Smooth muscle cell exosome for artificial skin as well as preparation method and application of smooth muscle cell exosome

Abstract

The invention discloses a smooth muscle cell exosome for artificial skin as well as a preparation method and application thereof, and relates to the technical field of biology, smooth muscle tissues are taken, cut into pieces, then cultured by adopting a complete culture medium until the smooth muscle tissues are adhered to 80% of the wall, the cut smooth muscle tissues are removed, then digested by 0.25% pancreatin, centrifuged at the rotating speed of 1200r / min for 10min, filtered, and dried to obtain the smooth muscle cell exosome for artificial skin. Sequentially culturing the obtained smooth muscle cell precipitate to the fourth generation by using a complete culture medium containing a gradient concentration modifier, so as to obtain a fourth generation modified smooth muscle cell culture solution; and extracting the smooth muscle cell exosome from the self-modified smooth muscle cell culture solution. The smooth muscle cell exosome for artificial skin prepared by the invention is rich in complex RNA (Ribonucleic Acid), protein and bioactive molecules such as lipid, and the bioactive molecules play an important role in promoting the growth, repair and regeneration of cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular to a smooth muscle cell exosome for artificial skin, and a preparation method and application thereof. Background Art

[0002] Artificial skin has excellent medical application prospects in the treatment of burns, scalds and other fields, and has thus received widespread attention and in-depth research.

[0003] The skin wounds of burn patients face a series of problems during the recovery process, such as tissue fluid infiltration and infection, especially Pseudomonas aeruginosa infection, which has a more serious impact, greatly prolongs the healing time of the wound and increases the difficulty of healing.

[0004] In the prior art, dressings are mostly used for treatment, but these dressings cannot effectively solve problems such as tissue fluid exudation and infection.

[0005] In order to solve the problem of tissue fluid leakage, artificial skin is used. However, the artificial skin prepared by the prior art still has the problem of poor biocompatibility with the wound surface.

[0006] Based on this, a smooth muscle cell exosome for artificial skin is provided to solve the existing technical problems. Summary of the invention

[0007] The purpose of the present invention is to provide a smooth muscle cell exosome for artificial skin 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:

[0009] A method for preparing smooth muscle cell exosomes for artificial skin comprises taking smooth muscle tissue, cutting it into pieces, and then using complete culture medium to culture it until 80% of the area is attached to the wall, removing the cut smooth muscle tissue, and then using 0.25% trypsin to digest it, and then centrifuging it at a speed of 1200r / min for 10min, filtering it to obtain a smooth muscle cell precipitate, and then using a complete culture medium containing a gradient concentration modifier to culture the obtained smooth muscle cell precipitate in sequence to the fourth generation to obtain a fourth-generation modified smooth muscle cell culture fluid;

[0010] Extracting smooth muscle cell exosomes from the culture medium of modified smooth muscle cells;

[0011] The specific extraction method is as follows: centrifuge the clarified 4th generation modified smooth muscle cell culture medium at 50,000×g and 4°C in an ultracentrifuge for 90 min, and take the supernatant;

[0012] The supernatant was centrifuged at 100,000 × g at 4°C for 90 min in an ultracentrifuge to precipitate exosomes.

[0013] As a further technical solution, the complete culture medium is obtained by adding fetal bovine serum to a serum-free culture medium and mixing them evenly;

[0014] The mass fraction of fetal bovine serum added to the complete culture medium is 10%.

[0015] As a further technical solution, the method for preparing the serum-free medium is:

[0016] Dissolve 1 bag of DMEM culture medium powder in 1L of ultrapure water, mix well, adjust the pH of the mixed system to 7.2, and then filter through a 0.22μm filter membrane to obtain a serum-free culture medium;

[0017] The pH was adjusted using sodium bicarbonate.

[0018] As a further technical solution, the complete medium is used for culturing until the area of ​​adhesion is 80%:

[0019] The cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0020] As a further technical solution, the complete culture medium containing the gradient concentration modifier is:

[0021] Adding the modifier to the complete medium obtained above to obtain a complete medium with a modifier concentration of 5 μg / mL, a complete medium with a modifier concentration of 15 μg / mL, a complete medium with a modifier concentration of 20 μg / mL, and a complete medium with a modifier concentration of 25 μg / mL in sequence;

[0022] The complete medium containing the gradient concentration modifier does not contain FBS.

[0023] As a further technical solution, the preparation method of the modifier is:

[0024] Firstly, pomegranate peel polysaccharide is added into formamide, the temperature is adjusted to 35°C, and the mixture is kept warm and stirred for 2 hours to obtain a pomegranate peel polysaccharide solution;

[0025] Under ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate;

[0026] Continue to add the intermediate dropwise into the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution;

[0027] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0028] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0029] As a further technical solution: the mixing ratio of the pomegranate peel polysaccharide and formamide is 150-180 mg: 80-90 mL;

[0030] The mixing mass ratio of pyridine and chlorosulfonic acid is 1:3-4;

[0031] The mixing mass ratio of the intermediate and the pomegranate peel polysaccharide solution is 3-5:30-40.

[0032] As a further technical solution, the following are cultured to the fourth generation:

[0033] The first, second, third and fourth generations were cultured in complete medium with a modifier concentration of 5 μg / mL, complete medium with a modifier concentration of 15 μg / mL, complete medium with a modifier concentration of 20 μg / mL and complete medium with a modifier concentration of 25 μg / mL, respectively;

[0034] All cells were cultured in a cell culture incubator at 37°C and 5% CO2. Each generation was passed down after 90% confluence and 0.25% trypsin digestion.

[0035] As a further technical solution, the method prepares smooth muscle cell exosomes for artificial skin.

[0036] Smooth muscle cell exosomes are used in artificial skin.

[0037] Beneficial effects:

[0038] The smooth muscle cell exosomes prepared by the present invention for artificial skin are rich in complex RNA and proteins, as well as bioactive molecules such as lipids, and these bioactive molecules have an important promoting effect on cell growth, repair and regeneration. By applying them to artificial skin, the smooth muscle cell exosomes participate in intercellular communication, regulate the biological activity of receptor cells, and promote the repair and regeneration of damaged skin cells.

[0039] The specific growth factors, cytokines and other bioactive substances contained in the smooth muscle cell exosomes prepared by the present invention can promote the proliferation and migration of skin cells, and in the application process of artificial skin, in conjunction with the effect of the smooth muscle exosomes, applied to the damaged skin area, the smooth muscle exosomes can stimulate the activation and proliferation of surrounding skin cells, thereby accelerating wound healing and tissue regeneration, and under the action of the smooth muscle exosomes, the biocompatibility between the artificial skin and the wound surface is greatly improved, and the rejection effect is reduced, thereby avoiding the problem of wound surface deterioration due to poor biocompatibility.

[0040] Compared with other exosomes, the smooth muscle cell exosomes used in the present invention have the ability to dynamically balance and regulate the secretion of inflammatory factors in the wound area, including pro-inflammatory factors and anti-inflammatory factors, and can effectively inhibit the inflammatory response of the wound surface, thereby providing a favorable external environment for the growth and repair of skin cells on the wound surface. At the same time, since the prepared smooth muscle cell exosomes can promote angiogenesis and collagen deposition, they can further improve the structure and function of skin tissue.

[0041] By combining the smooth muscle exosomes prepared by the present invention with artificially synthesized skin materials, the biocompatibility of artificial skin is improved, mainly enabling it to better simulate the structure and function of natural skin, thereby reducing rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a bar graph of wound healing time in each group of the experiment;

[0043] Figure 2 This is an electron microscopic image of the 4th generation cells with 90% confluence in Example 1;

[0044] Figure 3 This is a statistical chart of the time it takes for the wound to heal completely. DETAILED DESCRIPTION

[0045] 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.

[0046] 0.25% trypsin: 0.25% trypsin-EDTA digestion solution was purchased from Gibi co, USA;

[0047] DMEM culture medium and FBS fetal bovine serum were purchased from Gibco, USA;

[0048] Embodiment 1:

[0049] A method for preparing smooth muscle cell exosomes for artificial skin comprises taking smooth muscle tissue, cutting it into pieces, and then using complete culture medium to culture it until 80% of the area is attached to the wall, removing the cut smooth muscle tissue, and then using 0.25% trypsin to digest it, and then centrifuging it at a speed of 1200r / min for 10min, filtering it to obtain a smooth muscle cell precipitate, and then using a complete culture medium containing a gradient concentration modifier to culture the obtained smooth muscle cell precipitate in sequence to the fourth generation to obtain a fourth-generation modified smooth muscle cell culture fluid;

[0050] Cultivated to the 4th generation:

[0051] The first, second, third and fourth generations were cultured in complete medium with a modifier concentration of 5 μg / mL, complete medium with a modifier concentration of 15 μg / mL, complete medium with a modifier concentration of 20 μg / mL and complete medium with a modifier concentration of 25 μg / mL, respectively;

[0052] All cells were cultured in a cell culture incubator at 37°C and 5% CO2. Each generation was passed down after 90% confluence and 0.25% trypsin digestion.

[0053] Extracting smooth muscle cell exosomes from the culture medium of modified smooth muscle cells;

[0054] The specific extraction method is as follows: centrifuge the clarified 4th generation modified smooth muscle cell culture medium at 50,000×g and 4°C in an ultracentrifuge for 90 min, and take the supernatant;

[0055] The supernatant was centrifuged at 100,000 × g at 4°C for 90 min in an ultracentrifuge to precipitate exosomes.

[0056] Complete medium is obtained by adding fetal bovine serum to serum-free medium and mixing them evenly;

[0057] The mass fraction of fetal bovine serum added to the complete culture medium is 10%.

[0058] The method for preparing serum-free medium is as follows:

[0059] Dissolve 1 bag of DMEM culture medium powder in 1L of ultrapure water, mix well, adjust the pH of the mixed system to 7.2, and then filter through a 0.22μm filter membrane to obtain a serum-free culture medium;

[0060] The pH was adjusted using sodium bicarbonate.

[0061] Use complete medium to culture until 80% of the cells adhere to the wall:

[0062] The cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0063] The complete medium containing gradient concentration modifiers is:

[0064] Adding the modifier to the complete medium obtained above to obtain a complete medium with a modifier concentration of 5 μg / mL, a complete medium with a modifier concentration of 15 μg / mL, a complete medium with a modifier concentration of 20 μg / mL, and a complete medium with a modifier concentration of 25 μg / mL in sequence;

[0065] The complete medium containing the gradient concentration modifier does not contain FBS.

[0066] The preparation method of the modifier is:

[0067] First, add pomegranate peel polysaccharide to formamide, adjust the temperature to 35°C, keep warm and stir for 2 hours to obtain a pomegranate peel polysaccharide solution; the mixing ratio of pomegranate peel polysaccharide to formamide is 150 mg:80 mL;

[0068] In an ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; the mass ratio of pyridine to chlorosulfonic acid was 1:3;

[0069] Continue to add the intermediate to the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; the mixing mass ratio of the intermediate to the pomegranate peel polysaccharide solution is 3:30.

[0070] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0071] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0072] Example 2

[0073] Based on Example 1, the difference from Example 1 is that the preparation method of the modifier is:

[0074] First, add pomegranate peel polysaccharide to formamide, adjust the temperature to 35°C, keep warm and stir for 2 hours to obtain a pomegranate peel polysaccharide solution; the mixing ratio of pomegranate peel polysaccharide to formamide is 155 mg:85 mL;

[0075] In an ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; the mass ratio of pyridine to chlorosulfonic acid was 1:3.5;

[0076] Continue to add the intermediate to the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; the mixing mass ratio of the intermediate to the pomegranate peel polysaccharide solution is 3.5:32.

[0077] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0078] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0079] Example 3

[0080] Based on Example 1, the difference from Example 1 is that the preparation method of the modifier is:

[0081] First, add pomegranate peel polysaccharide to formamide, adjust the temperature to 35°C, keep warm and stir for 2 hours to obtain a pomegranate peel polysaccharide solution; the mixing ratio of pomegranate peel polysaccharide to formamide is 160 mg:85 mL;

[0082] In an ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; the mass ratio of pyridine to chlorosulfonic acid was 1:3.8;

[0083] Continue to add the intermediate to the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; the mixing mass ratio of the intermediate to the pomegranate peel polysaccharide solution is 4:35.

[0084] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0085] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0086] Example 4

[0087] Based on Example 1, the difference from Example 1 is that the preparation method of the modifier is:

[0088] First, add pomegranate peel polysaccharide to formamide, adjust the temperature to 35°C, keep warm and stir for 2 hours to obtain a pomegranate peel polysaccharide solution; the mixing ratio of pomegranate peel polysaccharide to formamide is 165 mg:85 mL;

[0089] In an ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; the mass ratio of pyridine to chlorosulfonic acid was 1:3.6;

[0090] Continue to add the intermediate to the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; the mixing mass ratio of the intermediate to the pomegranate peel polysaccharide solution is 4.5:38.

[0091] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0092] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0093] Example 5

[0094] Based on Example 1, the difference from Example 1 is that the preparation method of the modifier is:

[0095] First, add pomegranate peel polysaccharide to formamide, adjust the temperature to 35°C, keep warm and stir for 2 hours to obtain a pomegranate peel polysaccharide solution; the mixing ratio of pomegranate peel polysaccharide to formamide is 180 mg:90 mL;

[0096] In an ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; the mass ratio of pyridine to chlorosulfonic acid was 1:4;

[0097] Continue to add the intermediate to the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; the mixing mass ratio of the intermediate to the pomegranate peel polysaccharide solution is 5:40.

[0098] The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant;

[0099] The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

[0100] Comparative Example 1:

[0101] Based on Example 1, the complete culture medium containing the gradient concentration modifier was completely replaced with a complete culture medium containing 25 μg / mL modifier concentration, and the rest of the technical scheme remained unchanged.

[0102] Comparative Example 2:

[0103] Based on Example 1, the smooth muscle tissue is replaced by adipose tissue, and the rest of the technical solutions remain unchanged.

[0104] Comparative Example 3:

[0105] Based on Example 1, the modifier was replaced with pomegranate peel polysaccharide, and the rest of the technical scheme remained unchanged.

[0106] Comparative Example 4:

[0107] Based on Example 1, the pomegranate peel polysaccharide in the modifier is replaced with lentinan, and the rest of the technical scheme remains unchanged.

[0108] Comparative Example 5

[0109] Based on Example 1, the complete culture medium containing the gradient concentration modifier is completely replaced with a complete culture medium without the modifier, and the rest of the technical scheme remains unchanged.

[0110] test:

[0111] Establishment of skin defect model

[0112] One hundred and twenty five-week-old experimental SD rats, half male and half female, weighing 200 g ± 10 g, were used to make the skin defect model;

[0113] First, the rats were anesthetized, and then the backs of the experimental rats were depilated. Then, a full-thickness skin defect with a diameter of 1.5 cm was made at the same position of each experimental rat.

[0114] The mice were randomly divided into 12 groups, 10 in each group, one of which was used as a blank control group, without treatment and allowed to heal naturally;

[0115] The artificial skin made by the prior art was tested with reference to the application number 2018100730708. The prior artificial skin was mixed with the exosomes of the embodiment and the control (the exosomes of the embodiment and the control were mixed with PBS, each mL containing 4.5×10 10 The artificial skin was soaked in 40% exosomes for 30 minutes at 4°C, and then transplanted to the wound of the experimental rats. The wound was then sutured and fixed. A group of untreated artificial skin was used for the experiment as the model control group. The changes of each group were compared and observed:

[0116] After the experiment in each group, the number of deaths was counted (34 days):

[0117] Table 1

[0118]

[0119]

[0120] As can be seen from Table 1, the artificial skin treated with exosomes of the present invention can significantly reduce the mortality rate of experimental rats when used for rat wound treatment;

[0121] The wound healing status of each group was statistically analyzed, as shown in Table 2:

[0122] Table 2

[0123]

[0124]

[0125] As can be seen from Table 2, under the action of the exosomes of the present invention, the wound healing can be accelerated. When no modifier is used, the wound healing efficiency will be reduced to a certain extent. The use of pomegranate peel polysaccharide as a modifier to prepare a raw material has a better effect on promoting wound healing than other plant polysaccharides. After the pomegranate peel polysaccharide is modified, it can play a better role in accelerating wound healing; the use of the gradient concentration of the modifier of the present invention can more significantly improve the wound healing effect than the culture medium using a fixed concentration of the modifier.

[0126] The complete healing time of wounds in each group was statistically analyzed, as shown in Table 3:

[0127] Table 3

[0128]

[0129]

[0130] It can be seen from Table 3 that the exosomes prepared by the present invention are applied to artificial skin and can shorten the wound healing time.

[0131] 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 smooth muscle cell exosomes for artificial skin, characterized in that: Smooth muscle tissue was taken, cut into pieces, and then cultured with complete medium until 80% of the area was attached to the wall, the cut smooth muscle tissue was removed, and then digested with 0.25% trypsin, and then centrifuged at 1200r / min for 10min, filtered to obtain smooth muscle cell precipitates, and then the obtained smooth muscle cell precipitates were cultured in sequence with complete medium containing gradient concentration modifiers to the 4th generation to obtain the 4th generation modified smooth muscle cell culture fluid; Extracting smooth muscle cell exosomes from the culture medium of modified smooth muscle cells; The specific extraction method is as follows: centrifuge the clarified 4th generation modified smooth muscle cell culture medium at 50,000×g and 4°C in an ultracentrifuge for 90 min, and take the supernatant; The supernatant was centrifuged at 100,000 × g at 4°C for 90 min in an ultracentrifuge to precipitate exosomes.

2. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 1, characterized in that: The complete culture medium is obtained by adding fetal bovine serum to serum-free culture medium and mixing them evenly; The mass fraction of fetal bovine serum added to the complete culture medium is 10%.

3. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 2, characterized in that: The serum-free medium preparation method is: Dissolve 1 bag of DMEM culture medium powder in 1L of ultrapure water, mix well, adjust the pH of the mixed system to 7.2, and then filter through a 0.22μm filter membrane to obtain a serum-free culture medium; The pH was adjusted using sodium bicarbonate.

4. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 1, characterized in that: The area of ​​culturing with complete medium until 80% of the adhesion is: The cells were cultured in a cell culture incubator at 37°C and 5% CO2.

5. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 3, characterized in that: The complete culture medium containing the gradient concentration modifier is: Adding the modifier to the complete medium obtained above to obtain a complete medium with a modifier concentration of 5 μg / mL, a complete medium with a modifier concentration of 15 μg / mL, a complete medium with a modifier concentration of 20 μg / mL, and a complete medium with a modifier concentration of 25 μg / mL in sequence; The complete medium containing the gradient concentration modifier does not contain FBS.

6. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 5, characterized in that: The preparation method of the modifier is: Firstly, pomegranate peel polysaccharide is added into formamide, the temperature is adjusted to 35°C, and the mixture is kept warm and stirred for 2 hours to obtain a pomegranate peel polysaccharide solution; Under ice bath, pyridine was added dropwise to chlorosulfonic acid, and the mixture was stirred for 1 hour to obtain an intermediate; Continue to add the intermediate dropwise into the pomegranate peel polysaccharide solution under ice bath conditions, adjust the pH of the reaction system to 7.5, and then stir for 4 hours to obtain a mixed solution; The mixed solution was dialyzed with distilled water using a dialysis bag for 48 hours, and then distilled under reduced pressure to obtain a reactant; The obtained reactant is precipitated with anhydrous ethanol, filtered, and freeze-dried to obtain a modifying agent.

7. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 6, characterized in that: The mixing ratio of the pomegranate peel polysaccharide and formamide is 150-180 mg: 80-90 mL; The mixing mass ratio of pyridine and chlorosulfonic acid is 1:3-4; The mixing mass ratio of the intermediate and the pomegranate peel polysaccharide solution is 3-5:30-40.

8. The method for preparing smooth muscle cell exosomes for artificial skin according to claim 1, characterized in that: Cultivated to the 4th generation: The first, second, third and fourth generations were cultured in complete medium with a modifier concentration of 5 μg / mL, complete medium with a modifier concentration of 15 μg / mL, complete medium with a modifier concentration of 20 μg / mL and complete medium with a modifier concentration of 25 μg / mL, respectively; All cells were cultured in a cell culture incubator at 37°C and 5% CO2. Each generation was passed down after 90% confluence and 0.25% trypsin digestion.

9. The method according to any one of claims 1 to 8 is used to prepare smooth muscle cell exosomes for artificial skin.

10. The smooth muscle cell exosomes according to claim 9 are used in artificial skin.

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