Preparation method and application of extracellular vesicles derived from tremella fuciformis fermentation liquor

Abstract

The invention discloses a preparation method and application of extracellular vesicles derived from tremella fuciformis fermentation broth, and the prepared extracellular vesicles have the effects of promoting cell proliferation, cell migration and angiogenesis and can remarkably promote wound healing. Therefore, the extracellular vesicles derived from the tremella fuciformis fermentation liquor have the effect of promoting wound recovery and can be applied to the fields of skin injury repair, medical beauty and cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a preparation method of extracellular vesicles derived from Tremella fermentation liquid and application thereof. Background Art

[0002] Skin wounds are a type of mechanical damage to the skin and its internal tissues and organs caused by external forces, and are open injuries. The formation of skin wounds is mainly due to the action of external forces, burns, surgery, and complications caused by various diseases. After the skin is traumatized, treatment measures must be taken as soon as possible to promote the healing of the wound surface. Without intervention, adverse reactions such as inflammation, ischemia or necrosis, scars, pigmentation, ulcers, etc. are very likely to occur. With the rapid development of economy and technology, human treatment of skin wounds has gradually improved. Traditional skin healing therapies such as lasers, therapeutic dressings, negative pressure therapy, and skin transplants have great limitations when used in clinical practice. Rapid and seamless skin wound repair is still one of the difficult problems in clinical medicine.

[0003] Extracellular vesicles (EVs) are usually defined as vesicles containing cytoplasm surrounded by a lipid bilayer and released by cells into the extracellular space. Extracellular vesicles carry characteristic biological information molecules of maternal cells, such as proteins, lipids, DNA, and non-coding RNA. They have natural molecular transport properties and good biocompatibility, and can mediate intercellular communication. At the same time, extracellular vesicles are natural nanocarriers of endogenous cells with very low cytotoxicity and immunogenicity. In recent years, more and more reports have found that plants and fungi can also secrete extracellular vesicles and have significant activity, which has been shown to have great potential in disease treatment. The conventional plant or animal cell extraction method for preparing extracellular vesicles has problems such as raw material quality stability and limited material sources, resulting in high manufacturing costs and difficulty in industrialization.

[0004] Tremella fuciformis (TF) is the fruiting body of Tremella fuciformis, also known as white fungus. As a traditional medicinal and edible fungus, Tremella fuciformis has high nutritional value and can be used in both medicine and food. It is known as the "king of fungi". Studies have shown that Tremella fuciformis has unique biological activity and has significant effects in anti-aging, anti-tumor, anti-inflammatory, antioxidant and skin protection. Studies have found that the Tremella fermentation liquid obtained by liquid culture not only ensures the quality of Tremella's active ingredients, but also can be industrialized to shorten the growth cycle, and has industrial advantages such as cost savings, small footprint, and easy control.

[0005] At present, the research on Tremella fuciformis fermented broth mainly focuses on active ingredients such as polysaccharides, phenolic acids, catechins and flavonoids. There are no reports on whether Tremella fuciformis fermented broth contains extracellular vesicles and whether it promotes wound repair. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an extracellular vesicle derived from a Tremella fermentation liquid and an application thereof.

[0007] The present invention prepares Tremella fuciformis fermentation broth-derived extracellular vesicles (TFB-EVs), which can promote cell proliferation and migration, and promote angiogenesis. The present invention provides a new choice for drugs or special medical formulas for wound cell repair.

[0008] The first object of the present invention is to provide an application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of a product that promotes cell proliferation and / or migration.

[0009] The second object of the present invention is to provide the use of extracellular vesicles derived from Tremella fermentation liquid in the preparation of products promoting angiogenesis.

[0010] The third object of the present invention is to provide the use of extracellular vesicles derived from Tremella fermentation liquid in the preparation of products that promote skin wound recovery.

[0011] The fourth object of the present invention is to provide a product for promoting the recovery of skin wounds.

[0012] The present invention claims the following applications:

[0013] Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote cell proliferation and / or migration.

[0014] Preferably, the cells are skin tissue cells.

[0015] Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products promoting angiogenesis.

[0016] Application of extracellular vesicles derived from Tremella fuciformis fermentation liquid in the preparation of products promoting skin wound recovery.

[0017] Preferably, the strain number of Tremella fuciformis is CGMCC 5.466 (China General Microbiological Culture Collection Center).

[0018] Preferably, the method for preparing the extracellular vesicles derived from Tremella fermentation broth is:

[0019] The mycelium is removed from the Tremella fermented liquid, and the liquid is centrifuged at 8000-15000×g for 25-35 min at 3-5°C, the precipitate is discarded and the supernatant is obtained, and the high-speed centrifugation is repeated to obtain the supernatant; then, the supernatant is ultracentrifuged at 90000-110000×g for 65-75 min at 3-5°C, the supernatant is discarded, the precipitate is resuspended with pre-cooled PBS, the ultracentrifugation is repeated, and the supernatant is obtained; purification is completed.

[0020] More preferably, the preparation method of the extracellular vesicles derived from Tremella fermentation liquid is:

[0021] The mycelium is removed from the Tremella fermented liquid, centrifuged at 12000×g for 30 min at 4°C, the precipitate is discarded and the supernatant is obtained, and the supernatant is obtained by repeated high-speed centrifugation; the supernatant is ultracentrifuged at 110000×g for 70 min at 4°C, the supernatant is discarded, the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated; the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated to obtain the supernatant; the purification is completed.

[0022] Preferably, the purification is sterilized by 0.22 μm filtration.

[0023] Further preferably, the method for removing mycelium is filtering through eight layers of gauze.

[0024] The present invention also claims to protect a product for promoting skin wound recovery, which contains extracellular vesicles derived from Tremella fermentation liquid.

[0025] Preferably, the preparation method of the extracellular vesicles derived from Tremella fermentation liquid is:

[0026] The mycelium is removed from the Tremella fermented liquid, and the liquid is centrifuged at 8000-15000×g for 25-35 min at 3-5°C, the precipitate is discarded and the supernatant is obtained, and the high-speed centrifugation is repeated to obtain the supernatant; then, the supernatant is ultracentrifuged at 90000-110000×g for 65-75 min at 3-5°C, the supernatant is discarded, the precipitate is resuspended with pre-cooled PBS, the ultracentrifugation is repeated, and the supernatant is obtained; purification is completed.

[0027] More preferably, the preparation method of the extracellular vesicles derived from Tremella fermentation liquid is:

[0028] The mycelium is removed from the Tremella fermented liquid, centrifuged at 12000×g for 30 min at 4°C, the precipitate is discarded and the supernatant is obtained, and the supernatant is obtained by repeated high-speed centrifugation; the supernatant is ultracentrifuged at 110000×g for 70 min at 4°C, the supernatant is discarded, the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated; the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated to obtain the supernatant; the purification is completed.

[0029] Preferably, the strain number of Tremella fuciformis is CGMCC 5.466 (China General Microbiological Culture Collection Center).

[0030] Preferably, the method for removing mycelium is filtering through eight layers of gauze.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The invention provides an extracellular vesicle derived from Tremella fermented liquid and an application thereof, wherein the prepared extracellular vesicle has the effects of promoting cell proliferation, cell migration and angiogenesis, and can significantly promote wound healing. It is shown that the extracellular vesicle derived from Tremella fermented liquid has the effect of promoting wound recovery, and can be applied to skin injury repair, medical beauty and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 To identify extracellular vesicles derived from Tremella fuciformis fermentation broth.

[0034] Figure 2 Effects of extracellular vesicles derived from Tremella fuciformis fermentation broth on the proliferation (a) and mobility (b) of HaCaT cells, (40×).

[0035] Figure 3 Effects of extracellular vesicles derived from Tremella fuciformis fermentation broth on the proliferation (a) and mobility (b) of HUVEC cells, (40×).

[0036] Figure 4 The results of HUVEC cell angiogenesis (40×).

[0037] Figure 5 The wound healing results of mice from day 0 to day 14. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] Preparation of culture medium:

[0040] PDA medium: 2 g glucose, 0.3 g KH2PO4, 0.15 g MgSO4·7H2O, 1.5 g agar, 100 mL potato extract. Sterilize at 115°C for 30 min.

[0041] Liquid culture medium: 2g glucose, 0.5g yeast extract powder, 0.05g KH2PO4, 0.1g MgSO4·7H2O, 100mL purified water. The culture medium was filtered through a 0.22μm water system membrane to remove larger particles and then dispensed into conical bottles (250mL), sealed and sterilized at 115℃ for 30min.

[0042] Human immortalized keratinocytes (HaCaT cells) were from Wuhan Sean Biotechnology, and human umbilical vein endothelial cells (HUVEC cells) HaCaT cells were from Wuhan Sean Biotechnology.

[0043] Example 1 Extraction of extracellular vesicles (TFB-EVs) from Tremella fuciformis fermentation broth

[0044] 1. Bacteria activation

[0045] In the clean bench, a sterile inoculation loop was used to inoculate the Tremella fuciformis strain (purchased from the China General Microbiological Culture Collection Center, strain number CGMCC 5.466) into three culture dishes containing PDA culture medium, and the dishes were placed in a 28°C constant temperature incubator. After constant temperature culture for 3 days, the culture dishes were used as the strains for subsequent tests.

[0046] 2. Preparation of seed solution

[0047] In the clean bench, use a sterile inoculation loop to pick up a piece of activated mycelium and inoculate it into a conical flask containing 100 mL of liquid fermentation culture. Cultivate it at 27°C and 160 r / min for 3 days as seed solution for later use.

[0048] 3. Liquid fermentation of bacteria

[0049] The seed liquid was added into a conical flask containing liquid fermentation medium at an inoculum volume ratio of 2%, and the mixture was shaken and cultured at 27° C. and 160 r / min for 3 days to obtain a fermentation liquid.

[0050] 4. Extracellular vesicle extraction from Tremella fermentation broth

[0051] Take the fermentation liquid after 3 days of fermentation, remove the mycelium with 8 layers of gauze, put it into a 50mL centrifuge tube, balance it, and then centrifuge it at 4℃, 12000×g for 30min, take the supernatant, and repeat the centrifugation once. Put the supernatant into an ultracentrifuge bottle, balance it, and centrifuge it at 4℃, 110000×g for 70min, discard the supernatant, resuspend the precipitate with PBS, repeat the ultracentrifugation once, resuspend it with 1mL PBS, filter it with a 0.22μm water filter membrane, and store it at -80℃ after aliquoting.

[0052] Example 2 Characterization of extracellular vesicles derived from Tremella fuciformis fermentation broth

[0053] 1. Experimental Methods

[0054] 1. Transmission electron microscopy (TEM) detection

[0055] Use a pipette to take 20 μL of the TFB-EVs sample obtained in Example 1 and drop it on the carbon film copper mesh for 3 to 5 minutes, and use filter paper to absorb the excess liquid. Then drop 2% phosphotungstic acid on the carbon support film copper mesh for 1 to 2 minutes, use filter paper to absorb the excess liquid, and dry at room temperature. Finally, observe under a transmission electron microscope and collect image analysis.

[0056] 2. NTA particle size analysis

[0057] The TFB-EVs bubble sample obtained in Example 1 was appropriately diluted with 1×PBS buffer, and the particle size and concentration of the extracellular vesicles were measured using nanoparticle tracking analysis (NTA) using a Zeta View PMX110 instrument and corresponding software Zeta View 8.04.02 (the Zeta View system was calibrated with 110 nm polystyrene particles, and the temperature was maintained at around 23°C and 37°C).

[0058] 2. Experimental Results

[0059] like Figure 1 The results of NTA analysis of TFB-EVs showed that the average particle size of LEVs was 85.3 nm and the concentration was 2.35×10 9 Particles / mL( Figure 1 A), morphological observation of LEVs by transmission electron microscopy. Figure 1 As shown in B, the results showed that TFB-EVs are double-layer membrane-coated nanoparticles with a spherical morphology, which is consistent with the electron microscopic morphology of extracellular vesicles.

[0060] Example 3 Tremella fermentation broth-derived vesicles promote the proliferation of skin cells

[0061] 1. Experimental Methods

[0062] The TFB-EVs (0.2-0.3 mg / mL) obtained in Example 1 were administered according to the protein content after the protein concentration was measured by the BCA kit.

[0063] HaCaT cells and HUVEC cells in the logarithmic growth phase were collected and the cell density was adjusted to 5×10 4 / mL, take a 96-well cell culture plate, add the cell suspension with adjusted density into the 96-well cell culture plate, add 100 μL of cell suspension to each well, place the 96-well cell culture plate in a constant temperature incubator, and culture for 24 hours; set up experimental groups: blank well (culture medium), blank control group (cells + culture medium), vesicle administration group (cells + TFB-EVs-containing culture medium, the concentration of TFB-EVs calculated by the amount of protein is: 5 μg / mL, 10 μg / mL, 20 μg / mL), and set up three replicate wells in each group.

[0064] The specific method is: after discarding the original culture medium in the well, add the prepared TFB-EVs culture medium of different concentrations into each well, place the 96-well cell culture plate back in the constant temperature incubator, and culture for 24 hours.

[0065] Then, the number of live cells was detected using a CCK-8 kit and the operation was performed according to the instructions. The absorbance value (OD value) of each well was detected at a wavelength of 450 nm using an ELISA reader; the result analysis was as follows: cell viability (%) = (OD value of cells in the drug group - OD value of blank wells) / (OD value of blank control group - OD value of blank wells) × 100%.

[0066] 2. Experimental Results

[0067] like Figure 2 a and Figure 3 As shown in a, compared with normal culture (blank control group), extracellular vesicles derived from Tremella fuciformis fermentation broth have significant proliferative activity on HaCaT cells and HUVEC cells (p<0.05).

[0068] Example 4 Tremella fermentation broth-derived vesicles promote migration of skin cells

[0069] 1. Experimental Methods

[0070] Prepare a 24-well plate and use a marker to draw three horizontal lines across the bottom of the 24-well plate, approximately every 0.5 to 1 cm. Take cells in the logarithmic growth phase. HaCaT cells were cultured at 5×10 5 500 μL per well was inoculated into a 24-well culture plate at a cell density of 3.5×10 5The cell density of cells / mL was 500 μL per well and inoculated into a 24-well culture plate. The cells were cultured for one day, and subsequent experiments were performed after the cells were fully grown. The 24-well plate with cells growing on the bottom was scratched with a 200 μL gun tip perpendicular to the cell surface, and the scratches were perpendicular to the horizontal lines on the back of the 24-well plate. After scratching, the old culture medium was discarded, the cells were washed twice with PBS, and the cells in the middle of the scratches were washed away. The intersection of the upper and lower edges of each line at the bottom of the 24-well plate and the scratches was used as an observation point. Serum-free culture medium (normal group) and serum-free culture medium containing TFB-EVs obtained in Example 1 with different concentrations were added to the corresponding wells (administration group, TFB-EVs final concentration was 5 μg / mL and 20 μg / mL). Take a picture after scratching, the time point is 0h at this time, remember the position of each observation point, and take pictures at 0h, 24h, and 48h.

[0071] 2. Experimental Results

[0072] Compared with the normal group, the extracellular vesicles derived from Tremella fuciformis fermentation broth had significant migration-promoting activity on HaCaT cells and HUVEC cells ( Figure 2 b and Figure 3 b) effect (p<0.05).

[0073] Example 5 Effect of extracellular vesicles derived from Tremella fuciformis fermentation broth on HUVEC cell angiogenesis

[0074] 1. Experimental Methods

[0075] The melted matrix gel was diluted with pre-cooled serum-free medium at a ratio of 1:2, and 50 μL / well was added to the pre-cooled 96-well cell culture plate using a pipette, and placed in the incubator for 40 minutes until the matrix gel solidified. While waiting for solidification, the HUVEC cells were digested and the cell density was adjusted to 2×10 4 Each well was inoculated in a 96-well cell culture plate containing matrix gel, and TFB-EVs obtained in Example 1 were added to the drug administration group at a final concentration of 20 μg / mL calculated by protein concentration, and no vesicles were added to the control group. Three replicate wells were set in each group and cultured in an incubator for 4 hours. The cell tube formation was observed under a microscope and analyzed using Image-J software.

[0076] 2. Experimental Results

[0077] The results are as follows Figure 4 As shown, angiogenesis experiments proved that extracellular vesicles derived from Tremella fuciformis fermentation broth can promote HUVEC cell tube formation and significantly increase the vascular length of HUVEC cell blood vessels (p<0.05).

[0078] Example 6 Tremella fermentation broth derived vesicles promote the healing of mouse skin wounds

[0079] 1. Experimental Methods

[0080] Twelve 4-week-old KM mice were randomly selected and ear-tagged. After one week of normal feeding, 1 mL / kg of sodium pentobarbital was intraperitoneally injected into the mice for anesthesia. After the mice fell into a coma, the backs were depilated and the mice were then raised separately in cages. Wound modeling was performed every other day.

[0081] Modeling: The mice were randomly divided into three groups: control group (NC), positive control group (PC) and EVs group, with 4 mice in each group.

[0082] After grouping, the mice were anesthetized in turn. After the mice fell into coma, their backs were wiped with alcohol cotton and full-thickness skin was removed in the center of the back with a sterile round puncher to form a circular or nearly circular full-thickness skin wound. The size of the wound was recorded and the wound at this time was recorded as Day 0.

[0083] Administration: 50 μL of PBS was injected into the wound of the NC group, 50 μL of commercially available recombinant human epidermal growth factor topical solution (2000 IU / mL) was injected into the wound of the PC group, and 50 μL of TFB-EVs obtained in Example 1 with a concentration of 0.2 mg / mL was injected into the wound of the EVs group.

[0084] After administration, the wounds of the mice were bandaged with elastic bandages, and the wound sizes of each mouse were recorded on the 7th and 14th days.

[0085] 2. Experimental Results

[0086] like Figure 5 As shown, the wound area of ​​mice in the EVs group on the seventh day (compared to the wound healing rate of 29% on Day 0) and the wound area on the fourteenth day (compared to the wound healing rate of 3.8% on Day 0) were significantly smaller than those of mice in the NC group at the same period, and there was no significant difference with mice in the PC group. The healing rate is the wound area on the 7th day divided by the wound area on the 0th day. The above results indicate that extracellular vesicles derived from Tremella fuciformis fermentation broth promote the healing of mouse skin wounds.

Claims

1. Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote cell proliferation and / or migration.

2. The use according to claim 1, characterized in that: The cells are skin tissue cells.

3. Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote angiogenesis.

4. Application of extracellular vesicles derived from Tremella fuciformis fermentation liquid in the preparation of products that promote skin wound recovery.

5. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the extracellular vesicles derived from Tremella fermentation liquid is as follows: The mycelium is removed from the Tremella fermented liquid, and the liquid is centrifuged at 8000-15000×g for 25-35 min at 3-5°C, the precipitate is discarded and the supernatant is obtained, and the high-speed centrifugation is repeated to obtain the supernatant; then, the supernatant is ultracentrifuged at 90000-110000×g for 65-75 min at 3-5°C, the supernatant is discarded, the precipitate is resuspended with pre-cooled PBS, the ultracentrifugation is repeated, and the supernatant is obtained; purification is completed.

6. The use according to claim 5, characterized in that: The preparation method of the extracellular vesicles derived from Tremella fermentation liquid is as follows: The mycelium is removed from the Tremella fermented liquid, centrifuged at 12000×g for 30 min at 4°C, the precipitate is discarded and the supernatant is obtained, and the supernatant is obtained by repeated high-speed centrifugation; the supernatant is ultracentrifuged at 110000×g for 70 min at 4°C, the supernatant is discarded, the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated; the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated to obtain the supernatant; the purification is completed.

7. The use according to claim 5, characterized in that: The method for removing mycelium is filtering with eight layers of gauze.

8. A product for promoting skin wound recovery, characterized in that: Contains extracellular vesicles derived from Tremella fuciformis fermentation broth.

9. The product according to claim 8, characterized in that The preparation method of the extracellular vesicles derived from Tremella fermentation liquid is as follows: The mycelium is removed from the Tremella fermented liquid, and the liquid is centrifuged at 8000-15000×g for 25-35 min at 3-5°C, the precipitate is discarded and the supernatant is obtained, and the high-speed centrifugation is repeated to obtain the supernatant; then, the supernatant is ultracentrifuged at 90000-110000×g for 65-75 min at 3-5°C, the supernatant is discarded, the precipitate is resuspended with pre-cooled PBS, the ultracentrifugation is repeated, and the supernatant is obtained; purification is completed.

10. The product according to claim 9, characterized in that The preparation method of the extracellular vesicles derived from Tremella fermentation liquid is as follows: The mycelium is removed from the Tremella fermented liquid, centrifuged at 12000×g for 30 min at 4°C, the precipitate is discarded and the supernatant is obtained, and the supernatant is obtained by repeated high-speed centrifugation; the supernatant is ultracentrifuged at 110000×g for 70 min at 4°C, the supernatant is discarded, the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated; the precipitate is resuspended with precooled PBS, and the ultracentrifugation is repeated to obtain the supernatant; the purification is completed.

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