A combination of a small molecule peptide and a leukocyte extract and uses thereof

Abstract

The application provides a small molecule peptide and leukocyte extract composition and application thereof, and belongs to the medical technical field.The composition comprises leukocyte extract prepared by a specific method and a small molecule peptide ZKSA-P-1001 (amino acid sequence: RWRFKWKK), and the mass ratio of the two is 1:1 to 3:1.The experimental results show that the leukocyte extract and ZKSA-P-1001 have a significant synergistic effect in combination application.Even when ZKSA-P-1001 is used alone at a low concentration with limited activity, the composition can significantly improve the migration ability of skin fibroblasts.Animal model experiments further confirm that the medicine containing the composition of the application can significantly accelerate the healing of infectious skin damage, especially in the early healing stage.The application provides an efficient, synergistic and promising skin repair solution.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to a composition of small molecule peptides and leukocyte extracts and its application. Background Technology

[0002] With the development of biotechnology and regenerative medicine, utilizing bioactive ingredients to promote skin tissue repair and regeneration has become an important research direction in the fields of skincare, aesthetic medicine, and wound healing. As the body's first line of defense, the skin is frequently damaged by factors such as trauma, inflammation, ultraviolet radiation, or natural aging, leading to a decline in its structural integrity and function. Among these, infectious skin injuries, caused by persistent inflammatory responses triggered by pathogenic microorganisms, significantly delay the healing process. Therefore, developing safe, effective, and bioactive skin repair drugs is of significant practical importance.

[0003] In recent years, leukocytes and their derivatives have attracted widespread attention due to their natural immunomodulatory capabilities and tissue repair potential. Leukocytes are a core component of the human immune system, releasing various bioactive molecules during inflammatory responses and tissue repair, including growth factors, cytokines, chemokines, and enzymes. These components can regulate the proliferation and migration of fibroblasts, vascular endothelial cells, and other cells, thereby promoting wound healing and collagen synthesis. However, current technologies still face bottlenecks in terms of the retention rate of active ingredients in extracts and their application effects. Therefore, how to better develop leukocyte extracts to enhance their application effects in skin repair is a problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a composition of small molecule peptides and leukocyte extracts and their applications. Through the synergistic effect of leukocyte extracts prepared by a specific process (stimulated by IL-4 / IL-13 and lysed under ultrasonic protection) and peptide ZKSA-P-1001, the skin damage repair effect is significantly improved, especially solving the clinical problem of slow healing of infected wounds.

[0005] To achieve the above objectives, the present invention provides a composition of a small molecule peptide and a leukocyte extract for skin damage repair, the composition comprising a leukocyte extract and a small molecule peptide ZKSA-P-1001, the amino acid sequence of which is RWRFKWKK.

[0006] The mass ratio of the leukocyte extract to the small molecule peptide ZKSA-P-1001 is 1:1 to 3:1.

[0007] Preferably, the mass ratio of the leukocyte extract to the small molecule peptide ZKSA-P-1001 is 2:1.

[0008] Preferably, in the composition, the protein concentration of the leukocyte extract is 100 to 300 μg / mL, and the concentration of the small molecule peptide ZKSA-P-1001 is 100 to 200 μg / mL.

[0009] Preferably, the leukocyte extract is prepared by the following method:

[0010] (1) After washing the leukocytes separated from human peripheral blood, centrifuge to obtain leukocyte precipitate;

[0011] (2) Prepare a cell suspension from the leukocyte pellet using a culture medium containing at least 10 ng / mL IL-4 and at least 10 ng / mL IL-13, and inoculate it into a culture vessel for stimulation culture for 48 h;

[0012] (3) Collect cells and resuspend them using low-temperature lysis protection buffer to obtain a leukocyte suspension;

[0013] (4) After sonicating the leukocyte suspension, centrifuge and collect the supernatant;

[0014] (5) Pass the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, filter the permeate through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and collect the retentate from the 10 kDa ultrafiltration membrane.

[0015] (6) After dialysis to remove small molecule impurities, the retentate is filtered and sterilized to obtain leukocyte extract.

[0016] Preferably, the low-temperature lysis protection buffer contains 5% trehalose, 5% mannitol and 0.5% human serum albumin;

[0017] The parameters for the ultrasonic treatment were: temperature 4℃, pulse mode: 30s ultrasound / 1min pause, power 35W, frequency 20kHz, and total time 4.5 minutes.

[0018] The dialysis procedure involves placing the retentate in 10 times its volume of physiological saline containing 0.5% human serum albumin, dialyzing at 4°C for 6 hours, and changing the dialysis fluid every 2 hours.

[0019] Preferably, the concentration of IL-4 is 10 ng / mL and the concentration of IL-13 is 10 ng / mL.

[0020] Preferably, the skin damage repair is infectious skin damage repair.

[0021] Secondly, the present invention provides the application of the above composition in the preparation of a skin damage repair drug.

[0022] Preferably, the skin damage repair is infectious skin damage repair.

[0023] Preferably, the repair of infectious skin damage is the repair of Staphylococcus aureus-infected skin damage.

[0024] In addition, the present invention provides a drug for rapid repair of skin damage, the core components of which are: 100-300 μg / mL leukocyte extract and 100-200 μg / mL small molecule peptide ZKSA-P-1001;

[0025] The leukocyte extract was prepared by the preferred preparation method described above;

[0026] The amino acid sequence of the small molecule peptide is RWRFKWKK.

[0027] Preferably, the core components of the drug are: 200 μg / mL leukocyte extract and 100 μg / mL small molecule peptide ZKSA-P-1001.

[0028] Preferably, the adjuvant components of the drug are physiological saline, glycerol, and vitamin C.

[0029] Preferably, the skin damage repair is infectious skin damage repair.

[0030] The beneficial effects of this invention are as follows:

[0031] First, the leukocyte extract obtained by this invention using a unique preparation process, through IL-4 / IL-13 stimulation and low-temperature ultrasonic lysis, effectively improves the retention rate and activity of bioactive components in the extract. Experimental data show that this modified leukocyte extract can significantly promote the migration of human skin fibroblasts, with effects significantly superior to extracts obtained using traditional freeze-thaw methods and under unstimulated conditions, providing a stronger biological basis for skin repair.

[0032] Secondly, this invention ingeniously combines the leukocyte extract with the small molecule peptide ZKSA-P-1001, achieving a significant synergistic effect. ZKSA-P-1001 alone, at low concentrations, has little effect on promoting cell migration, but when used in combination with the leukocyte extract of this invention, it greatly enhances the repair effect. This approach effectively reduces the effective concentration of each component, thereby lowering costs and reducing the risk of potential toxic side effects.

[0033] Finally, in a mouse model of infectious skin lesions, the drug containing the composition of this invention exhibited excellent repair effects. Compared with the control group and the single-component treatment group, the composition significantly improved the wound healing rate, especially showing a faster repair speed in the early stage of wound healing, providing an innovative solution for the rapid and efficient repair of infectious skin lesions. Attached Figure Description

[0034] Figure 1 The results show the promoting effects of leukocyte extracts prepared by different methods on the migration of human skin fibroblasts.

[0035] Figure 2 The figure shows the results of the synergistic effect of the combination of leukocyte extract a and small molecule peptide ZKSA-P-1001 on the migration of human skin fibroblasts.

[0036] Figure 3 The repair effect of a skin repair drug containing the composition of the present invention in a mouse model of infectious skin lesions is presented;

[0037] in, Figure 3 (a) Actual test results of the repair effect. Figure 3 (b) is a statistical chart of the repair effect on day 6. Figure 3 (c) is a statistical chart of the repair effect on day 12. Detailed Implementation

[0038] Example 1

[0039] Preparation of modified leukocyte extract

[0040] (1) The white blood cells obtained by the red blood cell lysis method were washed three times with pre-cooled PBS and centrifuged to obtain white blood cell pellet;

[0041] (2) The leukocyte pellet was prepared into a suspension using RPMI-1640 medium containing 10 ng / mL IL-4 + 10 ng / mL IL-13 (1×10⁻⁶). 6 (cells / mL) were inoculated into culture dishes and cultured for 48 hours (at 37°C and 5% CO2). The white blood cells were collected by centrifugation and washed three times with pre-cooled PBS.

[0042] (3) The cell density was adjusted to 1×10⁻⁶ cells using pre-cooled cryolysis protection buffer (PBS containing 5% (w / v) trehalose, 5% (w / v) mannitol, and 0.5% (w / v) human serum albumin, pH 7.4). 6 cell / mL, to obtain leukocyte buffer;

[0043] (4) Add 10 mL of leukocyte buffer (1 × 10⁻⁶) to the solution. 6 The cells (per mL) were placed in a Sonics Vibra-Cell sonicator and sonicated under the following conditions:

[0044] Temperature 4℃, pulse mode: 30s ultrasonic treatment / 1min pause, effective power 35 W, frequency 20kHz, probe diameter 5mm, probe depth into liquid 10mm, total time 4.5 minutes;

[0045] (5) Centrifuge at 10000g for 20 minutes at 4℃ to remove unlysed cells and cell debris. Collect the supernatant and pre-filter it through a 0.45 μm PVDF membrane to obtain the primary lysis buffer.

[0046] (6) Filter the primary lysis buffer using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, collect the filtrate, and then filter the filtrate using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, collect the retentate.

[0047] (7) Transfer the retentate into a dialysis bag and place it in 10 times the volume of physiological saline containing 0.5% human serum albumin. Dialyze at 4°C for 6 hours, changing the dialysis fluid every 2 hours to obtain the retention solution.

[0048] (8) After sterilizing the retention solution by filtration through a 0.22 μm PES membrane, sterile leukocyte extract a was obtained.

[0049] Comparative Example 1

[0050] Extraction using traditional freeze-thaw method

[0051] (1) The white blood cells obtained by the red blood cell lysis method were washed three times with pre-cooled PBS and centrifuged to obtain white blood cell pellet;

[0052] (2) The leukocyte precipitate was prepared into a cell suspension with a concentration of 1×106 cells / mL using physiological saline containing 0.5% human serum albumin;

[0053] (3) Freeze the cell suspension in liquid nitrogen for 5 minutes, then thaw it rapidly in a water bath at 37°C. Repeat this process 5 times.

[0054] (4) Centrifuge at 10000g for 20 minutes, collect the supernatant, and obtain leukocyte extract b.

[0055] Comparative Example 2

[0056] Extraction by ultrasonic lysis without IL-4 / IL-13 stimulation

[0057] (1) The white blood cells obtained by the red blood cell lysis method were washed three times with pre-cooled PBS and centrifuged to obtain white blood cell pellet;

[0058] (2) The leukocyte pellet was adjusted to a cell density of 1×10⁻⁶ cells using pre-cooled cryoprotective lysis buffer (PBS containing 5% (w / v) trehalose, 5% (w / v) mannitol, and 0.5% (w / v) human serum albumin, pH 7.4). 6 cell / mL, to obtain leukocyte buffer;

[0059] (3) Add 10 mL of leukocyte buffer (1 × 10⁻⁶) to the solution. 6The cells (per mL) were placed in an ultrasound machine and ultrasound was performed under the following conditions:

[0060] Temperature 4℃, pulse mode: 30s ultrasonic treatment / 1min pause, effective power 35W, frequency 20kHz, probe diameter 5mm, probe depth into liquid 10mm, total time 4.5 minutes;

[0061] (4) Centrifuge at 4℃ and 10000g for 20 minutes to remove unlysed cells and cell debris. Collect the supernatant and pre-filter it through a 0.45 μm PVDF membrane to obtain the primary lysis buffer.

[0062] (5) Filter the primary lysis buffer using a 100 kDa ultrafiltration membrane, collect the filtrate, and then filter the filtrate using a 10 kDa ultrafiltration membrane, collecting the retentate.

[0063] (6) Transfer the retentate into a dialysis bag and place it in 10 times the volume of physiological saline containing 0.5% human serum albumin. Dialyze at 4°C for 6 hours, changing the dialysis fluid every 2 hours to obtain the retention solution.

[0064] (7) After sterilizing the retention solution by filtration through a 0.22 μm PES membrane, leukocyte extract c was obtained.

[0065] Comparative Example 3

[0066] Traditional freeze-thaw extraction combined with IL-4 / IL-13 stimulation

[0067] (1) The white blood cells obtained by the red blood cell lysis method were washed three times with pre-cooled PBS and centrifuged to obtain white blood cell pellet;

[0068] (2) The leukocyte pellet was prepared into a suspension (1×106 cells / mL) using RPMI-1640 medium containing 10 ng / mL IL-4 + 10 ng / mL IL-13 and inoculated into a culture dish. After 48 hours of stimulation culture, the leukocytes were collected by centrifugation and washed three times with pre-cooled PBS to obtain leukocyte extract 2.

[0069] (3) Prepare 1×10⁻⁶ leukocyte extract 2 using physiological saline containing 0.5% human serum albumin. 6 Cell suspension with cell / mL;

[0070] (4) Freeze the cell suspension in liquid nitrogen for 5 minutes, then thaw it rapidly in a water bath at 37°C. Repeat this process 5 times.

[0071] (5) Centrifuge at 10000g for 20 minutes, collect the supernatant, and obtain leukocyte extract d.

[0072] Example 2

[0073] Detecting the effects of leukocyte extracts prepared by different methods on promoting the migration of skin fibroblasts

[0074] (1) Human skin fibroblasts were prepared into a cell suspension using DMEM medium containing 10% FBS and 1% double antibiotics;

[0075] (2) Seed the cell suspension into a 6-well cell culture plate and culture until the cells reach 90% confluence;

[0076] (3) Discard the original culture medium, gently wash the cells once with PBS, and use a 200 μL pipette tip to gently draw a straight line at the bottom of the well plate;

[0077] (4) Rinse twice with PBS to remove exfoliated cells, and replace with fresh culture medium according to the following groups:

[0078] Control group: DMEM medium + 0.1% FBS;

[0079] Treatment group 1: DMEM medium + 0.1% FBS + 300 μg / mL leukocyte extract a (the concentration of leukocyte extract was determined by the BCA method and then diluted with DMEM medium).

[0080] Treatment group 2: DMEM medium + 0.1% FBS + 300 μg / mL leukocyte extract b;

[0081] Treatment group 3: DMEM medium + 0.1% FBS + 300 μg / mL leukocyte extract c;

[0082] Treatment group 4: DMEM medium + 0.1% FBS + 300 μg / mL leukocyte extract d;

[0083] Add 2 mL to each well, set up 3 replicate wells for each group, and repeat the experiment 3 times.

[0084] (5) Record cell migration at 0h and 24h, measure the scratch area using ImageJ, and calculate the scratch closure rate.

[0085] from Figure 1 The results show that the scratch closure rate of the control group was 39.33%, the closure rate of treatment group 1 was 77.87%, the closure rate of treatment group 2 was 49.77%, the closure rate of treatment group 3 was 58.77%, and the closure rate of treatment group 4 was 56.65%.

[0086] Compared to the control group, the cell migration promotion rate of treatment group 1 (migration promotion rate = (treatment group closure rate - control group closure rate) / control group closure rate × 100%) was 97.99%, the cell migration promotion rate of treatment group 2 was 26.55%, the cell migration promotion rate of treatment group 3 was 49.43%, and the cell migration promotion rate of treatment group 4 was 44.04%. This demonstrates that the leukocyte extract a prepared in Example 1 of this invention exhibits significantly better efficacy than other extraction methods in promoting skin fibroblast migration. Compared to the traditional freeze-thaw extraction method (treatment group 2), the extract prepared using the method of this invention without stimulation (treatment group 3), and the extract prepared using the traditional method after stimulation (treatment group 4), treatment group 1 shows a significant advantage in cell migration promotion ability, proving that IL-4 / IL-13 stimulation combined with low-temperature ultrasonic lysis can effectively preserve active ingredients and enhance skin repair potential.

[0087] Example 3

[0088] ZKSA-P-1001 is a peptide (amino acid sequence: RWRFKWKK) with skin repair potential disclosed in patent CN201810319050.4. However, experimental data show that it has no significant promoting effect on cell proliferation and migration at concentrations of 100 μg / mL or 250 μg / mL, and only shows certain activity at a concentration of 500 μg / mL. However, the use of high concentrations not only increases the application cost, but may also bring potential toxic side effects or cytotoxic risks.

[0089] Therefore, in order to reduce the effective concentration of ZKSA-P-1001 while enhancing its biological activity, this invention proposes to use it in combination with the leukocyte extract provided by this invention.

[0090] First, the effect of the leukocyte extract provided by this invention in combination with ZKSA-P-1001 on the migration of skin fibroblasts was tested.

[0091] (1) Human skin fibroblasts were prepared into a cell suspension using DMEM medium containing 10% FBS and 1% double antibiotics;

[0092] (2) Seed the cell suspension into a 6-well cell culture plate and culture until the cells reach 90% confluence;

[0093] (3) Discard the original culture medium, gently wash the cells once with PBS, and use a 200 μL pipette tip to gently draw a straight line at the bottom of the well plate;

[0094] (4) Rinse twice with PBS to remove exfoliated cells, and replace with fresh culture medium according to the following groups:

[0095] Control group: DMEM medium + 0.1% FBS;

[0096] Treatment group 1: DMEM medium + 0.1% FBS + 300 μg / mL leukocyte extract a;

[0097] Treatment group 2: DMEM medium + 0.1% FBS + 200 μg / mL leukocyte extract a;

[0098] Treatment group 3: DMEM medium + 0.1% FBS + 100 μg / mL leukocyte extract a;

[0099] Treatment group 4: DMEM medium + 0.1% FBS + 100μg / mL small molecule peptide ZKSA-P-1001 (synthesized by Nanjing Genscript Biotech, solid phase synthesis, purity ≥95%).

[0100] Treatment group 5: DMEM medium + 0.1% FBS + 200 μg / mL small molecule peptide ZKSA-P-1001;

[0101] Treatment group 6: DMEM medium + 0.1% FBS + 200 μg / mL leukocyte extract a + 100 μg / mL small molecule peptide ZKSA-P-1001;

[0102] Treatment group 7: DMEM medium + 0.1% FBS + 100 μg / mL leukocyte extract a + 200 μg / mL small molecule peptide ZKSA-P-1001;

[0103] Treatment group 8: DMEM medium + 0.1% FBS + 200 μg / mL leukocyte extract b;

[0104] Treatment group 9: DMEM medium + 0.1% FBS + 200 μg / mL leukocyte extract b + 100 μg / mL small molecule peptide ZKSA-P-1001;

[0105] Add 2 mL to each well, set up 3 replicate wells for each group, and repeat the experiment 3 times.

[0106] (5) Record cell migration at 0h and 24h, measure the scratch area using ImageJ, and calculate the scratch closure rate.

[0107] from Figure 2The results show that the scratch closure rate was 38.60% in the control group, 76.07% in treatment group 1, 70.31% in treatment group 2, 58.86% in treatment group 3, 39.42% in treatment group 4, 42.19% in treatment group 5, 84.64% in treatment group 6, 70.52% in treatment group 7, 44.43% in treatment group 8, and 45.51% in treatment group 9.

[0108] The results above show that ZKSA-P-1001 alone only exhibits a slight promoting effect, indicating that its biological activity is limited at low concentrations, which is consistent with previous studies.

[0109] Leukocyte extract a alone significantly promoted cell migration in the range of 100–300 μg / mL, showing a dose-dependent trend, but the effect gradually decreased.

[0110] Secondly, it can be seen that the combination of leukocyte extract a and ZKSA-P-1001 has a significant synergistic effect: In treatment group 6, the combination of 200 μg / mL leukocyte extract a + 100 μg / mL ZKSA-P-1001 resulted in a scratch closure rate of 84.64%, significantly higher than the 70.31% achieved by using leukocyte extract a alone at the same concentration, and also higher than the 76.07% achieved by using 300 μg / mL leukocyte extract a alone. Since 100 μg / mL ZKSA-P-1001 itself cannot promote the migration ability of fibroblasts, it promotes the migration ability of fibroblasts through a synergistic effect with leukocyte extract a.

[0111] In treatment group 7, the addition of 200 μg / mL ZKSA-P-1001 also increased the efficacy of 100 μg / mL leukocyte extract a from 58.86% to 70.52%.

[0112] Furthermore, the results from treatment groups 8 and 9 show that the combined effect of leukocyte extract b prepared using the conventional method and ZKSA-P-1001 did not show a significant improvement, indicating that the combination of leukocyte extract b and ZKSA-P-1001 did not exhibit a synergistic effect. This may be because the traditional freeze-thaw method results in insufficient content of active ingredients (such as growth factors) in extract b, failing to reach the synergistic threshold with ZKSA-P-1001, thus preventing it from producing a synergistic effect when used in combination with ZKSA-P-1001.

[0113] Example 4

[0114] Preparation of skin repair drugs containing small molecule peptide ZKSA-P-1001 and leukocyte extract

[0115] (1) Add 60 mL of physiological saline to a sterile beaker, slowly add 10 mL of 2000 μg / mL leukocyte extract, and stir well;

[0116] (2) Add 10 mL of 1000 μg / mL small molecule peptide ZKSA-P-1001 and stir until homogeneous;

[0117] (3) Add 10mg of vitamin C, dissolve it completely, then add 2mL of glycerin and stir well;

[0118] (4) Add physiological saline to 100mL, stir well, filter and sterilize using a 0.22μm microporous membrane to obtain skin repair drug.

[0119] Example 5

[0120] (1) Add 60 mL of physiological saline to a sterile beaker, slowly add 10 mL of 3000 μg / mL leukocyte extract, and stir well;

[0121] (2) Add 10 mL of 1000 μg / mL small molecule peptide ZKSA-P-1001 and stir until homogeneous;

[0122] (3) Add 10mg of vitamin C, dissolve it completely, then add 2mL of glycerin and stir well;

[0123] (4) Add physiological saline to 100mL, stir well, filter and sterilize using a 0.22μm microporous membrane to obtain skin repair drug.

[0124] Example 6

[0125] (1) Add 60 mL of physiological saline to a sterile beaker, slowly add 10 mL of 1000 μg / mL leukocyte extract, and stir well;

[0126] (2) Add 10 mL of 2000 μg / mL small molecule peptide ZKSA-P-1001 and stir until homogeneous;

[0127] (3) Add 10mg of vitamin C, dissolve it completely, then add 2mL of glycerin and stir well;

[0128] (4) Add physiological saline to 100mL, stir well, filter and sterilize using a 0.22μm microporous membrane to obtain skin repair drug.

[0129] Example 7

[0130] Preparation of skin repair drugs containing small molecule peptide ZKSA-P-1001 and leukocyte extract

[0131] (1) Add 60 mL of physiological saline to a sterile beaker, slowly add 10 mL of 2000 μg / mL leukocyte extract, and stir well;

[0132] (2) Add 10 mL of 2000 μg / mL small molecule peptide ZKSA-P-1001 and stir until homogeneous;

[0133] (3) Add 10mg of vitamin C, dissolve it completely, then add 2mL of glycerin and stir well;

[0134] (4) Add physiological saline to 100mL, stir well, filter and sterilize using a 0.22μm microporous membrane to obtain skin repair drug.

[0135] Example 8

[0136] The skin repair drug of the present invention was tested for its effect on repairing skin damage.

[0137] (1) Thirty-two male BALB / c mice aged 6-8 weeks were randomly divided into 4 groups:

[0138] Control group: Apply skin repair medication without leukocyte extract and small molecule peptide ZKSA-P-1001 (except for the lack of leukocyte extract and small molecule peptide ZKSA-P-1001, the other ingredients and preparation are exactly the same as the skin repair medication in Example 4).

[0139] Treatment Group 1: Apply a skin repair medication without leukocyte extract (except for the lack of leukocyte extract, the other ingredients and preparation are exactly the same as the skin repair medication in Example 4).

[0140] Treatment Group 2: Apply a skin repair drug that does not contain the small molecule peptide ZKSA-P-1001 (except for the lack of the small molecule peptide ZKSA-P-1001, the other ingredients and preparation are exactly the same as the skin repair drug in Example 4).

[0141] Treatment group 3: Apply the skin repair medication from Example 4;

[0142] (2) After injecting 1% sodium pentobarbital into the intraperitoneal area of ​​randomly grouped mice, the mice were fixed on the operating table and hair was removed with hair removal cream, covering an area of ​​about 3×3cm.

[0143] (3) After disinfecting the skin with iodine, remove the iodine with alcohol and cut off about 1 cm × 1 cm of full-thickness skin in the center of the mouse’s back with sterile surgical scissors;

[0144] (4) After applying gentle pressure with a hemostatic cotton ball to stop the bleeding, use 2μL of 2×10 7 Mice were infected with CFU / mL Staphylococcus aureus (ATCC6538) to simulate real wound infection and damage. The wounds were left to stand for 3 minutes to allow the bacterial solution to be absorbed, and then covered with sterile gauze.

[0145] (5) Starting from the first day after surgery, according to the group, apply 20 μL of the corresponding drug at regular intervals every day. Take photos on the first day, the sixth day and the twelfth day to calculate the healing rate.

[0146] from Figure 3 The results showed that on day 6, the wound healing rate was 55.83% in the control group and 58.46% in treatment group 1, an increase of 4.71% compared to the control group (treatment group 1 - control group / control group × 100%); the wound healing rate was 71.50% in treatment group 2, an increase of 28.07% compared to the control group; and the wound healing rate was 89.38% in treatment group 3, an increase of 60.09% compared to the control group.

[0147] On day 12, the wound healing rate was 84.06% in the control group, 86.56% in treatment group 1 (2.97% higher than the control group), 93.23% in treatment group 2 (10.91% higher than the control group), and 98.08% in treatment group 3 (16.68% higher than the control group).

[0148] from Figure 3 The results showed that, compared with the control group, the skin repair drug containing 100 μg / mL small molecule peptide ZKSA-P-1001 did not have a significant promoting effect on either day 6 or day 12.

[0149] Compared with the control group, the wound healing rate of treatment group 2 and treatment group 3 was significantly improved, indicating that the anti-inflammatory factors contained in the leukocyte extract prepared by the present invention can effectively promote wound repair after infectious skin injury.

[0150] Compared to treatment group 2, the wound healing rate of treatment group 3 was significantly improved on both day 6 and day 12. Especially in the early stages of wound healing, the improvement in treatment group 3 was twice that of the control group. At day 12, due to the upper limit of tissue repair, the difference between the two groups narrowed, but the effect of treatment group 3 was still significantly higher than that of treatment group 2. These results indicate that although low concentrations of the small molecule peptide ZKSA-P-1001 alone do not have a significant promoting effect on skin repair, when used in combination with the leukocyte extract prepared in this invention, it can fully exert a synergistic promoting effect, achieving rapid and efficient repair of infected skin injuries.

Claims

1. A composition of small molecule peptides and leukocyte extracts for skin damage repair, characterized in that, The composition comprises leukocyte extract and small molecule peptide ZKSA-P-1001, the amino acid sequence of which is RWRFKWKK; The mass ratio of the leukocyte extract to the small molecule peptide ZKSA-P-1001 is 1:1 to 3:1; The leukocyte extract was prepared by the following method: (1) After washing the leukocytes separated from human peripheral blood, centrifuge to obtain leukocyte precipitate; (2) Prepare a cell suspension from the leukocyte pellet using a culture medium containing at least 10 ng / mL IL-4 and at least 10 ng / mL IL-13, and inoculate it into a culture vessel for stimulation culture for 48 h; (3) Collect cells and resuspend them using low-temperature lysis protection buffer to obtain a leukocyte suspension; (4) After sonicating the leukocyte suspension, centrifuge and collect the supernatant; (5) Pass the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, filter the permeate through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and collect the retentate from the 10 kDa ultrafiltration membrane. (6) After dialysis to remove small molecule impurities, the retentate is filtered and sterilized to obtain leukocyte extract; The low-temperature lysis protection buffer is PBS containing 5% trehalose, 5% mannitol and 0.5% human serum albumin; The parameters for the ultrasonic treatment were: temperature 4℃, pulse mode: 30s ultrasound / 1min pause, power 35W, frequency 20kHz, and total time 4.5 minutes. The dialysis procedure involves placing the retentate in 10 times its volume of physiological saline containing 0.5% human serum albumin, dialyzing at 4°C for 6 hours, and changing the dialysis fluid every 2 hours.

2. The composition according to claim 1, characterized in that, In the composition, the protein concentration of the leukocyte extract is 100 to 300 μg / mL, and the concentration of the small molecule peptide ZKSA-P-1001 is 100 to 200 μg / mL.

3. The composition according to claim 1, characterized in that, The skin damage repair described is for infected skin damage.

4. The use of the composition according to any one of claims 1-3 in the preparation of a skin damage repair medicament.

5. The application according to claim 4, characterized in that, The skin damage repair described is for infected skin damage.

6. A drug for rapid repair of skin damage, characterized in that, The core components of the drug are: 100-300 μg / mL leukocyte extract and 100-200 μg / mL small molecule peptide ZKSA-P-1001; The leukocyte extract was prepared by the preparation method according to claim 1; The amino acid sequence of the small molecule peptide is RWRFKWKK.

7. The drug according to claim 6, characterized in that, The adjuvant components of the drug are physiological saline, glycerol, and vitamin C.

8. The medicament according to claim 7, characterized in that, The skin damage repair described is for infected skin damage.

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