Fibroblast exosome for repairing scars as well as preparation method and application of fibroblast exosome

Abstract

The invention discloses a fibroblast exosome for repairing scars and a preparation method and application thereof, and relates to the technical field of biology, and the preparation method comprises the following steps: S1, taking fibroblasts, and carrying out adaptive culture on the fibroblasts in a complete culture medium for 24-30 hours; s2, after the adaptive culture is completed, replacing a new culture medium, culturing in a complete culture medium containing 10% of fetal calf serum until the fusion degree is 80%, carrying out digestion treatment by adopting 0.25% of pancreatin, centrifuging, collecting supernate, and carrying out ultracentrifugation extraction on the supernate to obtain a primary product of the fibroblast exosome; s3, carrying out transfection treatment on the primary product of the fibroblast exosome to obtain the fibroblast exosome for repairing scars; the prepared fiber-forming exosome can also influence the movement of epidermal and dermal cells by regulating the expression of related factors in a TGF-beta / Smad signal channel, and the regulating effect is mainly to promote scar-free healing of the skin, so that the generation of scars is reduced from the source.

Description

Technical Field

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

[0002] The formation and repair of scars is an important process after skin damage. Scar formation is a natural response and repair mechanism of the human body to trauma. When the skin is damaged by external forces, such as cuts, burns, abrasions, etc., the normal structure of the skin will be destroyed. In order to repair these injuries, the body will produce collagen to fill the wound. However, during this repair process, if the synthesis and arrangement of collagen are abnormal, scars will be formed.

[0003] The formation of scars is a great psychological burden for people who pursue beauty. Therefore, reducing the occurrence of scars and fading existing scars are the issues that need to be addressed.

[0004] Based on this, a fibroblast exosome for repairing scars is provided to solve the existing technical problems. Summary of the invention

[0005] The purpose of the present invention is to provide a fibroblast exosome for repairing scars and a preparation method thereof, so as to make up for the deficiencies in the prior art.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for preparing fibroblast exosomes for repairing scars, comprising the following steps:

[0008] S1 fibroblasts were adapted to culture in complete medium for 24-30 hours;

[0009] After the adaptation culture of S2 was completed, the new culture medium was replaced and cultured in a complete culture medium containing 10% fetal bovine serum until the confluence was 80%, and then digested with 0.25% trypsin, centrifuged, and the supernatant was collected. The supernatant was extracted by ultracentrifugation to obtain the initial product of fibroblast exosomes;

[0010] S3 transfects the initial fibroblast exosomes to obtain fibroblast exosomes for scar repair.

[0011] As a further technical solution, the adaptation culture in S1 is: culture in a 37° C., 5% CO 2 incubator.

[0012] As a further technical solution, the method for preparing the complete culture medium is:

[0013] In a sterile container, add 1 bag of DMEM culture medium powder to 1L ultrapure water, adjust the temperature to 60°C, keep warm and stir at 150r / min for 15min, then use sodium bicarbonate to adjust the pH of the system to 7.2, and cool naturally to room temperature.

[0014] As a further technical solution, the culture in S2 is carried out in an incubator at 37°C and 5% CO2;

[0015] The centrifugation is:

[0016] Centrifuge at 1000×g for 10 min and collect the supernatant.

[0017] As a further technical solution, the ultracentrifugation extraction comprises the following steps:

[0018] Step a, centrifuge at 2000×g for 10 min, discard the precipitate, and collect the supernatant;

[0019] Step b, centrifuging at 5000×g for 10 min, discarding the precipitate, and collecting the supernatant;

[0020] Step c, ultracentrifuging at 10,000×g for 30 min, discarding the precipitate, and collecting the supernatant;

[0021] Step d, filtering the supernatant obtained in step c using a 0.22 μm filter membrane, removing the filter residue and retaining the filtrate;

[0022] Step e, ultracentrifuging the filtrate obtained in step d at 120,000×g for 50 min, discarding the supernatant, and collecting the precipitate;

[0023] The precipitate obtained in step e was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 60 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0024] As a further technical solution, the transfection treatment of fibroblast exosomes in S3 is specifically as follows:

[0025] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0026] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0027] Then, the uniform mixed solution obtained above is transferred into an electrotransfer cup by using a pipette to perform electrotransfer treatment;

[0028] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0029] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0030] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0031] The concentration of CD24 siRNA in the uniform mixed solution is 550-600 nmol / L;

[0032] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0033] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0034] As a further technical solution: the concentration of the electrotransfer solution is:

[0035] 0.02-0.06 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0036] As a further technical solution, the electroporation parameters are: electroporation twice at a voltage of 150-160V, 1100μs, and an interval of 804ms.

[0037] The method described herein prepares fibroblast exosomes for repairing scars.

[0038] Fibroblast exosomes for scar repair.

[0039] For new wounds, coating can be used to reduce the formation of scars. For scars that have already formed, injection can be used to continuously fade the existing scars.

[0040] Beneficial effects:

[0041] After skin damage, treatment with the fibroblast exosomes prepared by the present invention can help reduce scar formation, shorten wound healing time, and reduce skin tissue hyperplasia and fibrosis.

[0042] The fiber-forming exosomes prepared by the present invention can also affect the movement of epidermal and dermal cells by regulating the expression of related factors in the TGF-β / Smad signaling pathway. This regulatory effect is mainly to promote scar-free healing of the skin, thereby reducing the generation of scars at the source.

[0043] In terms of shortening the healing time of skin wounds, it is mainly through the ability of fibrinolytic exosomes to promote the proliferation, migration and tube formation of vascular endothelial cells, thereby promoting the formation of capillary networks, which has a more significant promoting effect on improving local microcirculation of wounds and accelerating wound healing.

[0044] The formation of scars is mainly due to the abnormal deposition of collagen. Compared with conventional exosomes, the active ingredients in the fibroblast exosomes prepared by the present invention can inhibit the abnormal proliferation of fibroblasts and the excessive deposition of collagen fibers, thereby significantly reducing the formation of scars.

[0045] For scars that have already formed, they are mainly repaired through injections. Fibroblast exosomes contain a variety of growth factors and cytokines. These bioactive substances can promote the proliferation and migration of skin cells, thereby accelerating the repair of scars, gradually improving the appearance of scars, making them gradually fade, and significantly shortening the fading time.

[0046] Since scar formation is often accompanied by inflammatory response, the fibroblast exosomes prepared by the present invention have the ability to regulate inflammation-related signaling pathways, can reduce inflammatory response, and prevent further damage to skin tissue caused by inflammation. In addition, this anti-inflammatory effect can greatly reduce inflammatory cell infiltration and vasodilation in scar tissue, thereby promoting the continuous fading and regression of scars.

[0047] At the same time, the present invention can further improve the performance of exosomes by preparing a specific electroporation solution, and electroporation solutions with different components will affect the electroporation effect to varying degrees. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a bar graph of the residual wound area in each experimental group;

[0049] Figure 2 This is the picture before scar repair;

[0050] Figure 3 This is the picture after scar repair. DETAILED DESCRIPTION

[0051] 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.

[0052] Fibroblasts were derived from human skin fibroblasts and were obtained from Shanghai Qingqi Biotechnology Development Co., Ltd.;

[0053] Among them, 0.25% trypsin: 0.25% trypsin-EDTA digestion solution was purchased from Gibico, USA;

[0054] DMEM medium was in the specification of 10 × 1L / pack, and FBS fetal bovine serum was purchased from Gibco, USA;

[0055] The electroporation cup used: ROUMAI electroporation cup 2mm, from Zhonglong Regenerative Medicine (Shenzhen) Co., Ltd.;

[0056] Embodiment 1:

[0057] A method for preparing fibroblast exosomes for repairing scars, comprising the following steps:

[0058] S1 Take fibroblasts and place them in complete medium in an incubator at 37°C and 5% CO2 for 24 hours for adaptation. The complete medium preparation method is as follows:

[0059] In a sterile container, add 1 bag of DMEM culture medium powder to 1L ultrapure water, adjust the temperature to 60°C, keep warm and stir at 150r / min for 15min, then use sodium bicarbonate to adjust the pH of the system to 7.2, and cool naturally to room temperature.

[0060] After the adaptation culture of S2 was completed, a new culture medium was replaced and cultured in a complete culture medium containing 10% fetal bovine serum until the confluence was 80%, and then digested with 0.25% trypsin. After centrifugation, the supernatant was collected and ultracentrifuged to extract the primary product of fibroblast exosomes; the culture was carried out in a 37°C, 5% CO2 incubator;

[0061] The centrifugation is:

[0062] Centrifuge at 1000×g for 10 min and collect the supernatant.

[0063] Ultracentrifugation extraction involves the following steps:

[0064] Step a, centrifuge at 2000×g for 10 min, discard the precipitate, and collect the supernatant;

[0065] Step b, centrifuging at 5000×g for 10 min, discarding the precipitate, and collecting the supernatant;

[0066] Step c, ultracentrifuging at 10,000×g for 30 min, discarding the precipitate, and collecting the supernatant;

[0067] Step d, filtering the supernatant obtained in step c using a 0.22 μm filter membrane, removing the filter residue and retaining the filtrate;

[0068] Step e, ultracentrifuging the filtrate obtained in step d at 120,000×g for 50 min, discarding the supernatant, and collecting the precipitate;

[0069] The precipitate obtained in step e was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 60 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0070] S3 performs transfection treatment on the initial fibroblast exosomes to obtain fibroblast exosomes for scar repair; the transfection treatment on the fibroblast exosomes is specifically as follows:

[0071] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0072] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0073] Then, the uniform mixed solution obtained above was transferred into an electroporation cup by using a pipette for electroporation. The electroporation parameters were as follows: electroporation was performed twice at a voltage of 150 V and 1100 μs, with an interval of 804 ms.

[0074] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0075] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0076] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0077] The concentration of CD24 siRNA in the uniform mixed solution is 550 nmol / L;

[0078] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0079] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0080] The concentration of electrotransfer solution is:

[0081] 0.02 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0082] Example 2

[0083] On the basis of Example 1, the difference from Example 1 is that the transfection treatment of fibroblast exosomes is specifically as follows:

[0084] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0085] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0086] Then, the uniform mixed solution obtained above was transferred into an electroporation cup by using a pipette for electroporation. The electroporation parameters were as follows: electroporation was performed twice at a voltage of 150-160 V and 1100 μs, with an interval of 804 ms.

[0087] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0088] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0089] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0090] The concentration of CD24 siRNA in the uniform mixed solution is 570 nmol / L;

[0091] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0092] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0093] The concentration of electrotransfer solution is:

[0094] 0.03 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0095] Example 3

[0096] On the basis of Example 1, the difference from Example 1 is that the transfection treatment of fibroblast exosomes is specifically as follows:

[0097] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0098] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0099] Then, the uniform mixed solution obtained above was transferred into an electroporation cup by using a pipette for electroporation. The electroporation parameters were as follows: electroporation was performed twice at a voltage of 155 V and 1100 μs, with an interval of 804 ms.

[0100] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0101] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0102] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0103] The concentration of CD24 siRNA in the uniform mixed solution is 580 nmol / L;

[0104] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0105] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0106] The concentration of electrotransfer solution is:

[0107] 0.04 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0108] Example 4

[0109] On the basis of Example 1, the difference from Example 1 is that the transfection treatment of fibroblast exosomes is specifically as follows:

[0110] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0111] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0112] Then, the uniform mixed solution obtained above was transferred into an electroporation cup by using a pipette for electroporation. The electroporation parameters were as follows: electroporation was performed twice at a voltage of 155 V and 1100 μs, with an interval of 804 ms.

[0113] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0114] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0115] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0116] The concentration of CD24 siRNA in the uniform mixed solution is 590 nmol / L;

[0117] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0118] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0119] The concentration of electrotransfer solution is:

[0120] 0.05 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0121] Example 5

[0122] On the basis of Example 1, the difference from Example 1 is that the transfection treatment of fibroblast exosomes is specifically as follows:

[0123] First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution;

[0124] Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture;

[0125] Then, the uniform mixed solution obtained above was transferred into an electroporation cup by using a pipette for electroporation. The electroporation parameters were as follows: electroporation was performed twice at a voltage of 160 V and 1100 μs, with an interval of 804 ms.

[0126] After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair;

[0127] The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL;

[0128] The volume ratio of electroporation solution to resuspension solution was 1:1;

[0129] The concentration of CD24 siRNA in the uniform mixed solution is 600 nmol / L;

[0130] The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate;

[0131] The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

[0132] The concentration of electrotransfer solution is:

[0133] 0.06 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

[0134] Comparative Example 1:

[0135] Based on Example 1, the exosomes were not subjected to transfection treatment, and the rest of the technical solutions remained unchanged.

[0136] Comparative Example 2:

[0137] Based on Example 1, the concentration of the electrotransfer solution is:

[0138] 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water, and the rest of the technical scheme remains unchanged.

[0139] Comparative Example 3:

[0140] Based on Example 1, the concentration of the electrotransfer solution is:

[0141] 0.02mol / L trehalose, 0.018mol / L D-mannitol, 0.021mol / L sodium succinate, 0.003mol / L potassium chloride, 0.08mol / L magnesium chloride, 0.06mol / L sodium hydrogen phosphate, and the rest is distilled water, and the rest of the technical scheme remains unchanged.

[0142] Comparative Example 4:

[0143] Based on Example 1, the concentration of the electrotransfer solution is:

[0144] 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and the rest is distilled water. The rest of the technical plan remains unchanged.

[0145] test:

[0146] A total of 100 SPF mice, half male and half female, aged 6 weeks, were selected and housed in a sterile, constant temperature, and dark-light alternating clean environment. They were allowed to eat, drink, and carry out daily activities freely for 2 days.

[0147] Establishment of mouse back trauma model

[0148] The mice were anesthetized with 10% chloral hydrate (0.15 mL / 10 g), and then the hair on the back area of ​​the mice was shaved and disinfected with 75% alcohol. A full-thickness skin defect of 1.5 cm × 1.5 cm was prepared in the depilated back area of ​​the mice. The wound was covered with hemostatic dressing for 1 day and then exposed. The mouse back trauma model was established.

[0149] The mice were randomly divided into 10 groups, with 10 mice in each group:

[0150] The exosomes of the example and the control were resuspended in PBS to obtain an exosome resuspension with a concentration of 0.1 mg / mL;

[0151] 0.1 mL of the heavy suspension of the secretions of the embodiment and the control was applied to the wound surface of each group of mice, 3 times a day for 10 consecutive days. One group was left as the control group and coated with the same amount of PBS for comparison:

[0152] Wound healing: The wound healing of mice in each group was observed and counted 7 days after treatment, and the percentage of residual wound area was calculated;

[0153] Table 1

[0154] Residual wound area% Example 1 4.21 Example 2 4.15 Example 3 4.08 Example 4 4.17 Example 5 4.26 Comparative Example 1 5.17 Comparative Example 2 4.65 Comparative Example 3 5.08 Comparative Example 4 5.13 Blank control group 6.05

[0155] It can be seen from Table 1 that the wound area can be shortened and the wound healing time can be increased after being treated with the exosomes of the present invention, and the electroporation liquid can significantly improve the performance of the exosomes.

[0156] Scar hyperplasia comparison: 30 days after treatment, the scar conditions of mice in each group were observed and recorded, and the Vancouver scar measurement table was used to score the scar conditions on the back of the mice, with a total score of 0-15 points. The higher the score, the more severe the scar hyperplasia, as shown in Table 2:

[0157] Table 2

[0158] Scar hyperplasia / point Example 1 3.32 Example 2 3.25 Example 3 3.26 Example 4 3.31 Example 5 3.58 Comparative Example 1 5.33 Comparative Example 2 3.72 Comparative Example 3 4.11 Comparative Example 4 5.28 Blank control group 9.27

[0159] It can be seen from Table 2 that the exosomes of the present invention have a more significant effect on scar repair, and the use of electroporation solutions with different components will affect the efficacy of the exosomes to a certain extent.

[0160] Taking Example 1 as the basic sample, the effects of different volume ratios of electroporation solution and resuspension solution on the scar repair effect of the prepared exosomes are compared, as shown in Table 3:

[0161] Table 3

[0162]

[0163]

[0164] It can be seen from Table 3 that different volume ratios of electroporation solution to resuspension solution during electroporation have different effects on the final exosomes obtained, thereby affecting their repair effect on scars.

[0165] The existing scars were lightened by injection. The sample of Example 1 was used for the treatment. The exosomes of Example 1 were resuspended in PBS to obtain an exosome resuspension with a concentration of 0.1 mg / mL. The scars were injected continuously for 20 days, with 100 μL each time. Figure 2 (before restoration) and Figure 3 (After repair), it can be seen that the scar repair effect after injection treatment is more obvious.

[0166] 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 fibroblast exosomes for repairing scars, characterized in that: The following steps are involved: S1 fibroblasts were adapted to culture in complete medium for 24-30 hours; After the adaptation culture of S2 was completed, the new culture medium was replaced and cultured in a complete culture medium containing 10% fetal bovine serum until the confluence was 80%, and then digested with 0.25% trypsin, centrifuged, and the supernatant was collected. The supernatant was extracted by ultracentrifugation to obtain the initial product of fibroblast exosomes; S3 transfects the initial fibroblast exosomes to obtain fibroblast exosomes for scar repair.

2. The method for preparing fibroblast exosomes for scar repair according to claim 1, characterized in that: Adaptation culture in S1 is carried out in a 37°C, 5% CO2 incubator.

3. The method for preparing fibroblast exosomes for repairing scars according to claim 2, characterized in that: The complete culture medium preparation method is: In a sterile container, add 1 bag of DMEM culture medium powder to 1L ultrapure water, adjust the temperature to 60°C, keep warm and stir at 150r / min for 15min, then use sodium bicarbonate to adjust the pH of the system to 7.2, and cool naturally to room temperature.

4. The method for preparing fibroblast exosomes for scar repair according to claim 1, characterized in that: The S2 culture is carried out in a 37° C., 5% CO2 incubator; The centrifugation is: Centrifuge at 1000×g for 10 min and collect the supernatant.

5. The method for preparing fibroblast exosomes for scar repair according to claim 1, characterized in that: The ultracentrifugation extraction comprises the following steps: Step a, centrifuge at 2000×g for 10 min, discard the precipitate, and collect the supernatant; Step b, centrifuging at 5000×g for 10 min, discarding the precipitate, and collecting the supernatant; Step c, ultracentrifuging at 10,000×g for 30 min, discarding the precipitate, and collecting the supernatant; Step d, filtering the supernatant obtained in step c using a 0.22 μm filter membrane, removing the filter residue and retaining the filtrate; Step e, ultracentrifuging the filtrate obtained in step d at 120,000×g for 50 min, discarding the supernatant, and collecting the precipitate; The precipitate obtained in step e was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 60 min, the supernatant was discarded, and the precipitate was collected again to obtain.

6. The method for preparing fibroblast exosomes for scar repair according to claim 1, characterized in that: The transfection treatment of fibroblast exosomes in S3 is specifically as follows: First, the fibroblast exosomes were resuspended in PBS to obtain a resuspension solution; Add the resuspension solution to the electroporation solution, mix well, and then add CD24 siRNA to obtain a uniform mixture; Then, the uniform mixed solution obtained above is transferred into an electrotransfer cup by using a pipette to perform electrotransfer treatment; After the electroporation treatment is completed, ultracentrifugation is performed to obtain fibroblast exosomes for scar repair; The initial concentration of fibroblast exosomes in the resuspension is 2.5×10 10 Pieces / mL; The volume ratio of electroporation solution to resuspension solution was 1:1; The concentration of CD24 siRNA in the uniform mixed solution is 550-600 nmol / L; The ultracentrifugation is to ultracentrifuge the filtrate obtained in step d at 120,000×g for 50 min, then discard the supernatant and collect the precipitate; The precipitate obtained above was resuspended with PBS to obtain a resuspension, and the resuspension was ultracentrifuged again at 150,000×g for 50 min, the supernatant was discarded, and the precipitate was collected again to obtain.

7. The method for preparing fibroblast exosomes for scar repair according to claim 6, characterized in that: The concentration of the electrotransfer solution is: 0.02-0.06 mol / L trehalose, 0.018 mol / L D-mannitol, 0.021 mol / L sodium succinate, 0.003 mol / L potassium chloride, 0.08 mol / L magnesium chloride, 0.06 mol / L sodium hydrogen phosphate, and 0.03% by mass of Pluronic F-68, and the rest is distilled water.

8. The method for preparing fibroblast exosomes for scar repair according to claim 1, characterized in that: The electroporation parameters are: electroporation twice at a voltage of 150-160 V and 1100 μs, with an interval of 804 ms.

9. Fibroblast exosomes for scar repair are prepared according to any one of the methods of claims 1-8.

10. The fibroblast exosomes according to claim 9 are used for repairing scars.

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