Microvesicles, preparation method thereof and application of microvesicles in myocardial injury repair

Abstract

The invention relates to a microvesicle which comprises miR-143-3p, miR-191a-5p, and one or more of miR-21-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22-3p and miR-34a-5p, and also comprises one or more of miR-143-3p, miR-191a-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22- A plurality of in-vitro and in-vivo experiments prove that after the microvesicles and myocardial cells are co-cultured under an oxidative stress model, the oxidative stress injury of the myocardial cells can be effectively improved, and the microvesicles are used as active ingredients to be prepared into medicines and have the effect of improving various clinical diseases caused by the oxidative stress injury of the myocardial cells.

Description

Technical Field

[0001] The present invention relates to a nucleic acid composition, in particular to a microvesicle containing multiple nucleic acids, a preparation method thereof, and an application thereof in repairing myocardial (cell or tissue) damage (such as oxidative stress). Background Art

[0002] Oxidative stress injury to myocardial cells or tissues is one of the main ways of injury in heart-related diseases. For example, significant oxidative stress injury to myocardial cells can occur in myocardial infarction / reperfusion, myocardial ischemia, inflammation-related myocardial injury, adriamycin-mediated myocardial injury, and even in the extracorporeal circulation stage during cardiac surgery. However, in the clinical diagnosis and treatment process, there is still no highly specific and effective intervention method and means.

[0003] The main underlying cause of oxidative stress damage in cardiomyocytes and tissues is the imbalance of oxidation / reduction function. Abnormal oxidation function is mainly manifested in the generation of a large number of reactive oxides in the cytoplasm and mitochondria, among which the surge of mitochondrial reactive oxygen species (mROS) caused by mitochondria in pathological processes is the main contradiction. Abnormal reduction function is manifested as the dysfunction of the two major reduction systems, namely glutathione and thioredoxin (TRX) systems, which jointly maintain the oxidation / reduction balance in mitochondria. Among them, TRX has two transcripts, TRX1 exists in the cytoplasm, and TRX2 exists in the mitochondria. Unlike TRX1, TRX2 has an additional active cysteine ​​residue, so it has a more significant anti-oxidative stress effect than TRX1, and the stability of mitochondrial function depends on the mitochondrial oxidation / reduction balance. Conversely, mitochondrial dysfunction is inevitably accompanied by the release of a large number of mitochondrial contents (i.e., mtDAMPs) caused by oxidation / reduction imbalance, which ultimately leads to apoptosis and necrosis of cells, which is generally considered to be one of the main mechanisms of oxidative stress damage to cardiomyocytes. Summary of the invention

[0004] One object of the present invention is to provide a microvesicle containing several nucleic acid compositions, which has the effect of improving myocardial cells and tissues damaged by oxidative stress.

[0005] Another object of the present invention is to provide a microvesicle prepared from adipose tissue.

[0006] Another object of the present invention is to provide a use of microcapsules in the preparation of drugs for treating oxidative stress damage of myocardial cells and tissues.

[0007] Another object of the present invention is to provide a medicine with microvesicles as the main active substance.

[0008] After myocardial cells and tissues are damaged by oxidative stress, their clinical manifestations include: apoptosis, necrosis, mitochondrial dysfunction, etc. at the cellular level, and mainly manifested as a decrease in myocardial contractility at the tissue level (i.e., under cardiac ultrasound detection, it is found that the heart's contractile activity and function are reduced). The treatment of myocardial cell and tissue oxidative stress damage referred to in the present invention should be understood as the improvement of these clinical manifestations (symptoms) or the restoration of normal physiological state (i.e., at the cellular level, improving myocardial cell apoptosis and necrosis; improving the energy metabolism function of mitochondria; at the tissue and in vivo level, improving myocardial contractile function). The normal physiological state is within the threshold range formed by current medicine. For example, an adult heart rate of 60 to 100 beats per minute is medically judged to be normal, and a heart rate outside this range is tachycardia or bradycardia.

[0009] A microvesicle comprising miR-143-3p.

[0010] Another type of microvesicles includes miR-143-3p and miR-191a-5p.

[0011] Another microvesicle comprises miR-143-3p and miR-191a-5p, and one or more of miR-21-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22-3p and miR-34a-5p.

[0012] Another type of microvesicles mainly includes miR-143-3p, miR-191a-5p, miR-21-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22-3p and miR-34a-5p.

[0013] The microvesicles of the present invention have relevant protein phenotypes: CD63, CD81 and Tsg101.

[0014] A method for preparing the microvesicles of the present invention comprises co-culturing a gamma-aminobutyric acid receptor agonist (such as baclofen) with fat cells (or tissues), and extracting (such as ultra-high-speed centrifugation) the generated extracellular vesicles.

[0015] Another method for preparing the microvesicles of the present invention is to co-culture GABA receptor agonists (such as baclofen) and GABA with fat cells (or tissues), and extract (such as ultra-high-speed centrifugation) the produced extracellular vesicles.

[0016] Another method for preparing the microvesicles of the present invention is to place adipose tissue in F-12 culture medium containing baclofen (final concentration of 1 μM) for intervention (temperature setting: 37° C., time: 48 h), then aspirate the culture supernatant, filter (such as: 0.4 μM filter), and centrifuge for the first time (such as: 4° C., 300 g, 10 minutes), take the supernatant obtained by the first centrifugation, centrifuge for the second time (such as: 4° C., 2,000 g, 10 minutes), take the supernatant obtained by the second centrifugation; centrifuge for the third time (such as: 4° C., 10,000 g, 60 minutes) at a speed, take the supernatant of the three centrifugations, centrifuge for the fourth time (such as: 4° C., 10,000 g, 70 minutes), discard the supernatant, resuspend the precipitate (such as: using PBS solution), and finally centrifuge for the fifth time (such as: 4° C., 10,000 g, 70 minutes), the resulting precipitate is the microvesicles of the present invention prepared from adipose tissue.

[0017] To prepare the microvesicles of the present invention, subcutaneous fat tissue of organisms is preferred.

[0018] The microvesicles were co-cultured with cardiomyocytes under an oxidative stress model using a Transwell co-culture system to verify that the microvesicles of the present invention can effectively improve oxidative stress damage to cardiomyocytes. The microvesicles were used as active substances (components) to prepare drugs for improving symptoms caused by oxidative stress damage to cardiomyocytes and tissues, such as chest pain, weakness in the limbs, palpitations, chest tightness, decreased appetite, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a Western Blot result diagram of the protein markers related to the microvesicles (EVs) and adipose (Adipose) of the present invention;

[0020] Figure 2 This is a graph showing the results of the volume and particle size detection of the microvesicles of the present invention;

[0021] Figure 3 The results of the AnnexinV / PI method for identifying the effect of microvesicles on antagonizing oxidative stress damage of cardiomyocytes in vitro; A is the result of the AnnexinV / PI flow cytometry experiment, and B is the statistical result diagram;

[0022] Figure 4 This is the result of Seahorse testing the oxidative respiration level of cardiomyocytes;

[0023] Figure 5 The electron microscope field of view of the mitochondrial structure of cardiomyocytes in each experimental group; the arrows in the figure point to the visible mitochondrial structure;

[0024] Figure 6 The color result map

[0025] Figure 7 This is a diagram of the sequencing results of small RNA in microvesicles prepared in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0027] Unless otherwise specified, other reagents used in the examples of the present invention were purchased from Sigma-Aldrich.

[0028] Example 1 Preparation of microvesicles

[0029] Take 4-6 week old C57 mice; kill them (under anesthesia) without trimming the hair; soak them in 75% alcohol for 10 minutes, but not too long to avoid affecting the activity of cells and tissues.

[0030] The rat was taken out of the alcohol solution in the fume hood and placed in a sterile culture dish in a prone position. The back skin was cut along the dorsal coronal axis from 1 cm above the rat's tail to the level of the chest cavity. Then, two 16 cm curved tweezers were used to clamp the skin on both sides with the curved part of the tweezers, and the back skin of the rat was pulled to the base of the rat's lower limbs to expose the subcutaneous tissue of the groin. It can be seen that there is a white fat mass of about 1 cm square near the dorsal side of the groin on both sides of the rat. Use ophthalmic tweezers to carefully peel it off without piercing the peritoneum to avoid contamination; the collected fat mass was placed in a sterile culture dish, and a few drops of complete F12 culture medium were dripped on it to prevent the fat tissue from drying out. The fat tissue was cut into very fine minced meat with ophthalmic scissors or ultrasonic tissue crusher. The above-mentioned chopped fat tissue was placed in a 10 cm culture dish, and 10 ml of F-12 culture medium was added. About 0.5 cm 3 Add baclofen to the culture medium and adjust the final concentration of baclofen to 1 μM based on the original volume of the culture medium.

[0031] Baclofen was dissolved in PBS, shaken to dissolve, and the solvent dosage was adjusted to maintain the stock concentration of baclofen at 1 mM.

[0032] The above operation must strictly comply with the aseptic operation specifications. After the subcutaneous adipose tissue is intervened in F-12 culture medium containing baclofen (final concentration of 1 μM) at 37°C for 48 hours, the culture supernatant is aspirated, the adipose tissue is discarded, and the supernatant is collected after filtering through a 0.4 μM filter. The filtrate is first centrifuged at 300g for 10 minutes (4°C) to take the supernatant; then centrifuged at 2,000g for 10 minutes (4°C) to take the supernatant; then centrifuged at 10,000g for 60 minutes (4°C) to take the supernatant; then centrifuged at 4°C 100,000g for 70 minutes (4°C), the supernatant is discarded, and the precipitate is resuspended in PBS; finally, the precipitate is centrifuged at 100,000g for 70 minutes (4°C), which is the microvesicles secreted by the adipose tissue under the intervention of baclofen.

[0033] We also tried to select mouse adipose tissue from various sources. After many attempts, we finally determined that microvesicles from subcutaneous adipose tissue would be the active ingredient for subsequent experiments.

[0034] The prepared vesicles were subjected to Western Blot protein electrophoresis analysis, and the unique related protein phenotypes were observed: CD63, CD81 and Tsg101. Figure 1 shown.

[0035] Nanodrop analysis of vesicle volume-particle size, the results are as follows Figure 2 As shown, it can be seen that the volume uniformity of the prepared microvesicles is high, and the average particle size is 80nm to 150nm.

[0036] Example 2 Flow cytometry in vitro verification of the effect of microvesicles on improving oxidative stress-induced apoptosis in cardiomyocytes

[0037] Rat cardiomyocyte cell line H9C2 was plated in a six-well plate. When the cell confluence reached 70-80%, the culture medium was replaced with DMEM-high glucose, and 3% hydrogen peroxide was added to a final concentration of 100 μM in the culture medium. After incubation for 6 hours, the DMEM-high glucose culture medium containing the prepared microvesicles (e.g., prepared according to the method of Example 1) was added, and the microvesicle concentration was 20 μg protein / 10 ml culture medium. After incubation for 12 hours, the cells were extracted, and the Annexin-V / PI method was used to determine that the microvesicles prepared in this example had the effect of improving cardiomyocyte function under cell oxidative stress damage. The results are shown in FIG. Figure 3 shown.

[0038] Example 3 In vitro detection of the effect of microvesicles on improving the mitochondrial aerobic oxidative respiratory chain of myocardial cells damaged by oxidative stress

[0039] The oxygen consumption rate (OCR) of cardiomyocytes was measured using the Seahorse XFe96 cell efflux analyzer combined with the Seahorse Cell Mitochondrial Stress Test Kit (Agilent). Cells were cultured on Seahorse cell culture plates and operated according to established protocols. Before the measurement, the culture medium was replaced with Seahorse XF DMEM medium supplemented with 5 μM glucose, 1 μM pyruvate and 10 μM glutamine for 1 hour. After incubation at 37°C in a CO2-free incubator for 1 hour, the OCR was measured under basal conditions and with the addition of oligomycin (1.5 μM), FCCP (1 μM) and rotenone / antimycin A (0.5 μM). At the end of the experiment, the total number of cells was determined by Hoechst33342 staining to normalize the results. The experimental results are shown in Figure 2. Figure 4 As shown, the prepared microvesicles can significantly improve the mitochondrial respiratory function of cardiomyocytes under oxidative stress.

[0040] Example 4 Electron microscopic observation of myocardial cell mitochondrial morphology

[0041] Primary cardiomyocytes of mice were extracted, hydrogen peroxide was added to construct an oxidative stress model, and microvesicles from adipose tissue without intervention and microvesicles obtained by the method of Example 1 were added. The difference in mitochondrial morphology was observed by scanning electron microscopy. The experimental results showed that under in vitro oxidative stress conditions, after adding the microvesicles obtained by the method of Example 1, the cristae structure of cardiomyocyte mitochondria was more complete than that of the positive control group (oxidative stress), see Figure 5 .

[0042] Example 5 In vivo verification of the protective effect of microvesicles on ischemia / reperfusion injury in vivo

[0043] A myocardial ischemia / reperfusion injury model was established in C57 mice, and the present invention was used for in vivo studies. The mice were divided into four groups, including a negative control group, an ischemia / reperfusion injury group, an ischemia / reperfusion injury + unintervention fat microvesicle group, and an ischemia / reperfusion injury + present invention group. The relevant microvesicles were injected into the mouse circulation through the tail vein 30 minutes after modeling, and the injection amount was 1ug microvesicle protein / g mouse body weight. Seven days after the operation, cardiac ultrasound was performed on each group of mice to evaluate the left ventricular contractile function of each group of mice. The results are as follows Figure 6As shown, the left ventricular ejection fraction (LVEF%) and left ventricular shortening fraction (LVFS%) of the myocardial ischemia reperfusion (I / R) group were significantly lower than those of the sham operation (Sham) group, indicating that the left ventricular contractile function was limited after myocardial infarction, resulting in a significant decrease in cardiac ejection function. The cardiac ultrasound results of the ischemia reperfusion + present invention group (I / R+GABA-EVs) showed that the LVEF% and LVFS% were significantly improved compared with the ischemia reperfusion (I / R) group, indicating that the present invention can improve the degree of myocardial ischemia reperfusion injury at the in vivo level.

[0044] This indicates that the microvesicles prepared according to the method of Example 1 can significantly reduce ischemia / reperfusion injury in mice and improve cardiac function in mice.

[0045] Example 6 Microvesicle Sequencing Results

[0046] In view of the results of the above examples, in order to further understand the information of the microvesicles prepared according to Example 1, the relevant small RNA in the microvesicles was further sequenced, and the results are as follows: Figure 7 As shown. Figure 7 It can be seen that the levels of miR-21-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22-3p, miR-143-3p, miR-191a-5p and miR-34a-5p RNA increased significantly, indicating that these RNA substances are related to the antagonism of oxidative stress damage.

Claims

1. A microvesicle, characterized in that Including miR-143-3p.

2. The microvesicle according to claim 1, characterized in that Also included is miR-191a-5p.

3. The microvesicle according to claim 2, characterized in that It also includes one or more of miR-21-5p, miR-23a-3p, miR-199a-3p, miR-21-5p, miR-24-3p, miR-22-3p and miR-34a-5p.

4. The microvesicle according to claim 1, characterized in that The microvesicles are produced by adipose tissue.

5. The microvesicle according to claim 4, characterized in that The adipose tissue is subcutaneous adipose tissue.

6. A method for preparing the microvesicles according to claim 1, characterized in that It is obtained by co-culturing adipocytes or tissues with γ-aminobutyric acid receptor agonists and extracting the produced extracellular vesicles.

7. The method according to claim 6, characterized in that The gamma-aminobutyric acid receptor agonist is baclofen, and gamma-aminobutyric acid is added to co-culture with fat cells or tissues.

8. Use of the microvesicles according to claim 1 in preparing a drug for repairing myocardial damage caused by oxidative stress.

9. Use of the microvesicles according to claim 1 in preparing a drug for improving myocardial oxidative stress injury.

10. A drug, characterized in that The microcapsule according to any one of claims 1 to 5 is used as an active ingredient.

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