Application of extracellular vesicles derived from airway basal stem cells in preparation of product for treating airway stenosis

Abstract

The invention discloses application of extracellular vesicles derived from airway basal stem cells in preparation of a product for treating airway stenosis. The invention proposes that the extracellular vesicles derived from airway basal stem cells have an excellent airway stenosis treatment effect for the first time. Under the condition that a support is not placed, extracellular vesicles derived from airway basal stem cells are directly injected into a narrow airway, so that collagen on the airway wall is less, remodeling of the airway wall is improved, granulation tissue hyperplasia is inhibited, airway lumen is expanded, the symptom of stenosis is remarkably relieved, and the life cycle is prolonged; a new technical choice is provided for curing airway stenosis, preventing recurrence of airway stenosis, reducing risk of stem cell therapy and improving prognosis of patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of extracellular vesicles derived from airway basal stem cells in the preparation of products for treating airway stenosis. Background Art

[0002] Airway stenosis is a common disease in interventional respiratory medicine. The main pathological changes are manifested as obvious granulation tissue hyperplasia, which affects the patient's respiratory function and even causes suffocation in severe cases. The treatment methods for airway stenosis include surgical treatment and bronchoscopic interventional treatment. Surgical treatment has high requirements for the patient's physical condition and the operator. Only a small number of patients can tolerate surgical treatment, and some patients have obvious granulation tissue hyperplasia at the anastomosis, causing secondary stenosis. For patients with airway stenosis who cannot undergo surgery, bronchoscopic interventional treatment is the main treatment method, which is to keep the airway open by placing a stent in the airway. The short-term efficacy of this therapy is obvious, but the stent continues to rub the airway, which is easy to form new granulation tissue, and cannot inhibit the continued proliferation of the granulation tissue that has been formed. The patient's airway often stenosis again within weeks or months, resulting in the patient needing to undergo bronchoscopic interventional treatment multiple times to maintain the diameter of the stenotic trachea. It can be seen that both surgical treatment and bronchoscopic interventional treatment are subject to many constraints and cannot effectively treat airway stenosis.

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

[0004] Therefore, there is an urgent need to develop treatments that do not require stent placement, can effectively cure airway stenosis, prevent the recurrence of airway stenosis, and reduce the risks of stem cell therapy. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides the use of extracellular vesicles derived from airway basal stem cells in the preparation of a product for treating airway stenosis.

[0006] The first objective of the present invention is to provide the use of extracellular vesicles derived from airway basal stem cells in the preparation of products for treating airway stenosis.

[0007] The second objective of the present invention is to provide the use of extracellular vesicles derived from airway basal stem cells in the preparation of a product for inhibiting the proliferation of airway granulation tissue.

[0008] The third object of the present invention is to provide the use of extracellular vesicles derived from airway basal stem cells in the preparation of products for maintaining the patency of airway lumen.

[0009] The fourth object of the present invention is to provide the use of extracellular vesicles derived from airway basal stem cells in the preparation of products for improving airway wall remodeling.

[0010] The fifth object of the present invention is to provide a method for preparing extracellular vesicles derived from airway basal stem cells.

[0011] The sixth object of the present invention is to provide a biological agent.

[0012] The seventh object of the present invention is to provide the use of the biological agent in preparing a product for treating airway stenosis.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

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

[0015] Therefore, the present invention requests protection for the following contents:

[0016] Application of extracellular vesicles derived from airway basal stem cells in the preparation of products 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, scar tissue formation after surgery, foreign body obstruction, congenital abnormalities and other reasons, and can usually be improved by medical intervention. The present invention intervenes in an animal model of benign airway stenosis with obvious granulation tissue hyperplasia by using extracellular vesicles derived from airway basal cells. It is found that the animals after intervention treatment show reduced fibroblast activation, reduced granulation tissue hyperplasia, more mature and stable collagen in the granulation tissue, and more unobstructed lumen, which indicates that extracellular vesicles derived from airway basal cells can reduce granulation tissue hyperplasia after tracheal injury, reduce tracheal stenosis, and improve the survival rate after tracheal injury, which has a significant therapeutic effect on benign airway stenosis. Therefore, as a preferred embodiment, the present invention also requests the use of extracellular vesicles derived from airway basal stem cells in the preparation of products for treating benign airway stenosis.

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

[0019] The use of extracellular vesicles derived from airway basal stem cells in the preparation of products for maintaining the patency of airway lumens should also be within the scope of protection of the present invention.

[0020] Airway remodeling refers to the long-term changes in airway structure in chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), including thickening of airway smooth muscle, thickening of basement membrane, hyperplasia of mucous glands and goblet cells, increased angiogenesis, and increased extracellular matrix deposition. Airway wall remodeling can cause or aggravate airway stenosis, increase airway reactivity, and may affect lung function. The present invention reduces the degree of fibrosis of the animal airway by intervening in an airway stenosis animal model with extracellular vesicles derived from airway basal cells, improves the remodeling of the extracellular matrix and the repair of tissues, and has less airway wall collagen, significantly improving airway wall remodeling. Therefore, the present invention also requests protection of the use of extracellular vesicles derived from airway basal stem cells in the preparation of products that improve airway wall remodeling, which should also be within the scope of protection of the present invention.

[0021] A method for preparing extracellular vesicles derived from airway basal stem cells comprises collecting cell supernatant of cultured airway basal stem cells and separating and obtaining 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 derived from human level 3 to 5 bronchial epithelium.

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

[0026] Preferably, the airway basal stem cells are cultured in a BSCs-specific culture medium, and the BSCs-specific culture medium is the proliferation culture medium described in the prior art "CN117778294A".

[0027] More preferably, the BSCs-specific culture medium is the culture medium 1 of Example 1 in the prior art "CN117778294A".

[0028] Preferably, the airway basal stem cells are cultured using a cell factory.

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

[0030] A biological preparation whose active ingredients include extracellular vesicles derived from airway basal stem cells.

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

[0033] Further preferably, the content of the extracellular vesicles derived from airway basal stem cells in the biological preparation is 1×10 11 particles / 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 agent in the preparation of a product for treating airway stenosis should also be within the scope of protection of the present invention.

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

[0038] The use of the biological agent in the preparation of products for maintaining the patency of the airway lumen should also be within the scope of protection of the present invention.

[0039] The use of the biological agent in the preparation of products for improving airway wall remodeling should also be within the protection scope of the present invention.

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

[0041] The present invention proposes for the first time that extracellular vesicles derived from airway basal stem cells have excellent therapeutic effects on airway stenosis. Without placing a stent, extracellular vesicles derived from airway basal stem cells are directly injected into the airway at the stenosis, resulting in less collagen deposition on the airway wall, improving airway remodeling, while inhibiting granulation tissue hyperplasia, thereby expanding the airway lumen, significantly alleviating stenosis symptoms, and prolonging the survival period, providing a new technical option for curing airway stenosis, preventing the recurrence of airway stenosis, reducing the risk of stem cell therapy, and improving patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0046] Figure 5 These are 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 to N3 represent 3 BSC-EVs total protein samples prepared in parallel.

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

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

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

[0050] Fig. 9 These are the survival analysis results of the animal model of benign airway stenosis in the PBS group and the BSC-EVs group.

[0051] Fig.10 These are the efficacy evaluation results of the benign airway stenosis animal model in the PBS group and the BSC-EVs group. From left to right are the results of bronchoscopic manifestations, HE staining, and MASSON staining.

[0052] Fig.11 These are the WB test results of airway tissue in the animal model of benign airway stenosis in the PBS group and the BSC-EVs group. A is the WB band graph; 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 present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels 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] The isolation of BSCs from humans includes the following steps:

[0057] (1) Perform brush examination of grade 3 to 5 bronchial epithelium using a fiberoptic bronchoscope. Use a 15-mL centrifuge tube to recover the tip of the brush, place it in a container filled with ice, and transport it to the laboratory within 1 hour.

[0058] (2) Use a BSCs-specific culture medium (i.e., culture medium 1 in Example 1 of the prior art "CN117778294A") to thoroughly rinse the brush, collect the rinse solution and centrifuge it to collect the cell precipitate.

[0059] (3) Resuspend the cell pellet in BSCs-specific culture medium, transfer the pellet to a T25 cell culture flask, and culture the flask in a cell culture incubator at 37°C and 5% carbon dioxide.

[0060] (4) During the first three days of culture, the culture medium is changed every day to remove red blood cells, non-adherent cells, and other impurities to obtain purified BSCs. When the BSCs grow to a density of about 50% to 70%, they can be subcultured.

[0061] 2. Cultivation of BSCs

[0062] The air-liquid interface culture technology is used to promote the differentiation of BSCs, which specifically includes the following steps:

[0063] (1) Proliferation culture

[0064] BSCs were cultured in a BSCs-specific culture medium until the growth density reached 90% to 95%, the cells were digested and the number of cells was counted, and then BSCs were plated at 2 × 10 5 Cells were inoculated into the upper chamber of a 24-well Transwell culture plate (Cat. No. 3470, Corning), 800 μL of proliferation medium was added to the lower chamber, and the plate was placed at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator and subcultured when the growth density reached 80% to 90%.

[0065] (2) Differentiation culture

[0066] Remove the culture medium in the upper chamber of the Transwell culture plate and keep the upper chamber in a dry state without liquid; at the same time, replace the culture medium in the lower chamber with differentiation induction medium (PneumaCult TM -ALI Medium, Stemcell, 05001) was used to continue the culture. The differentiation induction medium in the lower chamber was renewed every 48 hours, and the upper chamber was cleaned with PBS every 5 days, and the air-liquid interface culture environment was maintained for a total of 21 days.

[0067] 3. Identification of BSCs

[0068] (1) Immunofluorescence detection

[0069] The proliferation and differentiation potential of BSCs was identified by this method, which specifically included the following steps: after BSCs were cultured for 2 days, they were fixed with 4% paraformaldehyde, the fixative was removed, and the cell membrane was permeabilized with Triton X 100 for 30 minutes, followed by blocking with 5% BSA for 30 minutes, and then incubated with primary antibody at 4°C overnight; the next day, the primary antibody was removed, the secondary antibody was added and incubated at 37°C for 30 minutes; the secondary antibody was removed, DAPI was used for staining for 15 minutes, the DAPI was removed again, and PBS was used to prevent drying; the staining results were observed using a DMi8 fluorescence microscope and photographed and recorded. 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), among which 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 cells differentiating into goblet cells and ciliated cells.

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

[0071] (2) HE staining

[0072] On the 21st day of differentiation culture, the membrane of the upper chamber of the Transwell was cut off, and after gradient ethanol dehydration, gradient xylene transparency, and paraffin penetration, the specimen was placed in a paraffin mold for embedding to obtain a paraffin-embedded sample, which was then sliced ​​and baked for 30 minutes to 1 hour. After dewaxing with xylene, gradient ethanol hydration was performed, followed by hematoxylin staining, 0.2% ammonia blueing, and eosin staining, and finally dehydration, transparency, and sealing to complete the staining process. After staining, take photos and record under an ordinary optical microscope.

[0073] HE staining results Figure 2 As shown, BSCs were successfully differentiated into an in vitro model of airway pseudostratified ciliated columnar epithelium 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 the present example successfully obtains primary airway basal stem cells with the potential to self-proliferate and differentiate into airway epithelial cells.

[0075] Example 2 Preparation and Identification of Airway Basal Stem Cell-Derived Extracellular Vesicles (BSC-EVs)

[0076] 1. Preparation method of extracellular vesicles derived from airway basal stem cells

[0077] (1) Obtaining BSCs culture supernatant

[0078] BSCs were obtained according to the method of Example 1. 550 ml of BSCs-specific culture medium without exosomes (specific components: 445 ml of BSCs-specific culture medium + 50 ml of FBS without exosomes + 5 ml of penicillin-streptomycin double antibody) were used to culture 3×10 7 ~4×10 7 BSCs were inoculated into a five-layer cell factory (NEST, 771204) for culture. 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×g for 10 minutes at 4°C to obtain supernatant 1; then, supernatant 1 was centrifuged at 1000×g for 30 minutes at 4°C to obtain supernatant 2; in a sterile clean bench, supernatant 2 was filtered using a 0.22 μm sterile filter to remove any residual cell debris, then transferred to an ultracentrifuge tube and centrifuged at 10000×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 μl of PBS to obtain the BSC-EVs suspension. If not used immediately, it can be aliquoted and stored at -80°C (avoid repeated freezing and thawing).

[0081] 2. Identification of extracellular vesicles derived from airway basal stem cells

[0082] (1) TEM observation of BSC-EVs morphology

[0083] Take 10 μL of BSC-EVs suspension and drop it on a 200-mesh copper grid, let it stand for 5 minutes, then use filter paper to absorb the excess liquid, then drop 10 μL of uranium water and let it stand for 2 minutes and absorb the excess liquid again, then dry the copper grid and observe and take pictures under a transmission electron microscope. Figure 3 As shown, BSC-EVs present a typical cup-shaped vesicle structure with a diameter of less than 100 nm.

[0084] (2) Nano-flow cytometry (NanoFCM) detection of particle size and concentration of BSC-EVs

[0085] Take 10 μL of BSC-EVs suspension and add it to the NanoFCM detector for detection. Set appropriate detection parameters, including flow rate, etc. Use the analysis software supporting NanoFCM to analyze the detection data to obtain information such as the particle size distribution and concentration of exosomes. The NanoFCM results are as follows: Figure 4 As shown, the concentration of BSC-EVs was 8.25×10 11 particle / mL, the particle size was 70.8±17.1nm, and the median particle size was 70.8nm.

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

[0087] The BSC-EVs suspension was mixed with an equal volume of protein lysis buffer (mixed with PMSF:RIPA at a volume ratio of 1:100), shaken vigorously for 30 seconds, and then placed in an ice bath for 10 minutes. This process was repeated three times. Next, centrifuged at 12000g for 20 minutes at 4°C, the supernatant was transferred to a new EP tube, four volumes of Loading Buffer were added, mixed, boiled in boiling water for 10 minutes, then quickly transferred to ice for cooling, and stored in Store in refrigerator for use in Western blot analysis.

[0088] Use 10% electrophoresis gel, load 20 μL in each sample well, perform electrophoresis, initial voltage 80V for 30 minutes, then increase voltage to 120V and continue electrophoresis for 60 minutes. Transfer protein bands to PVDF membrane, transfer using 300mA current for 60 minutes. Afterwards, block with skim milk for 1 hour, add primary antibodies respectively, and incubate overnight at 4°C. Wash the membrane with TBST buffer for 10 minutes, three times. Next, use HRP-labeled goat anti-rabbit IgG (1:5000 dilution) as secondary antibody and incubate at room temperature for 1 hour. Wash the membrane again with TBST buffer for 10 minutes, three times. Finally, use ECL color development method for development. The primary antibodies used included 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, Abcam, ab17130), and P63 antibody (1:200, Abcam, ab124762).

[0089] like Figure 5As shown, BSC-EVs expressed exosomal marker proteins including CD9, CD63, CD81, HSP70, and TSG101, as well as the BSC-specific nuclear marker protein TP63, but the BSC-specific marker protein KRT5 was not detected.

[0090] The above results indicate that the present example successfully prepared extracellular vesicles derived from airway basal stem cells.

[0091] Example 3 BSC-EVs inhibit activation of primary human airway fibroblasts in vitro

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

[0093] The bronchial epithelium obtained by brushing was separated into human primary airway fibroblasts by differential centrifugation, and the cell pellet was resuspended in DMEM complete medium (i.e., DMEM containing 10% FBS), and transferred to a T25 cell culture flask and cultured in a cell culture incubator at 37°C and 5% carbon dioxide. When the cells grow to a density of about 70% to 80%, they can be subcultured.

[0094] Human primary airway fibroblasts were cultured at 4×10 5 cells / well and 8×10 5 The cells were inoculated into 12-well and 6-well cell plates at a seeding rate of 10 cells / well and cultured with DMEM complete medium.

[0095] BSC-EVs were prepared according to the method of Example 2. When the primary human airway fibroblasts were cultured to 70%, BSC-EVs with a final concentration of 200 particles / cell (i.e., 2e2), 2000 particles / cell (i.e., 2e3), 10000 particles / cell (i.e., 1e4), 20000 particles / cell (i.e., 2e4) and 200000 particles / cell (i.e., 2e5) were added for treatment, and the primary human airway fibroblasts treated with PBS were used as the control group (Control). The next step of detection was performed after 48 hours of treatment.

[0096] 2. Cell immunofluorescence detection of α-smooth muscle actin (α-SMA) expression

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

[0098] The specific steps of cell immunofluorescence detection in this example are as follows: wash each group of cells 3 times with PBS, fully cover the cells with 4% paraformaldehyde, and fix at room temperature for 15 minutes; wash each group of cells 3 times with PBST, each time for 5 minutes; cover the cells with 0.1% TritonX-100, and incubate at room temperature for 20 minutes; wash each group of cells 3 times with PBST, each time for 5 minutes; place the cell plate in a wet box, completely cover the sample with 5% BSA, and incubate at room temperature for 60 minutes; The primary antibody (α-SMA antibody, ab7817, 1:200) was diluted with BSA blocking solution, mixed and covered with the sample, and incubated at 4°C overnight; after rewarming at room temperature the next day, each group of cells was washed 3 times with PBST, each time for 5 minutes; anti-rabbit fluorescent secondary antibody ab150077 was used, incubated at room temperature in the dark for 1 hour, and each group of cells was washed 3 times with PBST, each time for 5 minutes; the residual liquid was absorbed with absorbent paper, and DAPI / anti-fading agent two-in-one sealing solution was added on the cells, and coverslips were covered to avoid bubbles.

[0099] Immunofluorescence results Figure 6 As shown in A and B, with the increase of BSC-EVs concentration, the expression of α-SMA in airway fibroblasts gradually decreased, especially at a concentration of 200,000 particles / cell, reaching a significant difference. This indicates that the activation of airway fibroblasts is inhibited under BSC-EVs treatment, and the transformation to myofibroblasts is reduced.

[0100] 3. Gel contraction test to detect the contractile ability of airway fibroblasts

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

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

[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. The primary antibodies used included: Collagen I (ab21286, 1:1000), FAP antibody (CST66562, 1:1000) and β-Tubulin antibody (30301, 1:1000)

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

[0106] After airway tissue injury, airway fibroblasts will be activated, migrate to the wound, 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 granulation proliferation. 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 Efficacy of BSC-EVs in benign airway stenosis animal model

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

[0109] New Zealand white rabbits (male or female, weighing about 3±0.2 kg) were given general anesthesia, and lidocaine was sprayed on the epiglottis. A hard nylon brush (0.2 mm bristle diameter, 6 mm outer diameter, 2 cm hair area) was placed through the tracheal tube, and the brush head of the nylon brush was extended outside the tracheal tube. The tracheal inner wall was quickly rotated and scraped 10 times to cause airway injury. The nylon brush was then pulled out and the injury was immediately observed under a bronchoscope. Epinephrine was given after the operation to stop bleeding, and BTS was constructed. The day 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, BTS were given airway secretion and necrosis removal treatment. On the 7th day of modeling, fresh granulation tissue was observed in the airway of BTS under bronchoscope. At this time, 100 μL of 6×10 10The suspension of particle BSC-EVs was resuspended in 500 μL of physiological saline to obtain 600 μL of BSC-EV injection solution (i.e., the concentration of BSC-EVs was 1×10 11 The rabbits were injected with 200 μL of saline solution into the granulation tissue of the inner wall of the BTS airway through bronchoscopy for 3 times (i.e., 200 μL each time). A total of three injection sites were injected, which were recorded as the BSC-EVs treatment group (i.e., the BSC-EVs group). The BTS injected with an equal volume of saline solution through bronchoscopy was used as the control group (i.e., the PBS group). The two groups of rabbits were then kept until the 42nd day of modeling.

[0112] 3. Survival analysis

[0113] Starting from the first day of modeling, the survival of the rabbits was monitored daily and the deaths were 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 the survival curves were drawn.

[0114] like Fig. 9 As shown in the figure, the survival rate of rabbits in the control group was only 20% on the 14th day of modeling, and all rabbits in this group died on the 29th day of modeling; while the survival rate of rabbits in the BSC-EVs treatment group was still as high as 75% on the 14th day of modeling, and one rabbit survived until the end of the experiment. This shows 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 the 14th, 21st and 28th day of modeling, and photographs were taken for record. Fig.10 As shown, at day 14, the treatment group had reduced granulation and improved lumen patency compared with the control group, and after treatment, the degree of stenosis slowly progressed over time.

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

[0118] According to the construction and treatment method of BTS in this example, a group of New Zealand white rabbits were treated in parallel. One rabbit was randomly selected from each group on the 14th, 21st and 28th day of modeling to isolate airway tissue, prepare paraffin sections, and then bake the sections in a 65°C oven for 2 h. The sections were taken out while hot, dewaxed and hydrated with TO and gradient ethanol in turn, and then HE staining and MASSON staining were performed respectively.

[0119] The specific operation steps of HE staining are as follows: place the dewaxed slices in hematoxylin staining solution (Sevier, China) for staining for 5 minutes; rinse the stained tissue slices with running water for 5 minutes; place the rinsed slices in hematoxylin color separation solution (Sevier, China) for differentiation for 5 seconds to selectively decolorize over-stained nuclei and tissue components that should not be stained; rinse the differentiated tissue slices with running water for 1 minute, then place them in blueing solution (Sevier, China) for blueing for 5 seconds and rinse them with running water again for 1 minute; then place the slices in eosin (alcohol-soluble) staining solution (Sevier, China) for staining for 5 minutes; rinse the slices with running water for 5 minutes.

[0120] The specific operation steps of MASSON staining are as follows: use Bouin solution to fix the dewaxed sections for 1 hour, and then rinse with running water for 5 minutes; then use iron-containing hematoxylin solution to stain for 5 minutes, and then rinse with deionized water; add alkaline fuchsin solution to stain for 5 minutes, and rinse with deionized water; use a mixed solution of phosphotungstic acid and phosphomolybdic acid for 5 minutes, avoiding rinsing; add aniline blue solution to stain for 5 minutes, and then rinse with deionized water; use alcohol gradient for dehydration, and finally use xylene and neutral gum for sealing.

[0121] The stenosis index was calculated based on the HE-stained images using the following formula: stenosis index = [1-(d1+d2) / (D1+D2)] × 100%, where d1 and d2 are the long and short axis measurements of the stenotic site, and D1 and D2 are the long and short axis measurements of the adjacent non-stenotic site.

[0122] like Fig.10 As shown in the figure, HE staining results showed that a large amount of proliferative granulation tissue was observed in the stenotic part of the control group, and these granulation tissues were mainly composed of new capillaries, fibroblasts and inflammatory cells; MASSON staining results showed that a large amount of proliferative collagen fibers were observed in the control group, forming thick fiber bundles, which was in contrast to the less and orderly fiber distribution in the treatment group; the stenosis index analysis results showed that the inner diameter of the stenotic airway lumen in the treatment group was significantly larger than that in the control group. This indicates that compared with the control group, the granulation tissue at the airway stenosis of the rabbits in the BSC-EVs treatment group was significantly reduced, the lumen was more unobstructed, and there was less collagen in the granulation tissue.

[0123] 6. Western blot analysis (WB) evaluation of therapeutic effects

[0124] For the BTS in the previous step, 5 rabbits were randomly selected from each group on the 14th day of modeling to isolate airway tissues, and WB detection 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 Fig.11 As shown in A and B in Figure 1, compared with the control group, the expression of Collagen I and FAP in the BSC-EVs treatment group was significantly decreased, and the expression of MMP2 and MMP9 was significantly increased. This indicates that the degree of fibrosis in the treatment group was reduced, and the remodeling of the extracellular matrix and tissue repair were improved. These changes may promote the healing process after airway injury, make tissue repair more effective and orderly, help alleviate airway stenosis, and improve airway patency.

[0126] The above results show that BSC-EVs have excellent therapeutic effects on airway stenosis, with less airway wall collagen, improved airway wall remodeling, and inhibited granulation tissue proliferation, thereby expanding the airway lumen, significantly alleviating stenosis symptoms, and prolonging survival period.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of extracellular vesicles derived from airway basal stem cells in the preparation of products for the treatment of airway stenosis.

2. The use according to claim 1, characterized in that: The airway stenosis is benign airway stenosis.

3. Application of extracellular vesicles derived from airway basal stem cells in the preparation of products for inhibiting the proliferation of airway granulation tissue.

4. Application of extracellular vesicles derived from airway basal stem cells in the preparation of products for maintaining patency of airway lumen.

5. Application of extracellular vesicles derived from airway basal stem cells in the preparation of products for improving airway wall remodeling.

6. A method for preparing extracellular vesicles derived from airway basal stem cells, characterized in that: The cell supernatant of the cultured airway basal stem cells was collected and the extracellular vesicles were isolated.

7. The preparation method according to claim 6, characterized in that: The cell supernatant is collected from airway basal stem cells in proliferation culture.

8. A biological agent, characterized in that: Its active ingredients include extracellular vesicles derived from airway basal stem cells.

9. The biological agent according to claim 9, characterized in that The content of the extracellular vesicles derived from airway basal stem cells in the biological preparation is 0.5×10 11 particle / mL~1.5×10 11 parti cle / mL.

10. Use of the biological preparation according to claim 8 or 9 in the preparation of a product for treating airway stenosis.

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