Application of human umbilical cord blood plasma exosome in promoting skin wound healing

Abstract

The invention discloses an application of a human umbilical cord blood plasma exosome in preparation of a medicine for promoting skin wound healing and / or reducing scar formation. The biological active molecules of the exosome are used for adjusting the wound healing process, increasing the healing speed, reducing scar formation and improving the prognosis of a patient, the biological active molecules have important clinical value, a novel and effective treatment method can be provided for healing of skin wounds, and the biological active molecules are particularly suitable for repairing skin injuries caused by postoperation, trauma, burn and the like, and have a wide application prospect. And moreover, scars possibly generated after wound healing can be remarkably reduced, and the wound dressing has a relatively high market prospect and wide application potential. According to the method, byproducts of hematopoietic stem cell storage are fully utilized, waste is turned into wealth, and a theoretical basis is provided for research and development of functional products for promoting skin wound healing.

Description

Technical Field

[0001] The present invention relates to application of human umbilical cord blood plasma exosomes in promoting skin wound healing and reducing scar formation, and belongs to the technical field of exosomes. Background Art

[0002] In recent years, many studies have shown that mesenchymal stem cells have broad application prospects in the field of regenerative medicine. In addition to stem cells themselves, exosomes (Exos) have also become a hot topic for researchers and have brought new ideas for the treatment of diseases. Exosomes are a type of extracellular vesicles, and it has been proven that exosomes are important carriers for the exchange of substances and information between cells.

[0003] Skin is the outermost layer of the human body and the largest organ in the human body. Its main function is to protect the body from the invasion of external pathogens, and it also has functions such as sweating and sensation. In the field of skin repair and regeneration, stem cell-centered therapies have achieved a variety of surprising therapeutic effects. Initial studies believed that the therapeutic effect of stem cells was related to their ability to differentiate into a variety of tissue cells, such as adipocytes, osteoblasts, chondrocytes, epithelial cells, etc. However, more and more studies have shown that stem cells mainly rely on paracrine methods to promote the repair of skin trauma in wound repair, while the transplantation and differentiation rate of stem cells themselves to the injured site is slightly low, and the effect is short-lived. In addition, the use of stem cells is also limited by a short shelf life. Therefore, it is very necessary to find a treatment method that has both the therapeutic effect of stem cells and overcomes the shortcomings of stem cells. Exosomes, as a subcellular component secreted by cells, are widely involved in the communication between cells and can act as paracrine factors of stem cells to exert biological effects, providing a new way to further realize cell-free therapy. Exosomes can not only be used as diagnostic and therapeutic tools for diseases, but also participate in a variety of pathophysiological processes, such as tissue repair, immune response and inflammation, tumor growth and metastasis, etc.

[0004] Most of the existing studies focus on the effects of exosomes derived from mesenchymal stem cells cultured in vitro on skin function. Umbilical cord blood contains some mesenchymal stem cells. Extracting exosomes directly from umbilical cord blood (human umbilicalcord plasma exosomes, UCB-Exos) has the characteristics of simple method and strong applicability. However, it is still unknown whether exosomes extracted from umbilical cord blood have the tissue repair function of mesenchymal stem cell exosomes cultured in vitro, and further verification is needed, which is the purpose and significance of this invention. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides the use of human umbilical cord blood plasma exosomes in the preparation of a drug for promoting skin wound healing and / or reducing scar formation.

[0006] The present invention is achieved through the following technical solutions:

[0007] Application of human umbilical cord blood plasma exosomes in the preparation of drugs for promoting skin wound healing and / or reducing scar formation.

[0008] Furthermore, the human cord blood plasma exosomes are prepared by the following method: human cord blood plasma is taken, diluted with PBS buffer, and centrifuged three times in sequence: 4°C, 2000g centrifugation for 30 minutes; 4°C, 10000g centrifugation for 45 minutes; 4°C, 110000g ultracentrifugation for 70 minutes; then the suspension is filtered with a 0.22μm sterilizing filter, and then ultracentrifuged, the supernatant is discarded, and the precipitate is the cord blood plasma exosomes.

[0009] Furthermore, the volume ratio of human umbilical cord blood plasma to PBS buffer is 1:1.

[0010] Furthermore, the ultracentrifugation is performed twice.

[0011] Furthermore, the parameters of the ultracentrifugation are: 4°C, 110,000 g, 70 min.

[0012] A drug for promoting skin wound healing and / or reducing scar formation, wherein the active ingredient is human umbilical cord blood plasma exosomes. The preparation method of the human umbilical cord blood plasma exosomes is the same as above.

[0013] A method for preparing human umbilical cord blood plasma exosomes, as described above.

[0014] In the present invention, exosomes were first extracted from human umbilical cord blood plasma and purified through a series of centrifugation and filtration steps to obtain highly pure exosomes. The extracted exosomes were then characterized by nanoparticle tracking analysis, transmission electron microscopy and other techniques to ensure that their morphology and size met expectations. The effects of exosomes on the proliferation and migration of human fibroendothelial cells were then evaluated by in vitro cell experiments. Finally, the purified exosomes were directly applied to a mouse trauma model, and the therapeutic effect of the exosomes was evaluated by observing the improvement in wound healing speed, scar area and tissue structure.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Promote wound healing: Exosomes can promote the proliferation and migration of fibroblasts and accelerate the wound healing process.

[0017] (2) Reduce scar formation: Exosomes can regulate inflammatory responses and reduce abnormal collagen deposition, thereby reducing scar formation.

[0018] (3) Improve treatment safety: Ensure the safety and effectiveness of treatment methods through strict exosome extraction and purification processes, as well as safety assessments.

[0019] The present invention regulates the wound healing process through the bioactive molecules of exosomes, improves the healing speed, reduces scar formation, and improves the patient's prognosis. It has important clinical value and can provide a new and effective treatment method for the healing of skin wounds. It is particularly suitable for repairing skin damage caused by surgery, trauma, burns, etc., and can significantly reduce the scars that may be produced after wound healing. It has a high market prospect and wide application potential. The present invention makes full use of the byproducts of hematopoietic stem cell storage, turning waste into treasure, and provides a theoretical basis for the research and development of functional products that promote skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of transmission electron microscopy results of exosomes.

[0021] Figure 2 : Schematic diagram of nanoparticle tracking analysis results of exosomes.

[0022] Figure 3 : Schematic representation of the results of exosome internalization by human fibroblasts.

[0023] Figure 4 : Schematic diagram of the effects of different concentration gradients of UCB-Exos on the proliferation of human dermal fibroblasts.

[0024] Figure 5 : Schematic diagram of the effects of different concentrations of UCB-Exos at different time gradients on the proliferation of human dermal fibroblasts.

[0025] Figure 6 : Schematic diagram of cell scratch test results.

[0026] Figure 7 : Schematic diagram of cell migration assay results.

[0027] Figure 8 : Schematic diagram of wound healing.

[0028] Fig. 9 : Schematic diagram of wound healing rate results.

[0029] Fig.10 : Schematic diagram of scar width results.

[0030] Fig.11 : Schematic diagram of re-epithelialization rate results.

[0031] Fig.12 : Results of immunohistochemical staining. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0033] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0034] In the statistical analysis involved in the present invention, each group of experiments was repeated at least 3 times, and the experimental data of each group were expressed as mean ± standard deviation (SD). The means between multiple groups were compared using one-way analysis of variance (ANOVA). The means between two groups were compared using independent sample t-test. GraphPadPrism software was used for statistical analysis, and P < 0.05 was considered statistically significant.

[0035] Experiment 1 Isolation, purification and identification of exosomes

[0036] (1) Ultra-high-speed centrifugation to separate exosomes

[0037] Take out the packaged frozen human umbilical cord blood plasma, dilute it with PBS buffer at 1:1 (volume ratio), and centrifuge it three times in sequence, as follows: 4℃, 2000g centrifugation for 30min; 4℃, 10000g centrifugation for 45min; 4℃, 110000g ultracentrifugation for 70min; then filter the suspension with a 0.22μm sterilizing filter, and perform ultracentrifugation twice (4℃, 110000g centrifugation for 70min), discard the supernatant, and the precipitate is the umbilical cord blood plasma exosomes. Resuspend the precipitate with 1.5-2mL PBS buffer, transfer it into a 2mL EP tube, and store it at -80℃ for later use.

[0038] (2) Identification of exosomes

[0039] The identification of exosomes was mainly carried out by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM and NTA were tested by Guangzhou Huayinkang Company.

[0040] TEM results are as follows Figure 1 As shown, the results showed that UCB-Exos presented a spherical or disk-shaped morphology.

[0041] NTA results as Figure 2 As shown, the results showed that the particle size range of UCB-Exos was between 100 and 200 nm, which was consistent with the particle size range of exosomes.

[0042] Experiment 2: Internalization of exosomes by human fibroblasts

[0043] (1) Culture of human fibroblasts

[0044] Cell recovery: Shake and thaw the cryotube containing 1 mL of human fibroblast suspension quickly in a 37°C water bath, add 5 mL of culture medium and mix well. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 4-6 mL of complete culture medium and blow evenly. Then add all the cell suspension to a 6 cm culture dish and culture overnight. Change the medium and check the cell density the next day.

[0045] Cell passaging: discard the culture supernatant and rinse the cells with D-PBS 1-2 times. Add 1-2 mL of digestion solution (0.25% Trypsin-0.53 mM EDTA) to the culture bottle and place it in a 37°C incubator for digestion for 1-2 minutes. Observe the digestion of the cells under a microscope; if most of the cells become round and fall off, quickly take it back to the operating table, tap the culture bottle a few times, and add 5 mL of complete culture medium containing 10% serum to stop digestion. Gently blow the cells, aspirate them after they fall off completely, centrifuge at 1000 rpm for 8-10 minutes, discard the supernatant, add 1-2 mL of culture medium and blow evenly. Passage the cell suspension to a new dish or bottle containing 5-6 mL of culture medium at a ratio of 1:2 to 1:5.

[0046] (2) Fluorescence tracing of exosomes

[0047] After the exosomes and human dermal fibroblast nuclei were stained with the optical dyes PKH67 and DAPI respectively, the two were co-cultured for 24 hours. After fixation, the internalization of exosomes by human dermal fibroblasts was observed under a fluorescence microscope.

[0048] The results are as follows Figure 3 As shown, the experimental results showed that UCB-Exos could be phagocytosed and internalized by human dermal fibroblasts when co-cultured with them for 24 hours, indicating that human dermal fibroblasts can phagocytize exosomes.

[0049] Experiment 3: Study on the effect of exosomes on the migration and proliferation of fibroblasts at the cellular level

[0050] (1) Cell scratch assay

[0051] Add 5 × 10 5Human dermal fibroblasts (3 replicates per group); after the cells were fully spread, a 200μL pipette tip was used to make cell scratches perpendicular to the well plate and line; after washing with D-PBS, serum-free medium was added to set up a control group (100μL / mL D-PBS) and a UCB-Exos group (100μg / mL); placed in a 37°C, 5% CO2 incubator for co-culture for 0, 12h, and 24h; photographed, and the area and average width of the scratches were measured using Image J software, and then the migration rate was calculated. The scratch closure area was calculated as follows: Migration area (%) = (A0-An) / A0×100%, where A0 is the initial scratch area and An represents the remaining area.

[0052] (2) Cell migration assay

[0053] In the cell migration experiment, 1×10 4 Fibroblasts / well (3 replicates per group) were suspended in low serum (1% fetal bovine serum) basal medium and inoculated into the upper chamber of a 24-well plate containing an 8μm pore filter. Complete medium without exosomes (containing 10% fetal bovine serum) was then added to the lower chamber, and UCB-Exos (100μg / mL) was added. After 24h, the cells attached to the upper surface of the filter were washed, and the cells migrating to the lower surface were fixed with 4% paraformaldehyde for 30min and stained with 0.5% crystal violet for 30min. The number of migrations was observed under an optical microscope.

[0054] (3) Cell proliferation assay

[0055] The cell proliferation / toxicity assay (CCK-8) was used to determine the effect of UCB-Exos on HDF-α cell proliferation. 3 Fibroblasts (6 replicates per group) were seeded into 96-well plates and cultured in medium containing different concentrations (10, 50, 80, 100, 120, 150 μg / mL) of UCB-Exos; a group without cells was used as a blank group. At 12h, 24h, 48h, and 72h, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 3h. The absorbance was observed at 450nm using an ELISA reader. Cell viability = (OD of the drug group - OD of the blank group) / (OD of the control group - OD of the blank group) × 100%.

[0056] Results: This study used the CCK-8 method to detect the effects of different concentrations of UCB-Exos co-cultured with human dermal fibroblasts for 24 hours on the proliferation of fibroblasts. Figure 4As shown in the results, the cell viability increased with the increase of exosome concentration and was concentration-dependent. In addition, the effects of different time gradients of UCB-Exos at concentrations of 80μg / mL, 100μg / mL, and 120μg / mL on the proliferation of human dermal fibroblasts were also tested. The results are shown in Figure 5 As shown, the results showed that the cell viability increased with time.

[0057] The results of cell scratch test were as follows Figure 6 As shown, the results showed that the UCB-Exos treatment group could significantly promote the migration area of ​​human dermal fibroblasts compared with the PBS treatment group, and the difference was statistically significant.

[0058] The results of cell migration assay were Figure 7 As shown, the results showed that the UCB-Exos treatment group could significantly increase the migration of human dermal fibroblasts compared with the PBS treatment group, and the number of cell migration in each group was quantitatively analyzed. The UCB-Exos treatment group could significantly increase the number of fibroblast migration.

[0059] The above experiments show that UCB-Exos can promote the proliferation and migration of human dermal fibroblasts.

[0060] Experiment 4: Animal experiment to observe the effect of exosomes on skin wound healing

[0061] (1) Establishment and treatment of mouse skin wound model

[0062] In this experiment, male 6-8 week-old clean-grade BALB / c mice (26-30 g) were used to establish a skin wound model. The back of the mice was shaved and depilated. After the mice adapted for a few hours, the mice were anesthetized by isoflurane inhalation and a full-thickness skin wound (10 mm in diameter) was created on the back. 30 mice were randomly divided into 2 treatment groups, with 15 mice in each group (n=15). UCB-Exos and an equal amount of exosome diluent (PBS) were subcutaneously injected around the wound starting from the 3rd day for 5 consecutive days (40 μg of exosomes diluted in 200 μl of PBS per day), and the wound conditions were photographed and recorded during the healing process. All wounds were measured with calipers, and the area of ​​each wound on days 3, 7, 11, 15, and 17 was evaluated using Image-Pro Plus 6 software. The reduction in wound size was calculated using the mathematical formula: wound healing rate (%) = (A0-At) / A0×100%, where A0 is the initial wound area and At is the wound area on postoperative days 3, 7, 11, 15, and 17. The mice were then killed on postoperative day 17 and skin samples from the wounds of each treatment group were collected for histopathological analysis.

[0063] Results: The results are as follows Figure 8 , Fig. 9 As shown, the results showed that the UCB-Exos treatment group could better promote wound healing compared with the PBS treatment group, and the wound area measured on days 3, 7, 11, 15, and 17 after injury was smaller. Quantitative analysis of the wound area at different times and different treatments showed that the UCB-Exos treatment group could significantly promote wound healing compared with the PBS treatment group, and there were significant differences on days 5, 7, 11, 15, and 17.

[0064] (2) Immunohistochemistry

[0065] After the mouse skin samples of each treatment group were collected, they were immediately placed in a 50mL centrifuge tube filled with 4% paraformaldehyde for fixation, and then rinsed and dehydrated with a series of different concentrations of alcohol, and then embedded in paraffin for sectioning. Hematoxylin-eosin staining (HE staining) was performed and photographed under an optical microscope to observe the histological changes, the new epidermis, dermis, regenerated hair follicles and adipocytes of the wound, and the scar width was measured and the percentage of new epithelium was evaluated. The calculation formula for the percentage of re-epithelialization (E%) is: E% = WN / WO × 100%, where WO is the original wound area and WN is the length of the newly generated epithelial cells passing through the wound surface. Masson trichrome staining was performed and photographed under an optical microscope to observe the degree of collagen maturation and the condition of wavy fibers in the wound.

[0066] Results: Fig.10 , Fig.11 As shown in Figure 2, quantitative analysis of re-epithelialization rate and scar width further confirmed that UCB-Exos enhanced epidermal regeneration and reduced scar formation in wounds (P < 0.05). Fig.12 As shown, the results showed that on the 17th day after injury, the skin tissue of the UCB-Exos-treated group had longer new epidermis and dermis, intact stratum corneum repair, and thicker epidermis compared with the skin tissue of the control group mice; a large number of fibroblasts, fibrocytes and collagen fibers were seen in the dermis, accompanied by a large number of lymphocyte infiltrations, capillary proliferation and hair follicle formation.

[0067] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of human umbilical cord blood plasma exosomes in the preparation of drugs that promote skin wound healing and / or reduce scar formation.

2. The use according to claim 1, characterized in that: The human umbilical cord blood plasma exosomes are prepared by the following method: human umbilical cord blood plasma is taken, diluted with PBS buffer, and centrifuged three times in sequence: 4°C, 2000g centrifugation for 30 minutes; 4°C, 10000g centrifugation for 45 minutes; 4°C, 110000g ultracentrifugation for 70 minutes; then the suspension is filtered with a 0.22μm sterilizing filter, and then ultracentrifuged, the supernatant is discarded, and the precipitate is the umbilical cord blood plasma exosomes.

3. The use according to claim 2, characterized in that: The volume ratio of human umbilical cord blood plasma to PBS buffer is 1:

1.

4. The use according to claim 2, characterized in that: The number of ultracentrifugation is twice; Alternatively, the ultracentrifugation parameters are: 4° C., 110,000 g, 70 min.

5. A drug for promoting skin wound healing and / or reducing scar formation, characterized in that: Its active ingredients are human umbilical cord blood plasma exosomes.

6. The drug for promoting skin wound healing and / or reducing scar formation according to claim 5, characterized in that: The human umbilical cord blood plasma exosomes are prepared by the following method: human umbilical cord blood plasma is taken, diluted with PBS buffer, and centrifuged three times in sequence: 4°C, 2000g centrifugation for 30 minutes; 4°C, 10000g centrifugation for 45 minutes; 4°C, 110000g ultracentrifugation for 70 minutes; then the suspension is filtered with a 0.22μm sterilizing filter, and then ultracentrifuged, the supernatant is discarded, and the precipitate is the umbilical cord blood plasma exosomes.

7. The drug for promoting skin wound healing and / or reducing scar formation according to claim 6, characterized in that: The volume ratio of human umbilical cord blood plasma to PBS buffer is 1:

1.

8. The drug for promoting skin wound healing and / or reducing scar formation according to claim 6, characterized in that: The number of ultracentrifugation is twice; Alternatively, the ultracentrifugation parameters are: 4° C., 110,000 g, 70 min.

9. A method for preparing human umbilical cord blood plasma exosomes, characterized in that: Human umbilical cord blood plasma was taken, diluted with PBS buffer, and centrifuged three times in sequence: 4°C, 2000g centrifugation for 30 min; 4°C, 10000g centrifugation for 45 min; 4°C, 110000g ultracentrifugation for 70 min; then the suspension was filtered with a 0.22μm sterilizing filter, and then ultracentrifuged, the supernatant was discarded, and the precipitate was the umbilical cord blood plasma exosomes.

10. The method for preparing human umbilical cord blood plasma exosomes according to claim 9, characterized in that: The volume ratio of human umbilical cord blood plasma to PBS buffer is 1:1; Or: the number of ultracentrifugation is twice; Alternatively, the ultracentrifugation parameters are: 4° C., 110,000 g, 70 min.

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