Human umbilical cord mesenchymal stem cell exosome for treating scars and preparation method thereof

Abstract

The invention discloses a human umbilical cord mesenchymal stem cell exosome for treating scars and a preparation method of the human umbilical cord mesenchymal stem cell exosome. The exosome is extracted from human umbilical cord mesenchymal stem cells pretreated by extracellular vesicles in scar tissues. The human umbilical cord mesenchymal stem cell exosome can be used for preparing medicines for improving and treating scars, has a potential action effect on early scar treatment, and has important significance on improving the scar treatment effect and improving the life quality of patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a human umbilical cord mesenchymal stem cell exosome for treating scars and a preparation method thereof. BACKGROUND

[0002] Scar formation is a classic problem in wound healing, which is often seen in skin tissue deep wounds, chronic ulcers and vascular insufficiency after wound healing. According to statistics, nearly 60% of patients have hyperpastic scars (HS) after receiving chest surgery. It is reported that about 100 million scars are produced every year, of which 11 million will become keloids. In addition to morphological changes, it can also cause itching and pain. At the same time, severe scars not only lead to disfigurement and dysfunction of patients, but also bring discomfort such as itching and pain to patients, seriously affecting the physical and mental health of patients. Although research on the treatment of scars has been carried out for several decades, the current treatment scheme still has limitations, and the number of studies on scars is still increasing year by year, and the research to solve this problem is still challenging. Scar formation after wound healing is a physiological response to skin damage. Wound healing is a complex, dynamic and continuous process, which includes hemostasis, inflammation, proliferation and shaping. If the wound healing process cannot appear continuously and orderly, it may lead to abnormal healing of the wound, such as long-term non-healing or excessive healing to form scars. Scar is a fibroproliferative disease of skin soft tissue, and its pathological hallmark is excessive proliferation of fibroblasts and excessive deposition of extracellular matrix (ECM) in the dermis.

[0003] At present, there are many kinds of treatment programs for scar, but satisfactory results have not been achieved. In recent years, a large number of studies have shown that mesenchymal stem cells (MSCs) take the pathophysiology of wound surface as the treatment target, such as improving the local pathological microenvironment, regulating inflammation, increasing vascularization, promoting cell proliferation and wound epithelialization, effectively promoting wound healing and reducing scar formation. In recent years, a large number of studies have found that MSCs-exosomes (MSCs-Exos) have almost the same biological effects as MSCs. Compared with MSCs, MSCs-Exos have high efficiency, can be stored for a long time and are easy to transport, and the dosage and time are easy to control, without the risk of tumor and thrombosis. It is considered to be a substitute for MSCs treatment in the future and has become a research hotspot in the field of tissue damage repair and regenerative medicine. Exosomes (Exos) is a subtype of extracellular vesicles (EVs), with a size of 30-150 nm.

[0004] Studies have found that the biological effects of MSCs are heterogeneous. In wound repair, MSCs promote the up-regulation of vascular endothelial growth factor (VEGF) expression, promote blood vessel regeneration and accelerate wound healing. Normal dermal fibroblasts co-cultured with MSCs promote the proliferation and migration of fibroblasts and promote healing, while the proliferation and migration of hypertrophic scar fibroblasts are inhibited. The above shows that the paracrine biological effects of MSCs are affected by the local microenvironment. Recent studies have shown that the paracrine factors of MSCs also have similar effects, and MSCs-Exos can reduce scar formation by controlling the deposition of ECM, reduce scar area, prevent fibroblastization into myofibroblasts, and increase the ratio of TGF-β3 to TGF-β1. In the early stage of wound healing, human adipose-derived mesenchymal stem cell exosomes play a role in promoting collagen expression and promoting wound healing, while in the late stage of healing, these exosomes play a role in inhibiting collagen expression and reducing scar formation. However, most of the current studies on MSCs-Exos are to culture MSCs under traditional culture conditions to isolate exosomes, and MSCs and exosomes of different sources show significantly different biological effects in similar microenvironments such as inflammation and hypoxia.

[0005] Based on the previous research and related research, the team proposes that the separation and extraction of MSCs-Exos needs to combine with the pathological microenvironment of the disease to be treated, and conceives that when culturing MSCs in vitro to separate MSCs-Exos, the pathological microenvironment of the disease to be treated is simulated as much as possible, and the in vitro cultured MSCs are induced or pretreated, so that the MSCs-Exos with targeted treatment effect for the disease can be obtained.

[0006] Therefore, the inventors explore the potential effect of scar tissue-derived extracellular vesicles (STEVs) pretreated human umbilical cord mesenchymal stem cell exosomes on early scar treatment, and it is of great significance to improve the scar treatment effect and improve the quality of life of patients. The innovation of the present application lies in the first use of scar tissue-derived extracellular vesicles to pretreat human umbilical cord mesenchymal stem cell exosomes, to explore its potential effect on early scar treatment, and to understand the effect of scar tissue-derived extracellular vesicles pretreatment on mesenchymal stem cell gene expression by combining transcriptomic analysis, to provide a new theoretical basis for precise treatment of scars and a new treatment option for patients. SUMMARY

[0007] The purpose of the present application is to provide a human umbilical cord mesenchymal stem cell exosome for treating scars, which is secreted by human umbilical cord mesenchymal stem cells pretreated by scar tissue-derived extracellular vesicles (STEVs), and has potential effect on early scar treatment, and is of great significance to improve the scar treatment effect and improve the quality of life of patients.

[0008] To achieve the purpose of the present application, the following embodiments are provided.

[0009] In some embodiments, the human umbilical cord mesenchymal stem cell exosome for treating scars of the present application is extracted from human umbilical cord mesenchymal stem cells pretreated by extracellular vesicles derived from scar tissue.

[0010] In some preferred embodiments, the exosome of the present application described above, the scar is mainly skin scar.

[0011] In some embodiments, the preparation method of the human umbilical cord mesenchymal stem cell exosome of the present application described above comprises the following steps:

[0012] 1) Isolate and extract extracellular vesicles (STEVs) from ex vivo scar tissue, and prepare STEVs-containing serum-free complete culture medium for human umbilical cord mesenchymal stem cells;

[0013] 2) Take the umbilical cord stripped of the umbilical vein, umbilical artery and outer membrane of the umbilical cord, cut into small pieces, culture, digest, and collect primary cells HUC-MSCs;

[0014] 3) The primary cells HUC-MSCs are placed in a culture box for culture, and the cell density reaches 80-90% for cell passage, until P4 generation HUC-MSCs;

[0015] 4) The cell density of the P4 generation HUC-MSCs in the above step is diluted to 1×10 5 / mL, the serum-free complete culture medium for human umbilical cord mesenchymal stem cells containing STEVs in step 1) is added, and it is placed in a culture box for culture for 24 hours, the original supernatant is discarded, and the supernatant is collected after the new non-preprocessed serum-free complete culture medium for human umbilical cord mesenchymal stem cells is added for culture for 48 hours. HUC-MSCs-Exos is separated and extracted by differential centrifugation combined with ultrahigh speed centrifugation.

[0016] In some specific embodiments, one of the above-mentioned preparation methods of the human umbilical cord mesenchymal stem cell exosomes of the present application comprises the following steps:

[0017] 1) Take the ex vivo scar tissue, and extract and separate the extracellular vesicles STEVs by tissue cutting treatment, enzymatic dissociation, gradient size exclusion, differential centrifugation combined with ultrahigh speed centrifugation, and prepare the serum-free complete culture medium for human umbilical cord mesenchymal stem cells containing STEVs;

[0018] 2) Take the umbilical cord tissue of a newborn, and after stripping the umbilical vein, umbilical artery and outer membrane of the umbilical cord, wash the remaining tissue (called Wharton's jelly) with sterile PBS, cut it into small pieces, put it into the culture medium for culture, and digest and collect primary cells HUC-MSCs;

[0019] 3) The primary cells HUC-MSCs are cultured in a cell culture box at 37℃ and 5% CO2 for 48-72 hours, and under the microscope, the HUC-MSCs are long spindle-shaped and fish school vortex-shaped, and the cell density reaches 80-90% for cell passage;

[0020] 4) The HUC-MSCs are washed with sterile PBS containing 1% penicillin / streptomycin, and stem cell mild digestion enzyme is added, and the serum-free culture medium for human umbilical cord mesenchymal stem cells is added for culture. When the cell fusion density reaches 80%-90%, the next passage is carried out;

[0021] 5) Take P4 generation HUC-MSCs with good cell state and density of 80%-90%, wash the cells with PBS, digest and centrifuge, and then count the cells. The cell density of the cell suspension is diluted to 1×10 5Resuspend and mix thoroughly, add the human umbilical cord mesenchymal stem cell special serum-free complete culture medium containing STEVs in step 1), resuspend and mix thoroughly;

[0022] 6) After resuspension and mixing, place in a 37℃, 5% CO2 cell incubator for 24h, discard the original supernatant, replace with new non-preprocessed human umbilical cord mesenchymal stem cell special serum-free complete culture medium and continue to culture for 48h;

[0023] 7) After 48h of culture, collect the corresponding supernatant, and separate and extract HUC-MSCs-Exos by differential centrifugation combined with ultrahigh-speed centrifugation.

[0024] In some embodiments, the preparation method of extracellular vesicles STEVs in scar tissue comprises:

[0025] a) Dissociate the ex vivo scar tissue with dissociation solution containing collagenase type IV and DNase I enzyme, filter, and collect the filtrate;

[0026] b) Differential centrifugation of the filtrate to remove tissues and impurities, collect the filtrate, separate by size exclusion through different size filter screens, and centrifuge the filtrate at 110,000xg for 70min, discard the supernatant, resuspend the precipitated particles with sterile PBS;

[0027] c) Transfer the resuspended precipitated particles to an ultrafiltration tube for purification, centrifuge at 5000xg for 15min, collect the upper chamber liquid, filter, centrifuge the filtrate at 110,000xg, discard the supernatant, resuspend the precipitated particles with PBS, and obtain the scar extracellular vesicles STEVs.

[0028] In a specific embodiment, the above preparation method of extracellular vesicles STEVs in scar tissue further comprises the following steps:

[0029] 1) Wash the ex vivo scar tissue with sterile PBS (containing 1% double antibody), absorb the water, and cut into small tissue pieces;

[0030] 2) Prepare the tissue dissociation solution: DMEM high-sugar medium containing collagenase type IV (2mg / mL) and DNase I enzyme (40U / mL) ;

[0031] 3) Add the tissue dissociation solution to the small tissue pieces, after dissociation is complete, centrifuge and filter, and collect the filtrate which is sequentially passed through 70μm and 45m funnel-shaped nylon filter screens;

[0032] 4) Differential centrifugation of the last collected filtrate at 4℃: 800xg for 10min, take the supernatant, 3,000xg for 20min, take the supernatant, 3,000xg for 20min again, discard the precipitate, and collect the supernatant;

[0033] 5) The supernatant collected in the previous step is passed through 20m, 10m funnel-shaped nylon filter screens in sequence, and the supernatant is allowed to flow into a centrifuge tube by gravity, and the supernatant is collected;

[0034] 6) The supernatant of the previous step is centrifuged at 16500xg for 30min at 4℃, the precipitate is removed, and the supernatant is collected;

[0035] 7) The supernatant of the previous step is further passed through a 2.5m funnel-shaped nylon filter screen, and the liquid is allowed to flow into a centrifuge tube, and the supernatant is collected.

[0036] 8) The supernatant of the previous step is transferred to a 30mL polypropylene ultracentrifuge tube, which is filled with PBS, sealed, and strictly balanced to 0.01mg, and an ultracentrifuge is used to centrifuge at 110,000xg for 70min at 4℃, the supernatant is discarded, and the precipitated particles are resuspended with 1mL of sterile PBS;

[0037] 9) The resuspended precipitated particles are transferred to an ultrafiltration tube, centrifuged at 5000xg for 15min at 4℃, the upper chamber liquid of the ultrafiltration tube is collected, washed with PBS, and the resuspended liquid is passed through 0.8m, 0.45m, and 0.22m needle filters in sequence, and the filtrate is collected;

[0038] 10) The filtrate of the previous step is again transferred to a polypropylene ultracentrifuge tube, which is filled with PBS, sealed, and a benchtop ultracentrifuge is used to centrifuge at 110,000xg for 70min at 4℃, the supernatant is discarded, and the precipitated particles are resuspended with 500L of sterile PBS, and the scar cell extracellular vesicles STEVs are obtained.

[0039] In some embodiments, the present application provides the use of human umbilical cord mesenchymal stem cell exosomes (STEVs-Exos) in the preparation of a medicament for improving and treating scars, which are extracted from human umbilical cord mesenchymal stem cells pretreated with extracellular vesicles derived from scar tissue.

[0040] Technical effects:

[0041] The STEVs-Exos of the present application has a significant improvement and treatment effect on early proliferative scars of rabbit ears, which is manifested as improving the appearance of scars, reducing the scar index, promoting collagen remodeling, and promoting scar maturation.

[0042] Studies have shown that STEVs carry pathological information related to scars, can represent the scar microenvironment, and activate the extracellular matrix remodeling and cell cycle-related signaling pathways in HUC-MSCs, thereby obtaining specific exosomes for scar treatment. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1Transmission electron microscope images of extracellular vesicles in the scar tissue of Example 1, in which A. 10000x, scale bar = 2.0 μm, B is a local enlarged view of A, 20000x, scale bar = 500 nm.

[0044] Figure 2 Transmission electron microscope images of STEVs of Example 1, in which A. STEVs under transmission electron microscope are a half-sphere or a tea tray-like structure with one side concave, scale bar = 100 nm; B. Nanoparticle size analysis of STEVs, the vesicle particle size distribution is 30-200 nm; C. WB detection of STEVs specific markers, CD9, CD63, TSG101 are all positively expressed, Calnexin expression is negative; D. Nanoflow cytometry detection of STEVs membrane surface proteins, CD9, CD81 are positively expressed.

[0045] Figure 3 Morphological observation of HUC-MSCs extracted in Example 2, in which A, P0 generation HUC-MSCs, scale bar = 200 μm; B, P4 generation HUC-MSCs, scale bar = 200 μm.

[0046] Figure 4 Flow cytometry identification result chart of HUC-MSCs extracted in Example 2.

[0047] Figure 5 Uptake staining chart of STEVs by HUC-MSCs extracted in Example 2.

[0048] Figure 6 Bar chart of the influence of different STEVs concentration intervention on the proliferation ability of HUC-MSCs in Example 2.

[0049] Figure 7 Transmission electron microscope morphological observation and particle size distribution chart of Exos in Example 2.

[0050] Figure 8 Uptake staining chart of STEV-Exos by fibroblasts (Fb) in Example 2.

[0051] Figure 9 Rabbit ear hypertrophic scar model and pathological tissue identification chart of Example 2

[0052] Figure 10 Bar chart of the change of HUC-MSC-Exos acting on rabbit ear HS and MSS score of each group in Example 2.

[0053] Figure 11 HE staining of HUC-MSC-Exos acting on rabbit ear HS of each group in Example 2 and statistical bar chart of the change of scar epidermis thickness and scar index.

[0054] Figure 12 Masson staining and statistical column chart of collagen volume ratio of rabbit ear HS treated by each group of HUC-MSC-Exos of Example 2.

[0055] Figure 13 Picrosirius staining and collagen statistical chart of rabbit ear HS treated by each group of HUC-MSC-Exos of Example 2.

[0056] Figure 14 TNF-α and TGF-β immunohistochemical staining and statistical column chart of staining intensity of rabbit ear HS treated by each group of HUC-MSC-Exos of Example 2.

[0057] Figure 15 Volcano plot of differential genes of STEV pretreated HUC-MSCs of Example 3.

[0058] Figure 16 Column chart of GO enrichment results of differential genes of STEV and Tra pretreated HUC-MSCs of Example 3.

[0059] Figure 17 Column chart of GO enrichment results of differential genes of STEV and STH pretreated HUC-MSCs of Example 3.

[0060] Figure 18 Column chart of GO enrichment results of differential genes of Tra and STH pretreated HUC-MSCs of Example 3.

[0061] Figure 19 Column chart of KEGG enrichment results of differential genes of STEV and Tra pretreated HUC-MSCs of Example 3.

[0062] Figure 20 Column chart of KEGG enrichment results of differential genes of STEV and STH pretreated HUC-MSCs of Example 3.

[0063] Figure 21 Column chart of KEGG enrichment results of differential genes of Tra and STH pretreated HUC-MSCs of Example 3. DETAILED DESCRIPTION

[0064] The following examples are provided to further illustrate the present application. However, the following examples are provided only for purposes of further understanding the present application and are not intended to limit the present application. It will be understood by those skilled in the art that equivalent substitutions or corresponding modifications of the present content, still within the scope of the present application.

[0065] Example 1 Extraction and identification of extracellular vesicles of scar tissue

[0066] 1Materials and Methods

[0067] 1.1Main experimental materials

[0068] Experimental samples The collection standard of the proliferative scar tissue samples used in this experiment: 1) female patients aged 20-60 years old; 2) visible pathological scars caused by surgical incisions, with a scar formation time greater than or equal to 1 year, and planned for scar excision surgery treatment. Exclusion criteria: 1) diagnosed as having keloid; 2) combined with scar ulcer and infection; 3) precancerous lesions or cancer. The proliferative scar tissue samples were derived from discarded scar tissue in the operating room of the Affiliated Hospital of Zunyi Medical University during proliferative scar excision surgery. After obtaining the verbal informed consent of the responsible physician and the patient, the samples were collected. All tissue samples were quickly placed in liquid nitrogen after collection and transported to a -80°C freezer for storage for subsequent research. The collection and use of the scar tissue specimens in this experiment comply with the ethical principles (Ethical Review Approval Number: KLLY-2023-151).

[0069] The main experimental instruments and consumables are shown in Table 1.

[0070] Table 1. Experimental instruments and consumables

[0071]

[0072]

[0073] The main experimental reagents are shown in Table 2.

[0074] Table 2. Experimental reagents

[0075]

[0076]

[0077] Preparation method of main experimental reagents

[0078] Reagents for Western Blot experiment:

[0079] ① TBS solution: Tris 4.844 g + NaCl 17.55 g, add deionized water to 2 L.

[0080] ② TBST solution: 400 mL TBS + 1 mL 20% Tween.

[0081] ③ Electrophoresis solution: Tris 15.1 g + Glycine 94 g + SDS 2 g, add deionized water to 2 L.

[0082] (4) Transference liquid: Tris 3g + Glycine 14.4g + SDS 0.2g + 200mL methanol, add deionized water to 1L.

[0083] (5) Skim milk blocking solution: TBS + 5% skim milk.

[0084] 1.2 Experimental method

[0085] 1.2.1 Bioelectric microscope observation of extracellular vesicles in scar tissue

[0086] Collect the discarded scar tissue just out of the body, take 3 pieces of tissue with a volume of 5mm 3 , and immediately put them into a 1.5mL small centrifuge tube containing 2.5% glutaraldehyde (4℃ pre-cooled) for fixation. Put the centrifuge tube into an ice box, then transfer it to a 4℃ refrigerator for fixation for 12h-24h. After fixation is completed, pour out the fixing solution, and wash the tissue with PBS for 3 times, each time for 15min. Then, use 1% osmium acid solution to fix the sample for 2h. After fixation is completed, gently remove the osmium acid waste liquid, and then wash the tissue with 0.1M phosphate buffer PBS (PH7.4) for 3 times, each time for 15min. Then, put the washed tissue sample into ethanol with a concentration gradient of 30%, 50%, 70%, 90% in order, and each time interval is 15min. After gradient dehydration, first put the tissue into 100% ethanol for 2 times, and then put it into 100% acetone for 2 times, each time for 20min. After dehydration is completed, put the tissue into pure embedding agent at 37℃ overnight, so that the embedding agent fully penetrates into the tissue gap. Then, pour the pure embedding agent into the embedding plate, insert the sample into the embedding plate, and then put it into a 70℃ oven for polymerization for 24h. Take out the resin block after polymerization, and use an ultramicrotome to cut the resin block containing the sample into ultrathin sections, and set the section thickness to 70nm. Finally, use a copper mesh to fish the sections, first stain the tissue with uranyl acetate for 15min, and then with lead citrate for 10min, and dry at room temperature. Observe the morphology under a transmission electron microscope, and take pictures to collect images for subsequent analysis.

[0087] 1.2.2 Extraction of STEVs

[0088] STEVs are mainly extracted by tissue cutting treatment, enzymatic dissociation, gradient size exclusion, differential centrifugation combined with ultrahigh speed centrifugation, and the specific method is as follows:

[0089] (1) Put the scar tissue into a 50mL centrifuge tube, and repeatedly wash it with pre-cooled sterile PBS (containing 1% double-antibody) until there is no obvious blood color. After absorbing the water, weigh the tissue.

[0090] (2) Use a sterile surgical knife to cut the scar tissue into small pieces, and then use sterile ophthalmic scissors to cut the small pieces of scar tissue into 1mm ^3Small pieces of tissue are placed in an internal sterile 50 mL centrifuge tube.

[0091] (3) Prepare the tissue dissociation solution: DMEM high glucose medium containing collagenase type IV (2 mg / mL) and DNase I enzyme (40 U / mL), and pre-cool in a 4°C refrigerator;

[0092] (4) Add 5 times the volume of pre-cooled tissue dissociation solution to the centrifuge tube containing the tissue, and place the centrifuge tube in a 37°C constant temperature shaker at a speed of 100-150 rpm for dissociation for 3 hours. During the dissociation period, observe and change the direction of the centrifuge tube at regular intervals to ensure that the dissociation solution fully and effectively acts on the surface of the tissue block, increasing the number of EVs released from the tissue gap, otherwise they may remain in the tissue fibers. When the tissue dissociation solution appears milky and the tissue fragments are not significant, the dissociation can be stopped.

[0093] After dissociation is complete, place a sterile 100 μm funnel-shaped nylon filter screen on the 50 mL centrifuge tube, transfer the remaining tissue fragments and dissociation solution to the filter screen, and allow the liquid to flow into the centrifuge tube by gravity; collect the filtrate, rinse the digestion centrifuge tube with about 5 mL of PBS, and repeatedly blow for about 10 times to ensure that all remaining dissociation solution is transferred to the nylon filter screen and is discharged to the centrifuge tube by gravity. The filtrate is sequentially passed through 70 μm and 45 m funnel-shaped nylon filter screens, and the above PBS rinsing steps are repeated.

[0094] The last collected filtrate is subjected to differential centrifugation at 4°C: 800 x g for 10 min, discard the tissue fragments, and collect the supernatant; 3,000 x g for 20 min, discard the cell fragments and apoptotic bodies, and collect the supernatant; 3,000 x g for 20 min again, discard the precipitate, and collect the supernatant.

[0095] The above supernatant is sequentially passed through 20 m and 10 m funnel-shaped nylon filter screens, allowing the liquid to flow into the centrifuge tube by gravity, and repeating the PBS rinsing step of step (5) to collect the final supernatant.

[0096] The above supernatant is centrifuged at 16500 x g for 30 min at 4°C to remove the precipitate, and the supernatant is collected.

[0097] The supernatant is further passed through a 2.5 m funnel-shaped nylon filter screen, allowing the liquid to flow into the centrifuge tube by gravity, and the supernatant is collected.

[0098] The supernatant is transferred to a 30 mL polypropylene ultracentrifuge tube, filled with PBS, and sealed. The polypropylene ultracentrifuge tube is carefully balanced to 0.01 mg. A benchtop ultracentrifuge is used, with acceleration and deceleration set to maximum speed, and vacuum suction started immediately. The sample is centrifuged at 110,000 x g for 70 min at 4°C, and the supernatant is discarded. The pellet is resuspended in 1 mL of sterile PBS.

[0099] The resuspension is transferred to an ultrafiltration tube for purification, and centrifuged at 5000 x g for 15 min at 4°C. The upper chamber liquid is collected, and 1 mL of PBS is added to rinse the upper chamber.

[0100] The purified resuspension is passed through 0.8 m, 0.45 m, and 0.22 m needle filters in sequence, and the filtrate is collected.

[0101] The filtrate is again transferred to a 30 mL polypropylene ultracentrifuge tube, filled with PBS, and carefully sealed. The polypropylene ultracentrifuge tube is balanced to 0.01 mg. A benchtop ultracentrifuge is used, and the sample is centrifuged at 110,000 x g for 70 min at 4°C. The supernatant is discarded, and the pellet is resuspended in 500 L of sterile PBS.

[0102] 100 L of the resuspension is taken for concentration detection and identification, and the remaining resuspension is aliquoted into 100 L and stored in a -80°C refrigerator.

[0103] 1.2.3 BCA method for determining the concentration of STEVs

[0104] The BCA method is used to determine the concentration. Protein standard with a concentration of 5 mg / mL is first diluted 10-fold with PBS, to a concentration of 0.5 mg / mL. The diluted protein standard is aliquoted and stored in a -20°C freezer.

[0105] The extracted STEVs were centrifuged for 30 s using a handheld centrifuge. 20 L of STEVs were taken and diluted by 10 times and 30 times with PBS. First, a standard curve and a sample layout were designed using a 96-well plate, and the number of wells required for the standard curve and the sample to be tested was counted, and the total volume of the BCA working solution was calculated, and the working solution was prepared according to the BCA kit instructions (BCA reagent: Cu reagent = 50: 1), and mixed well at room temperature. Then, the protein standard with a concentration of 0.5 mg / mL was diluted with PBS to a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 20 L of standard protein with different concentrations were taken and added to the corresponding positions of the standard curve marked in advance in the well plate, with three replicate wells for each concentration. Then, 20 L of STEVs solution diluted by different times was taken and added to the corresponding positions of the sample marked in advance in the well plate, with three replicate wells for each concentration. Finally, 200 L of working solution was added to the wells containing the standard and sample, and the protein sample and working solution were mixed well to avoid air bubbles. The well plate was covered and placed in a 37°C incubator for 30 min. Finally, the absorbance of each well of the 96-well plate was determined by full-wavelength microplate reader at 562 nm, and the standard curve was prepared according to the absorbance of the protein standard and the concentration of the STEVs was calculated.

[0106] 1.2.4 Transmission electron microscopy observation of the morphology of STEVs

[0107] 10 L of STEVs sample was taken from the -80°C refrigerator and dissolved at 4°C. After dissolution, the sample was centrifuged for about 15 s using a handheld centrifuge. 10 L of STEVs sample was dropped onto a copper grid, and the excess floating liquid was absorbed with a water-absorbing paper after 1 min. Then, 10 L of uranyl acetate was dropped onto the sample on the copper grid, and the floating liquid was absorbed with a water-absorbing paper after 1 min. After natural drying at room temperature, the imaging results were observed and photographed using a transmission electron microscope.

[0108] 1.2.5 Nanoparticle size analysis of the diameter of STEVs

[0109] 10 L of STEVs sample was taken from the -80°C refrigerator and thawed in a 25°C water bath, and then centrifuged for 15 s using a handheld centrifuge and placed on ice. The STEVs sample was diluted with 1x PBS, and the diluted sample was directly used for NTA detection.

[0110] 1.2.6 Nanoflow detection of membrane proteins of STEVs

[0111] Take out the STEVs sample 20 L from -80℃ refrigerator, place it in 4℃ condition for dissolution, after dissolution, centrifuge it for about 15 s by using a palm centrifuge. After diluting the STEVs to 60 L by using PBS, take 30 L of the diluted STEVs into a new centrifuge tube, add 20 L of fluorescently labeled CD9 antibody and CD81 antibody respectively, mix them by blowing, then incubate them for 30 min in 37℃ dark condition. Transfer the STEVs sample labeled by fluorescent antibody into a 2 mL polypropylene ultracentrifuge tube, fill the polypropylene ultracentrifuge tube with pre-cooled PBS to make it completely full, strictly balance it, then seal the tube by using a heat sealing gun. Use a benchtop ultracentrifuge to centrifuge it at 110,000 x g for 70 min in 4℃, discard the supernatant, then resuspend the precipitated particles with 1 mL of sterile PBS. Transfer the above resuspension into a new 2 mL polypropylene ultracentrifuge tube, fill the polypropylene ultracentrifuge tube with pre-cooled PBS to make it completely full, strictly balance it, then seal the tube by using a heat sealing gun. Again use a benchtop ultracentrifuge to centrifuge it at 110,000 x g for 70 min in 4℃, discard the supernatant, then resuspend the precipitated particles with 50 L of pre-cooled PBS. Test the instrument performance by using a standard sample before testing, after testing qualified, load the above resuspended STEVs sample containing fluorescent antibody for testing, then obtain the protein index test result. 1.2.7 WB identification of STEVs phenotype

[0112] WB experiment to detect the expression of specific markers of STEVs CD9, CD63, TSG101 and negative protein Calnexin. From the -80℃ refrigerator to take out the STEVs sample, placed on the ice box to dissolve, add sample volume 1 / 4 of protein loading buffer, vortex mix and centrifuge. Put in 98℃ constant temperature metal bath for 3 min, then put on ice box to cool. At the same time, according to the protein molecular weight of the sample to be tested, prepare 10% SDS-PAGE separation gel (for specific operation, refer to the instruction manual). Mix the separation gel thoroughly and pour it into the gel plate installed in advance at a constant speed. Seal with 1-2 mL of anhydrous ethanol, and after the separation gel solidifies, tilt the gel plate to pour off the anhydrous ethanol, and use a water-absorbing paper to absorb the residual anhydrous ethanol. Then, pour the prepared concentrated gel at a constant speed and slowly insert the comb. Wait for the gel to solidify at room temperature for about 30 min. Install the electrophoresis device, add electrophoresis liquid, and slowly pull out the comb. Use a pipette to add the STEVs sample and Maker into the lanes on the electrophoresis gel in the order designed to avoid air bubbles. Turn on the power and set 80V to run the gel until the bromophenol blue reaches the junction of the concentrated gel and the separation gel. Then, turn to 110V to run the gel until the bromophenol blue reaches about 1 cm from the bottom of the gel. Stop electrophoresis. Take out the electrophoresis gel, cut off the concentrated gel and excess part on the edge, and make a cut corner mark on the upper left corner. According to the size of the gel, cut a PVDF membrane of the same size, and make a cut corner mark on the upper left corner. Soak in methanol for 30 s, then soak in pre-cooled electrotransfer liquid. Similarly, soak the sponge and transfer filter paper in pre-cooled electrotransfer liquid. Then, make a transfer membrane "sandwich" structure in the order of sponge-transfer filter paper-electrophoresis gel-PVDF membrane-transfer filter paper-sponge, and place it in the electrotransfer tank. Add pre-cooled transfer liquid, put in an ice brick of appropriate size, and bury the device in an ice bath for transfer (250 mA, time calculated as 1 kDa = 1 min). After transfer, take out the PVDF membrane, and soak the membrane protein in TBS containing 5% skim milk with the front side facing up. Incubate on a shaker at room temperature for 2 h. After blocking, soak the membrane in prepared primary antibody, and the concentration of the primary antibody is CD9 1:1000, CD63 1:3000, TSG101 1:3000, and Calnexin 1:3000. Incubate overnight (about 14-16 h) in a 4℃ refrigerator on a shaker. Recover the primary antibody, wash the membrane with TBST at room temperature for 10 min, and repeat 3 times. According to the selection of the primary antibody, dilute the secondary antibody in 5% skim milk TBST solution (the concentration of the secondary antibody is 1:5000). Soak the membrane in the secondary antibody solution and incubate at room temperature for about 1 h. Discard the secondary antibody, wash the membrane with TBST at room temperature for 10 min, and repeat 3 times. Absorb the liquid on the surface of the PDVF membrane with filter paper, prepare 2 mL of ECL developing solution, and incubate in the dark for 15 s. Expose the PDVF membrane with an imager, adjust the brightness and contrast, and save the picture.

[0113] Take a small piece of scar tissue and place it in a 50 mL centrifuge tube. Wash repeatedly with pre-cooled sterile PBS (containing 1% penicillin and antibiotics) until no obvious blood is visible. After abstaining from the water, weigh the tissue. After weighing, use a sterile scalpel to cut the scar tissue into small pieces. Add PBS (containing 1% protease inhibitor) according to the weight of the scar tissue at a ratio of 1 mL of liquid per 100 mg of scar tissue. Next, add a grinding magnetic bead (2 steel beads and 1 magnet) to the tissue, balance it, and then place it in a tissue homogenizer for grinding at 4°C, 70 Hz, and 360 s until no obvious tissue is visible to the naked eye. Remove the magnetic bead, balance it again, and centrifuge at 4°C and 12000 rpm for 30 min using a low-temperature high-speed centrifuge. Gently aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube to obtain the obtained scar tissue homogenate (STH). The protein concentration of STH is determined using the BCA method (same method as above).

[0114] 2 Results

[0115] 2.1 Extracellular vesicles exist in the interstitial spaces of scar tissue.

[0116] After obtaining informed consent from the patient and attending physician, discarded hypertrophic scar tissue was collected after scar excision, prepared into sections, and observed under a biological transmission electron microscope. Figure 1 Microscopic examination revealed numerous round or oval vesicles of varying sizes, ranging from approximately 30-500 nm, within the scar tissue interstitial spaces. The vesicle membranes were clearly defined and uniform in thickness, confirming the presence of extracellular vesicles (STEVs) in the scar tissue. Microscopic examination also revealed differences in electron density within some vesicles, suggesting the possible presence of different biomolecules. The STEVs were widely and uniformly distributed throughout the interstitial spaces, without significant aggregation. These characteristics indicate the presence of typical extracellular vesicles in the interstitial spaces, providing a structural basis for intercellular communication and the regulation of the tissue microenvironment.

[0117] 2.2 Extraction and Identification of STEVs

[0118] Transmission electron microscopy revealed that STEVs exhibited a saucer-like structure with an intact membrane structure, consistent with the morphology of EVs. Figure 2 A). Nanoparticle size analysis results show that the average diameter of the vesicles is 173.7 nm, which is consistent with the diameter range of small EVs (A). Figure 2 B). Western blot results showed that the specific markers of EVs, TSG101, CD63, and CD9, were all positively expressed, while the negative marker Calnexin was negatively expressed. Figure 2C) Nano-flow cytometry detection of exosome surface protein expression showed that the positive rate of exosome surface protein CD9 expression was 31.8%, and the positive rate of CD81 expression was 9.0%, while the positive rate of the blank control group was only 0.3%. CD9 and CD81 are both classic markers of exosomes, and their positive expression further confirms that the extracted samples are EVs Figure 2 D).

[0119] Figure 2 A. STEVs showed a concave hemispherical or saucer-like structure under transmission electron microscopy, scale = 100 nm; B. Nanoparticle size analysis of STEVs, vesicle particle size distribution was 30-200 nm; C. WB detection of STEVs specific markers, CD9, CD63, TSG101 were positively expressed, and Calnexin expression was negative; D. Nano-flow cytometry detection of STEVs membrane surface protein, CD9, CD81 positive expression.

[0120] 3 DISCUSSION

[0121] EVs are lipid bilayer structured vesicles released by cells into the surrounding environment through different pathways, carrying active substances such as proteins and RNA. EVs play a role in information transmission and signal exchange between cells. Compared with the EVs extracted from cell supernatant, blood or urine, directly extracting EVs from tissues can more accurately reflect the true situation inside the tissue. Tissue EVs refer to all EVs secreted by cells in the tissue, which not only reflect the pathological and physiological characteristics of tissue cells in the three-dimensional environment of the tissue, but also mediate cell communication in the microenvironment of the diseased tissue. In our early research, we used scar tissue homogenate (STH) to simulate the scar microenvironment and confirmed that the pretreatment of HUC-MSCs with STH and inflammatory factors (IF) had a certain "guiding" effect on their paracrine effect in the treatment of scar. However, although scar tissue homogenate contains all the components of the tissue and can provide comprehensive biochemical information about the disease, it lacks specificity and dynamic information, making it difficult to identify the key factors that play a role and accurately reflect the true situation of the tissue microenvironment. In summary, this experiment determined the presence of extracellular vesicles in the interstitial microenvironment of scar tissue through biological transmission microscopy, and the EVs extracted from scar tissue can more accurately represent the pathological microenvironment of scar.

[0122] According to the 2023 ISEV guidelines (MISEV2023), we identified the extracted STEVs by TEM, NTA, WB and nanoflow cytometry to meet the classic exosome characterization. TEM showed that STEVs had a typical tea tray-like structure, and NTA analysis showed that the particle size range of STEVs met the particle size of exosomes. In the WB results, STEVs positively expressed CD9 and CD63, which are membrane protein markers of EVs, and TSG101, which is a cytoplasmic protein marker of EVs, while Calnexin, which is negatively expressed, can exclude the contamination of endoplasmic reticulum components in STEVs, indicating that the extracted STEVs in this experiment have satisfactory purity. Our results show that the EVs isolated from scar tissue in this experiment meet the experimental requirements. Based on the above extraction method, we then used STEVs to represent the scar microenvironment, pretreated HUC-MSCs, and isolated STEVs-Exos after pretreatment, aiming to explore the therapeutic effect of STEVs-Exos on early scar.

[0123] Example 2 Effect of Exosomes of Scar Tissue Extracellular Vesicles Pretreated Human Umbilical Cord Mesenchymal Stem Cells on Early Scar Treatment

[0124] 1 Materials and Methods

[0125] 1.1 Main experimental materials

[0126] 1.1.1 Experimental cells

[0127] The neonatal umbilical cords used in the experiment were obtained from normal full-term pregnant women after cesarean section in the operating room of the Affiliated Hospital of Zunyi Medical University in October 2023. The pregnant women and the attending physicians had given their verbal informed consent before the operation. The umbilical cord was immediately placed in a sterile glass bottle containing pre-cooled sterile PBS (containing 1% double-antibiotic) after being detached from the body. HUC-MSCs were obtained by tissue adhesion culture for subsequent experiments. The acquisition and use of umbilical cord samples in this study comply with ethics and are approved by the Biomedical Research Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Ethical Review Approval Number: KLLY-2020-176).

[0128] 1.1.2 Experimental animals

[0129] New Zealand white rabbits in this experiment were purchased from Chongqing Enswell Experimental Animal Sales Co., Ltd. (License: SCXK(YU)2022-0007), a total of 16, male, 3 months old, body weight: 2.0-2.5 kg, single-cage feeding in the experimental animal center of Zunyi Medical University. During feeding, the experimental animals were reasonably fed according to the welfare requirements, the animal experiment process conformed to the experimental animal ethics, and the experiment obtained the approval of the experimental animal welfare ethics committee of Zunyi Medical University Affiliated Hospital (Ethical Approval Number: zyfy-an-2024-0281).

[0130] 1.1.3 Main experimental instruments and consumables see Table 3

[0131] Table 3. Experimental instruments and consumables

[0132]

[0133] 1.1.4 Main experimental reagents, see Table 4

[0134] Table 4. Experimental instruments and consumables

[0135]

[0136]

[0137]

[0138] 1.2 Experimental method

[0139] 1.2.1 Obtaining and culturing of HUC-MSCs

[0140] 1.2.1.1 Isolation of HUC-MSCs

[0141] The biological safety cabinet was sterilized by UV for 30 min before use. During this period, the autoclaved surgical instruments, centrifuge tubes, cell culture bottles, cell culture dishes, pre-cooled sterile PBS, and human umbilical cord mesenchymal stem cell serum-free complete medium were prepared. Under sterile conditions, the umbilical cord sample was taken out in the biological safety cabinet, the ends of the umbilical cord were cut off with sterile surgical scissors, and the umbilical cord was cut into 2-3 cm small pieces. The atrophied, edematous, and coagulated parts of the umbilical cord were discarded. The small pieces of umbilical cord were washed 3 times with sterile PBS containing 1% penicillin / streptomycin, and then washed 3 times with sterile PBS without double antibodies. Then, the 2 umbilical arteries and 1 umbilical vein in the small piece of umbilical cord were found, the umbilical vein was cut open with ophthalmic scissors, the umbilical vein and intima were completely exposed, and the umbilical vein was fully stripped with ophthalmic forceps. Then, the umbilical cord was turned over, the outer membrane of the umbilical cord was gently stripped with ophthalmic forceps, and the 2 umbilical arteries were stripped. During this period, the umbilical cord tissue was kept moist. After the umbilical vein, umbilical artery, and umbilical cord outer membrane were completely stripped, the remaining tissue was smooth Wharton's jelly, which could be evenly spread. Then, the stripped Wharton's jelly was washed with sterile PBS, repeated 3 times, and the mucus on the surface of the tissue was removed. Then, the Wharton's jelly was cut into 3-5 mm pieces with a surgical knife, and washed with sterile PBS again for 3 times. The small pieces of washed Wharton's jelly tissue were filtered through a 100-mesh cell filter to remove excess PBS. Finally, the Wharton's jelly tissue pieces were transferred to a T75 cell culture bottle with a Pasteur pipette with the tip removed, and arranged evenly. About 30 pieces of Wharton's jelly tissue were attached to the wall of the culture bottle. The culture bottle was placed upside down in a cell culture incubator at 37°C and 5% CO2 for 12 h to adhere. After 12 h of adhesion, the cell culture bottle was slowly placed upright, the volume of the culture medium was slowly added to 13 mL, and then placed in the cell culture incubator. The culture medium was replaced after 7 days of culture. Then, the culture medium was replaced every 3 days, and the cell state was observed regularly during this period. After about 14 days of culture, the cell density reached about 80%, and the primary cells were collected for subculture.

[0142] 1.2.1.2HUC-MSCs of the subculture

[0143] HUC-MSCs were cultured in a cell incubator at 37°C with 5% CO2for 48-72h. Under the microscope, HUC-MSCs were long spindle-shaped and grew in a fish school vortex. When the cell density reached 80-90%, the cells could be passaged. The biological safety cabinet was sterilized by ultraviolet light for 30 min. The sterile PBS containing 1% penicillin / streptomycin, serum-free medium for human umbilical cord mesenchymal stem cells, and gentle cell digestion enzyme were preheated in a 37°C water bath. The cell culture bottle was taken out from the CO2incubator and sterilized with 75% ethanol. After opening the cell culture bottle, the supernatant was discarded, and about 5 mL of preheated sterile PBS containing 1% penicillin / streptomycin was added to the culture bottle along the side wall. Then, the culture bottle was moved slowly in the shape of "8" to rinse, and the PBS was aspirated. The rinsing step was repeated 3 times. Then, 3 mL of preheated gentle cell digestion enzyme was added to cover the cells, and the culture bottle was placed in the cell incubator for 3 min. During this time, the cell digestion was observed under an inverted microscope. When the cells were round and mobile, the culture bottle was transferred to the biological safety cabinet, 6 mL of complete medium was added to terminate digestion, and the cells were dispersed by gently blowing with a Pasteur pipette. The cell suspension in the culture bottle was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min at room temperature. The supernatant was discarded, 3 mL of complete medium was added, and the cells were resuspended by gently blowing. 1 mL of cell suspension was taken and plated in a new T75 cell culture bottle, 3 bottles were inoculated, and then serum-free medium for human umbilical cord mesenchymal stem cells was added to 12 mL. The bottle was shaken in the shape of "8" and then placed in the cell incubator for culture. After 24 h, the cell state was observed, and when the cell fusion density reached 80%-90%, the next passage was performed.

[0144] 1.2.1.3 Cryopreservation of HUC-MSCs

[0145] When the cell growth density reached 80%-90%, the cells could be cryopreserved when the cell passage was not continued. The preparation was the same as step 1.2.1.2. After the cells were rinsed with sterile PBS 3 times, the digestion enzyme was added for digestion and centrifugation (refer to the cell digestion and centrifugation steps in 1.2.1.2). After centrifugation, the supernatant was discarded, 1 mL of complete medium was added to resuspend the cell pellet, and 10 ul of the resuspended cell suspension was taken to a hemocytometer for cell counting. Finally, according to the cell counting results, the calculated volume of serum-free cell cryopreservation solution was added to the cell suspension to make the cell density 1 x 10 6 Finally, the cell suspension containing the cell cryopreservation solution was added to the cryopreservation tube, 1 mL per tube. The cell name, passage number, cryopreservation time, and operator were marked on the wall of the cryopreservation tube, and the tube was sealed with a sealing film and stored in a -80°C refrigerator.

[0146] 1.2.1.4 HUC-MSCs recovery

[0147] Bio-safety cabinet UV sterilization 30 min, in advance to the human umbilical cord mesenchymal stem cells special serum-free medium to 37℃ water bath pot preheating. From the -80℃ freezer, take out the cell cryopreservation tube, with forceps to hold the cryopreservation tube quickly into the 37℃ water bath pot gentle shaking, so that it is heated evenly melt. When the cryopreservation tube completely melted (about 1-1.5 min), with 75% ethanol disinfection cryopreservation tube wall after the put into the bio-safety cabinet. First, the stem cell special complete medium to 15 mL centrifuge tube, the volume of the cell suspension in the cryopreservation tube volume of 2 times. Then the pipette gun to take the cell suspension in the cryopreservation tube into the above centrifuge tube, resuspended 800 rpm normal temperature centrifugation 5 min. After removing the supernatant, add the appropriate complete medium to the cell pellet resuspended and gently blow. Finally, according to a tube of cryopreservation tube inoculation a bottle of T75 culture bottle ratio, the cell suspension plated to T75 culture bottle, add stem cell special serum-free complete medium to 12 mL, before and after, up and down, left and right gently shake, so that the cells in the culture bottle dispersed evenly. Finally in the culture bottle bottom mark after put into the cell culture box culture, after recovery the next day to observe the cell growth state, and replace the culture medium continue to culture 48 h, during the regular observation of cell growth state, when the cell growth density of 80-90% can be subcultured.

[0148] 1.2.2 Flow cytometry identification of HUC-MSCs phenotype

[0149] Bio-safety cabinet UV sterilization 30 min, in advance to the human umbilical cord mesenchymal stem cells special serum-free medium to 37℃ water bath pot preheating. From the -80℃ freezer, take out the cell cryopreservation tube, with forceps to hold the cryopreservation tube quickly into the 37℃ water bath pot gentle shaking, so that it is heated evenly melt. When the cryopreservation tube completely melted (about 1-1.5 min), with 75% ethanol disinfection cryopreservation tube wall after the put into the bio-safety cabinet. First, the stem cell special complete medium to 15 mL centrifuge tube, the volume of the cell suspension in the cryopreservation tube volume of 2 times. Then the pipette gun to take the cell suspension in the cryopreservation tube into the above centrifuge tube, resuspended 800 rpm normal temperature centrifugation 5 min. After removing the supernatant, add the appropriate complete medium to the cell pellet resuspended and gently blow. Finally, according to a tube of cryopreservation tube inoculation a bottle of T75 culture bottle ratio, the cell suspension plated to T75 culture bottle, add stem cell special serum-free complete medium to 12 mL, before and after, up and down, left and right gently shake, so that the cells in the culture bottle dispersed evenly. Finally in the culture bottle bottom mark after put into the cell culture box culture, after recovery the next day to observe the cell growth state, and replace the culture medium continue to culture 48 h, during the regular observation of cell growth state, when the cell growth density of 80-90% can be subcultured. 7 / mL. Next, prepare 7 flow cytometry special tube, of which 6 tubes are added with 100 L of the above cell suspension, and then 5 L of flow cytometry antibody is added respectively: HLA-DR-FITC, CD105-APC, CD45-PE, CD44-TITC, CD34-PE, CD29-PE. After gentle mixing, mark the tube with a self-identifiable mark. The remaining 1 tube is added with 5 uL of PBS (as a negative control group). The above tubes are placed in a 4℃ refrigerator in the dark for 30 min, and then centrifuged at 4℃ for 5 min at 1000 rpm. After centrifugation, the supernatant is removed, and the cell pellet is resuspended with 500 L of PBS. After centrifugation at 1000 rpm for 5 min, the supernatant is discarded and the washing step is repeated for a total of 3 times. Finally, 200 L of sterile PBS is added to each tube to resuspend the washed cell pellet, and the cell pellet is gently blown and resuspended. After resuspension, the cell pellet is placed in a flow cytometer for detection and data analysis.

[0150] 1.2.3 Uptake of STEVs by HUC-MSCs

[0151] The experiment of STEVs uptake by HUC-MSCs showed that STEVs can enter HUC-MSCs and affect the changes of its contents. The biological safety cabinet was ultraviolet sterilized for 30 min, and sterile PBS containing 1% penicillin / streptomycin, serum-free medium for human umbilical cord mesenchymal stem cells, and gentle stem cell digestion enzyme were preheated in a 37°C water bath. 100ul of STEVs sample was taken from the -80°C refrigerator and dissolved at 4°C. After dissolution, it was centrifuged for 15s using a handheld centrifuge. According to the concentration determined by the BCA method before, 50ug of STEVs sample was taken. According to the instructions of the red fluorescent labeling dye (PKH26), STEVs were stained: first, 50ug of STEVs sample was added to diluent C to make the final volume 500L and mixed well. Then, 2L of PKH26 was added to 500L of diluent C and mixed well. Then, the diluent C containing PKH26 was added to the diluent C containing STEVs, and the two were mixed well and protected from light, then placed in a cell incubator for 20 minutes for staining. After staining, an equal volume of PBS was added and mixed well to terminate the staining. Then, the above stained STEVs suspension was transferred to a high-speed special centrifuge tube, filled with PBS, and strictly balanced, sealed, and centrifuged at 4°C, 100000xg for 70min to remove excess dye. The precipitate after centrifugation was PKH26 fluorescently labeled STEVs. 200L of sterile PBS was added to resuspend the STEVs precipitate.

[0152] P4 generation HUC-MSCs with good cell state and density of 80%-90% were taken, and the cells were counted after routine PBS rinse, digestion and centrifugation. The PKH26-labeled STEVs were added to the serum-free complete medium for human umbilical cord mesenchymal stem cells, mixed well, and then filtered with a 0.22m filter. Then, the cell density of the cell suspension was diluted to 1x10 5HUC-MSCs and STEVs co-culture 24h after removing the culture medium, washed with PBS 3 times, add 2mL 4% cell tissue fixed liquid in room temperature for 10min. After discarding the fixing liquid, washed with PBS 3 times. Finally, add DAPI in room temperature for 10min, washed with PBS 3 times again, then take pictures under the laser confocal microscope. 1.2.4 Detection of the proliferation ability of HUC-MSCs by STEVs

[0153] In this experiment, the effect of different concentrations of STEVs pretreatment on the proliferation ability of HUC-MSCs was detected by CCK8 experiment, and the best STEVs pretreatment concentration was screened. First, take 4 96-well plates and design the layout in advance. This experiment is mainly divided into 5 groups, 5 replicates in each group: blank group, control group, STEVs (20 μg / mL) group, STEVs (40 μg / mL) group, STEVs (80 μg / mL) group. The concentration gradient of STEVs is designed according to relevant research literature. Take P4 generation HUC-MSCs with good growth state and density of 80-90% for digestion and centrifugation, and then count the cells. Add STEVs special serum-free complete culture medium for human umbilical cord mesenchymal stem cells to dilute the cell suspension to a cell density of 2×10 4 3 ​Blank group: each well only added 100 uL of human umbilical cord mesenchymal stem cell special serum-free complete medium. Cover the cover plate and mark, put it in the cell culture box for 6h. After 6h of culture, the cells adhere to the wall, the medium in each well is discarded and washed with PBS for 3 times. Prepare the medium in different groups in advance, and filter the medium with a 0.22 μm filter. Then, according to the marked groups, add the following different group medium to the corresponding wells of the plate, 100L per well: blank group and control group add human umbilical cord mesenchymal stem cell special serum-free medium, STEVs (20 μg / mL) group add human umbilical cord mesenchymal stem cell special serum-free medium containing STEVs 20 μg / mL, STEVs (40 μg / mL) group add human umbilical cord mesenchymal stem cell special serum-free medium containing STEVs 40 μg / mL, STEVs (80 μg / mL) group add human umbilical cord mesenchymal stem cell special serum-free medium containing STEVs 80 μg / mL. At this time, the three 96-well plates are marked as 0h of STEVs pretreated HUC-MSCs and start timing. At 12h, 24h, 48h, 72h after pretreatment, take one 96-well plate at each time point to add CCK8 for detection: after discarding the medium, wash with PBS for 3 times, add 100L of CCK8 reagent containing medium prepared in advance to each well, and react in the cell culture box for 1h. Finally, place the 96-well plate in the enzyme label instrument and detect the absorbance (OD) value at 450nm, arrange the data, remove the maximum and minimum values of the OD value of each group, and select the best STEVs intervention concentration for subsequent experiments.

[0154] 1.2.5 Pretreatment of HUC-MSCs under different conditions and extraction and identification of corresponding Exos

[0155] 1.2.5.1 Grouping of HUC-MSCs

[0156] The pretreatment of HUC-MSCs in this experiment was divided into three groups: STEVs group (adding human umbilical cord mesenchymal stem cell special serum-free medium containing STEVs 40 μg / mL concentration), STH group (adding human umbilical cord mesenchymal stem cell special serum-free medium containing STH 40 μg / mL concentration, the concentration is referred to the previous experimental results of the research group. Tra group (adding human umbilical cord mesenchymal stem cell special serum-free medium of traditional culture group) is the control group. Prepare the above different groups of medium, and use 0.22 μm filter to filter the medium, and mark the group. The P4 generation HUC-MSCs with satisfactory growth state were routinely digested, centrifuged and counted. After resuspension, equal volume of cell suspension was added to T75 cell culture bottle, and randomly divided into culture groups and marked. Then, the corresponding medium was added to each group of culture bottles, and then placed in the cell culture box for 24 h. After 24 h, the original cell supernatant was discarded, and after 3 times of PBS rinse, 15 mL of new human umbilical cord mesenchymal stem cell special serum-free complete culture medium without pretreatment was slowly added to each culture bottle for 48 h. After 48 h of culture, the corresponding supernatant was collected for exosome extraction, and the cell pellets after PBS rinse, digestion and centrifugation of each group of cells were stored at -80℃ for standby.

[0157] 1.2.5.2 Extraction of HUC-MSCs-Exos

[0158] The supernatant collected from each group of HUC-MSCs was separated into corresponding HUC-MSCs-Exos by differential centrifugation combined with ultrahigh-speed centrifugation. First, the 50 mL centrifuge tube containing the supernatant of each group was balanced, and a low-temperature high-speed centrifuge (4°C pre-cooling) was used to perform the following differential centrifugation operations in sequence: 600 x g centrifugation for 10 min to remove dead cells and retain the supernatant; 2000 x g centrifugation for 20 min to remove cell debris and retain the supernatant; 10000 x g centrifugation for 30 min to remove large vesicles and retain the supernatant. Next, the supernatant was transferred to a 100 kd ultrafiltration tube, and centrifuged at 2500 g for 15 min at 4°C to collect the concentrated supernatant containing Exos in the upper filter tube. Finally, the concentrated supernatant was transferred to an ultrahigh-speed special centrifuge tube, which was filled with sterile PBS to completely fill the special centrifuge tube, and the centrifuge tube was carefully sealed with a tube cap. The ultrahigh-speed special centrifuge tube was strictly balanced to 0.01 mg, and a benchtop ultrahigh-speed centrifuge was used with the acceleration and deceleration set to the maximum rate, and the vacuum suction and centrifugation were started immediately. The supernatant was discarded after centrifugation at 110,000 x g for 70 min at 4°C, and the precipitated particles were resuspended in sterile PBS. After strict balancing, the resuspended particles were centrifuged again at 110,000 x g for 70 min at 4°C, and the supernatant was discarded. Finally, the precipitated particles were resuspended in 500 ul of sterile PBS, and 100 L of the resuspension was used for concentration detection and identification. The remaining resuspension was aliquoted into 100 L and stored in a -80°C freezer. The Exos extracted from the supernatant collected from STEVs-preconditioned HUC-MSCs was named STEVs-Exos, the Exos extracted from the supernatant collected from STH-preconditioned HUC-MSCs was named STH-Exos, and the Exos extracted from the supernatant collected from HUC-MSCs cultured under traditional conditions was named Tra-Exos.

[0159] 1.2.5.3 Identification of HUC-MSCs-Exos

[0160] 1.2.5.3.1 Transmission electron microscope observation of the morphology of Exos in each group

[0161] The aliquoted HUC-MSCs-Exos in each group were taken out from the -80°C freezer, dissolved in a 4°C refrigerator, and centrifuged for about 15 s using a handheld centrifuge. 10 L of each group of Exos samples were aspirated and added dropwise onto a copper mesh. After 1 min, the excess floating liquid was absorbed with a water-absorbing paper. Then, 20 L of uranyl acetate (phosphotungstic acid staining solution) was added dropwise, and the excess floating liquid was absorbed with a water-absorbing paper after 5 min of continuous precipitation. Finally, the copper mesh was baked under an incandescent lamp for about 10 min, and observed and photographed using a transmission electron microscope.

[0162] 1.2.5.3.2 Nanoparticle size analysis of the diameter of Exos in each group

[0163] The divided HUC-MSCs-Exos were taken out from the-80℃ refrigerator, dissolved in the 4℃ refrigerator, centrifuged for about 15s by the palm centrifuge, and 10L of each group of Exos samples were taken and diluted with PBS for NTA detection.

[0164] 1.2.6 Construction of rabbit ear hypertrophic scar animal model and grouping

[0165] Two New Zealand white rabbits, both male, 3-4 months old, were checked for skin damage on both ears. First, after the rabbit was anesthetized with isoflurane inhalation, the head and limbs were fixed on the rabbit fixator in a prone position. The abdominal hair of both ears was removed with an animal hair clipper, and then the modeling point was selected by avoiding obvious blood vessels, with an interval of 1 cm between each wound and positioning. Then, the whole ear skin was disinfected with iodophor for 3 times, and the skin of the modeling area was disinfected with 75% alcohol for 1 time. After injection of 1% lidocaine at the modeling point, a high-pressure sterilized circular skin punch with a diameter of 1 cm was used to puncture the skin at the modeling point to the cartilage surface, and the whole layer of skin on the cartilage was removed with a sterile ophthalmic scissors to construct a 1 cm diameter skin full-thickness defect rabbit ear wound. According to the size of the ear, 4-6 wounds can be prepared on each side of the ear. Finally, the sterile periosteal knife was used to thoroughly remove the cartilage membrane of the wound. After modeling, the wound was pressed to stop bleeding, and the bloodstains on the wound and surrounding skin were washed with PBS, and erythromycin eye ointment was applied. After modeling, the rabbit's condition and wound were observed regularly, and the wound was disinfected and changed every 48h until the wound healed. After modeling, the rabbits were photographed every week, and the scars were taken for HE staining and Masson staining after 21 days of scar formation.

[0166] After the success of the rabbit ear hypertrophic scar model at 21 days, 14 New Zealand white rabbits were selected again to construct a 1 cm diameter skin full-thickness defect rabbit ear wound. On the 21st day after modeling, all the rabbit ear wounds were epithelialized and had local red raised scars. These 14 rabbits were numbered: I-XIV, each rabbit was divided into left and right ears, and the wounds of each ear were numbered from 1-6 in clockwise order. The rabbit ear scars were randomly grouped: PBS group, Tra-Exos group, STH-Exos group, STEVs-Exos group. The Exos of each group were thawed, the protein concentration was determined by BCA method, and the concentration of each group of Exos was adjusted to 1 μg / uL. PBS 100 μL and each group of Exos 100 μg were injected into the corresponding group of scars, and each scar was injected in 4 directions. After treatment, the state of the rabbits was observed, and the changes of the rabbit ear scars in each group were recorded and photographed at 1 week, 2 weeks, 3 weeks, and 4 weeks after injection of Exos, and the samples were taken regularly.

[0167] 1.2.7 Efficacy observation of each group of HUC-MSCs-Exos on early treatment of rabbit ear hypertrophic scar

[0168] 1.2.7.1 Manchester Scar Scale to evaluate rabbit ear scar

[0169] The Manchester Scar Scale (MSS) was used to quantitatively evaluate the appearance and texture of the rabbit ear scar at 1 week, 2 weeks, 3 weeks, and 4 weeks after injection of Exos. The effect of Exos on the appearance and texture of the rabbit ear scar at each time point was visually observed, and the therapeutic effect was evaluated. The specific scoring indicators are as follows (see Table 5). Add the scores of the items to get the total score. The lower the total score, the better the appearance and texture of the scar, and the scar is not obvious. The higher the total score, the more serious the scar hyperplasia.

[0170] Table 5. Manchester Scar Scale

[0171]

[0172]

[0173] 1.2.7.2 Rabbit ear scar sampling

[0174] After injection of Exos, the changes in the rabbit ear scar were observed, and the rabbit ear scar of each group was sampled at 2 weeks and 4 weeks after injection. After the rabbit was anesthetized by inhaling isoflurane, its head and limbs were fixed, the hair around the scar was shaved, and the sampling range was marked as 5 mm from the edge of the scar to the surrounding normal skin. The entire scar tissue and surrounding skin were removed with a sterile scalpel, and the depth reached the subcartilage. Each scar tissue was cut in half with a scalpel, and one half was placed in a tissue embedding box and labeled, and then placed in 10% formalin for 48 h. The other half was placed in a tissue freezing tube and labeled, and then immediately placed in a liquid nitrogen tank. The rabbit was sacrificed by air embolism under anesthesia after sampling.

[0175] 1.2.7.3 Tissue dehydration and paraffin embedding

[0176] After the rabbit ear scar was fixed in 10% formalin for 48 h, the formalin was discarded and the tissue was washed with running water for 2 h. The tissue was dehydrated and transparentized using a dehydration machine: ① Washing and dehydrating: the tissue block was placed in 65% ethanol, 80% ethanol, 90% ethanol, and 90% ethanol for 1.5 h each, and then in two cylinders of anhydrous ethanol for 1.5 h each; ② Transparentizing: the tissue block was placed in two cylinders of xylene for 1 h each; ③ Wax immersion: the tissue block was placed in two cylinders of paraffin for 1.5 h each. Finally, paraffin embedding was performed.

[0177] 1.2.7.4 HE staining

[0178] The rabbit ear scar tissue embedded with paraffin was paraffin sectioned and dyed. ①Sectioning and baking: when sectioning, first coarsely repair the tissue wax block to expose the tissue part, then adjust the sectioning thickness to 4 μm to start sectioning, then use the adherent glass slide to pick up the tissue and make a mark, place the sectioned tissue slice on the slice spreading machine to spread the slice and make it flat to reduce wrinkles, the spreading temperature is generally 48℃. Then place the tissue slice in a 65℃ oven to bake for 1 h; ②deparaffinization to water: 3 cylinders of xylene, No. I, No. II, and No. III, deparaffinization in turn, each for 8 min; 2 cylinders of 100% anhydrous ethanol, 2 cylinders of 95% ethanol, 1 cylinder of 85% ethanol, a total of 5 cylinders, each for 5 min; flow water washing for 5 min. ③dyeing: first dry the water of the slice after washing, then place it in the hematoxylin dyeing solution for 8 min, after the end, move the slice to the square dyeing cylinder containing water and wash with flowing tap water until the water flow is clean. Immerse the slice in the hydrochloric acid alcohol for differentiation for 5 s, after differentiation, quickly place the slice in the square cylinder containing water and wash with flowing tap water for 5-8 min to make the nucleus bluing (during the observation of the returning blue degree under the microscope to determine the returning blue time). Then place the slice in the eosin dyeing solution (water-soluble) for 5 min, after dyeing, place the slice in the square cylinder containing water and wash with flowing tap water for 3-5 min to stop the continuous dyeing of eosin. Finally, dehydrate, transparentize and mount the dyed slice, 1 cylinder of 85% ethanol (10 s), 2 cylinders of 95% ethanol (each for 15 s), 100% ethanol I (30 s), 100% ethanol II (1 min), 3 cylinders of xylene, each for 1 min, dry in the fume hood, then mount with neutral balsam.

[0179] 1.2.7.5 Masson staining

[0180] After paraffin sectioning, the sections were baked in a 65℃ oven for 3 hours. ① Dewaxing to water: 3 tanks, xylene I (15 min), xylene II (15 min), xylene III (15 min), 100% anhydrous ethanol (2 tanks), 95% ethanol (2 tanks), 85% ethanol (1 tank), for a total of 5 tanks, each for 10 min; rinse with running water for 5 min. ② Staining: After rinsing, the sections were first shaken dry, and circles were drawn around the tissue using an immunohistochemical pen. Staining was performed according to the instructions of the modified Masson trichrome staining kit: 100 L of mordant solution was added to the tissue within the circle to mordate the tissue. The sections were placed in a humidified chamber and placed in a 60℃ oven for 1 hour for mordation. Then, the sections were placed in a square staining jar and rinsed three times with pure water, 3 min each time. The water around the tissue was blotted dry with filter paper, and azurite blue staining solution was added to the tissue surface for staining. After 3 min, the sections were rinsed with pure water for 15 s, and this process was repeated twice. After aspirating the water around the tissue, continue staining with Mayer's hematoxylin solution for 3 minutes, then rinse the sections with pure water for 15 seconds, repeating twice. Add acidic differentiation solution to each section for 5 seconds, then quickly place it in a square staining jar and rinse with running tap water to terminate differentiation, rinsing for about 5-8 minutes until the cell nuclei turn blue (blue return). Once the blue return is satisfactory, treat with Ponceau S and Fuchsin solution for 10 minutes, then rinse the sections with pure water for 15 seconds, repeating twice. Next, after differentiating the tissue with phosphomolybdic acid solution for 10 minutes, directly discard the supernatant. Do not wash the sections with water; aspirate the water around the tissue and treat with aniline blue solution for 5 minutes. After staining, rinse the sections with pure water for 15 seconds, repeating twice. Continue adding a weak acid solution to cover the tissue for 3-5 minutes. Finally, dehydrate and clear the stained sections, then mount them. Quickly dehydrate with 95% ethanol for 10 seconds, then with 100% ethanol I (30 seconds), 100% ethanol II (1 minute), and clear with xylene twice, 1 minute each time. After air-drying in a fume hood, the slides were sealed with neutral resin. Slides were scanned and images processed using an OLYMPUS VS200 all-glass slide scanner. Three fields of view were selected for each slide, and ImageJ software was used to determine the collagen volume fraction (CVF), which is the percentage of collagen-positive blue area to the total tissue area.

[0181] 1.2.7.6 Sirius Scarlet Staining

[0182] ① Baking and dewaxing to water according to 1.2.7.5.

[0183] ② Staining: Staining was performed according to the instructions of the Sirius red staining kit: drop Sirius red staining solution, stain for 20 min; rinse with flowing tap water for about 5 min to remove excess staining solution; finally, dehydrate, transparentize and mount the sample according to the conventional method, 85% ethanol 1 jar (10 s), 95% ethanol 2 jars (15 s each), 100% ethanol I (30 s), 100% ethanol II (1 min), xylene 3 jars 1 min each. After air drying in the fume hood, the sample was sealed with neutral gum. The sample was observed and photographed under a polarizing microscope. The average optical density value of the type I collagen fibers in each slice was detected by ImageJ software, and the type I collagen content was calculated.

[0184] 1.2.7.7 Immunohistochemical staining

[0185] After paraffin section, the sample was baked in an oven at 65°C for 3 h.

[0186] ① Dewaxing to water: 3 jars, No. I xylene (20 min), No. II xylene (15 min), No. III xylene (15 min), 100% anhydrous ethanol 2 jars, 95% ethanol 2 jars, 85% ethanol 1 jar, a total of 5 jars 10 min each; rinse with flowing water for 5 min.

[0187] ② Antigen repair: immerse the slice in EDTA antigen repair solution, put it in a microwave oven, high heat for 6 min, low heat for 6 min; at room temperature, immerse the slice in the repair solution and cool it to room temperature, PBS immersion wash 3 times, 5 min each time.

[0188] ③ Hydrogen peroxide penetration: absorb the water around the tissue with filter paper, and mark the tissue around the slice with an immunohistochemical pen. According to the instructions of the immunohistochemical kit, add the reagents in turn: first, add reagent 1 endogenous peroxidase blocker to cover the tissue, incubate at room temperature for 30 min in the dark, the purpose is to eliminate the influence of endogenous enzymes and active substances in the tissue, improve specificity, reduce background, and after incubation, wash with PBS 3 times, 5 min each time.

[0189] ④ Goat serum blocking: prepare 10% goat serum blocking solution, add 10% goat serum blocking solution to the tissue in the circle, and put it in a wet box, block in an oven at 37°C for 1 h, the purpose is to block the non-specific binding sites on the tissue slice. ⑤ Primary antibody incubation: after blocking, pour the blocking solution, absorb the liquid around the tissue with a water-absorbing paper, continue to add the primary antibody TNF-α (1:200) or TGF-β (ab215715, 1:200), incubate at 4°C overnight for 15 h.

[0190] (6) Second antibody incubation: The sections were rewarming for 30 min, and TBST was infiltrated for 5 min, repeated 5 times. After the water around the tissue was absorbed, reagent 3, biotin-labeled goat anti-mouse / rabbit IgG polymer, was added dropwise, and the sections were incubated in a 37°C oven for 30 min. TBST was infiltrated for 5 min each time, 5 times in total.

[0191] (7) Color development: DAB reagent was prepared according to the instructions, and the sections were observed under a microscope after the addition of DAB color developing solution. When the color development effect was satisfactory, the sections were placed in a staining jar and washed with flowing tap water for about 5 min to terminate color development.

[0192] (8) Hematoxylin staining: Hematoxylin was added for 2 min, and flowing tap water was used for washing for 5 min. Hydrochloric acid alcohol differentiation solution was continuously added for 5 s, and tap water was used for washing for 5-10 min to reverse blue. Observation was carried out under a microscope.

[0193] (9) Dehydration, transparency, and mounting: Gradient alcohol dehydration was carried out, 1 jar of 85% ethanol, 2 jars of 95% ethanol, 2 jars of 100% anhydrous ethanol, 2 jars of xylene, each for 1 min. After being placed in a fume hood for air drying, neutral balsam was added dropwise for mounting. Under a microscope, the expression of TNF-a (brown) in the wound sections of each group was observed. Scanning and image processing were carried out using an OLYMPUS VS200 whole slide scanner. Three fields of view were randomly selected under a high-power lens for each tissue sample in each group. The color development intensity of TNF-a positive expression was counted using ImageJ software, and the average value was calculated for statistical difference, which was helpful for evaluating the inflammatory response and fibrosis degree after scar treatment in each group.

[0194] 1.3 Statistical analysis method

[0195] SPSS 29.0 (IBM Corp., Armonk, NY, USA, Version 29.0) and GraphPad Prism 9.5 software were used for statistical analysis of experimental data. For measurement data, first, normality test was performed on the data. If the data were normally distributed, the mean ± standard deviation was used to represent the data, and one-way ANOVA was used for further comparison between groups. Bonferroni correction was used for P value correction. After correction, P<0.05 was considered to have statistical significance between groups. If the data of the measurement data were not normally distributed, the interquartile range was used for description, and Kruskal-Wallis test was used for comparison between groups. P<0.05 was considered to have statistical significance between groups.

[0196] 2 Results

[0197] 2.1 Culture and identification of HUC-MSCs

[0198] 2.1.1 Morphological observation of HUC-MSCs

[0199] After the patient and the attending physician's informed consent, the umbilical cord tissue of the full-term cesarean section newborn of the primipara in the childbearing age was obtained, and the umbilical cord tissue was treated under aseptic conditions. Human umbilical cord mesenchymal stem cells (HUC-MSCs) were cultured by tissue adherence method. The P0 generation cells were short rod-like (see Figure 3 A). After the HUC-MSCs were stably subcultured to P4 generation, the cell morphology gradually showed long spindle-shaped arrangement of vortex and fish school (see Figure 3 B).

[0200] 2.1.2 Flow cytometry identification of HUC-MSCs phenotype

[0201] The mesenchymal stem cell markers of P4 generation HUC-MSCs were identified by flow cytometry. The results showed that CD29, CD44 and CD105 were positively expressed and greater than 90%, and the sum of HLA-DR, CD34 and CD45 was less than 2% and was negative, which was consistent with the phenotype characteristics of mesenchymal stem cells, confirming that we successfully isolated and cultured HUC-MSCs by umbilical cord tissue adherence method (see Figure 4 ).

[0202] 2.2 Effect of STEVs pretreatment on the proliferation ability of HUC-MSCs

[0203] 2.2.1 Uptake of STEVs by HUC-MSCs

[0204] First, STEVs were labeled with PKH-26, and after co-cultured with HUC-MSCs for 24 hours, the uptake of STEVs by HUC-MSCs was observed under confocal microscope and photographed. Under the microscope, DAPI staining of cell nucleus showed blue fluorescence, and PKH26 staining of STEVs showed red fluorescence (see Figure 5 ). After merging, red fluorescence constituting the cell outline was observed around the HUC-MSCs nucleus. The results showed that STEVs pretreated HUC-MSCs could successfully uptake STEVs, and this process could lead to changes in cell contents, thereby playing an intervention role.

[0205] 2.2.2 Detection of STEVs inhibiting the proliferation of HUC-MSCs by CCK8 method

[0206] The CCK8 results showed that the STEVs pretreatment did not affect the proliferation cycle of HUC-MSCs, but compared with the Control group, the cell proliferation ability of STEVs was weakened, which may be related to the fact that STEVs carried signal molecules related to the pathological microenvironment of scar, making the cells exist in a state of stress to the disease, resulting in a decrease in the proliferation ability of HUC-MSCs after STEVs pretreatment. This further illustrates that STEVs can represent the pathological microenvironment of scar. Therefore, in order to make HUC-MSC fully respond to the signal changes in the STEVs pretreatment process, we selected a pretreatment time of 24 h. The CCK8 experiment results showed that when pretreated for 24 h, the STEVs intervention concentration of 40 μg / mL had the lowest effect on the proliferation of HUC-MSCs, and the intervention concentration of 40 μg / mL was used in the subsequent experiments (see Figure 6 ).

[0207] 2.3 Grouping of HUC-MSCs pretreated under different conditions and identification of Exos extracted therefrom

[0208] 2.3.1 Identification of Exos

[0209] In order to isolate and identify the obtained exosomes (Exos), the present study used differential centrifugation combined with ultrahigh-speed centrifugation to isolate the corresponding exosomes from the cell supernatant collected under the traditional culture conditions of HUC-MSCs intervened by 40 g / mL STEVs, HUC-MSCs intervened by 40 g / mL STH, and HUC-MSCs without intervention. The transmission electron microscope observation results showed that the morphology of HUC-MSC-Exos in each group presented a typical double-concave disc or tea-tray shape ( Figure 7 A-C), which was consistent with the characteristic morphology of exosomes. Further detection by nanoparticle size analysis (NTA) found that the particle size of HUC-MSC-Exos in each group was mostly distributed between 50 nm and 200 nm ( Figure 7 D-F), which was within the standard particle size range of exosomes.

[0210] Figure 7 A-B are the morphological observation under transmission electron microscope of STEVs-Exos, STH-Exos, and Tra-Exos, respectively, and the scale bar = 100 nm; D-F are the nanoparticle size analysis of STEVs-Exos, STH-Exos, and Tra-Exos, respectively, and the Exos particle size is mainly distributed in the range of 50-200 nm.

[0211] 2.3.2 Exos tracing

[0212] To verify whether the isolated exosomes can be taken up by target cells and play a role in intercellular communication, the experiment used PKH-26 red fluorescence to label STEVs-Exos. After co-culturing the labeled STEVs-Exos with fibroblasts, a large number of scattered red fluorescent STEVs-Exos were observed around the DAPI blue-stained fibroblast nuclei under a microscope, and these fluorescent signals constituted the outline of the cells (see Figure 8 ). This result showed that the isolated exosomes not only met the typical characteristics of exosomes in terms of morphology and particle size, but also could be successfully taken up by cells, thereby playing an effective communication role between cells.

[0213] 2.4 Identification of rabbit ear hypertrophic scar model

[0214] A hypertrophic scar (HS) model was successfully established on the ventral side of the rabbit ear. At 3 weeks after modeling, it was observed that the scar tissue after wound healing was significantly higher than that of normal skin, red, significantly thickened, tough, and the scar surface capillary vessels significantly increased and could fade upon pressure. After taking the material, further histopathological analysis was performed on the normal rabbit ear tissue and the HS tissue after modeling by HE staining, and the results showed that compared with the normal rabbit ear skin, the epidermis and dermis of the HS tissue 3 weeks after modeling were significantly thickened, and there were a large number of inflammatory cell infiltrations. In addition, the Masson staining results showed that compared with the normal rabbit ear skin, the collagen deposition in the dermis of the HS tissue 3 weeks after modeling was significantly increased, and the collagen fibers were arranged in disorder (see Figure 9 ). These results showed that the early hypertrophic scar model of the rabbit ear was successfully constructed, providing a reliable experimental basis for subsequent research.

[0215] Figure 9 In the middle, A. Rabbit ear HS modeling layout; B. General observation of rabbit ear HS 3 weeks after modeling; C. HE staining of normal rabbit ear skin, scale bar = 200 μm; D. HE staining of rabbit ear HS 3 weeks after modeling, scale bar = 200 μm; E. Masson staining of normal rabbit ear skin, scale bar = 200 μm; F. Masson staining of rabbit ear HS 3 weeks after modeling, scale bar = 200 μm.

[0216] 2.5 Efficacy observation of HUC-MSCs-Exos in each group on early treatment of rabbit ear hypertrophic scar

[0217] 2.5.1 STEVs-Exos has a significant effect on improving the appearance of early hypertrophic scar in rabbit ear

[0218] After 3 weeks of rabbit ear modeling, the HS was randomly divided into groups, and PBS (100 L), Tra-Exos (100 g / cm 2 ), STH-Exos (100 g / cm2 ), STEVs-Exos (100 g / cm 2 ). Photographs were taken before injection and evaluated using the Manchester Scar Assessment Scale (MSS), and photographs were taken to observe the scar at different time points, such as 1 week, 2 weeks, 3 weeks, and 4 weeks after injection of Exos, and the Manchester Scar Assessment Scale (MSS) was used for quantitative evaluation. The changes of HS before and after intervention were compared. As shown in Figure 10 A, compared with the PBS control group, all Exos injection groups showed that the color of HS gradually faded, the scar gradually became smaller and softer, the scar gradually became flat, and the thickness gradually thinned, indicating that HUC-MSC-Exos obtained from different pretreatment groups had a significant improvement effect on the appearance of the scar. However, among them, the STEVs-Exos group showed the most significant changes in the color and thickness of the scar 2-4 weeks after injection of Exos. MSS score statistics showed that the PBS group had little change in score before and after injection, and the MSS score of each group after injection of Exos decreased. The score after injection of Tra-Exos showed a gradual decrease, indicating that this group had a certain effect on improving the scar, P < 0.05. The score after injection of STH-Exos showed a decrease, but the decrease was relatively stable, while the MSS score of HS tissue 1-4 weeks after injection of STEVs-Exos decreased significantly, especially during the 2-4 week period, the score decreased most obviously, P < 0.05 (see Figure 10 B). The above results show that injection of Exos has a certain therapeutic effect on improving the scar, however, STEVs-Exos is significantly superior to other groups in the early treatment of rabbit ear hypertrophic scar, and can significantly improve the appearance of the scar.

[0219] Figure 10 A. The general changes of rabbit ear HS before and after injection of each group of HUC-MSC-Exo (0W is before injection, 1-4W is the 1st-4th week after injection of Exos; B. The MSS score column chart of the HS changes of each group of rabbit ear HS before and after injection of Exos, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0220] 2.5.2 STEVs-Exos group reduces the epidermal thickness of rabbit ear HS, reduces the scar index, and improves collagen deposition

[0221] 2.5.2.1 Evaluation of HE staining results of HUC-MSC-Exos in each group acting on rabbit ear HS

[0222] MSS score showed that the score of STEVs-Exos group decreased significantly 2-4 weeks after injection of Exos, and the color and thickness of rabbit ear HS changed more significantly. The tissues at 2W and 4W after injection of Exos were taken for HE staining (see Figure 11 A-B), and the changes of scar epidermis thickness and scar index of each group were counted. HE staining results showed that more cell infiltration was observed in STH-Exo group and STEVs-Exos group at 2 weeks and 4 weeks after injection, indicating the role of inflammatory reaction. The epidermis thickness statistical results showed that compared with PBS group, the epidermis thickness of each group of HUC-MSC-Exos injected into rabbit ear HS was thinner, among which STEVs-Exos was more significant (P<0.05), especially at 4W after injection, the epidermis thickness was the lowest, which had statistical significance, indicating that it might have a stronger inhibitory effect in long-term treatment (see Figure 11 C). The scar index statistical results showed that the scar index of STEVs-Exos group was lower than that of the other groups at 2W after injection of Exos, and the scar index of STEVs-Exos group was significantly reduced at 4W after injection (P<0.05) (see Figure 11 D).

[0223] Figure 11 Among them, A, B are HE staining of rabbit ear HS at 2W, 4W after treatment of each group of HUC-MSC-Exos, scale = 500 μm, 100 μm; C, E are epidermis thickness statistical column chart of each group of HUC-MSC-Exos at 2W, 4W after treatment, n = 6; D, F are scar index statistical column chart of each group of HUC-MSC-Exos at 2W, 4W after treatment, n = 6, *: P<0.05; **: P<0.01; ***: P<0.001.

[0224] 2.5.2.2 Masson staining results of each group of HUC-MSC-Exos acting on rabbit ear HS

[0225] The rabbit ear HS tissues at 2W and 4W after injection of Exos were subjected to Masson staining, and the collagen production and collagen volume ratio of each group were counted. The collagen volume ratio statistical results showed (see Figure 12 B-C), compared with PBS group, the collagen volume of each group of HUC-MSC-Exos injected into rabbit ear HS was reduced at 2W and 4W, and STEVs-Exos was more significant (P<0.05). At the same time, the high-power lens observation of Masson staining (see Figure 12A), the collagen of PBS group was disordered and dense, and the collagen content was the highest, which was consistent with the collagen over-deposition performance of the hypertrophic scar tissue. The collagen arrangement of Tra-Exos group and STEVs-Exos group was more orderly compared with STH-Exos group, and more coarse strip-shaped collagen arrangement was observed in STEVs-Exos group, and the retraction was uniform. The above results showed that STEV-Exos group had a significant advantage in promoting collagen maturation and remodeling.

[0226] Figure 12 A. Masson staining of rabbit ear HS in each group 2W and 4W after treatment of HUC-MSC-Exos group, scale bar = 50 μm; B and C are CVF statistical column charts of each group 2W and 4W after treatment of HUC-MSC-Exos group, n = 6, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0227] 2.5.2.3 Evaluation of Sirius red staining results of HUC-MSC-Exos in each group on rabbit ear HS

[0228] Combined with the results of HE staining and Masson staining, 2W after injection of Exos, STEV-Exos group had shown anti-scarring effect, and we used the tissue 2W after injection of Exos for analysis.

[0229] The rabbit ear HS tissue 2W after injection of Exos was subjected to Sirius red staining, and the contents of type I collagen and type III collagen were calculated by calculating the average optical density values of type I collagen and type III collagen in Sirius red staining. The results of Sirius red staining are shown in Figure 13 A), the orange-red fluorescence of PBS group was more, and the collagen arrangement was more disordered, indicating that the type I collagen deposition was more, and the orange-red fluorescence of STEV-Exos group was reduced, indicating that the type I collagen deposition of the two groups was the least. In addition, compared with Tra-Exos and STH-Exos groups, the collagen fiber arrangement of STEV-Exos group was the most orderly, showing the best tissue remodeling effect. The average optical density value statistics chart of type I collagen showed (see Figure 13 B), the average optical density of type I collagen of Tra-Exos and STEVs-Exos groups was reduced, which indirectly confirmed that Exos could reduce the deposition of type I collagen in scar tissue. Among them, the average optical density of type I collagen of STEV-Exos group was the lowest, which was consistent with the previous Masson statistical analysis, which indicated that STEV-Exos had more significant effect on improving collagen deposition, which helped to reduce the hardness and thickness of scar tissue.

[0230] Figure 13Fig. 6. Hematoxylin-eosin staining of rabbit ear HS tissues at 2W after injection of Exos. A. Hematoxylin-eosin staining of rabbit ear HS tissues at 2W after injection of Exos in each group of HUC-MSC-Exos group, scale bar = 200 pm; B, C are the quantitative statistics of type I collagen and type III collagen at 2W after injection of Exos in each group of HUC-MSC-Exos group, n = 3, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0231] 2.5.3 TNF-a and TGF-b immunohistochemical staining of STEVs-Exos group

[0232] TNF-a immunohistochemical staining of rabbit ear HS tissues at 2W after injection of Exos showed that compared with PBS group, the staining intensity of TNF-a decreased after Exos treatment in each group, but there was no significant difference between groups (see Fig. 6A). Figure 14 C).

[0233] TGF-b immunohistochemical staining of rabbit ear HS tissues at 2W after injection of Exos showed that compared with PBS group, the staining intensity of TGF-b decreased after Exos treatment in Tra-Exos group and STEVs-Exos group, and the staining intensity of TGF-b in STH-Exos group was higher than that in the other groups, P < 0.05 (see Fig. 6B). Figure 14 D). We believe that STEVs-Exos group may remodel collagen by reducing TGF-b expression to improve scar effect.

[0234] Figure 14 Fig. 6. Hematoxylin-eosin staining of rabbit ear HS tissues at 2W after injection of Exos. A. Hematoxylin-eosin staining of rabbit ear HS tissues at 2W after injection of Exos in each group of HUC-MSC-Exos group, scale bar = 200 pm; B, C are the quantitative statistics of type I collagen and type III collagen at 2W after injection of Exos in each group of HUC-MSC-Exos group, n = 3, *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0235] 3 DISCUSSION

[0236] In the present application, we set up a control group (PBS), a Tra-Exos group, an STHExos group, and an STEVs-Exos group, respectively injecting the above Exos into the rabbit ear HS. Through MSS score, HE staining, Masson staining, and Sirius red staining, we evaluated the therapeutic effect of each group of Exos on the rabbit ear HS. The results showed that STEVs-Exos had a significant advantage in improving the appearance of the scar, reducing the thickness of the epidermis, reducing the scar index, and promoting collagen remodeling, significantly better than the other groups of Exos, proving that the STEVs-Exos group showed a more significant therapeutic effect in improving the scar. In this study, although Tra-Exos also had a certain therapeutic effect in improving the scar, due to the lack of specific simulation of the scar microenvironment, its effect was still different compared with the STEVs group. At the same time, the results of this study also showed that compared with STH, STEVs may carry specific signal molecules related to scar maturation, enabling STEVs to more accurately simulate the scar microenvironment.

[0237] In this study, compared with the PBS group, the Tra-Exos and STEVs group showed a decrease in epidermal thickness, a decrease in scar index, and a decrease in collagen deposition 2 weeks after injection of HS, indicating that the anti-fibrosis effect of Exos in each group was significant, which was consistent with the results of previous studies on exosomes improving scars. However, the STEVs group showed the most significant decrease in epidermal thickness and scar index after treatment of HS. During the process of scar formation, the activation and proliferation of fibroblasts lead to the excessive production and abnormal deposition of type I and III collagen, ultimately resulting in the formation of scar tissue (WANG B, ZHANG S, CHENG A, et al. Soluble Polymer Microneedles Loaded with Interferon Alpha 1b for Treatment of Hyperplastic Scar [J]. Polymers (Basel), 2023, 15(12)). Studies have shown that TGF-β1 inhibits the production of type I and III collagen. The TGF-β-mediated Smad signaling pathway is the most typical regulatory pathway for collagen formation in fibroblasts and myofibroblasts. In this study, the picrosirius staining results showed that the Tra-Exos and STEVs-Exos groups had lower type I collagen content, and the immunohistochemical staining results of TGF-β also showed that the Tra-Exos and STEVs-Exos groups had lower TGF-β staining intensity, indicating that the Tra-Exos and STEVs-Exos groups may remodel collagen by reducing TGF-β expression to improve scars. This is consistent with the mechanism reported in previous studies that the activation of the TGF-β / Smad pathway promotes collagen deposition. However, the high expression of TGF-β in the STH-Exos group may be related to the complex components in the tissue homogenate affecting the contents of HUC-MSC-Exos, which needs to be further analyzed by transcriptome sequencing or proteomics. In the Tra-Exos and STEVs-Exos groups, TNF-α was lowly expressed, but there was no statistical difference between the groups. Considering TNF-α as an early inflammatory marker, the expression of TNF-α in the rabbit ear scar may have fallen to the baseline level at the 2-week time point after injection of Exos in the rabbit ear HS in each group, so the regulatory effect after Exos intervention is difficult to highlight statistically. This result suggests that the anti-scar effect of STEVs-Exos may be mainly through fibrosis regulation. Further analysis of the HE staining and Masson staining results showed that the MSS score, scar index, and collagen deposition in the STEVs group were significantly reduced, and the collagen arrangement was more orderly, close to the collagen fiber arrangement results of normal skin, which was consistent with the results of picrosirius staining, further indicating that STEVs-Exos may regulate the proportion of collagen to promote the maturation of scars.

[0238] The above results show that STEVs can more effectively simulate the scar microenvironment, and using STEVs to represent the scar microenvironment for pretreatment of HUC-MSCs can obtain Exos with specificity for scar treatment, which has a significant effect on promoting scar maturation and remodeling. Due to the current experimental techniques and conditions, there are still challenges in large-scale extraction of STEVs and Exos extraction from cell supernatant after STEVs pretreatment, resulting in a small number of animal samples in this experiment and not enough observation time points after Exos injection. Therefore, we were unable to take samples for comparison every week after injection, which affected the evaluation of the dynamic changes in the pathological histological analysis of the therapeutic effect of Exos on scars in each group. In addition, due to the limitation of antibody specificity, the expression of molecules related to extracellular matrix remodeling and fibrosis could not be verified when corresponding molecular detection was performed in the rabbit ear HS tissue. In the next step, we used high-throughput sequencing technology to perform transcriptome sequencing on STEVs and HUC-MSCs pretreated by STH, analyzed the effect of STEVs and STH pretreatment on the gene expression of HUC-MSCs, and revealed the potential molecular mechanisms.

[0239] Example 3 Effect of Scar Tissue Extracellular Vesicles Pretreatment on the Transcriptome Expression Profile of HUC-MSCs

[0240] 1 Materials and Methods

[0241] 1.1 Transcriptomic Analysis Method

[0242] Transcriptomic sequencing technology refers to a technology that can study and analyze the gene transcription activities in tissues or cells at the whole level and reveal the existing regulatory mechanisms. The mRNA sequencing (mRNA-Seq) technology has become a key tool for exploring the changes in the transcriptome in the process of diseases and various biological processes. mRNA-Seq not only has high sensitivity and accuracy in gene expression determination, but also can identify existing transcripts in tissues or cells, find new transcript variants, detect gene fusions and distinguish transcripts from different alleles, and identify allele-specific expression. mRNA-Seq provides an unbiased transcriptome view, enabling researchers to have a comprehensive understanding of the overall picture of gene expression. In this study, transcriptomic analysis was used to evaluate the effect of STH and STEVs pretreated MSCs on the gene expression of HUC-MSCs, with MSCs not pretreated under traditional culture conditions as the control group. First, total RNA was extracted from STH-MSCs, STEV-MSCs, and Tra-MSCs, and then cDNA was synthesized using reverse transcriptase. Next, high-throughput sequencing technology was used to sequence the transcriptome to identify differentially expressed genes. The data obtained by sequencing will undergo strict bioinformatics analysis processes, including quality control, sequence alignment, expression evaluation, and differential expression analysis.

[0243] 1.2 Sample Grouping

[0244] HUC-MSCs of each group were collected according to the method of 1.2.5.1, and the HUC-MSCs of STH and STEV pretreatment groups were named as STH-MSC group and STEV-MSC group, respectively. The HUC-MSCs without pretreatment under traditional culture conditions were named as Tra-MSC (as a control group), and each group had 3 biological replicates.

[0245] 1.3 Extraction and quality control of RNA

[0246] 1.3.1 Extraction of RNA

[0247] We used Trizol reagent method to extract cell mRNA. The specific steps are as follows: first, add 1 mL Trizol reagent to each group of HUC-MSCs, resuspend and beat the cells to lyse them, and transfer the cells + Trizol reagent to a 1.5 mL enzyme-free EP tube, label it, and stand at room temperature for 5 min. Then, add 200 L chloroform and vortex mix for 15 seconds to mix them thoroughly, present a milkshake, and stand at room temperature for 5 min. Centrifuge at 4°C, 12,000 rpm / min for 15 min. After centrifugation, the sample is layered, a new enzyme-free EP tube is taken, and the upper transparent liquid is carefully transferred to the EP tube. Add an equal volume of isopropanol to the new EP tube, mix gently, and stand at room temperature for 10 min. Again, centrifuge at 4°C, 12,000 rpm / min for 15 min. Then, add 1 mL of 75% ethanol (DEPC water: anhydrous ethanol = 1:3), thoroughly wash the tube cover and tube wall, and gently tap the tube bottom to suspend the precipitate. Centrifuge at 4°C, 8,000 rpm / min for 5 min, carefully aspirate the supernatant, and again add 75% ethanol 1 mL to wash the precipitate, centrifuge, and repeat the washing step 2 times. Place at room temperature for 5-10 min to dry the remaining ethanol. Finally, add an appropriate amount of 50 L DEPC water to dissolve the precipitate. After determining the concentration, store the extracted mRNA in a -80°C refrigerator.

[0248] 1.3.2 Quality control of RNA

[0249] The three groups of RNA were detected by agarose gel electrophoresis to be free of contamination and degradation, and the RNA purity (OD260 / 280 ratio) was detected by NanoPhotometer spectrophotometer.

[0250] 1.4 Library construction and sequencing of RNA

[0251] The samples were collected by Chongqing Life Know Source Technology Co., Ltd. for detection. When constructing the transcriptome library, we chose NEBNext ultra RNA Library Prep Kit for Illumina. The kit uses fragmented mRNA as a template, and the primer is a random oligonucleotide. The first strand of cDNA is synthesized under the action of M-MulV reverse transcriptase. Subsequently, after the mRNA strand is degraded by RNaseH, the second strand of cDNA is synthesized using dNTPs under the action of DNA polymerase I. The purified double-stranded cDNA is subjected to end repair, A tail treatment, and then ligated with sequencing adapters. Use AMPure XP beads to screen 250-300 bp cDNA fragments, then perform PCR amplification, and again use AMPure XP beads to purify the PCR product, and finally complete the library construction.

[0252] After the library construction is completed, we first use Qubit 2.0 for preliminary quantification and dilute the library to 1.5 ng / L. Ideally, the average fragment length of the inserted fragments in the library should be between 420 bp and 650 bp, and the "tail" of the longer fragments should not exceed 1 KB. Next, we use Agilent 2100 to detect the distribution of inserted fragments in the library to ensure that the inserted fragments are satisfactory. The effective concentration required for sequencing is higher than 3 nM, therefore, we use qRT-PCR method to accurately quantify the effective concentration of the library to ensure that the quality of the library is satisfactory. After the library quality inspection is passed, in order to obtain high-quality sequencing results, we use Illumina NovaSeq PE150 sequencing strategy, and it is recommended that each sample should be sequenced at least 6G data.

[0253] 1.5 Data analysis

[0254] 1.5.1 Data quality control

[0255] In order to ensure the reliability of the subsequent analysis data, the original Fastq data is based on fastp (v0.20.0) for quality control, and the specified parameters are: -n 15 -q 20 -u 50 --detect_adapter_for_pe, and the rest are default parameters. The detailed quality control filtering steps include: (1) remove sequences with adapters; (2) remove sequences containing more than 15 N bases (N represents undetermined base information); (3) screen low-quality sequences (Qphred≤20 base number accounts for more than 50% of the entire read length); (4) for data with high rRNA content (10%), choose to remove rRNA sequences using bbduk. After the original data is filtered according to the above steps, it becomes effective data (Clean Reads), which ensures the high quality of the data and the accuracy of the subsequent analysis.

[0256] 1.5.2 Reference genome alignment

[0257] The quality controlled Clean Reads were aligned to the reference genome to determine the genomic coordinates of each fragment. Efficient and accurate alignment was performed using Hisat2 (v2.2.1) software to obtain the positioning information of reads on the reference genome, and then the results were sorted using samtools (v1.15.1) and bam format files were generated. For the selection of reference genome files (fasta files) and annotation files (gtf / gff files), Zhiyuan Gene prefers to use the reference files provided by the database, commonly used databases such as Ensembl, NCBI, GENECOD, etc. The Hisat2 specified parameters are: --met-stderr --new-summary --dta --threads 10 --no-mixed --no-discordant, and the rest are default parameters.

[0258] 1.5.3 Gene expression quantification

[0259] Based on the accurate and fast quantification of reads number to each gene by featureCount (v2.0.1), the original Count expression profile matrix was generated; based on stringtie (v2.1.6), the FPKM expression profile matrix of genes was obtained. Compared with other transcript assembly software, stringtie not only performs well in the accuracy of gene assembly and the estimation of gene expression, but also has more abundant assembled transcripts. The featureCounts specified parameters are: -T 10 -p -t exon -g gene_id, and the rest are default parameters, and stringtie uses default parameters for analysis.

[0260] 1.5.4 Differential expression analysis

[0261] We automatically performed differential analysis based on the original Count matrix data on whether the project has repeated experiments: 1. For data with biological replicates, use the DESeq2 R package (v1.32.0) to perform differential expression analysis on the original Count expression matrix. DESeq2 performs differential analysis based on the negative binomial distribution model. DESeq2 uses Wald test by default for calculation. 2. For no biological replicates, the edgeR package (v3.34.0) uses the weighted truncated mean of the log expression ratio between samples for TMM correction, and the Fisher test based on the negative binomial distribution is used for P value calculation. Finally, the P value needs to be corrected for multiple hypothesis testing to get the FDR value (false positive rate, i.e. the probability of P value error), which uses the Benjamini & Hochberg (BH) algorithm by default; the threshold for screening differential genes is:

[0262] |log2FoldChange|>0&pvalue<0.05.

[0263] 1.5.5 Differential expression gene enrichment analysis

[0264] 1.5.5.1 GO function analysis

[0265] GO (Gene Ontology) is a comprehensive database that classifies genes mainly through their essential functions, and then clearly describes the functions of genes and proteins. GO database divides the ontology of genes into three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). In this study, we used the clusterProfiler (4.0.0) R package for GO enrichment analysis, and we selected padj less than 0.05 as the significant enrichment result.

[0266] 1.5.5.2 KEGG pathway analysis

[0267] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database that integrates genomic, chemical, and system function information. The KEGG pathway database covers seven pathways: metabolism, genetic information processing, environmental information processing, cellular processes, organism systems, human diseases, and drug development. We used clusterProfiler to perform pathway enrichment of differential genes based on KEGG, and selected padj less than 0.05 as the significant enrichment result.

[0268] 2 Results

[0269] 2.1 Differential gene result statistics

[0270] The transcriptome of each group of HUC-MSCs was analyzed by high-throughput sequencing technology. After analyzing the differential significance of each comparison group, the differential gene statistics table was sorted out (see Table 6), and the top 10 differential gene results in each comparison analysis group were shown (see Table 7).

[0271] Table 6. Differential gene statistics table

[0272]

[0273]

[0274] Table 7. Differential gene list

[0275]

[0276] Volcano Plot is a scatter plot used for visualizing the data of differentially expressed genes. This scatter plot combines the significance level in statistical test and the change amplitude, and the data points (genes) with large change amplitude and statistically significant difference can be quickly and intuitively identified. We visualized the differential genes of each comparison group by Volcano Plot based on the results of the differential gene comparison analysis (see Figure 15 ).

[0277] Based on the above analysis, STEV pretreatment had the most significant effect on the gene expression of HUC-MSCs, involving the most up-regulated and down-regulated genes. These differentially expressed genes were mainly related to cell proliferation, differentiation and inflammation-related genes such as FOS, EGR1, CXCL12, etc., and their expression changes might directly affect the maturation and remodeling of scars.

[0278] 2.2 GO functional enrichment analysis

[0279] In this study, we used the clusterProfiler tool to perform functional annotation of differential genes at three levels of GO, and P value less than 0.05 was used as the standard for significant enrichment.

[0280] Figure 16 In this study, we used the clusterProfiler tool to perform functional annotation of differential genes at three levels of GO, and P value less than 0.05 was used as the standard for significant enrichment. Figure 16As shown, compared with the Tra-MSC group, the upregulated genes in the STEV-MSC group were mainly enriched in biological processes related to cell development, differentiation, and environmental adaptation; the construction of the extracellular environment, including the extracellular matrix, vesicles, and cell membranes; and molecular functions related to cell signaling and ion homeostasis. Conversely, the downregulated genes in the STEV-MSC group were mainly enriched in biological processes involved in maintaining intracellular homeostasis; the structure of the nucleus, including the nucleolus and cytoskeleton; and molecular functions related to gene expression regulation.

[0281] Figure 17 In Figure A, the STEV-MSC group vs. the STH-MSC group shows upregulated gene enrichment; in Figure B, the STEV-MSC group vs. the STH-MSC group shows downregulated gene enrichment. Figure 17 As shown, compared with the STH-MSC group, the upregulated genes in the STEV-MSC group were mainly enriched in biological processes regulating cell cycle progression, participating in the construction of the extracellular environment and cell membrane-related functions, and molecular functions involved in signal transduction and enzyme activity regulation. Conversely, compared with the STH-MSC group, the downregulated genes in the STEV-MSC group were mainly enriched in biological processes maintaining intracellular homeostasis and cell cycle regulation, participating in the construction of nuclear structure and cytoskeleton, and molecular functions involved in nucleic acid metabolism and energy metabolism.

[0282] Figure 18 Figure A shows the upregulated gene enrichment function in the STH-MSC group versus the Tra-MSC group; Figure B shows the downregulated gene enrichment function in the STH-MSC group versus the Tra-MSC group.

[0283] like Figure 18 As shown, compared with the Tra-MSC group, the genes upregulated in the STH-MSC group were mainly enriched in biological processes such as cell proliferation and differentiation, involved in the construction of the extracellular environment and cell membrane-related functions, and involved in molecular functions of nucleic acid metabolism and energy metabolism. Conversely, the genes downregulated in the STH-MSC group were mainly enriched in biological processes maintaining intracellular homeostasis and cell cycle regulation, involved in the construction of nuclear structure and cytoskeleton, and involved in molecular functions of enzyme activity regulation and energy metabolism.

[0284] 2.3 KEGG pathway enrichment analysis

[0285] KEGG excels in integrating metabolic pathway queries, covering the metabolism of carbohydrates, nucleosides, amino acids, and the biodegradation of organic matter. To visually demonstrate the results of KEGG enrichment of pathways, the top 20 most significant KEGG Pathways were selected, and bar charts were created to show the KEGG Pathway enrichment results for differentially expressed genes in each comparison group.

[0286] Figure 19In the study, A. STEV-MSC group vs. Tra-MSC group showed downregulated gene enrichment pathways; B. STEV-MSC group vs. Tra-MSC group showed upregulated gene enrichment pathways. (Example) Figure 19 As shown, compared with the Tra-MSC group, the STEV-MSC group showed that the upregulated gene pathways were mainly enriched in the PI3K-Akt signaling pathway, MAPK signaling pathway, extracellular matrix receptor interaction (ECM-receptor interaction), and focal adhesion. These pathways are related to cell proliferation, differentiation, signal transduction, and cell adhesion, and involve biological processes such as cell cycle regulation, extracellular matrix remodeling, and inflammatory responses, indicating that these upregulated genes promote cell development and adaptive responses. Conversely, the STEV-MSC group showed that the downregulated gene pathways were mainly enriched in the cell cycle, oocyte meiosis, Fanconi anemia pathway, homologous recombination, and p53 signaling pathway. These pathways are related to cell cycle regulation, DNA repair, and cellular stress responses, indicating that these downregulated genes are involved in maintaining normal cell function and structural stability.

[0287] Figure 20 In the study, A. STEV-MSC group vs. STH-MSC group showed downregulated gene enrichment pathways; B. STEV-MSC group vs. STH-MSC group showed upregulated gene enrichment pathways. For example... Figure 20 As shown, compared with the STH-MSC group, the STEV-MSC group showed enrichment of upregulated gene pathways mainly in the PI3K-Akt signaling pathway, MAPK signaling pathway, extracellular matrix receptor interaction (ECM-receptor interaction), and cancer-related pathways, involving biological processes such as signal transduction, extracellular matrix remodeling, and inflammatory responses, indicating that these upregulated genes promote cell development and adaptive responses. Compared with the STH-MSC group, the STEV-MSC group showed enrichment of downregulated gene pathways mainly in the cell cycle, DNA replication, Fanconi anemia pathway, homologous recombination, and p53 signaling pathway, related to cell cycle regulation, DNA repair, and cellular stress responses, indicating that these downregulated genes are involved in maintaining normal cell function and structural stability.

[0288] Figure 21 In the study, A. STH-MSC group vs. Tra-MSC group showed downregulated gene enrichment pathways; B. STH-MSC group vs. Tra-MSC group showed upregulated gene enrichment pathways. For example... Figure 21As shown, the pathways of up-regulated genes in the STH-MSC group were mainly enriched in DNA replication, Cell cycle, Focal adhesion, Gap junction, etc. compared with the Tra-MSC group. These pathways were related to cell proliferation, adhesion and signal transduction, and involved in biological processes such as ECM-receptor interaction, protein digestion and absorption, fluid shear stress and atherosclerosis, indicating that these up-regulated genes promote the development and adaptive response of cells. While the pathways of down-regulated genes in the STH-MSC group were mainly enriched in Cytokine-cytokine receptor interaction, Pertussis, Rheumatoid arthritis, Legionellosis, etc. These pathways were related to immune response, inflammation and infection, indicating that these genes may play a role in immune regulation and pathogen defense, and involved in biological processes such as lipid and atherosclerosis, inflammatory bowel disease, TGF-beta signaling pathway, IL-17 signaling pathway, etc., indicating that the down-regulated genes are related to the immune regulation and pathogen defense ability of cells.

[0289] 3Discussion and conclusion

[0290] Since the viability and bioactivity of MSCs are affected by donor status and in vitro culture conditions, and the survival and therapeutic capacity of MSCs are strongly dependent on their cellular behavior, such as cell proliferation, cell migration, and paracrine effects, there are certain challenges in the process of pre-treating HUC-MSCs with disease-derived EVs. The formation of scars involves a variety of biological processes, cellular components, and molecular functions. In this study, transcriptomic analysis showed that the differential genes of STEVs pre-treated HUC-MSCs were the most significant, with the largest number of up-regulated and down-regulated genes involved, indicating that STEVs pre-treatment significantly changed the gene expression profile of HUC-MSCs. These differentially expressed genes were mainly related to genes associated with cell proliferation, differentiation, and inflammatory response. (ZHANG L, QIN H, WU Z, et al. Identification of the potential targets for keloid and hypertrophic scar prevention [J]. J Dermatolog Treat, 2018, 29(6): 600-605) Zhang et al. have reported that FOS / ERG1 could be a new target for preventing skin scarring. In the differential genes of STEVs and STH groups, early response genes such as FOS, ERG1 were up-regulated, which are involved in cell response to changes in the culture microenvironment, enabling HUC-MSCs to have proliferative capacity in the simulated scar microenvironment pre-treatment. In this study, the results of CCK8 experiment also showed that STEVs pre-treatment affected the proliferative capacity of HUC-MSCs to some extent, but did not affect the cell morphology and passage cycle of HUC-MSCs.

[0291] In the differential genes of STEVs group, ECM-related genes such as COL14A1, CXCL12 were up-regulated, and CXCL12 was proved to improve the level of fibrosis (WU X, QIAN L, ZHAO H, et al. CXCL12 / CXCR4: An amazing challenge and opportunity in the fight against fibrosis [J]. Ageing Res Rev, 2023, 83: 101809). The increase of COL14A1 helps to improve the remodeling of ECM by regulating the formation of collagen fibers (KOLOKONGASSIE M L, DE VRIES M, BORGHUIS T, et al. Age-associated differences in the human lung extracellular matrix [J]. Am J Physiol Lung Cell Mol Physiol, 2023, 324(6):

[0292] Further GO functional enrichment analysis showed enrichment in "extracellular region", "vesicle" and other cell components, which enhanced the secretion process of HUC-MSCs.

[0293] In summary, we analyzed the exosomes secreted by STEVs group pretreated HUC-MSCs, which may directly affect the maturation and remodeling of the scar through the regulation of ECM. In this study, the down-regulation of HJURP and KIF2C in the STEVs group was significant, and these down-regulated genes were cell cycle-related genes, indicating that the cell proliferation ability in the scar tissue was inhibited. At the same time, the down-regulation of MKI67 further supported the inhibition of cell proliferation. At the same time, the down-regulation of IL1B and other inflammatory factors in the STEVs group reflected the presence of an anti-inflammatory phenotype. Further GO functional enrichment analysis and KEGG pathway enrichment analysis showed that these differentially expressed genes were mainly enriched in cell cycle regulation, cell homeostasis, extracellular matrix remodeling, signal transduction, and other aspects. At the same time, some signal pathways such as "cell cycle regulation" and "extracellular matrix receptor interaction" also had differential gene enrichment. In summary, the transcriptome analysis results showed that the up-regulated genes in the SETVs group in the extracellular matrix and cell membrane and other cell components may be related to the remodeling of the extracellular matrix of the scar. The down-regulated genes in the STEVs group in the cell cycle were involved in the regulation of the cell cycle and the inhibition of cell proliferation in HUC-MSCs, which enabled HUC-MSCs to inhibit the proliferation of the scar, and finally enabled the Exos secreted by HUC-MSCs to have specificity for the treatment of the scar. This is consistent with the results of the animal experiment.

[0294] Conclusion: The exosomes of human umbilical cord mesenchymal stem cells pretreated by extracellular vesicles of scar tissue have a significant improvement in the treatment of early proliferative scar in rabbit ears, which is manifested by improving the appearance of the scar, reducing the scar index, promoting collagen remodeling, and promoting the maturation of the scar.

[0295] The research of the present application shows that the extracellular vesicles of the scar tissue carry pathological information related to the scar, can represent the microenvironment of the scar, and can activate the related signal pathways of extracellular matrix remodeling and cell cycle regulation in human umbilical cord mesenchymal stem cells, thereby obtaining exosomes with specificity for the treatment of the scar.

Claims

1. Human umbilical cord mesenchymal stem cell exosomes for treating scars, extracted from human umbilical cord mesenchymal stem cells pre-treated with extracellular vesicles from scar tissue.

2. Use of human umbilical cord mesenchymal stem cell exosomes according to claim 1 in the preparation of a medicament for improving and treating scars.

3. Use according to claim 2, wherein the scars are skin scars.

4. A method for preparing human umbilical cord mesenchymal stem cell exosomes according to claim 1, comprising the following steps: 1) Isolating and extracting extracellular vesicles (STEVs) from ex vivo scar tissue, and preparing a serum-free complete medium for human umbilical cord mesenchymal stem cells containing STEVs; 2) Taking the umbilical cord stripped of the umbilical vein, umbilical artery and outer membrane, cutting it into small pieces, culturing, digesting, and collecting primary cells HUC-MSCs; 3) Culturing the primary cells HUC-MSCs in a cell incubator, and subculturing when the cell density reaches 80-90%, until P4 HUC-MSCs are obtained; 4) The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5 The cell density of the P4 generation HUC-MSCs of the previous step P4 is diluted to 1x10 5. The preparation method according to claim 4, comprising the following steps: 1) Taking ex vivo scar tissue, and isolating and extracting extracellular vesicles STEVs by tissue cutting treatment, enzymatic dissociation, gradient size exclusion, and differential centrifugation combined with ultrahigh-speed centrifugation, and preparing a serum-free complete medium for human umbilical cord mesenchymal stem cells containing STEVs; 2) Taking the umbilical cord tissue of a newborn, stripping the umbilical vein, umbilical artery and outer membrane, and then washing the remaining tissue (known as Wharton's jelly) with sterile PBS, cutting it into small pieces, and placing it in a culture medium to culture and digest to collect primary cells HUC-MSCs; 3) Culturing the primary cells HUC-MSCs in a cell incubator at 37°C and 5% CO2 for 48-72 h, and observing under a microscope that the HUC-MSCs grow in a long spindle shape and fish swarm vortex shape, and that the cells can be subcultured when the cell density reaches 80-90%; 4) Rinsing the HUC-MSCs with sterile PBS containing 1% penicillin / streptomycin, adding stem cell mild digestion enzyme, and adding a serum-free medium for human umbilical cord mesenchymal stem cells for culture, and subculturing when the cell fusion density reaches 80%-90%; 5) Take P4 generation HUC-MSCs with good cell state and density of 80% to 90%, wash the cells with PBS, digest and centrifuge, count the cells, dilute the cell density of the cell suspension to 1×10 5 6) Add the serum-free complete culture medium containing STEVs and human umbilical cord mesenchymal stem cells in step 1) to the cell suspension, mix thoroughly. 6) Resuspending and mixing uniformly, and then placing in a cell incubator at 37°C and 5% CO2 for 24 h, discarding the original culture medium, and replacing it with a new serum-free complete medium for human umbilical cord mesenchymal stem cells without pre-treatment for further culture for 48 h; 7) Collecting the corresponding supernatant after 48 h of culture, and isolating and extracting HUC-MSCs-Exos by differential centrifugation combined with ultrahigh-speed centrifugation.

6. The preparation method according to claim 4, wherein the isolating and extracting extracellular vesicles STEVs from ex vivo scar tissue in step 1) comprises: a) Dissociating the ex vivo scar tissue with a dissociation solution containing collagenase type IV and DNase I enzyme, filtering, and collecting the filtrate; b) Differential centrifugation of the filtrate to remove tissues and impurities, collecting the filtrate, and separating by different size filter screens, and centrifuging the filtrate at 110,000 x g for 70 min, discarding the supernatant, and resuspending the precipitate with sterile PBS; c) The resuspended pellet was transferred to an ultrafiltration tube and purified by centrifugation at 5000 x g for 15 min, the upper chamber liquid was collected, filtered, and the filtrate was centrifuged at 110,000 x g, the supernatant was discarded, and the resuspended pellet was obtained by resuspending in PBS.

7. The preparation method of claim 6, further comprising the following steps: 1) The ex vivo scar tissue was washed with sterilized PBS (containing 1% BSA), the water was absorbed, and the tissue was cut into small pieces; 2) The tissue dissociation solution was prepared: DMEM high-glucose medium containing collagenase IV (2 mg / mL) and DNase I enzyme (40 U / mL); 3) The tissue dissociation solution was added to the small tissue pieces, and after dissociation, centrifugal filtration was performed, and the filtrate was sequentially passed through 70 μm and 454 m funnel-shaped nylon filters, and the filtrate was collected; 4) The last collected filtrate was subjected to differential centrifugation at 4°C: 800 x g for 10 min, the supernatant was collected, 3,000 x g for 20 min, the supernatant was collected again, 3,000 x g for 20 min, the precipitate was discarded, and the supernatant was collected; 5) The collected supernatant was sequentially passed through 204 m and 104 m funnel-shaped nylon filters, and the supernatant was collected by gravity flow into a centrifuge tube; 6) The supernatant of the previous step was centrifuged at 16,500 x g for 30 min at 4°C, the precipitate was removed, and the supernatant was collected; 7) The supernatant of the previous step was further passed through a 2.54 m funnel-shaped nylon filter, and the liquid was collected into a centrifuge tube, and the supernatant was collected; 8) The supernatant of the previous step was transferred to a 30 mL polypropylene ultracentrifuge tube, filled with PBS, sealed, and balanced to 0.01 mg, and then centrifuged at 110,000 x g for 70 min at 4°C using an ultracentrifuge, the supernatant was discarded, and the precipitate was resuspended in 1 mL of sterile PBS; 9) The resuspended pellet was transferred to an ultrafiltration tube and centrifuged at 5000 x g for 15 min at 4°C, the upper chamber liquid was collected, washed with PBS, and the resuspension was sequentially passed through 0.84 m, 0.454 m, and 0.224 m needle filters, and the filtrate was collected; 10) The filtrate of the previous step was transferred to a polypropylene ultracentrifuge tube, filled with PBS, sealed, and centrifuged at 110,000 x g for 70 min at 4°C using a benchtop ultracentrifuge, the supernatant was discarded, and the precipitate was resuspended in 5004 L of sterile PBS, thereby obtaining the scar cell extracellular vesicles STEVs.

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