Application of recombinant III-type humanized collagen in heat-induced melanogenesis and keratinogenesis cell migration ability decline

Abstract

The invention provides an application of recombinant III type humanized collagen in thermal induction of melanogenesis and keratinogenesis cell migration capability reduction, wherein the amino acid sequence of the recombinant III type humanized collagen is as shown in SEQ ID NO.3. The invention also provides an application of the recombinant III type humanized collagen in thermal induction of melanogenesis and keratinogenesis cell migration capability reduction. Test results show that the recombinant III-type humanized collagen can significantly reduce melanogenesis under different heating environment conditions, and the repair effect of cell migration ability decline in the heating environment is improved. According to the invention, the application potential of the recombinant III-type humanized collagen in skin care products is expanded, and the multifunctionality of the recombinant III-type humanized collagen as a novel skin care component is shown. Besides, the skin care product containing the recombinant III-type humanized collagen provided by the invention also has the advantages of safety, effectiveness and no skin irritation or allergy, and the protection effect of the skin care product is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and cosmetics, and specifically relates to the application of recombinant type III humanized collagen in heat-induced melanin production and decreased migration ability of keratinocytes. Background Art

[0002] As global warming intensifies, people are increasingly concerned about the impact of high temperatures on skin health. Recent studies have found that skin is susceptible to various types of damage in high temperature environments.

[0003] Melanin cells are cells in the skin that are responsible for producing melanin. The melanin they produce can be transferred to the surrounding keratinocytes. Their main function is to protect the skin from UV damage by producing melanin. However, in a high temperature environment, even if the influence of UV rays is completely isolated, melanin will be overproduced, causing the skin to darken, affecting the appearance of the skin and people's mental health. Therefore, in the context of rising summer temperatures year by year, finding effective skin care ingredients to inhibit or slow down the production of melanin induced by high temperatures is bound to meet the relevant needs of consumers.

[0004] The migration ability of keratinocytes is crucial for wound healing, but under high temperature conditions, this migration ability may be inhibited, resulting in a decrease in wound healing ability, which will further aggravate the damage to the skin in high temperature environment.

[0005] However, there are few studies on heat-induced skin damage. Although there are reports on high temperature-induced melanocyte darkening in the prior art, they mainly focus on the description of the phenomenon of color changes of melanocytes caused by extreme high temperature conditions (such as melanocyte darkening), and fail to explore in detail the effect of slight temperature increases on melanocyte color changes. There is no report on any substance that can prevent and / or improve high temperature-induced melanocyte darkening, and the protective effect of skin care products in this area is also unclear. Existing skin care products mainly rely on traditional whitening skin care ingredients (such as kojic acid, arbutin, vitamin C and niacinamide, etc.), and the effect is limited.

[0006] The whitening ingredients in existing skin care products are mostly chemical inhibitors, such as kojic acid and arbutin, which reduce melanin production by inhibiting tyrosinase activity. There are also very few evaluations of scar repair ability under high temperature conditions. In addition, these chemical inhibitors may cause skin irritation and allergic reactions, and long-term use poses a safety hazard. At the same time, the effect of avoiding or alleviating skin darkening caused by high temperature is still unclear.

[0007] The efficacy of existing skin care products is mainly focused on preventing UV damage, and there is little research on the increased melanin production and scar repair ability caused by temperature increases in different scenarios (such as 39°C and 41°C) under the background of global warming. In addition, no existing commercially available skin care products claim to protect against skin damage caused by high temperatures.

[0008] Collagen is a type of protein widely distributed in human connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Its main functions are to maintain the extracellular environment, maintain the normal physiological functions of tissues and organs, and repair body damage. Collagen is a natural biological resource with bio-tissue compatibility, cell support elasticity and degradability that other polymer materials cannot match. As an important skin care ingredient, collagen has been widely used due to its good moisturizing, anti-wrinkle and repair functions.

[0009] Traditional collagen extraction methods have problems such as high cost, waste of resources, and potential allergic risks. Specifically, traditional collagen extraction methods are mainly extracted from animal tissues (such as cowhide and fish skin). Although these methods can obtain high-purity collagen, they are costly and there is a waste of resources and allergic risks. In recent years, the rapid development of synthetic biology has provided new methods for the production of collagen and brought new opportunities to the skin care industry. With the development and application of synthetic biology technology, recombinant collagen has been mass-produced, but its specific functions have not yet been fully explored.

[0010] Through microbial fermentation technology, type III collagen can be produced efficiently and environmentally friendly. Among them, recombinant humanized type III collagen has good cell adhesion, moisturizing, anti-wrinkle, and repair functions, but its specific function in preventing and treating melanin production caused by slight temperature increases is still unclear.

[0011] The patent application with application number CN201811438582.6 and invention name “Polypeptides, production methods and uses thereof” (CN109593126 A) records the recombinant type III humanized collagen previously studied and produced by the inventors of the present invention, but the patent application does not disclose the specific use of the recombinant type III humanized collagen in the medical field. Summary of the invention

[0012] Problem that the invention aims to solve

[0013] The research on the skin damage effect caused by temperature increase in the prior art is still unclear. First, the specific mechanism by which high temperature causes melanocytes to overproduce melanin is still unclear, and there is a lack of systematic research, which limits the development of effective protective measures. Secondly, under high temperature scenarios, the migration ability of keratinocytes decreases, resulting in a weakened wound healing ability, and this problem has not received sufficient attention. Traditional collagen extraction methods rely on animal tissues, which are costly and have the risk of waste of resources and allergies, limiting their application in skin care products. In addition, existing studies also have the defects of insufficient functional exploration and verification of recombinant type III humanized collagen in high temperature-induced melanocyte darkening and restoration of keratinocyte migration ability. The present invention aims to provide a recombinant type III humanized collagen for use in heat-induced melanin production and decreased keratinocyte migration ability, solve the above-mentioned technical problems, and expand its application potential in skin care products.

[0014] Solutions for solving problems

[0015] In view of the problems existing in the above-mentioned prior art, the inventors conducted in-depth research and repeated experiments, and used the recombinant type III humanized collagen in the patent application with application number CN201811438582.6 as a test material to evaluate its function in preventing and improving melanin production and decreased migration ability of keratinocytes caused by slight temperature increases, thereby completing the present invention. That is, the present invention is as follows: Specifically, the present invention provides an application of recombinant type III humanized collagen in heat-induced melanin production and decreased migration ability of keratinocytes. The results of the study showed that recombinant type III humanized collagen can significantly reduce melanin production under different temperature rise environments, and enhance the migration ability of keratinocytes, promote wound healing, and thus effectively improve the damage caused to the skin by high temperature environments. This invention not only expands the application potential of recombinant type III humanized collagen in skin care products, but also demonstrates its versatility and safety as a new skin care ingredient.

[0016] In a first aspect, the present invention provides a use of recombinant type III humanized collagen in the preparation of a composition for preventing and / or improving high temperature-induced melanocyte darkening, wherein the recombinant type III humanized collagen comprises n repetitions of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

[0017] In a second aspect, the present invention provides a use of recombinant type III humanized collagen in the preparation of a composition for preventing and / or improving a decreased scar repair ability caused by a decreased migration ability of keratinocytes induced by high temperature, wherein the recombinant type III humanized collagen comprises n repetitions of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

[0018] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the recombinant humanized type III collagen contains the sequence shown in SEQ ID NO.2 or does not contain the sequence shown in SEQ ID NO.2.

[0019] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the recombinant humanized type III collagen comprises:

[0020] a) the amino acid sequence of SEQ ID NO.3;

[0021] b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature;

[0022] c) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by decreased migration ability of keratinocytes induced by high temperature; or,

[0023] d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with the amino acid sequence encoding SEQ ID NO.3 under strict conditions, and the amino acid sequence retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature; the strict conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0024] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the composition is a medical device, a tissue engineering product, a cosmetic or a health product.

[0025] In some preferred technical solutions, the composition is a cosmetic.

[0026] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the high temperature is 37°C to 50°C; in some preferred technical schemes, the high temperature is 38°C to 45°C; in some most preferred technical schemes, the high temperature is 39°C to 41°C.

[0027] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the recombinant humanized type III collagen is prepared by a method comprising the following steps:

[0028] (1) culturing a host cell encoding the sequence shown in SEQ ID NO. 3 in a production medium and producing recombinant type III humanized collagen;

[0029] (2) harvesting the polypeptide; and

[0030] (3) Purifying recombinant humanized type III collagen by Ni column and / or anion exchange chromatography.

[0031] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the method further comprises the step of enzymatically cleaving the recombinant humanized type III collagen with TEV protease.

[0032] In some specific embodiments, in the use of the first aspect or the second aspect of the present invention, the purified recombinant humanized type III collagen contains endotoxins below 5 EU / ml.

[0033] Effects of the Invention

[0034] It can be seen from the technical solution of the present invention that, compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0035] 1) The present invention clarifies the melanin production effect caused by different temperature increase scenarios through experiments.

[0036] The present invention solves the technical problem in the prior art that the effect of different temperature increase scenarios (39° C. and 41° C.) on melanin production is unclear, and verifies and reveals the specific effect of this process through experimental data.

[0037] The present invention clarifies and reveals the melanin production effect caused by slight temperature increases (such as 39°C and 41°C) by describing the phenomenon of high temperature-induced melanin production and analyzing existing melanin prevention and control methods, filling the gap in the prior art and embodying the advantages of the present invention in solving the problem of melanin production caused by different temperature increase scenarios.

[0038] 2) The present invention discovered through experiments that the migration ability of keratinocytes decreases under elevated temperature conditions.

[0039] The present invention has experimentally clarified the negative impact of elevated temperature, such as 39°C, on the migration ability of keratinocytes, solving the problem of insufficient attention paid to the decreased migration ability of keratinocytes under elevated temperature conditions in the prior art. This discovery is of great significance for understanding the mechanism of skin damage under elevated temperature conditions.

[0040] 3) The present invention provides an effective means of preventing and improving skin damage caused by high temperature.

[0041] The skin care products in the prior art mainly rely on chemical inhibitors to achieve the whitening and repairing effects of the skin, which have limited effects and may have side effects, and cannot specifically prevent and improve the darkening of the skin and the decline in the repair ability caused by high temperature. The present invention provides the use of recombinant type III humanized collagen to effectively inhibit melanin production and enhance the migration ability of keratinocytes in a slightly elevated temperature environment, ensuring safety and effectiveness. The technical solution provided by the present invention solves the defect that the existing protective means have limited effects. The technical solution of the present invention is safe and effective, will not cause skin irritation or allergies, and significantly improves the protective effect of skin care products.

[0042] 4) The present invention explores the new efficacy of recombinant humanized type III collagen.

[0043] In response to the problem of insufficient functional exploration of recombinant humanized type III collagen in the field of skin care products, the present invention verifies for the first time its effectiveness in preventing and treating melanin production and enhancing the migration ability of keratinocytes under elevated temperature conditions, expanding its application potential in skin care products.

[0044] The present invention uses synthetic biology technology to efficiently and environmentally produce recombinant type III humanized collagen, which solves the problems of high cost, waste of resources and allergy risk of traditional extraction methods, reduces production costs, improves resource utilization efficiency, and meets environmental protection requirements. The recombinant type III humanized collagen produced by synthetic biology technology not only has moisturizing, anti-wrinkle, and repair functions, but also demonstrates potential application value in preventing and treating melanin production caused by slight temperature increases.

[0045] In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following preferred embodiments are specifically cited and described in detail with reference to the accompanying drawings: BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The blank control, positive control (arbutin) and recombinant type III humanized collagen inhibit the melanin production of melanoma B16F10 cells under different temperature conditions (39°C and 41°C).

[0047] Figure 2 The blank control, positive control (EGF), and recombinant humanized type III collagen affect the migration ability of keratinocytes (HaCaT cells) under elevated temperature conditions (39°C).

[0048] Figure 3 This is a quantitative data analysis of the effects of blank control, positive control (EGF), and recombinant type III humanized collagen on the migration ability of keratinocytes (HaCaT cells) under elevated temperature conditions (39°C). DETAILED DESCRIPTION

[0049] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0050] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0051] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0052] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0053] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0054] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0055] The recombinant humanized type III collagen described in the present invention is recorded in a patent application with application number CN201811438582.6 and invention name “Polypeptide, production method and use thereof”, and its content is incorporated into the present invention by reference.

[0056] In some specific embodiments, the recombinant humanized type III collagen of the present invention comprises n repetitions of the sequence shown in SEQ ID NO.1, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected. In a more preferred embodiment, the recombinant humanized type III collagen of the present invention comprises 16 repetitions of the sequence shown in SEQ ID NO.1, and each repeated sequence is directly connected.

[0057] SEQ ID NO. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0058] Furthermore, the recombinant humanized type III collagen contains the sequence shown in SEQ ID NO.2 or does not contain the sequence shown in SEQ ID NO.2.

[0059] SEQ ID NO. 2: GPPGPCCGGG.

[0060] Furthermore, the recombinant humanized type III collagen comprises:

[0061] a) the amino acid sequence of SEQ ID NO.3;

[0062] b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature;

[0063] c) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by decreased migration ability of keratinocytes induced by high temperature; or,

[0064] d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO.3 under strict conditions, and the amino acid sequence retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature, and the strict conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0065] SEQ ID NO.3:

[0066] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE

[0067] KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRG

[0068] PAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP

[0069] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE

[0070] KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRG

[0071] PAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP

[0072] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE

[0073] KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRG

[0074] PAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0075] The embodiments of the present invention will be described in detail below in conjunction with the examples, and those skilled in the art will appreciate. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if no specific conditions are specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0076] [Test materials]

[0077] Mouse skin melanoma cells (B16F10 cells) were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences.

[0078] Human epidermal keratinocytes (HaCaT cells) were purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences.

[0079] The Masson-Fontana melanin staining kit was purchased from Solarbio (Shanghai).

[0080] Epidermal growth factor (EGF) positive control was purchased from Solarbio Company (Shanghai).

[0081] L-DOPA (levodopa) reagent was purchased from Solarbio (Shanghai).

[0082] Arbutin was purchased from MedChemExpress (MCE) Company (Shanghai).

[0083] Filter-type multifunctional microplate reader, model: Tecan Infinite F200 Pro. Recombinant type III humanized collagen: recombinant type III humanized collagen with an amino acid sequence as shown in SEQ ID NO.3.

[0084] Example 1. Use of recombinant humanized type III collagen to inhibit melanin levels in B16F10 cells under elevated temperature conditions

[0085] Mouse skin melanoma cells (B16F10 cells) in the logarithmic growth phase were digested with 1 mL of 0.25% trypsin for 1 minute and then centrifuged. The supernatant was discarded and fresh culture medium (DMEM containing 10% FBS) was added. The cells were resuspended and the cell density was adjusted to 1.5×10 5 Pieces / mL.

[0086] The digested cells were inoculated into 6-well plates, 2 mL per well, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells adhered, the old culture medium was removed, and 2 mL of different treatment solutions (DMEM containing 2% FBS) were added to each well.

[0087] The treatment groups were set up as: distilled water (blank control), 100 μg / mL arbutin (positive control) and 100 μg / mL recombinant humanized type III collagen (experimental group). After 2 hours of treatment, the cells were transferred to 39°C and 41°C incubators, respectively, and exposed to heat for 4 hours every day for 2 days. The rest of the time was returned to the 37°C incubator for continued culture, in order to simulate the actual situation of human skin being affected by high temperatures in summer.

[0088] After 48 hours of culture, discard the culture medium and rinse the cells twice with PBS. Add 800 μL of 4% paraformaldehyde solution to each well to fix the cells for 20 minutes. After fixation, discard the paraformaldehyde and rinse the cells 3 times with distilled water. Add 800 μL of 10% ammonia silver solution to each well and place the cells in a 56°C incubator for staining for 30 minutes. After staining, discard the ammonia silver solution and rinse the cells 5 times with distilled water. Then add 5% sodium thiosulfate solution (Hypo solution) to each well and balance for 3-5 minutes, then observe under a microscope and take pictures for record.

[0089] like Figure 1 As shown: (i) Under 37℃, the cellular melanin content of the blank control group, arbutin group and recombinant type III humanized collagen group was low. (ii) Under the conditions of 39℃ and 41℃, the cellular melanin content of the blank control group increased significantly, showing obvious temperature dependence; while the melanin content of the arbutin group and recombinant type III humanized collagen group decreased significantly, among which the treatment effect of recombinant type III humanized collagen was better than that of the arbutin group.

[0090] The above results indicate that recombinant humanized type III collagen can effectively inhibit the production of melanin in B16F10 cells under elevated temperature conditions, and has potential skin care effects, which can prevent and improve skin darkening caused by high temperature.

[0091] Example 2. Recombinant humanized type III collagen is used to improve the repair effect of impaired migration ability of HaCaT cells under elevated temperature conditions

[0092] Human epidermal keratinocytes (HaCaT cells) were cultured at 2×10 5 / mL was evenly distributed in a 6-well culture plate to form a monolayer of cells. After culturing in the culture plate, it was marked and 2mL of fresh culture medium (MEM containing 10% FBS) was added to each well for 48 hours. A 1mL pipette tip was used to scratch a cell-free area, and the cells were washed twice with PBS solution to remove the detached cells. Subsequently, serum-free culture medium MEM was added as a negative control group (negative control, NC), the substance added to the positive control group (positive control, PC) was EGF (1ng / mL), and the substance added to the experimental group (PRO) was recombinant type III humanized collagen solution, with a concentration of (0.01mg / mL); all were dissolved in serum-free MEM culture medium for drug treatment. At the same time, images were collected under a microscope and the positions were marked, recorded as 0h. After 24h and 48h, the cell migration was observed and recorded, and photos were taken and recorded, recorded as 24h and 48h respectively. The area of ​​the scratch area in each image was analyzed using Image Pro Plus software. The relative migration area was calculated with the scratch area at 0h as 100%.

[0093] like Figure 2 As shown: (i) Under 39°C conditions, the cell migration ability of the negative control group (NC) was significantly reduced compared with that under 37°C conditions, and the positive control group (PC) showed the same trend. (ii) The addition of recombinant humanized type III collagen (PRO) significantly improved the cell migration ability that was inhibited in a high temperature environment.

[0094] Figure 3 for Figure 2 Quantitative data analysis showed that recombinant humanized type III collagen significantly improved the migration ability of HaCaT cells under high temperature conditions.

[0095] The above results indicate that recombinant humanized type III collagen can effectively repair the migration ability of HaCaT cells under elevated temperature conditions, has potential skin care effects, and can be used to improve skin damage caused by high temperature.

Claims

1. Use of recombinant humanized type III collagen in the preparation of a composition for preventing and / or improving hyperthermia-induced melanocyte darkening, in, The recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

2. Use of recombinant humanized type III collagen in the preparation of a composition for preventing and / or improving the decreased scar repair ability caused by decreased migration ability of keratinocytes induced by high temperature, in, The recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24, or 32, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

3. The use according to claim 1 or 2, characterized in that The recombinant humanized type III collagen contains the sequence shown in SEQ ID NO.2 or does not contain the sequence shown in SEQ ID NO.

2.

4. The use according to any one of claims 1 to 3, characterized in that The recombinant humanized type III collagen comprises: a) the amino acid sequence of SEQ ID NO.3; b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature; c) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO.3, which retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by decreased migration ability of keratinocytes induced by high temperature; or, d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with the amino acid sequence encoding SEQ ID NO.3 under strict conditions, and the amino acid sequence retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the darkening of melanocytes induced by high temperature, or retains the effect of the amino acid sequence of SEQ ID NO.3 in preventing and / or improving the decreased scar repair ability caused by the decreased migration ability of keratinocytes induced by high temperature; the strict conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

5. The use according to any one of claims 1 to 4, characterized in that: The composition is a medical device, a tissue engineering product, a cosmetic or a health product.

6. The use according to claim 5, characterized in that The composition is a cosmetic.

7. The use according to any one of claims 1 to 6, characterized in that The high temperature is 37°C to 50°C; preferably, the high temperature is 38°C to 45°C; most preferably, the high temperature is 39°C to 41°C.

8. The use according to any one of claims 1 to 7, characterized in that The recombinant humanized type III collagen is prepared by a method comprising the following steps: (1) culturing a host cell encoding the sequence shown in SEQ ID NO. 3 in a production medium and producing recombinant type III humanized collagen; (2) harvesting the polypeptide; and (3) Purifying recombinant humanized type III collagen by Ni column and / or anion exchange chromatography.

9. The use according to claim 8, characterized in that The method further comprises the step of enzymatically cleaving the recombinant humanized type III collagen with TEV protease.

10. The use according to claim 8, characterized in that The purified recombinant humanized type III collagen contains endotoxin below 5EU / ml.

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