A kind of extracellular vesicles with a defined size derived from cardiomyocytes, and a preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and specifically to a cardiomyocyte-derived extracellular vesicle of limited size, and a preparation method and application thereof. In the present invention, lipopolysaccharide (LPS) is used to stimulate cardiomyocyte H9C2 to induce the production of myocardial inflammatory cells, and differential centrifugation is used to separate extracellular vesicles of limited size from the induced myocardial inflammatory cells. Studies have found that the extracellular vesicles of limited size can inhibit the secretion of inflammatory factors by macrophages in an inflammatory state, and transform macrophages into an anti-inflammatory repair phenotype (M2). At the same time, in mice with myocarditis, large extracellular vesicles can reduce the myocardial inflammatory state and have the effect of relieving myocarditis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cardiomyocyte-derived extracellular vesicle of a limited size and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Extracellular vesicles (EVs) are lipid membrane-enclosed vesicles produced by the vast majority of cells. Extracellular vesicles contain proteins, nucleic acids, and lipids and serve as important intercellular communicators (facilitated by receptors on the membrane of the vesicles). Exosomes, microvesicles, and apoptotic bodies represent the major subtypes of extracellular vesicles. Exosomes are produced inside cells, released via the endosomal pathway, and range in diameter from about 30 to 100 nm. Microvesicles bud from the plasma membrane and range from about 50 to 1000 nm. Apoptotic bodies are released during cell death, contain various parts of the cell, and range from about 50 to 5000 nm.

[0004] Extracellular vesicles are a promising tool for medicine because they can deliver a variety of molecules into cells under normal and pathological conditions. Myocarditis is a life-threatening inflammation of the myocardium caused by a variety of etiologies, which can lead to serious consequences such as cardiac dysfunction and arrhythmias. Currently, symptomatic treatment of myocarditis is still the main treatment, and specific treatments are lacking. Cardiomyocytes are the main cells that make up the heart, while macrophages are the main immune cells infiltrating in myocarditis. However, the crosstalk mechanism between cardiomyocytes and macrophages has not been elucidated. Extracellular vesicles are an important pathway for intercellular communication. In myocarditis, it is not clear whether cardiomyocytes can communicate with macrophages through extracellular vesicles. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a cardiomyocyte-derived extracellular vesicle of limited size and a preparation method and application thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing extracellular vesicles of a limited size derived from cardiomyocytes, comprising the following steps:

[0008] (1) Cultivate cardiomyocytes and induce them to produce myocardial inflammatory cells;

[0009] (2) Continue to culture the induced myocardial inflammatory cells and then aspirate the supernatant in the culture medium;

[0010] (3) subjecting the supernatant to differential centrifugation to collect extracellular vesicles of a defined size;

[0011] Wherein, the cardiomyocytes described in step (1) are H9C2.

[0012] The particle size of extracellular vesicles of defined size ranges from 100 to 600 nm, with a peak value of approximately 175 nm.

[0013] The second aspect of the present invention provides a cardiomyocyte-derived extracellular vesicle of a limited size, which is prepared by the preparation method described in the first aspect.

[0014] The third aspect of the present invention provides use of the cardiomyocyte-derived extracellular vesicles of a defined size described in the second aspect in the preparation of a drug for treating myocarditis.

[0015] The fourth aspect of the present invention provides a pharmaceutical preparation comprising the cardiomyocyte-derived extracellular vesicles of defined size described in the second aspect.

[0016] The fifth aspect of the present invention provides a pharmaceutical composition comprising the cardiomyocyte-derived extracellular vesicles of defined size as described in the second aspect.

[0017] The beneficial effects of the present invention are:

[0018] In the present invention, lipopolysaccharide (LPS) is used to stimulate myocardial cells H9C2 to induce the production of myocardial inflammatory cells, and differential centrifugation is used to separate extracellular vesicles of limited size from the induced myocardial inflammatory cells. Studies have found that the extracellular vesicles of limited size can inhibit the secretion of inflammatory factors by macrophages under inflammatory conditions, and transform macrophages to an anti-inflammatory repair phenotype (M2). At the same time, in mice with myocarditis, large extracellular vesicles can reduce the state of myocardial inflammation and have the effect of alleviating myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 The state of the LPS-induced cardiomyocyte inflammation model in Example 1;

[0021] Figure 2 Western blotting was used to verify the extracellular vesicles C-1EV of limited size obtained in Example 1. LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS ;

[0022] Figure 3 The extracellular vesicle C-1EV of limited size obtained in Example 1 LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS Transmission electron microscope image of the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS , b is the extracellular vesicle C-1EV of limited size obtained in Example 1 LPS ;

[0023] Figure 4 The extracellular vesicle C-1EV of limited size obtained in Example 1 LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS The particle size detection results of the present invention are shown in Table 1, wherein a is the extracellular vesicle C-1EV obtained in Comparative Example 1. PBS , b is the extracellular vesicle C-1EV of limited size obtained in Example 1 LPS ;

[0024] Figure 5 qPCR detection of the extracellular vesicles C-1EV of limited size obtained in Example 1 LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS Inflammatory factors of macrophages after treatment, where a is IL-6, b is TNF-α, and c is IL-1β;

[0025] Figure 6 Western blotting to verify the extracellular vesicles C-sEV obtained in Comparative Example 2 LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS ;

[0026] Figure 7 Extracellular vesicles C-sEV obtained from Comparative Example 2 LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS Transmission electron microscopy images of the extracellular vesicles C-sEV PBS , b is extracellular vesicle C-sEV LPS ;

[0027] Figure 8 Extracellular vesicles C-sEV obtained from Comparative Example 2 LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS The particle size detection results, where a is the extracellular vesicle C-sEV PBS , b is extracellular vesicle C-sEV LPS ;

[0028] Fig. 9Extracellular vesicles C-sEV obtained by qPCR detection in comparative example 2 LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS Inflammatory factors of macrophages after treatment, where a is IL-6, b is TNF-α, and c is IL-1β;

[0029] Fig.10 Flow cytometry was used to detect the extracellular vesicles C-1EV of limited size obtained in Example 1. LPS Polarization states of macrophages after treatment, where A and D are M1 polarization effects, and B and C are M2 polarization effects;

[0030] Fig.11 The results of the mouse animal experiment verification, where A is the mouse treatment process, B is the mouse weight change, C is the surface appearance of the mouse heart, D is the mouse heart ultrasound result, E is the cardiac ejection fraction, and F is the left ventricular short axis shortening rate;

[0031] Fig.12 The results of mouse serum cTnT and inflammatory factors detection, where A is mouse serum cTnT, B is IL-6, C is TNF-α, and D is IL-1β;

[0032] Fig.13 HE staining and immunohistochemistry to detect extracellular vesicles C-lEV LPS Myocardial inflammatory factors in treated mice. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] A first typical embodiment of the present invention provides a method for preparing extracellular vesicles of a limited size derived from cardiomyocytes, comprising the following steps:

[0036] (1) Cultivate cardiomyocytes and induce them to produce myocardial inflammatory cells;

[0037] (2) Continue to culture the induced myocardial inflammatory cells and then aspirate the supernatant in the culture medium;

[0038] (3) subjecting the supernatant to differential centrifugation to collect extracellular vesicles of a defined size;

[0039] Wherein, the cardiomyocytes described in step (1) are H9C2.

[0040] In one or more embodiments, the particle size of the defined size extracellular vesicles is 100-600 nm, with a peak size of approximately 175 nm.

[0041] In one or more embodiments, the specific method in step (1) includes:

[0042] Culture the cardiomyocytes until they grow to a density of about 60-80%, replace the serum-free DMEM medium and continue culturing for 10-14 h. After culturing, change the medium and replace the serum-free DMEM medium again. At the same time, add lipopolysaccharide with a final concentration of 1-2 μg / mL and culture for 20-30 h to induce the production of cardiomyocyte inflammatory cells.

[0043] In one or more embodiments, the differential centrifugation method in step (3) includes:

[0044] Centrifuge at 280-320 g for 9-12 min at 3-5 °C and collect the supernatant;

[0045] Centrifuge at 1900-2200 g for 18-22 min at 3-5 °C and collect the supernatant;

[0046] Centrifuge at 9000-12000 g for 28-32 min at 3-5 °C and collect the precipitate;

[0047] After washing the precipitate, centrifuge at 9000-12000 g for 28-32 min at 3-5 °C to collect the precipitate and obtain extracellular vesicles of a specified size.

[0048] Preferably, the precipitate is washed with PBS buffer solution.

[0049] A second typical embodiment of the present invention provides a cardiomyocyte-derived extracellular vesicle of a limited size, which is prepared by the preparation method described in the first aspect.

[0050] A third typical embodiment of the present invention provides use of the cardiomyocyte-derived extracellular vesicles of a defined size described in the second aspect in the preparation of a drug for treating myocarditis.

[0051] A fourth typical embodiment of the present invention provides a pharmaceutical preparation comprising the cardiomyocyte-derived extracellular vesicles of a defined size as described in the second aspect.

[0052] A fifth typical embodiment of the present invention provides a pharmaceutical composition comprising the cardiomyocyte-derived extracellular vesicles of a defined size as described in the second aspect.

[0053] In the above-mentioned pharmaceutical composition, the dosage of the cardiomyocyte-derived extracellular vesicles of a limited size can be routinely adjusted according to factors such as the purpose of drug administration, the subject's condition, etc. In the above-mentioned pharmaceutical composition, the dosage of the cardiomyocyte-derived extracellular vesicles of a limited size should be an effective dosage, and the pharmaceutical composition adopts a dosage form that is easy to accurately administer.

[0054] When the pharmaceutical composition is used to prepare a pharmaceutical preparation for in vivo administration, the pharmaceutical preparation should be sterile, and methods for achieving sterility of the pharmaceutical preparation should be known to those skilled in the art, for example, it can be achieved by filtering through a sterile filter membrane, etc. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms, such as a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0055] Preferably, the above-mentioned dosage forms for administration via the gastrointestinal tract include powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, and the like.

[0056] Preferably, parenteral dosage forms include injection dosage forms (e.g., injections, including intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracavitary injections, and other injections); respiratory tract dosage forms (e.g., sprays, aerosols, powder aerosols, etc.); skin dosage forms (e.g., external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.); cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] Example 1

[0059] H9C2 cells were cultured until the density reached about 60-80%. The culture medium was discarded in the cell workbench and replaced with serum-free DMEM culture medium (starvation treatment). The cells were placed in the incubator and cultured for 12 hours. After 12 hours, the medium was changed to DMEM without fetal bovine serum and LPS (the stock concentration was 1 mg / mL) was added to make the final concentration of LPS 1.5 μg / mL. The cardiomyocytes were cultured for 24 hours after the addition of LPS.

[0060] Observe the cell morphology in Example 1 and under normal culture, extract total cell RNA, and examine the mRNA expression of inflammatory factors by PCR. The results are as follows Figure 1 As shown, from Figure 1 It can be seen that the inflammatory factors increased after LPS stimulation, indicating that the inflammatory model was successfully constructed.

[0061] The supernatant in the culture medium was aspirated, and the extracellular vesicles of a defined size were separated from the culture medium by a standard differential centrifugation protocol. Specifically, the cells were centrifuged at 300 g for 10 min at 4 °C and the precipitate was discarded; the cells were centrifuged at 2000 g for 20 min at 4 °C and the precipitate was discarded; the cells were centrifuged at 10000 g for 30 min at 4 °C and the supernatant was discarded; the precipitate was washed with PBS, and centrifuged at 10000 g for 30 min at 4 °C and the supernatant was discarded; the precipitate was diluted with PBS to obtain extracellular vesicles of a defined size, namely C-lEVs. LPS .

[0062] Comparative Example 1

[0063] Compared with the example, this comparative example uses PBS to stimulate the cardiomyocyte cell line (H9C2), and other conditions are the same as those in Example 1. At the same time, the same differential centrifugation conditions as in Example 1 are used to obtain extracellular vesicles C-1EV PBS .

[0064] The extracellular vesicles C-1EV of limited size obtained in Example 1 were analyzed using Western Blot, Nanoparticle Tracking Analysis (NTA), and transmission electron microscopy. LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS .

[0065] Western Blot analysis confirmed that the extracellular vesicles C-1EV of limited size obtained in Example 1 LPS And the extracellular vesicle C-1EV obtained in Comparative Example 1 PBS Expresses proteins TSG101, CD63, and Actin, but not Calnexin ( Figure 2). Transmission electron microscopy showed that C-lEV PBS and C-1EV LPS Typical membrane cup shape ( Figure 3 The particle size determined by NTA is mainly between 100 and 600 nm. Figure 4 ). The extracellular vesicles C-1EV of limited size obtained in Example 1 LPS The particle size is 100-600 nm, with a peak value of about 175 nm. Extracellular vesicles C-1EV were obtained in Example 1 PBS The particle size is 100~600nm, with a peak value of 158 nm.

[0066] Experimental Example 1

[0067] In order to study C-lEV LPS To investigate the anti-inflammatory effect of LPS, the mouse macrophage cell line RAW264.7 was used to establish an inflammatory injury model: RAW264.7 macrophages were cultured to a density of about 60-80%, the culture medium was discarded in the cell workbench, and the medium was replaced after rinsing with PBS. After the medium was replaced, LPS (the stock concentration was 1 mg / mL) was added to the culture medium, and the final concentration of LPS after addition was 1 μg / mL, and only an equal volume of PBS was added to the control group. RAW264.7 macrophages were cultured for 12 h after the addition of LPS.

[0068] Mouse macrophage cell line RAW264.7 was treated with LPS and then given C-1EV PBS or C-1EV LPS qPCR was used to detect changes in inflammatory factors. The results showed that in LPS-induced RAW264.7 cells, the expression levels of inflammatory factors IL-6, TNF-α and IL-1β increased significantly, while C-1EV LPS Treatment reduced the expression of these inflammatory factors. PBS There was no effect on the expression of inflammatory factors ( Figure 5 ).

[0069] Comparative Example 2

[0070] Compared with Example 1, this comparative example adopts a different differential centrifugation scheme to obtain extracellular vesicles. Specific differential centrifugation scheme: centrifuge at 300 g for 10 min at 4 °C, discard the precipitate; centrifuge at 2000 g for 20 min at 4 °C, discard the precipitate; centrifuge at 10000 g for 30 min at 4 °C, take the supernatant; ultracentrifuge at 140000 g for 90 min; remove the supernatant, wash the precipitate with PBS buffer and resuspend it, centrifuge again at 140000 g for 90 min, resuspend the precipitate with 100 μL PBS buffer, and freeze it at -80 °C for later use to obtain extracellular vesicles C-sEVLPS .

[0071] Comparative Example 3

[0072] Compared with Comparative Example 2, this comparative example uses PBS to stimulate the cardiomyocyte cell line (H9C2), and other conditions are the same as those in Comparative Example 2. At the same time, the same differential centrifugation conditions as those in Comparative Example 2 are used to obtain extracellular vesicles C-sEV PBS .

[0073] The extracellular vesicles C-sEV obtained in Comparative Example 2 were analyzed using Western Blot, Nanoparticle Tracking Analysis (NTA), and transmission electron microscopy. LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS .

[0074] Western Blot analysis confirmed that the extracellular vesicles C-sEV obtained in Comparative Example 2 LPS And the extracellular vesicle C-sEV obtained in comparative example 3 PBS Expresses proteins TSG101, CD63, and Actin, but not Calnexin ( Figure 6 ). Transmission electron microscopy showed C-sEV PBS and C-sEV LPS Typical membrane cup shape ( Figure 7 The particle size determined by NTA is mainly between 0 and 300 nm ( Figure 8 ). Among them, the extracellular vesicles C-sEV obtained in comparative example 2 LPS The particle size is 100-300 nm, with a peak value of 86 nm; the extracellular vesicles C-sEV obtained in comparative example 3 PBS The particle size is 100~300 nm, with a peak value of 73 nm.

[0075] Experimental Example 2

[0076] The inflammatory injury model of mouse macrophage cell line RAW264.7 was established in the same manner as in Experimental Example 1.

[0077] Mouse macrophage cell line RAW264.7 was treated with LPS and then given C-sEV PBS or C-sEV LPS qPCR was used to detect changes in inflammatory factors. The results showed that the expression levels of inflammatory factors IL-6, TNF-α and IL-1β increased significantly in RAW264.7 cells induced by LPS, while C-sEV LPSTreatment with EVPBS increased the expression of these inflammatory factors. In contrast, C-sEVPBS had no effect on the expression of inflammatory factors ( Fig. 9 ).

[0078] In summary, the extracellular vesicles C-lEV derived from cardiomyocytes with limited size under LPS stimulation LPS The inflammatory factors of macrophages under inflammatory conditions were reduced. Therefore, the next step of research was to focus on C-lEVLPS.

[0079] As the main inflammatory and infiltrating cells, macrophages play a crucial role in the early development of myocarditis. Depending on the changes in the cardiac environment, macrophages can polarize into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. LPS To investigate whether LPS treatment can reduce inflammation by regulating macrophage polarization, we evaluated the phenotype of macrophages by flow cytometry in vitro. The results showed that the proportion of CD86-positive M1 macrophages increased significantly after LPS treatment, while C-1EV LPS Treatment reduced the proportion of M1 macrophages ( Fig.10 In contrast, the proportion of CD206-positive M2 macrophages increased after LPS treatment and was significantly increased in C-1EV LPS After further treatment ( Fig.10 ).

[0080] Example 2

[0081] Four-week-old Balb / c male mice were randomly divided into three groups after three days of adaptive feeding: control group, viral myocarditis group, and viral myocarditis plus EV intervention group. 5 TCID 50 Coxsackievirus B3 (CVB3) virus solution. Mice in the EV intervention group were injected with one dose of C-lEV via tail vein on the day of virus injection and on the 4th day. LPS (150 μL10 8 C-1EV LPS ). Observe and record the weight and survival of mice. On the 7th day, perform cardiac ultrasound on mice, then kill the mice, and take the heart and peripheral blood for later use ( Fig.11 A). On day 7, C-1EV LPS Slightly reversed the weight loss of mice after CVB3 infection ( Fig.11 B). After anesthesia, the mice were killed and the heart tissue was taken. The macroscopic observation showed that the heart surface of the control mice was smooth, while the heart of the mice with viral myocarditis showed large areas of white patchy lesions. The area of ​​white patchy lesions on the heart of the mice in the EV intervention group was significantly reduced ( Fig.11C). Echocardiography was used to evaluate cardiac function and found that mice in the viral myocarditis group showed cardiac dysfunction as evidenced by decreased ejection fraction (EF) and fractional shortening (FS), while EV intervention significantly alleviated cardiac dysfunction in mice with viral myocarditis ( Fig.11 D~F). ELISA analysis showed that the plasma cTnT level in CVB3-infected mice was significantly increased, while CVB3+C-1EV LPS group significantly decreased ( Fig.12 ). RT-qPCR analysis showed that the expression of TNF-α, IL-6, and IL-1β in the hearts of mice in the viral myocarditis group was significantly increased compared with the control group ( Fig.12 ). H&E staining showed that the pathological scores of mice with myocarditis were significantly higher than those of the control group, while the pathological scores of mice in the EV intervention group were significantly lower than those in the myocarditis group ( Fig.13 Immunohistochemical staining and expression of these factors in CVB3+C-lEV LPS group was significantly lower than that of the CVB3 group ( Fig.13 ).

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a cardiomyocyte-derived extracellular vesicle of a limited size in the preparation of a drug for treating myocarditis; in, A method for preparing extracellular vesicles of limited size derived from cardiomyocytes comprises the following steps: (1) Cultivate cardiomyocytes and induce them to produce myocardial inflammatory cells; (2) Continue to culture the induced myocardial inflammatory cells and then aspirate the supernatant in the culture medium; (3) subjecting the supernatant to differential centrifugation to collect extracellular vesicles of a defined size; Wherein, the cardiomyocytes described in step (1) are H9C2; The particle size of the extracellular vesicles of the limited size is 100-600 nm, with a peak value of 175 nm; The specific method in step (1) includes: The cardiomyocytes were cultured until the growth density reached 60-80%, and the serum-free DMEM medium was replaced for 10-14 h. After the culture, the medium was changed and the serum-free DMEM medium was replaced again. At the same time, lipopolysaccharide with a final concentration of 1-2 μg / mL was added for 20-30 h to induce the production of myocardial inflammatory cells. The differential centrifugation method in step (3) includes: Centrifuge at 280-320 g for 9-12 min at 3-5 °C and collect the supernatant; Centrifuge at 1900-2200 g for 18-22 min at 3-5 °C and collect the supernatant; Centrifuge at 9000-12000 g for 28-32 min at 3-5 °C and collect the precipitate; After washing the precipitate with PBS buffer solution, centrifuge at 9000-12000 g for 28-32 min at 3-5 °C to collect the precipitate and obtain extracellular vesicles of a limited size.

Images