A recombinant v-type humanized collagen and application thereof

Abstract

The application belongs to the field of biological materials, and particularly relates to a recombinant V-type humanized collagen and application thereof. The application provides a recombinant V-type humanized collagen, wherein the amino acid sequence of the recombinant V-type humanized collagen comprises (repeat unit)n, the repeat unit comprises a sequence shown in SEQ ID NO. 1, each repeat unit is directly connected, and the number n of the repeat units is 10. The recombinant V-type humanized collagen can resist skin aging in all directions.

Description

Technical Field

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

[0002] Type V collagen (COL V) is a dermal collagen that regulates the formation of fibrils. It consists of at least three different homo or heterotrimers: α1(V)3, α1(V)2α2(V), and α1(V)α2(V)α3(V), composed of three different polypeptide α chains (α1, α2, and α3). Type V collagen is a relatively rare collagen in the extracellular matrix, accounting for only 2-4% of total collagen in adult skin. COL V provides more support. Type I collagen (COL I) / Type III collagen (COL III) (especially COL I) attaches to the COL V backbone to assemble fibrils. The collagen fibers subsequently formed by these fibrils provide mechanical support to the dermal layer, jointly resisting external stimuli and maintaining the overall stability of the skin. Literature shows that the importance of COL V in collagen fiber formation has been confirmed in gene knockout mouse models. Even with normal COL I secretion, the absence of COL V in these mice still leads to insufficient collagen fiber formation, causing various diseases. The lack of COL V can even result in some organs failing to form due to a lack of collagen fibers, leading to the death of young mice. In summary, COL V is essential for normal collagen fiber formation. Furthermore, type V collagen can bridge type VI collagen (COL VI) in the dermal matrix to type IV collagen (COL IV) in the epidermal basement membrane, thereby regulating the stability between the epidermis and dermis.

[0003] Studies have shown that collagen fiber bundles in young people aged 20 to 40 are thinner and their morphology and arrangement are more regular than those in older people. This is attributed to the significantly higher expression level of V-type collagen in the skin of young people compared to older people. COL V, as a type of FACIT collagen (Fibril-Associated Collagens with Interrupted Triple Helices), has a molecular structure containing discontinuous triple helical regions and globular domains. In the early stages of fiber assembly, COL V binds to integrins (such as α2β1) on the surface of fibroblasts through its globular domain, forming the initial fiber nucleus, which then self-assembles with COLI to form protofibrils. Furthermore, COL V encapsulates the surface of COLI fibers, limiting excessive fiber thickening through steric hindrance, ensuring that the fibers are long, thin, and uniform (abnormal thickening leads to skin stiffness). Existing literature indicates that protofibrils in young skin are thinner and firmer than those in aging skin, thus providing stable support and reducing the formation of wrinkles and fine lines. Aging skin lacks V-type and I-type collagen, which prevents it from properly assembling into healthy and stable fibrils. The inability of these fibrils to assemble tightly leads to an increased diameter, resulting in thicker and less dense collagen fibers. This weakens the skin's support, ultimately causing sagging and wrinkles. Effectively replenishing V-type collagen, which forms the backbone of collagen fibers, while simultaneously increasing the levels of I, III, and VI collagen, is crucial for the skin's resistance to aging. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] One cause of skin aging is the reduction of collagen. Currently, there is a wide market demand for products that can delay skin aging and have firming and anti-wrinkle effects.

[0006] During the in-depth study of the function of the recombinant V-type humanized collagen disclosed in ZL202210849498.3, it was unexpectedly discovered that it can enhance the expression level of various collagen proteins in the skin, maintain the fineness and firmness of collagen fibers, and has the effects of anti-wrinkle, firming and improving skin elasticity.

[0007] Based on this, the purpose of the present invention is to provide the use of the recombinant V-type humanized collagen in anti-skin aging.

[0008] Solution for solving the problem

[0009] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0010] [1]. A recombinant humanized V-type collagen; wherein the amino acid sequence of the recombinant humanized V-type collagen includes (repetitive unit) n, the repeating unit includes the sequence shown in SEQ ID NO.1; each repeating unit is directly connected and the number of repeating units n is 10.

[0011] [2]. The recombinant humanized V-type collagen according to [1], wherein the amino acid sequence of the recombinant humanized V-type collagen is as shown in SEQ ID NO.2.

[0012] [3]. Use of recombinant V-type humanized collagen as described in [1] or [2] in the preparation of products for anti-skin aging;

[0013] Preferably, the anti-skin aging includes skin firming and / or skin anti-wrinkle.

[0014] [4]. According to the use described in [3], the anti-skin aging includes regulating epidermal-dermal stability, enhancing skin support and / or improving skin density.

[0015] [5]. According to the use described in [3] or [4], the anti-skin aging includes any one or more of the following: reducing the diameter of collagen fibrils, increasing the content of collagen fibers, improving skin elasticity and increasing the expression level of collagen.

[0016] [6]. According to the use described in [5], the enhancement of collagen expression level includes enhancing the expression level of any one or more of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type XII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen and type XVIII collagen.

[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 and pressure;

[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 V-type humanized collagen in the product is greater than or equal to 0.3 ppm.

[0024] [9]. A product with anti-skin aging effects, wherein the product comprises recombinant humanized V-type collagen; wherein the amino acid sequence of the recombinant humanized V-type collagen comprises (repetitive unit) n, the repetitive unit comprises the sequence shown in SEQ ID NO.1; each repetitive unit is directly connected and the number of repetitive units n is 10;

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

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

[0027]

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

[0028] 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.

[0029] Preferably, the product comprises a pharmaceutically and / or cosmetically acceptable carrier;

[0030] Preferably, the product is a solid, liquid, or gel at room temperature and pressure;

[0031] Preferably, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation;

[0032] 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.

[0033] The effects of the invention

[0034] This invention provides the use of recombinant V-type humanized collagen in the preparation of products for anti-skin aging. The recombinant V-type humanized collagen used in this invention has excellent hydrophilicity and stability, and its amino acid composition is 100% identical to the corresponding parts of the amino acid sequence of natural collagen. It will not cause immune rejection or allergic reactions when applied to the human body, and can be widely used in the biopharmaceutical and cosmetic industries.

[0035] Experimental data show that recombinant V-type humanized collagen can significantly increase the expression levels of COLI and COLV in aging cell models, promote the spontaneous assembly of COLI and COLV into fibrils, improve the skin's support properties, and prevent sagging and wrinkles.

[0036] Meanwhile, experimental results from a 3D full-thickness skin model of aging showed that recombinant V-type humanized collagen, at different treatment concentrations, could reduce the diameter of fibrils and increase the content of collagen fibers, resulting in a more youthful skin appearance. Furthermore, the skin's elasticity coefficient R² test results further corroborated this effect. Therefore, recombinant V-type humanized collagen has the function of reshaping the collagen framework, promoting fibril generation and stabilization, thereby exerting an anti-aging effect on the skin.

[0037] Furthermore, experimental data showed that recombinant humanized collagen type V significantly increased the expression levels of 11 collagen proteins in fibroblasts. Recombinant humanized collagen type V significantly increased the expression levels of COL IV and COL VII genes. COL IV is a major structural protein of the skin's basement membrane (located at the junction of the epidermis and dermis), forming a reticular scaffold; COL VII constitutes anchoring fibers, extending from the dense layer of the basement membrane to the dermis, tightly connecting the collagen fiber bundles of the basement membrane and the dermis, anchoring the basement membrane to the dermis, and resisting shear forces. The effect of recombinant humanized collagen type V on increasing the content of these two collagen proteins can strengthen the basement membrane structure and epidermal-dermal adhesion, increase basement membrane thickness and the number of anchoring fibers, thereby improving skin firmness. Simultaneously, recombinant humanized collagen type V also increased the expression levels of COL V and COL VI. COL V regulates the stability between the epidermis and dermis by bridging COL VI (highly expressed) in the dermal matrix to COL IV (highly expressed) in the epidermal basement membrane, thus improving skin firmness. Simultaneously, recombinant humanized V-type collagen can increase the expression levels of COL I, COL III, COL XII, COL XIV, COL XV, COL XVI, and COL XVIII genes, especially COL VI and COL XII, with significantly higher levels than the positive control group. This increased expression of multiple collagen proteins helps increase collagen fiber content and facilitates collagen network formation, enhancing the mechanical strength and elasticity of the dermis. It also improves the extracellular matrix environment, promoting cell adhesion and proliferation. Therefore, recombinant humanized V-type collagen comprehensively enhances the skin's anti-wrinkle ability, both in terms of the biomechanical properties of the dermis and the dynamic balance of the extracellular matrix. Meanwhile, recombinant type V humanized collagen can also increase the expression level of type XVII collagen in keratinocytes. The increase in the expression level of type XVII collagen can increase the number of hemidesmosomes, increase cell adhesion, and promote the connection between the basement membrane and keratinocytes, thereby improving the adhesion ability of the epidermis and enhancing the stability of the basement membrane, thus resulting in a skin tightening effect. Attached Figure Description

[0038] Figure 1 The results of the detection of type I collagen content in Example 1.

[0039] Figure 2 Results of V-type collagen content detection in Example 1.

[0040] Figure 3 Observation results of fibrils in Example 2.

[0041] Figure 4 Analysis results of collagen fiber content in Example 2.

[0042] Figure 5 The test results of the elastic coefficient R2 in Example 2. Detailed Implementation

[0043] The embodiments of the present invention will be described below, 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. The embodiments and examples obtained by appropriately combining the technical means disclosed in different implementation schemes and examples are also included in the technical scope of the present invention.

[0044] 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.

[0045] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0046] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In this invention, "normal temperature and pressure" refers to an ambient temperature of 25°C under standard atmospheric pressure.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In this invention, the term "expression level" refers to the amount of gene product (including mRNA and proteins, etc.) present in the body or sample at a specific time point. Expression level can be measured or detected by any method known in the art, such as methods for detecting and measuring the target gene product that typically work by binding the target gene product to one or more different molecules or detection devices (e.g., primers, probes, antibodies, proteins, etc.) that are specific to the target gene product.

[0060] 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.

[0061] I. Recombinant V-type humanized collagen

[0062] The recombinant V-type humanized collagen described in this invention is described in the patent application number 202210849498.3, entitled "A method for preparing a biosynthetic human structural material", the contents of which are incorporated herein by reference.

[0063] In some embodiments, the amino acid sequence of the recombinant V-type humanized collagen includes (repetitive units) n, the repeating units including the sequence shown in SEQ ID NO.1; the repeating units are directly linked and the number of repeating units n is 10.

[0064] SEQ ID NO. 1: GKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPV.

[0065] In some embodiments, the amino acid sequence of the recombinant type V humanized collagen includes any one of the following (i)-(iv):

[0066] (i) A sequence as shown in SEQ ID NO.2;

[0067] (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-skin aging effect of the sequence shown in SEQ ID NO.2;

[0068] (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-skin aging effect of the sequence shown in SEQ ID NO.2;

[0069] (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.2 under stringent conditions, and said amino acid sequence retaining the anti-skin aging effect of the sequence shown in SEQ ID NO.2, said stringent conditions being moderately stringent, medium-high stringent, high stringent, or very high stringent.

[0070] SEQ ID NO.2: GKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGD PGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPG LRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPVGKEGTKGDPGPAGLPGKDGPPGLRGFPGDRGLPGPV.

[0071] In some embodiments, the amino acid sequence of the recombinant type V humanized collagen includes the sequence shown in SEQ ID NO. 2.

[0072] In some embodiments, the amino acid sequence of the recombinant type V humanized collagen is as shown in SEQ ID NO.2.

[0073] The recombinant humanized type V 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 humanized type V collagen; and (4) purification and optional enzymatic digestion of recombinant humanized type V collagen.

[0074] II. Uses of Recombinant Type V Humanized Collagen

[0075] This invention provides the use of the recombinant type V humanized collagen described in Part I in the preparation of products for anti-skin aging.

[0076] In some embodiments, the recombinant V-type humanized collagen described in Part I of this invention is used for anti-skin aging.

[0077] In some embodiments, the present invention also provides a method for combating skin aging, the method comprising the step of administering an effective amount of the recombinant type V humanized collagen to an individual in need thereof.

[0078] In some implementations, the anti-skin aging includes skin firming and / or skin wrinkle reduction.

[0079] In some implementations, the anti-skin aging measures include modulating epidermal-dermal stability, enhancing skin support, and / or improving skin firmness.

[0080] In some implementations, the anti-skin aging measures also include regulating epidermal-dermal stability, enhancing skin support, and improving skin firmness.

[0081] In some implementations, the anti-skin aging includes any one or more of the following: reducing collagen fibrillary diameter, increasing collagen fiber content, improving skin elasticity, and increasing collagen expression levels.

[0082] In some implementations, the anti-skin aging process includes reducing collagen fiber diameter, increasing collagen fiber content, improving skin elasticity, and increasing collagen expression levels.

[0083] In some embodiments, the enhancement of collagen expression levels includes enhancing the expression levels of any one or more of type I, type III, type IV, type V, type VI, type VII, type XII, type XIV, type XV, type XVI, type XVII, and type XVIII collagen.

[0084] In some embodiments, the enhancement of collagen expression levels includes simultaneously enhancing the expression levels of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type XII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, and type XVIII collagen.

[0085] In some specific implementations, the enhancement of collagen expression levels includes enhancing the expression levels of type I, type III, type IV, type V, type VI, type VII, type XII, type XIV, type XV, type XVI, and type XVIII collagen in fibroblasts, and / or enhancing the expression level of type XVII collagen in keratinocytes.

[0086] In some implementations, the product is selected from cosmetics, pharmaceuticals, and medical devices.

[0087] 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.

[0088] In some implementations, the product comprises a pharmaceutically and / or cosmetically acceptable carrier.

[0089] In some embodiments, the product is a solid, liquid, or gel at room temperature and pressure.

[0090] In some embodiments, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation.

[0091] 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.

[0092] In some embodiments, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 0.3 ppm; for example, the content of the recombinant V-type 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 V-type humanized collagen in the product is greater than or equal to 0.5 ppm; more preferably, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 5 ppm; even more preferably, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 20 ppm.

[0093] III. Products containing the aforementioned recombinant type V humanized collagen

[0094] This invention provides a product with anti-skin aging effects, which comprises the recombinant V-type humanized collagen described in Part I.

[0095] In some implementations, the product is selected from cosmetics, pharmaceuticals, and medical devices.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In some embodiments, the product is a solid, liquid, or gel at room temperature and pressure.

[0100] In some embodiments, the product is a solution, lyophilized powder, gel, sponge, or fiber formulation.

[0101] 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.

[0102] In some embodiments, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 0.3 ppm; for example, the content of the recombinant V-type 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 V-type humanized collagen in the product is greater than or equal to 0.5 ppm; more preferably, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 5 ppm; even more preferably, the content of the recombinant V-type humanized collagen in the product is greater than or equal to 20 ppm.

[0103] Example

[0104] 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.

[0105] The amino acid sequence of the recombinant V-type humanized collagen used in this embodiment of the invention is shown in SEQ ID NO.2.

[0106] The preparation and production of this recombinant V-type humanized collagen are described in the prior invention patent of the applicant: ZL202210849498.3.

[0107] Example 1: Detection of changes in the content of type I collagen (COL I) and type V collagen (COL V)

[0108] The extracellular matrix (ECM) of the dermis is the main structure providing tension, elasticity, and mechanical support to the skin. ECM-related proteins are expressed by fibroblasts. With skin aging, fibroblasts secrete matrix metalloproteinases to break down the ECM, and collagen synthesis decreases, leading to a reduction in the synthesis of supporting collagen fibrils. This causes the supporting structure to collapse, ultimately resulting in fine lines. Collagen fibers are bundles of fibers composed of fibrils, with COLI and COL V forming the core components. Therefore, based on aged fibroblasts, after the action of recombinant humanized type V collagen, changes in COLI and COL V can be detected to assess the effect of recombinant humanized type V collagen on collagen fiber assembly, thereby evaluating its firming and anti-wrinkle effects.

[0109] After resuscitating 40th generation fibroblasts, when the cell deposition rate reached approximately 60%, cells were seeded into 24-well plates and incubated overnight. The experiment consisted of a blank control group (BC) and sample groups (0.5 ppm recombinant humanized type V collagen, 5 ppm recombinant humanized type V collagen, and 50 ppm recombinant humanized type V collagen). According to the test groups, when the cell deposition rate in the 24-well plates reached approximately 15%, the cells were administered, with three replicates per group. 1 mL of fresh complete culture medium was added to each well of the blank control group, and 1 mL of fresh complete culture medium containing the corresponding concentration of the test sample was added to each well of the sample groups. After administration, the 24-well plates were incubated for 48 hours. After incubation, cells were fixed with 4% paraformaldehyde. First, incubate with COL V primary and secondary antibodies. After staining, incubate with COL I primary and secondary antibodies. After incubation, perform nuclear staining and mounting. Take pictures and observe under a fluorescence microscope, collect images and analyze them.

[0110] Lift rate calculation:

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[0113] The results of the type I collagen (COL I) content test are as follows: Figure 1 As shown in Table 1, the results of the V-type collagen (COL V) content detection are as follows: Figure 2As shown in Table 2, the experimental results showed that compared with the BC group, the COL I content in the 0.5 ppm V-type collagen group, the 5 ppm V-type collagen group, and the 50 ppm V-type collagen group all increased significantly, with increases of 19.00%, 37.00%, and 47.00%, respectively; the COLV content also increased significantly, with increases of 10.00%, 30.00%, and 33.00%, respectively. This indicates that recombinant humanized V-type collagen can promote the expression of COL I and COL V in fibroblasts, and can promote the spontaneous assembly of COL I and COL V to form fibrils, thereby contributing to collagen fiber assembly, providing good mechanical support for the skin, and further exhibiting skin tightening and anti-wrinkle effects.

[0114] Example 2: Detection of changes in fibril diameter, dermal collagen fiber content, and elasticity coefficient R2.

[0115] Based on an aging 3D full-thickness skin model, the anti-wrinkle effect of collagen was evaluated by detecting changes in fibrillary diameter, dermal collagen fiber content, and elasticity coefficient R2.

[0116] Constructing a 3D full-thickness aging skin model: Senescent fibroblast seed cells were induced, collected, and mixed with a collagen matrix to construct the dermis layer of the full-thickness aging model. Sample groups (0.5 ppm recombinant humanized V-type collagen, 5 ppm recombinant humanized V-type collagen, and 50 ppm recombinant humanized V-type collagen) were mixed simultaneously according to the test grouping conditions, with the corresponding sample concentrations. After the dermis layer matured, keratinocytes were added to the surface of all groups to construct the epidermis layer.

[0117] Sample collection: After the aging 3D full-thickness skin model has matured, the culture is stopped. The model is used for the determination of fibril diameter, collagen fiber staining and skin elasticity coefficient.

[0118] Lift rate calculation:

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[0120] When the concentration of COL V in the skin is high or its content in tissues is high, collagen fibrils are fully assembled. Compared with unhealthy skin, collagen fibrils in normal healthy skin have smaller diameters and are more tightly assembled. Therefore, by observing changes in fibril diameter, the effect of recombinant V-type humanized collagen on fibril assembly was evaluated, and its anti-wrinkle efficacy was assessed. The fibril diameter detection results are shown in Table 3. The experimental results show that compared with the BC group, the median fibril diameter in the full-thickness skin models constructed by the 0.5ppm V-type collagen group, 5ppm V-type collagen group, and 50ppm V-type collagen group was reduced, indicating that recombinant V-type humanized collagen has anti-wrinkle efficacy. Furthermore, through... Figure 3 Observing the fibrils, it is clear that the fibrils of the 0.5ppm V-type collagen group and the 50ppm V-type collagen group are thinner in diameter and more compactly arranged.

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[0122] Collagen fibers are bundles of collagen fibrils that cross-link with elastic fibers, maintaining the skin's elasticity and resilience. Typically, UV exposure leads to a reduction in collagen fibers. Therefore, by assessing changes in collagen fiber content after sample addition, the anti-wrinkle efficacy of the sample can be determined. The collagen fiber content analysis results are as follows: Figure 4 As shown in Table 4. The experimental results showed that, compared with group BC, the collagen fiber content in the dermal layer of the full-thickness skin model constructed with 0.5 ppm V-type collagen, 5 ppm V-type collagen, and 50 ppm V-type collagen were significantly increased, with increases of 32.00%, 58.00%, and 132.00%, respectively, indicating that this recombinant humanized V-type collagen has anti-wrinkle effects. Figure 4 It can be observed that the collagen fiber content of recombinant V-type humanized collagen at different concentrations not only increases, but also intertwines with each other to form a network structure, thus improving the mechanical properties of the skin.

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[0124] The test results of the elastic coefficient R2 are as follows Figure 5 As shown in Table 5, the experimental results showed that, compared with the BC group, the elastic coefficient R2 of the full-thickness skin model constructed by the 0.5ppm V-type collagen group, the 5ppm V-type collagen group, and the 50ppm V-type collagen group all increased significantly, with improvement rates of 23.18%, 33.86%, and 40.68%, respectively, indicating that V-type collagen has anti-wrinkle effects.

[0125] Example 3: Detection of gene expression levels of type I, III, IV, V, VI, VII, XII, XIV, XV, XVI, and XVIII collagen.

[0126] Based on fibroblasts, the firming and anti-wrinkle effects of recombinant type V humanized collagen were evaluated by detecting the gene expression levels of type I, III, IV, V, VI, VII, XII, XIV, XV, XVI, and XVIII collagen.

[0127] After fibroblast resuscitation, when the cell deposition rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight. The experiment consisted of a blank control group (BC), a positive control group (PC, 100 ng / ml TGF-β1), and a sample group (1% recombinant humanized type V collagen (1 g / 100 mL)). According to the experimental groups, when the cell deposition rate in the 6-well plates reached 40%–60%, the cells were administered to each group, with three replicates per group. 2 mL of culture medium was added to each well of the blank control group, 2 mL of culture medium containing TGF-β1 was added to each well of the positive control group, and 2 mL of culture medium containing the corresponding concentration of the test sample was added to each well of the sample group. After administration, the 6-well plates were placed in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0128] After incubation, discard the old solution, wash twice with PBS, add 1 mL of AG RNAex Pro Reagent to each well, lyse the cells by pipetting, and collect the samples. Extract RNA, reverse transcribe it into cDNA, and perform real-time PCR detection using a 2... -△△CT The method is used to calculate the results.

[0129] Upward adjustment rate calculation:

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[0141] The gene expression levels of collagen types I, III, IV, V, VI, VII, XII, XIV, XV, XVI, and XVIII are shown in Tables 6-16. Based on fibroblasts, compared with the blank control group, the gene expression levels of the above collagen types in the PC group were significantly increased, demonstrating the effectiveness and reliability of this experimental system and ensuring the accuracy and reproducibility of the experimental results. Simultaneously, compared with the blank control group, after 24 hours of incubation with recombinant type V humanized collagen, the expression levels of COLIV and COL VII genes were significantly upregulated, with upregulation rates of 96.00% and 49.00%, respectively, significantly higher than those in the PC group. For the upregulation rate of COL IV gene expression, the sample group was 33.00% higher than the PC group; for the upregulation rate of COL VII gene expression, the sample group was 7.00% higher than the PC group. The upregulation of COL IV and COL VII gene expression by recombinant type V humanized collagen can strengthen the basement membrane structure and epidermal-dermal adhesion, increase basement membrane thickness and anchor fiber number, and has a skin-tightening effect.

[0142] Compared with the blank control group (BC), the expression levels of COLⅠ, COLⅢ, COL V, COL VI, COL XII, COLXIV, COL XV, COL XVI, and COL XVIII genes were significantly upregulated in the sample group, with upregulation rates of 116.00%, 97.00%, 39.00%, 119.80%, 17.00%, 102.00%, 85.00%, 52.00%, and 50.00%, respectively. Furthermore, the upregulation rates of COL VI and COL XII gene expression were 38.61% and 2.00% higher in the sample group than in the PC group, respectively, showing a significant upward trend compared to both the BC and PC groups. Recombinant V-type humanized collagen can help form collagen networks, increase the mechanical strength and elasticity of the dermis, improve the extracellular matrix environment, promote cell adhesion and proliferation, and has skin-tightening and anti-wrinkle effects by upregulating the expression levels of COLⅠ, COL III, COL V, COL VI, COL XII, COL XIV, COL XV, COL XVI, and COL XVIII genes.

[0143] The upregulation rates of COL I, COL III, COL V, COL XIV, COL XV, COL XVI, and COL XVIII gene expression were lower in the sample group than in the PC group. This may be because TGF-β1, the drug used in the PC group, is secreted by fibroblasts. As a cytokine, it can activate the Smad signaling pathway, significantly promoting collagen synthesis (i.e., TGF-β1 is an upstream regulator of collagen expression) and the synthesis of extracellular matrix (ECM) such as fibronectin. In the PC group, TGF-β1 directly acted on fibroblasts, promoting the expression of the aforementioned collagen genes. Furthermore, TGF-β1 is a secreted cytokine, with physiological concentrations typically in the pg / mL range (e.g., approximately 1-100 pg / mL in serum), exhibiting a "high effect at low concentration" mechanism. The PC group used a TGF-β1 concentration of 100 ng / mL, resulting in a very significant effect.

[0144] Example 4: Detection of gene expression level of type XVII collagen (COL XVII)

[0145] Fibroblasts are mainly distributed in the dermis and are responsible for synthesizing type I and type III interstitial collagens. In their natural state, they almost never express type XVII collagen. Type XVII collagen is a transmembrane collagen (also known as BP180) specifically expressed in the hemidesmosomes of the epidermal basal cell layer. Its extracellular domain extends to the basement membrane compactum, and it is routinely validated using a keratinocyte model. Therefore, Example 4, based on keratinocytes, evaluated the firming effect of recombinant type V humanized collagen by detecting the gene expression level of COL XVII.

[0146] After reviving keratinocytes, when the cell deposition rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight. The experiment consisted of a blank control group (BC) and a sample group (0.125% recombinant humanized type V collagen (0.125 g / 100 mL)). According to the experimental groups, when the cell deposition rate in the 6-well plates reached 40%–60%, the cells were administered to each group, with three replicates per group. 2 mL of culture medium was added to each well in the blank control group, and 2 mL of culture medium containing the corresponding concentration of the test sample was added to each well in the sample group. After administration, the 6-well plates were incubated for 24 hours.

[0147] After incubation, discard the old solution, wash twice with PBS, add 1 mL of AG RNAex Pro Reagent to each well, lyse the cells by pipetting, and collect the samples. Extract RNA, reverse transcribe it into cDNA, and perform real-time PCR detection using a 2... -△△C The T method is used to calculate the results.

[0148] Upward adjustment rate calculation:

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[0150] The gene expression levels of type XVII collagen are shown in Table 17. Based on keratinocytes, compared with the control group, the expression level of COL XVII gene was significantly upregulated after 24 hours of incubation with recombinant type V humanized collagen, with an upregulation rate of 77.00%. The upregulation of COL XVII gene expression by recombinant type V humanized collagen can promote the connection between the basement membrane and keratinocytes, thereby improving the adhesion ability of the epidermis and enhancing the stability of the basement membrane, resulting in a skin-tightening effect.

Claims

1. Use of a recombinant V-type humanized collagen in the preparation of products for anti-skin aging; The amino acid sequence of the recombinant type V humanized collagen is as shown in SEQ ID NO.2; The anti-skin aging measures include regulating epidermal-dermal stability, enhancing skin support and / or improving skin firmness; The anti-skin aging measures include: It reduces the diameter of collagen fibers, increases collagen fiber content, improves skin elasticity, and enhances collagen expression levels. The enhancement of collagen expression levels includes enhancing the expression levels of any one or more of type I, type III, type IV, type V, type VI, type VII, type XII, type XIV, type XV, type XVI, type XVII, and type XVIII collagen.

2. The use according to claim 1, characterized in that, The anti-skin aging treatment includes skin firming and / or skin wrinkle reduction.

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

4. The use according to claim 1 or 2, characterized in that, The products include biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, or pharmaceutical excipients.

5. The use according to claim 1 or 2, characterized in that, The product contains a pharmaceutically and / or cosmetically acceptable carrier.

6. The use according to claim 1 or 2, characterized in that, The product is a solid, liquid, or gel at room temperature and pressure.

7. The use according to claim 1 or 2, characterized in that, The product is in the form of a solution, lyophilized powder, gel, sponge, or fiber.

8. The use according to claim 1 or 2, characterized in that, The product is for topical application.

9. The use according to claim 8, characterized in that, The term "topical application" refers to application to the skin.

10. The use according to claim 8, characterized in that, The topical application refers to the application of the product to the facial skin.

11. The use according to claim 1 or 2, characterized in that, The content of the recombinant V-type humanized collagen in the product is greater than or equal to 0.3 ppm.

12. A product with anti-skin aging effects, characterized in that, The product comprises recombinant humanized type V collagen; wherein the amino acid sequence of the recombinant humanized type V collagen is as shown in SEQ ID NO.2; The content of the recombinant V-type humanized collagen in the product is greater than or equal to 0.3 ppm; The anti-skin aging measures include regulating epidermal-dermal stability, enhancing skin support and / or improving skin firmness; The anti-skin aging measures include: reducing the diameter of collagen fibers, increasing collagen fiber content, improving skin elasticity, and increasing the expression level of collagen. The enhancement of collagen expression levels includes enhancing the expression levels of any one or more of type I, type III, type IV, type V, type VI, type VII, type XII, type XIV, type XV, type XVI, type XVII, and type XVIII collagen.

13. The product according to claim 12, characterized in that, The products are selected from cosmetics, pharmaceuticals, and medical devices.

14. The product according to claim 12, characterized in that, The products include biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, or pharmaceutical excipients.

15. The product according to claim 12, characterized in that, The product contains a pharmaceutically and / or cosmetically acceptable carrier.

16. The product according to claim 12, characterized in that, The product is a solid, liquid, or gel at room temperature and pressure.

17. The product according to claim 12, characterized in that, The product is in the form of a solution, lyophilized powder, gel, sponge, or fiber.

18. The product according to claim 12, characterized in that, The product is for topical application.

19. The product according to claim 18, characterized in that, The term "topical application" refers to application to the skin.

20. The product according to claim 18, characterized in that, The topical application refers to the application of the product to the facial skin.

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