Application of mesenchymal stem cell exosome in preparation of atomized preparation for treating pulmonary arterial hypertension

Abstract

The invention discloses an application of a mesenchymal stem cell exosome in preparation of an atomized preparation for treating pulmonary arterial hypertension, and relates to the field of biological medicines, the diameter peak value of the exosome is 125.4 nm, and the exosome expresses positive marker proteins CD9, TSG101 and CD63 and does not express Calnexin and GAPDH. The composition is delivered in an aerosol inhalation way, the administration dosage is 1 * 10 < 11 > particles per kilogram of body weight, and the composition is administered once every three days for at least one month. Compared with the prior art, the exosome is directly delivered to the lung tissue through aerosol inhalation, whole-body circulation loss is avoided, the concentration of focus medicine is remarkably improved, and the whole-body side effect risk is reduced; the pathological process can be reversed, and pulmonary arterial hypertension is effectively reduced; the thickness of the wall of the pulmonary arteriole can be obviously narrowed, collagen deposition is reduced, and pulmonary fibrosis is relieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of mesenchymal stem cell exosomes in preparation of a nebulized preparation for treating pulmonary arterial hypertension. BACKGROUND

[0002] Pulmonary arterial hypertension (PAH) is a severe and fatal disease. Existing treatment methods can alleviate symptoms, but the effect is limited and it is difficult to reverse the pathology.

[0003] In recent years, mesenchymal stem cells (MSCs) are considered as a potential therapy for PAH due to their anti-inflammatory, pro-angiogenic and immunomodulatory functions. However, MSCs transplantation itself has risks such as tumorigenicity, immune rejection and low retention rate in vivo. Studies have shown that exosomes secreted by MSCs (MSC-Exos) can mediate its key therapeutic effects by affecting the response of tissues to injury, infection and disease, regulating cell-cell interactions, mediating cell signal transduction and changing tissue metabolism. Compared with MSCs, MSC-Exos has the advantages of convenient storage, efficient delivery of active molecules, and avoidance of tumorigenicity and immunogenicity risks related to cell therapy. Therefore, MSC-Exos as a new cell-free therapy strategy shows broad prospects in the field of disease treatment.

[0004] However, there is no nebulized exosome preparation for PAH and its application scheme. SUMMARY

[0005] The present application aims to provide application of mesenchymal stem cell exosomes in preparation of a nebulized preparation for treating pulmonary arterial hypertension, so as to solve the problems in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] Application of mesenchymal stem cell exosomes in preparation of a nebulized inhalation preparation for treating pulmonary arterial hypertension.

[0008] Preferably, the exosomes have a peak diameter of 125.4 nm.

[0009] Preferably, the exosomes have at least one of the following characteristics: (a) express positive marker proteins CD9, TSG101 and CD63; (b) do not express negative marker proteins Calnexin and GAPDH.

[0010] Preferably, the exosomes are delivered to the lung by a nebulized inhalation route.

[0011] Preferably, the administration dose of the nebulized inhalation preparation is 1 x 10^11 exosome particles per kilogram of body weight, and the administration frequency is once every 3 days.

[0012] Preferably, the duration of the nebulized inhalation preparation treatment is at least one month.

[0013] Preferably, the exosomes used to prepare the preparation can reduce pulmonary arterial pressure and inhibit inflammatory response.

[0014] Preferably, the exosomes used to prepare the preparation can inhibit pulmonary vascular remodeling and alleviate pulmonary fibrosis.

[0015] A medicine for treating pulmonary arterial hypertension, comprising the exosomes as described above.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. Nebulized inhalation directly delivers exosomes to lung tissue, avoids systemic circulation loss, significantly increases lesion drug concentration, and reduces the risk of systemic side effects.

[0018] 2. Reverses pathological progression and effectively reduces pulmonary arterial pressure.

[0019] 3. Significantly narrows the pulmonary arteriole wall thickness, reduces collagen deposition (Masson staining), and alleviates pulmonary fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A: Picture of MSCs exosomes under TEM; B: Diameter distribution diagram of exosome vesicles; C: Exosome characterization protein result diagram.

[0021] Figure 2 A process diagram for treating pulmonary arterial hypertension in a pulmonary arterial hypertension model mouse.

[0022] Figure 3 A: Small animal echocardiogram of a pulmonary arterial hypertension model mouse before treatment; B: Small animal echocardiogram of a pulmonary arterial hypertension model mouse after treatment.

[0023] Figure 4 A: Comparison diagram of right ventricular pressure of a pulmonary arterial hypertension model mouse measured by right heart catheter; B: Statistical diagram of right ventricular pressure measured by right heart catheter.

[0024] Figure 5 A: Comparison diagram of H&E staining results of lung tissue of a pulmonary arterial hypertension model mouse; B: Comparison diagram of Masson staining results of lung tissue of a pulmonary arterial hypertension model mouse. DETAILED DESCRIPTION

[0025] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0026] The reagents or instruments used in the embodiments of the present application are not specified by the manufacturer, and are all conventional reagent products available on the market.

[0027] Embodiment 1

[0028] Preparation and characterization of MSCs exosomes

[0029] Preparation:

[0030] (1) MSCs culture medium was replaced with serum-free culture medium for 24 hours of culture;

[0031] (2) The culture supernatant was collected and centrifuged at 3000g at 4°C for 30 minutes to remove particulate matter;

[0032] (3) The supernatant was retained and centrifuged at 10000g at 4°C for 1 hour to remove larger vesicles;

[0033] (4) The supernatant was retained and filtered using a 0.45μm filter membrane, and the filtrate was collected;

[0034] (5) The filtrate was transferred to an ultracentrifuge tube and centrifuged at 50000rpm at 4°C for 3 hours;

[0035] (6) The supernatant was discarded, and the precipitate was resuspended with 100μL PBS buffer and stored at -80°C for long-term storage.

[0036] Characterization: please refer to Figure 1 The characterization of MSCs exosomes was identified by TEM (transmission electron microscope), NTA (nanoparticle tracking analysis) and Western Blot (protein immunoblotting). TEM showed that the exosomes presented a typical disc-shaped vesicle morphology ( Figure 1 A). NTA detection showed that the diameter size of the exosome vesicles was concentrated between 45-255nm, with a peak value of about 125.4nm ( Figure 1 B). WB results showed that the exosomes expressed positive marker proteins CD9, TSG101 and CD63, while no endoplasmic reticulum protein Calnexin and cytoplasmic protein GAPDH were detected ( Figure 1 C).

[0037] Embodiment 2

[0038] Construction of a pulmonary arterial hypertension animal model

[0039] 6-8 weeks old mice were placed in a hypoxia chamber with 10% oxygen concentration, and subcutaneously injected with SU5416 (20 mg / kg) at a fixed time every week for three weeks. Heart ultrasound examination was performed to determine whether the modeling was successful.

[0040] Example 3

[0041] Exosome nebulization treatment of pulmonary hypertension

[0042] The prior art suggests that MSCs exosomes can inhibit inflammatory response and show potential in reversing pulmonary hypertension in animal models. In addition, nebulization can directly deliver exosomes to the lungs, increase local drug concentration, and reduce systemic response.

[0043] For the above reasons, after the mice were randomly divided, exosome nebulization treatment was carried out. The mice were placed in a nebulization chamber, and nebulized at 1 x 10 11 particles / kg, once a day for 3 days, and the efficacy was determined by right heart catheterization and pathology after 1 month, and the process was as shown in Figure 2 .

[0044] Example 4

[0045] Efficacy determination of pulmonary hypertension treatment

[0046] After one month of MSCs exosome nebulization treatment of the mouse pulmonary hypertension model, the mouse pulmonary arterial pressure was dynamically and non-invasively evaluated by small animal echocardiography. The results showed that the pulmonary arterial pressure of the model mice was significantly reduced, as shown in Figure 3 .

[0047] The right ventricular pressure of the mice was measured by invasive right heart catheterization to indirectly estimate the pulmonary arterial pressure. The results showed that the average right ventricular pressure of the model mice reached 23.63 ± 4.39 mmHg, which was much higher than the normal mice of 13.27 ± 2.72 mmHg. After nebulization treatment, the right ventricular pressure of the mice decreased, and the average right ventricular pressure recovered to 18.87 ± 3.41 mmHg, as shown in Figure 4 .

[0048] MSCs exosome nebulization improved pulmonary microvascular remodeling and fibrosis in mice: Lung tissue HE staining results showed that nebulization treatment could significantly reduce the wall thickness and area of mouse pulmonary arterioles. Masson staining showed that nebulization treatment could reduce collagen deposition in lung tissue, and pulmonary fibrosis was significantly alleviated, as shown in Figure 5 .

[0049] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified or some of the technical features can be replaced by equivalent ones by those skilled in the art, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application, and the contents not described in detail in the specification are all the prior art known by those skilled in the art.

Claims

1. Use of mesenchymal stem cell exosomes in the preparation of an inhalation preparation for treating pulmonary arterial hypertension.

2. Use according to claim 1, characterized in that, The diameter of the exosomes is 45-255 nm.

3. Use according to claim 2, characterized in that, The peak diameter of the exosomes is 125.4 nm.

4. Use according to claim 1, characterized in that, The characterization of the exosomes comprises at least one of the following: (a) expressing positive marker proteins CD9, TSG101 and CD63; (b) not expressing negative marker proteins Calnexin and GAPDH.

5. The use according to claim 1, characterized in that, The exosomes are delivered to the lung by an inhalation route.

6. Use according to claim 1, characterized in that, The dosage of the inhalation preparation is 1 x 10^11 exosome particles per kilogram of body weight, and the administration frequency is once every 3 days.

7. Use according to claim 1, characterized in that, The duration of the inhalation preparation treatment is at least one month.

8. The use according to claim 1, characterized in that, The exosomes used to prepare the preparation can inhibit inflammatory response.

9. The use according to claim 1, characterized in that, The exosomes used to prepare the preparation can inhibit pulmonary vascular remodeling and relieve pulmonary fibrosis.

10. A medicament for treating pulmonary arterial hypertension, characterized by, The exosomes according to any one of claims 1-9.

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