Use of airway basal stem cell-derived extracellular vesicles in the preparation of a product for treating airway stenosis

Abstract

The application discloses application of airway basal stem cell-derived extracellular vesicles in preparation of products for treating airway stenosis. The application first proposes that the airway basal stem cell-derived extracellular vesicles have excellent airway stenosis treatment effect. In the case that no stent is placed, the airway basal stem cell-derived extracellular vesicles are directly injected into the airway at a stenosis position, so that the airway wall collagen is less, airway wall remodeling is improved, granulation tissue proliferation is inhibited, and then the airway lumen is enlarged, the stenosis symptoms are significantly relieved, and the survival cycle is prolonged, which provides a new technical selection for curing airway stenosis, preventing airway stenosis recurrence, reducing stem cell therapy risk and improving patient prognosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular, to the application of airway basal stem cell-derived extracellular vesicles in the preparation of products for treating airway stenosis. BACKGROUND

[0002] Airway stenosis is a common disease in interventional respiratory medicine, and the main pathological change is obvious granulation tissue proliferation, which affects the respiratory function of patients and even causes suffocation in severe cases. The treatment methods for airway stenosis are surgical treatment and bronchoscopic interventional treatment. Surgical treatment has high requirements for the physical condition of patients and the operation of the operator, and only a small number of patients can tolerate surgical treatment, and some patients have obvious granulation tissue proliferation at the anastomotic site, causing secondary stenosis. For patients with airway stenosis who cannot accept surgery, bronchoscopic interventional treatment is the main treatment method, which maintains the patency of the airway by placing a stent in the airway. The short-term effect of this treatment is obvious, but the stent continuously rubs the airway, which is easy to form new granulation tissue, and cannot inhibit the continued proliferation of the formed granulation tissue, so that the airway of the patient often becomes stenosis again within a few weeks or months, resulting in the need for the patient to receive bronchoscopic interventional treatment multiple times to maintain the diameter of the stenosis segment of the trachea. It can be seen that both surgical treatment and bronchoscopic interventional treatment are restricted by many aspects, and cannot effectively treat airway stenosis.

[0003] Airway basal stem cells (BSCs) as progenitor cells of airway epithelium exhibit strong proliferation and differentiation potential, and play a key role in the repair process of airway injury. The prior art (CN113827617B) has found that BSCs autologous transplantation can inhibit the proliferation of granulation tissue in the stenosis segment, but in this technical solution, a metal stent must be implanted in the airway in advance to stabilize the stenosis segment of the airway and support the colonization and growth of BSCs after transplantation, and the operation is complex, and there is a risk of immune rejection and introduction of exogenous infection sources for BSCs. On the other hand, the granulation proliferation caused by stent placement is one of the most common complications.

[0004] Therefore, it is urgent to develop a treatment method that does not place a stent, can effectively cure airway stenosis, prevent the recurrence of airway stenosis, and reduce the risk of stem cell therapy. SUMMARY

[0005] In order to solve the above-mentioned problems existing in the prior art, the present application provides the application of airway basal stem cell-derived extracellular vesicles in the preparation of products for treating airway stenosis.

[0006] The first object of the present application is to provide the application of airway basal stem cell-derived extracellular vesicles in the preparation of products for treating airway stenosis.

[0007] A second object of the present application is to provide the use of airway basal stem cell-derived extracellular vesicles in the preparation of a product for inhibiting the proliferation of airway granulation tissue.

[0008] A third object of the present application is to provide the use of airway basal stem cell-derived extracellular vesicles in the preparation of a product for maintaining the patency of airway lumen.

[0009] A fourth object of the present application is to provide the use of airway basal stem cell-derived extracellular vesicles in the preparation of a product for improving airway wall remodeling.

[0010] A fifth object of the present application is to provide a method for preparing airway basal stem cell-derived extracellular vesicles.

[0011] A sixth object of the present application is to provide a biological agent.

[0012] A seventh object of the present application is to provide the use of the biological agent in the preparation of a product for treating airway stenosis.

[0013] To achieve the above objects, the present application is implemented by the following scheme:

[0014] Extracellular vesicles (EVs) are key mediators of biological signal communication between cells. The present application found that airway basal stem cell-derived extracellular vesicles (BSC-EVs) play a key role in treating airway stenosis, inhibiting fibroblast activation in vitro, reducing granulation tissue proliferation, maintaining collagen maturation and stability in granulation tissue, and reducing airway stenosis in vivo.

[0015] Therefore, the present application claims the following:

[0016] The use of airway basal stem cell-derived extracellular vesicles in the preparation of a product for treating airway stenosis.

[0017] According to the cause, airway stenosis can be divided into benign (non-cancerous) and malignant (cancer-related). Benign airway stenosis is caused by inflammation, infection, trauma, postoperative scar tissue formation, foreign body obstruction, congenital abnormalities, etc., and can usually be improved by medical intervention. The present application intervenes in the animal model of benign airway stenosis with obvious granulation tissue proliferation by using airway basal cell-derived extracellular vesicles, and finds that the animals treated after intervention show reduced fibroblast activation, reduced granulation tissue proliferation, more mature and stable collagen in the granulation tissue, and more unobstructed lumen, which indicates that airway basal cell-derived extracellular vesicles can reduce granulation tissue proliferation after tracheal injury, reduce tracheal stenosis, and improve survival rate after tracheal injury, and have significant therapeutic effect on benign airway stenosis. Therefore, as a preferred scheme, the present application also claims the use of airway basal stem cell-derived extracellular vesicles in the preparation of products for treating benign airway stenosis.

[0018] The use of airway basal stem cell-derived extracellular vesicles in the preparation of products for inhibiting airway granulation tissue proliferation should also be within the scope of protection of the present application.

[0019] The use of airway basal stem cell-derived extracellular vesicles in the preparation of products for maintaining airway lumen patency should also be within the scope of protection of the present application.

[0020] Airway remodeling refers to the long-term changes in airway structure under chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), etc., including thickening of airway smooth muscle, thickening of basement membrane, proliferation of mucous glands and goblet cells, increased angiogenesis, and increased extracellular matrix deposition, etc. Airway remodeling can cause or aggravate airway stenosis, increase airway reactivity, and can affect lung function. The present application intervenes in the animal model of airway stenosis by using airway basal cell-derived extracellular vesicles, so that the degree of fibrosis of the animal airway is reduced, and the remodeling of extracellular matrix and tissue repair is improved, the airway wall collagen is less, and the airway wall remodeling is significantly improved. Therefore, the present application also claims the use of airway basal stem cell-derived extracellular vesicles in the preparation of products for improving airway wall remodeling should also be within the scope of protection of the present application.

[0021] A method for preparing airway basal stem cell-derived extracellular vesicles, collecting cell supernatant of cultured airway basal stem cells, and separating to obtain extracellular vesicles.

[0022] Preferably, the airway basal stem cells are primary airway basal stem cells.

[0023] More preferably, the primary airway basal stem cells are human primary airway basal stem cells.

[0024] Further preferably, the human primary airway basal stem cells are from human 3 to 5 grade bronchial epithelium.

[0025] Preferably, the cell supernatant is collected from the airway basal stem cells in proliferation culture.

[0026] Preferably, the airway basal stem cells are cultured with BSCs special medium, which is the proliferation medium described in the prior art “CN117778294A”.

[0027] More preferably, the BSCs special medium is medium 1 of Example 1 in the prior art “CN117778294A”.

[0028] Preferably, the airway basal stem cells are cultured with cell factories.

[0029] Preferably, the extracellular vesicles are isolated by ultracentrifugation method.

[0030] A biological preparation, the active ingredient of which comprises airway basal stem cell-derived extracellular vesicles.

[0031] Preferably, the content of the airway basal stem cell-derived extracellular vesicles in the biological preparation is 0.5×10 11 particle / mL~1.5×10 11 particle / mL.

[0032] More preferably, the content of the airway basal stem cell-derived extracellular vesicles in the biological preparation is 0.8×10 11 particle / mL~1.2×10 11 particle / mL.

[0033] Further preferably, the content of the airway basal stem cell-derived extracellular vesicles in the biological preparation is 1×10 11 particle / mL.

[0034] Preferably, the biological preparation further comprises a pharmaceutically acceptable carrier.

[0035] More preferably, the pharmaceutically acceptable carrier comprises PBS and / or physiological saline.

[0036] The use of the biological preparation in the preparation of a product for treating airway stenosis should also be within the protection scope of the present application.

[0037] The use of the biological preparation in the preparation of a product for inhibiting airway granulation tissue proliferation should also be within the protection scope of the present application.

[0038] The use of the biological preparation in the preparation of a product for maintaining the patency of the airway lumen should also be within the protection scope of the present application.

[0039] The use of the biological preparation in the preparation of a product for improving airway wall remodeling should also be within the protection scope of the present application.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] The present application first proposes that airway basal stem cell-derived extracellular vesicles have excellent airway stenosis treatment effects. In the absence of stent placement, airway basal stem cell-derived extracellular vesicles are directly injected into the airway at the stenosis site, so that less collagen is deposited in the airway wall, airway remodeling is improved, and granulation tissue proliferation is inhibited, thereby expanding the airway lumen, significantly relieving the stenosis symptoms, and prolonging the survival cycle. It provides a new technical choice for curing airway stenosis, preventing airway stenosis recurrence, reducing the risk of stem cell therapy, and improving the prognosis of patients. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The immunofluorescence identification results of the proliferation and differentiation ability of BSCs.

[0043] Figure 2 The HE staining identification results of the differentiation ability of BSCs.

[0044] Figure 3 The TEM identification results of BSC-EVs.

[0045] Figure 4 The NTA identification results of BSC-EVs.

[0046] Figure 5 The WB identification results of BSC-EVs, BSC represents the total protein sample of BSCs, Nucleus represents the nuclear protein sample of BSCs, Cytosol represents the cytoplasmic protein sample of BSCs, and N1-N3 represent three parallel prepared total protein samples of BSC-EVs.

[0047] Figure 6 The alpha-SMA cell immunofluorescence detection results of human primary airway fibroblasts treated with different concentrations of BSC-EVs; A is the fluorescence staining image of the cells in each group; B is the statistical result of the alpha-SMA cell positive rate in A.

[0048] Figure 7 The gel contraction experiment results of human primary airway fibroblasts treated with different concentrations of BSC-EVs; A is the bright field image of the cells in each group; B is the gel area statistical result in A.

[0049] Figure 8WB detection results of human primary airway fibroblasts treated with different concentrations of BSC-EVs; A is the WB strip chart; B-C are the protein expression level analysis results of Collagen I and FAP, respectively.

[0050] Figure 9 Survival analysis results of benign airway stenosis animal models in the PBS group and the BSC-EVs group.

[0051] Figure 10 Therapeutic evaluation results of benign airway stenosis animal models in the PBS group and the BSC-EVs group, from left to right are the results of bronchoscopic performance, HE staining and MASSON staining.

[0052] Figure 11 WB detection results of airway tissues of benign airway stenosis animal models in the PBS group and the BSC-EVs group, A is the WB strip chart; B is the protein expression level analysis results of FAP, Collagen I and extracellular matrix remodeling related proteins (MMP2, MMP3, MMP9, TIMP1, TIMP2). DETAILED DESCRIPTION

[0053] The application will be further described in conjunction with the drawings of the specification and specific embodiments, which are used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0054] Example 1 Isolation, purification, culture and identification of primary airway basal stem cells (BSCs)

[0055] 1. Isolation and purification of BSCs

[0056] BSCs are isolated from humans as the collection object, which specifically includes the following steps:

[0057] (1) Brush the 3-5 grade bronchial epithelium through the fiberoptic bronchoscope, use a 15ml centrifuge tube to recover the front end of the brush, and place it in a container with ice blocks, which is transported to the laboratory within 1 hour.

[0058] (2) Thoroughly rinse the brush with BSCs special medium (i.e. medium 1 in Example 1 of the prior art “CN117778294A”), collect the rinse liquid and centrifuge, and collect the cell precipitate.

[0059] (3) Resuspend the cell precipitate with BSCs special medium, and transfer it to a T25 cell culture flask, which is placed in a cell culture incubator at 37°C with 5% carbon dioxide for culture.

[0060] (4) During the first three days of culture, change the culture medium daily to remove red blood cells, non-adherent cells, and other impurities to obtain purified BSCs. Once the BSCs have grown to a density of approximately 50% to 70%, they can be passaged.

[0061] 2. Cultivation of BSCs

[0062] Promoting BSC differentiation using gas-liquid interface culture technology includes the following steps:

[0063] (1) Proliferation culture

[0064] BSCs were cultured in a dedicated BSC medium until the growth density reached 90%–95%. The cells were then digested and the cell count was determined. Afterward, the BSCs were cultured at a density of 2 × 10⁶ cells per well. 5 One cell was seeded into the upper chamber of a 24-well Transwell plate (catalog number: 3470, Corning), and 800 μL of proliferation medium was added to the lower chamber. The plate was then cultured in a 37°C, 5% CO2 cell culture incubator and passaged at a growth density of 80%–90%.

[0065] (2) Differentiation culture

[0066] Remove the culture medium from the upper chamber of the Transwell plate, leaving it in a dry, liquid-free state. Simultaneously, replace the culture medium in the lower chamber with differentiation-inducing medium (PneumaCult). TM -ALI Medium, Stemcell, 05001) continued to be cultured. The differentiation induction medium in the lower chamber was changed every 48 hours, and the upper chamber was washed with PBS every 5 days to maintain the gas-liquid interface culture environment. The culture was carried out for a total of 21 days.

[0067] 3. Identification of BSCs

[0068] (1) Immunofluorescence detection

[0069] The method for identifying the proliferation and differentiation potential of BSCs includes the following steps: After BSCs adhered to the culture vessel for 2 days, they were fixed with 4% paraformaldehyde. The fixative was removed, and the cells were treated with Triton X 100 for 30 minutes to permeate the cell membrane. Then, they were blocked with 5% BSA for 30 minutes, followed by overnight incubation with primary antibody at 4°C. The next day, the primary antibody was removed, secondary antibody was added, and the cells were incubated at 37°C for 30 minutes. The secondary antibody was removed, and the cells were stained with DAPI for 15 minutes. The DAPI was then removed, and PBS was used to prevent drying. The staining results were observed and photographed using a DMi8 fluorescence microscope. The primary antibodies used were as follows: KRT5 antibody (1:200, Abcam, ab17130), P63 antibody (1:200, Abcam, ab124762), Ki67 antibody (1:200, Abcam, ab15580), MUC5AC antibody (1:200, Abcam, ab3649) and AC-TUB antibody (1:200, Sigma, T6793). KRT5 and P63 are specific markers of airway basal stem cells, Ki67 is a proliferation marker, and MUC5AC and AC-TUB are markers of airway epithelial cell differentiation into goblet cells and ciliated cells.

[0070] Immunofluorescence staining results as follows Figure 1 As shown, the BSCs isolated and purified in this embodiment expressed P63, KRT5, Ki67, MUC5AC and ACTUB after 2 days of adherent culture.

[0071] (2) HE staining detection

[0072] On day 21 of differentiation culture, the membrane of the upper chamber of the Transwell was excised. After gradient dehydration with ethanol, gradient clearing with xylene, and paraffin infiltration, the specimen was embedded in a paraffin mold to obtain paraffin-embedded samples. The samples were then sectioned, baked for 30 minutes to 1 hour, dewaxed with xylene, and then hydrated with gradient ethanol. Hematoxylin staining, 0.2% ammonia solution blue staining, and eosin staining were then performed sequentially. Finally, dehydration, clearing, and mounting were performed to complete the staining process. After staining, photographs were taken under a regular optical microscope for recording.

[0073] HE staining results are as follows Figure 2 As shown, BSCs successfully differentiated into an in vitro model of pseudostratified ciliated columnar epithelium of the airway after 21 days of air-liquid interface culture, with clustered cilia and airway functional cells such as goblet cells, columnar cells, and ciliated cells.

[0074] The above results indicate that this embodiment successfully obtained primary airway basal stem cells with the potential to self-proliferate and differentiate into airway epithelial cells.

[0075] Preparation and identification of airway basal stem cell-derived extracellular vesicles (BSC-EVs)

[0076] 1. Preparation method of airway basal stem cell-derived extracellular vesicles

[0077] (1) Obtaining of BSCs culture supernatant

[0078] BSCs were obtained according to the method of Example 1. 3 x 10 7 ~ 4 x 10 7 BSCs were inoculated into a five-layer cell factory (NEST, 771204) for culture using 550 mL of BSCs special culture medium without exosomes (specific components: BSCs special culture medium 445 mL + exosome-free FBS 50 mL + penicillin-streptomycin double antibody 5 mL). After 3 to 4 days of culture, the cell supernatant was collected to obtain the BSCs culture supernatant.

[0079] (2) Extraction of BSC-EVs by ultracentrifugation

[0080] The BSCs culture supernatant was centrifuged at 300 x g for 10 minutes at 4°C to obtain supernatant 1. Then, supernatant 1 was centrifuged at 1000 x g for 30 minutes at 4°C to obtain supernatant 2. In a sterile clean bench, supernatant 2 was filtered through a 0.22-micron sterile filter to remove any residual cell debris, and then transferred to an ultracentrifuge tube and centrifuged at 10000 x g for 70 minutes at 4°C. At this time, a precipitate was formed at the bottom of the tube, and the supernatant was discarded. The precipitate was resuspended with 500 microliters of PBS to obtain the BSC-EVs suspension. If not used immediately, it can be divided and stored at -80°C (avoiding repeated freezing and thawing).

[0081] 2. Identification of airway basal stem cell-derived extracellular vesicles

[0082] (1) Observation of BSC-EVs morphology by transmission electron microscopy (TEM)

[0083] 10 μL of BSC-EVs suspension was dropped on a 200-mesh copper grid and left for 5 minutes. The excess liquid was absorbed with filter paper, and then 10 μL of water was dropped and left for 2 minutes and the excess liquid was again absorbed. The copper grid was then dried and placed under a transmission electron microscope for observation and photography. As shown in FIG. 1, BSC-EVs presented a typical cup-shaped vesicular structure with a diameter of less than 100 nm. Figure 3

[0084] (2) Detection of particle size and particle concentration of BSC-EVs by nano flow cytometry (Nano FCM)

[0085] ​Take 10 μL of BSC-EVs suspension into NanoFCM detector for detection, set appropriate detection parameters, including flow rate, etc., use the analysis software matched with NanoFCM to analyze the detection data, and get the particle size distribution and concentration of exosomes and other information. The NanoFCM results are shown in Figure 4 , the concentration of BSC-EVs is 8.25 x 10 11 particles / mL, the particle size is 70.8 ± 17.1 nm, and the median particle size is 70.8 nm.

[0086] (3) Western blot analysis (WB) to detect the protein expression of BSC-EVs

[0087] Mix BSC-EVs suspension with an equal volume of protein lysis buffer (mixed by volume ratio of 1:100 PMSF:RIPA), shake vigorously for 30 seconds, then place in ice bath for 10 minutes, repeat this process three times. Then, centrifuge at 12000g at 4°C for 20 minutes, transfer the supernatant to a new EP tube, add four times the volume of Loading Buffer, mix and boil in boiling water for 10 minutes, then quickly transfer to ice to cool, and store in the refrigerator for Western blot detection.

[0088] Use 10% electrophoresis gel, load 20 μL per sample well, electrophorese, initial voltage 80V for 30 minutes, then increase the voltage to 120V and continue electrophoresis for 60 minutes. Transfer the protein band to PVDF membrane, use 300mA current for 60 minutes. Then, block with skim milk for 1 hour, add primary antibody respectively, and incubate at 4°C overnight. Wash the membrane with TBST buffer for 10 minutes, three times. Then, use HRP-labeled goat anti-rabbit IgG (1:5000 dilution) as secondary antibody, incubate at room temperature for 1 hour. Wash the membrane with TBST buffer for 10 minutes, three times. Finally, use ECL color development method for development. The primary antibodies used include CD9 antibody (1:1000, SAB, 40708), CD63 antibody (1:1000, SAB, 44012), CD81 antibody (1:1000, SAB, 29677), TSG101 antibody (1:1000, SAB, 49270), HSP70 antibody (1:1000, SAB, 48597), KRT5 antibody (1:200, Abeam, ab17130), and P63 antibody (1:200, Abeam, ab124762).

[0089] As shown in Figure 5 ​As shown, BSC-EVs expressed exosome marker proteins including CD9, CD63, CD81, HSP70 and TSG101, and the nucleus marker protein TP63 specific to BSC, but no KRT5, a marker protein specific to BSC, was detected.

[0090] The above results show that the airway basal stem cell-derived extracellular vesicles are successfully prepared in the present embodiment.

[0091] Example 3 Inhibition of human primary airway fibroblast activation by BSC-EVs in vitro

[0092] 1. Culture and treatment of human primary airway fibroblasts

[0093] The bronchial epithelium obtained by the foregoing brushing was used to isolate human primary airway fibroblasts by differential centrifugation. The cell pellet was resuspended in DMEM complete medium (i.e. DMEM containing 10% FBS) and transferred to a T25 cell culture flask, which was then placed in a cell culture incubator at 37°C containing 5% carbon dioxide for culture. When the cell density reached about 70% to 80%, the cells were subcultured.

[0094] The human primary airway fibroblasts were seeded into 12- and 6-well cell plates at a seeding density of 4 x 10 5 cells / well and 8 x 10 5 cells / well, respectively, and cultured in DMEM complete medium.

[0095] BSC-EVs were prepared according to the method of Example 2. When the human primary airway fibroblasts were cultured to 70%, they were treated with BSC-EVs at a final concentration of 200 particles / cell (i.e. 2e2), 2000 particles / cell (i.e. 2e3), 10000 particles / cell (i.e. le4), 20000 particles / cell (i.e. 2e4) and 200000 particles / cell (i.e. 2e5), respectively, and the human primary airway fibroblasts treated with PBS were used as a control group (Control). The next step was performed 48 h after the treatment.

[0096] 2. Immunofluorescence detection of a-smooth muscle actin (a-SMA) expression

[0097] α-SMA is a protein expressed in smooth muscle cells, as well as activated fibroblasts and myofibroblasts. It plays an important role in cell contraction, migration, and extracellular matrix production, especially during tissue repair and remodeling. Fibroblasts typically do not express α-SMA or express it at low levels in a resting state, but α-SMA expression increases when they are activated to become myofibroblasts. The process of airway wall remodeling involves the conversion of fibroblasts to myofibroblasts, accompanied by an increase in α-SMA expression.

[0098] The specific steps for cell immunofluorescence detection in this embodiment are as follows: Wash each group of cells three times with PBS, fully cover the cells with 4% paraformaldehyde, and fix at room temperature for 15 min; wash each group of cells three times with PBST, 5 min each time; cover the cells with 0.1% Triton X-100 and incubate at room temperature for 20 min; wash each group of cells three times with PBST, 5 min each time; place the cell plate in a humidified chamber, completely cover the sample with 5% BSA, and incubate at room temperature for 60 min; use 5%... Dilute the primary antibody (α-SMA antibody, ab7817, 1:200) with BSA blocking buffer, mix well, and cover the sample. Incubate overnight at 4°C. The next day, after warming to room temperature, wash each group of cells three times with PBST for 5 min each time. Use anti-rabbit fluorescent secondary antibody ab150077 and incubate at room temperature in the dark for 1 h. Wash each group of cells three times with PBST for 5 min each time. Blot dry the residual liquid with absorbent paper, add a DAPI / antiquencher combination sealing solution to the cells, and cover with a coverslip to avoid air bubbles.

[0099] Immunofluorescence results as follows Figure 6 As shown in Figures A and B, the expression level of α-SMA in airway fibroblasts gradually decreased with increasing BSC-EV concentration, especially reaching a significant difference at a concentration of 200,000 particles / cell. This indicates that BSC-EV treatment inhibits the activation of airway fibroblasts and reduces their conversion into myofibroblasts.

[0100] 3. Gel shrinkage assay to detect the contractile ability of airway fibroblasts

[0101] Airway fibroblasts from each group were suspended in serum-free DMEM medium and infused with 3 mg / mL rat tail type I collagen (Corning, USA) at a volume ratio of 2:1, and cured at 37°C for 2 hours. Subsequently, 1 mL of DMEM complete medium was added to the wells, and the cells were cultured for another 8 hours. The formation of collagen gels in each group of airway fibroblasts was observed and photographed, and the area was measured using ImageJ software. The gel area (%) for each group was calculated using the formula: Gel area (%) = x / n * 100% (where x is the area of ​​each cell group, and n is the area of ​​the cell pores).

[0102] As shown in A and B of FIG. 6, with the increase of the concentration of BSC-EVs, the gel area increased significantly, indicating that the contraction ability of airway fibroblasts was weakened under the treatment of BSC-EVs. Figure 7

[0103] 4. Western blot analysis (WB)

[0104] The protein expression of airway fibroblasts in each group was detected according to the method in Example 2, and the primary antibodies used included Collagen I (ab21286, 1:1000), FAP antibody (CST66562, 1:1000), and β-Tubulin antibody (30301, 1:1000).

[0105] As shown in A-C of FIG. 7, with the increase of the concentration of BSC-EVs, the expression of FAP and Collagen I, the activation-related indicators of airway fibroblasts, gradually decreased, indicating that the activation of fibroblasts was inhibited under the treatment of BSC-EVs. Figure 8

[0106] After the injury of airway tissue, airway fibroblasts will be activated, migrate to the wound site, proliferate and secrete extracellular matrix (such as collagen), and form granulation tissue. Therefore, the activation of airway fibroblasts is an important basis and key step for the proliferation of granulation tissue. The above results show that BSC-EVs can inhibit the activation of airway fibroblasts in a concentration gradient-dependent manner, and prevent the proliferation of granulation tissue.

[0107] Example 4 Therapeutic effect of BSC-EVs on animal model of benign airway stenosis

[0108] 1. Construction of a benign airway stenosis rabbit model (BTS)

[0109] After general anesthesia, the New Zealand white rabbits (male or female, body weight about 3±0.2 kg) were sprayed with lidocaine at the epiglottis, a hard nylon brush (hair diameter 0.2 mm, outer diameter 6 mm, hair area 2 cm) was inserted through the tracheal tube, and the brush head was extended outside the tracheal tube, and the inner wall of the trachea was scraped 10 times by rapid rotation, causing airway injury. Then the nylon brush was pulled out, and the injury was observed immediately under the bronchoscope. After the operation, adrenaline was given to stop bleeding, and a BTS was constructed. The day when the above operation was completed was recorded as the first day of modeling.

[0110] 2. Treatment of BTS

[0111] On the 3rd and 5th day of modeling, the airway secretions and necrosis of BTS were removed. On the 7th day of modeling, fresh granulation tissue was observed in the airway of BTS under the bronchoscope. At this time, 100 μL of BSC-EVs containing 6×10 10 ​​The suspension of particle BSC-EVs was resuspended in 500 μL of physiological saline to obtain 600 μL of BSC-EVs injection solution (i.e., BSC-EVs concentration of 1×10⁻⁶). 11 Rabbits were injected with 200 μL of BTS (particles / mL) three times via bronchoscopy into the granulation tissue of the airway wall, at three injection sites. This group was designated as the BSC-EVs treatment group (i.e., the BSC-EVs group). Rabbits with BTS injected with an equal volume of physiological saline solution via bronchoscopy served as the control group (i.e., the PBS group). Both groups of rabbits were then continuously fed until day 42 of model establishment.

[0112] 3. Survival Analysis

[0113] From the first day of modeling, the survival of the rabbits was monitored daily, and mortality was recorded in a timely manner until the end of the experiment. The survival rates of the BSC-EVs treatment group and the control group were calculated and survival curves were plotted.

[0114] like Figure 9 As shown, the survival rate of rabbits in the control group was only 20% on day 14 of modeling, and all rabbits in this group died by day 29. In contrast, the survival rate of rabbits in the BSC-EVs treatment group was as high as 75% on day 14 of modeling, and one rabbit survived until the end of the experiment. This indicates that BSC-EVs treatment significantly prolongs the survival of BTS.

[0115] 4. Bronchoscopy

[0116] The airway stenosis of rabbits in each group was examined under bronchoscopy on days 14, 21, and 28 after modeling, and photographs were taken and recorded. Figure 10 As shown, on day 14, compared with the control group, the treatment group showed reduced granulation tissue growth and improved lumen patency, and the degree of stenosis progressed slowly over time after treatment.

[0117] 5. Histological analysis and evaluation of treatment efficacy

[0118] According to the construction and treatment method of BTS in this embodiment, another batch of New Zealand white rabbits were treated in parallel. On the 14th, 21st and 28th day of modeling, one rabbit was randomly selected from each group to separate airway tissue, prepare paraffin sections, and then bake them in a 65°C oven for 2 hours. They were then taken out while hot and dewaxed and hydrated with TO and graded ethanol in sequence, and then stained with HE and MASSON respectively.

[0119] The specific steps for HE staining are as follows: Place the dewaxed sections in hematoxylin staining solution (Saiwell, China) for 5 min; rinse the stained tissue sections with running water for 5 min; place the rinsed sections in hematoxylin separation solution (Saiwell, China) for 5 seconds to differentiate, selectively destaining overstained nuclei and tissue components that should not be stained; rinse the differentiated tissue sections with running water for 1 min, then place them in blueing solution (Saiwell, China) for 5 seconds and rinse again with running water for 1 minute; then place the sections in eosin (alcohol-soluble) staining solution (Saiwell, China) for 5 min; rinse the sections with running water for 5 min.

[0120] The specific steps for MASSON staining are as follows: Fix the dewaxed sections with Bouin's solution for 1 hour, then rinse with running water for 5 minutes; then stain with iron-containing hematoxylin solution for 5 minutes, and rinse with deionized water; stain with alkaline fuchsin solution for 5 minutes, and rinse with deionized water; treat with a mixed solution of phosphotungstic acid and phosphomolybdic acid for 5 minutes, avoiding rinsing; stain with aniline blue solution for 5 minutes, and rinse with deionized water; dehydrate using an alcohol gradient, and finally mount with xylene and neutral resin.

[0121] Based on the stenosis index of HE-stained images, the calculation formula is: Stenosis index = [1-(d1+d2) / (D1+D2)]×100%, where d1 and d2 are the major and minor diameter measurements of the stenotic part, respectively, and D1 and D2 are the major and minor diameter measurements of the adjacent non-stenotic part, respectively.

[0122] like Figure 10 As shown, HE staining results revealed abundant proliferating granulation tissue at the narrowed site in the control group. This granulation tissue was mainly composed of newly formed capillaries, fibroblasts, and inflammatory cells. MASSON staining results showed abundant proliferating collagen fibers in the control group, forming large fiber bundles, which contrasted with the less abundant and orderly fiber distribution in the treatment group. Narrowing index analysis showed that the luminal diameter of the narrowed airway in the treatment group was significantly larger than that in the control group. This indicates that compared to the control group, the BSC-EVs treatment group showed significantly less granulation tissue at the narrowed airway site, a more unobstructed lumen, and less collagen at the granulation site.

[0123] 6. Evaluation of treatment efficacy by Western blot analysis (WB)

[0124] For the BTS from the previous step, on the 14th day of modeling, 5 rabbits were randomly selected from each group to isolate airway tissue, and Western blot was performed according to the method in Example 2. The primary antibodies used included: Collagen I antibody (ab21286, 1:1000), FAP antibody (CST66562, 1:1000), β-actin antibody (CST4967, 1:1000), and Matrix Remodeling Antibody Sampler Kit (CST73959, 1:1000).

[0125] like Figure 11 As shown in Figures A and B, compared to the control group, the expression of Collagen I and FAP was significantly decreased in the BSC-EVs treatment group, while the expression of MMP2 and MMP9 was significantly increased. This indicates that the degree of fibrosis was reduced in the treatment group, and that extracellular matrix remodeling and tissue repair were improved. These changes, working together, may promote the healing process after airway injury, making tissue repair more effective and orderly, helping to alleviate airway narrowing and improve airway patency.

[0126] The above results indicate that BSC-EVs have excellent therapeutic effects on airway stenosis, with less collagen in the airway wall, improving airway wall remodeling, inhibiting granulation tissue proliferation, thereby expanding the airway lumen, significantly relieving stenosis symptoms, and prolonging survival.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of airway basal stem cell-derived extracellular vesicles in the manufacture of a product for treating benign airway stenosis.

2. Use of airway basal stem cell-derived extracellular vesicles in the manufacture of a product for inhibiting airway granulation tissue proliferation under benign airway stenosis.

3. Use of airway basal stem cell-derived extracellular vesicles in the manufacture of a product for maintaining airway lumen patency under benign airway stenosis.

4. Use of airway basal stem cell-derived extracellular vesicles in the manufacture of a product for improving airway wall remodeling under benign airway stenosis.

5. Use of a biological agent in the manufacture of a product for the treatment of benign airway narrowing, characterised in that, The active ingredient of the biological agent comprises airway basal stem cell-derived extracellular vesicles.

6. Use according to claim 5, characterized in that, The content of the airway basal stem cell-derived extracellular vesicles in the biological preparation is 0.5 x 10 11 particles / mL~1.5 x 10 11 particles / mL.

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