ ID NO.2;

[0099] (iv) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.2 under stringent conditions, and the amino acid sequence retaining the anti-wrinkle and / or skin-tightening and / or skin-repairing effects of the sequence shown in SEQ ID NO.2, wherein the stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent.

[0100] SEQ ID NO.2: GAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPG IPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGS KGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQ.

[0101] In some embodiments, the amino acid sequence of the recombinant type IV humanized collagen includes the sequence shown in SEQ ID NO. 2.

[0102] In some embodiments, the amino acid sequence of the recombinant type IV humanized collagen is as shown in SEQ ID NO.2.

[0103] The recombinant type IV humanized collagen of the present invention can be prepared by conventional methods in the art. For example, it can be prepared by the following steps: (1) construction of Escherichia coli genetically engineered bacteria; (2) fermentation culture of Escherichia coli genetically engineered bacteria; (3) induction expression of recombinant type IV humanized collagen; and (4) purification and optional enzymatic digestion of recombinant type IV humanized collagen.

[0104] II. Uses of Recombinant Type IV Humanized Collagen

[0105] This invention provides the use of the recombinant type IV humanized collagen described in Part I in the preparation of products for skin anti-wrinkle and / or skin firming and / or skin repair.

[0106] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin anti-wrinkle purposes.

[0107] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin firming.

[0108] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin repair.

[0109] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin anti-wrinkle and skin repair.

[0110] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin firming and skin repair.

[0111] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for skin anti-wrinkle, skin firming, and skin repair.

[0112] In some embodiments, the recombinant type IV humanized collagen of the present invention is used for skin anti-wrinkle and / or skin firming and / or skin repair.

[0113] In some embodiments, the present invention provides a method for skin anti-wrinkle and / or skin firming and / or skin repair, the method comprising the step of applying an effective amount of the recombinant type IV humanized collagen to an individual to whom it is needed.

[0114] In some embodiments, the product for anti-wrinkle and / or firming and / or repairing skin has transdermal absorption properties.

[0115] In some implementations, the skin anti-wrinkle and / or skin firming and / or skin repair includes dermal-epidermal junction repair.

[0116] In some embodiments, the present invention provides the use of the recombinant type IV humanized collagen in the preparation of products for dermal-epidermal junction repair.

[0117] In some embodiments, the skin anti-wrinkle and / or skin firming and / or skin repair includes any one or more of the following: improving the continuity of dermal-epidermal junction, increasing the thickness of the epidermal living cell layer, increasing the content of elastin, decreasing the content of matrix metalloproteinase 1, increasing the content of α-smooth muscle actin, increasing the content of epidermal morphogenetic proteins, increasing the content of integrin-interacting protein Kindlin-1, increasing the content of integrin β4, increasing the content of collagen fibers, and increasing the content of collagen.

[0118] In some embodiments, the skin anti-wrinkle and / or skin firming and / or skin repair simultaneously includes improving the continuity of dermal-epidermal junction, increasing epidermal thickness, increasing elastin content, decreasing matrix metalloproteinase 1 content, increasing α-smooth muscle actin content, increasing epidermal morphogenetic protein content, increasing integrin-interacting protein Kindlin-1 content, increasing integrin β4 content, increasing collagen fiber content, and increasing collagen content.

[0119] In some embodiments, increasing collagen content includes increasing the content of any one or more collagens from Collagen I, Collagen III, Collagen V, Collagen VI, Collagen VII, Collagen XVII, and Collagen XVIII.

[0120] In some embodiments, increasing collagen content includes increasing the content of Collagen I, Collagen III, Collagen V, Collagen VI, Collagen VII, Collagen XVII, and Collagen XVIII.

[0121] In some implementations, the product is selected from cosmetics, pharmaceuticals, and medical devices.

[0122] In some implementations, the product is a biological dressing, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material, a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, or a pharmaceutical excipient.

[0123] In some implementations, the product comprises a pharmaceutically and / or cosmetically acceptable carrier.

[0124] In some embodiments, the product is a solid, liquid, or gel at room temperature and pressure.

[0125] In some embodiments, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation.

[0126] In some embodiments, the product is a topical application product; preferably, the topical application is a topical application to the skin; more preferably, the topical application is a topical application to the facial skin.

[0127] In some embodiments, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.3 ppm; for example, the content of the recombinant type IV humanized collagen in the product may be about 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm, 40 ppm, 45 ppm, or 50 ppm, etc.; preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.5 ppm; more preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 5 ppm; even more preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 20 ppm.

[0128] III. Products containing the aforementioned recombinant type IV humanized collagen

[0129] The present invention provides a product having the effects of anti-wrinkle and / or skin firming and / or skin repair, comprising the recombinant type IV humanized collagen described in Part I.

[0130] In some implementations, the product is selected from cosmetics, pharmaceuticals, and medical devices.

[0131] In some implementations, the product is a biological dressing, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filler material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material, a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, or a pharmaceutical excipient.

[0132] The "biological dressing" described in this invention refers to an artificial material used to cover wounds, promoting wound healing, and may possess hemostatic, anti-infection, and tissue regeneration-promoting functions. The "human biomimetic material" described in this invention refers to a material that simulates the structure and function of natural tissues. The "plastic and cosmetic material" described in this invention refers to a material used to repair defects or improve appearance, possessing bioinertness, plasticity, and long-term stability. The "organoid culture material" described in this invention refers to a three-dimensional matrix material supporting organoid growth, providing a microenvironment close to that in vivo to promote cell self-organization. The "cardiovascular stent material" described in this invention refers to a device material used to support narrowed / occluded blood vessels, possessing both mechanical strength and endothelialization-promoting capabilities. The "coating material" described in this invention refers to a functional material coated on the surface of medical devices, used to improve the biocompatibility or durability of the substrate. The "tissue injection filler material" described in this invention refers to a material injected to fill soft tissue depressions or wrinkles, possessing injectability and low immunogenicity. The "ophthalmic material" described in this invention refers to a material used for intraocular or ocular surface repair. The "gynecological and obstetric biomaterials" described in this invention refer to materials applied to the female reproductive system, which possess tissue flexibility and anti-infection properties. The "nerve repair and regeneration materials" described in this invention refer to scaffold materials that promote nerve regeneration, often loaded with neurotrophic factors to guide the directional growth of nerve cells. The "liver tissue materials" described in this invention refer to materials used for liver injury repair or in vitro liver model construction, simulating the unique metabolic functions of the liver. The "vascular repair and regeneration materials" described in this invention refer to materials used for vascular replacement or regeneration, possessing anticoagulant properties and dynamic compliance. The "3D-printed artificial organ biomaterials" described in this invention refer to materials used to construct organ substitutes through 3D printing technology. The "cosmetic raw materials" described in this invention refer to active or matrix ingredients in skincare or makeup products. The "pharmaceutical excipients" described in this invention refer to inactive ingredients in pharmaceutical preparations.

[0133] In some embodiments, the product comprises a pharmaceutically and / or cosmetically acceptable carrier. Such carriers include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, etc.

[0134] In some embodiments, the product is a solid, liquid, or gel at room temperature and pressure.

[0135] In some embodiments, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation.

[0136] In some embodiments, the product is a topical application product; preferably, the topical application is a topical application to the skin; more preferably, the topical application is a topical application to the facial skin.

[0137] In some embodiments, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.3 ppm; for example, the content of the recombinant type IV humanized collagen in the product may be about 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm, 40 ppm, 45 ppm, or 50 ppm, etc.; preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.5 ppm; more preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 5 ppm; even more preferably, the content of the recombinant type IV humanized collagen in the product is greater than or equal to 20 ppm.

[0138] Example

[0139] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0140] The amino acid sequence of the recombinant type IV humanized collagen (recombinant human collagen Col_4) used in this invention is shown in SEQ ID NO.2.

[0141] The preparation and production of this recombinant type IV humanized collagen are described in the prior invention patent ZL202311391711.1 of the applicant.

[0142] Example 1

[0143] This embodiment uses an in vitro porcine skin-Franz cell system. Recombinant type IV humanized collagen (Col_4) was applied to the skin for 2 hours, 6 hours, and 24 hours. Single-spectral signals of Col_4 in the skin were collected using confocal Raman spectroscopy. Multi-point Raman scanning spectra of the entire skin layer and reconstructed Raman pseudocolor images after large-scale scanning were obtained for skin permeability analysis to evaluate the in vitro permeability of Col_4. If the intensity of the sample signal in all skin layers (especially the deep layers) increases with time, it indicates that the sample has transdermal absorption capacity and good permeability after application to the skin. If the signal is only present in the stratum corneum or superficial epidermis (e.g., no significant change after 2 hours), it suggests that the sample's transdermal capacity is insufficient, remaining only on the skin surface without transdermal absorption.

[0144] 1. Testing Method

[0145] 1) Test Grouping

[0146] The test group information is shown in Table 1 below:

[0147] Table 1 Test Group Information

[0148]

[0149] 2) Penetration Test

[0150] ① Skin fixation: Take out the pig skin stored at 20℃, select skin with no hair, no damage, and no wrinkles, cut it into 3.5cm×3.5cm pieces, rinse repeatedly with PBS buffer for 5s, and fix the skin taut between the supply chamber and the receiving chamber of the Franz diffusion cell;

[0151] ② Sample addition: Add 8.5L of PBS buffer to the receiving chamber to ensure close contact between the dermal layer of the skin and the receiving solution. Add 300μL of sample to distribute the sample evenly on the skin surface. Seal the supply chamber with sealing film.

[0152] ③ Infiltration: Place the Franz diffusion cell in the TK-12D transdermal absorption diffusion instrument, and simultaneously turn on the electromagnetic stirrer at a speed of 300 rpm, while maintaining a constant temperature water bath of 32±1℃.

[0153] ④ Sample collection: Rinse the skin surface repeatedly with PBS buffer for 15 seconds, and freeze at -80℃;

[0154] ⑤ Frozen sections: The skin was frozen and embedded into sections with a thickness of 16 μm. The sections were then mounted on gold-plated quartz slides for Raman spectroscopy.

[0155] 3) Raman spectroscopy detection experiment

[0156] ① The confocal Raman spectrometer was calibrated using a silicon wafer before data acquisition;

[0157] ② Place the gold-plated quartz slide with frozen sections under a microscope and record the white light image of the study area. Measure its single spectrum using a Raman spectrometer.

[0158] ③ Start scanning the sample from the point where the measured characteristic peak is prominent and the noise is relatively small, and obtain the Raman spectrum dataset.

[0159] 4) Statistical analysis of results

[0160] Raman spectral data were preprocessed using Witc data analysis software. The instrument response in the spectrum was corrected by subtracting black current, detector response, and optical system signal. Noise reduction was achieved using a 5-point Savitzky-Golay smoothing method, and baseline correction was performed using a 5th-order polynomial function, resulting in a clearer spectrum.

[0161] Using Origin software, we analyzed the single-spectrum dataset, plotted multi-point Raman scan spectra of the entire skin layer, and performed semi-quantitative analysis of the characteristic peaks of the substances.

[0162] GraphPad Prism was used for plotting, and the results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using the t-test method, and the statistical analysis was two-tailed. Significance compared to groups BC is indicated by *, with a p-value < 0.05 (*) indicating a significant difference and a p-value < 0.01 (**) indicating a highly significant difference.

[0163] 2. Test Results

[0164] 1) Full-thickness multi-point Raman scanning spectrum of skin

[0165] Full-thickness multi-point Raman spectroscopy of skin as shown in the image. Figure 1 As shown.

[0166] 2) Sample permeation analysis

[0167] The results of the sample permeation analysis are shown in Table 2 below. Figure 2 As shown.

[0168] Table 2 Summary of Total Integral Strength Results

[0169]

[0170] The permeability of Col_4 samples in the skin was detected by confocal Raman spectroscopy. Compared with group BC, after 2 hours of skin treatment, Col_4 mainly penetrated into the epidermis, with no significant difference in total integrated intensity and a permeability of 1.09%. After 6 hours and 24 hours of skin treatment, Col_4 could penetrate into the dermis, with a significant increase in total integrated intensity (P-value < 0.01), and permeability of 11.51% and 12.78%, respectively. The increased permeability indicates that Col_4 exhibits permeation behavior in the skin, that is, it has good transdermal absorption properties and can cross the skin epidermal barrier to act on deep skin tissues and cells.

[0171] 3. Conclusion

[0172] The skin has a multi-layered structure, with the outermost dense and hydrophobic stratum corneum providing the main barrier function. This effectively resists the invasion of foreign substances such as chemicals and pathogens, and is also the biggest obstacle to transdermal penetration. If the active ingredients in skin products remain only on the skin's surface and cannot penetrate the stratum corneum barrier, their efficacy and effects are greatly limited.

[0173] In this embodiment, based on the isolated pig skin-Franz cell system, recombinant type IV humanized collagen (Col_4) was applied to the skin for 2h, 6h, and 24h. Single-spectral signals of Col_4 in the skin were collected using a confocal Raman spectrometer to obtain multi-point Raman scanning spectra of the entire skin layer and reconstructed Raman pseudocolor images after large-scale scanning for skin permeability analysis, thus evaluating the in vitro permeability of Col_4.

[0174] Experimental results are as follows Figure 1 , Figure 2 As shown in Table 2, the experimental results show that after the sample is applied to the skin, the intensity of the sample signal in each layer of the skin (especially the deep layer) increases with time, and it is not only present in the stratum corneum or the superficial epidermis, indicating that the sample has transdermal absorption capacity and good penetration performance after being applied to the skin.

[0175] Col_4 penetrated into the dermis after 6 and 24 hours of application to the skin, with a significant increase in total intensity (P-value < 0.01) and permeability of 11.51% and 12.78%, respectively. This increased permeability indicates that Col_4 exhibits transdermal absorption, crossing the epidermal barrier and acting on deeper skin tissues and cells. This greatly ensures the effectiveness of the recombinant type IV humanized collagen in anti-wrinkle, firming, and repairing effects on the skin in actual human use.

[0176] Example 2

[0177] In this embodiment, ex vivo skin tissue (provided by Guangdong Boxi Biotechnology Co., Ltd.) was irradiated with a combination of UVA and UVB. The effects of Col_4 on firming, anti-wrinkle and repair were evaluated by detecting changes in the continuity of the dermal-epidermal junction (DEJ), tissue morphology, and the contents of elastin, collagen XVII, matrix metalloproteinase 1 (MMP-1), α-smooth muscle actin (α-SMA) and epidermal morphogenetic pigment 2.

[0178] The dermal-epidermal junction (DEJ) is a key structure connecting the epidermis and dermis, responsible for maintaining the overall stability and elasticity of the skin. Tissue morphology reflects the integrity and arrangement of the skin structure. Elastin is a crucial component for maintaining skin elasticity and resilience, giving the skin its resilience. Matrix metalloproteinase-1 (MMP-1) is an enzyme that breaks down collagen; excessive activity can lead to collagen degradation and skin aging. Inhibiting MMP-1 activity can reduce collagen degradation and maintain skin firmness. α-Smooth muscle actin (α-SMA) is a marker of fibroblast activation and participates in collagen synthesis and skin repair. Type XVII collagen is an important component of the DEJ, involved in maintaining the connection between the epidermis and dermis. Syntaxin-2, a protein associated with vesicle transport and membrane fusion, has an expression level related to skin barrier repair. These expression factors reflect the skin's structure, elasticity, and repair capacity from different perspectives, providing a scientific basis for evaluating the firming, anti-wrinkle, and repairing effects of collagen.

[0179] 1. Testing Method

[0180] 1) Test Grouping

[0181] The test group information is shown in Table 3 below:

[0182] Table 3 Test Group Information

[0183]

[0184] 2) Tissue processing: Freshly obtained skin tissue was immersed in 75% alcohol for 30 seconds, followed by washing three times with sterile PBS buffer; after completion, the skin was cut into 24±2 mm sections. 2 The tissue block, with the epidermis facing up and the dermis facing down, was placed in a culture mold. The culture mold was then transferred into a 6-well plate, and 3.7 mL of culture medium was added to each well. The plate was then incubated at 37°C in a 5% CO2 incubator, with the medium changed daily.

[0185] 3) Drug administration: Two days after model culture, irradiation and drug administration were initiated according to the test groups and corresponding treatment conditions in Table 3. The irradiation dose was UVA (30 J / cm²). 2 ) and UVB (50mJ / cm 2 The samples were continuously irradiated for 4 days, with fresh culture medium added after each irradiation and drug administration administered. The positive control group received subsurface drug administration, while the test samples received surface drug administration. After 4 days of continuous irradiation, irradiation was stopped, and the excised skin tissue was cultured for another 3 days, during which drug administration was continued.

[0186] 4) Transmission Electron Microscope (TEM) photography: After cleaning, fix with 2.5% glutaraldehyde fixative. After 24 hours of fixation, prepare the sample, embed and section it, and then take pictures using a transmission electron microscope.

[0187] 5) Tissue morphology test: Skin tissue for testing was taken, fixed with 4% paraformaldehyde, and after 24 hours of fixation, H&E staining was performed, and the tissue was photographed and observed under a microscope. The images were collected and analyzed.

[0188] 6) Immunohistochemical test: Skin tissue for testing was taken, fixed with 4% paraformaldehyde, and after 24 hours of fixation, immunohistochemical detection was performed. The images were photographed and analyzed under a microscope.

[0189] 7) Immunofluorescence test: Skin tissue for testing was taken, fixed with 4% paraformaldehyde, and after 24 hours of fixation, immunofluorescence detection was performed. The images were photographed and analyzed under a fluorescence microscope.

[0190] 8) Calculation of enhancement rate and inhibition rate:

[0191]

[0192] 9) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as mean ± standard deviation (Mean ± SD). t-tests were used for comparisons between groups. All statistical analyses were two-tailed. Significance compared to group BC is indicated by *, with a p-value < 0.05 (*) indicating a significant difference and a p-value < 0.01 (**) indicating a highly significant difference.

[0193] 2. Test Results

[0194] 1) Continuity of dermal-epidermal junction (DEJ)

[0195] The results of the dermal junction (DEJ) continuity test are as follows: Figure 3 As shown, images were taken using a transmission electron microscope (Hitachi). Compared to the BC group, the DEJ structure in the NC group showed significant fragmentation, indicating that the stimulation conditions in this test were effective. Compared to the NC group, the DEJ structure fragmentation in the PC group was significantly alleviated, indicating that the positive control in this test was effective. Compared to the NC group, the DEJ structure fragmentation in the Col_4 group was significantly alleviated at all concentrations.

[0196] 2) Organizational Form

[0197] Table 4 Summary of Epidermal Viable Cell Layer Thickness Detection Results

[0198]

[0199] Tissue morphology test results as follows Figure 4 , Figure 5 As shown in Table 4, compared with the BC group, the epidermal viable cell layer thickness in the NC group was significantly decreased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the epidermal viable cell layer thickness in the PC group was significantly increased, indicating that the positive control in this test was effective. Compared with the NC group, the epidermal viable cell layer thickness in the Col_4 group was significantly increased, with increases of 38.46%, 46.37%, and 64.74%, respectively. Furthermore, the epidermal viable cell layer thickness in the Col_4 group was higher or significantly higher than that in the PC group. Additionally, the concentrations of VC and VE administered in the PC group (100 μg / ml + 7 μg / ml) were 200 to 2 times higher than those in the sample groups Col_4-0.5ppm (0.5 μg / ml), Col_4-5ppm (5 μg / ml), and Col_4-50ppm (50 μg / ml), further demonstrating that Col_4 was significantly more effective than the PC group in increasing epidermal viable cell layer thickness.

[0200] 3) Elastin content

[0201] Table 5 Summary of Elastin Immunohistochemical Analysis Results

[0202]

[0203] The results of the elastin content test are as follows: Figure 6 , Figure 7 As shown in Table 5, compared with the BC group, the Elastin content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Elastin content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Elastin content in the Col_4 group increased significantly, with increase rates of 97.37%, 105.26%, and 192.11%, respectively. Furthermore, the Elastin content in the Col_4 group was higher or significantly higher than that in the PC group. Given that the drug concentration in the PC group was significantly higher than that in the sample group, this further demonstrates that the Col_4 group was significantly more effective than the PC group in increasing Elastin content.

[0204] 4) Matrix metalloproteinase 1 (MMP-1) content

[0205] Table 6 Summary of MMP-1 Immunohistochemical Analysis Results

[0206]

[0207] The results of the matrix metalloproteinase 1 (MMP-1) content test are as follows: Figure 8 , Figure 9 As shown in Table 6, compared with the BC group, the MMP-1 content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the MMP-1 content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the MMP-1 content in the Col_4 groups was significantly decreased, with inhibition rates of 22.10%, 71.06%, and 79.61%, respectively. Furthermore, the effect of Col_4 in reducing MMP-1 content showed a certain dose-dependent effect. When the Col_4 concentration in the sample groups reached 50 ppm (50 μg / ml, approximately 0.5 times the concentration in the PC group), the inhibition rate of Col_4 on MMP-1 content was higher than that in the PC group, proving that Col_4 was significantly more effective than the PC group in reducing MMP-1 content.

[0208] 5) α-Smooth muscle actin (α-SMA) content

[0209] Table 7 Summary of α-SMA Immunofluorescence Analysis Results

[0210]

[0211] The results of the α-smooth muscle actin (α-SMA) content test are as follows: Figure 10 , Figure 11 As shown in Table 7, compared with the BC group, the α-SMA content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the α-SMA content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the α-SMA content in the Col_4 groups increased significantly, with increase rates of 15.38%, 57.69%, and 150.00%, respectively. Furthermore, the effect of Col_4 in increasing α-SMA content showed a certain dose-dependent effect; when the Col_4 concentration in the sample groups reached 50 ppm (50 μg / ml, approximately 0.5 times the concentration in the PC group), the increase rate of Col_4 in α-SMA content was basically the same as that in the PC group. Meanwhile, the concentrations of VC and VE in the PC group (100 μg / ml + 7 μg / ml) were approximately 200 and 20 times higher than those in the sample groups (Col_4-0.5 ppm (0.5 μg / ml) and Col_4-5 ppm (5 μg / ml), respectively. However, the increase rate of α-SMA content in the PC group was only 10.0 times and 2.7 times that of the sample groups (Col_4-0.5 ppm and Col_4-5 ppm, respectively). This demonstrates that Col_4 is significantly more effective than the PC group in increasing α-SMA content.

[0212] 6) Syntaxin-2 content

[0213] Table 8 Summary of Syntaxin-2 Immunofluorescence Analysis Results

[0214]

[0215] The results of the epidermal morphogenetic element (Syntaxin-2) content test are as follows: Figure 12 , Figure 13 As shown in Table 8, compared with the BC group, the Syntaxin-2 content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Syntaxin-2 content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Syntaxin-2 content in the Col_4 groups increased significantly, with increase rates of 2050.00%, 3250.00%, and 1600.00%, respectively. Furthermore, the effect of Col_4 in increasing Syntaxin-2 content showed a trend of first increasing and then decreasing, i.e., there was a peak. When the Col_4 concentration in the sample groups reached 5 ppm (5 μg / ml, approximately 0.05 times the concentration in the PC group), the increase rate of Syntaxin-2 content by Col_4 was higher than that in the PC group.

[0216] 7) Collagen XVII content

[0217] Table 9 Summary of Immunofluorescence Analysis Results of Collagen XVII

[0218]

[0219] The test results for the content of type XVII collagen are as follows: Figure 14 , Figure 15 As shown in Table 9, compared with the BC group, the Collagen XVII content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen XVII content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen XVII content in the Col_4 groups increased significantly, with increase rates of 35.90%, 53.85%, and 120.51%, respectively. Furthermore, the effect of Col_4 in increasing Collagen XVII content showed a certain dose-dependent effect. When the Col_4 concentration in the sample groups reached 50 ppm (50 μg / ml, approximately 0.5 times the concentration in the PC group), the increase rate of Col_4 in Collagen XVII content was higher than that in the PC group, proving that Col_4 was significantly more effective than the PC group in increasing Collagen XVII content.

[0220] 3. Conclusion

[0221] Based on ex vivo skin tissue, compared with the control group, Col_4 at concentrations of 0.5ppm, 5ppm, and 50ppm significantly alleviated the dermal-epidermal junction (DEJ) structural breakage, and significantly increased the thickness of the epidermal viable cell layer, with increases of 38.46%, 46.37%, and 64.74%, respectively; the matrix metalloproteinase-1 (MMP-1) content was significantly decreased, with inhibition rates of 22.10%, 71.06%, and 79.61%, respectively; and α-smooth muscle actin (α- The contents of SMA (skin morphogenetic acid) increased significantly, with increases of 15.38%, 57.69%, and 150.00%, respectively; the contents of Syntaxin-2 (epidermal morphogenetic hormone) increased significantly, with increases of 2050.00%, 3250.00%, and 1600.00%, respectively; the contents of Elastin (elastin) increased significantly, with increases of 97.37%, 105.26%, and 192.11%, respectively; and the contents of Collagen XVII (type XVII) increased significantly, with increases of 35.90%, 53.85%, and 120.51%, respectively.

[0222] Col4, a protein homologous to type IV collagen, a core collagen component of the basement membrane, participates in the reconstruction of the basement membrane reticular structure, improving tissue morphology and dermal-epidermal junction (DEJ) damage. Col4 downregulates matrix metalloproteinase-1 (MMP-1) levels, which can help slow its degradation of natural proteins such as type IV collagen in the basement membrane region, thus helping to maintain the structural integrity of the basement membrane. Col4 increases α-smooth muscle actin (α-SMA) levels, indicating that Col4 can promote myofibroblast differentiation, thereby indirectly repairing the basement membrane and accelerating the skin repair process. While increasing α-SMA expression, Col4 also significantly increases elastin levels. Simultaneously, Col4 promotes the synthesis of type XVII collagen, which helps in the connection between the basement membrane and keratinocytes, and also facilitates the layered assembly of the basement membrane's compact and stratum lucidum. Col_4 can increase the content of Syntaxin-2, thereby promoting the secretion and synthesis of core components of the basement membrane, such as type IV collagen, as well as other proteins. It can fill the dermal reticular structure and reduce wrinkles caused by collagen loss. Col_4's multifaceted reconstruction and repair effects on the basement membrane result in excellent firming, anti-wrinkle, and repairing effects.

[0223] Example 3

[0224] This embodiment uses a UVA irradiated 3D full-thickness skin model. The effects of Col_4 on firming, anti-wrinkle and repair were evaluated by detecting changes in tissue morphology, collagen fibers, type I collagen (Collagen I), integrin-interacting protein (Kindlin-1), and integrin β4 content.

[0225] Collagen fibers are the main component of the dermis, responsible for providing skin strength, support, and elasticity. Type I collagen is the most abundant type of collagen in the skin, accounting for 80%-90% of the total dermal collagen, and is primarily responsible for skin strength and elasticity. Kindlin-1, a cytoskeleton-associated protein, participates in the interaction between cells and the extracellular matrix (such as collagen), and is crucial for maintaining skin cell adhesion and stability. Integrin β4 is a bridging molecule between the extracellular matrix and the intracellular cytoskeleton, participating in cell adhesion, signal transduction, and the maintenance of skin barrier function. These expression factors reflect the structural stability of the skin, the interaction between cells and the matrix, and the repair capacity from different perspectives, providing a scientific basis for evaluating the firming, anti-wrinkle, and repairing effects of collagen.

[0226] 1. Testing Method

[0227] 1) Test Grouping

[0228] The test group information is shown in Table 10 below:

[0229] Table 10 Test Group Information

[0230]

[0231] 2) Drug administration and irradiation

[0232] ① Pre-test preparation and drug administration: During the model construction stage, after the dermis layer was constructed, the sample to be tested was added and incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. After washing with PBS, the epidermis layer was constructed. The constructed 3D full-thickness skin model was transferred to a 6-well plate, and 2L of model culture medium was added to each well. The model was randomly divided into a blank control group (BC), a negative control group (NC), and a sample group, with 3 replicates in each group.

[0233] ② Irradiation: Except for the blank control group (BC), the negative control group (NC) and the sample group were subjected to UVA irradiation. The UVA irradiation dose was 35 J / cm². 2 .

[0234] ③ Model culture: The model was transferred to a petri dish and incubated in a CO2 incubator (37°C, 5% CO2). Irradiation was performed once daily for a total of four times. After the final irradiation, the model was cultured for another 24 hours. After the culture, residual test material was washed with sterile PBS solution, and any remaining liquid was gently wiped away with a sterile cotton swab.

[0235] 3) Tissue morphology detection: The model used for detection was fixed with 4% paraformaldehyde. After 24 hours of fixation, H&E staining was performed, and the images were taken and analyzed under a microscope.

[0236] 4) Collagen fiber detection: The model used for detection was fixed with 4% paraformaldehyde. After 24 hours of fixation, Masson staining was performed, and the images were photographed and analyzed under a microscope.

[0237] 5) Immunofluorescence detection: The model used for detection was fixed with 4% paraformaldehyde. After 24 hours of fixation, immunofluorescence detection was performed, and the images were photographed and analyzed under a microscope.

[0238] 6) Calculation of lift rate

[0239]

[0240] 7) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as mean ± standard deviation (Mean ± SD). t-tests were used for comparisons between groups. All statistical analyses were two-tailed. Significance compared to group BC is indicated by *, with a p-value < 0.05 (*) indicating a significant difference and a p-value < 0.01 (**) indicating a highly significant difference.

[0241] 2. Test Results

[0242] 1) Organizational structure

[0243] Table 11 Summary of Epidermal Viable Cell Layer Thickness Results

[0244]

[0245] Tissue morphology test results as follows Figure 16 , Figure 17 As shown in Table 11, compared with the BC group, the epidermal viable cell layer thickness in the NC group was significantly decreased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the epidermal viable cell layer thickness in the Col_4 groups was significantly increased, with increases of 15.48%, 18.18%, and 36.19%, respectively.

[0246] 2) Collagen fiber content

[0247] Table 12 Summary of Collagen Fiber Analysis Results

[0248]

[0249] Collagen fiber content test results are as follows Figure 18 , Figure 19 As shown in Table 12, the collagen fiber content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the collagen fiber content in the Col_4 groups was significantly higher, with increases of 15.15%, 33.33%, and 48.48%, respectively.

[0250] 3) Type I collagen content

[0251] Table 13 Summary of Collagen I Analysis Results

[0252]

[0253] The results of the type I collagen content test are as follows: Figure 20 , Figure 21 As shown in Table 13, the Collagen I content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen I content in the Col_4 groups was significantly higher, with increases of 17.14%, 37.14%, and 94.29%, respectively.

[0254] 4) Integrin-interacting protein (Kindlin-1) content

[0255] Table 14 Summary of Kindlin-1 Immunofluorescence Analysis Results

[0256]

[0257] Results of Kindlin-l integrin interactor protein content assay: Figure 22 , Figure 23 As shown in Table 14, the Kindlin-1 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Kindlin-1 content in the Col_4-5ppm and Col_4-50ppm groups was significantly higher, with increases of 71.43% and 200.00%, respectively.

[0258] 5) Integrin β4 content

[0259] Table 15 Summary of Integrin β4 Immunofluorescence Analysis Results

[0260]

[0261] Results of integrin β4 content test as follows Figure 24 , Figure 25As shown in Table 15, the β4 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the β4 content in the Col_4 groups was significantly higher, with increases of 25.00%, 41.67%, and 216.67%, respectively.

[0262] 3. Conclusion

[0263] Based on 3D full-layer skin model Compared with the control group, Col_4 at a concentration of 0.5 ppm significantly increased the thickness of the epidermal viable cell layer, with an increase rate of 15.48%; the contents of collagen fibers, type I collagen (Collagen I), and integrin β4 were all significantly increased, with increase rates of 15.15%, 17.14%, and 25.00%, respectively. At concentrations of 5 ppm and 50 ppm, Col_4 significantly increased the thickness of the epidermal viable cell layer, with increase rates of 18.18% and 36.19%, respectively; the contents of collagen fibers were significantly increased, with increase rates of 33.33% and 48.48%, respectively; the contents of type I collagen (Collagen I) were significantly increased, with increase rates of 37.14% and 94.29%, respectively; the contents of integrin β4 were significantly increased, with increase rates of 41.67% and 216.67%, respectively; and the contents of integrin interacting protein (Kindlin-1) were significantly increased, with increase rates of 71.43% and 200.00%, respectively.

[0264] Col_4 increases the expression levels of integrin-interacting proteins Kindlin-1 and integrin β4, which helps strengthen hemidesmosome connections between keratinocytes and the basement membrane, enhancing the mechanical anchoring force of the dermis. Col_4 also increases the content of type I collagen and collagen fibers, which interact with the basement membrane by anchoring fibers, jointly maintaining the structural stability and mechanical properties of the skin, providing elasticity, firmness, and repair to the skin.

[0265] Example 4

[0266] This embodiment uses a UVA and UVB combined irradiation of an ex vivo skin model (i.e., real ex vivo human skin) to evaluate the firming, anti-wrinkle, and repairing effects of Col_4 by detecting changes in the content of type III collagen (Collagen III), type V collagen (Collagen V), type VI collagen (Collagen VI), type VII collagen (Collagen VII), and type XVIII collagen (Collagen XVIII).

[0267] 1. Testing method:

[0268] 1) Test Grouping

[0269] The test group information is shown in Table 16 below:

[0270] Table 16 Test Group Information

[0271]

[0272] 2) Preparation before testing: Place the ex vivo skin model into the culture mold of a 6-well plate, add 3.7 mL of culture medium to each well, and incubate at 37℃ in a 5% CO2 incubator, changing the medium daily.

[0273] 3) Irradiation and drug administration: Two days after model culture, irradiation and drug administration were initiated according to the test groups and corresponding treatment conditions in Table 16. The irradiation dose was UVA (30 J / cm²). 2 ) and UVB (50mJ / cm 2 The culture medium was continuously irradiated for 4 days, with fresh medium added after each irradiation and drug administration administered. The positive control group received submersible drug administration, while the sample group received surface drug administration. After 4 days of continuous irradiation, the culture was continued for another 3 days without further irradiation, only drug administration.

[0274] 4) Immunofluorescence detection: Skin tissue for immunofluorescence detection was taken, fixed with 4% paraformaldehyde, and after 24 hours of fixation, immunofluorescence detection was performed. The images were photographed and analyzed under a microscope.

[0275] 5) Calculation of lift rate:

[0276]

[0277] 6) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as mean ± standard deviation (Mean ± SD). t-tests were used for comparisons between groups. All statistical analyses were two-tailed. Significance compared to group BC is indicated by *, with a p-value < 0.05 (*) indicating a significant difference and a p-value < 0.01 (**) indicating a highly significant difference.

[0278] 2. Test Results

[0279] 1) Collagen III content

[0280] Table 17 Summary of Collagen III Immunofluorescence Analysis Results

[0281]

[0282] Collagen III content test results are as follows: Figure 26 , Figure 27As shown in Table 17, compared with the BC group, the Collagen III content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen III content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen III content in the Col_4 groups increased significantly, with increases of 77.55% and 120.41%, respectively. Furthermore, the increase rate of Collagen III content in the Col_4-50ppm group was higher than that in the Col_4-5ppm group. The concentration of Col_4 administered in the Col_4-50ppm group (50 μg / ml) was approximately 0.5 times that of the concentrations of VC and VE administered in the PC group (100 μg / ml + 7 μg / ml), but the increase rates of Collagen III content in both groups were basically the same (the PC group was about 1.1 times that of the Col_4-50ppm group). This demonstrates that the Col_4 group is significantly more effective than the PC group in increasing Collagen III content.

[0283] 2) Collagen V content

[0284] Table 18 Summary of Collagen V Immunofluorescence Analysis Results

[0285]

[0286] Collagen V content test results are as follows: Figure 28 , Figure 29 As shown in Table 18, compared with the BC group, the Collagen V content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen V content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen V content in the Col_4 groups increased significantly, with increases of 75.44% and 112.28%, respectively. Furthermore, the increase rate of Collagen V content in the Col_4-50ppm group was higher than that in the Col_4-5ppm group. Meanwhile, the concentrations of VC and VE administered in the PC group (100μg / ml + 7μg / ml) were approximately 20 and 2 times that of the sample groups Col_4-5ppm (5μg / ml) and Col_4-50ppm (50μg / ml), respectively, while the increase rate of Collagen V content in the PC group was only about 2.1 times and 1.4 times that of the sample groups Col_4-5ppm and Col_4-50ppm, respectively. The results show that Col_4 is significantly better than the PC group in increasing Collagen V content.

[0287] 3) Collagen VI content

[0288] Table 19 Summary of Collagen VI Immunofluorescence Analysis Results

[0289]

[0290] Collagen VI content test results are as follows Figure 30 , Figure 31 As shown in Table 19, compared with the BC group, the Collagen VI content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen VI content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen VI content in the Col_4 groups increased significantly, with increases of 38.27% and 49.38%, respectively. Furthermore, the increase rate of Collagen VI content in the Col_4-50ppm group was higher than that in the Col_4-5ppm group. Meanwhile, the concentrations of VC and VE administered in the PC group (100μg / ml + 7μg / ml) were approximately 20 and 2 times that of the sample groups Col_4-5ppm (5μg / ml) and Col_4-50ppm (50μg / ml), respectively, while the increase rate of Collagen VI content in the PC group was only about 1.9 and 1.5 times that of the sample groups Col_4-5ppm and Col_4-50ppm, respectively. The results show that Col_4 is significantly better than the PC group in increasing Collagen VI content.

[0291] 4) Collagen VII content

[0292] Table 20 Summary of Immunofluorescence Analysis Results of Collagen VII

[0293]

[0294] The test results for Collagen VII content are as follows: Figure 32 , Figure 33As shown in Table 20, compared with the BC group, the Collagen VII content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen VII content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen VII content in the Col_4 groups increased significantly, with increases of 68.89% and 111.11%, respectively. Furthermore, the increase rate of Collagen VII content in the Col_4-50ppm group was higher than that in the Col_4-5ppm group. Meanwhile, the concentrations of VC and VE administered in the PC group (100μg / ml + 7μg / ml) were approximately 20 and 2 times that of the sample groups Col_4-5ppm (5μg / ml) and Col_4-50ppm (50μg / ml), respectively, while the increase rate of Collagen VII content in the PC group was only about 2.3 times and 1.4 times that of the sample groups Col_4-5ppm and Col_4-50ppm, respectively. The results show that Col_4 is significantly better than the PC group in increasing Collagen VII content.

[0295] 5) Collagen XVIII content

[0296] Table 21 Summary of Immunofluorescence Analysis Results of Collagen XVIII

[0297]

[0298] Collagen XVIII content test results are as follows: Figure 34 , Figure 35 As shown in Table 21, compared with the BC group, the Collagen XVIII content in the NC group decreased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the Collagen XVIII content in the PC group increased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the Collagen XVIII content in the Col_4 groups increased significantly, with increase rates of 47.14% and 68.57%, respectively. Furthermore, the increase rate of Collagen XVIII content in the Col_4-50ppm group was higher than that in the Col_4-5ppm group. The concentration of Col_4 administered in the Col_4-50ppm group (50 μg / ml) was approximately 0.5 times that of the concentrations of VC and VE administered in the PC group (100 μg / ml + 7 μg / ml), but the increase rates of Collagen XVIII content in both groups were basically the same (the PC group was about 1.1 times that of the Col_4-50ppm group). This demonstrates that the Col_4 group is significantly more effective than the PC group in increasing Collagen XVIII content.

[0299] 3. Conclusion

[0300] Based on the ex vivo skin model, compared with the control group, the content of type III collagen (Collagen III) in Col_4 at concentrations of 5 ppm and 50 ppm was significantly increased, with increases of 77.55% and 120.41%, respectively; the content of type V collagen (Collagen V) was significantly increased, with increases of 75.44% and 112.28%, respectively; the content of type VI collagen (Collagen VI) was significantly increased, with increases of 38.27% and 49.38%, respectively; the content of type VII collagen (Collagen VII) was significantly increased, with increases of 68.89% and 111.11%, respectively; and the content of type XVIII collagen (Collagen XVIII) was significantly increased, with increases of 47.14% and 68.57%, respectively. This indicates that Col_4 can increase the content of type III collagen, type V collagen, type VI collagen, type VII collagen, and type XVIII collagen, thus having firming and anti-wrinkle effects.

[0301] The experimental results from Examples 1-4 show that:

[0302] 1. Based on tests on ex vivo skin tissue, recombinant human collagen Col_4 exhibits excellent transdermal absorption properties. Furthermore, in terms of dermal-epidermal junction (DEJ) continuity, tissue morphology, elastin, collagen XVII, matrix metalloproteinase 1 (MMP-1), and epidermal morphogenetic pigment 2 (Syntaxin-2) content, the improvement effects achieved by a certain concentration of recombinant human collagen Col_4 are significantly higher than those in the positive control group. This demonstrates that recombinant human collagen Col_4 has unexpected technical effects in anti-wrinkle, firming, and repair.

[0303] 2. This recombinant human collagen Col_4 can reduce the effect of MMP-1, thereby decreasing the degradation effect of MMP-1 on type I and III collagen and elastin. Simultaneously, experimental results based on ex vivo skin and 3D full-thickness skin models show that this recombinant human collagen Col_4 can also promote the synthesis of type I and type III collagen. Under these two effects, the content of type I and type III collagen increases, thereby enhancing the collagen fiber content in skin tissue and achieving anti-wrinkle, firming, and repairing effects.

[0304] 3. The recombinant human collagen Col_4 has a significant promoting effect on Syntaxin-2. Syntaxin-2 promotes the secretion and synthesis of core components of the basement membrane, such as type IV collagen, as well as other proteins, thereby filling the dermal reticular structure and reducing wrinkles caused by collagen loss.

[0305] 4. α-SMA is a hallmark protein of myofibroblasts. Fibroblasts express α-SMA during differentiation into myofibroblasts, indicating the formation of fibrous tissue and thus ensuring the firmness of skin tissue. Recombinant human collagen Col_4 not only increases α-SMA expression but also significantly increases elastin levels (significantly exceeding the effect of the PC control group), thereby enhancing anti-wrinkle, firming, and repair effects through these two mechanisms.

[0306] 5. This recombinant human collagen Col_4 significantly increased the expression levels of various collagen proteins, particularly type XVIII collagen, with increases of 47.14% and 68.57%, respectively. For the basement membrane, type IV collagen, the main component, forms the reticular framework, providing elasticity and mechanical strength. Type XVIII collagen, as a secondary component, binds to type IV collagen, assisting in structural stability and regulating molecular interactions. Together, they maintain the structural and functional integrity of the basement membrane, achieving unexpected technical effects.

[0307] 6. This recombinant human collagen Col_4 can significantly increase the expression levels of various collagen proteins, especially type VII and type XVII collagen. Type VII collagen forms anchor fibers, with one end fixed to the basement membrane and the other end inserted into the dermis; type XVII collagen, as a transmembrane protein, is anchored to the hemidesmosomes of the basement membrane. The high expression of these two types of collagen can tightly connect the basement membrane and the dermis, resulting in the technical effects of skin tightening, smoothing, and repair.

[0308] 7. This recombinant human collagen, Col_4, possesses excellent transdermal absorption properties. After 6 hours and 24 hours of application to the skin, Col_4 can penetrate into the dermis, crossing the epidermal barrier and acting on deep skin tissues and cells. This greatly ensures the effectiveness of the recombinant type IV humanized collagen in anti-wrinkle, firming, and repairing effects on the skin in actual human use.

Claims

1. The use of a recombinant type IV humanized collagen in the preparation of products for skin anti-wrinkle, skin firming and / or skin repair; wherein, The amino acid sequence of the recombinant type IV humanized collagen is the sequence shown in SEQ ID NO.2; The products used for skin anti-wrinkle, skin firming and / or skin repair are transdermal absorbable; The skin anti-wrinkle, skin firming, and / or skin repair effects are any one or more of the following: improving the continuity of dermal-epidermal junction, increasing the thickness of the epidermal living cell layer, increasing the content of elastin, decreasing the content of matrix metalloproteinase 1, increasing the content of α-smooth muscle actin, increasing the content of epidermal morphogenetic proteins, increasing the content of integrin-interacting protein Kindlin-1, increasing the content of integrin β4, increasing the content of collagen fibers, and increasing the content of collagen. The increase in collagen content refers to increasing the content of any one or more collagens from Collagen I, Collagen III, Collagen V, Collagen VI, Collagen VII, Collagen XVII, and Collagen XVIII.

2. The use according to claim 1, characterized in that, The products are selected from cosmetics and medical devices.

3. The use according to claim 1, characterized in that, The product is a biological dressing.

4. The use according to claim 1, characterized in that, The product is a cosmetic surgery material.

5. The use according to claim 1, characterized in that, The product is a tissue injection filler material.

6. The use according to claim 1, characterized in that, The product in question is a cosmetic ingredient.

7. The use according to claim 1, characterized in that, The product contains a cosmetically acceptable carrier.

8. The use according to claim 1, characterized in that, The product is a solid, liquid, or gel at room temperature and pressure.

9. The use according to claim 1, characterized in that, The product is in the form of a solution, lyophilized powder, gel, sponge, or fiber.

10. The use according to claim 1, characterized in that, The product is for topical application.

11. The use according to claim 10, characterized in that, The term "topical application" refers to application to the skin.

12. The use according to claim 10, characterized in that, The topical application refers to the application of the product to the facial skin.

13. The use according to claim 1, characterized in that, The content of the recombinant type IV humanized collagen in the product is greater than or equal to 0.3 ppm.

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