Polypeptide kfll capable of promoting wound healing and application of polypeptide kfll

Abstract

The invention discloses a polypeptide kfll capable of promoting wound healing and application thereof, and relates to the technical field of bioactive peptides, the polypeptide is derived from Procaspase-8DED2-alpha 2 protein, and the amino acid sequence of the polypeptide kfll is as shown in SEQ ID NO.1; the polypeptide can promote proliferation and migration of human immortalized keratinocytes (HaCaT), and has no cytotoxicity; experiments prove that the polypeptide disclosed by the invention can promote wound repair of mouse full-thickness skin injury; therefore, the polypeptide kfll has a good application prospect in the field of wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a polypeptide kfll capable of promoting wound healing and applications thereof. Background Art

[0002] As the largest organ of the human body and a physical barrier to the external environment, the skin plays a vital role in temperature regulation, as well as protection, perception, excretion, absorption, metabolism and immunity. The structural integrity of the skin is also crucial for maintaining physiological functions. However, many injuries can lead to skin damage, tissue loss, and structural and functional disorders. After injury, the body typically initiates a series of highly complex and overlapping physiological processes to repair the wound: hemostasis, inflammation, proliferation and remodeling. Each stage is strictly controlled and involves the regulation of various cells (such as fibroblasts, macrophages, platelets and epithelial keratinocytes) and substances (such as fibroblast-derived, platelet-derived, transforming, endothelial and epidermal growth factors).

[0003] Currently, research on peptides for wound healing is continuously developing and being explored in depth. Peptides are small proteins composed of amino acids that possess diverse biological activities, including promoting cell proliferation, apoptosis, differentiation, and repair. In recent years, researchers have discovered a number of peptides with wound healing properties, such as those that promote angiogenesis, collagen synthesis, and cell migration and proliferation. These peptides can promote various aspects of the wound healing process through diverse mechanisms, accelerating wound healing, reducing scar formation, and improving healing quality.

[0004] Therefore, in order to solve the above problems, this paper proposes a polypeptide kfll that promotes wound healing and its application. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide that can promote the proliferation and migration of human immortalized keratinocytes (HaCaT) and has no cytotoxicity and can promote wound healing.

[0006] In order to achieve the above technical effects, the present invention is implemented by the following technical solutions: a polypeptide kfll having the function of promoting wound healing, characterized in that: the amino acid sequence of the polypeptide kfll is as shown in SEQ ID NO.1;

[0007] SEQ ID NO. 1: Lys-Phe-Leu-Leu.

[0008] Another object of the present invention is to provide a use of a polypeptide kfll that promotes wound healing in the preparation of a preparation that promotes skin wound healing.

[0009] Furthermore, the polypeptide kfll promotes skin wound healing, specifically by promoting skin cell proliferation and migration.

[0010] Furthermore, the skin cells are human immortalized keratinocytes.

[0011] Furthermore, the concentration of the polypeptide kfll used in promoting skin cell proliferation and migration is 10 to 40 μg / mL.

[0012] The beneficial effects of the present invention are:

[0013] The present invention screened out the polypeptide kfll based on the Procaspase-8DED2-α2 protein; the polypeptide can promote the proliferation and migration of human immortalized keratinocytes (HaCaT) and is non-cytotoxic. Experiments have shown that the polypeptide of the present invention can promote wound repair in full-thickness skin injuries in mice; therefore, the polypeptide kfll has good application prospects in the field of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 This is a comparison of the effects of different concentrations of the polypeptide of the present invention on the proliferation of HaCaT cells;

[0016] Figure 2 This is a graph showing the changes in scratch area after 24h and 48h of action of different concentrations of the polypeptide of the present invention;

[0017] Figure 3 This is a bar graph showing the changes in scratch area after 24 hours and 48 hours of action of different concentrations of the polypeptide of the present invention;

[0018] Figure 4 This is a graph showing changes in wound surface area after different concentrations of the polypeptide were applied to mice of the present invention;

[0019] Figure 5 This is a graph showing changes in wound healing rates in mice after administration of different concentrations of the polypeptides of the present invention. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0021] Example 1

[0022] Screening for the peptide kfll that promotes wound healing based on the Procaspase-8DED2-α2 protein, the steps are as follows:

[0023] Procaspase-8DED2-α2 proteins were fragmented. The KFLL protein, which promotes HaCaT cell proliferation at low concentrations, was used as an experimental target, considering that the D-type peptide is not easily degraded by proteases. The amino acid sequence of the helical procaspase-8DED2-α2 is: RSELRSFKFLLQEEIS.

[0024] To investigate the effect of peptide kfll on HaCaT cell activity, the steps are as follows:

[0025] Step 1: Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). When the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin, and the cells were pipetted with culture medium and transferred to a centrifuge tube. The cells were centrifuged at room temperature to retain the cells, and the culture medium was added and pipetted;

[0026] Step 2: Remove the counting plate from the 75% alcohol tank, add an appropriate amount of suspension, and count under a microscope; 7000 cells per well of a 96-well plate, with a culture volume of 180 μL / well. Because the liquid around the 96-well plate is easy to evaporate, do not add cells to the 36 wells around the perimeter, only add culture medium, and incubate the plate in an incubator for 12 hours;

[0027] Step 3: Prepare different concentrations of polypeptide (0, 10, 20, 30, 40 μg / mL) in DMEM medium without serum and double antibody, add 180 μL of polypeptide of different concentrations to each well of a 96-well plate, incubate the 96-well plate in an incubator for 20 h, add 20 μL of MTT to each well, incubate in a CO2 incubator in the dark for 4 h, discard the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake at room temperature for 15 minutes, and detect the absorbance at 490 nm using a microplate reader.

[0028] Step 4: Calculate cell viability according to cell viability = OD test group / OD control group × 100%, and obtain the following: Figure 1Proliferation comparison of HaCaT cells shown; based on Figure 1 It can be seen that compared with the blank control group of 0 μg / mL, 10, 20, and 30 μg / mL of polypeptide kfll can significantly promote the proliferation of HaCaT cells after 48 hours of action.

[0029] Example 2

[0030] To investigate the healing activity of the peptide kfll on the HaCaT cell scratch model, the steps are as follows:

[0031] Step 1: Hacat cells were seeded into 6-well plates at a density of 50 × 104 cells per well and then cultured in a 37°C, 5% CO2 incubator until the cells were fully attached.

[0032] Step 2: After the cells attach to the wall, observe their growth density. When the cell density reaches 80%, perform a scratch test.

[0033] Step 3: First, discard the turbid cell culture medium and use a 200 μL pipette tip to make a "cross" scratch in each well of cells to facilitate accurate positioning during subsequent photography. Then, wash the cells three times with 1× PBS to remove dead cells and impurities that may affect subsequent photography.

[0034] Step 4: After washing, the culture medium was replaced with fresh low serum concentration (1%) culture medium containing different concentrations of polypeptide kfll (0, 10, 20, 30, 40 μg / mL). The scratch area was recorded with a microscope at 0, 24 and 48 hours after the treatment with polypeptide kfll; the following results were obtained: Figure 2 The changes in scratch area after 24h and 48h of action of different concentrations of peptides are shown; Figure 2 It can be seen that compared with the blank control group of 0 μg / mL, the scratch area was significantly reduced after 24h and 48h of treatment with 10, 20, 30, and 40 μg / mL of polypeptide kfll.

[0035] Example 3

[0036] The effect of polypeptide kfll on full-thickness skin defect wounds in mice was studied in the following steps:

[0037] Step 1: After 1 week of adaptive feeding, 30 4-week-old male Kunming mice were randomly divided into a blank control group and groups with different concentrations of polypeptide kfll (10, 20, 30, 40 μg / mL), with 6 mice in each group.

[0038] Step 2. Before the experiment, anesthetize the mice by intraperitoneal injection of sodium pentobarbital, then shave the hair on their backs and disinfect with alcohol cotton balls; fix the mice on a constant temperature operating table, and use a skin punch to create a circular wound with a diameter of 10 mm; after the wound is dry, take a photo and use Image J software to calculate the area of ​​the mouse skin wound, which is the initial wound area.

[0039] Step 3. After the full-thickness skin defect wound model was successfully established in mice, 20 μL of the corresponding concentration (10, 20, 30, 40 μg / mL) of kfll solution was applied to the wound every 24 hours in the mice in the different kfll groups, while 20 μL of sterile PBS buffer was applied to the blank control group.

[0040] Step 4: Determination of wound healing rate: Observe the changes in the wound every day and take pictures of the mouse wounds on the 3rd, 5th, 7th, 9th, 11th and 13th days (e.g. Figure 4 The wound area was calculated using Image J software, which is the area of ​​the unhealed wound. The calculation formula is as follows: Wound healing rate (%) = (initial wound area - wound area at a certain time point) ÷ initial wound area × 100%; the following can be obtained: Figure 5 The wound healing rate change diagram shown is Figure 5 It can be seen that compared with the blank control group, the wound healing rate of mice treated with polypeptide kfll was significantly improved.

[0041] In summary, the present invention provides a polypeptide kfll having wound healing promoting activity; the polypeptide has good biocompatibility and has good application prospects in the treatment of skin wounds.

[0042] The above description is only a preferred embodiment of the present invention, but the implementation of the present invention is not limited to the above embodiment. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polypeptide kfll having the function of promoting wound healing, characterized in that: The amino acid sequence of the polypeptide kfll is shown in SEQ ID NO.

1.

2. Use of the polypeptide kfll having the function of promoting wound healing according to claim 1 in the preparation of a preparation for promoting skin wound healing.

3. The use according to claim 2, characterized in that The polypeptide kfll promotes skin wound healing, specifically by promoting skin cell proliferation and migration.

4. The use according to claim 3, characterized in that The skin cells are human immortalized keratinocytes.

5. The use according to claim 3, characterized in that The concentration of the peptide kfll used in promoting skin cell proliferation and migration is 10 to 40 μg / mL.

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