Preparation method of platelet exosome and application thereof in wound healing

Abstract

The application provides a preparation method of platelet exosomes and application thereof in wound healing, and belongs to the technical field of biological medicine. The application provides a complete method for preparing animal platelet exosomes PDEs by freezing and thawing, and provides application of the animal platelet exosomes PDEs in drugs for treating skin wound healing across species. Researches of the application prove that sheep-derived PDEs treated by activation can effectively promote skin wound healing of mice, and can significantly promote formation of new blood vessels in skin tissue; the application fills the research blank of heterogenous PDEs in the field of wound healing treatment, provides a new drug selection and treatment approach for skin wound treatment, and has a wide application prospect in the field of clinical skin burn treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the preparation of platelet-derived exosomes (PDEs) formulations for animals (such as deer, pigs, cattle, sheep, etc.) based on the freeze-thaw method, and the application of such formulations in wound repair in pets or humans. Background Technology

[0002] With the continued expansion of the global pet pharmaceutical market, the clinical need for repair and treatment of common pet diseases such as skin lesions and arthritis is becoming increasingly urgent. Currently, although platelet-rich plasma (PRP) has been used for pet tissue repair, it still faces challenges such as limited autologous platelet supply, heterogeneous formulation components, susceptibility to interference from circulating exosomes, poor reproducibility of preparation, and limitations in room temperature storage and transportation, making it difficult to meet the needs of large-scale, standardized clinical applications.

[0003] Therefore, there is an urgent need in this field to overcome the aforementioned technical bottlenecks and seek key strategies and methods to solve the clinical repair problems of pet skin damage. Summary of the Invention

[0004] This invention aims to fill this gap by developing a wound healing therapeutic agent based on platelet exosomes from xenogeneic animals. This invention overcomes the limitations of autologous platelet exosomes, investigates the feasibility of xenogeneic exosome therapy, and is applicable to the repair of skin injuries in various animals and humans. It provides a new solution for the development of xenogeneic platelet exosome PDEs and for the repair of skin injuries in pets and potentially in humans.

[0005] The first objective of this invention is to provide a complete method for preparing animal platelet exosomes (PDEs) using a freeze-thaw process.

[0006] A second objective of this invention is to provide the use of animal platelet exosomes (PDEs) in medicaments for cross-species treatment of skin wound healing.

[0007] To achieve the above-mentioned objectives, this invention establishes a freeze-thaw method for preparing platelet exosomes (PDEs) and applies it to a mouse skin wound healing model. The study investigates the expression of growth factors, regulation of collagen synthesis, and inflammatory response levels in wound-affected skin tissue, assesses its safety and efficacy, and forms a complete and efficient repair and treatment plan applicable to wound repair, tissue regeneration, and other scenarios. This provides key technical support and theoretical reference for pet clinical trauma treatment and related basic translational research.

[0008] Therefore, this invention provides a method for preparing platelet exosomes, comprising the following steps: separating platelets from platelet-rich plasma (PRP), and activating the platelets using a freeze-thaw method; the freeze-thaw method involves rapidly freezing the platelets in an ultra-low temperature freezer at -80 ℃ for 24 h, followed by rapid thawing in a 37 ℃ water bath for 3 min, repeating the above "freeze-thaw" cycle three times; centrifuging the activated platelet-rich plasma suspension at 4 ℃ and 2000×g for 15 min to remove incompletely lysed platelet fragments and cell debris, collecting the supernatant as the platelet lysis buffer. The buffer is then filtered through a 0.22 μm filter membrane to remove cell debris, large molecular impurities such as proteins, and incubated with platelet exosome extract, followed by ultracentrifugation and washing to specifically isolate and purify platelet exosomes (PDEs), ultimately obtaining a high-purity platelet exosome (PDEs) suspension.

[0009] The source of platelets used in this invention is not limited, including but not limited to animals such as pigs, cattle, sheep, deer, horses, and dogs.

[0010] This invention uses a freeze-thaw method to activate platelets. By repeatedly freezing and thawing (freezing at -80 ℃ and thawing at 37 ℃), the platelet cell membrane structure is destroyed, allowing intracellular platelet exosomes (PDEs) to be released, which can significantly improve the platelet activation efficiency and the extraction quality of PDEs.

[0011] This invention utilizes small RNA high-throughput sequencing technology to analyze the expression profile of exosomal miRNAs in sheep platelets. It was found that oar-miR-148a was the most abundant miRNA, accounting for 61.78%, and it may serve as a marker miRNA, playing a central role in platelet physiological and pathological processes and intercellular communication. Other highly abundant miRNAs, such as oar-miR-21, may also be involved in functional regulation. This study not only provides fundamental data for further research into the biological functions, mechanisms, and clinical applications of platelet exosomal miRNAs, but also provides standardized technical references for research on exosomal miRNAs in sheep and other livestock.

[0012] This invention provides an in-depth study on the role of sheep platelet exosomes (PDEs) in skin wound healing. The study shows that local injection of sheep platelet exosome PDEs solution significantly promotes the healing of full-thickness skin defects in BALB / c mice, accelerates scab formation and epithelialization, and improves wound healing rate and quality, indicating that PDEs have a role in promoting skin wound healing. This is because sheep platelet exosomes (PDEs) contain abundant growth factors (such as EGF, FGF-2, VEGF, etc.), cytokines, and bioactive substances. These substances can be absorbed by cells surrounding the wound through local injection, activating the proliferation and migration of skin fibroblasts and keratinocytes, promoting angiogenesis, accelerating the growth of granulation tissue and epithelialization, thereby shortening wound healing time and improving healing quality.

[0013] This invention, through the detection of growth factor expression, collagen synthesis regulation, and inflammatory response levels in mouse wound skin tissue, revealed that sheep platelet exosomes (PDEs) can significantly upregulate the expression of growth factor genes such as EGF, FGF-2, and VEGF-A in wound tissue, promoting cell proliferation and angiogenesis; regulate collagen synthesis and granulation tissue maturation by upregulating α-SMA and COL1A1 gene expression and downregulating COL3A1 gene expression; and alleviate wound inflammation by inhibiting the expression of pro-inflammatory factors such as IL-1β and TNF-α and promoting the expression of anti-inflammatory factor TGF-β, thereby synergistically promoting skin wound healing. It is evident that sheep platelet exosomes (PDEs) promote wound healing through multiple pathways by regulating the expression of the aforementioned genes. This further elucidates the mechanism by which sheep platelet exosomes (PDEs) promote wound healing.

[0014] This invention also investigated the effects of sheep exosome PDEs on HaCat cell function, finding that sheep exosome PDEs promoted HaCaT cell proliferation and angiogenesis by upregulating EGF, FGF-2, and VEGF-A genes; upregulated α-SMA and COL1A1 and downregulated COL3A1 to regulate collagen synthesis; inhibited IL-1β and TNF-α and promoted TGF-β to reduce inflammation, and synergistically promoted wound healing.

[0015] Therefore, the platelet exosomes prepared by the freeze-thaw method of this invention can be widely used in the treatment of various wounds, especially skin wounds, including mechanical wounds, traumatic wounds, chemical wounds, ulcerative wounds, and radiation wounds. These skin wounds can be from animals such as rats, pigs, cattle, sheep, deer, horses, and dogs, or from human skin wounds.

[0016] Preferably, the present invention provides the use of sheep platelet exosomes in a drug or pharmaceutical composition for healing skin wounds in mice.

[0017] On the other hand, the present invention provides a platelet exosome preparation, which is prepared from platelet exosomes and pharmaceutically acceptable carriers or excipients; the dosage form of the preparation includes injections, ointments, powders, sprays, etc.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention has confirmed that sheep-derived PDEs after activation treatment can effectively promote the healing of skin wounds in mice and can significantly promote the formation of new blood vessels in skin tissue; the present invention fills the research gap of xenogeneic PDEs in the field of wound healing treatment, provides new drug options and treatment approaches for skin wound treatment, and has broad application prospects in the field of clinical skin burn treatment.

[0019] Compared to traditional PRP therapy and allogeneic platelet exosomes, the freeze-thaw method for preparing sheep platelet exosome PDEs in this invention exhibits significant technical advantages: First, this invention standardizes and optimizes the preparation process of platelet exosome PDEs, employing repeated freeze-thaw cycles to induce secondary release of exosomes from platelets. Therefore, the resulting PDE formulations have significantly higher homogeneity, effectively avoiding interference from circulating exosomes mixed in PRP, and greatly improving the stability of the preparation process and the reproducibility of experimental results; Second, experimental data results (Figure 3 and...) Figure 4 The intervention and treatment effects of xenogeneic PDEs are significantly better than those of autologous treatment; it fills the research gap in the field of xenogeneic PDEs in wound healing treatment, and provides new drug options and treatment approaches for skin wound treatment; thirdly, the platelet exosome PDEs prepared by the process of this invention naturally have low immunogenicity, do not cause apoptosis but promote cell proliferation; physiological and biochemical indicators and pathological results show that there is no significant damage to liver and kidney function, and it does not cause pathological damage to major organs such as the heart, liver, spleen, lungs and kidneys. Figure 10 and Figure 11 This indicates that PDEs have good safety at the intervention dose in this experiment and can be used for skin wound healing applications. No immune rejection was induced in xenogeneic animal wound healing experiments, and they have been preliminarily applied in allogeneic clinical interventions, providing important safety evidence for xenogeneic PDEs therapy. Attached Figure Description

[0020] Figure 1 Example 2: Morphology and characteristics of sheep platelet exosomes (PDEs). A: Morphology of sheep platelet exosomes (PDEs) observed by transmission electron microscopy; B: Particle size distribution of sheep platelet exosomes (PDEs) determined by nanoparticle size analysis; C: Western blot analysis of surface biomarkers TSG101, CD9, CD63, and Calnexin of sheep platelet exosomes (PDEs).

[0021] Figure 2 Example 2. Structure diagram of miRNA components in sheep platelet exosomes (PDEs).

[0022] Figure 3 Example 4: Effect of sheep platelet exosomes (PDEs) on skin wound healing. The wound healing status at various time points during the experiment.

[0023] Figure 4 Example 4. Effects of sheep platelet exosomes (PDEs) on skin wound healing. The cumulative healing rate and healing speed of the wounds were observed in the experiment. Figure A shows the cumulative healing rate (%) at each time point; Figure B shows the healing speed at each time point.

[0024] Figure 5 Example 5: H&E staining of wound tissue at various time points in the histomorphological observation and analysis of sheep platelet exosomes (PDEs) after skin wound treatment.

[0025] Figure 6 Example 5: CD31 immunohistochemical results of sheep platelet exosomes (PDEs) in the histomorphological observation and analysis of skin wound treatment. Figure A shows a representative image of CD31 immunohistochemical staining of the wound; Figure B shows the quantitative analysis of CD31 microvessel density.

[0026] Figure 7 Example 5: Immunohistochemical results of VEGF-A in the histomorphological observation and analysis of sheep platelet exosomes (PDEs) after skin wound treatment. Figure A shows a representative image of VEGF-A immunohistochemical staining in the wound; Figure B shows the quantitative analysis of VEGF-A H-score.

[0027] Figure 8 Example 5: Immunohistochemical results of α-SMA in the histomorphological observation and analysis of sheep platelet exosomes (PDEs) after skin wound treatment.

[0028] Figure 9 Example 5: Masson staining of wound tissue sections from sheep platelet exosomes (PDEs) used in the histomorphological observation and analysis of skin wounds after treatment. Figure A shows Masson staining in each group; Figure B shows the quantitative analysis of collagen fiber area percentage.

[0029] Figure 10 Example 6: Safety assessment of sheep exosome PDEs: Serum liver and kidney function indicators in mice.

[0030] Figure 11 Example 6: Safety assessment experiment of sheep exosome PDEs. Histopathological observation of major organs stained with HE.

[0031] Figure 12Example 7. Expression levels of collagen synthesis-related genes and proteins in mouse wound skin tissue. Figure A shows the effect of sheep PDEs on the expression of collagen synthesis-related proteins; Figure B shows the protein grayscale value analysis; Figure C shows the effect of sheep PDEs on the mRNA expression level of collagen synthesis-related genes.

[0032] Figure 13 Example 7. Detection of serum inflammatory factor levels in mouse wound skin tissue. Figure A shows serum TNF-α level; Figure B shows serum TGF-β level; Figure C shows serum IL-1β level.

[0033] Figure 14 Example 7. Expression levels of inflammatory factor genes in mouse wound skin tissue.

[0034] Figure 15 Example 8: Sheep exosomes PDEs promote HaCat cell proliferation.

[0035] Figure 16 Example 8: Sheep exosomes PDEs enhance HaCat cell migration. Detailed Implementation

[0036] The present invention is further illustrated below with reference to specific embodiments, but these embodiments do not limit the scope of the invention. Unless otherwise specified, the reagents and materials involved in the embodiments are all commercially available products. Unless otherwise specified, the experimental methods involved in the embodiments are methods generally used in the art. The experimental animals were purchased from the Animal Experiment Center of Yanbian University, and the sheep were raised on the ranch of the College of Agriculture of Yanbian University. The experiment was approved by the Animal Ethics Committee.

[0037] In the experiment, sheep were placed in a fasting state, and 50 mL of whole blood was aseptically collected via the jugular vein. Using 3.2% sodium citrate as an anticoagulant, the blood was centrifuged at 400×g for 10 min at 4 ℃, collecting the upper plasma layer and the middle platelet layer. The collected liquid was then placed back at 4 ℃ and centrifuged at 800×g for 10 min. The upper plasma layer was discarded, retaining only 1 mL of plasma and the bottom platelet precipitate, which was the platelet mixture. The platelet mixture was frozen at -80 ℃ for 24 h and then thawed in a 37 ℃ water bath for 3 min. This cycle was repeated 3 times to activate the platelet mixture. After activation, the mixture was centrifuged at 4 ℃ and 2000×g for 15 min to obtain the platelet lysate.

[0038] Platelet lysis buffer was filtered through a 0.22 μm filter, and an equal volume of platelet exosome extraction buffer (PEG10000-Dextran extraction buffer as described in patent CN202410054453.6: A method for extracting exosomes from platelet-rich plasma) was added. The mixture was incubated at 4 °C for 12 h to induce exosome precipitation. The extract was centrifuged at 15000×g for 1 h at 4 °C, and the supernatant was discarded; the gelatinous precipitate at the bottom of the tube was the crude exosome extract. The precipitate was gently resuspended in pre-cooled, pH 7.4 sterile 1×PBS, and ultracentrifuged at 100000×g for 70 min at 4 °C. The mixture was washed twice to remove impurities and soluble contaminants. The concentration was adjusted, and the purified exosomes were resuspended in 100 μL of sterile PBS, aliquoted, and stored at -80 °C for later use.

[0039] This embodiment uses the "centrifugation-freeze-thaw method" to successfully obtain a high-purity PDE suspension, laying the foundation for subsequent PDE identification and functional experiments.

[0040] Experimental Methods: Core identification of PDEs prepared in Example 1 was performed using the following methods: Transmission Electron Microscopy (TEM): The purified PDEs suspension was dropped onto a copper grid coated with a Formvar membrane, rinsed three times with distilled water, stained with 2% phosphotungstic acid for 10 min, and allowed to air dry. TEM images were then observed and captured. Particle Size and Concentration Detection: The PDEs stock solution was diluted to a suitable concentration with sterile PBS. The field of view was captured using a nanoparticle tracking analyzer (NTA) at 25 ℃, and the particle size distribution and concentration of PDEs were analyzed using the instrument's built-in software. Western blot Detection: A 12% SDS-PAGE gel was prepared, and the PDEs protein samples were separated by gel electrophoresis. The samples were then transferred to a PVDF membrane, blocked with 5% skim milk powder at 4 ℃ for 2 h, incubated overnight with primary antibodies containing exosome-specific markers (CD63, CD81, TSG101), rinsed three times with TBST, and incubated for 1 hour with the corresponding secondary antibodies. h, the ECL chemiluminescence reagent kit was used for color development, the image was captured after exposure, and Western blot detection was completed.

[0041] Experimental results are as follows Figure 1 The purified sheep PDEs were identified using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Transmission electron microscopy revealed that the sheep PDEs exhibited a cup-shaped morphology. Figure 1 A), nanoparticle tracking analysis results show that the particle size of sheep PDEs is mainly distributed at around 110 nm ( Figure 1 B). Western blot results showed that sheep PDEs expressed surface marker proteins CD9, CD63, and TSG101, while no signal was detected for the endoplasmic reticulum marker protein Calnexin. Figure 1C).

[0042] Morphological, particle size, concentration, and specific marker identification confirmed that the PDEs prepared in Example 1 of this invention have intact morphology, uniform particle size, stable concentration, and high purity, which are consistent with the core characteristics of exosomes and can be used for subsequent skin wound healing and safety-related experiments.

[0043] Experimental Methods: Sheep platelet exosome samples were sent to a biotechnology company for library construction and high-throughput sequencing. After passing quality control, small RNA sequencing libraries were constructed. Raw sequencing data were quality-controlled to obtain clean reads, which were then aligned to sheep and mouse reference genomes. The reference genome information is as follows: mm39 for mice (Mus musculus); and ARS-UI_Ramb_v3.0 (RefSeq assembly accession: GCF_016772045.2) for sheep (Ovis aries). The miRdeep2 software was used for known miRNA annotation and new miRNA prediction, with known miRNA annotation based on the miRBase database.

[0044] Experimental results are as follows Figure 2 Small RNA sequencing results from sheep platelet exosomes showed that their miRNA expression was highly specific, exhibiting a distribution pattern of a few high-abundance dominant miRNAs and most low-abundance miRNAs. Among them, oar-miR-148a accounted for 61.78%, making it the absolutely dominant high-abundance miRNA; followed by oar-miR-21, oar-miRNAs of all types, oar-miR-27a, the let-7 family, and oar-miR-30d, etc. Newly predicted novel-miRNAs accounted for 1.78%, while the overall expression abundance of the remaining miRNAs was relatively low.

[0045] This study employed small RNA high-throughput sequencing technology to analyze the expression profile of sheep platelet exosomal miRNAs. It confirmed that oar-miR-148a was the most abundant miRNA and may serve as a marker miRNA, playing a central role in platelet physiological and pathological processes and intercellular communication. Other highly abundant miRNAs, such as oar-miR-21, may also be involved in functional regulation. This study not only provides fundamental data for further research into the biological functions, mechanisms, and clinical applications of platelet exosomal miRNAs but also provides standardized technical references for research on exosomal miRNAs in sheep and other livestock.

[0046] Experimental Methods: Thirty healthy male 6-8 week old BALB / c mice, weighing 20-25 g, were used and acclimatized for 3 days before the experiment began. Mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (0.02 ml / g). After complete anesthesia, the hair on the backs of the mice was removed using a pet shaver and depilatory cream, covering an area of ​​approximately 2 cm × 2 cm, ensuring complete skin exposure. The depilated area was then disinfected with 75% ethanol. Using an 8 mm diameter circular skin punch, uniform pressure was applied vertically on both sides of the midline of the mouse's back to remove the entire skin layer in one go, forming a standardized circular full-thickness skin defect model, ensuring no bleeding or infection at the wound site. A sheep full-thickness skin resection model was established according to the literature method. Three 6-month-old male Yanbian semi-fine wool sheep were used, and the modeling sites were locally anesthetized with 1 mL of 1% lidocaine hydrochloride solution. Hair was removed from the experimental area, and six wounds (three on each side) were created on both sides of the midline of the back of each sheep. A self-controlled design was used, with one side of the wound randomly assigned to the control group and the other side assigned to the sheep platelet exosome (PDEs) treatment group. A full-thickness skin defect model was established using a 2 cm diameter circular skin punch, and equal amounts of the intervention were injected into the wound edges on days 0, 1, and 2.

[0047] Grouping and Intervention: All successfully modeled mice were randomly divided into two groups of 10 mice each, ensuring that the weight, wound size, and health status of the mice in each group were consistent. The NC group (negative control group) received 100 μL of 1×PBS solution injected at four points around the wound edge. The sheep platelet exosome group (treatment group) received 100 μL of 100 μg / mL PDEs solution injected at four points around the wound edge. All interventions were performed on the day of model establishment (day 0) and on the subsequent days 1 and 2, with consistent intervention times each day. After each intervention, the wound was gently covered with sterile gauze to prevent the mice from scratching.

[0048] The sheep that successfully developed the model were used in a self-controlled design. One side of the wound was randomly assigned to the control group NC (negative control group), and 1 mL of 1×PBS solution was injected around the wound edge. The other side was assigned to the sheep platelet exosome PDEs group (treatment group), and 1 mL of sheep exosome PDEs solution with a concentration of 100 μg / mL was injected around the wound edge.

[0049] Wound healing observation: On days 0, 3, 7, 10, and 14 after model establishment, photos of the wounds of mice / sheep in each group were taken with a digital camera under the same light source and distance. The wound area was measured using Image-Pro Plus software, and the wound healing rate was calculated (wound healing rate = (initial wound area - current wound area) / initial wound area × 100%). The wound healing status of each group was recorded, including scab formation, epithelialization, and presence or absence of infection.

[0050] Experimental results are as follows Figure 3 , 4 As shown: Figure 3 Results showed that in the mouse model, the wound area in the treatment and control groups was basically the same at each time point on postoperative day 0, with no significant difference. On postoperative day 3, the wound edges in the treatment group began to shrink towards the center, the wound was relatively moist and clean, and a small amount of granulation tissue was visible, while the wound area in the control group did not shrink significantly, and inflammatory exudate was still visible. On postoperative day 7, the wound in the treatment group shrank significantly, while the wound in the control group still had a large unhealed area, and the epithelialization process was significantly delayed. On postoperative day 10, the wound in the treatment group was basically completely closed, while the wound in the control group, although smaller than on day 7, still showed a small amount of unclosed area, and epithelialization was not yet complete. On postoperative day 14, the wound in the treatment group was completely closed, and the newly formed epithelium matured. The wound in the control group was basically closed, but the healing quality was not as good as that in the treatment group. Figure 3 The above results indicate that sheep PDEs treatment can significantly accelerate wound contraction and epithelialization in mice.

[0051] Figure 3 B shows the healing status of the treatment and control groups of the sheep model at various time points. Gross photographs of the sheep wounds were taken on postoperative days 0, 3, 7, 10, and 14. Results showed that the wound areas were basically the same in both groups on postoperative day 0. On day 3, the wounds in the control group showed little contraction, and significant inflammatory exudate was visible in the wound bed; the wound edges in the treatment group began to contract towards the center, the wounds were cleaner, and there was less exudate. On day 7, the wounds in the control group still had a large unhealed area; the wounds in the treatment group shrank significantly. On day 14, the wounds in the control group still showed complete scab coverage, and healing beneath the scab was not yet complete; the scabs in the treatment group had completely fallen off, and new epithelium had formed, but the new area was pale red or pink, indicating that the epithelial layer was still in the early stages of maturation, and the overall healing quality was significantly better than that in the control group.

[0052] Figure 4 The figures show the cumulative healing rate and healing speed of the wounds; Figure A shows the cumulative healing rate (%) of the mouse wounds at each time point; Figure B shows the healing speed of the mouse wounds at each time point. Figure 4 Results A and 4B showed that there was no significant difference in healing rate between the two groups of mice on postoperative day 3; from day 7 onwards, the healing rate in the treatment group was significantly higher than that in the control group, and the difference was highly statistically significant. P <0.001); on day 10, the healing rate in the treatment group was still significantly higher than that in the control group ( P<0.01); By day 14, both groups of wounds were essentially closed, with no significant difference in healing rate. Further analysis of wound healing speed revealed that the average time to complete closure in the treatment group was significantly shorter than that in the control group. Stage healing rate analysis showed that the daily increase in healing rate in the treatment group was significantly higher than that in the control group from day 3 to 7 (P<0.01); from day 7 to 10, the treatment group maintained a faster healing rate, with a statistically significant difference compared to the control group (P<0.05); from day 10 to 14, the healing rate slowed down in both groups, with no significant difference between the groups. These results indicate that sheep platelet exosome (PDE) treatment can significantly improve wound healing rate and accelerate the early to mid-stage healing process.

[0053] Figure 4 C and 4D results showed that although the healing rate in the treatment group was slightly higher than that in the control group, the difference was not statistically significant. P >0.05). Analysis of healing speed showed that the average time to complete closure in the treatment group was slightly shorter than that in the control group, but the difference was not statistically significant. P >0.05). This indicates that PDEs treatment in sheep mainly promotes healing in the early stages of the sheep model.

[0054] Because platelet exosomes contain abundant growth factors (such as EGF, FGF-2, VEGF, etc.), cytokines, and bioactive substances, these substances can be absorbed by cells around the wound through local injection, activating the proliferation and migration of skin fibroblasts and keratinocytes, promoting angiogenesis, accelerating the growth of granulation tissue and epithelialization of the wound, thereby shortening the wound healing time and improving the healing quality; while PBS, as a negative control, contains no bioactive substances and cannot promote wound healing, so the healing speed of the NC group was significantly slower than that of the PDEs group.

[0055] The above results indicate that local injection of a 100 μg / mL PDEs solution significantly promotes the healing of full-thickness skin defects in BALB / c mice, accelerates the process of scab formation and epithelialization, and improves the wound healing rate and quality. This demonstrates that PDEs have a role in promoting skin wound healing. Furthermore, sheep exosome PDEs showed better wound healing effects in mice than in sheep.

[0056] Experimental Methods: Mice modeled and intervened in Example 4 were selected. On days 7 and 14 after model establishment, 5 mice from each group were euthanized. Tissue samples from the wound area and surrounding 0.5 cm of healthy skin, as well as tissues from the mouse's heart, liver, spleen, lungs, and kidneys, were collected and fixed in 4% paraformaldehyde solution for 24 h. Subsequent treatment followed these steps: (1) H&E staining: The fixed tissue was dehydrated in a gradient (70%, 80%, 90%, 95%, 100% ethanol in sequence), cleared with xylene, impregnated with paraffin, and embedded in paraffin to prepare continuous sections with a thickness of 5 μm. After dewaxing and hydration (xylene dewaxing and gradient ethanol hydration), the sections were stained with hematoxylin for 5 min, differentiated with hydrochloric acid ethanol for 30 s, blued with distilled water for 5 min, stained with eosin for 30 s, dehydrated with gradient ethanol, cleared with xylene, mounted with neutral resin, observed and photographed under an optical microscope, and the degree of epithelialization, granulation tissue growth and inflammatory cell infiltration of the wound tissue were analyzed, and the morphology of organ tissues was analyzed.

[0057] (2) Immunohistochemical staining: The tissue sections were baked at 60°C for 2 h, dewaxed and hydrated, and then subjected to high-temperature antigen retrieval for 15 min with citrate buffer (pH=6.0). They were then incubated at room temperature with 3% H2O2 for 10 min to block endogenous peroxidase, and blocked with 5% skim milk powder at 4°C for 1 h. Ki67 (cell proliferation marker) primary antibody was added and incubated overnight. After washing 3 times with TBST, secondary antibody was added and incubated for 1 h. DAB staining was performed, hematoxylin counterstaining was performed, and the sections were dehydrated, cleared, and mounted. The Ki67 positive cell rate was quantitatively analyzed using Image-Pro Plus software.

[0058] (3) Masson staining and Sirius red staining: After baking and dewaxing the tissue sections, Masson staining was performed according to the kit instructions. After hematoxylin staining, Ponceau S staining, phosphomolybdic acid staining, and aniline blue staining, the sections were differentiated, dehydrated, cleared, and mounted. The distribution of collagen fibers was observed under a microscope. After Sirius red staining, the sections were stained with Sirius red dye solution, counterstained with hematoxylin, dehydrated, cleared, and mounted. The type of collagen fibers was observed under a polarized light microscope. The area ratio of collagen fibers was quantitatively analyzed using Image-Pro Plus software.

[0059] Experimental results: Figure 5 Results showed that wound tissue was collected on postoperative days 7 and 14, and H&E staining was used to observe epidermal regeneration, granulation tissue formation, and skin appendage reconstruction. On day 7, the control group (NC) had a larger area of ​​unepithelialized wound, with abundant inflammatory cell infiltration and a thin granulation tissue layer; the treatment group (PDEs) had relatively complete new epidermis at the wound edge, significantly advanced epithelialization, thickened granulation tissue layer, and dense fibroblasts. On day 14, the control group's wound was basically epithelialized, but the epidermal layer was thin, and appendages such as hair follicles and sebaceous glands were rare; the treatment group (PDEs) had an intact and thickened epidermal layer, with visible new hair follicles and sebaceous gland structures, and the healing quality was significantly better than that of the control group (NC).

[0060] Figure 6CD31 immunohistochemical results showed that on postoperative days 7 and 14, the number of CD31-positive microvessels in the granulation tissue of the treated group (PDEs) was significantly higher than that in the control group (NC). P <0.001).

[0061] Figure 7 Immunohistochemical results for VEGF-A showed no significant difference in VEGF-A expression levels between the two groups on postoperative day 7. P >0.05); On postoperative day 14, the VEGF-A expression level in the control group (NC) was significantly higher than that in the treatment group (PDEs) ( P <0.05)( Figure 7 ).

[0062] Figure 8 Immunohistochemical results of α-SMA showed that PDEs treatment in sheep significantly promoted early angiogenesis and vascular maturation in the treatment group (PDEs), with CD31 microvessel density being higher than that in the control group (NC) at both 7 and 14 days. Overall results indicated that VEGF-A and α-SMA levels were higher in the control group (NC) than in the treatment group (PDEs) at 14 days, suggesting that the healing process in the treatment group (PDEs) had entered the remodeling phase earlier, and angiogenesis-related signals were naturally downregulated.

[0063] Figure 9 Masson staining of wound tissue sections showed that on day 7, collagen deposition in the control group (NC) was sparse and lightly stained, with a large proportion of red muscle fibers and sparse, disordered collagen fibers. In the treatment group (PDEs), more blue-stained collagen fibers were visible, mainly distributed in the granulation tissue area, and the collagen deposition area was significantly larger than that in the control group (NC). On day 14, collagen deposition in the control group (NC) increased, but remained predominantly loose collagen fibers; collagen deposition in the treatment group (PDEs) was denser, with thicker, more orderly collagen fibers and a significantly increased staining depth. On postoperative days 7 and 14, the area of ​​blue collagen fiber deposition in the granulation tissue of the wound in the treatment group (PDEs) was significantly larger than that in the control group (NC). This indicates that sheep PDEs treatment in the treatment group (PDEs) can promote collagen deposition in the wound and improve collagen fiber structure.

[0064] Experimental Methods: Mice modeled and intervened in Example 4 were selected. On days 7 and 14 after the establishment of the skin defect model, five mice from each group were euthanized by intraperitoneal injection of an overdose of 1.25% tribromoethanol anesthetic. Blood was collected from the eyeballs. Immediately after blood collection, a painless cervical dislocation was performed to confirm death and avoid causing pain to the mice. The collected blood was placed in centrifuge tubes and centrifuged at 3000×g for 15 min to obtain serum. The serum samples were sent to the testing center, where a fully automated biochemical analyzer was used to detect liver function indicators (alanine aminotransferase ALT, aspartate aminotransferase AST) and kidney function indicators (serum creatinine Cr, blood urea nitrogen BUN). At the same time, the mice underwent complete organ dissection, and the five major organs—heart, liver, spleen, lung, and kidney—were removed. The organs were rinsed with physiological saline to remove surface bloodstains and fixed in 4% paraformaldehyde solution for subsequent systematic pathological and histological analysis to observe whether there were any abnormalities such as damage or inflammation in the organs.

[0065] Figure 10 The results of serum liver and kidney function indicators in mice showed that there were no significant differences in any liver and kidney function indicators among mice treated with sheep platelet exosomes (PDEs). P The values ​​were >0.05, and all test values ​​were within the normal physiological range. This indicates that local application of sheep platelet exosomes (PDEs) did not cause significant liver and kidney function damage during the observation period, demonstrating good short-term safety.

[0066] Figure 11 Histopathological observation of major organs using HE staining showed that the organs in both the control group (NC) and the treatment group treated with sheep PDEs (PDEs) had intact structures and normal cell morphology, with no obvious pathological changes such as degeneration, necrosis, inflammatory cell infiltration, or fibrosis. There were no significant differences in the histological manifestations of organs between the two groups. This indicates that during the observation period for skin defect repair, local application of sheep PDEs did not cause significant organic damage to major organs, demonstrating good biocompatibility.

[0067] Experimental results showed that local injection of 100 μg / mL PDEs solution did not significantly damage liver or kidney function in BALB / c mice, nor did it cause pathological damage to major organs such as the heart, liver, spleen, lungs, and kidneys. This indicates that PDEs have good safety at the experimental intervention dose and can be used for skin wound healing.

[0068] The core of safety assessment is to detect the effects of drugs / biologics on the function and tissue morphology of vital organs. Elevated liver function indicators ALT and AST suggest hepatocellular damage, while elevated kidney function indicators Cr and BUN suggest renal impairment. Histopathological observation can directly reflect changes in the microstructure of organs. In this experiment, PDEs are platelet-derived exosomes with good biocompatibility and no significant immunogenicity. After local injection, they do not accumulate in large quantities in the bloodstream and do not burden metabolic organs such as the liver and kidneys. Therefore, they do not cause organ dysfunction or tissue damage, and their safety is good.

[0069] EGF (Epidermal Growth Factor) promotes keratinocyte proliferation and migration, accelerating epithelialization; FGF-2 promotes fibroblast proliferation and collagen synthesis; VEGF-A promotes angiogenesis, providing nutrients and oxygen for wound healing. α-SMA is a marker of fibroblast activation; COL1A1 and COL3A1 are the encoding genes for type I and type III collagen, respectively. Upregulation of COL1A1 and downregulation of COL3A1 can improve collagen ratio and reduce scarring. IL-1β and TNF-α are major pro-inflammatory factors; increased expression of these genes exacerbates wound inflammation and delays healing; TGF-β can inhibit inflammatory responses and promote fibroblast proliferation and collagen synthesis. This experiment aims to investigate whether PDEs promote wound healing through multiple pathways by regulating the expression of these genes.

[0070] Experimental methods: (1) Primer design: Based on the NCBI sequences of genes related to growth factors (EGF, FGF-2, VEGF-A), collagen (α-SMA, COL1A1, COL3A1) and inflammatory factors (IL-1β, TNF-α, TGF-β), specific primers were designed using Oligo 7.0 software. GAPDH was used as the internal reference gene. The primer sequences were verified by sequencing to ensure specificity. (2) Total RNA extraction and cDNA reverse transcription: Skin tissue from the wounds of mice in each group in Example 3 on day 7 and day 14 was taken, minced and added to Trizol lysis buffer, and thoroughly ground into homogenate. The total RNA was obtained by chloroform extraction, isopropanol precipitation and washing with 75% ethanol. The purity (A260 / A280=1.8-2.0) and concentration of RNA were detected by Nanodrop instrument to ensure that the RNA was not degraded and the purity was qualified. The total RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit and stored at -20 ℃ for later use. (3) Real-time quantitative PCR (qPCR) detection: Using cDNA as a template, a real-time quantitative PCR kit was used. The reaction was performed according to the set system (20 μL system: 2 μL cDNA, 0.8 μL each of forward and reverse primers, 10 μL SYBR Green Mix, 6.4 μL ddH2O) and program (95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 10 s, 60 ℃ annealing for 30 s, 40 cycles; melting curve analysis). Each sample was set up with 3 replicates. The 2-ΔΔCt method was used to calculate the relative expression levels of each target gene (EGF, FGF-2, VEGF-A, α-SMA, COL1A1, COL3A1, IL-1β, TNF-α, TGF-β) relative to the internal reference gene GAPDH, and the differences in gene expression between the two groups were compared.

[0071] Figure 12 The results showed that, compared with the control group, the expression levels of Col1a1 and Col3a1 mRNA were significantly upregulated in the sheep PDEs-treated group on postoperative days 7 and 14. Western blot analysis further confirmed that the protein expression levels of COL1A1 and COL3A1 in the treatment group were significantly higher than those in the control group at the corresponding time points. The expression levels of α-SMA gene and protein were significantly higher in the treatment group than in the control group on postoperative day 7, but lower on day 14. Figure 13 The results showed that on postoperative day 7, compared with the control group, the treatment group had significantly lower serum TNF-α and IL-1β levels and significantly higher TGF-β levels. P <0.05. On postoperative day 14, the levels of TNF-α, IL-1β, and TGF-β in the treatment group were significantly lower than those in the control group ( P <0.05). For example... Figure 14The results showed that on postoperative day 7, the mRNA expression levels of Tnf and Il1b in the treatment group were significantly downregulated compared with those in the control group, while the mRNA expression level of Tgfb was significantly upregulated. P <0.05). On postoperative day 14, the expression levels of Tnf, Il1b, and Tgfb mRNA in the treatment group were significantly downregulated compared to the control group ( P <0.05). This result is basically consistent with the trend of changes in serum inflammatory factors.

[0072] Experimental results show that PDEs can promote cell proliferation and angiogenesis by significantly upregulating the expression of growth factor genes such as EGF, FGF-2, and VEGF-A in wound tissue; regulate collagen synthesis and granulation tissue maturation by upregulating the expression of α-SMA and COL1A1 genes and downregulating the expression of COL3A1 gene; and reduce wound inflammation by inhibiting the expression of pro-inflammatory factors such as IL-1β and TNF-α and promoting the expression of anti-inflammatory factor TGF-β, thereby synergistically promoting skin wound healing. This further reveals the mechanism by which PDEs promote wound healing.

[0073] Experimental Methods: Two experiments were conducted using HaCaT cells, as follows: 1. CCK-8 proliferation assay: Four experimental groups were set up with 0, 10, 50, and 100 μg / mL sheep PDEs, with 6 replicates in each group; single-cell suspensions were prepared from logarithmic growth phase HaCaT cells, and 5,000 cells per well with a final volume of 100 μL (serum-free DMEM high-glucose medium) were seeded into 96-well plates. After cell adhesion, sheep PDEs of corresponding concentrations were added to each group, and a blank control group (equal volume of cell-free medium, used for ELISA reader calibration) was set up. After culturing for 24 h and 48 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 ℃ and 5% CO2 in the dark for 2 h. The absorbance was measured at 450 nm using an ELISA reader, and the optical density value (positively correlated with the number of viable cells) was recorded to assess cell proliferation. ODa is the absorbance of experimental wells, ODb is the absorbance of control wells, and ODC is the absorbance of blank wells. 2. Scratch assay: Two experimental groups were set up: the NC group and the 100 μg / mL sheep PDEs group, with three replicates in each group. HaCaT cells were seeded at an appropriate density in 6-well plates and cultured in serum-containing complete medium until 90% confluence to form a monolayer. A straight scratch was made vertically to the bottom of each well using a sterile 200 μL pipette tip. The old medium was discarded, and the cells were washed twice with preheated 1×PBS to remove floating cells and debris. The medium was then replaced with serum-free DMEM and treated with sheep PDEs. Images were taken under a microscope immediately after scratching and at 12 h and 24 h of culture. The scratch area was measured using ImageJ software, and the wound healing rate was calculated using the formula: Wound healing rate (%) = (A0 - A t ) / A0× 100% Where A0 represents the area of ​​the scratch at the time of the initial shooting, A t This represents the area of ​​the scratch after 12 or 24 hours of cultivation.

[0074] Experimental results: Figure 15 The results showed that, compared with the control group, treatment with sheep PDEs at concentrations of 10, 50, and 100 μg / mL significantly increased HaCat cell proliferation activity after 24 h and 48 h. The 10 μg / mL treatment group showed statistically significant increases at both 24 h and 48 h. P <0.01), while the 50 and 100 μg / mL treatment groups showed more significant proliferative effects at both time points ( P <0.001). Further analysis showed that the promoting effect of sheep PDEs on HaCat cell proliferation was significantly dose-dependent, and the overall proliferation-promoting effect of the 48 h treatment group was better than that of the 24 h group. These results indicate that treatment with 10-100 μg / mL sheep PDEs for 24-48 h can effectively promote HaCat cell proliferation. 100 μg / mL sheep PDEs was selected as the optimal treatment condition for subsequent experiments.

[0075] Figure 16 The results showed that compared with the control group, the scratch area in the sheep PDEs treatment group was significantly reduced at 12 h and 24 h after scratching, and the cell migration rate was significantly increased. The differences were statistically significant. P <0.01). The above results indicate that sheep PDEs treatment can effectively promote the migration of HaCat cells.

Claims

1. A method for preparing platelet exosomes, characterized in that the steps include: The following steps were taken: Platelets were separated from platelet-rich plasma and activated using a freeze-thaw method; the activated platelet-rich plasma suspension was centrifuged at 4 ℃ and 2000×g for 15 min to remove incompletely lysed platelet fragments and cell debris, and the supernatant was collected to obtain platelet lysate. Macromolecular impurities were removed by filtration, and the platelet exosomes were incubated with platelet exosome extract and then washed by ultracentrifugation to specifically separate and purify platelet exosomes, obtaining a high-purity platelet exosome suspension.

2. The method for preparing platelet exosomes as described in claim 1, characterized in that, The freeze-thaw method involves rapidly freezing platelets in an ultra-low temperature freezer at -80 ℃ for 24 h, followed by rapid thawing in a 37 ℃ water bath for 3 min, and repeating the freeze-thaw cycle three times.

3. The use of platelet exosomes obtained by the preparation method of claim 1 or 2 in the preparation of medicaments or pharmaceutical compositions for skin wound healing.

4. The application according to claim 3, characterized in that, The platelet exosomes are sheep platelet exosomes.

5. The application according to claim 3, characterized in that, The application is the use of sheep platelet exosomes in drugs or drug compositions for the healing of skin wounds in non-sheep animals.

6. The application according to claim 5, characterized in that, The non-sheep animals include, but are not limited to, rats, pigs, cattle, sheep, deer, horses, and dogs.

7. The application according to claim 3, characterized in that, The skin wounds include mechanical wounds, traumatic wounds, chemical wounds, ulcerative wounds, and radiation wounds.

8. The application according to claim 3, characterized in that, The concentration of platelet exosomes in the pharmaceutical composition is 100 μg / mL.

9. A platelet exosome preparation, characterized in that, The platelet exosome preparation is prepared from platelet exosomes and a pharmaceutically acceptable carrier; the platelet exosomes are obtained by the preparation method described in claim 1 or 2; the dosage form of the preparation includes injections, ointments, powders, and sprays.

10. The platelet exosome preparation of claim 9, characterized in that, The concentration of platelet exosomes in the drug formulation is 100 μg / mL.

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