Application of 3D hypoxia mesenchymal stem cell derived exosome preparation in preparation of immune cell proliferation inhibitor or medicine for treating acute myocardial infarction

Abstract

The invention discloses application of a 3D hypoxia mesenchymal stem cell derived exosome preparation in preparation of an immune cell proliferation inhibitor or a medicine for treating acute myocardial infarction, belongs to the technical field of biology, and solves the problems of low cell survival rate and immunological rejection in application of a cell treatment method in the prior art. The 3D hypoxia mesenchymal stem cell derived exosome is used as a medium for treating acute myocardial infarction, and the problem of immunological rejection can be solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of a 3D hypoxic mesenchymal stem cell-derived exosome preparation in the preparation of an immune cell proliferation inhibitor or a drug for treating acute myocardial infarction. Background Technology

[0002] The conventional treatments for acute myocardial infarction (AMI) mainly include the following: Drug therapy: This involves using anticoagulants, antiplatelet drugs, and anti-ischemic drugs to relieve symptoms and prevent complications. For example, antiplatelet drugs such as aspirin are used to prevent thrombosis, and nitroglycerin is used to relieve angina symptoms. Interventional therapy: This includes percutaneous coronary intervention (PCI), which restores blood flow and reduces myocardial damage by placing stents or other devices within the blood vessels. Surgical procedures: Such as coronary artery bypass grafting, which improves myocardial blood supply by establishing new vascular access. However, for already damaged myocardial cells, drugs can only relieve symptoms and cannot repair or regenerate them, thus limiting their therapeutic effect. Interventional therapy and surgical procedures also carry higher risks and are not suitable for all patients.

[0003] Cell therapy, as an emerging approach, overcomes the drawbacks of drug therapy, interventional therapy, and surgical treatment, while offering advantages such as promoting myocardial repair and regeneration, good universality, and low risk, thus attracting widespread attention. However, existing cell therapy methods suffer from problems such as low cell survival rates and immune rejection reactions. Summary of the Invention

[0004] This invention provides an application of 3D hypoxic mesenchymal stem cell-derived exosomes to address the problems of low cell survival rate and immune rejection in existing cell therapy methods.

[0005] In a first aspect, the present invention provides the application of a 3D hypoxic mesenchymal stem cell-derived exosome preparation in the preparation of an immune cell proliferation inhibitor or a drug for treating acute myocardial infarction, wherein the 3D hypoxic mesenchymal stem cell-derived exosome preparation is the sole active ingredient.

[0006] It should be noted that the application of this invention is for non-therapeutic and non-diagnostic purposes.

[0007] As one possible implementation, the 3D hypoxic mesenchymal stem cell-derived exosomes in the 3D hypoxic mesenchymal stem cell-derived exosome preparation are obtained by a preparation method comprising the following steps:

[0008] Mesenchymal stem cells were seeded into a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein the seeding density was 4000-8000 cells / cm³. 2In the 3D hypoxic culture system, the oxygen partial pressure is 3%-8%, the pH is 6.8-7.6, the culture medium is serum-free mesenchymal stem cell culture medium containing L-ascorbic acid, and the microcarriers are spherical silica microcarriers modified with poly-L-lysine, with a microcarrier packing density of 0.4-0.6 kg / L. The amplification culture temperature is 36℃-38℃, and the time is 6-9 days. The intermittent heat shock culture program is as follows: 42℃ for 0.5 h, 37℃ for 1.5 h constitutes one heat shock cycle, and a total of 12 heat shock cycles are performed.

[0009] The 3D hypoxic mesenchymal stem cell-derived exosomes were obtained from the culture supernatant by tangential flow ultrafiltration.

[0010] The present invention does not impose any particular limitation on the specific preparation method of the mesenchymal stem cells, which can be obtained by conventional methods or by the methods provided in the embodiments of the present invention.

[0011] In this invention, the mesenchymal stem cells are P2 to P4 generation mesenchymal stem cells passaged in vitro, preferably P3 generation mesenchymal stem cells.

[0012] In this invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, or dental pulp mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.

[0013] In this invention, both the amplification culture and the intermittent heat shock culture are carried out in a bioreactor.

[0014] As one possible implementation, the tangential flow ultrafiltration method includes the following steps:

[0015] The culture supernatant was clarified and filtered sequentially through 0.45 μm and 0.22 μm filters to obtain the filtrate;

[0016] The filtrate was concentrated by tangential flow ultrafiltration through a 100-500kD hollow fiber membrane column at a concentration ratio of 1:20, and then replaced with physiological saline to obtain an exosome suspension.

[0017] The exosome suspension was sterilized by passing it through a 0.22 μm filter to obtain the 3D hypoxic mesenchymal stem cell-derived exosomes.

[0018] As one possible implementation, the immune cell proliferation inhibitor is used to suppress the proliferation of immune cells.

[0019] As one possible implementation, the drug for treating acute myocardial infarction is used to improve cardiac ejection fraction.

[0020] As one possible implementation, the drug for treating acute myocardial infarction is used to reduce the infarct size and the degree of myocardial fibrosis.

[0021] As one possible implementation, the drug for treating acute myocardial infarction is used to reduce inflammatory cell infiltration in myocardial tissue.

[0022] As one possible implementation, the drug for treating acute myocardial infarction is used to promote cardiac angiogenesis and reduce cardiomyocyte apoptosis.

[0023] As one possible implementation, the drug for treating acute myocardial infarction is used to promote the polarization of M1 macrophages into M2 macrophages.

[0024] Secondly, the present invention provides a medicament for treating acute myocardial infarction, comprising the aforementioned 3D hypoxic mesenchymal stem cell-derived exosome preparation.

[0025] As one possible implementation, the dosage form of the drug for treating acute myocardial infarction is an injection.

[0026] This invention utilizes extracellular vesicles (in this embodiment, 3D hypoxic mesenchymal stem cell-derived exosomes) as a medium to treat acute myocardial infarction, which can overcome the problem of immune rejection.

[0027] The 3D hypoxia mesenchymal stem cell-derived exosomes (3D-Exos) provided by this invention can be internalized by cardiomyocytes, endothelial cells, and cardiac fibroblasts, resisting cardiomyocyte apoptosis induced by oxygen and glucose deprivation, promoting HUVEC tube formation and migration in vitro, and inhibiting the secretion of Col-I and α-SMA by fibroblasts. 3D-Exos also have the ability to inhibit the proliferation of immune cells BV2. Furthermore, after injecting 3D hypoxic mesenchymal stem cell-derived exosomes (3D-Exos) into the hearts of mice with acute myocardial infarction (AMI), echocardiographic results showed that 3D-Exos significantly improved the left ventricular ejection fraction; MASSON staining of cardiac sections showed that 3D-Exos significantly reduced the infarct area and the level of myocardial fibrosis; HE staining of pathological sections showed that 3D-Exos significantly reduced inflammatory cell infiltration in myocardial tissue; immunofluorescence staining of pathological sections showed that 3D-Exos effectively promoted the polarization of M1 macrophages to M2 macrophages, thereby effectively controlling inflammation and maintaining macrophages in a beneficial state; CD31 immunofluorescence staining showed that 3D-Exos promoted cardiac angiogenesis; TUNEL staining showed that the apoptosis rate of cardiomyocytes in the 3D-Exos group was significantly lower than that in the AMI group, suggesting that 3D-Exos has anti-apoptotic ability. These findings indicate that 3D-Exos can effectively treat acute myocardial infarction in mice, providing more options for the preparation of drugs to treat acute myocardial infarction. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The 3D-Exos characterization results provided in this embodiment of the invention are shown, where A is the TEM result, B is the NTA result, and C is the WB result.

[0030] Figure 2 The results of the 3D-Exos internalization experiment provided in the embodiments of the present invention.

[0031] Figure 3The experimental results of the protective effect of 3D-Exos on damaged cells provided in the embodiments of the present invention are as follows: A represents the activity of HUVECs in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)); B represents the activity of CMs in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)); C represents the LDH activity in HUVECs in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)); and D represents the LDH activity in CMs in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)).

[0032] Figure 4 This invention relates to the effect of 3D-Exos on the mRNA expression of apoptosis-related genes, where A represents the Bcl2 gene expression in HUVECs of each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)); B represents the Bcl2 gene expression in CMs of each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)); and C represents the effect of 3D-Exos on the mRNA expression of apoptosis-related genes of each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)). D shows the expression of the Bax gene in HUVECs of the experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia); E shows the Western blot results of AKT phosphorylation levels in HUVECs of the experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia); F shows the AKT / GAPDH signaling pathway; G shows the pAKT / AKT signaling pathway.

[0033] Figure 5 The results of the 3D-Exos tubular structure formation experiment and in vitro wound healing experiment provided in the embodiments of the present invention are shown in the figure. In the figure, A is the cumulative tube length of HUVEC; B is the quantitative statistics of A; C is the migration area of ​​HUVEC; and D is the quantitative statistics of C.

[0034] Figure 6The results of the in vitro anti-fibrotic ability investigation experiment of 3D-Exos provided in the embodiments of the present invention are as follows: A represents the expression of Col-I and α-SMA proteins in CF under different treatments under the induction of TGF-β1, where + indicates that the substance was added during cell culture and - indicates that the substance was not added during cell culture; B is the quantitative statistics of A, where the upper figure shows the expression of Col-I protein and the lower figure shows the expression of α-SMA protein, where + indicates that the substance was added during cell culture and - indicates that the substance was not added during cell culture; C is the fluorescence staining result of Col-I and D is the fluorescence staining result of α-SMA.

[0035] Figure 7 The results of the experiment exploring the immunomodulatory capacity of 3D-Exos provided in the embodiments of the present invention are shown. In this figure, A represents the relative viability of BV2 cells under different treatments; B represents the relative viability of PBMC cells under different treatments. + indicates that the substance was added during cell culture, and - indicates that the substance was not added during cell culture.

[0036] Figure 8 This is a schematic diagram of the mouse acute myocardial infarction (AMI) model and treatment regimen provided in an embodiment of the present invention. In this diagram, A represents the mouse AMI model; B represents the treatment regimen; Establishment of AMI Exosome injection indicates the establishment of AMI exosome injection; Echocardiography indicates echocardiography; and Histological analysis indicates histological analysis.

[0037] Figure 9 The following are experimental results of the application of 3D-Exos provided in the embodiments of the present invention, wherein A is the ultrasound result image under different treatments; B is the LVEF result under different treatments; C is the LVFS result under different treatments; D is the LVEDV result under different treatments; and E is the LVESV result under different treatments.

[0038] Figure 10 The images show the masson staining results of mouse heart sections with different treatments provided in the embodiments of the present invention. A represents the staining results images on Day 7 and Day 28; B represents the quantitative statistics on Day 7; and C represents the quantitative statistics on Day 28.

[0039] Figure 11 HE staining results of mouse heart sections with different treatments provided in embodiments of the present invention.

[0040] Figure 12The images show the immunofluorescence staining results of mouse heart sections under different treatments provided in the embodiments of the present invention. In this image, A represents the CD206 staining results under different treatments; B represents the quantitative statistics of A; C represents the iNOS staining results under different treatments; and D represents the quantitative statistics of C.

[0041] Figure 13 The images show the CD31 staining results of mouse heart slices with different treatments provided in the embodiments of the present invention. The left image shows the staining results, and the right image shows the quantitative statistics of the left image.

[0042] Figure 14 The images show the TUNEL fluorescence staining results of mouse heart sections with different treatments provided in the embodiments of the present invention. The left image shows the staining results, and the right image shows the quantitative statistics of the left image. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] To address the issues of low cell survival rates and immune rejection in existing cell therapy methods, this invention provides a method for preparing and characterizing 3D hypoxic mesenchymal stem cell-derived exosomes. The 3D-Exos prepared in this invention exhibits a clear bilayered membrane structure, with a saucer-shaped or one-sidedly concave hemispherical morphology; the particle size is between 30 and 150 nm, consistent with the size of MSC-exos; its surface marker proteins CD81, CD63, and CD9, as well as its internal marker protein TSG101, are positively expressed, while the negative marker calnexin is not detected. Furthermore, this invention demonstrates that the prepared 3D-Exos can be internalized by HUVECs, CMs, and CFs.

[0045] Furthermore, the embodiments of the present invention demonstrate that 3D-Exos has the function of protecting cells from damage, has in vitro anti-apoptotic effects, and has the ability to promote HUVEC angiogenesis and HUVEC migration in vitro.

[0046] Furthermore, embodiments of the present invention demonstrate that 3D-Exos can inhibit the differentiation of fibroblasts into myofibroblasts and can inhibit the proliferation of immune cells.

[0047] Furthermore, the embodiments of the present invention demonstrate that 3D-Exos can improve cardiac function in AMI mice; reduce cardiac fibrosis in AMI mice; reduce inflammatory response in the heart of AMI mice; effectively promote the polarization of M1 macrophages to M2 macrophages, thereby effectively controlling inflammation and maintaining macrophages in a beneficial state; promote cardiac angiogenesis in AMI mice; and have anti-apoptotic ability.

[0048] This invention applies 3D hypoxia-scale production of mesenchymal stem cell exosomes to the treatment of acute myocardial infarction. It can effectively improve cardiac function in AMI mice, reduce myocardial infarction area and myocardial fibrosis level, reduce inflammatory cell infiltration, promote the polarization of M1 macrophages to M2 macrophages, thereby effectively controlling inflammation and maintaining macrophages in a beneficial state, promoting cardiac angiogenesis, and reducing cardiomyocyte apoptosis, providing more options for the preparation of drugs for the treatment of acute myocardial infarction.

[0049] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0050] In the following examples, the bioreactor used is a stainless steel bioreactor BRSTMCELL-2.6L, sourced from BiRui biotech; L-ascorbic acid (HY-B0166, medchemexpress); serum-free mesenchymal stem cell culture medium (SC2013-G, TBD); spherical silica microcarriers (1.1 mm in diameter, 5001-89-S1, Tiantai Precision); and poly-L-lysine solution (0.1 mg / mL, 60717ES50, Yisheng Biotechnology).

[0051] Example 1

[0052] This embodiment provides an experiment for the preparation and characterization of 3D hypoxic mesenchymal stem cell-derived exosomes.

[0053] Preparation of P3 generation umbilical cord mesenchymal stem cells: The two ends of the human umbilical cord tissue were tied off and immersed in a bottle containing physiological saline. The tube was then transported to the laboratory. The bottle was opened in a clean bench, and the preservation solution was collected (if cells were contaminated, this liquid was used for testing to locate the source of contamination). After discarding the excess preservation solution, 20 mL of 75% alcohol was added to the bottle (enough to submerge the umbilical cord). The bottle was tightened, and the tube was shaken 7 times from side to side before soaking for 2 minutes. The alcohol was discarded, and the tube was washed twice with 20 mL of physiological saline to remove any alcohol residue. The umbilical cord was removed with sterile forceps and placed in a sterile 100 mm culture dish. 10 mL of physiological saline was added. Using sterile scissors, divide the umbilical cord into 3-4 cm segments. Grasp the center of the cord with forceps and push it towards both ends to wash away blood. Locate the umbilical vein, bluntly peel the cord apart from the vein, and remove the vein to obtain a smooth tissue block. Separate the umbilical cord mesenchyme (gelatinous), and promptly place the separated mesenchyme into physiological saline. Remove the umbilical arteries (2). Place the tissue block into a 50 mL centrifuge tube and cut it into 1-3 mm pieces with long scissors. 3 Small segments. Take serum-free culture medium (containing serum substitute) and dispense 10 mL / bottle into T75 cell culture flasks. Dispense the chopped tissue pieces into each flask; approximately 10 flasks can be dispensed from each umbilical cord. Shake the tissue pieces evenly to distribute them, then place them in an incubator at 37°C, 5% CO2, and saturated humidity for 5 days, changing the medium afterward. Observe the area around the tissue pieces under a microscope. Change the culture medium every 5 days. After 9 days, cells will emerge from around the tissue pieces. Once 80% of the cells have merged, collect the cells for passage and culture in normoxic conditions for 2D up to the 3rd generation to obtain P3 generation umbilical cord mesenchymal stem cells.

[0054] The P3 generation umbilical cord mesenchymal stem cells were seeded into a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein, the seeding density was 5000 cells / cm³. 2In the 3D hypoxic culture system, the oxygen partial pressure was 5%, the pH was 7.2, and the culture medium used was serum-free mesenchymal stem cell culture medium containing 50 μmol / L L-ascorbic acid. The microcarriers used were spherical silica microcarriers modified with poly-L-lysine, with a packing density of 0.5 kg / L. The amplification culture was conducted at 37°C for 7 days. The intermittent heat shock culture program was as follows: 0.5 h at 42°C followed by 1.5 h at 37°C constituted one heat shock cycle, for a total of 12 heat shock cycles. Both the amplification culture and the intermittent heat shock culture were carried out in a BRSTMCELL-2.6L stainless steel bioreactor. The serum-free culture medium for mesenchymal stem cells containing L-ascorbic acid was prepared by the following steps: 500 mL of serum-free culture medium for mesenchymal stem cells was taken, and L-ascorbic acid was added to a final concentration of 50 μmol / L; the poly-L-lysine-coated spherical silica microcarriers were prepared by the following steps: the spherical silica microcarriers were placed in a poly-L-lysine solution and soaked at 37°C for 2 h, and the coating solution was discarded to obtain poly-L-lysine-coated spherical silica microcarriers.

[0055] The culture supernatant was sequentially filtered through 0.45 μm and 0.22 μm filters to remove cell debris and large impurity particles, yielding a filtrate. The filtrate was then concentrated by tangential flow ultrafiltration through a 300 kD hollow fiber membrane at a concentration ratio of 1:20, followed by replacement with physiological saline to obtain an exosome suspension. The exosome suspension was then sterilized by filtration through a 0.22 μm filter to obtain 3D hypoxic mesenchymal stem cell-derived exosomes (3D-Exos).

[0056] The prepared 3D-Exos was characterized and analyzed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB) techniques, yielding the following results: Figure 1 The results are shown. (By...) Figure 1 As shown in A, 3D-Exos has a clear double-layered capsule structure, with a saucer-shaped or hemispherical shape with one side concave; Figure 1 As indicated by B, the particle size of 3D-Exos is between 30-150 nm, which is consistent with the size of mesenchymal stem cell-derived exosomes (MSC-exos); Figure 1 As shown in C, the surface marker proteins CD81, CD63, and CD9 of 3D-Exos, as well as the internal marker protein TSG101, are positively expressed, while the negative marker calnexin is not detected.

[0057] The 3D-Exos prepared in Example 1 were labeled with PKH-67 (a fluorescent dye) and co-cultured with HUVECs (human umbilical vein endothelial cells), CMs (cardiomyocytes), and CFs (cardiomyocyte fibroblasts) labeled with phalloidin, respectively, to obtain the following results: Figure 2 The results are shown. (By...) Figure 2 It can be seen that PKH-67 labeled 3D-Exos (green fluorescence) is effectively taken up by cells into the cytoplasm (red fluorescence) and accumulates around the cell nucleus, indicating that 3D-Exos can be internalized by HUVECs, CMs, and CFs.

[0058] Example 2

[0059] This embodiment provides an experimental study to explore the mechanism of action of 3D hypoxic mesenchymal stem cell-derived exosomes.

[0060] Establish a cell OGD (Oxygen-Glucose Deprivation) model (OGD treatment): Cells (human umbilical vein endothelial cells (HUVECs) or cardiomyocytes (CMs)) were cultured in glucose-free DMEM medium at 37 °C for 10 h in an environment containing 94% N2, 5% CO2 and 1% O2 to simulate cell damage. Experimental group (OGD+3D-Exos): Cells were cultured in glucose-free DMEM medium at 37 ℃ for 10 h in an environment containing 94% N2, 5% CO2 and 1% O2, and then 3D-Exos was added to a final concentration of 50 μg / mL; Control group (OGD+PBS): Cells were cultured in glucose-free DMEM medium at 37 ℃ for 10 h in an environment containing 94% N2, 5% CO2 and 1% O2, and then PBS solution was added (the volume of PBS solution was the same as the volume of 3D-Exos added in the experimental group); Normal group (Normoxia): Cells were cultured under normal aerobic and normal glucose conditions.

[0061] The protective effect of 3D-Exos on damaged cells was investigated using the CCK-8 assay, and the results were as follows: Figure 3 The results shown in A and B are from... Figure 3 As shown in A and B, HUVECs or CMs exhibited significantly reduced relative cell viability after OGD treatment, while 3D-Exos co-culture significantly improved the viability of HUVECs or CMs, promoting cell proliferation and protecting damaged cells. The release of lactate dehydrogenase (LDH) was used to detect cell apoptosis in each group, yielding the following results: Figure 3 The results shown in C and D are derived from... Figure 3 As shown in C and D, compared with the control group (OGD+PBS), the experimental group (OGD+3D-Exos) showed a significant decrease in LDH activity in HUVECs or CMs, indicating reduced cell damage.

[0062] The mRNA expression levels of anti-apoptosis-related Bcl2 and pro-apoptosis-related Bax genes in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)) were detected, and the results were as follows: Figure 4 The results are shown in AD. (By...) Figure 4 As shown in A and B, compared with the control group (OGD+PBS), the experimental group (OGD+3D-Exos) upregulated the expression of the Bcl2 gene in HUVECs or CMs treated with OGD; Figure 4 As shown in C and D, compared with the control group (OGD+PBS), the experimental group (OGD+3D-Exos) downregulated the expression of the Bax gene in HUVECs or CMs treated with OGD. These results indicate that 3D-Exos can protect against OGD-induced cell damage by upregulating the anti-apoptotic gene Bcl2 and downregulating the pro-apoptotic gene Bax, thereby inhibiting OGD-induced apoptosis.

[0063] AKT protein is believed to play an important role in cell survival. Phosphorylated AKT protein can promote cell proliferation and growth by activating various signaling molecules and pathways. Western blotting (WB) was used to detect the AKT phosphorylation level in HUVECs in each group (experimental group (OGD+3D-Exos), control group (OGD+PBS), and normal group (Normoxia)). The results are as follows: Figure 4 The results are shown in EG. (From...) Figure 4 The EG data showed that the AKT phosphorylation level in HUVECs of the experimental group (OGD+3D-Exos) was significantly higher than that of the control group (OGD+PBS), indicating that 3D-Exos may activate the intracellular pAKT / AKT signaling cascade, suggesting the potential mechanism of the cell proliferation and growth-promoting effect of 3D-Exos.

[0064] The therapeutic effects of 3D-Exos were analyzed through tubular structure formation experiments and in vitro wound healing experiments, yielding the following results: Figure 5 The results are shown. (By...) Figure 5 As shown in A and B, compared with the control group (OGD+PBS), the experimental group (OGD+3D-Exos) had a significantly higher cumulative HUVEC tube length (cumulative tube length). Figure 5 As shown in C and D, the experimental group (OGD+3D-Exos) had a larger HUVEC migration area compared to the control group (OGD+PBS). These results indicate that 3D-Exos possesses the ability to promote HUVEC tube formation and migration in vitro.

[0065] Example 3

[0066] This embodiment provides an in vitro effect study on 3D hypoxic mesenchymal stem cell-derived exosomes.

[0067] (1) In order to investigate the in vitro anti-fibrotic ability of 3D-Exos, primary cardiac fibroblasts were induced to transdifferentiate into myofibroblasts by transforming growth factor-β1 (TGF-β1 or TGFβ1).

[0068] Preparation of complete culture medium: Add 15 mL of FBS and 1 mL of penicillin-streptomycin solution to 84 mL of DMEM-F12 medium, mix well, and prepare a complete culture medium containing 1% penicillin-streptomycin and 15% serum.

[0069] TGF-β1 group: Primary cardiac fibroblasts of SD rats were cultured in complete culture medium with a final concentration of 10 ng / mL of TGF-β1 to induce the transdifferentiation of fibroblasts (CF) into myofibroblasts to establish a fibrosis model.

[0070] The TGF-β1+3D-Exos group; primary cardiac fibroblasts of SD rats were cultured in complete culture medium with a final concentration of 10 ng / mL TGF-β1 and 50 μg / mL 3D-Exos to observe the regulatory and intervention effects of exosomes on TGF-β1-induced CF transdifferentiation.

[0071] Blank group: Primary cardiac fibroblasts of SD rats were cultured in complete culture medium without the addition of TGF-β1 and 3D-Exos, serving as a baseline control to reflect the normal state of the cells.

[0072] After 48 hours of culture, cells from each group were collected. The expression of type I collagen (Col-I) and α-smooth muscle actin (α-SMA) in each group was detected using Western blotting (WB) and immunofluorescence staining. The results were as follows: Figure 6 The results are shown. (By...) Figure 6 As shown in A and B, compared with the TGF-β1 group, the expression levels of Col-I and α-SMA proteins were significantly downregulated in the CF group treated with 3D-Exos (TGF-β1+3D-Exos group). Figure 6 From C and D, we can see that after TGF-β1 treatment (TGF-β1 group), α-SMA ( Figure 6 C) and Col-I ( Figure 6 The fluorescence intensity of α-SMA (in the group D) was significantly enhanced; while after co-culture with TGF-β1 and 3D-Exos (TGF-β1+3D-Exos group), the fluorescence intensity of α-SMA (in the group D) was significantly enhanced; Figure 6 C) and Col-I ( Figure 6The fluorescence signal of D in the D group was reduced to varying degrees. These results indicate that 3D-Exos has the ability to inhibit the secretion of Col-I and α-SMA by fibroblasts, and can inhibit the differentiation of fibroblasts into myofibroblasts.

[0073] (2) The immunomodulatory capacity of 3D-Exos was investigated by culturing BV2 cells (mouse microglia) and PBMC cells (peripheral blood mononuclear cells) in vitro.

[0074] Control group: BV2 cells were cultured normally in DMEM medium containing 10% FBS for 72 h without the addition of exosomes, to reflect the basic activity of cells under normal conditions.

[0075] 3D-Exos group (3D-Exos): BV2 cells were cultured normally in DMEM medium containing 10% FBS for 24 h, and then 3D-Exos was added to a final concentration of 50 μg / ml. The cells were cultured for another 48 h to observe the effect of exosomes on cell viability.

[0076] PHA-M group (PHA-M): PBMC cells were cultured normally in RPMI-1640 medium containing 10% FBS for 4 h, and then collected by centrifugation; the collected cells were then divided into 3×10⁶ cells per well. 4 The cells were re-seeded at a density of 100 cells / well in a 96-well plate, and PHA-M was added to a final concentration of 5 µg / mL. The plate was then incubated for 24 h.

[0077] PHA-M+3D-Exos group (PHA-M+3D-Exos): PBMC cells were cultured normally in RPMI-1640 medium containing 10% FBS for 4 h, and then collected by centrifugation; the collected cells were then divided into 3×10⁶ cells per well. 4 The cells were re-seeded at a density of 100 cells / well in a 96-well plate, and PHA-M and 3D-Exos were added to a final concentration of 5 µg / mL and 50 µg / mL, respectively. The plates were then incubated for 24 h.

[0078] The proliferation activity of BV2 cells and PBMC cells in each group was detected using the CCK-8 assay, and the results were as follows: Figure 7 The results are shown. (By...) Figure 7 As shown in A, 3D-Exos can effectively inhibit the proliferation of BV2 cells. Figure 7 As indicated by B in the data, PHA-M is the most commonly used mitogen, which can stimulate PBMC cell proliferation, while 3D-Exos has the ability to reverse this proliferation. The above data suggest that 3D-Exos can inhibit immune cell proliferation in vitro, exhibiting both immunomodulatory and immunosuppressive effects, and can exert anti-inflammatory or immune-balancing effects by inhibiting immune cell proliferation.

[0079] Example 4

[0080] This embodiment provides an application experiment of 3D hypoxic mesenchymal stem cell-derived exosomes.

[0081] Experimental animals: C57BL / 6 mice (male, 22-25g, 8 weeks old).

[0082] Establishment of a mouse model of acute myocardial infarction (AMI): Eighteen mice were randomly divided into three groups of six mice each. The three groups were as follows:

[0083] Sham group: Mice underwent thoracotomy without ligation;

[0084] AMI group (AMI): Acute myocardial infarction (AMI) mice were created by ligating the left anterior descending coronary artery of the mouse heart;

[0085] 3D-Exos group (3D-Exos): Acute myocardial infarction (AMI) mice were created by ligating the left anterior descending coronary artery of the mouse heart. Immediately afterwards, 3D-Exos was injected at three points in the infarct border area (10 µL at each point, 20 µg / 10µL).

[0086] Cardiac function in each group was observed by echocardiography at different time points, including Day 3 (D3), Day 7 (D7), Day 14 (D14), and Day 28 (D28), and histological analysis was performed to obtain the following results: Figure 9 The results are shown.

[0087] Depend on Figure 9 In the A-cell echocardiography, 3D-Exos was shown to improve cardiac function in AMI mice. Figure 9 In groups B and C, the 3D-Exos group showed sustained improvement in LVEF (left ventricular ejection fraction) and LVFS (left ventricular fractional shortening) throughout the 4-week monitoring period. Figure 9 As shown in D and E, although the left ventricle of mice in the 3D-Exos group showed a continuous expansion trend, the increase trend of LVEDF (left ventricular end-systolic volume) and LVESV (left ventricular end-diastolic volume) was slowed down in the 3D-Exos group compared with the AMI group.

[0088] Masson staining was performed on heart sections from each group of mice to obtain the following results: Figure 10 The results are shown. (By...) Figure 10The AC data showed that the hearts of AMI group mice exhibited extensive collagen deposition and large areas of blue fibrous connective tissue, along with thinning of the left ventricle and enlargement of the heart chambers. The infarct area reached 31.45% on day 7 and 38.88% on day 28. In the 3D-Exos group, the blue area was significantly reduced, with infarct areas of 10.45% and 13.97% on days 7 and 28, respectively, indicating a significant decrease in infarct area and thus a reduced degree of myocardial fibrosis. Therefore, 3D-Exos can effectively alleviate cardiac fibrosis in AMI mice.

[0089] HE staining was performed on heart sections from each group of mice to obtain the following results: Figure 11 The results are shown. (By...) Figure 11 Significant cardiomyocyte necrosis was observed in the myocardial tissue of AMI group mice. Microscopic observation of the necrotic cardiomyocytes revealed pathological features including the disappearance of cytoplasmic striations and varying staining intensities. Furthermore, a large number of inflammatory cells infiltrated the myocardial tissue. The cardiac function of mice in the 3D-Exos group showed improvement; inflammatory cell infiltration in the myocardial tissue was significantly reduced, the inflammatory response was milder, and the myocardial fibers were arranged normally and clearly defined. In addition, the boundary between the infarcted and non-infarcted areas was more distinct. Therefore, 3D-Exos can effectively alleviate the inflammatory response in the heart of AMI mice.

[0090] Immunofluorescence staining was used to assess the polarization of macrophages in the hearts of mice in each group, and the results were as follows: Figure 12 The results are shown. (By...) Figure 12 As can be seen from A and B in the diagram, compared with the AMI group, the 3D-Exos group had CD68 in the infarct area. + CD206 (a marker of M2 macrophages) expression is upregulated in macrophages; by Figure 12 As shown in C and D, compared with the AMI group, the expression of iNOS (a marker of M1 macrophages) was downregulated in the 3D-Exos group. This indicates that 3D-Exos can effectively promote the polarization of M1 macrophages to M2 macrophages, thereby effectively controlling inflammation and maintaining macrophages in a beneficial state.

[0091] CD31 staining was performed on heart sections from each group of mice to investigate the effect of 3D-Exos on promoting cardiac angiogenesis in AMI mice. Results were obtained as follows: Figure 13 The results are shown. (By...) Figure 13 It is evident that, compared to the AMI group, the 3D-Exos group was able to observe more CD31. + Blood vessels (CD31) + Vessels); Through quantitative analysis of tubular neovascularization density, it was found that CD31 formed in the 3D-Exos group +The number of blood vessels was significantly higher in the 3D-Exos group than in the AMI group, indicating that 3D-Exos promotes cardiac angiogenesis in AMI mice.

[0092] Persistent myocardial ischemia induces apoptosis in a large number of cardiomyocytes. TUNEL fluorescence staining was performed on heart sections from mice in each group to investigate the anti-apoptotic effect of 3D-Exos in infarcted hearts. Results were as follows: Figure 14 The results are shown. (By...) Figure 14 It can be seen that, compared with the AMI group, the red TUNEL in the 3D-Exos group + cardiomyocytes (TUNEL) + CMs) were significantly reduced; through TUNEL + Quantitative analysis of cardiomyocytes revealed that TUNEL in the 3D-Exos group + The apoptosis rate of cardiomyocytes was significantly lower than that of the AMI group, indicating that 3D-Exos has anti-apoptotic ability.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a 3D hypoxic mesenchymal stem cell-derived exosome preparation in the preparation of an immune cell proliferation inhibitor or a drug for treating acute myocardial infarction, characterized in that, The 3D hypoxic mesenchymal stem cell-derived exosome preparation is the sole active ingredient.

2. The application according to claim 1, characterized in that, The 3D hypoxic mesenchymal stem cell-derived exosomes in the aforementioned 3D hypoxic mesenchymal stem cell-derived exosome preparation are obtained through a preparation method comprising the following steps: Mesenchymal stem cells were seeded into a 3D hypoxic culture system for expansion culture and intermittent heat shock culture, and the culture supernatant was collected; wherein the seeding density was 4000-8000 cells / cm³. 2 In the 3D hypoxic culture system, the oxygen partial pressure is 3%-8%, the pH is 6.8-7.6, the culture medium is serum-free mesenchymal stem cell culture medium containing L-ascorbic acid, and the microcarriers are spherical silica microcarriers modified with poly-L-lysine, with a microcarrier packing density of 0.4-0.6 kg / L. The amplification culture temperature is 36℃-38℃, and the time is 6-9 days. The intermittent heat shock culture program is as follows: 42℃ for 0.5 h, 37℃ for 1.5 h constitutes one heat shock cycle, and a total of 12 heat shock cycles are performed. The 3D hypoxic mesenchymal stem cell-derived exosomes were obtained from the culture supernatant by tangential flow ultrafiltration.

3. The application according to claim 2, characterized in that, The tangential flow ultrafiltration method includes the following steps: The culture supernatant was clarified and filtered sequentially through 0.45 μm and 0.22 μm filters to obtain the filtrate; The filtrate was concentrated by tangential flow ultrafiltration through a 100-500kD hollow fiber membrane column at a concentration ratio of 1:20, and then replaced with physiological saline to obtain an exosome suspension. The exosome suspension was sterilized by passing it through a 0.22 μm filter to obtain the 3D hypoxic mesenchymal stem cell-derived exosomes.

4. The application according to claim 1, characterized in that, The immune cell proliferation inhibitor is used to suppress the proliferation of immune cells.

5. The application according to claim 1, characterized in that, The medication used to treat acute myocardial infarction is used to improve cardiac ejection fraction.

6. The application according to claim 1, characterized in that, The medication used to treat acute myocardial infarction is intended to reduce the infarct size and the degree of myocardial fibrosis.

7. The application according to claim 1, characterized in that, The medication used to treat acute myocardial infarction is intended to reduce inflammatory cell infiltration in myocardial tissue.

8. The application according to claim 1, characterized in that, The medication used to treat acute myocardial infarction is intended to promote cardiac angiogenesis and reduce myocardial cell apoptosis.

9. The application according to claim 1, characterized in that, The drug used to treat acute myocardial infarction is used to promote the polarization of M1 macrophages into M2 macrophages.

10. A drug for treating acute myocardial infarction, characterized in that, Includes the 3D hypoxic mesenchymal stem cell-derived exosome preparation as described in any one of claims 1-9; the dosage form of the drug for treating acute myocardial infarction is an injection.

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