Therapeutic compositions for chronic lung disease comprising exosomes derived from thrombin-treated stem cells

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of chronic pulmonary disease comprising exosomes derived from stem cells treated with thrombin as an active ingredient, a pharmaceutical preparation containing the same, and a method for preparing the same. The exosome-based therapeutic agent according to the present invention has the following advantages: it is a cell-free preparation, thus having a low risk of oncogenicity, and there is no problem of graft rejection; there is no concern of capillary obstruction when administered systemically; since it is an isolated material rather than a cell, the agent can be developed in the form of a finished product, reducing production costs; and an excellent therapeutic effect on chronic pulmonary disease is obtained with a low concentration of exosomes by treatment with thrombin.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition for the prevention or treatment of chronic pulmonary disease, comprising exosomes derived from stem cells treated with thrombin as an active ingredient, a pharmaceutical preparation containing the same, and a method for preparing the same. BACKGROUND

[0002] Bronchopulmonary dysplasia (BPD) mainly occurs in premature infants and is a progressive chronic lung disease caused by respiratory failure and artificial airway treatment therefrom. Recently, as treatment of extremely low birth weight infants (less than 1000 g) who are particularly susceptible to pulmonary hypoplasia and have a higher risk of BPD is actively performed, the incidence of the disease has also increased sharply. BPD is not only a major cause of death in newborns, especially premature infants, but also requires long-term hospitalization for surviving infants, and can induce serious sequelae such as pulmonary hypertension, and even after discharge, more than 50% of cases require re-hospitalization due to viral acute bronchiolitis and pneumonia. Moreover, there are many cases of bronchial asthma due to an increase in bronchial hypersensitivity over a long period of time, and eventually lead to serious neurological sequelae such as cerebral palsy.

[0003] Currently, as a method for treating BPD in premature infants, physical efforts such as reducing the pressure of positive pressure ventilation, brain volume injury, and oxygen concentration during artificial ventilation treatment of newborns and premature infants are mainly used, and steroid therapy for reducing pulmonary inflammatory damage is also used, but its use is limited for premature infants because it can cause neurological adverse outcomes, particularly an increase in cerebral palsy. Therefore, since BPD in newborns / premature infants is difficult to treat, there is an urgent need to develop an effective and definitive treatment method.

[0004] It is well known that mesenchymal stem cells, which are cells involved in tissue regeneration, therapy, and immune response due to their pluripotency, are isolated and cultured from umbilical cord blood, bone marrow, and the like, and thus development of therapeutic agents for chronic lung diseases such as BPD has been continuously conducted. However, the above-mentioned treatment method using stem cells has had the following limitations and side effects.

[0005] First, basically, cell therapeutic agents cannot exclude the possibility of tumorogenicity due to DNA transfer,

[0006] Second, since stem cells are large in size, they can induce vascular obstruction or myocardial infarction (see Circ Heart Fail. 2010; 3:e5-e6),

[0007] Third, when the same cells (e.g., umbilical cord blood) are used for transplantation (allogeneic transplantation), there is a problem of rejection due to surface antigens,

[0008] Fourth, generally, the preparation process of a cell therapeutic agent is harsh, and there are many constraints in storage and transportation, and there is a limitation of high production cost.

[0009] Due to the innate limitations of stem cells, as a solution to reduce side effects while improving therapeutic effects, a method of improving efficacy through genetic manipulation has been developed, but the reality is that there is no clear solution.

[0010] On the other hand, exosomes are small vesicles (about 30-100 nm in diameter) with a membrane structure secreted from various cells, and in studies using an electron microscope, it was observed that exosomes are not directly separated from the plasma membrane, but are derived from a specific region in the cell called multivesicular bodies (MVBs) and are released and secreted to the outside of the cell. That is, when fusion of the multivesicular body with the plasma membrane occurs, the vesicle is released to the extracellular environment, and it is called an exosome. There is no clear study on what molecular mechanism produces such exosomes, but it is known that not only red blood cells, but also various immune cells including B lymphocytes, T lymphocytes, dendritic cells, platelets, macrophages, and tumor cells, stem cells, etc. can produce and secrete exosomes in a viable state.

[0011] In particular, exosomes derived from stem cells contain not only receptors and proteins, but also nuclear components, and thus can play a role in intercellular communication. Also, the above-mentioned exosomes derived from stem cells contain relatively less animal serum than stem cells, and thus can exclude the risk of symptoms of animal serum infection (zoonosis). Considering the characteristics of the above-mentioned exosomes, it can be expected that a cell therapy method using exosomes can become a new model for overcoming the limitations of existing stem cell therapy methods. SUMMARY

[0012] TECHNICAL PROBLEM

[0013] To this end, the inventors of the present application confirmed the fact that exosomes derived from stem cells, particularly exosomes derived from stem cells treated with thrombin, can significantly enhance the protective effect against apoptosis and the effect of angiogenesis, as a result of research to overcome the limitations of stem cell therapeutic agents while improving the therapeutic effect on chronic lung diseases including BPD, thereby completing the present application.

[0014] Accordingly, the present application is to provide a pharmaceutical composition for the prevention or treatment of chronic pulmonary disease comprising exosomes derived from stem cells treated with thrombin.

[0015] However, the technical solution to be achieved by the present application is not limited to the above-mentioned problems, and other problems not mentioned can also be clearly understood by those skilled in the art through the following content.

[0016] Technical solution

[0017] The present application provides a pharmaceutical composition for the prevention or treatment of chronic pulmonary disease comprising exosomes derived from stem cells treated with thrombin as an active ingredient.

[0018] One embodiment of the present application is characterized in that the stem cell is a stem cell selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, pluripotent stem cells, and amniotic epithelial cells.

[0019] Another embodiment of the present application is characterized in that the mesenchymal stem cell is derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0020] Still another embodiment of the present application is characterized in that the chronic pulmonary disease is bronchopulmonary dysplasia, chronic bronchitis, emphysema, cystic fibrosis, or peripheral small airway diseases.

[0021] Still another embodiment of the present application is characterized in that the chronic pulmonary disease is bronchopulmonary dysplasia.

[0022] Yet another embodiment of the present application is characterized in that the pharmaceutical composition is administered into the airway or blood vessel of the subject.

[0023] Yet another embodiment of the present application is characterized in that the pharmaceutical composition further comprises an auxiliary ingredient selected from the group consisting of a culture medium, a cytokine, a growth factor, and a gene.

[0024] Yet another embodiment of the present application is characterized in that the expression of a growth factor, an immunomodulatory factor, an antioxidant factor, or a regenerative factor in the exosome is increased.

[0025] Yet another embodiment of the present application is characterized in that the growth factor is brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), or vascular endothelial growth factor (VEGF).

[0026] Also, the present application provides a pharmaceutical preparation for the prevention or treatment of chronic pulmonary disease, comprising the composition.

[0027] An embodiment of the present application is characterized in that the preparation is in the form of an injection, an infusion, or a spray.

[0028] An embodiment of the present application is characterized in that the preparation further comprises a pharmaceutically acceptable carrier.

[0029] Also, the present application provides a method of preparing the pharmaceutical composition,

[0030] which comprises the steps of:

[0031] Step a, treating thrombin after culturing stem cells;

[0032] Step b, isolating exosomes from the culture solution of the step a; and

[0033] Step c, preparing a composition comprising the exosomes isolated in the step b as an effective ingredient.

[0034] An embodiment of the present application is characterized in that the thrombin of the step a is contained in the culture medium at a concentration of 1-1000 units / ml.

[0035] Another embodiment of the present application is characterized in that the exosomes of step c are separated using centrifugation.

[0036] Still another embodiment of the present application is characterized in that the centrifugation is performed at 5,000-500,000 g for 10 minutes to 5 hours.

[0037] Also, the present application provides a method for treating Chronic Pulmonary Disease, comprising the step of administering to a subject an exosome derived from a thrombin-treated stem cell.

[0038] Further, the present application provides a use of an exosome derived from a thrombin-treated stem cell for manufacturing a preparation for preventing or treating Chronic Pulmonary Disease.

[0039] Effects of the Invention

[0040] The exosome-based therapeutic agent according to the present application is a cell-free preparation and does not contain DNA, thus having a lower risk of oncogenicity, and does not have cell surface antigens, thus having no problem of graft rejection.

[0041] Also, the size is very small compared to cells, thus having no concern of capillary obstruction when administered systemically, and, since it is an isolated substance rather than a cell, the agent can be developed in the form of a finished product, reducing production costs.

[0042] Further, the exosome derived from a thrombin-treated stem cell has an excellent therapeutic effect on Chronic Pulmonary Disease even in a small amount compared to an untreated stem cell, thus enabling a significant reduction in the amount of stem cells required to produce a therapeutic amount of exosomes, having the advantage of significantly reducing the production cost of the therapeutic agent.

[0043] Accordingly, according to the present application, while solving the problems of the existing stem cell therapeutic agent, it is also possible to significantly improve the therapeutic efficacy, and thus can be effectively used in the treatment of various Chronic Pulmonary Diseases including bronchopulmonary dysplasia (BPD). BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 TEM image analysis results to confirm the activation of exosome secretion when thrombin is treated to stem cells.

[0045] Figure 2 Western blotting results to confirm whether exosome markers CD63 and CD9 are normally found in exosomes derived from thrombin-treated stem cells.

[0046] Figure 3 Fig. 6 is a result of H&E staining showing the alveolar damage treatment effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo,

[0047] Figure 4 Fig. 7 is a result of TUNEL analysis showing the apoptosis protection effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo,

[0048] Figure 5 Fig. 8 is a result of analysis of von Willebrand factor (vWf) showing the angiogenesis induction effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo,

[0049] Fig. 6 is a result of H&E staining showing the alveolar damage treatment effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo, Figure 6a is a histological staining photomicrograph, Figure 6b is a graph quantifying the degree of alveolar damage.

[0050] Fig. 7 is a result of TUNEL analysis showing the apoptosis protection effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo, Figure 7a is a TUNEL staining photomicrograph, Figure 7b is a graph quantifying the number of benign cells of TUNEL.

[0051] Fig. 8 is a result of analysis of von Willebrand factor (vWf) showing the angiogenesis induction effect of the exosome derived from the thrombin-treated stem cell in the bronchopulmonary dysplasia (BPD) animal model in vivo, Figure 8a is an anti-vWf immunofluorescence staining photograph, Figure 8b is a graph quantifying the same. DETAILED DESCRIPTION

[0052] The present application provides a pharmaceutical composition for the prevention and treatment of chronic pulmonary disease, which comprises an exosome derived from a thrombin-treated stem cell as an effective ingredient.

[0053] The "stem cell" in the present application refers to an undifferentiated cell, which has the ability to self-replicate as well as the ability to differentiate into two or more different types of cells. The stem cell of the present application can be autologous or allogeneic, and can be derived from any type of animal including humans and mammals other than humans, regardless of whether the stem cell is derived from an adult or an embryo, and the present application is not limited thereto.

[0054] The stem cell of the present application includes an embryonic stem cell or an adult stem cell, preferably an adult stem cell. The adult stem cell can be a mesenchymal stem cell, a human tissue-derived mesenchymal stromal cell, a human tissue-derived mesenchymal stem cell, a pluripotent stem cell, or an amniotic epithelial cell, preferably a mesenchymal stem cell, but is not limited thereto. The mesenchymal stem cell can be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, placenta, etc., but is not limited thereto.

[0055] In the present application, "umbilical cord blood" refers to blood collected from the umbilical vein connecting the placenta and the fetus. Umbilical cord blood is a natural byproduct produced at childbirth, and is easier to collect than general mesenchymal tissue such as bone marrow, which requires multiple surgeries; compared to bone marrow transplantation, the storage industry of umbilical cord blood is more active, and thus has established infrastructure, and thus it is easier to find donors. Further, umbilical cord blood-derived cells are cells that do not induce rejection reactions in tissue or organ transplantation, which are the most important cause of histocompatibility antigens HLA-DR (class II), and can avoid or minimize immune reactions such as rejection reactions induced in existing transplantation surgery, and thus not only autologous umbilical cord blood but also allogeneic umbilical cord blood can be used.

[0056] In the present application, "exosome" refers to a small vesicle (about 30-100 nm in diameter) having a membrane structure secreted by various cells, and is a small vesicle released to the extracellular environment due to fusion of a multivesicular body with a plasma membrane. The exosome includes a naturally secreted exosome or an artificially secreted exosome.

[0057] In the present application, "chronic lung disease" refers to a respiratory disease in which airflow limitation gradually occurs due to abnormal inflammatory responses in the lungs, resulting in decreased lung function and induced dyspnea. For example, it can include bronchopulmonary dysplasia, chronic bronchitis, emphysema, cystic fibrosis, or peripheral small airway disease, but is not limited thereto, and is preferably bronchopulmonary dysplasia.

[0058] The "prevention or treatment of chronic lung disease" of the present application includes alleviation, remission, and improvement of symptoms of chronic lung disease, and includes the meaning of reducing the likelihood of developing chronic lung disease.

[0059] The stem cells treated by "thrombin" of the present application improve paracrine performance, which is a main mechanism of stem cells, without bringing changes in cell stability such as cell viability, oxidative function, etc. compared to untreated stem cells, and thus can strengthen the function / efficacy of stem cells. Further, through thrombin treatment, not only the therapeutic efficacy of stem cell-derived exosomes can be strengthened, but also the secretion amount of exosomes can be increased.

[0060] At this time, the paracrine can be an increase in growth factors, immunomodulatory factors, antioxidant factors, or regenerative factors, and in particular, the growth factors are physiologically active substances of protein nature that promote the division or growth, differentiation of cells, and can include brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), etc.

[0061] The pharmaceutical composition of the present application can be administered to a subject in various routes without particular limitation, for example, can be administered orally or non-orally, but is preferably administered into the airway or blood vessel.

[0062] The pharmaceutical composition of the present application, together with the exosomes derived from stem cells, can further include one or more well-known auxiliary ingredients having a chronic lung disease treatment effect. For example, one or more auxiliary ingredients selected from the group consisting of a gene (e.g., an anti-inflammatory cytokine gene, an siRNA or an antisense primer against an inflammatory cytokine) or an expression vector containing the same, which is effective in treating chronic lung disease, or a cytokine (e.g., interleukin-10) or a growth factor (e.g., keratinocyte growth factor) that provides autocrine or paracrine effects, and combinations thereof.

[0063] The preferred administration amount of the pharmaceutical composition of the present application varies depending on the subject's condition and body weight, the degree of disease, the form of the drug, the route of administration, and the period, and can be appropriately selected by those skilled in the art. The administration of the above composition can be once a day, or divided into several times, and is not limited thereto.

[0064] For the treatment of chronic lung disease, the pharmaceutical composition of the present application can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.

[0065] The composition of the present application can also appropriately include carriers generally used in the preparation of pharmaceutical compositions. For example, for injections, preservatives, analgesics, solubilizers, or stabilizers, etc. can also be included; for preparations for topical administration, bases, excipients, lubricants, or preservatives, etc. can also be included.

[0066] The composition of the present application can be administered by formulating it into a unit administration form suitable for administration into the body of a subject by general methods in the pharmaceutical field. The dosage form that meets the above purpose, for non-oral administration preparations, is preferably, for example, injections such as ampoules for injection, infusions such as infusion bags, and sprays such as aerosols, etc. The above ampoule bottle can be formulated by mixing with an injection solution before use, and the injection solution can use physiological saline, dextrose, Ringer's solution, etc. Also, the infusion bag can use polyvinyl chloride or polyethylene materials. The administration of the present application means providing a predetermined composition of the present application to a subject in any appropriate method.

[0067] The preferred administration amount of the pharmaceutical composition of the present application varies depending on the subject's condition and body weight, the degree of disease, the form of the drug, the route of administration, and the period, and can be appropriately selected by those skilled in the art. The administration of the above composition can be once a day, or divided into several times, and is not limited thereto.

[0068] Also, the present application provides a method of preparing the above pharmaceutical composition, comprising: step a, a step of culturing stem cells and then performing a thrombin treatment; step b, isolating exosomes from the culture solution of the step a; and step c, preparing a composition containing the exosomes isolated in the step b as an effective ingredient.

[0069] In the present application, the thrombin treatment concentration is an appropriate concentration to enhance the efficacy of stem cells / exosomes, and is not particularly limited, but is preferably contained in the culture medium at a concentration of 1-1000 units / ml.

[0070] The present application is not limited to the method of isolating the exosome, for example, the exosome can be isolated by centrifugation, ultracentrifugation, filter filtration, gel filtration chromatography, free flow electrophoresis, capillary electrophoresis, and polymer separation, and a combination thereof in a culture solution, and the centrifugation / ultracentrifugation is preferred. At this time, the centrifugation / ultracentrifugation is preferably performed at 4°C for 10 minutes to 5 hours at 5,000-500,000g.

[0071] The medium for cell culture in the present application refers to a mixture for growing and proliferating cells such as stem cells in vitro, which contains sugars, amino acids, various nutrients, serum, growth factors, inorganic substances, and other essential nutrients required for cell growth and proliferation. The medium that can be used in the present application is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10 (DMEM / F-10), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), Isocove's Modified Dulbecco's Medium (IMDM), and KnockOut DMEM, and the like, which are commercially available or artificially synthesized, but are not limited thereto.

[0072] Hereinafter, examples are presented to facilitate the understanding of the present application. However, the following examples are only for easier understanding of the present application, and the content of the present application is not limited by the examples.

[0073] [Examples]

[0074] Example 1: Induction of exosome secretion and enhancement of efficacy by thrombin treatment of stem cells

[0075] 1-1. Induction of exosome secretion by thrombin

[0076] Human umbilical cord blood-derived mesenchymal stem cells (3 x 10 5 were injected into a 60 mm culture dish (orange scientific cat # 4450200) and cultured for 1 week. After confirming that the cells were proliferating satisfactorily in the culture dish, the medium was replaced with serum-free medium (MEM alpha medium) diluted with 50 units / ml of thrombin (REYON Pharmaceutical. Co., LTD) and cultured for another 6 hours.

[0077] At this time, to confirm whether exosomes are actively secreted from mesenchymal stem cells by thrombin treatment, the secretion process of exosomes was confirmed by transmission electron microscopy (TEM, Transmission Electronic Microscopy) images. As a result, as shown in FIG. 1B, it was found that the secretion of exosomes was induced by stimulation with thrombin. Figure 1

[0078] After that, the culture solution was aliquoted into centrifugation tubes, centrifuged at 100,000 rpm for 30 minutes at 4°C, and the supernatant was moved to a new tube to remove cell debris. Again, after ultracentrifugation of the supernatant at 100,000 rpm for 2 hours at 4°C, the exosomes were obtained by further removing the supernatant (final concentration: 15 μg / ml).

[0079] At this time, to confirm whether the obtained product is an exosome, the expression of CD63 and CD9 (System Bioscience, Mountain View, CA, USA), which are known as exosome markers, was verified by immunoblotting. As a result, as shown in FIG. 2B, it was confirmed that the exosomes obtained from thrombin-treated stem cells normally expressed CD63 and CD9, confirming that they were exosomes. Figure 2

[0080] 1-2. Enhancement of exosome efficacy by thrombin

[0081] It was confirmed whether the exosomes obtained in Example 1-1 increased the expression of anti-inflammatory cytokines such as growth factors or IL-6, etc. by thrombin treatment.

[0082] Specifically, after dissolving the exosome membrane with a soluble lysis buffer, the proteins in the exosomes were separated, and the amounts of BDNF, FGF, HGF, NGF, VEGF, and IL-6 in the exosomes were measured using a Procarta immunoassay kit (affymatrix, USA).

[0083] As a result, as shown in FIG. 3B, it was found that the expression of BDNF, FGF, HGF, NGF, VEGF, and IL-6 in the exosomes was increased by thrombin treatment.​​Figure 3 As shown, by the thrombin treatment, the expression of brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and Interleukin-6 (IL-6) in the exosomes was increased compared to the exosomes obtained from the stem cells not treated with thrombin (control, normal).

[0084] Accordingly, the cell regeneration, blood vessel regeneration, and anti-inflammatory effects of the exosomes derived from the thrombin-treated stem cells were enhanced.

[0085] Example 2: Inhibitory effect on lung cell apoptosis in vitro for exosomes derived from thrombin-treated stem cells

[0086] 2-1. Inhibition effect of lung cell apoptosis

[0087] The L2 cell line, which is a rat pulmonary epithelial cell line (Korean Cell Line Bank), was subjected to H2O2 treatment for 1 hour to induce oxidative injury, thereby making an in vitro model of hyperoxic lung disease.

[0088] After the exosomes obtained in Example 1 (i.e., exosomes obtained from thrombin-treated stem cells (10 ug)) were treated with the in vitro model, the survival rate of lung cells was measured by MTT analysis.

[0089] As a result, as shown in FIG. 2, the exosomes obtained from the thrombin-treated stem cells (Th exo) significantly inhibited lung cell apoptosis compared to the exosomes obtained from the stem cells not treated with thrombin (NC exo) or the exosomes obtained from fibroblasts (fibro exo), thereby verifying that the lung cell protection effect of the thrombin-treated exosomes of the present application was the most excellent. Figure 4

[0090] 2-2. Effect based on exosome concentration

[0091] ​Using the in vitro model of hyperoxic lung disease described in Example 2-1, it was confirmed whether the inhibitory effect of exosomes on lung cell apoptosis varies depending on concentration.

[0092] Specifically, the exosomes obtained in Example 1 (i.e., exosomes obtained from stem cells treated with thrombin) were used to treat the in vitro model at concentrations of 2.5, 5, 10 and 20 μg, respectively, and the survival rate of lung cells was measured by MTT assay.

[0093] The result is as follows Figure 5 As shown, it can be confirmed that exosomes inhibit lung cell apoptosis in a concentration-dependent manner (i.e., play a protective role for lung cells).

[0094] Example 3: Therapeutic effect in vivo for exosomes derived from thrombin-treated stem cells

[0095] 3-1. Establishment of an animal model of bronchopulmonary dysplasia

[0096] All animal experiments were approved by the Research Animal Laboratory Committee of Samsung Biomedical Research Institute (Korea) and followed the institution's guidelines.

[0097] First, to create an animal model of bronchopulmonary dysplasia, pregnant Spregodoray rats (Biolink, South Korea) of accurately confirmed gestational age were purchased and housed in an experimental animal facility. They were kept in sealed resin-glass cages (69.5 × 50.0 × 32.0 cm) in acrylic tubs, maintaining adequate humidity (40-60%) and temperature (23-26°C) at 1 atmosphere.

[0098] Subsequently, newborn white mice born naturally to their mothers were continuously supplied with high-concentration oxygen at an oxygen saturation level of 85-90% for 14 days, starting immediately after birth (within 10 hours). To prevent pulmonary edema caused by oxygen toxicity in the mother mice, they were moved to indoor air or oxygen conditions every 24 hours, and this method was continued for 14 days.

[0099] 3-2. Verify the therapeutic effect after exosome administration

[0100] 20 μg of exosomes obtained by the method of Example 1 (i.e., exosomes whose efficacy is enhanced by thrombin treatment) were suspended in FBS-free α-MEM, and 0.05 ml of the suspension was administered into the airway of 5-day-old mice exposed to high concentrations of oxygen in Example 3-1 using a 26G needle.

[0101] Subsequently, on day 14 of the experiment, newborn mice were anesthetized by intraperitoneal injection of pentobarbital. After limb fixation, the thorax was opened to expose the heart and lung tissue. In some mice, the heart and lungs were simultaneously removed after cardiac perfusion with ice-cold PBS solution. A tube was inserted into the bronchus and securely fixed, then placed in 4% formalin fixative and inflated at a pressure of 25 cmH2O. This fixative was then maintained overnight. Following this, the following experiments were conducted.

[0102] Histological observation: H&E staining

[0103] Lung tissue sections fixed in 4% formalin for 24 hours were embedded in paraffin, cut into 4 μm thicknesses, stained with hematoxylin / eosin, and then observed under an optical microscope to compare and evaluate the therapeutic effects of stem cells and stem cell-derived exosomes on lung tissue damaged by hyperoxia.

[0104] The result, such as Figure 6a As shown, compared with the lung tissue (NC) of normal mice, the lung tissue (HC) of mice with induced bronchopulmonary dysplasia exhibited more severe alveolar damage. Furthermore, for this alveolar damage, the exosomes derived from thrombin-induced stem cells (H+MSC-EV) and the stem cell group (H+MSC) showed similar alveolar protective / therapeutic effects. Conversely, the exosomes derived from fibroblasts (H+Fibro-EV), unlike stem cell-derived exosomes, showed almost no therapeutic effect. This demonstrates that the therapeutic effect of exosomes depends on the specificity of the cell from which they originate.

[0105] Furthermore, the results of quantitative analysis of alveolar damage using the mean linear index, such as through... Figure 6b As shown, compared with the normal group (NC), the bronchopulmonary dysplasia model group (HC) exhibited impaired alveolar development, resulting in a significantly higher mean linear index. However, in the bronchopulmonary dysplasia model, the group treated with stem cells (HM) or the group treated with exosomes derived from thrombin-induced stem cells (HM-ev) showed improvement in lung damage, leading to a significant decrease in the mean linear index. Conversely, the group treated with exosomes derived from fibroblasts (HF-ev), unlike stem cell-derived exosomes, showed almost no therapeutic effect.

[0106] Apoptosis analysis: TUNEL analysis

[0107] As is well known, TUNEL assay (Terminal deoxynucleotidyl-mediated dUTP nick-end labeling assay) is a staining method for measuring the degree of apoptosis. The DNA of apoptotic cells differs from that of normal cells, possessing fragmented DNA segments with exposed 3'-OH DNA ends. Therefore, an enzyme called terminal deoxynucleotidyl transferase (TdT) can be used to label the 3'-OH DNA ends with fluorescein-12-dUTP (nucleotides), thus distinguishing apoptotic cells from normal cells for measurement. Therefore, the more cells with benign TUNEL staining, the more apoptotic cells are represented.

[0108] Specifically, after preparing dewaxed 5μm lung sections, analysis was performed using an in situ apoptosis detection kit (S7110ApopTag, Chemicon, Temecula, CA, USA) according to the manufacturer's instructions.

[0109] The result, such as Figure 7a and 7b As shown, the results are consistent with those of the H&E tissue staining. Specifically, compared to normal mouse lung tissue (NC), lung tissue induced by bronchopulmonary dysplasia (HC) exhibited more severe apoptosis. Regarding this apoptosis, the exosomes derived from thrombin-induced stem cells (H+MSC-EV) and the stem cell group (H+MSC) showed similar inhibitory effects on apoptosis. Conversely, the exosomes derived from fibroblasts (H+Fibro-EV), unlike stem cell-derived exosomes, showed almost no inhibitory effect on apoptosis. This indicates that the therapeutic effect of exosomes is determined by the specificity of the cell source from which they originate.

[0110] Angiogenesis analysis: von Willebrand factor (vWF)

[0111] To confirm whether the exosomes derived from thrombin-induced stem cells of the present invention can induce angiogenesis and exert a therapeutic effect on bronchopulmonary dysplasia, the degree of angiogenesis was analyzed by observing the activity of von Willebrand factor (vWf) synthesized and secreted when mesenchymal cells differentiate into vascular endothelial cells.

[0112] Specifically, after preparing dewaxed 5μm lung sections, immunofluorescence staining was performed using primary antibodies against vWF (endothelial cell markers, rabbit polyclonal antibodies, Dako, Glostrup, Denmark) and biotinylated secondary antibodies to track vWF. Subsequently, the amount of vWF present in the lung sections was assessed by measuring the fluorescence intensity of the immunofluorescence staining using Image J (National Institutes of Health, USA).

[0113] The result, such as Figure 8a and 8b As shown, compared to normal mouse lung tissue (NC), lung tissue (HC) from mice with induced bronchopulmonary dysplasia exhibits reduced angiogenesis. Regarding this reduced angiogenesis, the exosome administration group derived from thrombin-induced stem cells (H+MSC-EV) showed similar angiogenic effects to the stem cell administration group (H+MSC). Conversely, the exosome administration group derived from fibroblasts (H+Fibro-EV), unlike stem cell-derived exosomes, showed almost no angiogenic effect. This demonstrates that the therapeutic effect of exosomes is determined by the specificity of the cell source from which they originate.

[0114] The above description of the present invention is illustrative. Those skilled in the art can easily modify it into other specific forms without changing the technical concept and essential features of the invention. Therefore, the embodiments described above are merely examples in any respect and are not intended to limit the scope of the invention.

[0115] Industrial applicability

[0116] The exosome-based therapeutic agent of the present invention can solve the problems of transplant rejection or high production cost of existing stem cell therapeutic agents, and at the same time, can significantly improve the therapeutic effect and can be beneficially used for the treatment of a variety of chronic lung diseases, including bronchopulmonary dysplasia (BPD).

Claims

1. Use of exosomes derived from thrombin-treated mesenchymal stem cells as an active ingredient in the preparation of pharmaceutical compositions for the prevention or treatment of hyperoxia-induced bronchopulmonary dysplasia; The pharmaceutical composition is prepared by the following steps: Step (a): Culture the stem cells and treat the stem cells with thrombin; Step (b) involves isolating exosomes from the culture medium of step (a); and Step (c) prepares a composition comprising the exosomes isolated from step (b) as an active ingredient.

2. The use according to claim 1, characterized in that, The mesenchymal stem cells are derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, or placenta.

3. The use according to claim 1, characterized in that, The pharmaceutical composition is applied to the airway or blood vessels of the subject.

4. The use according to claim 1, characterized in that, The pharmaceutical composition also includes auxiliary components selected from the following: culture medium, cytokines, growth factors and genes.

5. The use according to claim 1, characterized in that, The expression of growth factors, immunomodulatory factors, antioxidant factors, or regenerative factors in the exosomes is increased.

6. The use according to claim 5, characterized in that, The growth factor is brain-derived neurotropic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), or vascular endothelial growth factor (VEGF).

7. The use according to claim 1, characterized in that, The pharmaceutical composition is in the form of an injection, infusion, or spray.

8. The use according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The use according to claim 1, characterized in that, The thrombin in step (a) is contained in the culture medium at a concentration of 1-1000 units / ml.

10. The use according to claim 1, characterized in that, The exosomes from step (c) were separated by centrifugation.

11. The use according to claim 10, characterized in that, The centrifugation was carried out at 5,000-500,000g for 10 minutes to 5 hours.

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