Application of engineered 14-3-3 zeta exosome in targeted promotion of scar-free wound healing

Abstract

The invention discloses an application of an engineered 14-3-3 zeta exosome in targeted promotion of scar-free wound healing, and the engineered 14-3-3 zeta exosome can be used for class I surgical wounds, normal skin scratches / injuries and II-degree burn wounds, can shorten the healing time and does not leave scars; the engineered 14-3-3 zeta exosome disclosed by the invention is used for improving and intervening a fibrosis process in other organs; the engineered 14-3-3 zeta can regulate YAP so as to reduce the injury of renal tubular epithelial cells, reduce renal interstitial fibrosis and block the transformation from acute kidney injury to chronic kidney diseases; engineered 14-3-3 zeta exosomes are useful for the prevention or treatment of skin light damage and aging caused by ultraviolet radiation.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an application of engineered 14-3-3ζ exosomes in targeted promotion of scarless wound healing. Background Art

[0002] The skin is the first barrier to protect the body and is also the largest organ in the human body. Every year, millions of people are left with scars due to accidental trauma such as surgery, burns, and car accidents. Although some progress has been made in the clinical treatment of scars, such as skin transplantation, botulinum toxin injection, phototherapy, hormone and 5-fluorouracil injection, and pressure therapy, the efficacy is limited, the cost is high, and the treatment cycle is long. Scar treatment remains a major challenge facing the healthcare field. It is predicted that by 2026, the size of China's scar intervention and treatment market will reach 110 million US dollars. In the era of precision medicine, how to reduce wound scars and achieve scar-free wound healing is an urgent problem to be solved. How to quickly fade scars is one of the issues that patients are most concerned about after trauma or surgery, and it is also a major challenge facing doctors.

[0003] 14-3-3zeta (ζ) is a protein encoded by the YWHAZ gene, and its full name in Chinese is tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein zeta. This protein mediates signal transduction by binding to phosphoserine-containing proteins and is involved in many important cellular processes such as metabolism, protein transport, signal transduction, apoptosis, and cell cycle regulation. This highly conserved protein family exists in plants and mammals, and this protein is 99% identical to its orthologs in mice, rats, and sheep.

[0004] HEK293 cells derived from human embryonic kidney cells have the characteristics of high transfection efficiency and easy culture, and are widely used in biomedical research. Currently, there are no reports on HEK293 exosomes (HEK293-Exos) promoting wound healing and inhibiting scarring. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide the use of engineered 14-3-3ζ exosomes in targeted promotion of scarless wound healing.

[0008] To solve the above technical problems, the present invention provides the following technical solution: application of engineered 14-3-3ζ exosomes in targeted promotion of scarless wound healing, wherein: the target of the engineered 14-3-3ζ exosomes is the YAP protein in skin fibroblasts.

[0009] As a preferred embodiment of the application of the present invention, the wound includes one or more of a Class I surgical wound, a normal skin abrasion / injury, and a Class II burn wound.

[0010] As a preferred embodiment of the application of the present invention, the application concentration of the engineered 14-3-3ζ exosomes at the cellular level is 100 μg / mL.

[0011] As a preferred embodiment of the application of the present invention, the engineered 14-3-3ζ exosomes inhibit the homeobox protein transcription factor EN1 in the wound. + Transformation of fibroblasts, which reduces EN1 + The number of fibroblasts is reduced, scar formation is inhibited, and scar-free wound healing is promoted.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of engineered 14-3-3ζ exosomes in the treatment of inflammatory arthritis.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of engineered 14-3-3ζ exosomes in improving the process of organ fibrosis.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of engineered 14-3-3ζ exosomes in blocking the transformation of acute kidney injury to chronic kidney disease.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and to apply engineered 14-3-3ζ exosomes in skin antioxidant activities.

[0016] Beneficial effects of the present invention:

[0017] 1. The present invention can be used for Class I surgical wounds, normal skin abrasions / injuries, and Class II burn wounds, and can shorten the healing time without leaving scars.

[0018] 2. The engineered 14-3-3ζ exosomes of the present invention can be used to improve the fibrosis process in other organs. Related studies have also shown that 14-3-3ζ plays an important role in inflammatory arthritis and is an inhibitory factor of inflammatory arthritis.

[0019] 3. The engineered 14-3-3ζ exosomes of the present invention can regulate YAP to reduce the damage of renal tubular epithelial cells, reduce renal interstitial fibrosis, and block the transformation of acute renal injury to chronic kidney disease.

[0020] 4. The engineered 14-3-3ζ exosomes of the present invention can be used to prevent or treat skin photodamage and aging caused by ultraviolet radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0022] Figure 1 It is the 14-3-3ζ (Homo) overexpressing lentiviral vector used for transfection in Example 1 of the present invention; wherein A is a schematic diagram of the 14-3-3ζ (Homo) overexpressing lentiviral vector, and B is a flow chart of lentiviral transfection.

[0023] Figure 2 This is the process of harvesting exosomes in Example 2 of the present invention.

[0024] Figure 3 The markers, morphology and size of exosomes in Example 2 of the present invention were identified; wherein A is the expression of surface markers and 14-3-3ζ protein detected by WB, B is a transmission electron microscopy image of exosomes, and C is the particle diameter and distribution of HEK293-Exos and HEK293 Exos-oe determined by NTA.

[0025] Figure 4 The mouse wound model was prepared in Example 3 of the present invention.

[0026] Figure 5 The engineered exosomes were taken up by the mouse wound in Example 3 of the present invention.

[0027] Figure 6 These are macroscopic images of wound healing in mice in different treatment groups in Example 3 of the present invention; wherein A is the macroscopic image of splint wounds on the backs of mice in each group at different time points; and B is the analysis of the healing rate of wounds on the backs of mice.

[0028] Figure 7The pathological tissue structure of the wound skin of mice in different treatment groups in Example 3 of the present invention; wherein, A is the HE staining image of the wound at POD 14 of each group, 40×, scale bar = 500μm, 200×, scale bar = 50μm, B is the statistics of scar width, epidermal thickness and the number of hair follicles at the wound at POD 14, n=3 for each group, compared with the PBS group, ***P<0.001, ****P<0.0001.

[0029] Figure 8 The collagen fibers in the wounds of mice in Example 4 of the present invention are shown; wherein, A is a Masson staining image of the wound, 400×, scale bar = 20 μm, B is the collagen volume fraction in Figure A, n = 3 per group, compared with the PBS group, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0030] Fig. 9 The expression of type I collagen and type III collagen in the wound of mice in Example 4 of the present invention; wherein, A is the immunohistochemical staining of type I collagen and type III collagen in each group at POD 14, 400×, scale bar = 20 μm; B is the relative quantitative analysis of type I collagen and type III collagen staining, C is the ratio analysis of type I collagen to type III collagen, n = 3 in each group, compared with the PBS group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Fig.10 The expression of α-smooth muscle actin in the wound of mice in Example 4 of the present invention.

[0032] Fig.11 The expression of the homeobox protein Engrailed-1 (EN-1) in mouse wound fibroblasts in Example 4 of the present invention; wherein, A is the expression of EN1 (red) and PDGFRα (green) in the wound of POD 14 detected by IF, 400×, scale bar = 20 μm, B is the fluorescence quantitative analysis of the co-expression of EN1 (red) and PDGFRα (green) in the wound, n = 3 in each group, compared with the PBS group, **P < 0.01.

[0033] Fig.12 The engineered 14-3-3ζ exosomes in Example 5 of the present invention target and bind to YAP to improve fibrosis; wherein, A is the String database analysis that the 14-3-3 protein family has a protein interaction relationship with YAP, and B is a Co-IP experiment. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] The raw materials and reagents used in the present invention are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041]

[0042] Unless otherwise stated, all experimental methods disclosed in the present invention adopt conventional techniques in biology and related fields conventional in the art.

[0043] Example 1 Engineering transformation to obtain HEK293 cells overexpressing 14-3-3ζ

[0044] 1. Lentiviral transfection:

[0045] HIV virus is used to integrate the gene sequence of the target protein into the host genome, so that the target cell overexpresses / knocks down the target protein. The target protein gene sequence can be passed to daughter cells as the host cell divides. Puromycin can be used for screening to obtain purified host cells with successful stable transfection.

[0046] (1) This study used 14-3-3ζ (Homo) overexpressing lentivirus EX-C0032-Lv201 purchased from GeneCopoeia ( Figure 1 A).

[0047] (2) Cell preparation and plating: 24 h before lentiviral transfection, HEK293 cells were digested with 0.25% trypsin and cells less than P30 were used.

[0048] (3) Terminate digestion and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the cells in the corresponding complete medium and count the cells. Inoculate 5×104 cells per well in a 24-well plate and add 0.5 mL of medium to each well. Culture in a 5% CO2, 37°C incubator.

[0049] (4) Lentivirus dilution and preparation: Transfer the lentivirus stock solution from -80°C to ice in advance and slowly thaw at 4°C. Calculate the amount of lentivirus to be used based on the multiplicity of infection (MOI), where MOI = virus titer (TU / mL) × virus volume (mL) / number of cells. The titer of the lentivirus stock solution is 2.44×108TU / mL.

[0050] (5) The human kidney epithelial cell line HKE293 was transfected at an MOI of 1.

[0051] (6) Dilute the virus solution with culture medium and make up to 0.5 mL. Add the transfection reagent Polybrene to a final concentration of 5 μg / mL (2.5 μg per well). Gently invert the 1.5 mL centrifuge tube upside down.

[0052] (7) Lentivirus transfection: discard the old culture medium in the 24-well plate and add the culture medium containing the diluted virus. Gently shake the culture plate to mix it and place it in a 37°C constant temperature incubator (5% CO 2 ) and cultured overnight.

[0053] (8) The next day, replace the old culture medium with 0.5 mL of fresh complete culture medium. Place in a 5% CO2, 37°C incubator and culture overnight. 48 hours after transfection, observe the expression of eGFP under an inverted fluorescence microscope to confirm that the infection was successful ( Figure 1 B).

[0054] (9) Screening positive cells: Observe the cell status every day. When the cell death rate of the MOCK group reaches 90% (basically about 12h to 24h), discard the culture medium containing puromycin, add 0.5mL complete culture medium, mix gently, and place in 5% CO 2 , and cultured in a 37°C incubator. At this time, the surviving cells can be identified as the cells that have been successfully transfected.

[0055] (10) Screening of stable strains: To obtain stably transfected cells, the culture medium was replaced with fresh culture medium containing puromycin every 3 days. Stable strains were harvested after 7 to 14 days and were called HEK293-oe (oe: overexpression).

[0056] (11) Cell passaging and supernatant collection: After obtaining a stable strain, the cells in the 6-well plate are digested and transferred into a T175 or bioreactor for expansion culture. The cell culture fluid is collected after 48 hours of culture and the cells are further expanded and passaged.

[0057] Example 2 Extraction and identification of engineered 14-3-3ζ exosomes

[0058] Exosomes were harvested using ultrahigh-speed differential centrifugation and detected by western blotting (WB), transmission electron microscopy (TEM), and nanoparticle analysis (NTA) according to the International Society of Extracellular Vesicles (ISEV) definition of exosomes.

[0059] (1) Ultra-high-speed differential centrifugation to extract exosomes: Take out the collected cell culture medium in advance and thaw it in a 4°C refrigerator overnight. Precool the low-temperature centrifuge to 4°C, centrifuge at 300rcf for 10 minutes to remove residual cells. Transfer the supernatant to a new 50mL centrifuge tube and discard the remaining 5mL of residual liquid. Centrifuge at 4°C, 2000rcf for 10 minutes to remove dead cells. Transfer the supernatant to a new 50mL centrifuge tube and discard the remaining 5mL of residual liquid. Balance the supernatant (accurate to 0.1) and place it in a centrifuge, centrifuge at 4°C, 10000rcf for 30 minutes to remove cell debris. In the clean bench, transfer the supernatant to a sterile disposable filter device (0.22μm) for filtration, and discard the remaining 5mL of residual liquid in the last centrifuge tube. Prepare the ultra-high-speed centrifuge tube 24 hours in advance, soak the ultra-high-speed centrifuge tube in 0.1M NaOH solution for 4 hours, and filter it with ddH 2 O for 5 times, and then fill with phosphate buffered saline (DPBS) for later use. Pour out the DPBS before use, add the filtered supernatant to the ultracentrifuge tubes, and balance with a precision balance (accurate to 0.001). Centrifuge at 4℃, 100000rcf for 2h15min. Discard the residual liquid, resuspend the precipitate with 200μL DPBS, and after determining the protein concentration, dispense it into PCR tubes at a dose of 100μg / tube, and store it in a -80℃ ultra-low temperature refrigerator for a long time. Do not freeze and thaw repeatedly.

[0060] The extraction process of exosomes can be found in Figure 2 , exosomes harvested from HEK293 cells overexpressing 14-3-3ζ were named HEK293-Exos-oe, and exosomes harvested from untreated HEK293 cells were named HEK293-Exos (as a control in subsequent experiments).

[0061] (2) Identification of exosomes: Western blot (WB) was used to detect the positive markers CD9, CD81, CD63 and the negative marker Calnexin of exosomes. It was found that both HEK293-Exos-oe and HEK293-Exos met the marker detection standards for exosomes. The content of 14-3-3ζ in HEK293-Exos-oe was further detected, and the expression of 14-3-3ζ in HEK293-Exos-oe was increased compared with that in HEK293-Exos ( Figure 3 A). Transmission electron microscopy (TEM) showed that HEK293-Exos and HEK293-Exos-oe presented a tray-shaped structure, which was in line with the requirements of the International Extracellular Vesicle Association ( Figure 3 B). Nanoparticle analysis (NTA) showed that the modal particle size of HKE293-Exos was 118.3 nm, accounting for 95.9% of the total particle size. The particle concentration of this diameter was 2.5×10 6 / mL. The modal particle size of HEK293-Exos-oe is 121.5nm, accounting for 94.9% of the total, and the particle concentration of this diameter is 2.5×10 6 / mL( Figure 3 C).

[0062] Example 3: Engineered 14-3-3ζ exosomes promote scarless wound healing

[0063] After determining the protein concentration, a working concentration solution of 0.03 mg / mL was prepared, and the working solution volume was 30 μL. Two full-thickness skin incisions with a diameter of 10 mm were made on the back of the mouse, and a 0.5 mm thick circular silicone gasket (inner diameter 12 mm, outer diameter 20 mm) was applied with a small amount of Krazy Glue and attached to the back skin of the mouse. Tweezers were used to ensure that the splint fits tightly to the skin. 6-0 nylon non-absorbable sutures were used to suture the splint to the back skin intermittently, with at least 4 suture fixation points ( Figure 4 HEK293-Exos-oe was fluorescently labeled with PKH26 and applied to the wound. The wound skin of mice was collected at 6h, 12h and 24h, and fibroblasts were labeled with platelet-derived growth factor receptor α (PDGFRα). Immunofluorescence staining (IF) results showed that red fluorescently labeled HEK293-Exos-oe obviously gathered in the wound and was taken up by fibroblasts, and the red fluorescence intensity increased 12h to 24h after application ( Figure 5 On the first day after surgery (Post Of Damage 1, POD 1), the wounds were smeared every day according to different groups (PBS, aFGF, HEK293-Exos, HEK293-Exos-oe).

[0064] A digital camera was used to take pictures of the wounds and calculate the wound healing rate on POD 0, POD 4, POD 7, POD 10, and POD 14 on the day of surgery. Figure 7 A and B), the results showed that engineered 14-3-3ζ exosomes (HEK293-Exos-oe) could significantly promote wound healing, and the effect was significantly better than that of HEK293-Exos and PBS treatment groups, and similar to that of the positive drug control group (aFGF). Mice were killed by cervical dislocation on POD 14, and the back wound skin was taken for pathological section and immunohistochemical analysis.

[0065] HE staining results of wound tissue sections on POD 14 showed ( Figure 7 A), in the PBS control group, the new epidermis was thickened, the stratum corneum was dense, the granular layer and the spinous layer were thickened, and there was spongiform edema; in the dermis, horizontally arranged fiber bundles and vertical microvessels were seen, but no hair follicle structure was seen, and a small number of inflammatory cells were present; although the aFGF-treated group also showed epidermal thickening and stratum corneum density, basket-net fiber structure and horizontal blood vessels appeared in the dermis, indicating a better tissue reconstruction process, but no hair follicle structure was seen, and a small number of inflammatory cells were observed; the scar width of the HEK293-Exos intervention group did not change significantly compared with the PBS control group at POD14, and the epidermis did not thicken significantly; the blood vessels were distributed horizontally; no hair follicle structure was seen; the HEK293-Exos-oe treatment group showed similar effects at POD 14, compared with the PBS control group, the scar width was significantly reduced, the epidermis did not thicken significantly, the blood vessels were distributed horizontally, and hair follicle regeneration was observed at the wound.

[0066] Scar width, epidermal thickness, and wound hair follicle counts Figure 7 As shown in B, the HEK293-Exos intervention group promoted scar-free healing of skin wounds.

[0067] Example 4: Engineering 14-3-3ζ exosomes to improve wound fibrosis

[0068] 1.Masson staining

[0069] Masson staining is an authoritative and classic technical method for collagen fiber staining.

[0070] Specifically, the sections were stained with Wdigert's iron hematoxylin staining solution in the Masson staining kit, rinsed with distilled water, stained with Ponceau acid fuchsin solution, rinsed with distilled water, and finally counterstained with aniline blue staining solution. After dehydration with ethanol and transparentization with xylene, the sections were observed under a microscope.

[0071] The results showed that compared with the PBS control group at POD 14, the HEK293-Exos-oe treatment group and the aFGF group significantly promoted the increase in collagen deposition. The collagen arrangement in the HEK293-Exos-oe treatment group showed a basket-like structure ( Figure 8 A and B), this structure may help provide better support and elasticity, thereby promoting more effective wound healing.

[0072] 2. IHC staining of type I collagen and type III collagen

[0073] Immunohistochemistry (IHC) is an antibody-based technique used to detect protein expression in tissues whose structure and organization have been preserved.

[0074] Specifically, the tissue sections were placed in a wet box and a circle was drawn around the tissue with a histochemical pen. After sealing, 50 μL of primary antibody dilution was added to each section and incubated overnight at 4°C. After washing the sections with PBS, 50 μL of secondary antibody was added and incubated at room temperature for 60 min. After washing, the sections were counterstained with hematoxylin solution, dehydrated and transparentized, and observed under a microscope.

[0075] IHC staining of type I collagen and type III collagen Fig. 9 As shown in A, compared with the PBS control group, the HEK293-Exos-oe intervention group could significantly promote the expression of type I collagen and type III collagen at POD 14, and reduce the ratio of type I collagen to type III collagen ( Fig. 9 B, C), in wound repair, the skin with a high proportion of type III collagen is less likely to form scars; the results show that when HEK293-Exos-oe promotes collagen deposition, it improves the quality of skin healing by regulating the synthesis of different collagens.

[0076] 3. α-Smooth muscle actin (α-SMA) IHC staining

[0077] The staining results are as follows Fig.10 As shown in A, compared with the PBS control group, the HEK293-Exos-oe treatment group was able to significantly downregulate α-SMA in scars; the quantitative results are shown in Fig.10 B shows that the expression of α-SMA in the HEK293-Exo and aFGF-treated groups was significantly lower than that in the PBS-treated group, and there was no significant difference between the two groups.

[0078] 4. Immunofluorescence staining of homeobox transcription factor (EN1) and fibroblast marker (PDGFRα)

[0079] Immunofluorescence (IF) is a method that uses fluorescently labeled antibodies to detect and locate antigens.

[0080] Specifically, the tissue sections were placed in a wet box and a circle was drawn around the tissue using a histochemical pen. After sealing, 50 μL of primary antibody diluent was added to each section at 4°C overnight, and the secondary antibody was incubated for 1 hour in the dark after rinsing with PBS. After rinsing, DAPI was added to stain the cell nucleus, and finally anti-fluorescence quenching sealing medium was dripped in, and observation was performed after sealing.

[0081] EN1-activated fibroblasts have a high ability to synthesize extracellular matrix and collagen and are considered to be the main cell population for scar formation. The results of IF staining of fibroblast markers PDGFRα and EN1 are as follows: Fig.11 A, Compared with the PBS control group, the number of EN1-positive fibroblasts in the HEK293-Exos-oe group decreased significantly at POD 14 ( Fig.11 B) The results showed that HEK293-Exos-oe inhibited EN1 in wound + Transformation of fibroblasts, which reduces EN1 + The number of fibroblasts can be reduced, scar formation can be inhibited, and scar-free wound healing can be promoted.

[0082] In summary, by analyzing collagen deposition, α-SMA and EN1 indicators, it was further proved that HEK293-Exos-oe exosomes can effectively promote scar-free wound healing compared with the control group. Among them, HEK293-Exos-oe exosomes can improve the arrangement of collagen fibers in the wound to a woven basket shape, and down-regulate the expression of type I collagen, and improve the collagen ratio to normal skin; at the same time, the scar indicator α-SMA was detected, and HEK293-Exos-oe exosomes reduced the expression of α-SMA, indicating that there was no excessive granulation tissue and scar formation in the wound; finally, the wound EN1-positive fibroblasts were detected, and HEK293-Exos-oe exosomes significantly inhibited the expression of EN1, and the intervention effect of HEK293-Exos-oe was better than that of the aFGF treatment group.

[0083] Example 5: Engineered 14-3-3ζ exosomes target YAP to exert anti-fibrotic effects

[0084] 1. Bioinformatics prediction of 14-3-3ζ targets

[0085] The String database (https: / / string-db.org / ) is a database for retrieving interactions between known proteins and predicted proteins. YAP is a star molecule in the field of anti-fibrosis and regenerative medicine in recent years, and is also a key effector in the Hippo signaling pathway related to wound healing, organogenesis, and tissue regeneration.

[0086] The potential interactions between 14-3-3 protein family members and YAP were retrieved through the String database, and the protein interaction network showed that ( Fig.12 A) Six members of the 14-3-3 protein family have potential protein interactions with YAP.

[0087] 2. Immunoprecipitation to verify the interaction between 14-3-3ζ and its target YAP

[0088] Co-Immunoprecipitation (CO-IP) is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens to determine the physiological interaction between two proteins. In the experiment, the monoclonal antibody MouseAnti-YAP1 (1:250) was added to the cell protein sample, and an equal volume of Mouse IgG was added as a control. The samples were incubated overnight at 4°C, and then ProteinA and Protein G agarose beads in the immunoprecipitation (IP / CoIP) kit were added and continued to be incubated overnight. After centrifugation at 12000rcf for 1min, the precipitate was retained and the samples were analyzed by western blotting.

[0089] The results of co-immunoprecipitation showed that ( Fig.12 B) 14-3-3ζ interacts with YAP and the two bind to each other, which further proves that engineered 14-3-3ζ exosomes can target and bind to YAP to promote scar-free wound healing and improve fibrosis.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of engineered 14-3-3ζ exosomes in targeted promotion of scarless wound healing, characterized in that: The target of the engineered 14-3-3ζ exosomes is the YAP protein in skin fibroblasts.

2. The use according to claim 1, characterized in that: The wound includes one or more of a Class I surgical wound, a normal skin abrasion / injury, and a Class II burn wound.

3. The use according to claim 1, characterized in that: The engineered 14-3-3ζ exosomes were applied at the cellular level at a concentration of 100 μg / mL.

4. The use according to claim 1, characterized in that: The engineered 14-3-3ζ exosomes inhibit the homeobox transcription factor EN1 in wounds + Transformation of fibroblasts, which reduces EN1 + The number of fibroblasts is reduced, scar formation is inhibited, and scar-free wound healing is promoted.

5. Application of engineered 14-3-3ζ exosomes in the treatment of inflammatory arthritis.

6. Application of engineered 14-3-3ζ exosomes in improving the process of organ fibrosis.

7. Application of engineered 14-3-3ζ exosomes in blocking the transformation of acute kidney injury to chronic kidney disease.

8. Application of engineered 14-3-3ζ exosomes in skin antioxidant activity.

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