High-fat-content fungus-derived outer vesicle, preparation method thereof and application of outer vesicle in skin and mucous membrane injury repair products
The high-fat fungal exosomes were prepared by the rice fermentation method of the cicadae spores, and combined with the PEG co-precipitation filtration centrifugation extraction method to solve the problems of limited exosome sources and complex extraction, and achieve efficient and low-cost skin and mucosal damage repair effects.
Patent Information
- Application Number
- CN202510625273.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-09-26
AI Technical Summary
Existing sources of extracellular vesicles are limited, and the extraction methods are complex and costly, making them difficult to apply on a large scale to repair skin and mucosal injuries. Traditional drug treatments have limited effects and may cause side effects.
The high-fat fungal exosomes were prepared by the rice fermentation method with the fungus Myxocara cicadae. The PEG co-precipitation filtration centrifugation method was then used to extract the exosomes to improve the yield and quality of the exosomes for the repair of skin and mucosal injuries.
The efficient preparation and large-scale application of extracellular vesicles have been achieved, which significantly promotes skin and mucosal repair, reduces inflammatory response, and reduces immunogenicity. It is easy to operate and low-cost.
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Figure CN120695044A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an exosome derived from a high-fat fungus, a preparation method thereof, and an application thereof in skin and mucosal damage repair products. Background Art
[0002] In recent years, extracellular vesicles (EVs) have garnered widespread attention as a novel biotherapeutic material. Extracellular vesicles (EVs) are cell-secreted particles encapsulated by a lipid bilayer and lacking the ability to self-replicate. Their formation mechanism is complex, and they are primarily classified into three subtypes: exosomes, microvesicles, and apoptotic bodies. Exosomes are formed through cell membrane invagination and maturation into multivesicular bodies (MVBs), microvesicles are formed through cell membrane protrusions, and apoptotic bodies are formed by cell membrane invaginations during apoptosis. EVs contain a variety of bioactive molecules, including proteins, nucleic acids, lipids, and metabolites. These molecules play a key role in intercellular communication and possess important functions in both physiological and pathological states, demonstrating significant potential in the biomedical field. In particular, the discovery that adipose-derived stem cell-derived EVs can modulate immune and inflammatory responses in wound repair has garnered significant attention in the fields of skin and mucosal injury repair. However, research on EVs has focused on miRNAs, with limited research on lipids.
[0003] Extracellular vesicles (EVs) come from a wide range of sources, with mammalian mesenchymal stem cells being the most commonly used. While bacterial and plant-derived EVs are abundant, their content is low and their application is limited. Stem cell EVs also present ethical concerns. Insufficient understanding of their production mechanisms and cellular uptake limits their clinical application. Improving the efficiency and quality of EV preparation for clinical application is an urgent task.
[0004] The skin is the largest organ in the human body and serves as the first line of defense against external stimuli such as ultraviolet rays, pathogens, chemical and physical damage. Wound healing after skin damage is crucial. Mucosal injury refers to the damage of the thin film covering the surface of the body's inner cavity due to various reasons, resulting in damage to its structure and function. Skin and mucosal injuries are common clinical diseases that not only affect the patient's appearance and function, but may also cause serious complications. Timely and effective treatment and repair are necessary for the patient's recovery. Although current treatment methods are diverse, there are many problems: traditional drug treatments have limited effects and may cause side effects; specific biological agents have certain therapeutic effects, but they are complex to prepare, costly, and difficult to use on a large scale. Extracellular vesicles have become the first choice for the repair and treatment of skin and mucosal injuries and have been used in the cosmetics field.
[0005] Traditional ultracentrifugation remains the gold standard for extracellular vesicle extraction. However, this method is complex, time-consuming, expensive, and produces low yields, limiting its large-scale application. Density gradient centrifugation, size-exclusion chromatography, ultrafiltration, polymer precipitation, and immunomagnetic bead methods have been developed. In recent years, microfluidics, tangential flow filtration, asymmetric field flow fractionation, anion exchange chromatography, and the latest nanotechnology have also been developed. However, these methods each have their own limitations, and large-scale production still faces numerous challenges. Therefore, the search for a new source of extracellular vesicles and the development of efficient, high-yield extraction processes are of guiding significance and practical value for their application. Summary of the Invention
[0006] Technical problems to be solved: In response to the above technical problems, the present invention provides an exosome derived from a high-fat fungus, a preparation method thereof, and an application in skin and mucosal injury repair products. By adding a high-fat raw material during the solid fermentation process of rice with the spores of Cicada cicadae, the lipid content of the exosomes is increased, and the exosomes are extracted by PEG co-precipitation filtration centrifugation, which makes up for the current defects of animal-derived exosomes in the application of repairing skin and mucosal injuries, such as low yield, high immunogenicity, and difficult preservation. A new source of exosomes from fungi is explored, and compared with the ultra-high-speed centrifugation extraction method, the PEG co-precipitation of the present invention has the advantages of convenient operation, low cost, and high yield.
[0007] Technical solution: A method for preparing high-lipid-content fungal exosomes, comprising the following steps:
[0008] Step 1, preparing the seed liquid of Cicada spore;
[0009] Step 2: preparing a high-fat rice fermentation medium: by weight, taking 10-25 parts of rice, 0-5 parts of rice bran or wheat bran, 1-20 parts of silkworm pupa powder, 0-0.2 parts of potassium dihydrogen phosphate, 0-1 parts of peptone, 0-3 parts of yeast powder, and 10-30 parts of water, sterilizing and cooling;
[0010] Step 3, preparing the rice fermentation product of C. cicadae: inoculating C. cicadae seed liquid into high-fat rice fermentation medium under sterile conditions and then naturally fermenting;
[0011] Step 4: Prepare exosomes derived from high-lipid fungi: add PBS buffer to the rice fermentation product of Cicada cicadae, crush it and perform the first centrifugation, take the supernatant, add polyethylene glycol and let it stand overnight, centrifuge again, take the precipitate and add PBS buffer to resuspend it to obtain the exosomes.
[0012] Preferably, the preparation process of the Cicada spore seed liquid in the step 1 is as follows: 0.1-1 parts of bran, 1-3 parts of peptone, and 1-5 parts of glucose are taken by weight, and after sterilization, the Cicada spore is inoculated as a culture medium, and the Cicada spore seed liquid is obtained by shaking culture.
[0013] Furthermore, in the step 1, the temperature of the shaking culture is 10-30° C., the shaking frequency is 50-200 r / min, and the culture time is 3-6 days.
[0014] Preferably, the temperature of the natural fermentation in step 3 is 10-30° C. and the time is 5-60 days.
[0015] Preferably, the first centrifugation condition in step 4 is 1000-1500g centrifugation for 5-20 minutes.
[0016] Preferably, the polyethylene glycol in step 4 is a 2-40 wt% PEG aqueous solution.
[0017] Preferably, the centrifugation condition in step 4 is 5000-15000 g for 5-40 min.
[0018] High-lipid content fungal-derived exosomes prepared by the above method.
[0019] The application of the above-mentioned high-lipid content fungal-derived exosomes in the preparation of skin and mucosal damage repair products.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0021] Source Advantages: Fungal exosomes offer unique advantages over existing exosomes derived from plants and animals. Rice fermentation products derived from Isaria cicadae are rich in bioactive ingredients that can effectively promote the repair of skin and mucous membranes. They also exhibit good biocompatibility and low immunogenicity.
[0022] Raw material advantages: The Isaria cicadae rice solid fermentation exosomes induced by high-fat raw materials have no osmotic pressure problems, high concentration, high lipid content, light density, easy purification and easy mass production.
[0023] Advantages of the extraction method: The PEG co-precipitation centrifugal filtration method is used to extract extracellular vesicles. This method has high yield, simple operation and low cost. It can effectively solve the problems of low yield, complex operation and expensive equipment in the existing ultra-high-speed centrifugation method for extracting extracellular vesicles, and provides the possibility for large-scale application of extracellular vesicles.
[0024] Advantages of therapeutic effect: The exosomes of the present invention show significant therapeutic effects in the repair of skin and mucosal injuries. They can accelerate wound healing, reduce inflammation, especially endothelial inflammatory response, promote tissue regeneration, reduce scars and fibrosis, and are not prone to causing adverse reactions during use, so they are highly safe.
[0025] Economical and accessible: The extraction method and application scheme of the present invention are simple to operate, low in cost, and easy to mass produce and promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Transmission electron microscopy (100 nm) identification image of ICEVs;
[0027] Figure 2 Transmission electron microscopy (200 nm) identification image of ICEVs;
[0028] Figure 3 This is the ICEVs nanoparticle size analysis diagram;
[0029] Figure 4 ICEVs uptake by RAW and Caco-2 cells;
[0030] Figure 5 A diagram of the free radical scavenging ability of ICEVs;
[0031] Figure 6 The results of the cytotoxicity test of ICEVs on Caco-2 cells are shown;
[0032] Figure 7 The results of the ICEVs cytotoxicity test on RAW cells are shown;
[0033] Figure 8 This is the result of H2O2-induced oxidative damage in Caco-2 cells;
[0034] Figure 9 for the protective effect of ICEVs on Caco-2 cells;
[0035] Figure 10 The effect of ICEVs on SOD levels in Caco-2 cells;
[0036] Figure 11 The effect of ICEVs on MDA levels in Caco-2 cells;
[0037] Figure 12 The effect of ICEVs on the NO secretion of cells after LPS induction;
[0038] Figure 13 The effect of ICEVs on the migration rate of Caco-2 cells;
[0039] Note: ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P < 0.001, ＊＊＊＊ P<0.0001. DETAILED DESCRIPTION
[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0041] Example 1
[0042] Extraction and preparation of ICEVs
[0043] Experimental methods:
[0044] Step 1: Prepare the C. cicadae seed solution: Dispense 1 part by weight of bran, 1 part by weight of peptone, and 1 part by weight of glucose into conical flasks, wrap and sterilize at 121°C for 30 minutes, and then cool to below 20°C. Aseptically inoculate the culture medium with C. cicadae. Incubate at 30±2°C with an oscillation frequency of 100 rpm for 3 days until numerous mycelial pellets appear in the culture medium. This is the C. cicadae seed solution.
[0045] Step 2: preparing a high-fat rice fermentation medium: preparing the medium according to a ratio of 10 parts by weight of rice, 1 part by weight of rice bran or wheat bran, 1 part by weight of silkworm pupa powder, 0 parts by weight of potassium dihydrogen phosphate, 1 part by weight of peptone, 1 part by weight of yeast powder, and 10 parts by weight of water, and placing the medium into a culture box; placing the culture box in a sterilizer at a sterilization temperature of 121° C. and a sterilization time of 20 minutes; after the sterilization is completed, waiting for the solid culture medium to cool to below 20° C.
[0046] Step 3: preparing the rice fermentation product of C. cicadae: inoculating the cooled C. cicadae seed liquid into a high-fat rice fermentation medium under sterile conditions, and fermenting naturally at 27±2° C. for 15 days.
[0047] Step 4: Preparation of high-lipid fungal exosomes: The rice fermentation product of C. cicadae was added with PBS and crushed in a juicer for 40 minutes, then divided into 50 mL centrifuge tubes and centrifuged at 1500 g for 10 minutes. The supernatant was added with an equal volume of 3% PEG 10000 aqueous solution, allowed to stand at 4°C overnight, and centrifuged at 8000 g for 10 minutes. The precipitate was taken and resuspended in a small amount of PBS to obtain the final product.
[0048] Identification of ICEVs:
[0049] Nanoparticle analysis: Dilute the resuspended ICEVs extract 1000-fold before testing. Turn on the computer and NTA instrument. Before testing, clean the cover plate and the laser interface with ultrapure water and wipe dry with lint-free paper. Aspirate the sample with a 1mL syringe and inject it into the sample cell, avoiding the formation of bubbles during the injection process. Click "Capture" to capture the particle image. Adjust the focal length and light intensity to optimize the focus. Once the particles in the image are stable, capture the image again. Repeat the image three times at different concentrations, switching viewing angles, and record the data.
[0050] Transmission electron microscopy identification: Use a pipette to aspirate 10 μL of ICEVs sample extracted by PEG coprecipitation onto a carbon-coated copper mesh and let it sit for 3-5 minutes. Remove excess liquid with filter paper. Then, drip 2% phosphotungstic acid onto a carbon-coated copper mesh and let it sit for 1-2 minutes. Remove excess liquid with filter paper and rinse the staining solution two to three times with pure water. Finally, observe under a transmission electron microscope and collect images for analysis.
[0051] Experimental results:
[0052] The morphological observation results of ICEVs under transmission electron microscopy are as follows: Figure 1 、 2 The results showed that the extracellular vesicles had a spherical double-layer membrane-coated nanoparticle structure, which was consistent with the electron microscopic morphology of extracellular vesicles. The NTA technology was used to analyze the particle size of ICEVs. Figure 3 As shown, the results showed that the average particle size was 115 nm and the concentration was 2.35×10 9 Particles / mL.
[0053] Example 2
[0054] In vitro tracking of ICEVs
[0055] Experimental methods:
[0056] (1) Pkh26 staining of ICEVs
[0057] Dissolve Pkh26 dye in anhydrous ethanol or DMSO to prepare a stock solution. Resuspend the exosomes in PBS buffer and add an appropriate amount of Pkh26 dye. Incubate at 37°C in the dark for 15-30 minutes to ensure adequate labeling of the exosomes. Wash the exosomes with PBS buffer 2-3 times to remove unbound Pkh26 dye. Examine the labeled exosomes using a fluorescence microscope to confirm staining.
[0058] (2) DAPI staining of cell nuclei
[0059] Cells were seeded onto coverslips and allowed to adhere before incubation with Pkh26-labeled EVs. The medium was aspirated and the cells were gently washed three times with ice-cold PBS. The cells were fixed with 4% PFA at room temperature for 15 minutes, washed three times with PBS, permeabilized with 0.1% Triton X-100 / PBS at room temperature for 10 minutes, and washed three times with PBS. The cells were stained with 1 μg / mL DAPI for 5-10 minutes in the dark, followed by three 5-minute washes with PBS to remove free DAPI. Finally, the coverslips were mounted upside down on glass slides, and anti-quencher was added to the coverslips for mounting and observation under a confocal microscope.
[0060] Experimental results:
[0061] Figure 4 It was shown that Pkh26-labeled ICEVs were obviously internalized and absorbed by RAW cells and Caco-2 cells, and these extracellular vesicles were mainly distributed around the cell nucleus.
[0062] Example 3:
[0063] ICEVs activity detection
[0064] Experimental methods:
[0065] (1) DPPH antioxidant test
[0066] The ICEVs samples were diluted with PBS to different concentrations, and the positive control vitamin C was diluted with PBS to an appropriate concentration. In a 96-well plate, 100 μL of 0.1 mmol / L DPPH solution was added to each well, and 100 μL of ICEVs samples of different concentrations were added respectively. 100 μL PBS was added to the negative control well. Three replicates were set for each sample and control. The 96-well plate was incubated in the dark at room temperature for 30 minutes. Finally, the absorbance (OD value) of each well was measured at a wavelength of 517 nm using a microplate reader.
[0067] (2) Cytotoxicity test
[0068] Cells were seeded in 96-well plates (about 5 × 10 3 Cells were cultured for 24 hours until the cells adhered. A control group received normal culture medium (without extracellular vesicles); experimental groups received ICEVs at varying concentrations, with triplicate wells per group. After 24, 48, and 72 hours of culture, 20 μL of 0.5% MTT solution was added to each well and incubated at 37°C for 4 hours to allow the MTT to be reduced to purple formazan crystals by mitochondrial dehydrogenases in the living cells. The supernatant was carefully aspirated to avoid removing any formazan crystals. 150 μL of DMSO was added to each well and gently shaken for 10 minutes to fully dissolve the formazan crystals. The absorbance (OD) was measured at 570 nm using a microplate reader, and the OD value for each well was recorded.
[0069] (3) Establishment of H2O2-induced Caco-2 cell oxidative damage model
[0070] Remove the frozen Caco-2 cells from liquid nitrogen, thaw quickly in a 37°C water bath, and inoculate them into a culture flask containing complete medium. Incubate in a 37°C, 5% CO2 incubator. Subculture when the cell density reaches 80%-90%. Digest the cells with trypsin, centrifuge, and resuspend in complete medium. Adjust the cell density and inoculate the cells into 96-well plates (approximately 1×10 cells per well). 4Cells were cultured for 24-48 hours until the cells adhered. H2O2 was diluted with PBS to various concentrations (e.g., 100μM, 200μM, 500μM, 1mM, etc.). The cell culture medium was aspirated and culture medium containing various concentrations of H2O2 was added. Three replicates were set up for each group and the cells were incubated in a 37°C, 5% CO2 incubator for a specified period of time. After the incubation period, the culture medium was aspirated and the cells were washed twice with PBS. Cell viability was assayed using the MTT assay to determine the optimal H2O2 induction concentration and time.
[0071] (4) Effects of ICEVs on Caco-2 cell viability after H2O2 stimulation
[0072] Group design: control group normal culture medium (without H2O2 and extracellular vesicles); H2O2 group only added H2O2; ICEVs group added 10μg / mL, 50μg / mL, 100μg / mL ICEVs) and H2O2.
[0073] The culture medium of the adherent cells was aspirated and culture medium containing different concentrations of ICEVs was added respectively. The cells were pre-incubated for 2 hours. H2O2 was added and incubated for another 4 hours. The changes in cell activity were detected by MTT method. The culture medium was aspirated and 100 μL of fresh culture medium and 10 μL of MTT solution were added to each well. The cells were incubated at 37°C for 4 hours. The culture medium was aspirated and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance (OD value) was measured at a wavelength of 570 nm using a microplate reader, and the results were finally calculated.
[0074] (5) ICEVs detection of SOD and MDA in Caco-2 cells after H2O2 stimulation
[0075] Cell lysis and sample preparation
[0076] Aspirate the culture medium from adherent cells, wash cells twice with pre-chilled PBS, trypsinize cells, centrifuge, and discard the supernatant. Add the appropriate amount of lysis buffer and lyse on ice for 30 minutes. Centrifuge at 12,000 × g for 10 minutes at 4°C. Collect the supernatant and determine the protein concentration of the cell lysate using the BCA assay.
[0077] SOD activity detection
[0078] Prepare the reaction solution according to the SOD assay kit instructions. Add 20 μL of cell lysate and 200 μL of the reaction solution to each well of a 96-well plate and incubate at 37°C for 20 minutes. Measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate SOD activity according to the kit instructions and express it as U / mg protein.
[0079] MDA content detection
[0080] Prepare the reaction solution according to the MDA assay kit instructions. Add 100 μL of cell lysate and 200 μL of the reaction solution to a centrifuge tube. Heat in a boiling water bath for 15 minutes. Cool and centrifuge. Collect the supernatant and measure the absorbance (OD) at 532 nm using a microplate reader. Calculate the MDA content according to the kit instructions and express it in nmol / mg protein.
[0081] Experimental results:
[0082] DPPH itself is a stable free radical, dark purple, with a maximum absorption value at 517nm, and its fading degree is proportional to the free radical scavenging rate. Figure 5 It can be seen that the number of particles is 5-11×10 12 / mL ICEVs extract can produce about 55% to 69% DPPH free radical scavenging rate, and the scavenging rate increases with the increase of concentration.
[0083] like Figure 6 and 7 As shown, the concentration of ICEVs extract was 5×10 5 -1×10 9 When the number of particles / mL was 2.5, it had no effect on Caco-2 cells and RAW cells themselves, and there was no significant difference in the cell survival rate between the experimental group and the control group.
[0084] In this study, the MTT method was used to determine the survival rate of Caco-2 cells exposed to different concentrations of H2O2 to determine the degree of damage to Caco-2 cells by H2O2 and thus select the optimal concentration for modeling. Figure 8 As shown in the figure, the survival rate of Caco-2 cells decreased with increasing H2O2 concentration. When the H2O2 concentration was 1.2mmol / L, the survival rate of Caco-2 cells was 60%, which was significantly different from the blank control group. Therefore, 1.2mmol / L H2O2 was selected as the modeling concentration for subsequent experiments.
[0085] Depend on Figure 9 It can be seen that the survival rate of cells in the H2O2 group decreased significantly after stimulation, while the survival rate of cells in the drug group increased in a dose-dependent manner, which was statistically significant compared with the H2O2 group. 8 -1×10 9 The cell survival rate of cells treated with ICEVs extract was over 90%, and there was a significant difference, indicating that ICEVs extract can effectively protect Caco-2 cells from H2O2 stimulation, and the protective effect increases with increasing concentration, and has a strong ability to resist oxidative damage.
[0086] SOD is a type of antioxidant enzyme with metal as the catalytic center. It mainly reduces the cell damage caused by oxidative stress by promoting the decomposition of hydrogen peroxide and lipid peroxides in the body's cells. SOD can convert O2-free radicals in the body into H2O2, which can be further decomposed into water by catalase. Figure 10 It can be seen that compared with the control group, the SOD activity of Caco-2 cells in the H2O2 group was significantly decreased, 1×10 6 The effect of ICEVs extract at the concentration of 1×10 particles / mL on the enhancement of SOD activity was not significant, but with the increase of its concentration, SOD activity increased accordingly. 7 The ICEVs extracts with a particle number / mL concentration above 1×10 9 The number of particles / mL concentration was most significant.
[0087] MDA has certain toxicity, and its level is usually measured to reflect the degree of cellular oxidative damage, such as Figure 11 As shown in the results, the MDA content of Caco-2 cells without any treatment was about 0.79 nmol / mg prot. After H2O2 induction, the MDA content in the cells increased sharply, much higher than that in the control group (P < 0.01), indicating that the oxidative damage was serious. Different concentrations of ICEVs extracts can reduce the MDA level in cells, 1×10 7 The particle number / mL concentration of the extract had the most significant effect.
[0088] Example 4:
[0089] Study on the anti-inflammatory activity of ICEVs in vitro
[0090] Experimental methods:
[0091] Effects of ICEVs on NO secretion in RAW cells stimulated by LPS
[0092] The LPS-stimulated RAW cell inflammation model was established. RAW cells in logarithmic growth were digested and diluted to 4×10 4 Each well was inoculated with 160 μL of culture medium per well in a 96-well plate. The cells were divided into a control group, an LPS group, and an experimental group, with six replicates per well. Except for the control group, all other groups were treated with 20 μL of LPS solution (1 μg / mL). After 4 hours of pre-induction, 20 μL of ICEVs extract at different concentrations was added to each well of the experimental group. The control group received complete culture medium instead of LPS and ICEVs extract, and the cells were cultured in a CO2 incubator for another 24 hours. Cell supernatants were collected, and the NO release of RAW cells under different conditions was determined using the Griess method.
[0093] Experimental results:
[0094] Figure 12 The results showed that compared with the control group, the release of NO from RAW cells increased significantly after LPS stimulation, and the content changes were significantly different (P<0.001). Under the treatment of different concentrations of ICEVs extracts, the NO release of the experimental group decreased. 6 The effects of the extract at concentrations above 0.05 (number of particles / mL) were significantly different from those in the LPS group (P<0.001). The experimental results indicate that the cellular inflammation model was successfully established, and that the ICEVs extract was able to reduce NO secretion and inhibit the LPS-induced inflammatory response, demonstrating its anti-inflammatory activity.
[0095] Example 5:
[0096] Study on the activity of ICEVs in promoting cell proliferation and migration
[0097] Experimental methods:
[0098] Cell scratch assay
[0099] For cells in good condition, trypsin was used to digest the cells to be used for the scratch test, and after centrifugation, the cells were resuspended and counted in complete culture medium. The cells were seeded into six-well plates, with approximately 1x10 cells per well. 5 cells to ensure that the cell density is close to 100% monolayer cell state during the experiment. Use a marker to draw horizontal lines on the back of the six-well plate, approximately every 0.5-1 cm, and at least 5 lines across each well. After the cells are full, use a 20μL pipette tip to scratch perpendicularly to the marked line on the back of the well plate, so that the scratches intersect with the marked lines to form multiple fixed detection points. Aspirate the cell culture medium and gently rinse the well plate 2-3 times with PBS to wash away the cell debris produced by the scratches. Add serum-free culture medium as a negative control group and different concentrations of ICEVs as drug stimulation groups, and gently shake to mix. Take a picture and record it as the scratch condition at 0 hours. Then place the culture plate in a 37℃, 5% CO2 incubator for further culture. Remove the culture plate at the 24-hour point and observe and photograph the changes in the scratch width at the same position under a microscope.
[0100] Experimental results:
[0101] Through the scratch test, RAW cells were treated with different concentrations of ICEVs. Figure 13 Observing the images recorded at different time points, it can be found that cells can migrate without any treatment. After experimental intervention, the migration rate increases. The migration rate of cells 24 hours after scratching was quantified. When the concentration was 1×10 8 There was a significant increase in the migration rate when the number of particles / mL was greater than 1×10 7 When the significance increased to 0.01, the concentration reached 5×106 -1×10 9 When the number of particles / mL was 2.5, the migration rate of cells increased more significantly (P<0.001). Overall, ICEVs can promote cell repair by increasing the migration rate of RAW cells and have the potential to repair damaged mucosa.
Claims
1. A method for preparing exosomes derived from fungi with high lipid content, characterized in that: The steps are as follows: Step 1, preparing the seed liquid of Cicada spore; Step 2: preparing a high-fat rice fermentation medium: by weight, taking 10-25 parts of rice, 0-5 parts of rice bran or wheat bran, 1-20 parts of silkworm pupa powder, 0-0.2 parts of potassium dihydrogen phosphate, 0-1 parts of peptone, 0-3 parts of yeast powder, and 10-30 parts of water, sterilizing and cooling; Step 3, preparing the rice fermentation product of C. cicadae: inoculating C. cicadae seed liquid into high-fat rice fermentation medium under sterile conditions and then naturally fermenting; Step 4: Prepare exosomes derived from high-lipid fungi: add PBS buffer to the rice fermentation product of Cicada cicadae, crush it and perform the first centrifugation, take the supernatant, add polyethylene glycol and let it stand overnight, centrifuge again, take the precipitate and add PBS buffer to resuspend it to obtain the exosomes.
2. The method for preparing exosomes derived from high-fat fungi according to claim 1, characterized in that: The preparation process of the Cicada spore seed liquid in the step 1 is as follows: 0.1-1 parts of bran, 1-3 parts of peptone, and 1-5 parts of glucose are taken by weight, sterilized, and used as culture liquid to inoculate Cicada spore, and shake culture is performed to obtain the Cicada spore seed liquid.
3. The method for preparing exosomes derived from high-fat fungi according to claim 2, characterized in that: The shaking culture temperature in step 1 is 10-30° C., the shaking frequency is 50-200 r / min, and the culture time is 3-6 days.
4. The method for preparing exosomes derived from high-fat fungi according to claim 1, characterized in that: The temperature of the natural fermentation in step 3 is 10-30° C. and the time is 5-60 days.
5. The method for preparing exosomes derived from high-fat fungi according to claim 1, characterized in that: The first centrifugation condition in step 4 is 1000-1500g centrifugation for 5-20 minutes.
6. The method for preparing exosomes derived from high-fat fungi according to claim 1, characterized in that: The polyethylene glycol in step 4 is a 2-40wt% PEG aqueous solution.
7. The method for preparing exosomes derived from high-fat fungi according to claim 1, characterized in that: The centrifugation condition in step 4 is 5000-15000 g for 5-40 min.
8. Exosomes of high-lipid content fungus origin prepared by the method according to any one of claims 1 to 7.
9. Use of the high-fat fungus-derived exosomes according to claim 8 in the preparation of skin and mucosal damage repair products.