Epithelial cell exosome for repairing scalds as well as preparation method and application of epithelial cell exosome

Abstract

The invention discloses an epithelial cell exosome for repairing scalds and a preparation method and application thereof, and relates to the technical field of biology, and the preparation method comprises the following steps: a, taking skin tissues, cutting into pieces, soaking in PBS (Phosphate Buffer Solution) containing 0.1% mycillin for 30 minutes, then taking out, culturing by adopting 8% FBS L-DMEM culture solution, replacing the culture solution once every three days, and after replacing for three times, removing tissue blocks, the adherent cells are digested with pancreatin, then the adherent cells are cultured with a composite culture solution, and after 70% fusion, pancreatin digestion passage is conducted for three generations, and an epithelial cell culture solution is obtained; b, performing centrifugal treatment on the epithelial cell culture solution twice, and sequentially collecting supernate; c, obtaining an epithelial cell exosome for repairing scalds; the epithelial cell exosome prepared by the invention can transmit genetic information, reprogram host cells, participate in biological processes of regulating cell growth, differentiation, migration and the like, and can be transported to another cell as a nutrient substance to participate in biological processes of repairing, regenerating and the like of skin tissues.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular to epithelial cell exosomes for repairing burns, and a preparation method and application thereof. Background Art

[0002] Exosomes are small membrane vesicles (30-150 nm) containing complex RNA and proteins. Nowadays, they specifically refer to disc-shaped vesicles with a diameter of 40-100 nm.

[0003] Epithelial exosomes are extracellular vesicles secreted by epithelial cells with a diameter of approximately 30-150 nanometers and are rich in biological molecules such as RNA, proteins, and lipids.

[0004] Burns are a common type of disease in dermatology and surgery, with a high incidence rate. They are usually caused by careless contact with hot liquids or metals, and are considered accidental personal injuries. Especially for children, due to their age and life experience, their safety awareness is not very high, and they may come into contact with high-temperature objects more frequently, making burns more likely. Burns are very harmful. If they are not treated promptly and effectively, they are likely to cause serious consequences, leading to skin infection and necrosis.

[0005] Conventional burns are usually treated with chemical burn ointments, which have a certain effect, but are relatively irritating and take a relatively long time to heal, and cannot meet the needs of specific groups of people.

[0006] Based on this, an epithelial cell exosome for repairing burns is provided to solve the existing technical problems. Summary of the invention

[0007] The purpose of the present invention is to provide an epithelial cell exosome for repairing burns and a preparation method thereof, so as to make up for the deficiencies in the prior art.

[0008] The technical solution adopted by the present invention is as follows: A method for preparing epithelial cell exosomes for repairing burns, comprising the following steps: a. Take skin tissue, cut it into pieces, soak it in PBS containing 0.1% penicillin-streptomycin for 30 minutes, then take it out and culture it in 8% FBS L-DMEM culture medium. Change the culture medium every 3 days. After changing it 3 times, remove the tissue pieces, digest the adherent cells with trypsin, and then culture them with composite culture medium. After 70% confluence, digest it with trypsin and pass it for 3 generations to obtain epithelial cell culture medium; Cultured at 37°C, 5% CO2, saturated humidity; b. Centrifuge the epithelial cell culture medium twice and collect the supernatant successively; c. PBS buffer was mixed with the second supernatant at a mass ratio of 1:1-1.2, and then ultracentrifuged at 130000g for 1.5-1.8h, the precipitate was collected and resuspended with PBS buffer solution to obtain a crude product, which was purified to obtain epithelial cell exosomes for repairing burns; The purification is as follows: The crude product was passed through a Focurose 4FF gel chromatography column, the medium of the gel chromatography column was dextran with a dry particle size of 100 μm, the sample loading amount was 1.5% of the column volume, and eluted with PBS buffer at a flow rate of 12-15 cm / h, and the sample was collected to obtain the product.

[0009] As a further technical solution, the composite culture medium is an H-DMEM culture medium containing 0.1-0.3% of a composite extract of Phellinus linteus and Ampelopsis pilosula; The complex culture medium does not contain FBS.

[0010] As a further technical solution, the preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0011] As a further technical solution, the mulberry linterus powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0012] As a further technical solution, the preparation method of the crude extract of Phellinus igniarius is: The mulberry powder and water are mixed at a solid-liquid ratio of 1:10-12, the temperature is adjusted to 60°C, reflux extraction is performed for 4 hours, and then high-speed centrifugation is performed for 10 minutes, standing for 30 minutes, filtering, removing the filter residue, and obtaining a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0013] As a further technical solution, the preparation method of the crude extract of Bletilla striata is: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:10-15, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0014] As a further technical solution: the purification process is: First, the crude extract of Phellinus igniarius is added to a methanol solution with a mass fraction of 50-60%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate is spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 60-70%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0015] As a further technical solution, the epithelial cell culture fluid is centrifuged twice including: The first centrifugation was performed at 50,000 g for 20 min; The second centrifugation was performed at 100,000 g for 1 h.

[0016] The method described herein prepares epithelial cell exosomes for repairing burns.

[0017] The epithelial cell exosomes are used to repair burns.

[0018] Beneficial effects: The epithelial cell exosomes prepared by the present invention can transmit genetic information, reprogram host cells, participate in regulating biological processes such as cell growth, differentiation, migration, etc., and can be transported to another cell as nutrients, participating in biological processes such as repair and regeneration of skin tissue.

[0019] The epithelial cell exosomes prepared by the method of the present invention contain rich components such as lipids, proteins and nucleic acids. These components have specific biological functions, can affect the growth, division, migration and other behaviors of other cells, and participate in various physiological and pathological processes such as immune regulation, angiogenesis and tissue repair.

[0020] In the process of burn repair, the proliferation and migration of epidermal cells and dermal fibroblasts are crucial to filling wound gaps and promoting healing. The epithelial cell exosomes prepared by the present invention can promote the proliferation and migration of these cells, thereby increasing the healing speed of burn wounds and shortening the healing time of burn wounds.

[0021] Since after being scalded, the local tissue of the skin will have an acute inflammatory reaction, and excessive inflammatory reaction may lead to aggravated skin tissue damage and prolonged healing time, the epithelial cell exosomes prepared by the composite extract of Phellinus igniarius and Bletilla striata assisted in cultivation according to the present invention can reduce the degree of inflammatory reaction by regulating the balance of pro-inflammatory cytokines and anti-inflammatory cytokines, specifically by reducing the total number of white blood cells and down-regulating the expression of proteins such as TLR4, NF-κB / P65, thereby preventing the release of inflammatory factors such as IL-1β and TNF-α, thereby achieving the effect of reducing inflammatory reaction, thereby accelerating the healing speed of the scald wound and shortening the healing time. In addition, by reducing and eliminating the inflammatory reaction, the irritation can be reduced, giving the patient a more comfortable feeling.

[0022] The exosomes prepared by introducing the composite extract of Phellinus linteus and Bletilla striata into the culture medium during the culture process can increase the density of new blood vessels by promoting the proliferation and migration of vascular endothelial cells, can promote the proliferation and migration of endothelial cells, accelerate the healing speed of burn wounds, and can increase the synthesis and deposition of collagen by promoting the proliferation and secretion ability of fibroblasts, thereby promoting the remodeling and repair of skin tissue.

[0023] The present invention introduces the composite extract of Phellinus igniarius and Ampelopsis pilosulae, and the cooperation of the active ingredients of the two can achieve the effects of antibacterial and whitening. The antibacterial principle is mainly to directly kill pathogens by interfering with the synthesis of bacterial proteins and genetic materials, inhibiting their growth and metabolism, and can also reduce the inflammatory response of the skin, thus achieving the effects of antibacterial and anti-inflammatory. At the same time, the cooperation of the active ingredients of the two can also moisturize the skin and reduce pigmentation, and can play the role of soothing and repairing damaged skin. Under the cooperation of multiple active ingredients, the copper ions on the hydroxylase site can be competitively inhibited to achieve the effect of inhibiting the activity of tyrosine hydroxylase, thereby reducing the skin melanization caused by ultraviolet rays, thereby achieving a significant whitening effect. In addition, the transfer of melanin particles to keratinocytes can be inhibited, thereby reducing pigmentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a bar graph of burn healing time in each test group; Figure 2 This is an electron micrograph of exosomes in Example 1. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Trypsin digestion was performed using: 0.25% trypsin-EDTA digestion solution was purchased from China Sangon Biotechnology (Shanghai) Co., Ltd.; L-DMEM medium, H-DMEM medium, and FBS fetal bovine serum were purchased from Gibco, USA; Embodiment 1: A method for preparing epithelial cell exosomes for repairing burns, comprising the following steps: a. Take skin tissue, cut it into pieces, soak it in PBS containing 0.1% penicillin-streptomycin for 30 minutes, then take it out and culture it in 8% FBS L-DMEM culture medium. Change the culture medium every 3 days. After changing it 3 times, remove the tissue pieces, digest the adherent cells with trypsin, and then culture them with composite culture medium. After 70% confluence, digest it with trypsin and pass it for 3 generations to obtain epithelial cell culture medium; Cultured at 37°C, 5% CO2, saturated humidity; The composite culture medium is H-DMEM culture medium containing 0.1% composite extract of Phellinus linteus and Ampelopsis pilosula; The complex culture medium does not contain FBS.

[0027] The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0028] Phellinus igniarius powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0029] The preparation method of Phellinus linteus crude extract is as follows: The mulberry powder and water were mixed at a solid-liquid ratio of 1:10, the temperature was adjusted to 60°C, refluxed for 4 hours, and then subjected to high-speed centrifugation for 10 minutes, allowed to stand for 30 minutes, filtered, and the residue was removed to obtain a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0030] The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:10, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0031] Purification treatment: First, the crude extract of Phellinus igniarius was added to a methanol solution with a mass fraction of 50%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate was spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 60%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0032] b. Centrifuging the epithelial cell culture fluid twice and collecting the supernatant in turn; centrifuging the epithelial cell culture fluid twice includes: The first centrifugation was performed at 50,000 g for 20 min; The second centrifugation was performed at 100,000 g for 1 h.

[0033] c. PBS buffer was mixed with the second supernatant at a mass ratio of 1:1, and then ultracentrifuged at 130,000 g for 1.5 h. The precipitate was collected and resuspended with PBS buffer solution to obtain a crude product, which was purified to obtain epithelial cell exosomes for repairing burns; The purification is as follows: The crude product was passed through a Focurose 4FF gel chromatography column, the medium of the gel chromatography column was dextran with a dry particle size of 100 μm, the sample loading amount was 1.5% of the column volume, and eluted with PBS buffer at a flow rate of 12 cm / h, and the sample was collected to obtain the product.

[0034] Example 2 On the basis of Example 1, the difference from Example 1 is that the composite culture medium is H-DMEM culture medium containing 0.15% composite extract of Phellinus linteus and Ampelopsis pilosula.

[0035] The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0036] Phellinus igniarius powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0037] The preparation method of Phellinus linteus crude extract is as follows: The mulberry powder and water were mixed at a solid-liquid ratio of 1:10.5, the temperature was adjusted to 60°C, refluxed for 4 hours, and then subjected to high-speed centrifugation for 10 minutes, allowed to stand for 30 minutes, filtered, and the residue was removed to obtain a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0038] The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:12, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0039] Purification treatment: First, the crude extract of Phellinus igniarius was added to a methanol solution with a mass fraction of 55%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate was spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 62%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0040] Example 3 On the basis of Example 1, the difference from Example 1 is that the composite culture medium is H-DMEM culture medium containing 0.2% composite extract of Phellinus linteus and Bletilla striata.

[0041] The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0042] Phellinus igniarius powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0043] The preparation method of Phellinus linteus crude extract is as follows: The mulberry powder and water were mixed at a solid-liquid ratio of 1:11, the temperature was adjusted to 60°C, refluxed for 4 hours, and then subjected to high-speed centrifugation for 10 minutes, allowed to stand for 30 minutes, filtered, and the residue was removed to obtain a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0044] The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:13, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0045] Purification treatment: First, the crude extract of Phellinus igniarius was added to a methanol solution with a mass fraction of 58%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate was spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 66%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0046] Example 4 On the basis of Example 1, the difference from Example 1 is that the composite culture medium is H-DMEM culture medium containing 0.20% composite extract of Phellinus linteus and Bletilla striata.

[0047] The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0048] Phellinus igniarius powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0049] The preparation method of Phellinus linteus crude extract is as follows: The mulberry powder and water were mixed at a solid-liquid ratio of 1:11.5, the temperature was adjusted to 60°C, refluxed for 4 hours, and then subjected to high-speed centrifugation for 10 minutes, allowed to stand for 30 minutes, filtered, and the residue was removed to obtain a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0050] The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:13, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0051] Purification treatment: First, the crude extract of Phellinus igniarius was added to a methanol solution with a mass fraction of 56%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate was spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 65%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0052] Example 5 On the basis of Example 1, the difference from Example 1 is that the composite culture medium is H-DMEM culture medium containing 0.25% composite extract of Phellinus linteus and Ampelopsis pilosula.

[0053] The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The mulberry linterus powder is extracted by water extraction to obtain the crude extract of mulberry linterus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

[0054] Phellinus igniarius powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

[0055] The preparation method of Phellinus linteus crude extract is as follows: The mulberry powder and water were mixed at a solid-liquid ratio of 1:12, the temperature was adjusted to 60°C, refluxed for 4 hours, and then subjected to high-speed centrifugation for 10 minutes, allowed to stand for 30 minutes, filtered, and the residue was removed to obtain a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

[0056] The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:15, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

[0057] Purification treatment: First, the crude extract of Phellinus igniarius was added to a methanol solution with a mass fraction of 60%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate was spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 70%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

[0058] Comparative Example 1: On the basis of Example 1, the composite extract of Phellinus linteus and Bletilla striata was replaced with Phellinus linteus extract, which was added at a mass fraction of 0.1%. The preparation method and other technical solutions remained unchanged.

[0059] Comparative Example 2: Based on Example 1, the composite extract of Phellinus linteus and Bletilla striata was replaced by Bletilla striata extract, which was added at a mass fraction of 0.1%. The preparation method and other technical solutions remained unchanged.

[0060] Comparative Example 3 Based on Example 1, the composite extract of Phellinus linteus and Bletilla striata was not added, and the rest of the technical solutions remained unchanged.

[0061] test: The experiment selected 90 10-week-old SPF adult SD rats, half male and half female, with an average body weight of (230±10) g. The rats were raised in an SPF environment with good ventilation, temperature (24±2)℃, and humidity of 50%. Adult SD rats were not restricted in diet. After getting familiar with the environment for 1 week, they were divided into 9 groups, with 5 rats in each group, and the 9 groups were modeled: Establishment of skin burn model After anesthetizing the rats in each group, the back was depilated and then routinely disinfected. The rats were scalded with 85°C steam for 10 seconds to create a circular scald wound with a diameter of 5 mm on the back of the rats. Then, 8 of the groups were injected with 100 μL of the exosome injection solution of the PBS embodiment and the comparative example, and the other group was injected with an equal amount of normal saline. The wound healing time of each group was compared. The exosome injection solution of PBS Example and Comparative Example contained 3×10 10 exosome particles; Table 1

[0062] It can be seen from Table 1 that the exosomes prepared by the present invention can have an excellent burn repair effect.

[0063] Taking Example 1 as the basic sample, the effects of the mass ratio of the pure extract of Phellinus linteus and the pure extract of Bletilla striata on wound healing time were compared, as shown in Table 2: Table 2

[0064] It can be seen from Table 2 that the addition of different mass ratios of the pure extract of Phellinus igniarius to the pure extract of Bletilla striata has a certain degree of influence on the wound healing effect of the subsequent extracted exosomes.

[0065] Taking Example 1 as the basic sample, the effects of the concentrations of the composite extracts of Phellinus linteus and Bletilla striata in different composite culture solutions on the healing effect of the prepared exosomes on the scald wound were compared, as shown in Table 3: Table 3

[0066] It can be seen from Table 3 that with the increase of the concentration of the composite extract of Phellinus linteus and Bletilla striata in the composite culture medium, the healing effect of the exosomes on the scald wound first increased and then gradually decreased, indicating that within a certain concentration range, the scald wound healing effect of the exosomes can be improved. However, excessive introduction will affect the scald wound healing effect of the exosomes.

[0067] Antibacterial test: A 0.20% aqueous solution containing a composite extract of Phellinus linteus and Bletilla striata was prepared as Group 1; A water solution containing 0.20% of Phellinus linteus extract was prepared as group 2; An aqueous solution containing 0.20% of the compound extract of Ampelopsis pilosula was prepared as group 3; Culture medium: 0.3 g beef extract, 1.0 g peptone, 0.5 g NaCl, 1.5 g agar, 100 mL water, pH 7.2; Take a sterilized filter paper with a diameter of 6 mm and soak it in the solution of group 1-3 for 20 hours. Take 0.5m of Escherichia coli suspension and Staphylococcus aureus suspension and culture medium to make a bacterial plate. Use sterile tweezers to stick the soaked filter paper on the bacterial plate, 2 pieces per plate, perform 5 parallel experiments, take the average value, and place it in a 37℃ constant temperature incubator for 24 hours. Measure the diameter of the inhibition zone of the filter paper: Table 4

[0068] It can be seen from Table 4 that the composite extract of Phellinus igniarius and Bletilla striata can exert a certain antibacterial effect through the coordinated action of active ingredients.

[0069] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A method for preparing epithelial cell exosomes for repairing burns, characterized in that: The following steps are involved: a. Take skin tissue, cut it into pieces, soak it in PBS containing 0.1% penicillin-streptomycin for 30 minutes, then take it out and culture it in 8% FBSL-DMEM culture medium. Change the culture medium every 3 days. After changing it 3 times, remove the tissue blocks, digest the adherent cells with trypsin, and then culture them with composite culture medium. After 70% confluence, digest it with trypsin and pass it for 3 generations to obtain epithelial cell culture medium; b. Centrifuge the epithelial cell culture medium twice and collect the supernatant successively; c. PBS buffer was mixed with the second supernatant at a mass ratio of 1:1-1.2, and then ultracentrifuged at 130000g for 1.5-1.8h, the precipitate was collected and resuspended with PBS buffer solution to obtain a crude product, which was purified to obtain epithelial cell exosomes for repairing burns; The purification is as follows: The crude product was passed through a Focurose 4FF gel chromatography column, the medium of the gel chromatography column was dextran with a dry particle size of 100 μm, the sample loading amount was 1.5% of the column volume, and eluted with PBS buffer at a flow rate of 12-15 cm / h, and the sample was collected to obtain the product.

2. The method for preparing epithelial cell exosomes for repairing burns according to claim 1, characterized in that: The composite culture medium is H-DMEM culture medium containing 0.1-0.3% composite extract of Phellinus linteus and Ampelopsis pilosula; The complex culture medium does not contain FBS.

3. The method for preparing epithelial cell exosomes for repairing burns according to claim 2, characterized in that: The preparation method of the composite extract of Phellinus linteus and Bletilla striata is as follows: First, prepare Phellinus linteus and Bletilla striata separately; Grinding and sieving Phellinus linteus and Bletilla striata respectively to obtain Phellinus linteus powder and Bletilla striata powder; The water extraction method is used to extract Phellinus linteus powder to obtain a crude extract of Phellinus linteus; The powder of Bletilla striata was extracted by alcohol extraction to obtain the crude extract of Bletilla striata. Purifying the crude extract of Phellinus igniarius and the crude extract of Bletilla striata respectively to obtain the purified extract of Phellinus igniarius and the purified extract of Bletilla striata; The obtained pure extract of Phellinus linteus and pure extract of Bletilla striata were mixed evenly in a mass ratio of 3:1 to obtain a composite extract of Phellinus linteus and Bletilla striata.

4. The method for preparing epithelial cell exosomes for repairing burns according to claim 3, characterized in that: The mulberry linter powder is obtained by passing through a 120-mesh sieve; The white ampel powder is obtained by passing through a 100-mesh sieve.

5. The method for preparing epithelial cell exosomes for repairing burns according to claim 3, characterized in that: The preparation method of the Phellinus linteus crude extract is as follows: The mulberry powder and water are mixed at a solid-liquid ratio of 1:10-12, the temperature is adjusted to 60°C, reflux extraction is performed for 4 hours, and then high-speed centrifugation is performed for 10 minutes, standing for 30 minutes, filtering, removing the filter residue, and obtaining a filtrate; The filter residue was extracted repeatedly for 3 times, and all the filtrates were combined and spray-dried to obtain a crude extract of Phellinus igniarius. The rotation speed of the high-speed centrifugal treatment is 8000r / min.

6. The method for preparing epithelial cell exosomes for repairing burns according to claim 3, characterized in that: The preparation method of the crude extract of Bletilla striata is as follows: Mix the white peony powder and the ethanol solution at a solid-liquid ratio of 1:10-15, adjust the temperature to 45°C, stir for 2 hours, then high-speed centrifuge for 20 minutes, let stand for 30 minutes, filter, and remove the filter residue and the filtrate; The filter residue was extracted twice repeatedly, all the filtrates were combined, and then spray-dried to obtain a crude extract of Bletilla striata. Among them, the mass fraction of ethanol solution is 85%.

7. The method for preparing epithelial cell exosomes for repairing burns according to claim 3, characterized in that: The purification process is as follows: First, the crude extract of Phellinus igniarius is added to a methanol solution with a mass fraction of 50-60%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.4 μm filter membrane to obtain a Phellinus igniarius filtrate, and then the Phellinus igniarius filtrate is spray-dried; The crude extract of Bletilla striata is added to a methanol solution with a mass fraction of 60-70%, and then stirred at a speed of 500 r / min for 30 minutes, and then filtered through a 0.35 μm filter membrane to obtain a Bletilla striata filtrate, and then the Bletilla striata filtrate is spray-dried.

8. The method for preparing epithelial cell exosomes for repairing burns according to claim 1, characterized in that: The epithelial cell culture medium was centrifuged twice including: The first centrifugation was performed at 50,000 g for 20 min; The second centrifugation was performed at 100,000 g for 1 h.

9. Epithelial cell exosomes for repairing burns are prepared according to any one of the methods of claims 1-8.

10. The epithelial cell exosomes according to claim 9 are used to repair burns.

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