Composition containing recombinant triple-helix collagen as well as preparation and application of composition

Abstract

The invention provides a composition containing recombinant collagen with a triple helix structure, and belongs to the technical field of biology. The recombinant humanized III type collagen and the recombinant humanized XVII type collagen in the composition can synergistically promote cell adhesion, promote collagen regeneration, up-regulate expression of genes related to skin hydration and down-regulate expression of inflammatory factor genes, and can be applied to products for resisting aging, tightening, moisturizing, repairing and / or relieving inflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a composition containing recombinant triple-helix collagen, and its preparation and application. Background Art

[0002] The formation of skin laxity and wrinkles is related to various changes in the epidermis, dermis and subcutaneous tissues. The thinning of the epidermis, the enlargement and incomplete keratinization of keratinocytes lead to water loss in the skin. When the water content of the stratum corneum is less than 10%, the skin becomes rough, dry and wrinkled. In the dermis, the number of fibroblasts decreases, the volume becomes smaller and the arrangement is irregular, and the collagen fibers and elastic fibers decrease and degenerate, affecting skin elasticity. The reduction and deformation of subcutaneous adipose tissue cause the reticular layer of the dermis to lose support. These changes together cause the skin to lose elasticity, become loose and form wrinkles. The principle of anti-wrinkle repair includes promoting the regeneration of collagen and elastic fibers and strengthening the barrier function. Bioactive compositions are increasingly widely used in the fields of cosmetics, medicine, etc., but their development faces key technical challenges such as the stability of active ingredients, bioavailability and multi-component synergistic effects.

[0003] Skin care products often add exogenous collagen to enhance anti-aging, repair and other effects. Especially in recent years, with the development of the synthetic biology industry, recombinant collagen has been favored by the market due to its risk-free animal origin. However, the effects of existing skin care products with recombinant collagen as the active ingredient still need to be improved. On the one hand, the quality of recombinant collagen raw materials is uneven, because different amino acid sequences bring differences in the higher-order structure. Many commercially available recombinant collagen raw materials still remain in the single-chain structure. However, the unique triple-helix structure is the basis for collagen to exert biological activity and mobilize in vivo activities. Previous experiments found that the activity of single-chain recombinant collagen (such as promoting cell proliferation, adhesion, etc.) is significantly lower than that of triple-helix recombinant collagen. Therefore, there is still room for further improvement in the effect of improving skin quality of commercially available recombinant collagen raw materials in the existing market. On the other hand, the application of recombinant collagen in biomedicine and cosmetics continues to expand, but the development of multi-protein complex systems faces technical barriers of structural stability and functional synergy. Summary of the Invention

[0004] Based on this, the present invention provides a composition containing recombinant triple-helix collagen that can synergistically enhance the effect, and also provides a preparation method of the composition, which is of great significance to the development of related industries such as the daily chemical industry and the medical beauty industry.

[0005] The first object of the present invention is to provide a composition containing recombinant collagen with a triple-helix structure, and the components of the recombinant collagen are as follows:

[0006] (i) Recombinant humanized type III collagen (rhCOL3);

[0007] (ii) Recombinant humanized type XVII collagen (rhCOL17).

[0008] Preferably, the rhCOL3 in the composition is purchased from Dongguan Hengyi Biotechnology Co., Ltd.

[0009] Preferably, for the rhCOL17 in the composition, its amino acid sequence is as shown in SEQ ID NO:1.

[0010] The examples of the present invention demonstrate certain implementations of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases. In fact, good effects can be achieved at any concentration of each component between the two endpoint values shown in the examples. The working concentration of the recombinant triple-helix collagen composition is 0.01 ppm to 100 ppm, such as 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 50 ppm or 100 ppm, etc., and can also be other values within the range of 0.01 ppm to 100 ppm.

[0011] Preferably, the working concentration of the recombinant triple-helix collagen composition is 0.1 ppm to 10 ppm.

[0012] More preferably, the working concentration of the recombinant triple-helix collagen composition is 1 ppm to 10 ppm.

[0013] Regarding the ratio of the active ingredients in the composition, preferably, the mass ratio of rhCOL3 to rhCOL17 is (1 - 20):(1 - 20). Specifically, in the composition of the present invention, the mass ratio of rhCOL3 to rhCOL17 can be any of the following ratios: 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1.

[0014] The second object of the present invention is to provide the application of the composition.

[0015] In a specific example, the composition is used to promote cell adhesion or for preparing a product that promotes cell adhesion so as to play an anti-wrinkle and firming role.

[0016] Cell adhesion refers to the contact and interaction of cells with adjacent cells through direct or indirect actions. The ways of action include direct action through cell surface contact and indirect action through the extracellular matrix. Most cells in the body (such as epithelial cells) adhere and fix in a certain place to perform their functions. For example, integrin can bind to components of the extracellular matrix such as collagen and fibronectin, attaching the cell to the matrix. Integrin is interconnected with the actin cytoskeleton, and this interaction is stabilized by the binding of focal adhesion proteins (such as talin, tensin, and vinculin). Therefore, the composition of the present invention can maintain the integrity of tissue structure and form skin barrier function by promoting cell adhesion.

[0017] In a specific example, the composition of the present invention is used to promote collagen regeneration or to prepare a product for promoting collagen regeneration so as to play an anti-wrinkle and firming role.

[0018] The determination of type I collagen content is a laboratory method for evaluating the anti-wrinkle efficacy of cosmetics. This method is based on measuring the effect of cosmetics on the type I collagen secreted by human dermal fibroblasts cultured in vitro. Type I collagen is the main structural protein in the dermis of the skin and plays a key role in the firmness and elasticity of the skin. With the increase of age, the synthesis of type I collagen in the skin will decrease, resulting in wrinkles and sagging of the skin.

[0019] Type IV collagen forms a three-dimensional reticular scaffold of the basement membrane at the epidermal-dermal junction, maintaining the tightness of the skin structure. Its reticular structure forms a mechanical barrier by binding laminin, nestin, etc., delaying the formation of skin relaxation and wrinkles caused by aging. It can neutralize free radicals, reduce ultraviolet (UV)-induced oxidative damage, and protect the integrity of the basement membrane. The basement membrane interacts with cell surface integrin through type IV collagen to regulate the balance of keratinocyte proliferation and differentiation, preventing epidermal atrophy (commonly seen in aging skin).

[0020] The present invention evaluates the effect of the composition on collagen synthesis by measuring relative gene expression. For details, please refer to the examples. In addition, those skilled in the art can also use the enzyme-linked immunosorbent assay (ELISA) method to measure the content of collagen. By comparing the type I collagen content in the treatment group and the control group, the ability of cosmetics to promote collagen synthesis can be evaluated.

[0021] In a specific example, the composition of the present invention is used to up-regulate the expression of genes related to skin hydration or to prepare a moisturizing product.

[0022] Preferably, HaCaT cells can synthesize biomolecules related to skin moisturization such as hyaluronic acid (HA), aquaporin (AQP), tight junction protein (TP), and filaggrin (FLG).

[0023] HA is a natural polysaccharide present in the intercellular matrix of skin cells. Its molecular structure contains a large number of hydroxyl groups, which can form intermolecular hydrogen bonds with water, have a strong water retention effect, increase the moisture content of the stratum corneum, and play an important role in skin moisturization. Hyaluronan synthase (HAS) is a class of enzymes that play an important role in the synthesis of HA and can be divided into three types: HAS-1, HAS-2, and HAS-3. Therefore, the effect of cosmetic moisturizing raw materials on the ability of cells to synthesize HA can be evaluated by detecting the gene expression levels of the HAS family members HAS-1, HAS-2, and HAS-3.

[0024] In a specific example, the composition of the present invention is used to down-regulate the expression of inflammatory factor genes or to prepare products for relieving sensitivity.

[0025] The damage of ultraviolet irradiation to biological cell membranes can lead to the release of a series of inflammatory mediators. One of the induction pathways: up-regulating nuclear transcription factor NF-kB, promoting the expression of more secondary inflammatory factors such as tumor necrosis factor TNF-α and inflammatory factor interleukin IL-α and pro-inflammatory factors in the nucleus DNA, and inducing different inflammatory manifestations of the skin through signal transduction and regulatory mechanisms; the activation of IL-1α, the expression and activity of matrix metalloproteinases (MMPs) increase, further degrading collagen and elastin, resulting in a decrease in the elasticity and density (tightness) of the skin and the appearance of skin aging.

[0026] The activation of skin inflammatory factors will lead to a series of skin allergic reactions, such as erythema, edema, burning, pain, itching, etc., which will bring extremely uncomfortable and poor visual effects to people. At the same time, in order to reduce itching, continuous scratching will lead to a vicious cycle and penetrate into the dermis layer, which will cause pigment deposition and scars. Inflammatory factors will also accelerate the aging of the skin, that is, the so-called inflammatory aging.

[0027] Tumor necrosis factor α (TNF-α) is a trimeric structural protein composed of 157 amino acid residues and plays a key role in inflammation regulation. This cytokine mediates various biological processes, such as inflammatory responses and programmed cell death, by binding to receptors such as TNFR1 and TNFR2. However, excessive TNF-α can lead to chronic inflammation and tissue damage (such as rheumatoid arthritis), systemic inflammatory responses (such as septic shock), and the progression of autoimmune diseases. In addition, the activation of the NF-κB signaling pathway mediated by the TNFR2 receptor can promote the proliferation, invasion, and metastatic ability of tumor cells.

[0028] Interleukin-1α (IL-1α) is a key initiator of the inflammatory cascade and is closely related to tumorigenesis, development, and immune regulation. Multiple tumor-related cells can secrete IL-1α and express its receptor IL-1R1, forming an autocrine / paracrine regulatory network. This molecule promotes the expression of stemness genes, proliferation ability, and epithelial-mesenchymal transition of tumor stem cells by regulating the chronic inflammatory microenvironment in the early stage of cancer development. At the same time, the IL-1R1 signaling pathway can induce the high expression of chemokines / adhesion molecules, accelerate the infiltration of immune cells and the secretion of matrix metalloproteinases, thereby enhancing the potential of tumor invasion and metastasis.

[0029] Interleukin-8 (IL-8) is a polypeptide with a low molecular weight and is secreted by various cells, including monocytes, lymphocytes, fibroblasts, endothelial cells, etc. It plays a crucial role in biological processes such as inflammatory responses, cellular immunity, and tumor development. In chronic inflammatory diseases such as rheumatoid arthritis and psoriasis, the production of IL-8 may be abnormal and is involved in the occurrence and development of the diseases. Most tumors can secrete IL-8 to promote their own growth, such as lung cancer, breast cancer, colorectal cancer, etc. The overexpression of IL-8 is closely related to malignant biological behaviors such as the proliferation, invasion, and metastasis of tumor cells.

[0030] Matrix metalloproteinases (MMPs) are a family of endopeptidases whose biological activity depends on zinc ions and have the ability to degrade the extracellular matrix (ECM). An increase in the secretion of MMPs will accelerate the degradation of collagen, resulting in the loss of skin collagen, skin relaxation, decreased elasticity, and the appearance and deepening of fine lines. Elastase, as a proteolytic enzyme with extremely high selectivity and specificity, can break down elastin in connective tissue proteins, leading to skin aging. Therefore, the anti-wrinkle efficacy of cosmetics can be evaluated by inhibiting the activities of metalloproteinases and elastase.

[0031] In a practical scenario, the present invention provides an anti-aging and anti-wrinkle cosmetic, and the preparation raw materials of the cosmetic include the composition and acceptable excipients.

[0032] Preferably, it also contains other active ingredients, stabilizers, and / or preservatives;

[0033] Preferably, the cosmetic contains an acceptable solvent and the acceptable excipients; more preferably, the excipients include at least one of emollients, emulsifiers, thickeners, humectants, skin protectants and preservatives.

[0034] Preferably, the cosmetic is lotion, essence, emulsion, cream, spray, powder or mask.

[0035] In another practical scenario, the present invention provides a medical device with a repair function, including the composition as the main component.

[0036] The cosmetics of the present invention include, but are not limited to, skin care lotion, skin care cream, essence water, mask, scalp essence, shampoo.

[0037] The composition of the present invention can be applied locally to the skin to reduce skin damage or promote the repair of damaged skin. For example, the composition can protect the skin from ultraviolet damage, protect skin cells from the influence of exposure to oxidation, etc., and promote the repair of damaged skin by increasing cell viability and / or increasing the synthesis of procollagen when applied to the skin, and / or promote the viability of skin cells.

[0038] Based on this, the present invention can select a composition including a cosmetically or pharmaceutically effective amount of at least one of the aforementioned proteins and at least one excipient or a cosmetically or pharmaceutically acceptable adjuvant according to the actual application. The dosage forms of the composition include, but are not limited to, creams, emulsions, aqueous solutions, gels, oils, powders, muds, patches, films or freeze-dried products; further, in order to promote the transdermal absorption of collagen molecules, solid carriers such as non-woven fabrics can be used to prepare patch products, which are applied to the skin surface to extend the action time of the collagen solution.

[0039] From the perspective of terminal products, the collagen of the present invention can be applied to daily chemical products. The composition containing recombinant collagen may further contain at least one additional ingredient, which includes other active ingredients, topical carriers, and / or preservatives. Other active ingredients include sodium hyaluronate, tocopherol (vitamin E), tocopheryl acetate, citric acid, panthenol, centella asiatica extract, squalane, simmondsia chinensis seed oil, butyrospermum parkii fruit butter, glycyrrhiza glabra root extract, ectoine, lysate of saccharomyces cerevisiae fermentation products, salicylic acid, hydroxypropyltetrahydropyrantriol, retinol, portulaca oleracea extract, tranexamic acid, limnanthes alba seed oil, retinol palmitate, trehalose, ceramide NP, 3 - O - ethyl ascorbic acid, dipotassium glycyrrhizate, olea europaea leaf extract, allantoin, bisabolol, helianthus annuus seed oil, ascorbyl palmitate, acetyl hexapeptide - 8, arginine, olea europaea fruit oil, ascorbic acid (vitamin C), prunus amygdalus dulcis oil, ascorbyl glucoside, hydrogenated lecithin, glycyrrhiza inflata root extract, citrus aurantium dulcis peel oil, camellia sinensis leaf extract, rosa damascena flower water, phenylethyl resorcinol, carnosine, ascorbyl tetraisopalmitate, filtrate of saccharomyces cerevisiae fermentation products, vitis vinifera seed oil, rosa damascena flower oil, aloe barbadensis leaf water, camellia japonica seed oil, filtrate of galactomyces fermentation products, adenosine, citrus paradisi peel oil, citrus aurantium bergamia fruit oil, erythritol, copper tripeptide - 1, melaleuca alternifolia leaf oil, glycyrrhiza uralensis root extract, dipeptide diamino butyroyl benzylamide diacetate, α - arbutin, madecassoside, ergothioneine.Topical carriers include topical carriers selected from liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerol, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, waxes, sorbitan monostearate, polysorbates, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water. Preservatives include preservatives selected from tocopherol, diiodomethyl p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyl oxazolidine, 7-ethylbicyclo oxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0040] Specifically, the composition containing recombinant collagen is a personal care product, which may contain other cosmetic ingredients suitable for human use. The personal care product can be used to prevent or treat the damage of the external environment to human skin or hair. The application scenarios can be skin cleansing and repair products, hair cleansing and repair products, such as facial masks, serums, creams, soaps, cleansing pastes, cleansing milks, facial cleansers, shampoos, conditioners, skin lotions, and body washes, etc.

[0041] The humectants include at least one of glycerol, propylene glycol, butylene glycol, sodium hyaluronate, saccharides, and polyethylene glycol; the thickeners include at least one of xanthan gum, carbomer, cellulose, carrageenan, gellan gum, polyacrylic acid copolymers, and magnesium aluminum silicate.

[0042] The anti-wrinkle effect is essentially a refined branch of the anti-aging effect, so there are many commonalities in the in vitro efficacy evaluation methods. The in vitro evaluation methods include biochemical methods, cell biology methods, and three-dimensional reconstructed skin model substitution methods, etc. At present, the research on anti-aging has penetrated from the apparent morphology to the changes in the deep skin structure before and after using the product. In the anti-wrinkle cell experiments of cosmetics, human skin keratinocytes, fibroblasts, or three-dimensional reconstructed skin models are usually used to study the effects of anti-wrinkle ingredients on cell growth and proliferation. Fibroblasts can be normal cells or cells damaged by ultraviolet rays or H 2 O 2 2O2. After culturing for a period of time under the condition of the presence of a certain concentration of anti-wrinkle agent, the keratinocytes evaluate the efficacy of the anti-wrinkle agent by testing the hyaluronic acid (HA) index, and the fibroblasts evaluate the efficacy of the anti-wrinkle agent by testing indexes such as cell viability, related enzyme activities, cell antioxidant capacity, and collagen production promotion.

[0043] Biochemical methods generally include free radical scavenging experiments, superoxide anion radical experiments, hydroxyl radical experiments, oxygen radical absorbance capacity experiments, and experiments on inhibiting metalloproteinases and elastases.

[0044] The ability of cosmetic raw materials to scavenge free radicals can be evaluated by detecting the contents of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX) and apoptosis indexes in HaCaT cells, which is widely used in the efficacy evaluation of antioxidant cosmetic raw materials.

[0045] The application of the composition described in the present invention in the preparation of medical devices.

[0046] The medical materials described in the present invention include, but are not limited to, filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, drug delivery carriers, and the repair materials include, but are not limited to, bone repair materials, wound dressings, and sutures. More specifically, there are mainly collagen dressings, dura (spinal) mater patches, artificial corneas, oral patches, hernia patches, breast plastic surgery patches, etc., and the uses include, but are not limited to, wound healing, skin repair, dermatitis, eczema, hemorrhoids, oral mucositis, oral ulcers, scars, and allergic rhinitis, etc.

[0047] In the present invention, "comprising", "including", "containing" and "having" are generalizations of technical solutions, meaning that the described solutions may have other elements of the same type or elements that can achieve the same or similar effects in addition to the listed elements. When "and / or" is used herein, it includes the meanings of "and", "or" and "any other combination of all or part of the elements linked by the term".

[0048] In the present invention, "collagen" has the same meaning as "COL" (Collagen), that is, "type I collagen", "COL1", and "COLⅠ" can be used interchangeably; "recombinant humanized collagen" has the same meaning as "rhCOL" (Recombinant Humanized Collagen), that is, "recombinant humanized type III collagen", "rhCOL3", and "rhCOLⅢ" can be used interchangeably.

[0049] The numerical ranges and parameters involved in the present invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations caused by individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all numerical ranges or specific data used in the present disclosure may have certain reasonable deviations within a certain range.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The composition containing recombinant triple-helix collagen provided by the present invention can play a synergistic effect on various biological functions, providing a better active ingredient for the preparation of skin care products, cosmetics and medical devices. Description of the Drawings

[0052] Figure 1 Effects of single components at different concentrations and the composition of the present invention on the adhesion activity of 3T3 cells.

[0053] Figure 2 Effects of single components at different concentrations and the composition of the present invention on the expression of type I and type IV collagen in 3T3 cells.

[0054] Figure 3 Effects of single components at different concentrations and the composition of the present invention on the expression of type I, type III and type IV collagen in HFF-1 cells.

[0055] Figure 4 Effects of single components at different concentrations and the composition of the present invention on the gene expression of hyaluronic acid (HAS2) and aquaporin (AQP3) in HFF-1 cells.

[0056] Figure 5 Effects of single components at different concentrations and the composition of the present invention on the gene expression of inflammatory factors (IL-1α, IL-8, TNF-α) in the HaCaT cell injury model. Detailed Embodiments

[0057] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] In specific embodiments, unless otherwise specified, biological materials such as cells, reagents and consumables involved can be obtained by purchase.

[0059] The recombinant humanized type III collagen (rhCOL3) selected in the following examples was purchased from Dongguan Hengyi Biotechnology Co., Ltd. The protein purity was ≥95% detected by HPLC. An appropriate amount of collagen lyophilized powder was accurately weighed, dissolved in phosphate buffer, and magnetically stirred (500 rpm, 25 °C) until completely dissolved. The final concentration was 20 mg / mL, and it was filtered through a 0.22 μm sterile filter membrane and stored for later use. The recombinant humanized type XVII collagen (rhCOL17) was produced by Liying Bio, and its amino acid sequence was as shown in SEQ ID NO:1. It was also prepared into a solution with a final concentration of 20 mg / mL by the same method.

[0060] Example 1 Detection of the cell adhesion promoting activity of the composition of the present invention

[0061] Cell adhesion function is a fundamental biological mechanism for maintaining tissue morphological integrity. By regulating cell-cell junctions and cell-extracellular matrix interactions, it directly affects key physiological processes such as cell migration, differentiation, and signal transduction. The present invention discovers that recombinant humanized type III collagen (rhCOL3, purchased from Hengyi Biotech) and type XVII collagen (rhCOL17, produced by Liying Biotech) can synergistically activate the focal adhesion pathway mediated by integrin receptors when formulated in a specific ratio, significantly enhancing the binding efficiency of extracellular matrix receptors. The specific steps are as follows:

[0062] 1. Substrate coating: Take rhCOL3, rhCOL17, and the formulated composition of the present invention (the mass ratio of rhCOL3 to rhCOL17 is 1:1, 2:1, 4:1, 5:1, 10:1, 20:1), and dilute them with phosphate buffer (PBS) to a final concentration of 5 μg / mL respectively. Set a control group of bovine serum albumin (BSA) solution (5 μg / mL, diluted with PBS), and add each solution to a 96-well cell culture plate at 100 μL / well, and let it stand and coat at 4°C for 16 - 18 hours.

[0063] 2. Blocking non-specific binding: Discard the coating solution, and add DMEM medium (Pronocyte) containing 1% BSA to each well at 200 μL / well. After incubating at 37°C for 1 hour, wash with PBS 3 times (5 minutes each time, shaker speed 50 rpm).

[0064] 3. Cell seeding and culture: Digest 3T3 fibroblasts (purchased from Haixing Biotech) and resuspend them in DMEM medium containing 10% fetal bovine serum, and adjust the density to 5×10^4 cells / mL. Seed them into the pre-coated culture plate at 5000 cells / well (i.e., 100 μL / well), and culture them at 37°C and 5% CO 2 under constant humidity for 24 hours.

[0065] 4. Removal of non-adherent cells: Tilt the culture plate to 30°, slowly aspirate the medium, gently add pre-warmed PBS (37°C) along the well wall, let it stand for 1 minute and then aspirate, repeat 3 times.

[0066] 5. Quantitative analysis of adherent cells: Add 100 μL of medium containing 10% CCK-8 reagent (Beyotime, catalog number C0040) to each well, incubate at 37°C in the dark for 2 hours, and mix by oscillation every 20 minutes (amplitude 3 mm, frequency 60 times / minute). Use a multi-functional microplate reader (MD) for dual-wavelength detection. The main detection wavelength is 450 nm (measuring the absorbance of formazan products), and the reference wavelength is 630 nm (subtracting background interference). Set up a blank control well containing only CCK-8 medium without cells.

[0067] Adhesion efficiency calculation formula: Adhesion rate = (A 实验组 - A 空白 ) / (A 对照组 - A 空白 ) × 100%

[0068] Where:

[0069] A 实验组 : Absorbance value of the compound group (main wavelength - reference wavelength)

[0070] A 对照组 : Absorbance value of the well containing cells and culture medium

[0071] A 空白 : Absorbance value of the cell-free well

[0072] Experimental results show (as Figure 2 shown), when the mass ratio of rhCOL3 to rhCOL17 is between 5:1 and 10:1, the adhesion rate reaches the peak (45 - 62% higher than that of the single component).

[0073] Example 2 Quantitative analysis of the regulatory effect of the composition of the present invention on collagen genes in 3T3 cells

[0074] Resuscitate mouse 3T3 fibroblasts (HaiXing Bio), and passage and culture them using DMEM high-glucose medium (PuNuoSai) containing 10% fetal bovine serum (Gibco). When the cell confluence reaches 90%, digest with 0.25% trypsin-EDTA (Gibco) to prepare a single-cell suspension. Inoculate into a 6-well plate (BIOFIL) at a density of 4×10^5 cells / well, and culture under the conditions of 37°C and 5% CO 2 for 12 hours to ensure that the cells enter the logarithmic growth phase (confluence 80 ± 3%). Aspirate the culture medium, and gently wash twice with pre-cooled PBS (4°C) (2 mL / well each time, vortex oscillation frequency 30 rpm). Add serum-free medium containing 5 μg / mL of the test substance, with the blank control being PBS solution. The experimental groups are divided into single components of rhCOL3 or rhCOL17, and the mass ratios of rhCOL3 to rhCOL17 in the compound group are 1:1, 2:1, 4:1, 5:1, 10:1, and 20:1 in sequence. Continuously intervene at 37°C for 24 hours, and record the incubator parameters (temperature fluctuation ≤ 0.2°C, CO 2 concentration deviation ≤ 0.1%) every hour during this period. After protein pretreatment, wash 3 times with PBS and aspirate the residual liquid. Add 0.5 mL of TRIzol TMReagent (Novizan). Use a sterile cell scraper to mechanically scrape adherent cells and aspirate and blow repeatedly until the solution is homogenized. Transfer the lysate to an RNase-free centrifuge tube (Nest) and operate according to the instructions of the Novizan RNA extraction kit (RC101-01): chloroform phase separation (12000g, 4°C, 15 minutes), RNA precipitation (isopropanol addition ratio 1:1, stand at -20°C for 30 minutes), wash with 75% ethanol (centrifugation conditions 7500g, 4°C, 5 minutes), dissolve RNA in 20 μL of DEPC water, and measure A260 / A280 = 1.92 ± 0.05 with Nanodrop. For quantitative PCR analysis, take 1 μg of total RNA, use HiScript III RT SuperMix (Aikerui) to synthesize cDNA, and set the program as follows: 25°C for 5 minutes (genomic DNA removal), 50°C for 15 minutes (reverse transcription reaction), 85°C for 5 minutes (enzyme inactivation). The qPCR reaction system is: 10 μL of SYBR Green Master Mix (Aikerui), 0.8 μL each of forward / reverse primers (10 μM), 2 μL of cDNA template, and 6.4 μL of RNase-free water. Amplification program: pre-denaturation at 95°C for 30 seconds, cycling stage 95°C for 5 seconds → 60°C for 34 seconds, 40 cycles, melting curve from 60°C to 95°C, heating rate 0.3°C / second. Calculate the relative gene expression level using the 2^(-ΔΔCt) method, and select GAPDH as the internal reference gene.

[0075] 2^(-ΔΔCt) is a method for calculating relative expression levels, where ΔΔCt represents the change in the cycle threshold (Ct) after baseline correction. The following are the calculation steps for 2^(-ΔΔCt):

[0076] Calculate ΔCt for a single sample: For each sample, first calculate the Ct difference between the target gene and the reference gene.

[0077] The formula is: ΔCt = Ct_Target - Ct_Reference

[0078] Here, Ct_Target is the cycle threshold of the target gene, and Ct_Reference is the cycle threshold of the reference gene.

[0079] Calculate the average ΔCt of the control samples: Select a set of control samples (usually calibration samples or blank controls) and calculate the average of their ΔCt values.

[0080] The formula is: ΔCt_mean = mean(ΔCt_controls)

[0081] Calculate ΔΔCt: For each sample to be tested, calculate the difference between its ΔCt and the average ΔCt of the control samples.

[0082] The formula is: ΔΔCt = ΔCt - ΔCt_mean

[0083] Calculate the relative expression level (2^(-ΔΔCt)): Finally, use the formula 2^(-ΔΔCt) to calculate the relative expression level of the target gene relative to the control samples. This formula is based on the assumption that the cycle threshold (Ct) is inversely proportional to the initial template amount, and the 2^x function can reflect this inverse relationship. By this method, the relative expression levels of the target gene in different samples can be quantified. It should be noted that this calculation method assumes that the expression of the reference gene is stable in all samples and the amplification efficiency is the same. In practical applications, it should be ensured that these assumptions hold or appropriate corrections are made.

[0084] The results are as Figure 2 shown. The compound group of the present invention upregulates the expression of COL1, COL3, and COL4 genes in 3T3 cells and has the effect of promoting collagen regeneration.

[0085] Example 3 Quantitative analysis of the regulatory effect of the composition of the present invention on collagen genes in HFF-1 cells

[0086] Refer to the methods and steps of Example 2 to conduct experiments. Resuscitate and passage HFF-1 cells (Cell Bank of the Chinese Academy of Sciences). After the cells are plated for 24 h, add serum-free medium containing 5 μg / mL of the test substance. The blank control is PBS solution. The experimental groups are divided into single-component groups of rhCOL3 or rhCOL17, and the compound group with the mass ratio of rhCOL3 to rhCOL17 being 4:1 and 5:1 in sequence.

[0087] The results are as Figure 3 shown. The composition of the present invention upregulates the expression of COL1 and COL3 genes in HFF-1 cells compared with the single component, further verifying that the composition of the present invention has the regulatory effect of promoting collagen regeneration.

[0088] Example 4 Quantitative analysis of the regulatory effect of the composition of the present invention on hyaluronan synthase and aquaporin genes in HFF-1 cells

[0089] Hyaluronan synthase (HAS) and aquaporin (AQP) are the core molecular targets for maintaining skin hydration and barrier function, and their expression is co-regulated by mechanical stress, osmotic pressure, and the extracellular matrix microenvironment. The present invention reveals that the compound of rhCOL3 and rhCOL17 can regulate the gene expression profiles of hyaluronan synthase (HAS) and aquaporin (AQP) in HFF-1 cells.

[0090] Perform experiments according to the methods and steps of Reference Example 2. Resuscitate and passage HFF-1 cells (Cell Bank of the Chinese Academy of Sciences). After the cells are plated for 24 hours, add serum-free medium containing 5 μg / mL of the test substance. The blank control is a PBS solution, and the experimental groups are compound groups with the mass ratios of rhCOL3 to rhCOL17 being 4:1, 5:1, 10:1, and 20:1 in sequence.

[0091] The results are as Figure 4 shown. The composition of the present invention has a significant regulatory effect on upregulating hyaluronan synthase (HAS2) and aquaporin (AQP3) in HFF-1 cells.

[0092] Example 5 Quantitative analysis of the regulatory effect of the composition of the present invention on inflammatory factors in HaCaT cells

[0093] Oxidative stress-induced inflammatory damage is the core pathological mechanism of skin barrier dysfunction, which triggers the cascade release of pro-inflammatory factors through the ROS-NF-κB / NLRP3 signaling axis. The present invention finds that the compound system of rhCOL3 and rhCOL17 can alleviate the inflammatory damage of HaCaT cells caused by H 2 O 2 .

[0094] Perform experiments according to the methods and steps of Reference Example 2. Resuscitate and passage human immortalized keratinocytes HaCaT (Cell Bank of the Chinese Academy of Sciences), and inoculate them into 6-well plates (BIOFIL) at a density of 4.5×10^5 cells / well. Incubate at 37°C and 5% CO 2 for 24 hours until the logarithmic growth phase (confluence 85±2%). Add serum-free medium containing 5 μg / mL of the test substance. The blank control is a PBS solution, and the damage control is 800 μM H 2 O 2 . The experimental groups are divided into single-component groups of rhCOL3 or rhCOL17, and compound groups with the mass ratios of rhCOL3 to rhCOL17 being 1:1, 4:1, and 5:1 in sequence. After pretreatment at 37°C for 24 hours, except for the negative control group, 800 μM H 2 O 2 is added to the other groups to induce damage for 4 hours.

[0095] The results are as Figure 5 shown. In the damage control group, the levels of inflammatory factors IL-1α, IL-8, and TNF-α are significantly upregulated, indicating that the cell damage model is successfully established. Compared with the single component, the composition of the present invention downregulates the expression of inflammatory factors and alleviates the inflammatory response.

Claims

1. A composition comprising recombinant collagen having a triple helix structure, characterized in that: The recombinant collagen ingredients are as follows: (i) recombinant humanized type III collagen (rhCOL3); (ii) recombinant humanized type XVII collagen (rhCOL17); The rhCOL3 was purchased from Dongguan Hengyi Biotechnology Co., Ltd., and the rhCOL17 in the composition has an amino acid sequence as shown in SEQ ID NO:

1.

2. The composition according to claim 1, characterized in that The mass ratio of the recombinant humanized type III collagen to the recombinant humanized type XVII collagen is (1-20):(1-20).

3. The composition of claim 1 or 2 is used for promoting cell adhesion or for preparing a product that promotes cell adhesion and thus has an anti-wrinkle and firming effect.

4. The composition of claim 1 or 2 is used for promoting collagen regeneration or for preparing a product that promotes collagen regeneration and thus has an anti-wrinkle and firming effect.

5. The composition of claim 1 or 2 is used for up-regulating the expression of genes related to skin hydration or for preparing moisturizing products.

6. The composition of claim 1 or 2 is used for down-regulating the expression of inflammatory factor genes or for preparing allergy-relieving products.

7. A cosmetic, characterized in that: The preparation materials of the cosmetics include a cosmetically effective amount of the composition and an excipient or cosmetically acceptable adjuvant.

8. The cosmetic according to claim 7, characterized in that: It also contains other biologically active ingredients, including one or more of Bossein, Ecdoin, Annoin, and sodium hyaluronate.

9. A medical device, characterized in that: The medical device comprises the composition according to claim 1 or 2 as a main component.

10. The medical device according to claim 9, characterized in that: The medical device is a filling material, a repair material, an implant, a tissue engineering scaffold, a hemostatic agent or a drug release carrier.

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