Medicine prepared from apoptotic fibroblast-derived vesicles and used for preventing and treating myocardial ischemia-reperfusion injury as well as preparation method and application of medicine

Abstract

The invention discloses a medicine prepared from apoptotic fibroblast-derived vesicles and used for preventing and treating myocardial ischemia-reperfusion injury as well as a preparation method and application of the medicine, and relates to the technical field of biological medicines. The method comprises the following steps: digesting an in-vitro animal heart apex tissue to obtain a digested cell suspension; the myocardial fibroblasts are obtained; the method comprises the following steps: culturing myocardial fibroblasts in a DMEM (dulbecco's modified eagle medium) solution through apoptosis inducer H2O2 to obtain a culture solution; according to the method, apoptosis of fibroblasts is induced to generate specific extracellular vesicles, the surface of the extracellular vesicles carries CD22 as a marker, the content of the extracellular vesicles is enriched in miR-1246 and is taken by myocardial cells, expression of p53 is down-regulated, mitochondrial homeostasis is maintained, and the extracellular vesicles can be used for detecting apoptosis of the fibroblasts. The myocardial cell apoptosis is inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a drug for preventing and treating myocardial ischemia-reperfusion injury prepared by using vesicles derived from apoptotic fibroblasts, as well as a preparation method and application thereof. Background Art

[0002] Reperfusion injury after myocardial infarction (MI) is a widespread, unresolved clinical problem requiring timely identification and intervention. As more and more patients survive the early ischemic event, reperfusion-induced cardiac dysfunction and potential heart failure are becoming increasingly common and a significant prognostic issue for these patients. Cardiac fibroblasts (CFbs), the primary cellular agents responsible for cardiac healing after myocardial ischemia-reperfusion (MI / R), have emerged as an attractive therapeutic target. The heart is composed of both cardiomyocytes and non-cardiomyocytes, with non-cardiomyocytes accounting for 70% of the total body volume, of which over 90% are composed of cardiac fibroblasts. CFbs primarily provide structural and functional support to cardiomyocytes, aiding the healing of damaged myocardial tissue in the early stages of injury and, to a certain extent, possessing a protective effect on the myocardium. Myocardial damage occurs immediately within hours after MI, while the proliferation and differentiation of CFbs, key support cells, do not occur until two days later. Subsequently, aberrant activation of CFbs leads to ventricular injury, remodeling, and potentially fibrosis. However, after myocardial injury and before CFbs are activated, many factors such as oxidative stress, excessive preload and postload, calcium homeostasis imbalance and mitochondrial dysfunction promote the production of key mediators of cell apoptosis. Therefore, the simultaneous apoptosis of cardiomyocytes and CFbs in the early stage is also a key component of early cardiac injury and repair. The important question that follows is: Is there a direct mechanistic link between apoptotic fibroblasts and cardiomyocytes? Elucidating how apoptotic fibroblasts cause a response in cardiomyocytes, clarifying the specific triggering mechanism, and then using fibroblasts to continue to study the function of the heart in depth will provide new research basis and hope for the development of new and effective therapies to improve patients' heart disease.

[0003] Under conditions of hypoxia and mechanical stress, fibroblasts undergo massive apoptosis, and the extracellular vesicles (EVs) they produce may become new biomarkers of the cellular microenvironment in conditions such as cardiovascular disease. These specialized EVs, shed under different circumstances, can carry specific contents, including microRNAs, proteins, and lipids, and affect surrounding tissue cells. A growing body of research indicates that apoptotic cells can release metabolic molecules that maintain stability and exit the cell, transmitting anti-inflammatory effects, promoting tissue repair in surrounding cells, and alleviating inflammatory responses. However, the role of these signals in heart disease is poorly understood.

[0004] It is well known that the regenerative capacity of cardiomyocytes is limited, and preventing myocardial damage and loss is the essence of treating MI / R injury. The applicant confirmed the intercellular communication between cardiomyocytes and fibroblasts in MI / R. So, do the EVs secreted by apoptotic fibroblasts have a certain effect on cardiomyocytes by changing the surrounding cellular microenvironment? There are no reports on exploring the relevant phenotypes of "apoptotic fibroblast-derived EVs subtypes (CFbs-Apoptotic vesicles, CFbs-apoVs)" and developing drug treatments to protect the myocardium.

[0005] Extracellular vesicles (EVs) are a general term for various membrane-bound vesicles secreted and released by most cell types. As carriers of signaling molecules or biological information, EVs act like "cargo warehouses" to carry and transport cytokines, proteins, lipids, mRNA, miRNA, and organelle structures, influencing the surrounding environment. Therefore, EVs can serve as drug carriers and replace cells in their therapeutic effects. EVs are diverse, and the identification, characterization, and quantification of fibroblast-derived EVs in the heart, or their use as biologics, is particularly challenging and has yet to be reported. Through exploratory research, the applicants discovered a new approach to induce fibroblast apoptosis by producing specific extracellular vesicles—CFbs-apoVs. CFbs-apoVs carry the specific marker CD22 on their surface. The contents of CFbs-CD22+apoVs are enriched with miR-1246. Cardiomyocytes can take up CFbs-CD22+apoVs, downregulating p53 expression and maintaining mitochondrial homeostasis, reducing myocardial damage, and producing a protective effect. The isolation, extraction and identification of CFbs-CD22+apoVs, a functional extracellular vesicle subtype specifically secreted by fibroblasts of apoptotic origin, remain technically difficult and rarely reported. The engineered production of CFbs-CD22+apoVs and its translation into clinical practice will provide new ideas and treatment methods for improving the microenvironmental strategy of MI / R injury. Summary of the Invention

[0006] To address the shortcomings of the aforementioned background technology, the present invention provides a drug for preventing and treating myocardial ischemia-reperfusion injury using vesicles derived from apoptotic fibroblasts, as well as a preparation method and application. This method induces fibroblast apoptosis to produce specific extracellular vesicles. These vesicles carry CD22 on their surface and are enriched with miR-1246, which can be taken up by cardiomyocytes. This vesicles downregulate myocardial p53 expression, maintain mitochondrial homeostasis, and inhibit apoptosis. This method defines the pathophysiological process by which fibroblasts protect cardiomyocytes from a novel perspective of intercellular communication.

[0007] The first object of the present invention is to provide a method for preparing vesicles derived from apoptotic fibroblasts, comprising the following steps:

[0008] The isolated animal cardiac apex tissue was digested in stages using a mixed digestion solution of trypsin and collagenase II, and then the digestion was terminated using a DMEM solution containing FBS to obtain a digested cell suspension;

[0009] The digested cell suspension was filtered and centrifuged, and the precipitated cells were resuspended in DMEM and cultured for 1 to 2 hours. Then, the cells were separated and purified by differential adherence culture to obtain cardiac fibroblasts.

[0010] The cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 to obtain culture medium;

[0011] The culture fluid is separated and purified to obtain vesicles derived from apoptotic fibroblasts.

[0012] Preferably, the volume ratio of pancreatic enzyme to collagenase II in the mixed digestive fluid is 1:1-3, wherein the mass volume percentage of pancreatic enzyme is 0.05-0.2%;

[0013] The content of FBS in the FBS-containing DMEM solution is 8-12% by volume;

[0014] The filtration is performed using a 200-mesh sieve;

[0015] After filtration, centrifugation is performed at 1-5°C, at 600-1000 pr / min for 2-4 times, with each centrifugation lasting 3-8 minutes.

[0016] Preferably, the concentration of the apoptosis inducer H2O2 is 0.2-0.8 mM;

[0017] Cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 for 12 to 48 h;

[0018] The culture fluid separation process comprises:

[0019] The culture medium was centrifuged at 300 × g for 10 min, and the supernatant was centrifuged at 1000 × g for 15 min to remove cells and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 30 min at 1-5°C and filtered through a 0.22 μm filter to remove subcellular structures. The supernatant was then ultracentrifuged at 100,000 × g for 70 min to collect the EV pellet, which was washed with PBS and centrifuged at 100,000 × g for 70 min. The EV pellet was resuspended in 100 μL PBS.

[0020] The purification process of the culture fluid after separation includes:

[0021] The resuspended EVs pellet is spread on a sucrose solution and centrifuged at 100,000 g for 70 min. Protein aggregates will settle to the bottom of the centrifuge tube, while EVs containing lipid structures will settle to the isopycnic area, resulting in higher purity EVs.

[0022] The higher purity EVs were washed with PBS and centrifuged at 100,000 g for 70 min. The EVs were resuspended in 100 μL PBS to obtain vesicles derived from apoptotic fibroblasts.

[0023] Preferably, it is characterized in that the sucrose solution is a system in which two sucrose solutions with concentrations of 0.25 mol / L and 2.5 mol / L are prepared into a continuous density gradient.

[0024] The second object of the present invention is to provide a vesicle derived from apoptotic fibroblasts, wherein the vesicle contains apoptosis-specific proteins p53, Bcl-2, Bax, caspase-3, and Apaf-1 and has phosphatidylserine exposure;

[0025] The vesicles carry CD22 on their surface and are enriched with miR-1246.

[0026] Preferably, flow cytometry is used to characterize the expression of fibroblast markers in the vesicles, analyze the probability of α-SMA expression events in the vesicles, and determine that the specific EVs subtype is derived from fibroblasts.

[0027] The third object of the present invention is to provide a method for identifying surface CD22 in vesicles derived from apoptotic fibroblasts, comprising:

[0028] Based on high-throughput HNCIB detection, the vesicle marker CD22 was screened;

[0029] Alternatively, flow cytometry was used to analyze CD22 fluorescence on the vesicle surface;

[0030] Alternatively, immunogold labeling and transmission electron microscopy were used to observe the expression of CD22 on the surface of EVs.

[0031] Preferably, the expression of CD22 on the surface of EVs is observed using immunogold-labeled transmission electron microscopy, including:

[0032] Vesicles were labeled with anti-rabbit IgGGold2 CD22 antibody and a water film was formed on a quantitative foil R2 / 2 grid. The water film grid was permeabilized by injecting it into ethane cooled to the melting point by liquid nitrogen.

[0033] The specimens were transferred to a Gatan cryostat mount for a transmission electron microscope, and the expression of CD22 on the surface of EVs was observed using immunogold labeling and transmission electron microscopy.

[0034] A fourth object of the present invention is to provide a method for identifying miR-1246 in vesicles derived from apoptotic fibroblasts, comprising the following steps:

[0035] Total RNA was extracted from the vesicles, and the RNA concentration was measured using a nucleic acid quantifier. Target gene primers were then added to amplify the target gene and internal reference gene. The expression of miR-1246 was quantified using ABI 7500 real-time fluorescence.

[0036] The fifth object of the present invention is to provide a use of vesicles derived from apoptotic fibroblasts in the preparation of a drug for preventing and treating myocardial ischemia-reperfusion injury.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a drug for preventing and treating myocardial ischemia-reperfusion injury using vesicles derived from apoptotic fibroblasts, as well as a preparation method and application. The method induces apoptosis and promotes the secretion of nanovesicles by fibroblasts. Compared with small extracellular vesicles with exosomes as the main component, the nanovesicles are abundant and easy to obtain, have low immunogenicity, have a specific marker CD22 on the surface, have good targeting to cardiomyocytes, are enriched in miR-1246, and have good biocompatibility. The extraction, separation and identification of the vesicles are convenient and fast, with strong specificity and targeting, and are suitable for clinical promotion and application. The fibroblast-derived nanovesicles of the present invention can be effectively taken up by cardiomyocytes and downregulate p53 expression, maintain mitochondrial homeostasis, reduce myocardial damage and produce a cardioprotective effect, and have very good application and research value in the field of cardiovascular medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Phenotypic analysis of EV production by apoptotic fibroblasts;

[0040] Figure 2 CD22 can be used as a specific marker for CFbs-apoVs;

[0041] Figure 3 The specific CD22 antigen expression on the surface of EV subtypes may be an important molecular marker for cardiomyocytes to recognize and internalize EVs;

[0042] Figure 4 Myocardial infarction can induce fibroblasts to produce CD22 cells enriched with miR-1246 +- CFbs-apoVs and are delivered into cardiomyocytes;

[0043] Figure 5Targeting miR-1246 to regulate p53 expression;

[0044] Figure 6 CFbs-CD22 + ApoVs transport miR-1246 to downregulate p53 expression, maintain myocardial mitochondrial homeostasis, and inhibit cell apoptosis. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0046] The present invention is directed to the defects and deficiencies of the prior art:

[0047] 1) Limitations of EV subtype separation technology.

[0048] The present invention combines high-resolution density gradient analysis with flow cytometry to isolate a new subtype of EVs secreted by apoptotic fibroblasts. The subtype differs from the normal EV subtype of cells in terms of vesicle number, size, and concentration—CFbs-apoVs. This method breaks through the bottleneck of subtype resolution in traditional single high-speed centrifugation methods.

[0049] 2) There is a gap in the understanding of functionally specific EVs subtypes.

[0050] Existing studies have generally viewed apoptotic cell EVs as a homogeneous population, without focusing on the heterogeneity of their surface molecular markers. This study, for the first time, identified the specific expression of the CD22 antigen on the surface of apoptotic fibroblast EVs (CD22+apoVs). Prior studies have neither revealed the existence of this subtype nor elucidated the molecular mechanisms of its interaction with cardiomyocytes.

[0051] 3) Lack of specific EV content molecular delivery system.

[0052] Existing strategies for myocardial protection often rely on direct delivery of miRNA or gene editing technologies, but these strategies suffer from limitations such as poor targeting and low bioavailability. The present invention discovered that CD22+ apoVs naturally accumulate miR-1246 and achieve cardiomyocyte-specific uptake through CD22 signaling. This discovery of a delivery system fills a gap in existing technologies for naturally targeted vectors.

[0053] 4) The regulatory mechanism of EVs in maintaining mitochondrial homeostasis is unclear.

[0054] While existing research has shown that EVs can regulate cell apoptosis, molecular evidence specifically for their regulation of myocardial mitochondrial function is lacking. This study demonstrates for the first time that CD22+ apoVs maintain mitochondrial homeostasis by delivering miR-1246, a mechanism that transcends the superficial understanding of the anti-apoptotic effects of EVs.

[0055] 5) Technical bottlenecks in the application of vesicles in the treatment of myocardial injury.

[0056] Existing EV-based cardioprotective therapies suffer from issues such as subtype impurity, weak targeting, and poor stability. This invention, by establishing a standardized CFbs-apoVs preparation process and combining its natural cardiomyocyte targeting properties, provides a novel solution for the development of novel biological agents against myocardial ischemia, overcoming key barriers to clinical translation of existing technologies. There are no prior reports of fibroblast-secreted nanovesicles for cardiac protection.

[0057] The purpose of this invention is to provide a novel EV subtype: specific extracellular vesicles (CFbs-apoVs) that induce fibroblast apoptosis, providing new experimental evidence for studying the role of apoptosis in cardiovascular disease. Based on the discovery that apoptosis-mediated CFbs-apoVs carry damaged miR-1246 and are taken up by cardiomyocytes, downregulating p53 expression, maintaining mitochondrial homeostasis, and inhibiting apoptosis, this invention defines the pathophysiological process by which fibroblasts protect cardiomyocytes from a novel perspective of intercellular communication.

[0058] In order to achieve the above objectives, the present invention provides a first aspect of a method for preparing vesicles derived from apoptotic fibroblasts, comprising the following steps:

[0059] The isolated animal cardiac apex tissue was digested in stages using a mixed digestion solution of trypsin and collagenase II, and then the digestion was terminated using a DMEM solution containing FBS to obtain a digested cell suspension;

[0060] The digested cell suspension was filtered and centrifuged, and the precipitated cells were resuspended in DMEM and cultured for 1 to 2 hours. Then, the cells were separated and purified by differential adherence culture to obtain cardiac fibroblasts.

[0061] The cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 to obtain culture medium;

[0062] The culture fluid is separated and purified to obtain vesicles derived from apoptotic fibroblasts.

[0063] The volume ratio of pancreatic enzyme to collagenase II in the mixed digestive solution is 1:1-3, wherein the mass volume percentage of pancreatic enzyme is 0.05-0.2%; preferably, the volume ratio of pancreatic enzyme to collagenase II in the mixed digestive solution is 1:1, wherein the mass volume percentage of pancreatic enzyme is 0.125%.

[0064] The content of FBS in the FBS-containing DMEM solution is 8-12% by volume, preferably 10% by volume.

[0065] The filtration is performed using a 200-mesh sieve;

[0066] After filtration, centrifugation was performed at 1-5°C and 800 pr / min for 2 times, with each centrifugation lasting 5 minutes.

[0067] The concentration of the apoptosis inducer H2O2 was 0.2-0.8 mM. Cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 for 12-48 hours. The apoptosis rate of the fibroblasts was determined according to the instructions of the Annex v-FITC / PI cell apoptosis kit.

[0068] The culture fluid separation process includes:

[0069] The culture medium was centrifuged at 300 × g for 10 min, and the supernatant was centrifuged at 1000 × g for 15 min to remove cells and cell debris; the supernatant was collected and centrifuged at 10,000 × g for 30 min at 1-5°C and filtered through a 0.22 μm filter membrane to remove subcellular structures; the supernatant was then ultracentrifuged at 100,000 × g for 70 min, the EV pellet was collected, washed with PBS, and centrifuged at 100,000 × g for 70 min, and the EVs pellet was resuspended in 100 μL PBS.

[0070] The purification process of the culture fluid after separation includes:

[0071] The resuspended EVs pellet is spread on a sucrose solution and centrifuged at 100,000 g for 70 min. Protein aggregates will settle to the bottom of the centrifuge tube, while EVs containing lipid structures will settle to the isopycnic area, resulting in higher purity EVs.

[0072] The higher purity EVs were washed with PBS and centrifuged at 100,000 g for 70 min. The EVs were resuspended in 100 μL PBS to obtain vesicles derived from apoptotic fibroblasts.

[0073] The sucrose solution is a system in which sucrose solutions with two concentrations of 0.25 mol / L and 2.5 mol / L are prepared into a continuous density gradient.

[0074] The second aspect of the present invention provides a vesicle derived from apoptotic fibroblasts, wherein the vesicle contains apoptosis-specific proteins p53, Bcl-2, Bax, caspase-3, and Apaf-1 and has phosphatidylserine exposure;

[0075] The vesicles carry CD22 on their surface and are enriched with miR-1246.

[0076] Among them, flow cytometry was used to characterize the expression of fibroblast markers in vesicles, analyze the probability of occurrence of α-SMA expression events in vesicles, and determine that special EVs subtypes originated from fibroblasts.

[0077] A third aspect of the present invention provides a method for identifying surface CD22 in vesicles derived from apoptotic fibroblasts, comprising:

[0078] Based on high-throughput HNCIB detection, the vesicle marker CD22 was screened;

[0079] Alternatively, flow cytometry was used to analyze CD22 fluorescence on the vesicle surface;

[0080] Alternatively, immunogold labeling and transmission electron microscopy were used to observe the expression of CD22 on the surface of EVs.

[0081] Among them, immunogold labeling transmission electron microscopy was used to observe the expression of CD22 on the surface of EVs, including:

[0082] Vesicles were labeled with anti-rabbit IgGGold2 CD22 antibody and a water film was formed on a quantitative foil R2 / 2 grid. The water film grid was permeabilized by injecting it into ethane cooled to the melting point by liquid nitrogen.

[0083] The specimens were transferred to a Gatan cryostat mount for a transmission electron microscope, and the expression of CD22 on the surface of EVs was observed using immunogold labeling and transmission electron microscopy.

[0084] A fourth aspect of the present invention provides a method for identifying miR-1246 in vesicles derived from apoptotic fibroblasts, comprising the following steps:

[0085] Total RNA was extracted from the vesicles, and the RNA concentration was measured using a nucleic acid quantifier. Target gene primers were then added to amplify the target gene and internal reference gene. The expression of miR-1246 was quantified using ABI 7500 real-time fluorescence.

[0086] Exemplary, a method for preparing vesicles derived from apoptotic fibroblasts comprises:

[0087] (1) Isolation and culture of cardiac fibroblasts (CFbs) and primary cardiomyocytes (CMs);

[0088] Cardiac apex tissues of 1-3 day old C57BL / 6 pups were removed under sterile conditions and digested five times with a 0.125% trypsin: collagenase II = 1:1 mixed digestion solution. Digestion was terminated with DMEM containing 10% FBS by volume, filtered through a 200-mesh sieve, and centrifuged twice at 800 pr / min for 5 min at 4°C. The precipitated cells were resuspended in DMEM and cultured routinely for 1.5 h. Myocardial fibroblasts (CFbs) and primary cardiomyocytes (CMs) were isolated and purified using the differential adherent culture method.

[0089] Among them, cardiac fibroblasts grow rapidly and attach to the wall quickly, with large cell bodies and nuclei and no spontaneous beating, and they become fused in 2 to 3 days; primary cardiomyocytes begin to attach to the wall 2-4 hours after culture, and after 3 to 4 days of culture, it is observed that cardiomyocytes can fill more than 95% of the field of view and beating in sheets.

[0090] Adjust the cell concentration of both cells to 2 × 10 6 / mL, routine culture, change the culture medium once every 24h.

[0091] (2) Experimental groups

[0092] Fibroblasts were randomly divided into two groups: Normal and apoptosis inducer H2O2. All drugs were dissolved in high-glucose DMEM. The normal group was added with the same volume of PBS. After the drug treatment, the culture supernatant and cells of each group were collected by conventional methods.

[0093] Among them, the apoptosis inducer H2O2 was set up with a concentration gradient of 0, 0.2, 0.4, 0.8, 1 (mM) and different time points: 0, 6, 12, 24, 48h to screen out the optimal concentration and time for producing apoptotic vesicles.

[0094] Preferably, the concentration of the apoptosis inducer H2O2 is 0.2-0.8 mM; the cardiac fibroblasts are cultured in a DMEM solution with the apoptosis inducer H2O2 for 12-48 hours.

[0095] The optimal concentration of the apoptosis inducer H2O2 is 0.4 mM; the cardiac fibroblasts are cultured in a DMEM solution with the apoptosis inducer H2O2 for 24 hours.

[0096] (3) Flow cytometry detection of cell apoptosis rate

[0097] After drug treatment, cells were digested with 0.25% trypsin, washed twice with PBS, and detected according to the instructions of Annex v-FITC / PI cell apoptosis kit.

[0098] (4) Isolation and purification of EVs

[0099] The cell culture medium was collected and centrifuged at 300 × g for 10 minutes. The supernatant was centrifuged at 1000 × g for 15 minutes to remove cells and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 30 minutes at 4°C and filtered through a 0.22 μm filter to remove subcellular structures. The supernatant was ultracentrifuged at 100,000 × g for 70 minutes to collect the EV pellet, which was washed once with PBS and centrifuged at 100,000 × g for 70 minutes. The EV pellet was resuspended in 100 μL of PBS.

[0100] Two concentrations of sucrose solution (0.25 mol / L and 2.5 mol / L) were prepared into a continuous density gradient system and placed in an ultracentrifuge tube. EV samples were then layered on the sucrose solution and centrifuged at 100,000 g for 70 minutes. Protein aggregates sedimented to the bottom of the tube, while EVs containing lipid structures settled to an isopycnic region (1.10-1.18 kg / L), resulting in higher-purity EVs. The EV-containing pellet was collected, washed with PBS, and centrifuged at 100,000 g for 70 minutes. The EVs were resuspended in 100 μL of PBS and immediately stored at -80°C until further use. A protein standard curve was established using a BCA protein quantification kit, and the mass of the EV suspension was calculated.

[0101] It should be noted that EVs produced by normal cells are recorded as Nor-EVs, and EVs produced by H2O2-induced apoptotic cells are recorded as CFbs-apoVs.

[0102] (5) EVs morphological analysis using transmission electron microscopy;

[0103] Take 10 μL of EVs suspension and drop it on a 200-mesh copper grid. Let it stand at room temperature for 10 minutes. Use filter paper to absorb the liquid from the side of the filter. Stain it with 2% phosphotungstic acid negative staining solution for 5 minutes. Dry it at room temperature and observe and photograph it with a transmission electron microscope.

[0104] (6) NTA detection of EVs size distribution and concentration analysis;

[0105] The collected EVs were diluted with PBS to a particle concentration of 10 6 / mL, inject into the nanoparticle tracking analyzer with a 1mL syringe for analysis, and save the analysis data.

[0106] (7) Western blot was used to determine the protein expression of marker proteins and apoptosis-related factors in EVs.

[0107] Total EV protein was extracted using a protein extraction kit according to the operating instructions, and protein quantification was performed using a BCA protein quantification kit. The membranes were denatured by boiling, subjected to SDS-PAGE gel electrophoresis, and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 hour, and the primary antibody was added and incubated overnight at 4°C. The membranes were washed three times, and the secondary antibody was added and incubated at room temperature for 1 hour. The membranes were washed and fluorescently detected using an ECL-plus immunoassay kit. The expression of Alix, CD63, CD81, Tsg101, p53, Bcl-2, Bax, caspase-3, and Apaf-1 proteins was quantitatively analyzed using Bio-Image (Bio-Rad) analysis software. The exposure of the apoVs-specific marker, phosphatidylserine (PtdSer), was detected to determine that EVs were highly correlated with apoptosis.

[0108] (8) Analysis of miRNA expression in EVs by high-throughput miRNA sequencing;

[0109] EV total RNA samples were extracted and sequenced using Illumina's next-generation sequencer HiSeq 2500. After library construction, hybridization, ligation, signal collection and other sequencing reaction steps, differential miRNAs were screened and the data were analyzed.

[0110] (9) qRT-PCR detection of differential miRNA gene expression in EVs of each group;

[0111] Total EV RNA was extracted, and RNA concentration was measured using a nucleic acid quantifier. Target gene primers were added, and the target gene and internal reference gene were amplified in each group of samples according to the instructions of the miRNA RT-qPCR kit and the reaction conditions in relevant literature. The data were analyzed and processed using an ABI7500 real-time fluorescence quantitative PCR instrument.

[0112] (10) Detection and analysis of EV surface biomarkers by high-throughput HNCIB;

[0113] HNCIB uses a nanobiochip to capture and detect target membrane proteins. First, a suspension of EVs dissolved in PBS is added to the chip and incubated. During incubation, negatively charged EVs are attracted by electrostatic forces and fuse with cationic nanoparticles, allowing the contents of the two particles to mix thoroughly. After hybridization with target RNA, EV surface membrane proteins are detected using immunostaining. Data analysis identifies the EV marker CD22.

[0114] (11) Verify the expression of EVs surface marker CD22 using flow cytometry;

[0115] EVs were isolated using CD22 EV capture beads according to the kit instructions. 20 μL of the PBS-EV suspension was added to 10 μL of the CD22 EV magnetic beads, vortexed, and incubated overnight at room temperature in the dark. Following incubation, the CD22-AF488 fluorescent antibody was added to the mixture at a 1:100 volume ratio and incubated at 4°C in the dark for 2 hours. EV surface CD22 fluorescence was analyzed by flow cytometry.

[0116] (12) Immunogold labeling and transmission electron microscopy observation of CD22 expression on the surface of EVs

[0117] CD22 was labeled with anti-rabbit IgG Gold2 antibody (anti-CD22) (20 nM). A 2.5 μl aliquot of sample was applied to a thin water film on a quantitative foil R2 / 2 grid (quantitative foil GMbH, Jena BRD). The grid with the thin water film was permeabilized by injecting liquid nitrogen into ethane cooled to its melting point (-180°C). The specimen was transferred to a Gatan cryostat on a transmission electron microscope (FEI-T12 Biotwin), and transmission electron microscopy images were captured using a FEI-Eagle-4K-CCD camera.

[0118] (13) confirmed that the isolated apoVs were of fibroblast origin;

[0119] Flow cytometry was used to characterize the expression of myocardial markers (troponin T) and fibroblast markers (transforming growth factor β receptor I: TGFβR1, α-smooth muscle actin: α-SMA) in apoVs; the probability of α-SMA expression events in CFbs-apoVs was analyzed to further determine that the specific EVs subtype originated from fibroblasts, and then CD22-labeled cardiac EVs were sorted for subsequent CD22 + Mechanistic studies of EVs.

[0120] The CFbs-apoVs provided by the present invention are taken up by surrounding myocardial cells and target and regulate p53 to maintain mitochondrial homeostasis;

[0121] (1) Dual luciferase reporter gene assay to detect the relationship between miR-1246 and p53;

[0122] HEK-293 tool cells were routinely cultured, and luciferase reporter gene plasmids containing wild-type 3'-UTR (pGL3-p53-3'-UTR-WT) and mutant 3'-UTR (pGL3-p53-3'-UTR-Mutant) were constructed. After co-transfection of the plasmids with miR-1246, the cells were washed twice with PBS. An appropriate amount of Passive Lysis Buffer was added to each well, and the cells were lysed and centrifuged. The supernatant was transferred to a black 96-well plate, and the intensity of firefly and Renilla fluorescence was measured on a chemiluminescence analyzer.

[0123] (2) Targeting miR-1246 and p53, construct cardiomyocyte cell lines that upregulate or downregulate their gene expression;

[0124] Up-regulation and down-regulation plasmids were constructed for the mouse p53 gene sequence, and siRNA (50nM) targeting the gene was transfected into cardiomyocytes using Lipofectamine RNAiMAX (13778-150, Invitrogen). For p53 gene overexpression, plasmids were transfected using Lipofectamine 3000 (L3000-008, Invitrogen). 48-72 hours after transfection, cells were collected for experiments. For the mouse miR-1246 gene, 50nmol / L miR-1246 mimic, miR-1246 inhibitor, or negative scramble control (Qiagen) were transfected into cardiomyocytes using HiPerfect transfection reagent (Qiagen).

[0125] (3) Cell grouping, drug administration, and H / R model establishment;

[0126] Cardiomyocytes were cultured routinely, and the culture medium was replaced every 24 hours. When the cells filled 80% of the visual field, they were used for experiments. During the hypoxia process (H), the cells were placed in a 1% O2, 5% CO2, 94% N2 37℃ three-gas incubator without sugar and serum and cultured for 6 hours. During the reoxygenation process (R), the cells were replaced with high-glucose DMEM containing 10% fetal bovine serum and cultured normally for 12 hours. The Nor-EVs and CD22 isolated and extracted above were given separately. + EVs(CFbs-CD22 + apoVs) or CD22 - EVs were pretreated with a final concentration of 50 μg / mL using the same method as before.

[0127] (4) Cellular uptake experiments;

[0128] EVs were labeled using the PKH67 Green Fluorescent Cell Membrane Labeling Kit according to the manufacturer's instructions. 0.5 mL of Diluent C was added to 100 μL of PBS-EV suspension, mixed, and the EVs were resuspended to form a labeled mixture. Separately, 0.5 mL of Diluent C was added to 4 μL of PKH67 and mixed to prepare the labeling buffer. The labeling buffer was added to the labeled EV mixture and incubated at room temperature for 4 minutes. The staining was terminated by adding 1 mL of 0.5% BSA. The cells were washed three times with PBS to remove unbound dye, resulting in the PKH67-EV marker, which was then used for further analysis. The PKH67-EV marker was added to the cell culture medium at the designated dosage, incubated for 24 hours, fixed, stained with DAPI, and observed for cellular uptake of EVs using a confocal laser scanning microscope. As a negative control, EVs not stained with PKH67 were added to the control group of cells.

[0129] (5) qRT-PCR detection of miR-1246 and p53 gene expression;

[0130] Total cellular RNA was extracted using Trizol reagent, and RNA concentration was determined using a nucleic acid quantifier. Target gene primers were added, and the target gene and internal reference gene were amplified in each group of samples according to the PCR kit instructions and the reaction conditions in the literature (preliminary experiment). The data were analyzed and processed using an ABI 7500 real-time fluorescence quantitative PCR instrument.

[0131] (6) Western blot was used to detect the expression of mitochondrial-related proteins such as p53, TOM20 and TFAM.

[0132] The method is the same as before.

[0133] (7) Mitochondrial function testing;

[0134] Agilent Seahorse XF96 Analyzer (XF96 ExtracellularFlux Cellular oxygen consumption rate (OCR) was measured using a Fibroblast Analyzer (USA). Cells were stimulated with 1 μM oligomycin, 0.5 μM FCCP, and 1 μM antimycin A plus rotenone, and ATP-dependent OCR, maximum OCR, and non-mitochondrial OCR were measured. Intracellular protein concentrations were determined and all OCR data were normalized. Mitochondrial complex I-IV activity was assessed using the Mitochondrial Respiratory Chain Complex Activity Assay Kit (Solarbio, Beijing, China) according to the protocol.

[0135] Cardiomyocytes were collected, washed three times with 1× PBS, and then treated with 5 μM DCFH-DA-A or MitoSOX. TMThe cells were incubated in the dark for 45 minutes. The cells were then washed three times with 1× PBS and detached with trypsin / EDTA. The relative levels of cellular fluorescence were quantified and analyzed by flow cytometry.

[0136] The present invention is based on in vivo verification of the mechanism by which CFbs-apoVs exert protective effects on the myocardium;

[0137] (1) Animal selection and in vivo miRNA interference;

[0138] 18-25g male C57BL / 6 mice were selected. According to the experimental design, in vivo miRNA interference technology was used to inoculate some C57BL / 6 mice with AAV9 vector containing miR-1246 antagomir into 5x10 11 Before establishing the model, the rats were injected with 100 μL of vector / mL stock solution for 3 consecutive days (50 μL / time / rat) via the tail vein.

[0139] (2) Establishment of in vivo MI / R injury model;

[0140] Mice were intraperitoneally injected with sodium pentobarbital (60 mg / kg), immobilized in the supine position, and a midline cervical incision was performed. The trachea was intubated and connected to a small animal ventilator (respiratory rate 60 breaths / min, tidal volume 16 mL / kg, I:E ratio 1:2.5). Electrocardiogram (ECG) was continuously monitored using an MS4000 biosignal quantitative recording and analysis system. A longitudinal thoracotomy was performed approximately 0.5 cm to the left of the sternum to expose the heart. A 6 / 0 suture was passed under the left anterior descending coronary artery, approximately 2 mm from the origin of the left ventricular branch, and a silicone tube was placed over it for later use. After 15 minutes of ECG stabilization, the ligature was tightened in all groups except the sham group, where no ligature was applied. Successful ischemia was defined as increased QRS amplitude, ST segment elevation, and tall or inverted T waves. The ligature was cut 30 minutes after ischemia, and reperfusion was considered successful if the ECG recovered. Samples were collected 24 hours after reperfusion.

[0141] (3) grouped administration;

[0142] Normal and gene-interference mice were randomly divided into six groups (n=6): sham-operated (without surgical suture but without ligation of the anterior descending coronary artery), model, Nor-EV, CFbs-apoVs, CFbs-apoVs + GW4869, and GW4869 (2.5 μg / g, intraperitoneally injected twice weekly for 3 weeks). The sham and model groups received the same volume of saline. Except for the sham-operated group, each group was treated with an MI / R model. Immediately after model establishment, drugs were administered intramyocardially as follows:

[0143] The extracted EVs were dissolved in PBS to prepare a 1 μg / μL EV stock solution. The aorta and pulmonary arteries were clamped with an arterial clamp. Using a microsyringe, 200 μg of the EV / PBS mixture was injected from the left ventricular apex into the aortic root. The aorta and pulmonary artery clamps were maintained for 10 seconds after injection to ensure that the mixture perfused into the entire heart via the coronary arteries. After 10 seconds, the arterial clamps were released, the chest was sutured, and care was taken to maintain warmth and prevent infection. GW4869 is an inhibitor of extracellular vesicle synthesis and release.

[0144] (4) observe the uptake of CFbs-apoVs by fibroblasts and cardiomyocytes;

[0145] α-actinin was used to label cardiomyocytes (red), α-SMA was used to label fibroblasts (red), and PKH-67 (green) was used to label EVs in each group. Laser confocal microscopy was used to observe the uptake of CFbs-apoVs by fibroblasts and cardiomyocytes (yellow).

[0146] (5) TTC staining to detect myocardial infarction area;

[0147] The frozen heart was removed and serially sectioned along the long axis of the left ventricle using a pre-chilled blade, with each slice being 2-3 mm thick. The slices were placed in a 2% TTC solution and incubated at 37°C for 15-30 minutes. After fixation, the slices were observed and photographed under a light microscope, and analyzed using Image-ProPlus software to calculate the percentage of myocardial infarction area (%).

[0148] (6) Echocardiography to measure cardiac function;

[0149] Mice were anesthetized with 2% isoflurane, and left ventricular diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (EF), and left ventricular fractional shortening (FS) were measured using a small animal echocardiogram. All the above parameters were measured for three cardiac cycles and the average value was calculated.

[0150] (7) TUNEL staining to detect myocardial cell apoptosis;

[0151] At the end of the experiment, myocardial tissues from the infarcted area were collected, rinsed, fixed with paraformaldehyde, and sliced. The expressions of CD31 and α-SMA were detected by immunofluorescence using the same method as above.

[0152] (8) Transmission electron microscopy was used to observe the damage of myocardial mitochondria;

[0153] After the left ventricular myocardial tissue was collected, fixed, dehydrated, infiltrated, embedded, polymerized, sectioned and stained, the ultrastructure of the mitochondria in the myocardial cells was observed under a transmission electron microscope.

[0154] (9) qRT-PCR detection of miR-1246 and p53 gene expression;

[0155] (10) Western blot was used to detect the expression of mitochondrial-related proteins and p53.

[0156] For example, inducing fibroblast apoptosis can generate a new EV subtype, CFbs-apoVs, including:

[0157] Extract, separate and purify primary fibroblasts from C57BL / 6 neonatal mice, culture them in high-glucose DMEM (containing 10% EVs-free fetal bovine serum), and use 10 cm 2 The cells were cultured in a conventional manner in a cell culture dish. The cells were randomly divided into: Normal, apoptosis inducer H2O2 (final concentration of 400μM H2O2 incubated for 24h). After the end of the medication, the EVs in the culture supernatant of each group were separated, purified and identified, and labeled as Nor-EVs and CFbs-apoVs respectively; the EV morphology was observed by transmission electron microscopy (TEM); the NTA technology was used to detect the EV particle size distribution, number and concentration; Western blot was used to determine the expression of EV specific proteins (Alix, CD81, etc.). Figure 1 The results showed that the EV morphology in each group was round, with no significant differences. However, compared with the Normal group, the EV particle size and size in the H2O2 group increased, and the expression of EVs-specific proteins increased. More importantly, fluorescence staining and flow cytometry were used to detect the exposure of the apoptosis marker phosphatidylserine (PtdSer), and the apoptosis-related vesicles were marked by the apoptosis-specific protein caspase-3, confirming that the release of CFbs-apoVs was highly correlated with apoptosis. The results suggest that inducing myocardial apoptosis may produce a new subtype of EVs, which is different from normal EVs in terms of vesicle number and size.

[0158] A fifth aspect of the present invention provides a use of vesicles derived from apoptotic fibroblasts in the preparation of a drug for preventing and treating myocardial ischemia-reperfusion injury.

[0159] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0160] Example 1

[0161] A method for preparing vesicles derived from apoptotic fibroblasts, comprising:

[0162] (1) Isolation and culture of cardiac fibroblasts (CFbs);

[0163] Cardiac apex tissue was removed from 2-day-old C57BL / 6 pups under sterile conditions and digested five times with a 1:1 volume ratio of 0.125% trypsin to collagenase I. Digestion was terminated with DMEM containing 10% FBS, filtered through a 200-mesh sieve, and centrifuged twice at 800 rpm for 5 minutes at 4°C. The pelleted cells were resuspended in DMEM and cultured routinely for 1.5 hours. Cardiac fibroblasts (CFbs) were isolated and purified using the differential adherence method.

[0164] Among them, myocardial fibroblasts grow rapidly and attach to the wall quickly, with large cell bodies and nuclei and no spontaneous beating, and they become fused in 2 to 3 days.

[0165] Adjust the concentration of cardiac fibroblasts to 2×10 6 / mL, routine culture, change the culture medium once every 24h.

[0166] (2) Cardiac fibroblasts were treated with the apoptosis inducer H2O2. The drugs were dissolved in high-glucose DMEM, and the culture supernatant and cells were collected by conventional methods.

[0167] The concentration of the apoptosis inducer H2O2 was set to 0.4 mM, and the cardiac fibroblasts were cultured in a DMEM solution with the apoptosis inducer H2O2 for 24 hours.

[0168] (3) Flow cytometry was used to detect cell apoptosis rate;

[0169] After drug treatment, cells were digested with 0.25% trypsin, washed twice with PBS, and detected according to the instructions of Annex v-FITC / PI cell apoptosis kit.

[0170] (4) Isolation and purification of EVs

[0171] The cell culture medium was collected and centrifuged at 300 × g for 10 minutes. The supernatant was centrifuged at 1000 × g for 15 minutes to remove cells and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 30 minutes at 4°C and filtered through a 0.22 μm filter to remove subcellular structures. The supernatant was ultracentrifuged at 100,000 × g for 70 minutes to collect the EV pellet, which was washed once with PBS and centrifuged at 100,000 × g for 70 minutes. The EV pellet was resuspended in 100 μL of PBS.

[0172] Sucrose solutions at two concentrations, 0.25 mol / L and 2.5 mol / L, were prepared into a continuous density gradient system and placed in an ultracentrifuge tube. EV samples were then layered on the sucrose solution and centrifuged at 100,000 g for 70 minutes. Protein aggregates sedimented to the bottom of the tube, while EVs containing lipid structures settled to an isopycnic region (1.10–1.18 kg / L), yielding EVs of higher purity. The EV-containing pellet was collected, washed with PBS, and centrifuged at 100,000 g for 70 minutes. The EVs were resuspended in 100 μL of PBS and immediately stored at -80°C until further use. A protein standard curve was established using a BCA protein quantification kit, and the mass of the EV suspension was calculated.

[0173] It should be noted that the EVs produced by H2O2-induced apoptotic cells are recorded as: CFbs-apoVs.

[0174] Example 2

[0175] A method for preparing vesicles derived from apoptotic fibroblasts, comprising:

[0176] (1) Isolation and culture of cardiac fibroblasts (CFbs);

[0177] The cardiac apex tissue of 3-day-old C57BL / 6 mice was removed under sterile conditions and digested in batches with a 0.125% trypsin: collagenase П=1:1 mixed digestion solution. The digestion was terminated with DMEM containing 10% FBS, filtered through a 200-mesh sieve, and centrifuged twice at 800 pr / min for 5 min at 4°C. The precipitated cells were resuspended in DMEM and cultured routinely for 1.5 h. Myocardial fibroblasts (CFbs) were isolated and purified using the differential adherent culture method.

[0178] Among them, cardiac fibroblasts grow rapidly and attach to the wall quickly, with large cell bodies and nuclei and no spontaneous beating, and they become fused in 2 to 3 days. The concentration of cardiac fibroblasts was adjusted to 2×10 6 / mL, routine culture, change the culture medium once every 24h.

[0179] (2) Cardiac fibroblasts were treated with H2O2, an apoptosis inducer. All drugs were dissolved in high-glucose DMEM. After the treatment, the culture supernatant and cells were collected by conventional methods.

[0180] The concentration of the apoptosis inducer H2O2 was set at 0.8 mM, and the cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 for 48 h.

[0181] (3) Flow cytometry was used to detect cell apoptosis rate;

[0182] After drug treatment, cells were digested with 0.25% trypsin, washed twice with PBS, and detected according to the instructions of Annex v-FITC / PI cell apoptosis kit.

[0183] (4) Isolation and purification of EVs;

[0184] The cell culture medium was collected and centrifuged at 300 × g for 10 minutes. The supernatant was centrifuged at 1000 × g for 15 minutes to remove cells and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 30 minutes at 4°C and filtered through a 0.22 μm filter to remove subcellular structures. The supernatant was ultracentrifuged at 100,000 × g for 70 minutes to collect the EV pellet, which was washed once with PBS and centrifuged at 100,000 × g for 70 minutes. The EV pellet was resuspended in 100 μL of PBS.

[0185] Two concentrations of sucrose solution (0.25 mol / L and 2.5 mol / L) were prepared into a continuous density gradient system and placed in an ultracentrifuge tube. EV samples were then layered on the sucrose solution and centrifuged at 100,000 g for 70 minutes. Protein aggregates sedimented to the bottom of the tube, while EVs containing lipid structures settled to an isopycnic region (1.10-1.18 kg / L), resulting in higher-purity EVs. The EV-containing pellet was collected, washed with PBS, and centrifuged at 100,000 g for 70 minutes. The EVs were resuspended in 100 μL of PBS and immediately stored at -80°C until further use. A protein standard curve was established using a BCA protein quantification kit, and the mass of the EV suspension was calculated.

[0186] It should be noted that the EVs produced by H2O2-induced apoptotic cells are recorded as: CFbs-apoVs.

[0187] Example 3

[0188] Same as Example 1, except that,

[0189] The concentration of the apoptosis inducer H2O2 was set to 0.2 mM, and the cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 for 12 hours.

[0190] Comparative Example 1

[0191] The same as Example 1, except that the EVs produced by normal cells were not cultured with the apoptosis inducer H2O2, and were recorded as: Nor-EVs.

[0192] Comparative Example 2

[0193] Isolation and culture of primary cardiomyocytes (CMs);

[0194] One-day-old C57BL / 6 mouse pups were obtained, and the apical cardiac tissues were removed under sterile conditions. The tissues were digested in batches with a 0.125% trypsin: collagenase П=1:1 mixed digestion solution, and the digestion was terminated with DMEM containing 10% FBS. The cells were filtered through a 200-mesh sieve and centrifuged twice at 800 pr / min for 5 min at 4°C. The precipitated cells were resuspended in DMEM and cultured routinely for 1.5 h. Primary cardiomyocytes (CMs) were isolated and purified using the differential adherence culture method.

[0195] Among them, primary cardiomyocytes begin to adhere to the wall after 2-4 hours of culture. After 3-4 days of culture, it is observed that the cardiomyocytes can fill more than 95% of the visual field and beat in sheets. The concentration of primary cardiomyocytes is adjusted to 2×10 6 / mL, routine culture, change the culture medium once every 24h.

[0196] Comparative Example 3

[0197] Isolation and culture of endothelial cells (CMECs) and macrophages (cMacs);

[0198] Cardiac microvascular endothelial cells (CMEC) and macrophages (cMacs) were revived in high-glucose DMEM medium (containing 10% EV-free fetal bovine serum) and cultured with 10 cm 2 The cells were cultured in a conventional manner in a culture dish and used for experiments when the cells filled 80% of the visual field.

[0199] To illustrate the relevant properties of the vesicles derived from apoptotic fibroblasts provided by the present invention, the relevant properties of the vesicles derived from apoptotic fibroblasts prepared in Example 1 are described with reference to the accompanying drawings, and are also described with reference to comparative examples.

[0200] (1) Morphological analysis of EVs using transmission electron microscopy: 10 μL of EV suspension was dropped onto a 200-mesh copper grid, and allowed to stand at room temperature for 10 min. The liquid was then absorbed from the side of the grid with filter paper, and the EVs were stained with 2% phosphotungstic acid negative stain for 5 min. The EVs were dried at room temperature, and then observed and photographed using a transmission electron microscope.

[0201] NTA detection of EVs particle size distribution and concentration analysis: The collected EVs were diluted with PBS to a particle concentration of 10 6 / mL, inject into the nanoparticle tracking analyzer with a 1mL syringe for analysis, and save the analysis data.

[0202] Western blot was used to determine the protein expression of marker proteins and apoptosis-related factors in EVs: total EV protein was extracted using a protein extraction kit according to the operating instructions, and protein quantification was performed using a BCA protein quantification kit. The membranes were denatured by boiling, subjected to SDS-PAGE gel electrophoresis, and electrotransferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 hour, the primary antibody was added, incubated at 4°C overnight, and the membranes were washed three times. The secondary antibody was added, incubated at room temperature for 1 hour, and the membranes were washed. Fluorescence detection was performed using an ECL-plus immunoassay kit, and the expression of Alix, CD63, CD81, Tsg101, p53, Bcl-2, Bax, caspase-3, and Apaf-1 proteins was quantitatively analyzed using Bio-Image (Bio-Rad) analysis software. The exposure of the apoVs-specific marker, phosphatidylserine (PtdSer), was detected to confirm that EVs were highly correlated with apoptosis.

[0203] See also Figure 1 As shown, phenotypic analysis of EV production by apoptotic fibroblasts ( n=3) (Note: *P<0.05); Figure 1 A. Transmission electron microscopy observation of EV morphology; Figure 1 BD.NTA was used to detect the size and concentration distribution of EVs; Figure 1 E. Western blot was used to determine the expression of specific proteins in EVs; Figure 1 FG. Fluorescence staining and flow cytometry were used to detect the exposure of apoptosis marker phosphatidylserine (PtdSer); Figure 1 H. Western blot was used to detect the expression of apoptosis marker protein caspase-3 in EVs.

[0204] from Figure 1 It can be seen that the EV morphology in each group is round, and no significant difference is observed. However, compared with the Normal group, the EV particle size and size in the H2O2 group increased, and the expression of EVs-specific proteins increased. More importantly, fluorescence staining and flow cytometry were used to detect the exposure of the apoptosis marker phosphatidylserine (PtdSer), and the apoptosis-related vesicles were marked by the apoptosis-specific protein caspase-3, confirming that the release of CFbs-apoVs is highly correlated with apoptosis. The results suggest that inducing myocardial apoptosis may produce a new EVs subtype, which is different from the normal EV in terms of vesicle number and size.

[0205] (2) CFbs-apoVs can be labeled by CD22;

[0206] Ginseng Figure 2 As shown, CD22 can be used as a specific marker for CFbs-apoVs ( n=3) (Note: *P<0.05); Figure 2 A. KEGG pathway enrichment analysis of differential protein expression pathways. Figure 2 B. Dataset (GSE159657) analyzed the expression of CD22 in EVs of patients with myocardial infarction. Figure 2 C. Heat map and pathway enrichment map of differentially expressed genes in EVs in the plasma of healthy individuals or patients with myocardial infarction; Figure 2 D. Analysis of EV fluorescence signals using CD22 antibody staining. Bar, 10 μm. Figure 2 E. Flow cytometry analysis of the occurrence probability of CD22-positive EVs in different apoptotic states.

[0207] The present invention analyzes the molecular signatures / signals for identifying CFbs-apoVs. GEO 2R was used to analyze the extracellular vesicle expression profile (GSE159657) and detect mRNA in patients with myocardial infarction. The analysis found a total of 21,328 genes, and 579 upregulated mRNAs were screened out. Then, through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the pathways enriched for upregulated proteins were studied in detail; the results showed that these proteins were enriched in apoptosis-related pathways, such as "multi-species apoptosis signaling pathway", "MAPK signaling pathway", "p53 signaling pathway", etc. ( Figure 2 A). CD molecules are the material basis for mutual recognition between cells or between cells and the matrix, and are also cell surface markers. In the above dataset, four CD molecules related to the apoptosis pathway were selected: CD276, CD24, CD72, and CD22. It has been reported that CD22 is closely related to apoptosis of mouse and human B cells. Further bioinformatics differential analysis showed that the expression of CD22 is increased in patients with myocardial infarction (GSE159657, Figure 2 B); Heat map of differentially expressed genes in EVs in the plasma of healthy individuals or patients with myocardial infarction, confirming that the expression of CD22 is increased in the plasma of patients with myocardial infarction ( Figure 2 C). EVs surface biomarkers were detected and analyzed by high-throughput HNCIB. The results showed that CFbs-apoVs stained with CD22 antibody showed high-intensity fluorescence signal (the signal intensity was 9 times that of the EV control group). Figure 2 D); Flow cytometry was used to verify the expression of CD22, a surface marker of CFbs-apoVs. The results showed that more than 70% of CFbs-apoVs expressed CD22 on their surfaces ( Figure 2 E). The results showed that CD22 may serve as a specific marker for CFbs-apoVs.

[0208] (3) The specific CD22 antigen expression on the surface of EV subtypes may be an important molecular marker for cardiomyocytes to recognize and internalize CFbs-apoVs;

[0209] See also Figure 3 As shown in Figure 2, the specific CD22 antigen expression on the surface of EV subtypes may be an important molecular marker for cardiomyocytes to recognize and internalize EVs ( n=3) (Note: *P<0.05); Figure 3 A. Electron micrograph of EVs after immunogold labeling with anti-CD22. Single CD22 markers of 1-10 nm are indicated by white arrows. Figure 3 B. Total protein was extracted from the CFbs-apoVs membrane, and the expression of CD22 in the membrane was detected by Western blot; Figure 3 C. Flow cytometry detection of CD22 + Expression of cardiac troponin T, TGFβR1, and α-SMA in CFbs-apoVs (black line), with gray filling as control; Figure 3 D. PKH-67 (green) labeled EVs were collected and then incubated with cardiomyocytes for 24 hours. Laser confocal microscopy was used to observe the expression of CD22 in cardiomyocytes. + Uptake of CFbs-apoVs. Bar, 10 μm.

[0210] In order to confirm that CD22 is indeed expressed on the surface of CFbs-apoVs, the present invention took transmission electron microscopy images of CFbs-apoVs after immunogold labeling of CD22 and extracted CFbs-apoVs membrane proteins. Western blot analysis confirmed that CD22 was aggregated on the apoVs membrane ( Figure 3 AB). In order to further analyze the origin of CFbs in apoVs, flow cytometry was used to analyze apoVs. + apoVs are characterized by higher levels of fibroblast markers α-SMA and TGFβR1, and lower expression of cardiac troponin T, a cardiomyocyte-specific marker, indicating that CD22 + ApoVs is more likely to originate from fibroblasts and less likely to originate from myocardium ( Figure 3 C) To investigate the target cells of CD22+ apoVs and confirm their molecular mechanism of involvement in cardiomyocyte repair, the uptake of different EVs by cardiomyocytes was evaluated. Fluorescence microscopy showed that the uptake efficiency of green fluorescence by cardiomyocytes could reach over 70%. However, cardiomyocytes did not uptake normal EVs or CD22+ apoVs. - The uptake rate of EVs is less than 10% ( Figure 3 D) The results showed that CD22 +The uptake of apoVs is higher than that of other types of EVs. It is inferred that myocardial cells can recognize and internalize CFbs-apoVs. The specific CD22 antigen expression on the surface of EV subtypes may be an important marker for myocardial recognition of CFbs-apoVs.

[0211] (4) Myocardial infarction can induce fibroblasts to produce CD22 cells enriched with miR-1246 +- CFbs-apoVs and are delivered into cardiomyocytes;

[0212] See also Figure 4 As shown, myocardial infarction can induce fibroblasts to produce CD22 cells enriched with miR-1246. +- CFbs-apoVs are delivered into cardiomyocytes ( n=3) (Note: *P<0.05); Among them, the miRNAs contained in EVs of each group were extracted and subjected to bioinformatics analysis and analysis. Figure 4 A. Volcano plot was used to obtain significantly differentially expressed miRNAs from the GEO database (GSE185729). Figure 4 B. The heat map generated by correlation distance is based on the average expression level of miRNA detected by qRT-PCR. qRT-PCR showed that CD22 + miR-1246 was significantly elevated in apoVs. The relative abundance of each miRNA is represented by a color gradient from red (highest abundance) to blue (lowest abundance) (n=10). Figure 4 C. Analysis of differential expression of miR-1246 in extracellular vesicles from healthy individuals or patients with myocardial infarction (GSE185729). Figure 4 DF.qRT-PCR showed that miR-1246 was significantly increased in CFbs-apoVs. After normalization of the amount of CD22 + miR-1246 was also significantly elevated in apoVs. Figure 4 G. Cy3 fluorescent labeling of miR-1246 in CFbs-apoVs and incubation with cardiomyocytes revealed that miR-1246 can be internalized by cardiomyocytes. Bar, 50 μm.

[0213] The EVs provided by the present invention carry various signaling molecules involved in intercellular communication, but increasing evidence indicates that miRNAs are the most important molecules that EVs regulate the function of recipient cells. Therefore, it is inferred that identifying miRNAs responsible for the pro-apoptotic effect of CFbs-apoVs may have new therapeutic value. First, normal or CD22 +-apoVs and purified. Extract miRNAs and analyze them by bioinformatics. The differential miRNAs between healthy patients and AMI patients were analyzed by GEO database (GSE185729). Figure 4 A). Global miRNA analysis showed that compared with normal EVs, 36 miRNAs were found to be upregulated and 40 miRNAs were downregulated in EVs from the blood of AMI patients. We further screened 11 miRNAs related to apoptosis in plasma exosomes from patients with STEMI for 3-6 months and found the six miRNAs with the greatest differences, including miR-1246, miR-122-5p, miR-244-5p, miR-885-5p, miR-1290, and miR-4433b-5p ( Figure 4 B), qRT-PCR and data set analysis showed that miR-1246 had the highest expression level and the greatest difference ( Figure 4 BC). Secondly, the expression of the top 6 miRNAs was detected by miScript PCR system. Among the 6 pro-apoptotic miRNAs identified in CD22+ EVs, 5 miRNAs were slightly increased in CD22+ EVs, while miR-1246 increased 3-fold ( Figure 4 DF), indicating that miR-1246 is involved in CD22 + The biological effects of EVs may alleviate H / R injury. Next, to confirm that miR-1246 can + EVs enter H / R injured cardiomyocytes and express miR-1246 on CD22 + EVs were labeled with Cy3 and incubated with cardiomyocytes. Red fluorescence was clearly detected in cardiomyocytes ( Figure 4 G). qRT-PCR detection showed that CD22+apoVs treatment could upregulate the expression of miR-1246 in H / R injured cardiomyocytes ( Figure 4 H). These results suggest that CD22 + apoVs-miR-1246 can be delivered into cardiomyocytes.

[0214] (5) p53 is a target gene of miR-1246;

[0215] See also Figure 5 As shown, miR-1246 targets and regulates p53 expression ( n=3) (Note: *P<0.05); Figure 5 A. The target genes of miR-1246 were analyzed using the multiMiR, DIANA-TarBase, and RNAInte databases. Venn diagram analysis revealed 64 mRNAs shared by the three databases. Figure 5B. The potential relationship among miR-1246, CD22, and p53 was shown by constructing a PPI network; Figure 5 C. RNAhybrid website software predicts the pairing sites and binding free energy between human and mouse miR-1246 and p53; Figure 5 D. Dual luciferase gene reporter assay confirmed that p53 is the target gene of miR-1246.

[0216] MiR-1246 has been shown to play a regulatory role in various cancers, but its biological function and mechanism of action in cardiomyocyte apoptosis and mitochondrial damage have not been well explored. In order to identify new miR-1246 target genes that may directly inhibit cardiomyocyte apoptosis and MI / R injury, the target genes of miR-1246 were analyzed using the multiMiR, DIANA-TarBase and RNAInte databases. The Venn diagram analysis identified 64 mRNAs shared by the three databases ( Figure 5 A). By screening these 64 mRNAs, we noticed that p53 was highly correlated with the mitochondrial apoptosis pathway. A PPI network diagram was constructed to show the potential relationship between miR-1246, CD22, and p53 ( Figure 5 B). RNAhybrid website software predicts that both human and mouse miR-1246 have paired binding with p53 and the free energy is low ( Figure 5 C). Notably, dual-luciferase reporter gene analysis showed that the expression of miR-1246 significantly reduced the expression of TP53, indicating that p53 can serve as a target of miR-1246 ( Figure 5 D). More importantly, p53 has been shown to play a key role in the pathogenesis of coronary heart disease by regulating multiple cell biological processes.

[0217] (6)CFbs-CD22 + ApoVs transport miR-1246 to downregulate p53 expression, maintain myocardial mitochondrial homeostasis, and inhibit cell apoptosis;

[0218] See also Figure 6 As shown, CFbs-CD22 + ApoVs transport miR-1246 to downregulate p53 expression, maintain myocardial mitochondrial homeostasis, and inhibit cell apoptosis ( n=3) (Note: *P<0.05); Among them, myocardial cells were transfected with miR-1246 mimic or inhibitor to establish myocardial cell H / R model, and CD22 + EV(CD22 +apoVs) were pretreated, and cardiomyocytes were pretreated with the p53 activator DPBQ (1 μM, 48 h) and the p53 inhibitor PFTα (20 μM, 24 h). Figure 6 p53 protein and gene expressions in cardiomyocytes of each group after AH / R injury; Figure 6 Gene expression of miR-1246 after treatment with p53 activators and inhibitors under BH / R injury; Figure 6 C.CD22 + Effects of apoVs pretreatment on p53 protein expression in H / R cardiomyocytes; Figure 6 DE. Flow cytometry analysis of cardiomyocyte apoptosis rate; Figure 6 F. Flow cytometry detection of MitoSOX in cardiomyocytes TM The fluorescence intensity indicates the production of reactive oxygen species in mitochondria. Figure 6 GH. Flow cytometry analysis of mitochondrial status. The gate represents cells with damaged mitochondria.

[0219] Compared with the control group, overexpression of miR-1246 significantly inhibited the high expression of p53 in H / R-treated cardiomyocytes, while miR-1246 inhibitors had the opposite effect. Figure 6 As shown in A. In contrast, DPBQ significantly reduced the expression of miR-1246 through negative feedback regulation, while PFTα increased the expression of miR-1246, see Figure 6 Thus, these findings suggest that p53 3′-UTR is directly regulated by miR-1246 in cardiomyocytes. + ApoVs can rescue mitochondrial damage and reduce cell apoptosis by inhibiting the p53 signaling pathway. Overexpression of p53 was performed in cardiomyocytes. Figure 6 As shown in C, DPBQ increased the expression of p53 protein. + Compared with the EVs group, H / R+CD22 + In the EVs+DPBQ group, mitochondrial damage was significantly aggravated and the proportion of apoptotic cells was significantly increased. + EVs inhibit the p53 signaling pathway by upregulating miR-1246, rescue mitochondrial damage, and reduce cell apoptosis. Figure 6 As shown in DH.

[0220] The embodiments of the present invention are shown and described above. For those skilled in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing vesicles derived from apoptotic fibroblasts, characterized in that: The following steps are involved: The isolated animal cardiac apex tissue was digested in stages using a mixed digestion solution of trypsin and collagenase II, and then the digestion was terminated using a DMEM solution containing FBS to obtain a digested cell suspension; The digested cell suspension was filtered and centrifuged, and the precipitated cells were resuspended in DMEM and cultured for 1 to 2 hours. Then, the cells were separated and purified by differential adherence culture to obtain cardiac fibroblasts. The cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 to obtain culture medium; The culture fluid is separated and purified to obtain vesicles derived from apoptotic fibroblasts.

2. The method for preparing vesicles derived from apoptotic fibroblasts according to claim 1, characterized in that: The volume ratio of pancreatic enzyme to collagenase II in the mixed digestive solution is 1:1-3, wherein the mass volume percentage of pancreatic enzyme is 0.05-0.2%; The content of FBS in the FBS-containing DMEM solution is 8-12% by volume; The filtration is performed using a 200-mesh sieve; After filtration, centrifugation is performed at 1-5°C, at 600-1000 pr / min for 2-4 times, with each centrifugation lasting 3-8 minutes.

3. The method for preparing vesicles derived from apoptotic fibroblasts according to claim 1, characterized in that: The concentration of apoptosis inducer H2O2 was 0.2-0.8 mM; Cardiac fibroblasts were cultured in DMEM solution with the apoptosis inducer H2O2 for 12 to 48 h; The separation process of the culture solution comprises: The culture medium was centrifuged at 300 × g for 10 min, and the supernatant was centrifuged at 1000 × g for 15 min to remove cells and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 30 min at 1-5°C and filtered through a 0.22 μm filter to remove subcellular structures. The supernatant was then ultracentrifuged at 100,000 × g for 70 min to collect the EV pellet, which was washed with PBS and centrifuged at 100,000 × g for 70 min. The EV pellet was resuspended in 100 μL PBS. The purification process of the culture fluid after separation includes: The resuspended EVs pellet is spread on a sucrose solution and centrifuged at 100,000 g for 70 min. Protein aggregates will settle to the bottom of the centrifuge tube, while EVs containing lipid structures will settle to the isopycnic area, resulting in higher purity EVs. The higher purity EVs were washed with PBS and centrifuged at 100,000 g for 70 min. The EVs were resuspended in 100 μL PBS to obtain vesicles derived from apoptotic fibroblasts.

4. The method for preparing vesicles derived from apoptotic fibroblasts according to claim 3, characterized in that: The sucrose solution is a system in which sucrose solutions with two concentrations of 0.25 mol / L and 2.5 mol / L are prepared into a continuous density gradient.

5. A vesicle derived from apoptotic fibroblasts prepared by the method according to any one of claims 1 to 4, characterized in that: The vesicles contained expression of apoptosis-specific proteins p53, Bcl-2, Bax, caspase-3, and Apaf-1 and had phosphatidylserine exposure; The vesicles carry CD22 on their surface and are enriched with miR-1246.

6. The vesicles derived from apoptotic fibroblasts according to claim 5, characterized in that Flow cytometry was used to characterize the expression of fibroblast markers in vesicles, analyze the probability of α-SMA expression events in vesicles, and determine that specific EVs subtypes originated from fibroblasts.

7. A method for identifying surface CD22 in vesicles derived from apoptotic fibroblasts according to claim 5, characterized in that: include: Based on high-throughput HNCIB detection, the vesicle marker CD22 was screened; Alternatively, flow cytometry was used to analyze CD22 fluorescence on the vesicle surface; Alternatively, immunogold labeling and transmission electron microscopy were used to observe the expression of CD22 on the surface of EVs.

8. The method for identifying surface CD22 in vesicles derived from apoptotic fibroblasts according to claim 7, characterized in that: Immunogold-labeled transmission electron microscopy was used to observe the expression of CD22 on the surface of EVs, including: Vesicles were labeled with anti-rabbit IgGGold2 CD22 antibody and a water film was formed on a quantitative foil R2 / 2 grid. The water film grid was permeabilized by injecting it into ethane cooled to the melting point by liquid nitrogen. The specimens were transferred to a Gatan cryostat mount for a transmission electron microscope, and the expression of CD22 on the surface of EVs was observed using immunogold labeling and transmission electron microscopy.

9. A method for identifying miR-1246 in vesicles derived from apoptotic fibroblasts according to claim 5, characterized in that: The following steps are involved: Total RNA was extracted from the vesicles, and the RNA concentration was measured using a nucleic acid quantifier. Target gene primers were then added to amplify the target gene and internal reference gene. The expression of miR-1246 was quantified using ABI 7500 real-time fluorescence.

10. Use of the vesicles derived from apoptotic fibroblasts according to claim 5 in the preparation of a drug for preventing and treating myocardial ischemia-reperfusion injury.

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