Cardiopulmonary progenitor cell exosome, preparation method therefor and use thereof

Abstract

The present invention relates to a cardiopulmonary progenitor cell exosome and a preparation method therefor. A cardiopulmonary progenitor cell culture solution is prepared using a developed method for preparing cardiopulmonary progenitor cells, the supernatant is collected, and separation and extraction are performed using an ultracentrifugation method to obtain the cardiopulmonary progenitor cell exosome. Further disclosed is the use of the cardiopulmonary progenitor cell exosome in terms of reducing the area of cardiac necrosis and fibrosis, and promoting the improvement of cardiac function, the proliferation of cardiomyocytes, and angiogenesis in a damaged heart, indicating that the cardiopulmonary progenitor cell exosome has great potential in preventing and treating cardiovascular diseases.

Description

Cardiopulmonary progenitor cell exosomes and their preparation method and application

[0001] The present invention claims priority to Chinese patent application No. 2023118366859, filed with the Patent Office of China on December 28, 2023, entitled “Exosomes of cardiopulmonary progenitor cells and their preparation methods and applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biotechnology, and specifically relates to cardiopulmonary progenitor cell exosomes and a preparation method and application thereof. Background Art

[0003] Progenitor cells refer to undifferentiated multipotent or specialized stem cells, which usually exist in adult tissues. They can be mobilized and activated, proliferate in large numbers and migrate to damaged areas, and then differentiate and replace injured tissues. They have the function of helping tissues and organs repair and regenerate. Therefore, scientists are trying to cultivate progenitor cells and transplant them into patients to treat various degenerative diseases.

[0004] The inventors of this invention previously developed a method for preparing cardiopulmonary progenitor cells, enabling the stable production of these cells. However, the effects of cardiopulmonary progenitor cells on damaged hearts and their mechanisms have not been thoroughly explored, nor has the preparation of exosomes derived from these cells been reported. Furthermore, it remains unknown whether and what biological activities these exosomes possess.

[0005] Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a cardiopulmonary progenitor cell exosome and a preparation method thereof, and its application in promoting myocardial cell proliferation.

[0007] The technical solutions for achieving the above-mentioned objectives include the following.

[0008] The first aspect of the present invention is to provide a method for preparing exosomes of cardiopulmonary progenitor cells.

[0009] In some embodiments, the preparation method comprises the following steps:

[0010] S1. preparing cardiopulmonary progenitor cells and culturing them in cardiopulmonary progenitor cell culture medium;

[0011] S2. The culture medium of the cardiopulmonary progenitor cells cultured in step S1 is separated by ultracentrifugation to obtain cardiopulmonary progenitor cell exosomes.

[0012] In some embodiments, step S2 includes:

[0013] The supernatant of the cardiopulmonary progenitor cell culture medium in step S1 was centrifuged at 300±10g for 10±2 minutes, 2,000±10g for 10±2 minutes, and 10,000±100g for 30±5 minutes. The supernatant was taken and centrifuged at 100,000±100g for 70±8 minutes. The supernatant was removed and resuspended with PBS to extract the cardiopulmonary progenitor cell exosomes.

[0014] In some embodiments, step S1 includes the following:

[0015] SA) Day 9.5 mouse embryos were taken to isolate the region where the cardiopulmonary progenitor cells reside;

[0016] SB) digesting the area containing the cardiopulmonary progenitor cells obtained in step SA) with a digestion solution, centrifuging, and collecting the cells;

[0017] SC) Hanging drop culture was performed using the embryoid body induction method for 46-52 hours, with each drop of suspension having a volume of 15 μl and a cell number of 2000 ± 30;

[0018] SD) suspension culture, after culturing in differentiation medium for 24 ± 2 hours, change to basal medium and culture for 24 ± 2 hours, then digest the cells into single cell suspension and seed them on culture dishes covered with gelatin;

[0019] SE) Continue culturing with ABC medium, changing the medium every two days, until the cell confluence is greater than 90%, and then perform subculture.

[0020] In some of these embodiments, the passages were performed at a ratio of 1:3-4.

[0021] In some embodiments, the culture medium for the cardiopulmonary progenitor cells is ABC medium.

[0022] In some embodiments, the ABC medium comprises 2±0.5% B-27 without vitamin A, 2±0.5 mM L-glutamine, 1±0.2% non-essential amino acids, 0.1±0.02 mM β-mercaptoethanol, 1±0.2 μM A83-01, 50±5 ng / ml bFGF, 12±2 μM CHIR-99021, and 2.5-5.0% human platelet lysate.

[0023] In some embodiments, the ABC medium includes 2±0.1% B-27 without vitamin A, 2±0.1 mM L-glutamine, 1±0.1% non-essential amino acids, 0.1±0.01 mM β-mercaptoethanol, 1±0.1 μM A83-01, 50±0.5 ng / ml bFGF, 12±1 μM CHIR-99021, and 2.5±0.1% human platelet lysate.

[0024] The second aspect of the present invention is to provide cardiopulmonary progenitor cell exosomes.

[0025] In some embodiments, the particle size of the cardiopulmonary progenitor cell exosomes is 136.9±5.9 nm.

[0026] The present invention prepares exosomes from cardiopulmonary progenitor cells by selecting ultracentrifugation, separates them from many other extracellular vesicle structures distributed in a complex body fluid environment, studies their biological activity, and obtains relevant valuable medical applications.

[0027] The third aspect of the present invention is to provide the use of the above-mentioned cardiopulmonary progenitor cell exosomes or the above-mentioned method for preparing cardiopulmonary progenitor cell exosomes in the preparation of drugs for preventing and treating cardiovascular diseases in mammals.

[0028] The fourth aspect of the present invention is to provide the use of the above-mentioned cardiopulmonary progenitor cell exosomes or the above-mentioned method for preparing cardiopulmonary progenitor cell exosomes in improving the cardiac function of mammals.

[0029] In some embodiments, the cardiovascular disease is myocardial infarction.

[0030] In some embodiments, the above application is to promote the proliferation of mammalian cardiac tissue cells.

[0031] In some preferred embodiments, the cardiac tissue cells include at least one of cardiomyocytes, pericytes, myofibroblasts, smooth muscle cells, and endothelial cells.

[0032] In some embodiments, the above application is to promote angiogenesis in mammals.

[0033] In some embodiments, the above application is to increase cardiovascular density in a mammal.

[0034] In some embodiments, the above application is to reduce the area of ​​necrosis and / or fibrosis in the heart of a mammal.

[0035] In some embodiments, the above application is to increase the left ventricular ejection fraction and / or left ventricular fractional shortening of a mammal.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention found that cardiopulmonary progenitor cell exosomes have a significant effect on promoting the proliferation of cardiac tissue cells, and also have good performance in improving cardiac function, reducing the area of ​​cardiac necrosis and fibrosis, and promoting angiogenesis in damaged hearts. This indicates that cardiopulmonary progenitor cell exosomes have great potential in repairing infarcted myocardium, promoting the regeneration of myocardial fibroblasts, and preventing and treating cardiovascular diseases. In addition, it was found that the function of promoting cardiac function improvement brought about by cardiopulmonary progenitor cells (CPPs) is not achieved through the proliferation or differentiation of CPPs themselves, but should be achieved through paracrine pathways, especially secreted exosomes. The present invention provides cardiopulmonary progenitor cell exosomes and a preparation method thereof, and their use in the preparation of drugs for preventing and treating cardiovascular diseases in animals, such as myocardial infarction, and in the preparation of drugs for improving cardiac function in mammals. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows immunofluorescence staining of Isl1, Wnt2, and Gli1, markers for detecting CPPs (a), and a schematic diagram of flow cytometry detection results (b); the scale bar of the upper row of images is 100 μm, and the scale bar of the lower row of images is 50 μm.

[0039] Figure 2 is a schematic diagram of the in vitro differentiation process of CPPs. CPPs differentiated into cardiomyocytes (a), endothelial cells (b), fibroblasts (c), smooth muscle cells (d) and alveolar epithelial cells (e) under different induction conditions.

[0040] Figure 3 shows the extraction process of cardiopulmonary progenitor cell exosomes (CPPs-Exo), the nanoparticle tracking analysis results (a) and electron microscopy analysis images (b) of CPPs-Exo, and Western blot detection of exosome markers Alix and Hsp70 (c).

[0041] Figure 4 shows the results of CPPs-Exo characterization, including the analysis of the particle size and concentration of CPPs-Exo by nanoparticle tracking (a), the analysis of the morphology of CPPs-Exo by electron microscopy, which showed a typical cup-shaped appearance of microvesicles (b), and the detection of exosome markers Alix and Hsp70 by Western blot.

[0042] Figure 5 shows the teratoma formation risk assessment and karyotype analysis results of CPPs, the grouping of nude mice after subcutaneous injection of CPPs and mESCs (a), the teratoma formation after 3 months (b), and the karyotype analysis results of CPPs (c).

[0043] Figure 6 shows representative images (a) of left ventricular short-axis ultrasound results of MI mice after grouping and treatment, and their data analysis results (b is ejection fraction, c is left ventricular short-axis shortening rate); CPPs promote the improvement of cardiac function in MI mice through their exosomes.

[0044] Figure 7 shows the Masson staining results of heart tissue sections after grouping and treatment of MI mice (a) and the necrotic area analysis results (c). CPPs reduced the necrotic area of ​​the heart of MI mice through its exosomes; the Sirus red staining results of heart tissue sections after grouping and treatment of MI mice (b) and the data analysis results of the fibrosis ratio (d). CPPs reduced the fibrosis area of ​​the heart of MI mice through its exosomes.

[0045] Figure 8 shows the staining results of the smooth muscle marker α-SMA (a) (scale bar: 500 μm) and vascular density analysis (b) in MI mice after grouping and treatment. CPPs promote angiogenesis in the injured heart through their exosomes (c).

[0046] Figure 9 shows the staining results of proliferating cardiomyocyte markers cTnT and Ki67 after grouping and treatment of MI mice. The upper scale bar of each group is 500 μm, and the lower scale bar of each group is 250 μm. CPPs promote the proliferation of cardiomyocytes through their exosomes.

[0047] Figure 10 shows the analysis results of the proportion of proliferating cardiomyocytes in MI mice after grouping and treatment. The effect of promoting cardiomyocyte proliferation in the MI+CPPs-Exo group was significantly better than that in the MI+CPPs group.

[0048] Figure 11 shows hybridization of mouse heart sections using a Y chromosome probe. The scale bar is 20 μm. DETAILED DESCRIPTION

[0049] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0050] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] Myocardial infarction, also known as myocardial infarction, refers to ischemic necrosis of the myocardium. On the basis of coronary artery disease, the blood flow of the coronary artery is sharply reduced or interrupted, causing severe and persistent acute ischemia of the corresponding myocardium, and ultimately leading to ischemic necrosis of the myocardium.

[0053] The present invention is further described in detail below with reference to specific embodiments.

[0054] Example 1

[0055] 1. First, prepare cardiopulmonary progenitor cells. The method and results are as follows:

[0056] Under a dissecting microscope, the heads and tails of 9.5-day C57BL / 6 (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) mouse embryos were removed, and the regions containing CPPs (Cardiopulmonary progenitors) were isolated and digested with a digestive solution (0.04% Trypsin and 0.05% Collagenase IV) at 37°C for 10 minutes, gently inverting several times during the process. After terminating the digestion with FBS-containing medium (RPMI 1640 medium), the cells were centrifuged at 200×g for 5 minutes. After counting the collected cells, they were cultured in hanging drops using the embryoid body (EB) induction method (for example, see: Isolation and Functional Characterization of Pluripotent Stem Cell-Derived Cardiac Progenitor Cells, Curr Protoc Stem Cell Biol. 2010 September; DOI: 10.1002 / 9780470151808.sc01f10s14; page 4, steps 5 and 6). The volume of each drop of suspension was 15 μl, and the number of cells was 2000. After two days of hanging drop culture, cells were switched to suspension culture. Cells were cultured for one day in basal medium supplemented with 12 μM CHIR99021. The culture was then switched to basal medium (RPMI 1640 supplemented with 2% B-27 without insulin, 2 mM L-glutamine, 1% NEAA, 1% penicillin / streptomycin, and 0.1 mM β-mercaptoethanol) and cultured for another day. The cells were then digested into a single cell suspension and seeded onto a 0.2% gelatin-coated dish. Culture was continued in ABC medium (Table 1), with the medium changed every two days until the cells reached a confluence greater than 90%, at which point they were passaged at a 1:3 ratio. The detailed culture process is shown in Figure 3. The composition (volume ratio) of ABC medium is as follows:

[0057] Table 1 Reagents and concentrations added to prepare ABC medium in DMEM / F12 basal medium

[0058] Cardiopulmonary progenitor cells were obtained by the above-described passage. Immunofluorescence and flow cytometry confirmed that the above-described preparation method produced highly pure cardiopulmonary progenitor cells that simultaneously expressed Isl1, Wnt2, and Gli1. The results are shown in Figure 1.

[0059] 2. Identification of Differentiation Ability of Cardiopulmonary Progenitor Cell Culture Results

[0060] CPPs were induced to differentiate in vitro according to the induction method described in Figure 2(ae). Cells were harvested on day 6 or 12 and characterized using flow cytometry, immunofluorescence, and RT-qPCR. The results confirmed that the CPPs could differentiate into fibroblasts, cardiomyocytes, endothelial cells, smooth muscle cells, and alveolar epithelial cells. In this example, the percentages of successfully differentiated cells were 74.7%, 75.4%, 92.7%, 88.5%, and 47.9%, respectively. Therefore, the CPPs isolated and cultured here demonstrate the ability to differentiate into cardiomyocytes, fibroblasts, endothelial cells, smooth muscle cells, and alveolar epithelial cells in vitro.

[0061] 3. Preparation of exosomes from cardiopulmonary progenitor cells. The method and results are as follows:

[0062] Exosomes were isolated and extracted from the culture medium (supernatant) of cardiopulmonary progenitor cells (CPPs) by ultracentrifugation, while maintaining the centrifugation temperature at 4°C throughout the process: 300g centrifugation for 10 minutes to remove dead cells; 2000g centrifugation for 10 minutes to remove cell debris; 10,000g centrifugation for 30 minutes, and then 100,000g centrifugation for 70 minutes. The supernatant was removed, and the exosomes were resuspended in PBS and stored in aliquots at -80°C.

[0063] 4. Identification of cardiopulmonary progenitor cell exosomes using Western Blot assay:

[0064] (1) Extraction of total cell protein

[0065] 1) Prepare an ice box and collect the cells from the 6-well plate to be protein extracted. Discard the culture medium from the plate and rinse the plate twice with PBS buffer. Add 100 μL of RIPA cell lysis buffer with PMSF and lyse on ice for half an hour.

[0066] 2) After cell lysis, scrape the cells with a clean cell scraper and concentrate the cell lysate on one side of the well plate. Then, use a pipette to transfer the protein suspension into a 1.5 mL EP tube. To fully release the proteins from the cells, sonicate the protein suspension using an ultrasonic cell disruptor at 80W for 30 seconds, with a 5-second on / 2-second off cycle.

[0067] 3) After sonication, balance all samples and centrifuge at 12,000 rpm at 4°C for 20 minutes. Carefully transfer the supernatant to a new 1.5 mL EP tube to extract the total cellular protein.

[0068] (2) Protein concentration determination and protein denaturation

[0069] 1) Prepare a 50× protein standard using the purchased BCA protein concentration assay kit to obtain a 0.5 mg / mL BSA protein standard.

[0070] 2) Prepare a protein concentration standard curve by adding PBS and BSA according to Table 2.

[0071] Table 2. Configuration of protein standards.

[0072] 3) Add protein samples and standards to a 96-well plate according to the kit instructions, then add BCA working solution and incubate at 37°C for 30 minutes.

[0073] 4) Measure the absorbance of the reaction plate at wavelength A562 using a microplate reader. Create a protein standard curve based on the absorbance values, derive a linear trend equation, and then calculate the sample concentration.

[0074] 5) Add 5× loading buffer and PBS according to the calculated protein concentration so that the protein amount of each sample is 30-50 μg.

[0075] 6) Subsequently, all samples were heated in a 95°C water bath for 5 minutes to denature the proteins, and then stored at -80°C until use.

[0076] (3) SDS-PAGE gel electrophoresis

[0077] 1) Prepare separation gel and stacking gel according to Table 3.

[0078] Table 3 SDS-PAGE gel recipe.

[0079] 2) Sample loading: Place the solidified gel into the electrophoresis tank as required, add the prepared 1× electrophoresis transfer buffer, add the prepared protein samples and electrophoresis markers in the order of groups, and mark them.

[0080] 3) Electrophoresis: Run at a constant voltage of 80V for approximately 30 minutes, then adjust the voltage to 100V and continue for 90-100 minutes. 4) Transfer: After electrophoresis, remove the glass plate and sandwich the PVDF membrane, gel, and sponge, soaked in methanol, in a transfer tank. Add 1L of transfer solution, apply a constant current of 200mA, and transfer for 90-120 minutes.

[0081] 5) Blocking: After the transfer is completed, remove the PVDF membrane and place it in WB blocking solution with the front side facing up. Shake on a shaker at 55 rpm and block at room temperature for 1-2 hours.

[0082] 6) Primary Antibody Incubation: After blocking, remove excess blocking solution with TBST buffer. Cut the PVDF membrane according to protein size and insert the corresponding antibody (Gli1, Isl1, Wnt2, Tbx5, GAPDH). Incubate overnight at 4°C on a shaker.

[0083] 7) Secondary Antibody Incubation: The next day, remove the PVDF membrane and recover the primary antibody. Wash the PVDF membrane three times with TBST at high speed (10 min / wash). Then, add the anti-rabbit and anti-mouse secondary antibodies corresponding to the primary antibodies and incubate on a shaker at 55 rpm for 1 hour at room temperature.

[0084] 8) ECL Chemiluminescence Detection: After secondary antibody incubation, wash the PVDF membrane with TBST (120 rpm, three times for 10 min each). Then, apply ECL chemiluminescence solution to the front of the PVDF membrane and image it using an ultrasensitive multifunctional imager. Save the image for subsequent analysis.

[0085] The extraction process of cardiopulmonary progenitor cell exosomes (CPPs-Exo) is shown in Figure 3. We extracted exosomes from the supernatant of CPPs culture medium by ultracentrifugation at 4°C, and then used nanoparticle tracking analysis (NTA) technology to determine the particle size of the exosomes to be 136.9±5.9nm (see Figure 4a) and the concentration to be 4.37×10 9 / mL, transmission electron microscopy was used to examine the morphology of exosomes and identified them as having a typical cup-shaped appearance of microvesicles (see Figure 4b), and Western blotting was used to identify the exosome surface markers Alix and Hsp70 (see Figure 4c). These results are consistent with the basic characteristics of exosomes and indicate that the extracted exosomes are indeed exosomes.

[0086] Example 2

[0087] By exploring the functions of exosomes from cardiopulmonary progenitor cells, we found that CPPs promote myocardial repair after injury through their exosomes.

[0088] 1. Tumorigenicity experiment of CPPs:

[0089] Ten mice were randomly divided into two groups (as shown in Figure 5a) for subcutaneous tumor transplantation experiments. One group of nude mice received subcutaneous injections of mouse embryonic stem cells (mESCs), while the other group received subcutaneous injections of cardiopulmonary progenitor cells (CPPs) prepared by the aforementioned method to assess the tumorigenicity of CPPs. Teratoma formation three months later is shown in Figure 5b. No teratoma was observed after CPPs injection, indicating that CPPs could be used for transplantation. Figure 5c shows the karyotype analysis of the injected CPPs, demonstrating that the isolated and cultured CPPs contain a Y chromosome. Therefore, female mice were selected for the MI model to facilitate in vivo transdifferentiation of CPPs using Y chromosome probe hybridization.

[0090] All MI mice described below were female.

[0091] 2. Effects of cardiopulmonary progenitor cell exosomes on cardiac function in MI mice

[0092] Myocardial infarction (MI) mice were modeled and randomly divided into groups and treated as follows:

[0093] SHAM group: sham operation group;

[0094] MI+PBS group: intramyocardial injection of PBS after MI;

[0095] MI+CPPs group: intramyocardial injection of CPPs after MI;

[0096] MI+CPPs(GW4869) group: intramyocardial injection of CPPs treated with the inhibitor GW4869 after MI;

[0097] MI+CPPs Fragments group: intramyocardial injection of CPPs cell fragments after MI;

[0098] MI+CPPs-Exo group: intramyocardial injection of CPPs-Exo after MI;

[0099] Figure 6a shows the results of left ventricular short-axis ultrasound 42 days after MI mouse modeling. Figures 6b-6c show that compared with the MI+PBS group, the left ventricular ejection fraction and fractional shortening were significantly increased in the MI+CPPs group, indicating that cardiac function was restored. Compared with the MI+CPPs group, the left ventricular ejection fraction (b) and left ventricular fractional shortening (c) were significantly decreased in the MI+CPPs(GW4869) group, indicating that inhibiting the secretion of CPPs exosomes significantly inhibited the cardiac repair function of CPPs. CPPs exosomes promoted the improvement of cardiac function in MI mice.

[0100] 3. Effects of cardiopulmonary progenitor cell exosomes on cardiomyocytes

[0101] The following data are shown as mean ± SEM, with 5 biological replicates in each group, ns P>0.05, ***P<0.001 (t test).

[0102] Figure 7 shows the results of Masson staining (a) and Sirus red staining (b) of myocardial cells in MI mice treated according to the above grouping and digital analysis (c and d). The results show that the infarct area (12.37% and 12.12%, respectively) and fibrosis size (12.56% and 12.77%, respectively) in the MI+PBS and MI+CPPs (GW4869) groups were significantly different from those in the SHAM group, suggesting worsening cardiac function in both groups. However, the measured values ​​in the MI+CPPs, MI+CPPs fragments, and MI+CPPs-Exo groups were all below 5%, significantly lower than those in the MI+PBS group. This suggests that CPPs and CPPs-Exo can reduce scar formation. CPPs exosomes reduced the area of ​​cardiac necrosis and fibrosis in MI mice.

[0103] All of the above results indicate that CPP, CPPs fragments, and CPPs-Exo groups can improve cardiac function. When delivered to the infarcted area of ​​mice, the infarct size and degree of fibrosis were significantly reduced. However, injection of GW4869-treated CPPs reversed the repair effect of CPPs due to the inhibition of their exosome synthesis and release. These results demonstrate that CPPs exosomes promote functional improvement in damaged hearts.

[0104] To evaluate the effects of CPPs on angiogenesis and cardiomyocyte proliferation, we performed immunofluorescence staining on cardiac tissue sections with the vascular smooth muscle cell marker α-SMA (see Figure 8a, scale bar 500μm) and the proliferative cell marker Ki67 (see Figure 8b). The results are shown in Figure 8b. In the MI+CPPs group, the MI+CPPs fragment group, and the MI+CPPs-Exo group, the density of α-SMA+ vessels increased to nearly 40 vessels / mm 2 , while in the SHAM, MI+PBS and MI+CPPs(GW4869) groups it was less than 20 / mm 2 CPPs exosomes promote angiogenesis in the injured heart.

[0105] Figure 9 shows the staining results of proliferating cardiomyocyte markers cTnT and Ki67 in MI mice after grouping and treatment. Scale bar (upper row, each group): 500 μm; scale bar (lower row, each group): 250 μm. CPPs promote cardiomyocyte proliferation in MI mice through their exosomes.

[0106] As shown in Figure 10, the proportion of proliferating cardiomyocytes (identified by co-staining with cTnT and Ki67 antibodies) was significantly higher in the MI+CPPs, MI+CPPs fragments, and MI+CPPs-Exo groups (1.03%, 0.71%, and 1.18%, respectively), compared to only approximately 0.06% in the SHAM, MI+PBS, and MI+CPPs (GW4869) groups. This suggests that CPPs can promote cardiomyocyte proliferation. However, inhibiting exosome secretion with GW4869 reversed this pro-proliferative effect. Therefore, CPPs promote cardiomyocyte proliferation through their secreted exosomes. The MI+CPPs-Exo group exhibited significantly greater cardiomyocyte proliferation than the MI+CPPs group (Ki67-positive cardiomyocytes were 1.03% and 1.18%, respectively). Therefore, these results suggest that CPPs exosomes promote cardiomyocyte proliferation in MI mice.

[0107] MI mice were divided into: a. SHAM sham operation group, b. PBS intramyocardial injection group after MI, c. CPPs intramyocardial injection group after MI, d. CPPs intramyocardial injection treated with GW4896 after MI. The heart sections of the mice after grouping were hybridized with Y chromosome probes, and the results were compared with those of the positive control group e. male mice. As shown in ae of Figure 11 (scale bar 20 μm), the heart sections of the mice after grouping were hybridized with Y chromosome probes. It can be seen from Figure 11 that no signal was detected in the heart of the MI female mice 42 days after CPPs injection, indicating that 42 days after CPPs injection, the CPPs injected into the myocardium did not survive or transdifferentiate into other cells. Therefore, the above-mentioned function of promoting cardiac function improvement brought about by CPPs is not achieved through the proliferation or differentiation of CPPs themselves, but should be achieved through paracrine pathways, such as the exosomes secreted by them.

[0108] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing cardiopulmonary progenitor cell exosomes, characterized in that, The steps include the following: S1. Prepare cardiopulmonary progenitor cells and culture them in a cardiopulmonary progenitor cell culture medium. S2. Separate the cardiopulmonary progenitor cell exosomes from the culture medium of the cardiopulmonary progenitor cells in step S1 by ultracentrifugation.

2. The preparation method according to claim 1, characterized in that, Step S2 includes: Centrifuge the culture medium of the cardiopulmonary progenitor cells in step S1 at 300±10 g for 10±2 minutes, 2,000±10 g for 10±2 minutes, and 10,000±100 g for 30±5 minutes. Then, take the supernatant and centrifuge it at 100,000±100 g for 70±8 minutes. Discard the supernatant and resuspend it with PBS to extract the cardiopulmonary progenitor cell exosomes.

3. The preparation method according to claim 1, wherein The culture medium of the cardiopulmonary progenitor cells is ABC medium.

4. The preparation method according to claim 1, characterized in that, The ABC medium includes 2±0.5% B-27 without vitamin A, 2±0.5 mM L-glutamine, 1±0.2% non-essential amino acids, 0.1±0.02 mM β-mercaptoethanol, 1±0.2 μM A83-01, 50±5 ng / ml bFGF, 12±2 μM CHIR-99021, and 2.5 - 5.0% human platelet lysate.

5. The preparation method according to any one of claims 1-4, characterized in that, Step S1 includes: SA) Isolate the region where cardiopulmonary progenitor cells are located from a mammalian embryo at 9.5 days. SB) Digest the region where cardiopulmonary progenitor cells are obtained in step SA) with a digestive solution, centrifuge, and collect the cells. SC) Perform hanging drop culture for 46 - 52 hours by the method of inducing embryoid bodies, with the number of cells in each 15 μl hanging drop being 2000±30. SD) Perform suspension culture. After culturing in a differentiation medium for 24±2 hours, change to a basal medium and culture for 24±2 hours. Then, digest the cells into a single cell suspension and seed them on a gelatin-coated culture dish. SE) Continue to culture with ABC medium, changing the medium every two days until the cell confluence is greater than 90%, and then perform subculture.

6. Cardiopulmonary progenitor cell exosomes obtained by the preparation method according to any one of claims 1 - 5.

7. The cardiopulmonary progenitor cell exosome according to claim 5, characterized in that The particle size of the cardiopulmonary progenitor cell exosomes is 136.9±5.9 nm.

8. Use of the cardiopulmonary progenitor cell exosomes according to claim 6 in the preparation of a drug for preventing and / or treating cardiovascular diseases in mammals.

9. According to the use of claim 8, the cardiovascular disease is myocardial infarction.

10. Use of the cardiopulmonary progenitor cell exosomes according to claim 6 in the preparation of a drug for improving the heart function of mammals.

11. The application according to any one of claims 8 - 10, characterized in that, The use includes at least one of the following: a. Promote cardiovascular angiogenesis and increase cardiovascular density in mammals; b. Reduce the area of heart necrosis and / or fibrosis in mammals; c. Promote the proliferation of heart tissue cells in mammals, and the heart tissue cells include at least one of cardiomyocytes, pericytes, myofibroblasts, smooth muscle cells, and endothelial cells.

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