Application of an antimicrobial peptide in the preparation of a medical care solution for accelerating injury repair

Abstract

The present invention provides an application of an antimicrobial peptide in preparing a medical care solution for accelerating injury repair, and belongs to the technical field of care solutions. The antimicrobial peptide provided by the present invention is a Ranatuerin‑1C antimicrobial peptide, and its amino acid sequence is SMLSVLKNLGKVGLGLVACKINKQC. The present invention finds that the Ranatuerin‑1C antimicrobial peptide has a new function of promoting the proliferation and migration of skin fibroblasts and promoting angiogenesis of vascular endothelial cells. At the same time, the Ranatuerin‑1C antimicrobial peptide has an antibacterial and disinfecting effect, so it can be used to prepare a medical care solution for accelerating injury repair.

Description

Technical Field

[0001] The invention belongs to the technical field of nursing solutions, and in particular relates to the application of an antimicrobial peptide in the preparation of a medical nursing solution for accelerating damage repair. Background Art

[0002] The skin is the first line of defense between our body and the external environment, and plays an important function of protecting the human body from invasion by pathogens such as bacteria and viruses. It is like a solid barrier that not only blocks the invasion of external pathogens, but also plays a key role in preventing infection. However, when the skin is damaged, this solid barrier will be broken, making it easier for pathogens such as bacteria and viruses to invade the human body. This damage can be a scratch, rupture, burn or other form of injury, no matter how big or small, it will reduce the body's resistance and provide an opportunity for pathogens to take advantage. Therefore, we need to pay attention to protecting the integrity of the skin and promptly handle and treat any damage to ensure that our body can effectively resist external invasion.

[0003] At present, in clinical practice, iodine, alcohol, iodine tincture, potassium permanganate solution, erythromycin and chlorhexidine are often used for skin disinfection and debridement. However, through clinical application practice, we found that some of these drugs are too irritating, so they cannot be used on a large area, and there are unsatisfactory debridement in debridement, sometimes even leading to allergic reactions. Therefore, in order to overcome these shortcomings, we need to find new care solutions or improve existing care solutions to meet clinical needs.

[0004] Ranatuerin-1C antimicrobial peptide is a natural polypeptide derived from the North American green frog, and its amino acid sequence is SMLSVLKNLGKVGLGLVACKINKQC. Current studies have shown that Ranatuerin-1C antimicrobial peptide has the effect of inhibiting Gram-positive bacteria, Gram-negative bacteria, fungi and Candida albicans, but there is no research on the role of Ranatuerin-1C in damage repair. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an antimicrobial peptide in the preparation of a medical care solution for accelerating injury repair.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of an antimicrobial peptide in the preparation of a medical care solution for accelerating injury repair. The amino acid sequence of the antimicrobial peptide is as follows:

[0008] SMLSVLKNLGKVGLGLVACKINKQC.

[0009] Furthermore, the antimicrobial peptide improves the damage repairing ability of the medical care solution by promoting the proliferation of skin fibroblasts.

[0010] Furthermore, the antimicrobial peptide improves the damage repairing ability of the medical care solution by promoting the migration of skin fibroblasts.

[0011] Furthermore, the antimicrobial peptide improves the damage repairing ability of the medical care solution by promoting the angiogenesis ability of vascular endothelial cells.

[0012] Furthermore, the concentration of the antimicrobial peptide in the medical care solution is greater than 10 μg / mL.

[0013] Furthermore, the concentration of the antimicrobial peptide in the medical care solution is 1 mg / mL.

[0014] Secondly, the present invention provides a medical care solution having the function of accelerating damage repair, and the preparation method of the medical care solution comprises the following steps:

[0015] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0016] (2) After uniform mixing, add the Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution having a Ranatuerin-1C antimicrobial peptide concentration greater than 10 μg / mL;

[0017] The amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows:

[0018] SMLSVLKNLGKVGLGLVACKINKQC.

[0019] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the medical care solution is 1 mg / mL.

[0020] Secondly, the present invention provides the use of the Ranatuerin-1C antimicrobial peptide in the preparation of an agent for promoting the proliferation ability of skin fibroblasts in vitro, characterized in that the amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows:

[0021] SMLSVLKNLGKVGLGLVACKINKQC.

[0022] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is greater than 10 μg / mL;

[0023] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is 1 mg / mL.

[0024] Secondly, the present invention provides the use of the Ranatuerin-1C antimicrobial peptide in the preparation of an agent for promoting the migration ability of skin fibroblasts in vitro, characterized in that the amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows:

[0025] SMLSVLKNLGKVGLGLVACKINKQC.

[0026] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is greater than 10 μg / mL;

[0027] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is 1 mg / mL.

[0028] Secondly, the present invention provides the use of the Ranatuerin-1C antimicrobial peptide in the preparation of a reagent for promoting the angiogenesis ability of vascular endothelial cells in vitro, characterized in that the amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows:

[0029] SMLSVLKNLGKVGLGLVACKINKQC.

[0030] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is greater than 10 μg / mL;

[0031] Furthermore, the concentration of the Ranatuerin-1C antimicrobial peptide in the reagent is 1 mg / mL.

[0032] The beneficial effects of the present invention are:

[0033] The present invention finds that the Ranatuerin-1C antimicrobial peptide has the new function of promoting the proliferation and migration of skin fibroblasts and promoting angiogenesis of vascular endothelial cells. At the same time, the Ranatuerin-1C antimicrobial peptide has antibacterial and disinfecting effects, so it can be used to prepare a medical care solution that accelerates damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the tests on the regulation of proliferation of human skin fibroblasts by different antimicrobial peptides;

[0035] Figure 2 The results of the test on the regulation of the proliferation ability of human skin fibroblasts by the antimicrobial peptide Ranatuerin-1C;

[0036] Figure 3The results of the test on the regulation of the migration ability of human skin fibroblasts by the antimicrobial peptide Ranatuerin-1C.

[0037] Figure 4 These are the test results of the regulation of the angiogenic ability of human umbilical vein endothelial cells by the antimicrobial peptide Ranatuerin-1C. DETAILED DESCRIPTION

[0038] Example 1 Screening of antimicrobial peptides that can promote skin fibroblasts

[0039] 1. The present invention selects the following five antimicrobial peptides to screen antimicrobial peptides that can effectively promote skin fibroblasts:

[0040] Table 1 Antimicrobial peptide types and sequences

[0041]

[0042] 2. Use DMEM culture medium to prepare Bactenecin antimicrobial peptide, Bactericidin B-2 antimicrobial peptide, Combi-1 antimicrobial peptide, Ranatuerin-1C antimicrobial peptide and Crabrolin antimicrobial peptide into 10 mg / mL antimicrobial peptide solutions respectively;

[0043] 3. Human skin fibroblasts were digested with trypsin and prepared into 1×10 5 / ml cell suspension;

[0044] 4. Inoculate the cells into 96-well plates with 100 μL of cell suspension per well and culture in a cell culture incubator for 24 hours;

[0045] 5. Remove the original culture medium and replace it with an antimicrobial peptide solution containing Bactenecin antimicrobial peptide, Bactericidin B-2 antimicrobial peptide, Combi-1 antimicrobial peptide, Ranatuerin-1C antimicrobial peptide and Crabrolin antimicrobial peptide. The control group was replaced with DMEM culture medium without antimicrobial peptides and cultured in a cell culture incubator for 24 hours. Each antimicrobial peptide was treated with 3 replicate wells.

[0046] 6. Add 10 μL CCK-8 to each well and measure the absorbance at 450 nm using a microplate reader to screen for antimicrobial peptides that can promote the proliferation of human skin fibroblasts.

[0047] The test results are as follows Figure 1 As shown, from Figure 1 We can see that the OD values ​​of cells treated with Combi-1 antimicrobial peptide and Crabrolin antimicrobial peptide increased to a certain extent compared with the control group, but the difference was not statistically significant;

[0048] The cells treated with the Ranatuerin-1C antimicrobial peptide were significantly improved compared with the control group, so the present invention selected the Ranatuerin-1C antimicrobial peptide for subsequent research.

[0049] Example 2 Screening for the appropriate concentration of Ranatuerin-1C antimicrobial peptide

[0050] 1. Dilute the Ranatuerin-1C antimicrobial peptide into the following different concentrations using DMEM medium:

[0051] 1μg / mL, 10μg / mL, 100μg / mL, 1mg / mL, 10mg / mL, 100mg / mL antimicrobial peptide solutions;

[0052] 2. Human skin fibroblasts were digested with trypsin and prepared into 1×10 5 / ml cell suspension;

[0053] 3. Inoculate the cells into 96-well plates with 100 μL of cell suspension per well and culture in a cell culture incubator for 24 hours;

[0054] 4. Remove the original culture medium and replace it with the above antimicrobial peptide solution containing different concentrations of Ranatuerin-1C. The control group is replaced with DMEM culture medium without antimicrobial peptides and placed in a cell culture incubator for further culturing for 24 hours. Set up 3 replicate wells for each antimicrobial peptide treatment;

[0055] 5. Add 10 μL CCK-8 to each well and measure the absorbance at 450 nm using a microplate reader.

[0056] The test results are as follows Figure 2 As shown, from Figure 2 It can be seen that when 1μg / mL Ranatuerin-1C antimicrobial peptide solution is used to treat human skin fibroblasts, it has a certain promoting effect on cell proliferation, but it is not statistically significant. When 10μg / mL Ranatuerin-1C antimicrobial peptide solution is used to treat skin fibroblasts, it has a significant promoting effect on cell proliferation, and the difference is statistically significant.

[0057] When the concentration of Ranatuerin-1C antimicrobial peptide solution was greater than 10 μg / mL, the proliferation ability of the treated skin fibroblasts was better than that of the control group. Therefore, it can be considered that when the concentration of Ranatuerin-1C antimicrobial peptide solution was greater than 10 μg / mL, it had a promoting effect on skin fibroblasts.

[0058] At the same time, it can be found that when the concentration of Ranatuerin-1C antimicrobial peptide solution is 1 mg / mL, the effect is optimal.

[0059] Example 3: Exploring whether the antimicrobial peptide Ranatuerin-1C can effectively promote the migration ability of skin fibroblasts

[0060] 1. Human skin fibroblasts were inoculated into cell culture plates. The experimental treatment groups were pretreated with 10 μg / mL (group a), 100 μg / mL (group b), and 1 mg / mL (group c) of the Ranatuerin-1C antimicrobial peptide solution for 24 h, respectively. The control group was replaced with DMEM medium without antimicrobial peptides.

[0061] 2. After digesting the human skin fibroblasts, wash the cells with PBS and suspend the cells in serum-free DMEM medium to prepare a cell suspension, and count the cells;

[0062] 3. Add 600 μL of DMEM medium containing 10% serum to the lower chamber of the Transwell chamber, and add 100 μL of 2×10 4 The cell suspension of 10 cells was prepared and the culture plate was placed in a cell culture incubator for 24 hours.

[0063] 4. Take out the Transwell chamber, aspirate the upper liquid, transfer the Transwell chamber to the fixative, and fix at room temperature for 20 minutes;

[0064] 5. Take out the Transwell chamber and gently wash it 3 times with clean water. Carefully wipe off the cells in the upper chamber of the Transwell chamber, place it under a microscope to take pictures and randomly select 5 fields of view for counting.

[0065] The test results are as follows Figure 3 As shown, from Figure 3 It can be seen that compared with the control group, the number of migrating skin fibroblasts pretreated with 10μg / mL, 100μg / mL, and 1mg / mL of Ranatuerin-1C antimicrobial peptide increased significantly, indicating that Ranatuerin-1C antimicrobial peptide can effectively promote the migration ability of skin fibroblasts;

[0066] Similar to the proliferation promotion, 1 mg / mL of Ranatuerin-1C antimicrobial peptide has the best effect on promoting the migration of skin fibroblasts.

[0067] Example 4: Exploring whether the antimicrobial peptide Ranatuerin-1C can effectively promote angiogenesis

[0068] 1. Human umbilical vein endothelial cells (HUVEC) were inoculated into cell culture plates. The experimental treatment group was pretreated with 1 mg / mL of Ranatuerin-1C antimicrobial peptide solution for 24 h, and the control group was replaced with DMEM medium without antimicrobial peptides.

[0069] 2. After digesting the HUVEC cells, wash the cells with PBS and suspend the cells in serum-free DMEM medium to prepare a cell suspension, and count the cells;

[0070] 3. Place Matrigel at 4°C overnight, and pre-cool the 96-well cell culture plate and pipette tips required for the experiment at 4°C;

[0071] 4. On ice, add 50 μL of matrix gel to each well of a 96-well plate, and then place the 96-well plate in a cell culture incubator for 2 hours until the matrix gel is completely solidified;

[0072] 5. According to 1×10 per well 4 100 μL of HUVEC cells were seeded in a 96-well cell culture plate covered with Matrigel;

[0073] 6. After placing the 96-well cell culture plate in an incubator and incubating for 6 hours, observe the formation of tubules and calculate the tube formation rate (tube formation rate = area of ​​tubular structure / culture well area × 100%).

[0074] The experimental results are as follows Figure 4 As shown, from Figure 4 It can be seen that compared with the control group, the tube formation rate of human umbilical vein endothelial cells pretreated with Ranatuerin-1C antimicrobial peptide was significantly increased, indicating that pretreatment with Ranatuerin-1C antimicrobial peptide can effectively promote angiogenesis.

[0075] In medical wound care, effective sterilization and rapid wound healing are the main functions of the care solution. In the present invention, Ranatuerin-1C antimicrobial peptide, as an antimicrobial peptide, has excellent sterilization and disinfection effects. In addition, the present invention first discovered that Ranatuerin-1C antimicrobial peptide has the ability to promote the proliferation and migration of skin fibroblasts and the ability to promote angiogenesis, thereby contributing to the rapid healing of wounds, and is safe and non-irritating.

[0076] Example 5 A medical care solution 1 having the effect of accelerating injury repair

[0077] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0078] (2) After mixing evenly, add Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution 1 containing 10 μg / mL Ranatuerin-1C.

[0079] Example 6 A medical care solution having the effect of accelerating damage repair 2

[0080] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0081] (2) After mixing evenly, add Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution 2 containing 100 μg / mL Ranatuerin-1C.

[0082] Example 7 A medical care solution having the effect of accelerating injury repair 3

[0083] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0084] (2) After uniform mixing, add Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution 3 containing 1 mg / mL Ranatuerin-1C.

[0085] Example 8 A medical care solution having the effect of accelerating damage repair 4

[0086] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0087] (2) After uniform mixing, add Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution 4 containing 10 mg / mL Ranatuerin-1C.

[0088] Example 9 A medical care solution with the effect of accelerating injury repair

[0089] (1) Prepare the medical care solution pre-mix according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water;

[0090] (2) After mixing evenly, add Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution 5 containing 100 mg / mL Ranatuerin-1C.

Claims

1. An application of an antimicrobial peptide in the preparation of a medical care solution for accelerating the repair of skin damage, characterized in that: The amino acid sequence of the antimicrobial peptide is as follows: SMLSVLKNLGKVGLGLVACKINKQC.

2. The use according to claim 1, characterized in that: The antimicrobial peptide improves the skin damage repairing ability of the medical care solution by promoting the proliferation of skin fibroblasts.

3. The use according to claim 2, characterized in that: The antimicrobial peptide improves the skin damage repairing ability of the medical care solution by promoting the migration of skin fibroblasts.

4. The use according to claim 3, characterized in that: The antimicrobial peptide improves the skin damage repairing ability of the medical care solution by promoting the angiogenesis ability of vascular endothelial cells.

5. The use according to claim 4, characterized in that: The concentration of the antimicrobial peptide in the medical care solution is greater than 10 μg / mL.

6. The use according to claim 5, characterized in that: The concentration of the antimicrobial peptide in the medical care solution is 1 mg / mL.

7. A medical care solution having the function of accelerating the repair of skin damage, characterized in that: The preparation method of the medical care solution comprises the following steps: (1) Prepare a pre-made medical care solution according to the following ingredients: 10% glycerol, 20% isopropyl palmitate, 2% xanthan gum, 15% sodium hyaluronate, and 53% sterile water; (2) After uniform mixing, add the Ranatuerin-1C antimicrobial peptide to the pre-prepared medical care solution to prepare a medical care solution having a Ranatuerin-1C antimicrobial peptide concentration greater than 10 μg / mL; The amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows: SMLSVLKNLGKVGLGLVACKINKQC.

8. The medical care solution according to claim 7, characterized in that: The concentration of Ranatuerin-1C antimicrobial peptide in the medical care solution is 1 mg / mL.

9. Use of the antimicrobial peptide Ranatuerin-1C in the preparation of an agent for promoting the proliferation of skin fibroblasts in vitro, characterized in that: The amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows: SMLSVLKNLGKVGLGLVACKINKQC.

10. Use of the antimicrobial peptide Ranatuerin-1C in the preparation of an agent for promoting the migration ability of skin fibroblasts in vitro, characterized in that: The amino acid sequence of the Ranatuerin-1C antimicrobial peptide is as follows: SMLSVLKNLGKVGLGLVACKINKQC.

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