WO2024021024A1 - Heart-protecting factor mir-139-3p and use thereof - Google Patents

Heart-protecting factor mir-139-3p and use thereof Download PDF

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WO2024021024A1
WO2024021024A1 PCT/CN2022/108991 CN2022108991W WO2024021024A1 WO 2024021024 A1 WO2024021024 A1 WO 2024021024A1 CN 2022108991 W CN2022108991 W CN 2022108991W WO 2024021024 A1 WO2024021024 A1 WO 2024021024A1
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mir
exosomes
macrophages
expression
msc
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PCT/CN2022/108991
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Chinese (zh)
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杨跃进
宁瑜
龚钊婷
徐俊彦
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中国医学科学院阜外医院
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Priority to PCT/CN2022/108991 priority Critical patent/WO2024021024A1/en
Priority to CN202280028807.9A priority patent/CN117157083A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the present invention relates to the cardioprotective factor miR-139-3p and its application, especially regarding the role of miR-139-3p in inhibiting macrophage infiltration into the myocardium, promoting the polarization of infiltrated macrophages to M2, inhibiting inflammation and/or Promote myocardial repair effect and other applications.
  • AMI Acute myocardial infarction
  • MSC mesenchymal stem cells
  • Exosomes are secreted by parental cells and carry a large amount of parental-derived mRNA, non-coding RNA (such as miRNA), proteins, lipids, etc. These bioactive molecules and their important role in intercellular communication have led researchers to Begin to pay attention and explore the use of stem cell-derived exosomes to treat ischemic heart disease.
  • M1 and M2 This process is called polarization.
  • interleukin 4 IL-4
  • IL-10 IL-13
  • TGF- ⁇ transforming growth factor ⁇
  • Stat6 signal transducer and activator of transcription1
  • LPS lipopolysaccharide
  • IFN- ⁇ interferon ⁇
  • M1 macrophages secrete cytokines, chemokines, growth factors and matrix metalloproteinases (MMPs), such as TNF ⁇ , IL-1 ⁇ , IL-6, IL-12, iNOS, etc., which can promote inflammation and eliminate cells.
  • MMPs matrix metalloproteinases
  • M2 macrophages produce anti-inflammatory, pro-angiogenic, and pro-repair factors, such as CD206, Arg1, IL-10, vascular endothelial growth factor, TGF- ⁇ , etc., and have the ability to phagocytose apoptotic cells and promote angiogenesis and scar repair. Effect (Koudstaal S, Jansen of Lorkeers S J, Gaetani R, et al. Concise review: heart regeneration and the role of cardiac stem cells [J]. Stem Cells Transl Med, 2013, 2(6): 434-43). Therefore, regulating the balance of macrophages to polarize them from M1 to M2 can effectively improve post-infarction myocardial repair and improve cardiac function.
  • One object of the present invention is to provide a miRNA that can serve as a cardioprotective factor.
  • Another object of the present invention is to provide related applications of the miRNA.
  • miR-139-3p is a cardioprotective factor related to macrophage polarization.
  • exosomes MSC ATV -Exo
  • exosomes MSC ATV -Exo miR inhibitor
  • MSC ATV -Exo miR inhibitor
  • miR mimic overexpressing miR-139-3p can increase the expression levels of M2 markers in macrophages.
  • Stat1 signal transducer and activator of transcription 1
  • miR-139-3p can specifically bind to the 3'UTR region of Stat1 and inhibit its expression.
  • miR-139-3p mimic or inhibitor reduces or increases the total Stat1 expression level in macrophages; miR-139-3p mimic reduces the phosphorylation level of Stat1 in macrophages stimulated by LPS.
  • the present invention provides relevant applications of miR-139-3p as a cardioprotective factor.
  • the present invention provides the use of miR-139-3p in preparing a preparation for regulating macrophage polarization.
  • the present invention provides the use of a reagent material that overexpresses miR-139-3p in exosomes in the preparation of a preparation for promoting the polarization of macrophages toward the M2 type.
  • the preparation for promoting the polarization of macrophages toward the M2 type includes exosomes overexpressing miR-139-3p.
  • the exosomes are exosomes derived from bone marrow mesenchymal stem cells (mesenchymal stem cells, MSC).
  • the reagent materials for overexpressing miR-139-3p in exosomes include: reagent materials for overexpressing miR-139-3p in exosomes through genetic technology, and/or through drug pretreatment.
  • the drug used to pretreat stem cells may be, for example, a statin drug, such as atorvastatin (ATV).
  • the present invention provides the use of reagent materials that inhibit and/or knock down the expression of miR-139-3p in exosomes in the preparation of preparations for inhibiting the polarization of macrophages toward the M2 type. .
  • reagent materials for inhibiting and/or knocking down the expression of miR-139-3p in exosomes include: miR-139-3p inhibitors, knockdown of miR-139-3p in exosomes through genetic technology - One or more reagent materials for the expression of -139-3p.
  • the present invention provides related applications of miR-139-3p as a regulatory factor for macrophage polarization toward the M2 type.
  • the reagent materials that promote the polarization of macrophages toward the M2 type include: miR-139-3p mimics, reagent materials that increase the expression of miR-139-3p in macrophages through genetic technology. one or more of them.
  • the present invention also provides the application of miR-139-3p in the preparation of preparations for regulating the expression of signal transducer and activator of transcription 1 (Stat1).
  • the present invention provides the use of reagent materials that overexpress miR-139-3p in the preparation of preparations that reduce the total Stat1 expression level in macrophages.
  • the invention provides the use of reagent materials that inhibit and/or knock down the expression of miR-139-3p in exosomes in the preparation of a preparation for increasing the total Stat1 expression level in macrophages. .
  • the present invention provides the use of a reagent material that overexpresses miR-139-3p in the preparation of a preparation for increasing the total Stat1 expression level in macrophages.
  • the present invention also provides a method for increasing the expression level of M2 markers in myocardial tissue, which method includes: administering exosomes overexpressing miR-139-3p to a subject in need.
  • the present invention also provides a method for treating acute myocardial infarction, which method includes: administering exosomes overexpressing miR-139-3p to a subject in need.
  • administering exosomes overexpressing miR-139-3p to a subject in need.
  • overexpressing exosomes of miR-139-3p at least one of the following effects can be achieved: reducing macrophage infiltration in the myocardium in the acute phase of myocardial infarction, controlling the inflammatory response, and promoting the transformation of infiltrating macrophages from M1 type to M2 type.
  • Transform promote myocardial repair, reduce myocardial infarction size, and improve cardiac function; and/or deliver cardioprotective miR-139-3p to macrophages to promote M2 macrophage polarization by inhibiting the expression and activation of Stat1.
  • exosome treatment overexpressing miR-139-3p can more effectively reduce macrophage infiltration in the myocardium in the acute phase of myocardial infarction, control inflammatory responses, and promote the transformation of infiltrated macrophages from M1 type to M1 type.
  • M2 type transformation promotes myocardial repair, reduces myocardial infarction size, and improves cardiac function.
  • Exosomal MSC ATV -Exo overexpressing miR-139-3p can deliver cardioprotective miR-139-3p to macrophages, which promotes M2 macrophage polarization by inhibiting the expression and activation of Stat1.
  • Figure 1A to Figure 1G show the extraction and identification of exosomes derived from rat bone marrow mesenchymal stem cells.
  • Figure 1A Schematic scheme for the ex vivo isolation of rat bone marrow mesenchymal stem cells with or without ATV pretreatment.
  • Figure 1B Flow cytometric analysis of rat bone marrow mesenchymal stem cell surface markers.
  • Figure 1C Observation of the morphology of bone marrow mesenchymal stem cells treated with dimethyl sulfoxide or ATV under a microscope.
  • Figure 1D Transmission electron microscopy scanning of exosome morphology.
  • Figure 1E Western blot analysis of exosome protein markers.
  • Figure 1F Western blot analysis of the protein expression of M2 macrophage marker Arg1 after treatment with MSC-Exo or MSC ATV -Exo at different concentrations (2 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL) for 24 hours.
  • Figure 1G Rat bone marrow-derived macrophages (BMDM) were stimulated with LPS for 6 hours, and then incubated with 40 ⁇ g/mL MSC-Exo or MSC ATV -Exo for another 48 hours. Western blot analysis of Arg1 protein expression in BMDM.
  • BMDM Rat bone marrow-derived macrophages
  • Figure 2A to Figure 2I show the superior effects of MSC ATV -Exo on ventricular function, myocardial infarction size, macrophage infiltration and M2 polarization in rats with acute myocardial infarction.
  • Figure 2A Representative M-mode echocardiograms of each group on day 28 after AMI.
  • Figure 2C Representative cross-section of the heart stained with Masson's trichrome on day 28.
  • Figure 2E Typical confocal images of CD68+ or CD206+ macrophages in the infarct margin zone on day 3 after acute myocardial infarction.
  • Figure 2G Western blot analysis of iNOS and Arg1 expression in infarcted myocardium in each group.
  • Figures 3A to 3D show the effects of MSC ATV -Exo and MSC-Exo on macrophage polarization in vitro.
  • Figure 3A PKH-67 labeled MSC ATV -Exo or MSC-Exo (green) is taken up by rat BMDM (red).
  • Figure 3B Western blot analysis of iNOS and Arg1 protein expression in BMDM.
  • M1 macrophage markers
  • M2 Arg1, IL-10, CD206
  • Figure 4A to Figure 4B show differentially expressed miRNAs between MSC ATV -Exo and MSC-Exo.
  • Figure 4A Volcano plot shows all differentially expressed miRNAs between MSC ATV -Exo and MSC-Exo.
  • Figure 4B qRT-PCR verified the expression of miRNA in MSC ATV -Exo and MSC-Exo.
  • Figures 5A to 5L show that miR-139-3p is an effector of MSC ATV -Exo-mediated macrophage polarization targeting Stat1 in vitro.
  • Figure 5A Schematic diagram of the protocol for isolating MSC ATV -Exo-miR inhibitor and MSC-Exo-miR mimics and incubating LPS-pretreated rat BMDM.
  • Figure 5B Protein expression of macrophage M1 marker iNOS and M2 marker Arg1 in BMDM.
  • Figure 5E Schematic diagram of the protocol for direct transfection of miR-139-3p mimics into LPS-pretreated BMDM.
  • Figure 5F Protein expression of macrophage M1 marker iNOS and M2 marker Arg1 in BMDM.
  • Figures 6A to 6I show that MSC ATV -Exo enhances M2 macrophage polarization in the infarct area of AMI rats by transferring miR-139-3p.
  • Figure 6A Representative confocal images of CD68 + (M1) and CD206 + (M2) macrophages in the infarct area on day 3 after AMI.
  • Figure 6C Representative M-mode echocardiograms of each group on day 28 after AMI.
  • Figure 6E Representative cross-section of an AMI heart on day 28 using Masson's trichrome staining.
  • Figure 6G Western blot analysis of the expression of iNOS, Arg1 and p-Stat1 in infarcted myocardium in each group.
  • Figure 7 shows a schematic summary of the main findings of the present invention.
  • Intramyocardial injection of rat bone marrow MSC ATV- Exo in the infarct border zone delivers upregulated miR-139-3p to infiltrating macrophages and promotes M2 macrophage polarization by inhibiting Stat1 expression and activation, promoting post-infarction heart repair.
  • the bone marrow cells extracted from the two hind limbs of one rat can be seeded into four 10 cm dishes; or they can be directly inoculated into a 12-well culture plate for culture to facilitate subsequent experiments. ;
  • Group M1 LPS with a final concentration of 100ng/mL was added to macrophages;
  • M2 group Add final concentrations of 10ng/ml IL-4 and 10ng/ml IL-13 to macrophages;
  • Exosome RNA extraction and quality inspection After using gradient ultracentrifugation to extract exosomes MSC-Exo and MSC ATV -Exo derived from rat bone marrow mesenchymal stem cells, considering the low RNA content in exosomes, For the detection of small RNA expression profile in exosomes, the HiPure Liquid RNA Mini Kit of Meiji Biotech was used to extract exosomal RNA. Please refer to the product instructions for specific steps. Use Qubit or Agilent 2200 to perform quality inspection on the samples. The quality inspection content includes the concentration, total amount and RNA fragment length distribution peak diagram of exosomal RNA. Among them, if the Agilent 2200 peak chart has a large fragment peak of >500 nucleotides, it means that the exosome sample may be contaminated by ribosomes or mycoplasma.
  • miRNAs For significantly different miRNAs, four software, namely miRWalk, TargetScan, miRDB and miRTarBase, were used to further predict the target genes of the miRNAs, and gene function (GO) and KEGG biological pathway enrichment analysis were performed on the target genes.
  • miRWalk For significantly different miRNAs, four software, namely miRWalk, TargetScan, miRDB and miRTarBase, were used to further predict the target genes of the miRNAs, and gene function (GO) and KEGG biological pathway enrichment analysis were performed on the target genes.
  • WT 3’UTR+NCmimic WT is wild type
  • Example 1.miR-139-3p mediates the promotion of macrophage M2 polarization
  • Isolate and culture rat bone marrow MSC to passage 3-4 When the cells grow to a sub-confluent state, 1 ⁇ mol/L DMSO or 1 ⁇ mol/L ATV is given for 24 hours. The medium is changed to IMDM medium without fetal bovine serum and continued. After culturing for 48 hours, the cell conditioned medium was collected and extracted by gradient ultracentrifugation to obtain exosomes MSC-Exo and MSC ATV -Exo ( Figure 1A). Flow cytometry identified that the extracted primary MSC expressed a high proportion of surface markers CD29 and CD90, and a very low proportion of cells expressing CD45 and CD11b/c, indicating that the MSC extracted in the present invention had higher purity ( Figure 1B).
  • the concentration of 1 ⁇ mol/L ATV is the optimal concentration determined by the inventor of this case in previous studies. There was no obvious difference in the morphology of MSCs treated with 1 ⁇ mol/L ATV or DMSO under the microscope (Figure 1C). Transmission electron microscopy observed that MSC-derived exosomes were saucer-like or hemispherical membrane structures with one side depressed, with a diameter of about 100 nm ( Figure 1D). MSC-derived exosomes expressed exosome marker proteins such as Alix, CD63, TSG101, and CD81 ( Figure 1E).
  • Intramyocardial injection of MSC ATV -Exo effectively improves cardiac function in rats with acute myocardial infarction, and significantly promotes macrophage infiltration and macrophage M2 polarization.
  • Masson staining results showed that compared with the AMI model group, the myocardial infarction area of the MSC-Exo or MSC ATV -Exo treatment group was significantly reduced; and the myocardial infarction area of the MSC ATV -Exo treatment group was smaller than that of the MSC-Exo treatment group.
  • Figure 2C, Figure 2D The above results show that MSC ATV -Exo treatment can reduce infarct size and improve ventricular function more effectively than ordinary MSC-Exo treatment.
  • the present invention detected the expression of macrophages in the myocardium in the peri-infarct area on the third day of myocardial infarction.
  • the protein or mRNA expression levels were significantly reduced; compared with the MSC-Exo group, the MSC ATV -Exo group had higher protein or mRNA expression levels of Arg1, IL-10, and CD206, and higher protein or mRNA expression levels of iNOS, IL-12, and TNF- ⁇ . The levels are lower ( Figure 3B, Figure 3C, Figure 3D).
  • the above results show that MSC ATV -Exo significantly reduces the expression of M1 markers and promotes the expression of M2 markers in macrophages stimulated by LPS, and the effect is stronger than that of ordinary MSC-Exo.
  • the present invention uses the inhibitor (inhibitor) or mimic (mimic) of miR-139-3p to transfect MSC ATV or MSC, and then extracts the corresponding exosomes MSC ATV -Exo (inhibitor) and MSC-Exo (mimic). Exosomes act on macrophages stimulated by LPS ( Figure 5A). Western blot and qPCR results showed that compared with the LPS+MSC ATV -Exo (NC inhibitor) group, the LPS+MSC ATV -Exo (miR inhibitor) group had lower protein or mRNA expression levels of Arg1, IL-10, and CD206, and lower levels of iNOS, IL -12.
  • the present invention uses miR-139-3p mimic to directly transfect macrophages after LPS intervention (Figure 5E).
  • Western blot and qPCR results found that: compared with the M1 group, the protein or mRNA expression levels of Arg1, IL-10, and CD206 were increased in the LPS+miR-139-3p mimic group, and the protein or mRNA expression levels of iNOS, IL-12, and TNF- ⁇ were increased. levels decreased (Figure 5F, Figure 5G, Figure 5H).
  • the present invention In order to comprehensively analyze the target genes related to miR-139-3p and M2 polarization, the present invention simultaneously uses four software, namely miRWalk, TargetScan, miRDB and miRTarBase, to predict its target genes.
  • TargetScan predicted that Stat1 is a target gene of miR-139-3p. Since Stat1 can promote M1 polarization after activation, the present invention proposes Stat1 as the target gene of miR-139-3p, and proposes a scientific hypothesis: high expression of miR-139-3p will inhibit the expression and activation of Stat1, thereby inhibiting macrophages. Polarize to M1 and promote polarization to M2.
  • a luciferase reporter gene experiment was performed to verify the paired binding of miR-139-3p to the 3'UTR target fragment of the Stat1 transcript.
  • the constructed pmiR-RB-Report TM dual-luciferase plasmid vector and miR-139-3p mimic were co-transfected into 293T cells.
  • the luciferase activity detection results showed that: miR-139-3p mimic reduced Stat1 wild-type WT 3
  • the luciferase activity of the 'UTR group but had no significant effect on the luciferase activity of the mutant Mut 3'-UTR group, indicating that miR-139-3p can pair with the 3'UTR target fragment of the Stat1 transcript.
  • miR-139-3p mimic can reduce the expression level of Stat1 total protein, and using miR-139-3p inhibitor reduces Stat1 The total protein level did not decrease ( Figure 5I, Figure 5J).
  • Macrophages were stimulated with LPS and then transfected with miR-139-3p mimic or inhibitor. It was found that overexpression of miR-139-3p could significantly reduce the phosphorylation level of Stat1, promote Arg1 protein expression, and inhibit iNOS protein expression; while knockdown of miR-139-3p could significantly reduce the phosphorylation level of Stat1, promote Arg1 protein expression, and inhibit iNOS protein expression.
  • the phosphorylation level of Stat1 and the expression level of iNOS protein in macrophages stimulated by LPS were still high, while the expression level of Arg1 protein was still low (Figure 5K, Figure 5L).
  • miR-139-3p mainly promotes M2 and inhibits M1 by targeting the expression and activation of Stat1.
  • Example 2.miR-139-3p mediates the function of MSC ATV -Exo in promoting cardiac macrophage M2 polarization
  • This example verified the role of miR-139-3p in animals.
  • acute myocardial infarction model rats were given intramyocardial injection of exosomes with disturbed expression levels of miR-139-3p, namely MSC ATV -Exo (miR inhibitor) or MSC-Exo (miR mimic), to detect acute myocardial infarction.
  • the phase is the polarization of macrophages in myocardial tissue on day 3.
  • Masson staining results showed that compared with the MSC ATV -Exo group, the myocardial infarction area of the MSC ATV -Exo-miR inhibitor group was significantly increased; and the myocardial infarction area of the MSC Exo-miR mimic group was significantly higher than that of the MSC ATV Exo-miR inhibitor group. The area was significantly reduced ( Figure 6C, Figure 6F).
  • the above results indicate that miR-139-3p plays an important role in MSC ATV -Exo's ability to treat myocardial infarction and reduce infarct size.
  • ATV pretreatment causes high expression of miR-139-3p in MSC-derived exosomes.
  • MSC ATV - Exo After intramyocardial injection of MSC ATV - Exo, it inhibits more macrophages by delivering miR-139-3p to the injured myocardium. Infiltrate the myocardium and promote the polarization of infiltrated macrophages to M2, exerting the effect of inhibiting inflammation and promoting myocardial repair (Figure 7).

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Abstract

A heart-protecting factor miR-139-3p and use thereof. Provided is use of miR-139-3p in the preparation of a formulation for regulating and controlling macrophage polarization. Also provided are use of a reagent material that overexpresses miR-139-3p in exosomes in the preparation of a formulation for promoting the polarization of macrophages to type M2, use of a reagent material that inhibits and/or knocks down miR-139-3p expression in exosomes in the preparation of a formulation for inhibiting the polarization of macrophages to type M2, and use of miR-139-3p in the preparation of a formulation for regulating and controlling signal transduction and the expression of transcription activator 1. Exosomes that overexpress miR-139-3p can deliver heart-protecting miR-139-3p into macrophages, and miR-139-3p inhibits Stat1 expression and activation to promote M2 macrophage polarization.

Description

心脏保护因子miR-139-3p及其应用Cardioprotective factor miR-139-3p and its applications 技术领域Technical field
本发明是关于心脏保护因子miR-139-3p及其应用，特别是关于miR-139-3p在抑制巨噬细胞浸润心肌、促进已浸润的巨噬细胞向M2极化、发挥抑制炎症和/或促进心肌修复效应等方面的应用。The present invention relates to the cardioprotective factor miR-139-3p and its application, especially regarding the role of miR-139-3p in inhibiting macrophage infiltration into the myocardium, promoting the polarization of infiltrated macrophages to M2, inhibiting inflammation and/or Promote myocardial repair effect and other applications.
背景技术Background technique
急性心肌梗死(acute myocardial infarction,AMI)是一种致命性疾病。由于心肌细胞再生能力极弱，干细胞尤其是间充质干细胞(mesenchymal stem cell,MSC)移植有望成为修复受损心脏的替代疗法。尽管动物实验结果表明干细胞移植疗法可以有效改善AMI动物的心脏功能，但临床试验的结果却未见明显疗效和长期预后获益。这表明目前对AMI相关机制的了解并不透彻，细胞治疗策略仍需优化。Acute myocardial infarction (AMI) is a fatal disease. Due to the extremely weak regeneration capacity of cardiomyocytes, stem cell transplantation, especially mesenchymal stem cells (MSC), is expected to become an alternative therapy to repair damaged hearts. Although animal experimental results show that stem cell transplantation therapy can effectively improve the cardiac function of AMI animals, clinical trial results have not shown obvious efficacy and long-term prognostic benefits. This shows that the current understanding of AMI-related mechanisms is not thorough, and cell therapy strategies still need to be optimized.
近几年的一些观点认为，旁分泌作用是干细胞发挥心脏修复作用的重要机制，主要由干细胞衍生的外泌体(exosome)介导该过程。外泌体由亲本细胞分泌并携带大量源自亲本的mRNA、非编码RNA(例如miRNA)、蛋白质、脂质等，这些生物活性分子及其在细胞间通讯中发挥的重要作用，使得研究者们开始关注并探索使用干细胞衍生的外泌体治疗缺血性心脏病。Some views in recent years believe that paracrine action is an important mechanism for stem cells to perform cardiac repair, and that this process is mainly mediated by stem cell-derived exosomes. Exosomes are secreted by parental cells and carry a large amount of parental-derived mRNA, non-coding RNA (such as miRNA), proteins, lipids, etc. These bioactive molecules and their important role in intercellular communication have led researchers to Begin to pay attention and explore the use of stem cell-derived exosomes to treat ischemic heart disease.
另一方面，巨噬细胞功能对急性心肌梗死的影响一直吸引着研究者们进行探索。急性心肌梗死后一周内，大量来自外周血的单核细胞浸润到缺血和周边区心肌中，进而分化为巨噬细胞。这些单核细胞主要来源于骨髓和脾脏。急性心肌梗死后的前5天是炎症反应的爆发阶段，随后炎症逐渐消退。炎症反应对于心梗后心肌修复是必要的，但过度炎症会使损伤殃及正常心脏细胞，因此炎症的及时消退和控制对心肌修复非常有利。目前控制炎症的手段有：靶向单核细胞减少其聚集心肌，维持巨噬细胞吞噬功能，调节巨噬细胞平衡促进M2极化等。On the other hand, the impact of macrophage function on acute myocardial infarction has always attracted researchers to explore. Within one week after acute myocardial infarction, a large number of monocytes from peripheral blood infiltrate into the ischemic and peripheral myocardium and differentiate into macrophages. These monocytes originate mainly from bone marrow and spleen. The first 5 days after acute myocardial infarction are the outbreak stage of the inflammatory response, and then the inflammation gradually subsides. The inflammatory response is necessary for myocardial repair after myocardial infarction, but excessive inflammation can cause damage to normal heart cells. Therefore, timely resolution and control of inflammation is very beneficial to myocardial repair. Current methods to control inflammation include: targeting monocytes to reduce their accumulation in the myocardium, maintaining the phagocytic function of macrophages, regulating macrophage balance and promoting M2 polarization, etc.
巨噬细胞在受到刺激因素或外界环境的影响后，会分化为两个主要亚型：M1和M2，这个过程被称之为极化。在体外实验中，常用白细胞介素4(interleukin 4,IL-4)、IL-10、IL-13、或转化生长因子β(transforming growth factorβ,TGF-β)等刺激巨噬细胞诱导M2极化，伴随信号转导与转录激活因子6(signal transducer and activator of transcription1,Stat6)的激活；用脂多糖(lipopolysaccharide,LPS)或干扰素γ(interferonγ,IFN-γ) 刺激巨噬细胞诱导M1极化，伴随Stat1的激活(Khan J N,Mccann G P.Cardiovascular magnetic resonance imaging assessment of outcomes in acute myocardial infarction[J].World J Cardiol,2017,9(2):109-133；Makkar R R,Smith R R,Cheng K,et al.Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction(CADUCEUS):A prospective,randomized phase 1trial[J].The Lancet,2012,379(9819):895-904)。急性心肌梗死后，心肌微环境中既存在促M1因子(如IFN-γ)也存在促M2因子(如IL-10,TGF-β)，导致心肌巨噬细胞是M1和M2共存的异质混合体。在心梗后的第1-3天，心肌中主要是M1型巨噬细胞，第5-7天则以M2型巨噬细胞为主(Majka M,
Figure PCTCN2022108991-appb-000001
M,Badyra B,et al.Concise Review:Mesenchymal Stem Cells in Cardiovascular Regeneration:Emerging Research Directions and Clinical Applications[J].Stem Cells Translational Medicine,2017,6(10):1859-1867)。M1型巨噬细胞分泌细胞因子、趋化因子、生长因子和基质金属蛋白酶(matrix metalloproteinase,MMP)，如TNFα、IL-1β、IL-6、IL-12、iNOS等，具有促进炎症、清除细胞碎片、降解细胞外基质的作用，但M1巨噬细胞的持续存在会导致梗死扩大、妨碍炎症消退和疤痕形成。M2型巨噬细胞生成抗炎、促血管生成、促修复的因子，如CD206、Arg1、IL-10、血管内皮生长因子、TGF-β等，具有吞噬凋亡细胞，促进血管新生和疤痕修复的作用(Koudstaal S,Jansen of Lorkeers S J,Gaetani R,et al.Concise review:heart regeneration and the role of cardiac stem cells[J].Stem Cells Transl Med,2013,2(6):434-43)。因此，通过调节巨噬细胞平衡使其从M1向M2极化可有效改善梗死后心肌修复、提高心功能。 After being affected by stimulating factors or external environment, macrophages will differentiate into two main subtypes: M1 and M2. This process is called polarization. In in vitro experiments, interleukin 4 (IL-4), IL-10, IL-13, or transforming growth factor β (TGF-β) are commonly used to stimulate macrophages to induce M2 polarization. , accompanied by the activation of signal transducer and activator of transcription1 (Stat6); stimulating macrophages with lipopolysaccharide (LPS) or interferon γ (IFN-γ) to induce M1 polarization , accompanied by the activation of Stat1 (Khan J N,Mccann G P. Cardiovascular magnetic resonance imaging assessment of outcomes in acute myocardial infarction[J]. World J Cardiol, 2017, 9(2):109-133; Makkar R R, Smith R R, Cheng K, et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): A prospective, randomized phase 1trial[J]. The Lancet, 2012, 379(9819):895-904). After acute myocardial infarction, both pro-M1 factors (such as IFN-γ) and pro-M2 factors (such as IL-10, TGF-β) exist in the myocardial microenvironment, resulting in myocardial macrophages being a heterogeneous mixture of M1 and M2. body. On days 1-3 after myocardial infarction, the myocardium is dominated by M1 macrophages, and on days 5-7, M2 macrophages are dominant (Majka M,
Figure PCTCN2022108991-appb-000001
M, Badyra B, et al. Concise Review: Mesenchymal Stem Cells in Cardiovascular Regeneration: Emerging Research Directions and Clinical Applications[J]. Stem Cells Translational Medicine, 2017, 6(10):1859-1867). M1 macrophages secrete cytokines, chemokines, growth factors and matrix metalloproteinases (MMPs), such as TNFα, IL-1β, IL-6, IL-12, iNOS, etc., which can promote inflammation and eliminate cells. fragmentation and degradation of the extracellular matrix, but the persistence of M1 macrophages can lead to infarct expansion, hinder inflammation resolution, and scar formation. M2 macrophages produce anti-inflammatory, pro-angiogenic, and pro-repair factors, such as CD206, Arg1, IL-10, vascular endothelial growth factor, TGF-β, etc., and have the ability to phagocytose apoptotic cells and promote angiogenesis and scar repair. Effect (Koudstaal S, Jansen of Lorkeers S J, Gaetani R, et al. Concise review: heart regeneration and the role of cardiac stem cells [J]. Stem Cells Transl Med, 2013, 2(6): 434-43). Therefore, regulating the balance of macrophages to polarize them from M1 to M2 can effectively improve post-infarction myocardial repair and improve cardiac function.
发明内容Contents of the invention
本发明的一个目的在于提供一种可作为心脏保护因子的miRNA。One object of the present invention is to provide a miRNA that can serve as a cardioprotective factor.
本发明的另一目的在于提供所述miRNA的相关应用。Another object of the present invention is to provide related applications of the miRNA.
本案发明人在研究中发现，miR-139-3p是一种与巨噬细胞极化相关的心脏保护因子。与高表达miR-139-3p的外泌体(MSC ATV-Exo)相比，抑制外泌体中miR-139-3p的高表达后的外泌体MSC ATV-Exo(miR inhibitor)使巨噬细胞M2型标志物表达水平降低。过表达miR-139-3p的外泌体MSC-Exo(miR mimic)可增加巨噬细胞中M2型标志物的表达水平。发明人的研究还发现，信号转导与转录激活因子1(signal transducer and activator of transcription 1,Stat1)是miR-139-3p的一个靶基因。双荧光素酶报告基因实验表明miR-139-3p能与Stat1的3’UTR区特异结合后抑制其表达。miR-139-3p mimic或inhibitor降低或增加巨噬细胞中总Stat1表达水平；miR-139-3p mimic降低LPS刺激后的巨噬细 胞中Stat1磷酸化水平。心梗大鼠心肌内注射MSC ATV-Exo(miR inhibitor)后，心肌组织M2型标志物表达水平较MSC ATV-Exo治疗组降低。心肌内注射MSC-Exo(miR mimic)后，心肌组织中M2型标志物表达水平与MSC ATV-Exo治疗组相当。 The inventor of this case discovered in his research that miR-139-3p is a cardioprotective factor related to macrophage polarization. Compared with exosomes (MSC ATV -Exo) that highly express miR-139-3p, exosomes MSC ATV -Exo (miR inhibitor) that inhibits the high expression of miR-139-3p in exosomes makes macrophages The expression levels of cell M2 type markers were reduced. Exosomal MSC-Exo (miR mimic) overexpressing miR-139-3p can increase the expression levels of M2 markers in macrophages. The inventor's research also found that signal transducer and activator of transcription 1 (Stat1) is a target gene of miR-139-3p. Dual-luciferase reporter gene experiments showed that miR-139-3p can specifically bind to the 3'UTR region of Stat1 and inhibit its expression. miR-139-3p mimic or inhibitor reduces or increases the total Stat1 expression level in macrophages; miR-139-3p mimic reduces the phosphorylation level of Stat1 in macrophages stimulated by LPS. After intramyocardial injection of MSC ATV -Exo (miR inhibitor) in rats with myocardial infarction, the expression levels of M2 markers in myocardial tissue were lower than those in the MSC ATV -Exo treatment group. After intramyocardial injection of MSC-Exo (miR mimic), the expression levels of M2-type markers in myocardial tissue were comparable to those in the MSC ATV -Exo treatment group.
从而，本发明提供了miR-139-3p作为心脏保护因子的相关应用。Therefore, the present invention provides relevant applications of miR-139-3p as a cardioprotective factor.
具体而言，一方面，本发明提供了miR-139-3p在制备调控巨噬细胞极化的制剂中的应用。Specifically, in one aspect, the present invention provides the use of miR-139-3p in preparing a preparation for regulating macrophage polarization.
根据本发明的具体实施方案，本发明提供了在外泌体中过表达miR-139-3p的试剂材料在制备用于促进巨噬细胞向M2型极化的制剂中的应用。According to a specific embodiment of the present invention, the present invention provides the use of a reagent material that overexpresses miR-139-3p in exosomes in the preparation of a preparation for promoting the polarization of macrophages toward the M2 type.
根据本发明的具体实施方案，其中，用于促进巨噬细胞向M2型极化的制剂包括过表达miR-139-3p的外泌体。优选地，所述外泌体为骨髓间充质干细胞(mesenchymal stem cell,MSC)衍生的外泌体。According to a specific embodiment of the present invention, the preparation for promoting the polarization of macrophages toward the M2 type includes exosomes overexpressing miR-139-3p. Preferably, the exosomes are exosomes derived from bone marrow mesenchymal stem cells (mesenchymal stem cells, MSC).
根据本发明的具体实施方案，其中，在外泌体中过表达miR-139-3p的试剂材料包括：通过基因技术在外泌体中过表达miR-139-3p的试剂材料，和/或通过药物预处理干细胞而使得干细胞分泌高表达miR-139-3p的外泌体的试剂材料。优选地，所述药物预处理干细胞的药物例如可为他汀类药物，例如阿托伐他汀(atorvastatin,ATV)。According to specific embodiments of the present invention, the reagent materials for overexpressing miR-139-3p in exosomes include: reagent materials for overexpressing miR-139-3p in exosomes through genetic technology, and/or through drug pretreatment. A reagent material that treats stem cells to cause them to secrete exosomes that highly express miR-139-3p. Preferably, the drug used to pretreat stem cells may be, for example, a statin drug, such as atorvastatin (ATV).
根据本发明的具体实施方案，本发明提供了抑制和/或敲低外泌体中miR-139-3p的表达的试剂材料在制备用于抑制巨噬细胞向M2型极化的制剂中的应用。According to specific embodiments of the present invention, the present invention provides the use of reagent materials that inhibit and/or knock down the expression of miR-139-3p in exosomes in the preparation of preparations for inhibiting the polarization of macrophages toward the M2 type. .
根据本发明的具体实施方案，其中，抑制和/或敲低外泌体中miR-139-3p的表达的试剂材料包括：miR-139-3p抑制剂、通过基因技术敲低外泌体中miR-139-3p的表达的试剂材料中的一种或多种。According to specific embodiments of the present invention, reagent materials for inhibiting and/or knocking down the expression of miR-139-3p in exosomes include: miR-139-3p inhibitors, knockdown of miR-139-3p in exosomes through genetic technology - One or more reagent materials for the expression of -139-3p.
根据本发明的具体实施方案，本发明提供了miR-139-3p作为巨噬细胞向M2型极化的调控因子的相关应用。According to specific embodiments of the present invention, the present invention provides related applications of miR-139-3p as a regulatory factor for macrophage polarization toward the M2 type.
根据本发明的具体实施方案，其中，促进巨噬细胞向M2型极化的试剂材料包括：miR-139-3p模拟物、通过基因技术提高巨噬细胞中miR-139-3p的表达的试剂材料中的一种或多种。According to specific embodiments of the present invention, the reagent materials that promote the polarization of macrophages toward the M2 type include: miR-139-3p mimics, reagent materials that increase the expression of miR-139-3p in macrophages through genetic technology. one or more of them.
另一方面，本发明还提供了miR-139-3p在制备调控信号转导与转录激活因子1(signal transducer and activator of transcription 1,Stat1)的表达的制剂中的应用。On the other hand, the present invention also provides the application of miR-139-3p in the preparation of preparations for regulating the expression of signal transducer and activator of transcription 1 (Stat1).
根据本发明的具体实施方案，本发明提供了过表达miR-139-3p的试剂材料在制备降低巨噬细胞中总Stat1表达水平的制剂中的应用。According to a specific embodiment of the present invention, the present invention provides the use of reagent materials that overexpress miR-139-3p in the preparation of preparations that reduce the total Stat1 expression level in macrophages.
根据本发明的具体实施方案，本发明提供了抑制和/或敲低外泌体中miR-139-3p的 表达的试剂材料在制备用于增加巨噬细胞中总Stat1表达水平的制剂中的应用。According to a specific embodiment of the invention, the invention provides the use of reagent materials that inhibit and/or knock down the expression of miR-139-3p in exosomes in the preparation of a preparation for increasing the total Stat1 expression level in macrophages. .
根据本发明的具体实施方案，本发明提供了过表达miR-139-3p的试剂材料在制备用于增加巨噬细胞中总Stat1表达水平的制剂中的应用。According to a specific embodiment of the present invention, the present invention provides the use of a reagent material that overexpresses miR-139-3p in the preparation of a preparation for increasing the total Stat1 expression level in macrophages.
另一方面，本发明还提供了一种提高心肌组织M2型标志物表达水平的方法，该方法包括：向需要的受试者给予过表达miR-139-3p的外泌体。On the other hand, the present invention also provides a method for increasing the expression level of M2 markers in myocardial tissue, which method includes: administering exosomes overexpressing miR-139-3p to a subject in need.
另一方面，本发明还提供了一种治疗急性心肌梗死的方法，该方法包括：向需要的受试者给予过表达miR-139-3p的外泌体。通过过表达miR-139-3p的外泌体，可实现以下至少一种效果：减少心梗急性期心肌中巨噬细胞浸润、控制炎症反应，并促进浸润的巨噬细胞从M1型向M2型转化，促进心肌修复，减小心梗面积，改善心功能；和/或传递心脏保护性miR-139-3p到巨噬细胞中，通过抑制Stat1的表达与激活促进M2巨噬细胞极化。On the other hand, the present invention also provides a method for treating acute myocardial infarction, which method includes: administering exosomes overexpressing miR-139-3p to a subject in need. By overexpressing exosomes of miR-139-3p, at least one of the following effects can be achieved: reducing macrophage infiltration in the myocardium in the acute phase of myocardial infarction, controlling the inflammatory response, and promoting the transformation of infiltrating macrophages from M1 type to M2 type. Transform, promote myocardial repair, reduce myocardial infarction size, and improve cardiac function; and/or deliver cardioprotective miR-139-3p to macrophages to promote M2 macrophage polarization by inhibiting the expression and activation of Stat1.
本发明的具体实验表明，过表达miR-139-3p的外泌体治疗能更加有效地减少心梗急性期心肌中巨噬细胞浸润、控制炎症反应，并促进浸润的巨噬细胞从M1型向M2型转化，促进心肌修复，减小心梗面积，改善心功能。过表达miR-139-3p的外泌体MSC ATV-Exo可传递心脏保护性miR-139-3p到巨噬细胞中，后者通过抑制Stat1的表达与激活促进M2巨噬细胞极化。 Specific experiments of the present invention show that exosome treatment overexpressing miR-139-3p can more effectively reduce macrophage infiltration in the myocardium in the acute phase of myocardial infarction, control inflammatory responses, and promote the transformation of infiltrated macrophages from M1 type to M1 type. M2 type transformation promotes myocardial repair, reduces myocardial infarction size, and improves cardiac function. Exosomal MSC ATV -Exo overexpressing miR-139-3p can deliver cardioprotective miR-139-3p to macrophages, which promotes M2 macrophage polarization by inhibiting the expression and activation of Stat1.
附图说明Description of drawings
图1A至图1G显示大鼠骨髓间充质干细胞衍生的外泌体提取与鉴定。其中，图1A：经ATV预处理或未经ATV预处理的大鼠骨髓间充质干细胞外体分离的示意方案。图1B：流式细胞术分析大鼠骨髓间充质干细胞表面标志物。图1C：显微镜下观察经二甲基亚砜或ATV处理的骨髓间充质干细胞的形态。图1D：透射电子显微镜扫描外小体的形态。图1E：外泌体蛋白质标记的Western blot分析。图1F：用不同浓度(2μg/mL、20μg/mL、40μg/mL)的MSC-Exo或MSC ATV-Exo处理24小时后，对M2巨噬细胞标记物Arg1的蛋白表达进行Western blot分析。图1G：用LPS刺激大鼠骨髓源巨噬细胞(BMDM)6小时，然后与40μg/mL MSC-Exo或MSC ATV-Exo再孵育48小时，Western blot分析Arg1在BMDM中的蛋白表达。 Figure 1A to Figure 1G show the extraction and identification of exosomes derived from rat bone marrow mesenchymal stem cells. Among them, Figure 1A: Schematic scheme for the ex vivo isolation of rat bone marrow mesenchymal stem cells with or without ATV pretreatment. Figure 1B: Flow cytometric analysis of rat bone marrow mesenchymal stem cell surface markers. Figure 1C: Observation of the morphology of bone marrow mesenchymal stem cells treated with dimethyl sulfoxide or ATV under a microscope. Figure 1D: Transmission electron microscopy scanning of exosome morphology. Figure 1E: Western blot analysis of exosome protein markers. Figure 1F: Western blot analysis of the protein expression of M2 macrophage marker Arg1 after treatment with MSC-Exo or MSC ATV -Exo at different concentrations (2μg/mL, 20μg/mL, 40μg/mL) for 24 hours. Figure 1G: Rat bone marrow-derived macrophages (BMDM) were stimulated with LPS for 6 hours, and then incubated with 40 μg/mL MSC-Exo or MSC ATV -Exo for another 48 hours. Western blot analysis of Arg1 protein expression in BMDM.
图2A至图2I显示MSC ATV-Exo对急性心肌梗死大鼠心室功能、心肌梗死大小、巨噬细胞浸润和M2极化的优越作用。其中，图2A：各组在AMI后第28天的代表性M型超声心动图。图2B：各组在AMI后第0、3、7、28天LVEF、LVFS、LVESV和LVEDV的动态变化，n＝6-7/组。图2C：第28天Masson三色染色的心脏横截面代表。图2D： 图2C中各组梗死面积的量化，n＝6-7/组。图2E：急性心肌梗死后第3天梗死边缘区内CD68+或CD206+巨噬细胞的典型共焦图像。图2F：图2E中CD68+或CD206+巨噬细胞的定量，n＝6-7/组。图2G：各组梗死心肌中iNOS和Arg1表达的Western blot分析。图2H：图2G中iNOS和Arg1表达的定量，每组n＝3。图2I：通过qPCR测量梗死区M1(iNOS，IL-12，TNF-α)和M2(Arg1，IL-10，CD206)巨噬细胞标志物的mRNA表达，每组n＝3-4。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。 Figure 2A to Figure 2I show the superior effects of MSC ATV -Exo on ventricular function, myocardial infarction size, macrophage infiltration and M2 polarization in rats with acute myocardial infarction. Among them, Figure 2A: Representative M-mode echocardiograms of each group on day 28 after AMI. Figure 2B: Dynamic changes in LVEF, LVFS, LVESV and LVEDV in each group on days 0, 3, 7, and 28 after AMI, n=6-7/group. Figure 2C: Representative cross-section of the heart stained with Masson's trichrome on day 28. Figure 2D: Quantification of infarct size in each group in Figure 2C, n=6-7/group. Figure 2E: Typical confocal images of CD68+ or CD206+ macrophages in the infarct margin zone on day 3 after acute myocardial infarction. Figure 2F: Quantification of CD68+ or CD206+ macrophages in Figure 2E, n=6-7/group. Figure 2G: Western blot analysis of iNOS and Arg1 expression in infarcted myocardium in each group. Figure 2H: Quantification of iNOS and Arg1 expression in Figure 2G, n=3 per group. Figure 2I: Measurement of mRNA expression of macrophage markers in M1 (iNOS, IL-12, TNF-α) and M2 (Arg1, IL-10, CD206) in the infarct area by qPCR, n=3-4 in each group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
图3A至图3D显示MSC ATV-Exo和MSC-Exo对体外巨噬细胞极化的影响。其中，图3A：PKH-67标记的MSC ATV-Exo或MSC-Exo(绿色)通过大鼠BMDM摄取(红色)。图3B：免疫印迹分析BMDM中iNOS和Arg1的蛋白表达。图3C：图3B中iNOS和Arg1表达的定量，每组n＝3。图3D：定量PCR分析图3B中所述BMDM中巨噬细胞标记物(M1:iNOS、IL-12、TNF-α；M2:Arg1、IL-10、CD206)的mRNA表达，每组n＝4。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。 Figures 3A to 3D show the effects of MSC ATV -Exo and MSC-Exo on macrophage polarization in vitro. Among them, Figure 3A: PKH-67 labeled MSC ATV -Exo or MSC-Exo (green) is taken up by rat BMDM (red). Figure 3B: Western blot analysis of iNOS and Arg1 protein expression in BMDM. Figure 3C: Quantification of iNOS and Arg1 expression in Figure 3B, n=3 per group. Figure 3D: Quantitative PCR analysis of the mRNA expression of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in BMDM as described in Figure 3B, n=4 in each group . *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
图4A至图4B显示MSC ATV-Exo和MSC-Exo差异表达的miRNA。其中，图4A：火山图显示MSC ATV-Exo和MSC-Exo所有差异表达的miRNA。图4B：qRT-PCR验证miRNA在MSC ATV-Exo和MSC-Exo中的表达情况。 Figure 4A to Figure 4B show differentially expressed miRNAs between MSC ATV -Exo and MSC-Exo. Among them, Figure 4A: Volcano plot shows all differentially expressed miRNAs between MSC ATV -Exo and MSC-Exo. Figure 4B: qRT-PCR verified the expression of miRNA in MSC ATV -Exo and MSC-Exo.
图5A至图5L显示miR-139-3p是体外靶向Stat1的MSC ATV-Exo介导的巨噬细胞极化效应器。其中，图5A：分离MSC ATV-Exo-miR inhibitor和MSC-Exo-miR mimics并孵育LPS预处理的大鼠BMDM的方案示意图。图5B：BMDM中巨噬细胞M1标记物iNOS和M2标记物Arg1的蛋白质表达。图5C：图5B中iNOS和Arg1表达的定量，n＝3/组。图5D：定量PCR分析图5A中所述BMDM中巨噬细胞标记物(M1:iNOS、IL-12、TNF-α；M2:Arg1、IL-10、CD206)的mRNA表达，每组n＝3-4。图5E：miR-139-3p模拟物直接转染LPS预处理的BMDM的方案示意图。图5F：BMDM中巨噬细胞M1标记物iNOS和M2标记物Arg1的蛋白表达。图5G：如图5F中iNOS和Arg1表达的定量，n＝3/组。图5H：定量PCR分析图5E中所述BMDM中巨噬细胞标记物(M1:iNOS、IL-12、TNF-α；M2:Arg1、IL-10、CD206)的mRNA表达，每组n＝3-4。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。图5I、图5J：转染miR-139-3p mimic或inhibitor的大鼠BMDM中Stat1的蛋白表达，每组n＝3。图5K、图5L：用LPS刺激并用miR-139-3p mimic或inhibitor转染的BMDM中iNOS、Arg1和p-Stat1的蛋白表达，每组n＝3。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。 Figures 5A to 5L show that miR-139-3p is an effector of MSC ATV -Exo-mediated macrophage polarization targeting Stat1 in vitro. Among them, Figure 5A: Schematic diagram of the protocol for isolating MSC ATV -Exo-miR inhibitor and MSC-Exo-miR mimics and incubating LPS-pretreated rat BMDM. Figure 5B: Protein expression of macrophage M1 marker iNOS and M2 marker Arg1 in BMDM. Figure 5C: Quantification of iNOS and Arg1 expression in Figure 5B, n=3/group. Figure 5D: Quantitative PCR analysis of the mRNA expression of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in BMDM described in Figure 5A, n=3 for each group -4. Figure 5E: Schematic diagram of the protocol for direct transfection of miR-139-3p mimics into LPS-pretreated BMDM. Figure 5F: Protein expression of macrophage M1 marker iNOS and M2 marker Arg1 in BMDM. Figure 5G: Quantification of iNOS and Arg1 expression as in Figure 5F, n=3/group. Figure 5H: Quantitative PCR analysis of the mRNA expression of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in BMDM as described in Figure 5E, n=3 for each group -4. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 5I, Figure 5J: Stat1 protein expression in rat BMDM transfected with miR-139-3p mimic or inhibitor, n=3 for each group. Figure 5K, Figure 5L: Protein expression of iNOS, Arg1 and p-Stat1 in BMDM stimulated with LPS and transfected with miR-139-3p mimic or inhibitor, n=3 in each group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
图6A至图6I显示MSC ATV-Exo通过转移miR-139-3p增强AMI大鼠梗死区M2巨 噬细胞极化。其中，图6A：AMI后第3天梗死区内CD68 +(M1)和CD206 +(M2)巨噬细胞的代表性共焦图像。图6B：图6A中CD68 +或CD206 +巨噬细胞的定量，n＝6-7/组。图6C：各组在AMI后第28天的代表性M型超声心动图。图6D：根据超声心动图图像比较各组左室射血分数和左室射血分数，每组n＝6-7*P<0.05，**P<0.01，***P<0.001。图6E：第28天用Masson三色染色法对AMI心脏的代表性横截面进行观察。图6F：各组梗死面积的量化E，n＝6-7/组。图6G：Western blot分析各组梗死心肌中iNOS、Arg1和p-Stat1的表达。图6H：iNOS、Arg1和p-Stat1表达的量化(G)，每组n＝3。图6I：通过qPCR测量梗死区巨噬细胞M1(iNOS、IL-12、TNF-α)和M2(Arg1、IL-10、CD206)标记物的mRNA表达，每组n＝3-4。*P<0.05，**P<0.01，***P<0.001，****P<0.0001。 Figures 6A to 6I show that MSC ATV -Exo enhances M2 macrophage polarization in the infarct area of AMI rats by transferring miR-139-3p. Among them, Figure 6A: Representative confocal images of CD68 + (M1) and CD206 + (M2) macrophages in the infarct area on day 3 after AMI. Figure 6B: Quantification of CD68 + or CD206 + macrophages in Figure 6A, n=6-7/group. Figure 6C: Representative M-mode echocardiograms of each group on day 28 after AMI. Figure 6D: Comparison of left ventricular ejection fraction and left ventricular ejection fraction in each group based on echocardiographic images, n=6-7*P<0.05, **P<0.01, ***P<0.001 for each group. Figure 6E: Representative cross-section of an AMI heart on day 28 using Masson's trichrome staining. Figure 6F: Quantification of infarct size in each group E, n=6-7/group. Figure 6G: Western blot analysis of the expression of iNOS, Arg1 and p-Stat1 in infarcted myocardium in each group. Figure 6H: Quantification of iNOS, Arg1 and p-Stat1 expression (G), n=3 per group. Figure 6I: Measurement of mRNA expression of M1 (iNOS, IL-12, TNF-α) and M2 (Arg1, IL-10, CD206) markers in macrophages in the infarct area by qPCR, n=3-4 in each group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
图7显示本发明主要发现的示意性总结。在梗死边缘区心肌内注射大鼠骨髓MSC ATV-Exo将上调的miR-139-3p输送到浸润的巨噬细胞，并通过抑制Stat1表达和激活促进M2巨噬细胞极化，促进梗死后的心脏修复。 Figure 7 shows a schematic summary of the main findings of the present invention. Intramyocardial injection of rat bone marrow MSC ATV- Exo in the infarct border zone delivers upregulated miR-139-3p to infiltrating macrophages and promotes M2 macrophage polarization by inhibiting Stat1 expression and activation, promoting post-infarction heart repair.
具体实施方式Detailed ways
下面通过具体实施例进一步详细说明本发明的特点及所具有的技术效果，但本发明并不因此而受到任何限制。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。The characteristics and technical effects of the present invention will be further described in detail through specific embodiments below, but the present invention is not subject to any limitation thereby. In the examples, each original reagent material is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
各实施例中，通用的实验材料和方法如下：In each embodiment, the common experimental materials and methods are as follows:
1.原代骨髓来源巨噬细胞的分离和培养1. Isolation and culture of primary bone marrow-derived macrophages
(1)选用雄性、体重60-80g的SD大鼠，颈椎脱臼法处死后置于75％酒精中浸泡3分钟左右；(1) Select male SD rats weighing 60-80g, sacrifice them by cervical dislocation and soak them in 75% alcohol for about 3 minutes;
(2)在生物安全柜中，沿大鼠后肢外侧剪开皮肤，钝性分离皮肤和肌肉，取下股骨和胫骨，浸于冷PBS中；(2) In a biological safety cabinet, cut the skin along the outside of the rat's hind limbs, bluntly separate the skin and muscles, remove the femur and tibia, and immerse them in cold PBS;
(3)进一步剔除附着在骨骼上的肌肉、软骨等；(3) Further remove the muscles, cartilage, etc. attached to the bones;
(4)剪去骨骼两端少量骨骺，方便冲出骨髓腔中骨髓细胞；用2毫升注射器针头吸取IMDM完全培养基，插入骨骼两端，反复冲洗，收集冲出的骨髓细胞；(4) Cut off a small amount of epiphysis at both ends of the bone to facilitate flushing out the bone marrow cells in the bone marrow cavity; use a 2 ml syringe needle to absorb IMDM complete culture medium, insert it into both ends of the bone, rinse repeatedly, and collect the flushed out bone marrow cells;
(5)配制巨噬细胞培养液：高糖DMEM培养基+10％胎牛血清+1％双抗+20ng/ml CSF；(5) Prepare macrophage culture medium: high sugar DMEM medium + 10% fetal calf serum + 1% double antibody + 20ng/ml CSF;
(6)冲出的骨髓细胞液用70微米无菌滤网过滤掉组织残渣，500g离心5分钟，弃上清，用巨噬细胞培养基重悬细胞；(6) Use a 70-micron sterile filter to filter out the tissue residue from the washed bone marrow cell fluid, centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend the cells in macrophage culture medium;
(7)将细胞悬液接种于10厘米培养皿中，1只大鼠的2条后肢提取的骨髓细胞 可种4个10厘米皿；或者直接接种在12孔培养板中进行培养，方便后续实验；(7) Inoculate the cell suspension into a 10 cm culture dish. The bone marrow cells extracted from the two hind limbs of one rat can be seeded into four 10 cm dishes; or they can be directly inoculated into a 12-well culture plate for culture to facilitate subsequent experiments. ;
(8)放于37℃、5％CO 2细胞培养箱中培养； (8) Cultivate in a 37°C, 5% CO 2 cell culture incubator;
(9)每2天更换培养基，观察细胞分化情况；(9) Change the culture medium every 2 days and observe the cell differentiation;
(10)6-7天后可见已分化好的巨噬细胞，即可用于后续实验。(10) Differentiated macrophages can be seen after 6-7 days and can be used for subsequent experiments.
2.大鼠巨噬细胞极化实验2. Rat macrophage polarization experiment
(1)实验中设置了M1型、M2型巨噬细胞组作为对照：(1) In the experiment, M1 type and M2 type macrophage groups were set up as controls:
M1组：向巨噬细胞中加入终浓度为100ng/mL的LPS；Group M1: LPS with a final concentration of 100ng/mL was added to macrophages;
M2组：向巨噬细胞中加入终浓度为10ng/ml IL-4和10ng/ml IL-13；M2 group: Add final concentrations of 10ng/ml IL-4 and 10ng/ml IL-13 to macrophages;
(2)后续体外实验中，各种类型外泌体的浓度均为40μg/mL。(2) In subsequent in vitro experiments, the concentrations of various types of exosomes were 40 μg/mL.
3.外泌体中miRNA的提取3. Extraction of miRNA from exosomes
由于外泌体中暂无合适的内参，故在提取外泌体miRNA时要引入外参，本发明中使用的是化学合成RNA单链、线虫来源的cel-miR-39-3p(锐博，广州)作为外参。具体步骤如下：Since there is currently no suitable internal reference in exosomes, an external reference must be introduced when extracting exosome miRNA. In the present invention, chemically synthesized RNA single-stranded cel-miR-39-3p derived from nematodes (Ribo, Guangzhou) as an external reference. Specific steps are as follows:
(1)根据不同组别外泌体蛋白浓度，取同等蛋白量的外泌体，分别加入500微升TRIZOL，混匀静置10分钟后使其完全裂解，彻底混匀；(1) According to the protein concentration of exosomes in different groups, take exosomes with the same protein amount, add 500 microliters of TRIZOL respectively, mix and let stand for 10 minutes to completely lyse, and mix thoroughly;
(2)加入200nM、55微升的外参，彻底混匀，静置5-10分钟；(2) Add 200nM, 55 microliters of external ginseng, mix thoroughly, and let stand for 5-10 minutes;
(3)加入100微升氯仿，上下颠倒混匀数次后，静置于冰上5分钟待其分层；(3) Add 100 microliters of chloroform, mix it by inverting it several times, and then place it on ice for 5 minutes until it stratifies;
(4)以4℃、12000g离心15分钟；(4) Centrifuge at 4°C and 12000g for 15 minutes;
(5)小心吸取最上层的透明液体(含RNA)，加入等体积的异丙醇，颠倒混匀后，静置沉淀30分钟；(5) Carefully absorb the uppermost transparent liquid (containing RNA), add an equal volume of isopropyl alcohol, mix by inverting, and let stand for 30 minutes;
(6)以4℃、12000g离心15分钟；(6) Centrifuge at 4°C and 12000g for 15 minutes;
(7)弃上清，加入500微升75％乙醇(无水乙醇和双蒸水配制)，颠倒混匀重悬沉淀；(7) Discard the supernatant, add 500 microliters of 75% ethanol (prepared with absolute ethanol and double distilled water), mix by inverting and resuspend the pellet;
(8)以4℃、8000g离心5分钟；(8) Centrifuge at 4°C and 8000g for 5 minutes;
(9)弃上清，加入500微升75％乙醇，颠倒混匀重悬沉淀；(9) Discard the supernatant, add 500 μl of 75% ethanol, mix by inverting and resuspend the pellet;
(10)以4℃、8000g离心5分钟；(10) Centrifuge at 4°C and 8000g for 5 minutes;
(11)弃上清，室温下自然风干EP中多余的液体；(11) Discard the supernatant and let the excess liquid in the EP air dry naturally at room temperature;
(12)加入10-25微升DEPC水(可根据沉淀量调整)溶解沉淀，可在56℃水浴锅中促溶15分钟；(12) Add 10-25 microliters of DEPC water (can be adjusted according to the amount of precipitation) to dissolve the precipitate, and promote dissolution in a 56°C water bath for 15 minutes;
(13)取1-2微升样本用Nanodrop测定总RNA浓度。(13) Take 1-2 microliters of sample and use Nanodrop to determine the total RNA concentration.
4.外泌体小RNA(small RNA)测序4. Exosome small RNA (small RNA) sequencing
(1)外泌体RNA提取及质检采用梯度超速离心法提取大鼠骨髓间充质干细胞来源的外泌体MSC-Exo和MSC ATV-Exo后，考虑到外泌体中RNA含量较低，对于外泌体中small RNA表达谱检测，采用美基生物的HiPure Liquid RNA Mini Kit提取外泌体RNA，具体步骤见商品说明书。采用Qubit或Agilent 2200对样品质检，质检内容包括外泌体RNA的浓度、总量及RNA片段长度分布峰图。其中，若Agilent 2200峰图有>500个核苷酸的大片段峰，代表外泌体样本可能存在核糖体或支原体污染。 (1) Exosome RNA extraction and quality inspection After using gradient ultracentrifugation to extract exosomes MSC-Exo and MSC ATV -Exo derived from rat bone marrow mesenchymal stem cells, considering the low RNA content in exosomes, For the detection of small RNA expression profile in exosomes, the HiPure Liquid RNA Mini Kit of Meiji Biotech was used to extract exosomal RNA. Please refer to the product instructions for specific steps. Use Qubit or Agilent 2200 to perform quality inspection on the samples. The quality inspection content includes the concentration, total amount and RNA fragment length distribution peak diagram of exosomal RNA. Among them, if the Agilent 2200 peak chart has a large fragment peak of >500 nucleotides, it means that the exosome sample may be contaminated by ribosomes or mycoplasma.
(2)文库制备与质检提取到的外泌体RNA，先后连接3’端和5’的接头，逆转录成cDNA，再进行PCR扩增，切胶回收目的片段文库，质检合格的文库上机测序。(2) Library preparation and quality inspection. The extracted exosome RNA is connected to the 3' end and 5' adapters, reverse transcribed into cDNA, and then PCR amplified. The target fragment library is recovered by gel cutting, and the library passes the quality inspection. On-machine sequencing.
(3)上机测序使用Illumina HiSeqTM 2500进行上机测序。(3) On-machine sequencing Use Illumina HiSeqTM 2500 for on-machine sequencing.
(4)信息分析Illumina HiSeqTM 2500测序所得的原始数据，首先进行过滤：去掉数据序列两端的接头，去除片段长度<17个核苷酸的序列和低质量的序列等，完成数据的初步过滤并获得高质量数据。将高质量数据与大鼠参考基因组比对获得全基因组序列分布图谱，并与非编码RNA数据库(miRBase，Rfam和piRNABank)进行比对及分类注释。对鉴定出来的miRNA进行表达量计算、miRNA表达聚类和样品间差异表达miRNA分析，以差异倍数|log2(FoldChange)|>1且显著性水平P值<0.05为标准。对于差异显著的miRNA，采用miRWalk、TargetScan、miRDB和miRTarBase四个软件进一步预测miRNA的靶基因，并对靶基因进行基因功能(gene ontology，GO)和KEGG生物通路富集分析。(4) Information analysis of the raw data obtained by Illumina HiSeqTM 2500 sequencing, first filtering: remove the adapters at both ends of the data sequence, remove sequences with fragment length <17 nucleotides and low-quality sequences, etc., complete the preliminary filtering of the data and obtain High quality data. The high-quality data were compared with the rat reference genome to obtain the whole-genome sequence distribution map, and compared with non-coding RNA databases (miRBase, Rfam and piRNABank) for classification and annotation. Calculate the expression of the identified miRNAs, perform clustering of miRNA expression, and analyze differentially expressed miRNAs between samples, with the difference fold |log2(FoldChange)|>1 and the significance level P value <0.05 as the standard. For significantly different miRNAs, four software, namely miRWalk, TargetScan, miRDB and miRTarBase, were used to further predict the target genes of the miRNAs, and gene function (GO) and KEGG biological pathway enrichment analysis were performed on the target genes.
5.细胞转染miRNA模拟物或抑制剂5. Cells are transfected with miRNA mimics or inhibitors
(1)将待转染的细胞种植于12孔细胞板中，待细胞密度达60％汇合时开始转染；(1) Plant the cells to be transfected in a 12-well cell plate, and start transfection when the cell density reaches 60% confluence;
(2)取5微升lipo 3000和100微升opti-MEM混合，室温孵育5分钟；(2) Mix 5 μl lipo 3000 and 100 μl opti-MEM and incubate at room temperature for 5 minutes;
(3)取10微升浓度为100nM的miRNA mimic或miRNA inhibitor、100微升opti-MEM混合，室温孵育5分钟；(3) Mix 10 microliters of miRNA mimic or miRNA inhibitor with a concentration of 100nM and 100 microliters of opti-MEM, and incubate at room temperature for 5 minutes;
(4)将(2)和(3)溶液混合，室温孵育20分钟；(4) Mix the solutions (2) and (3) and incubate at room temperature for 20 minutes;
(5)将(4)中得到的转染混合液加入12孔板中，在细胞培养箱中孵育；(5) Add the transfection mixture obtained in (4) to the 12-well plate and incubate it in a cell culture incubator;
(6)6-10小时后更换培养基，继续培养或进行其他药物干预后实验。(6) Change the culture medium after 6-10 hours and continue culturing or conduct other post-drug intervention experiments.
6.双荧光素酶报告基因实验6. Dual luciferase reporter gene assay
(1)实验分组(1) Experimental grouping
1)WT 3’UTR+NCmimic：WT为野生型(wild type)；1)WT 3’UTR+NCmimic: WT is wild type;
2)WT 3’UTR+miR-139-3p mimic；2)WT 3’UTR+miR-139-3p mimic;
3)Mut 3’UTR+NC mimic：Mut为突变型(mutated)；3) Mut 3’UTR+NC mimic: Mut is mutated;
4)Mut 3’UTR+miR-139-3p mimic。4)Mut 3’UTR+miR-139-3p mimic.
(2)实验过程(2) Experimental process
1)将293T细胞系接种在96孔培养板中，细胞密度为1-2×10 4个/毫升，置于37℃、5％CO 2细胞培养箱中培养，细胞密度达50-80％时即可开始转染； 1) Inoculate the 293T cell line in a 96-well culture plate at a cell density of 1-2×10 4 cells/ml, and culture it in a 37°C, 5% CO 2 cell culture incubator. When the cell density reaches 50-80% Transfection can begin;
2)用7.5微升1X riboFECT TM CP Buffer稀释0.25微升20μM mimic储存液和1微升100ng/μl Stat1基因3’UTR双荧光素酶报告载体质粒，轻轻混匀，室温孵育5分钟； 2) Dilute 0.25 μl of 20 μM mimic stock solution and 1 μl of 100ng/μl Stat1 gene 3'UTR dual-luciferase reporter vector plasmid with 7.5 μl of 1X riboFECT TM CP Buffer, mix gently, and incubate at room temperature for 5 minutes;
3)混合液制备：向2)中加入0.75微升riboFECT TM CP Reagent，轻轻吹打混匀，室温孵育0-15分钟； 3) Mixed solution preparation: Add 0.75 μl riboFECT TM CP Reagent to 2), mix gently by pipetting, and incubate at room temperature for 0-15 minutes;
4)将riboFECT TM CP混合液加入到90.50微升细胞培养基中，轻轻混匀； 4) Add riboFECT TM CP mixture to 90.50 μl of cell culture medium and mix gently;
5)将培养板置于37℃的CO 2培养箱中培养24h； 5) Place the culture plate in a CO 2 incubator at 37°C for 24 hours;
6)取出待检测的培养板，吸去培养基，每孔加入75微升新鲜培养基，并加入75微升配制的luciferase底物，涡旋振荡10分钟，用酶标仪测定萤火虫荧光素酶基因(hLuc)荧光值；6) Take out the culture plate to be tested, aspirate the culture medium, add 75 μl of fresh culture medium to each well, and add 75 μl of the prepared luciferase substrate, vortex for 10 minutes, and measure firefly luciferase with a microplate reader. Gene (hLuc) fluorescence value;
7)加入配制的75微升stop regent，振荡10分钟，用酶标仪测定海肾荧光素酶基(hRluc)荧光值；7) Add 75 microliters of the prepared stop regent, shake for 10 minutes, and measure the Renilla luciferase (hRluc) fluorescence value with a microplate reader;
8)数据分析：将各孔hRluc的荧光值与hLuc的荧光值比较，将比值与对照孔的比值进行统计分析。8) Data analysis: Compare the fluorescence value of hRluc in each well with the fluorescence value of hLuc, and perform statistical analysis on the ratio with the ratio of the control well.
实施例1.miR-139-3p介导促进巨噬细胞M2极化Example 1.miR-139-3p mediates the promotion of macrophage M2 polarization
1.大鼠骨髓MSC来源外泌体鉴定1. Identification of exosomes derived from rat bone marrow MSCs
分离、培养大鼠骨髓MSC至第3-4代，待细胞生长至亚汇合状态时，给予1μmol/L DMSO或1μmol/L ATV干预24h，换液为不含胎牛血清的IMDM培养基后继续培养48h，收集细胞条件培养基后经梯度超速离心提取分别获得外泌体MSC-Exo和MSC ATV-Exo(图1A)。流式细胞术鉴定出提取的原代MSC高比例表达表面标志物CD29和CD90， 表达CD45和CD11b/c的细胞比例非常低，表明本发明提取的MSC纯度较高(图1B)。1μmol/L ATV的浓度为本案发明人前期研究确定的最佳浓度。显微镜下经1μmol/L ATV或DMSO处理的MSC形态学上无明显差异(图1C)。透射电子显微镜观察到MSC衍生的外泌体为茶托样或一侧凹陷的半球形膜结构，直径在100nm左右(图1D)。MSC衍生的外泌体表达Alix、CD63、TSG101和CD81等外泌体标志蛋白(图1E)。摸索体外模型最适合的外泌体浓度，发现用40μg/ml浓度的外泌体能达到最佳的促进M2极化的效果，后续体外实验选用该浓度(图1F)。体外模型中相同浓度下(40μg/ml)，MSC ATV-Exo相对于MSC-Exo有着更好的促进巨噬细胞M2极化的效果(图1G)。 Isolate and culture rat bone marrow MSC to passage 3-4. When the cells grow to a sub-confluent state, 1 μmol/L DMSO or 1 μmol/L ATV is given for 24 hours. The medium is changed to IMDM medium without fetal bovine serum and continued. After culturing for 48 hours, the cell conditioned medium was collected and extracted by gradient ultracentrifugation to obtain exosomes MSC-Exo and MSC ATV -Exo (Figure 1A). Flow cytometry identified that the extracted primary MSC expressed a high proportion of surface markers CD29 and CD90, and a very low proportion of cells expressing CD45 and CD11b/c, indicating that the MSC extracted in the present invention had higher purity (Figure 1B). The concentration of 1 μmol/L ATV is the optimal concentration determined by the inventor of this case in previous studies. There was no obvious difference in the morphology of MSCs treated with 1 μmol/L ATV or DMSO under the microscope (Figure 1C). Transmission electron microscopy observed that MSC-derived exosomes were saucer-like or hemispherical membrane structures with one side depressed, with a diameter of about 100 nm (Figure 1D). MSC-derived exosomes expressed exosome marker proteins such as Alix, CD63, TSG101, and CD81 (Figure 1E). After exploring the most suitable exosome concentration for the in vitro model, we found that using exosomes at a concentration of 40 μg/ml can achieve the best effect of promoting M2 polarization. This concentration was selected for subsequent in vitro experiments (Figure 1F). In the in vitro model, at the same concentration (40 μg/ml), MSC ATV -Exo had a better effect on promoting M2 polarization of macrophages than MSC-Exo (Figure 1G).
2.心肌内注射MSC ATV-Exo有效改善急性心梗大鼠的心功能，并显著促进巨噬细胞浸润和巨噬细胞M2极化 2. Intramyocardial injection of MSC ATV -Exo effectively improves cardiac function in rats with acute myocardial infarction, and significantly promotes macrophage infiltration and macrophage M2 polarization.
急性心梗大鼠模型前降支结扎30分钟后，在梗死周边区分3点心肌内注射100μl共105μg外泌体(MSC-Exo或MSC ATV-Exo)或等体积PBS。小动物超声结果显示：在心梗后第28天，相对于AMI模型组，MSC-Exo或MSC ATV-Exo治疗组的LVEF值和LVFS值显著提高，LVEDV和LVESV值较AMI组的低，表明这两种外泌体治疗都可改善心功能；但MSC ATV-Exo治疗组的LVEF值、LVFS值、LVEDV值和LVESV值较MSC-Exo治疗组的改善更加显著，表明MSC ATV-Exo治疗的心肌保护效果更优(图2A、图2B)。Masson染色结果表明，相对于AMI模型组，MSC-Exo或MSC ATV-Exo治疗组的心肌梗死面积显著减小；且MSC ATV-Exo治疗组的心肌梗死面积较MSC-Exo治疗组的减小更显著(图2C、图2D)。以上结果表明，MSC ATV-Exo治疗比普通MSC-Exo治疗能更加有效地减小梗死面积、改善心室功能。 Thirty minutes after anterior descending branch ligation in the rat model of acute myocardial infarction, 100 μl of 105 μg of exosomes (MSC-Exo or MSC ATV -Exo) or an equal volume of PBS was injected intramyocardially at 3 points around the infarction. The results of small animal ultrasound showed that on the 28th day after myocardial infarction, compared with the AMI model group, the LVEF value and LVFS value of the MSC-Exo or MSC ATV -Exo treatment group were significantly higher, and the LVEDV and LVESV values were lower than those of the AMI group, indicating that Both exosome treatments can improve cardiac function; however, the LVEF value, LVFS value, LVEDV value and LVESV value of the MSC ATV -Exo treatment group are more significant than those of the MSC-Exo treatment group, indicating that the MSC ATV -Exo treatment group The myocardial protection effect is better (Figure 2A, Figure 2B). Masson staining results showed that compared with the AMI model group, the myocardial infarction area of the MSC-Exo or MSC ATV -Exo treatment group was significantly reduced; and the myocardial infarction area of the MSC ATV -Exo treatment group was smaller than that of the MSC-Exo treatment group. Significantly (Figure 2C, Figure 2D). The above results show that MSC ATV -Exo treatment can reduce infarct size and improve ventricular function more effectively than ordinary MSC-Exo treatment.
为了进一步明确MSC ATV-Exo治疗对心梗急性期心肌组织中巨噬细胞浸润及亚型的影响，本发明检测了心梗第3天梗死周边区心肌中巨噬细胞的表达情况。免疫荧光结果显示，AMI组梗死周边区心肌CD68+巨噬细胞数量较假手术组明显增多；相对于AMI组，外泌体组的梗死周边区CD68+巨噬细胞数量减少；相对于MSC-Exo组，MSC ATV-Exo组的CD68+巨噬细胞数量减少更加明显(图2E-图2F)。进一步荧光标记M2型巨噬细胞(CD206+)结果显示，MSC ATV-Exo组梗死周边区心肌中CD206+巨噬细胞数量较AMI组或MSC-Exo组都显著增多(图2E、图2F)。Western blot和qPCR结果显示，MSC ATV-Exo组心肌组织中M2巨噬细胞标志物Arg1、IL-10、CD206的蛋白或mRNA表达量较AMI组或MSC-Exo组显著增高，而M1巨噬细胞标志物iNOS、IL-12、TNF-α的蛋白或mRNA表达量显著降低(图2G、图2H、图2I)。以上结果表明，心肌内注射MSC ATV-Exo较 普通MSC-Exo更加显著地减少心梗急性期梗死周边区心肌中CD68+巨噬细胞数量，但增加CD206+巨噬细胞数量。 In order to further clarify the effect of MSC ATV -Exo treatment on macrophage infiltration and subtypes in myocardial tissue in the acute phase of myocardial infarction, the present invention detected the expression of macrophages in the myocardium in the peri-infarct area on the third day of myocardial infarction. Immunofluorescence results showed that the number of myocardial CD68+ macrophages in the peri-infarct area of the AMI group was significantly increased compared with the sham operation group; compared with the AMI group, the number of CD68+ macrophages in the exosome group was reduced; compared with the MSC-Exo group, The number of CD68+ macrophages in the MSC ATV -Exo group decreased more obviously (Figure 2E-Figure 2F). Further fluorescent labeling of M2 macrophages (CD206+) showed that the number of CD206+ macrophages in the myocardium around the infarction in the MSC ATV -Exo group was significantly higher than that in the AMI group or MSC-Exo group (Figure 2E, Figure 2F). Western blot and qPCR results showed that the protein or mRNA expression of M2 macrophage markers Arg1, IL-10, and CD206 in the myocardial tissue of the MSC ATV -Exo group was significantly higher than that of the AMI group or MSC-Exo group, while the M1 macrophage The protein or mRNA expression levels of markers iNOS, IL-12, and TNF-α were significantly reduced (Figure 2G, Figure 2H, and Figure 2I). The above results show that intramyocardial injection of MSC ATV -Exo significantly reduces the number of CD68+ macrophages in the myocardium in the peri-infarct area in the acute phase of myocardial infarction, but increases the number of CD206+ macrophages compared with ordinary MSC-Exo.
3.MSC ATV-Exo促进体外培养的巨噬细胞从M1型向M2型极化 3.MSC ATV -Exo promotes the polarization of macrophages cultured in vitro from M1 type to M2 type
在体外实验探索了MSC ATV-Exo对巨噬细胞极化的影响。首先用PKH-67标记外泌体后加入到大鼠骨髓巨噬细胞的培养基中，免疫荧光结果可观察到巨噬细胞能有效摄取外泌体(图3A)。接着先用100ng/mL的LPS刺激巨噬细胞6小时，模拟急性心梗时体内炎症微环境、诱导巨噬细胞向M1极化，然后换液加入外泌体作用48小时。Western blot和qPCR结果显示，相对于空白对照组，M1组巨噬细胞中iNOS、IL-12、TNF-α的蛋白或mRNA表达水平增高，M2组巨噬细胞中Arg1、IL-10、CD206的蛋白或mRNA表达水平增高，表明M1和M2模型建立成功；相对于M1组，外泌体组Arg1、IL-10、CD206的蛋白或mRNA表达水平明显增高，iNOS、IL-12、TNF-α的蛋白或mRNA表达水平明显降低；相对于MSC-Exo组，MSC ATV-Exo组Arg1、IL-10、CD206的蛋白或mRNA表达水平较高，iNOS、IL-12、TNF-α的蛋白或mRNA表达水平较低(图3B、图3C、图3D)。以上结果表明，MSC ATV-Exo显著降低LPS刺激后的巨噬细胞表达M1型标志物、促进其表达M2型标志物，且较普通MSC-Exo的效应更强。 In vitro experiments explored the effect of MSC ATV -Exo on macrophage polarization. First, exosomes were labeled with PKH-67 and then added to the culture medium of rat bone marrow macrophages. Immunofluorescence results showed that macrophages could effectively uptake exosomes (Figure 3A). Then, 100ng/mL LPS was used to stimulate macrophages for 6 hours to simulate the inflammatory microenvironment in the body during acute myocardial infarction and induce macrophage polarization toward M1. Then, the medium was changed and exosomes were added for 48 hours. Western blot and qPCR results showed that compared with the blank control group, the protein or mRNA expression levels of iNOS, IL-12, and TNF-α in macrophages of the M1 group were increased, and the expression levels of Arg1, IL-10, and CD206 in the macrophages of the M2 group were increased. The protein or mRNA expression levels increased, indicating that the M1 and M2 models were successfully established; compared with the M1 group, the protein or mRNA expression levels of Arg1, IL-10, and CD206 in the exosome group were significantly increased, and the levels of iNOS, IL-12, and TNF-α were significantly higher. The protein or mRNA expression levels were significantly reduced; compared with the MSC-Exo group, the MSC ATV -Exo group had higher protein or mRNA expression levels of Arg1, IL-10, and CD206, and higher protein or mRNA expression levels of iNOS, IL-12, and TNF-α. The levels are lower (Figure 3B, Figure 3C, Figure 3D). The above results show that MSC ATV -Exo significantly reduces the expression of M1 markers and promotes the expression of M2 markers in macrophages stimulated by LPS, and the effect is stronger than that of ordinary MSC-Exo.
4.MSC ATV-Exo中miRNA的表达谱分析 4.Expression profile analysis of miRNA in MSC ATV -Exo
进行外泌体长链非编码RNA测序，以差异倍数|log2(FoldChange)|>1且显著性水平P值<0.05为标准，miRNA表达差异分析发现了上调表达的miR-139-3p(图4A)。用qPCR实验再次验证外泌体MSC-Exo和MSC ATV-Exo中miR-139-3p的表达量，结果(图4B)与测序结果一致。 Exosome long non-coding RNA sequencing was performed, and using the difference fold |log2(FoldChange)|>1 and the significance level P value <0.05 as the standard, the miRNA expression difference analysis found the up-regulated expression of miR-139-3p (Figure 4A ). The expression of miR-139-3p in exosomes MSC-Exo and MSC ATV -Exo was again verified using qPCR experiments, and the results (Figure 4B) were consistent with the sequencing results.
5.miR-139-3p介导促进巨噬细胞M2极化5.miR-139-3p mediates the promotion of M2 polarization of macrophages
本发明用miR-139-3p的抑制剂(inhibitor)或模拟物(mimic)转染MSC ATV或MSC后提取相应的外泌体MSC ATV-Exo(inhibitor)和MSC-Exo(mimic)，用此外泌体作用于LPS刺激后的巨噬细胞(图5A)。Western blot和qPCR结果发现：相对于LPS+MSC ATV-Exo(NC inhibitor)组，LPS+MSC ATV-Exo(miR inhibitor)组Arg1、IL-10、CD206的蛋白或mRNA表达水平降低，iNOS、IL-12、TNF-α的蛋白或mRNA表达水平升高；LPS+MSC-Exo(miR mimic)组Arg1、IL-10、CD206、iNOS、IL-12、TNF-α的蛋白或mRNA表达水平则和LPS+MSC ATV-Exo组相近(图5B、图5C、图5D)。 The present invention uses the inhibitor (inhibitor) or mimic (mimic) of miR-139-3p to transfect MSC ATV or MSC, and then extracts the corresponding exosomes MSC ATV -Exo (inhibitor) and MSC-Exo (mimic). Exosomes act on macrophages stimulated by LPS (Figure 5A). Western blot and qPCR results showed that compared with the LPS+MSC ATV -Exo (NC inhibitor) group, the LPS+MSC ATV -Exo (miR inhibitor) group had lower protein or mRNA expression levels of Arg1, IL-10, and CD206, and lower levels of iNOS, IL -12. The protein or mRNA expression levels of TNF-α increased; the protein or mRNA expression levels of Arg1, IL-10, CD206, iNOS, IL-12, and TNF-α in the LPS+MSC-Exo(miR mimic) group were the same as The LPS+MSC ATV -Exo group was similar (Figure 5B, Figure 5C, Figure 5D).
本发明用miR-139-3p mimic直接转染LPS干预后的巨噬细胞(图5E)。Western blot和qPCR结果发现：相对于M1组，LPS+miR-139-3p mimic组Arg1、IL-10、CD206的 蛋白或mRNA表达水平增高，iNOS、IL-12、TNF-α的蛋白或mRNA表达水平降低(图5F、图5G、图5H)。The present invention uses miR-139-3p mimic to directly transfect macrophages after LPS intervention (Figure 5E). Western blot and qPCR results found that: compared with the M1 group, the protein or mRNA expression levels of Arg1, IL-10, and CD206 were increased in the LPS+miR-139-3p mimic group, and the protein or mRNA expression levels of iNOS, IL-12, and TNF-α were increased. levels decreased (Figure 5F, Figure 5G, Figure 5H).
以上结果表明，无论是用干扰miR-139-3p表达后的外泌体还是直接用miR-139-3p干扰物去作用于LPS干预后的巨噬细胞，miR-139-3p过表达后会使M2型标志物表达升高、M1标志物降低，反之，miR-139-3p敲低后会使M2型标志物表达降低、M1标志物升高，表明MSC ATV-Exo通过传递miR-139-3p实现促进体外巨噬细胞向M2极化的作用。 The above results show that whether exosomes that interfere with the expression of miR-139-3p are used or miR-139-3p interfering substances are directly used to act on macrophages after LPS intervention, overexpression of miR-139-3p will cause The expression of M2 markers increased and M1 markers decreased. On the contrary, knockdown of miR-139-3p resulted in decreased expression of M2 markers and increased M1 markers, indicating that MSC ATV -Exo delivers miR-139-3p. Achieve the effect of promoting the polarization of macrophages to M2 in vitro.
为了全面分析miR-139-3p与M2极化相关的靶基因，本发明同时用miRWalk、TargetScan、miRDB和miRTarBase四个软件对其靶基因进行预测。其中，TargetScan预测到Stat1是miR-139-3p的一个靶基因。由于Stat1激活后可促进M1极化，故本发明拟定Stat1为miR-139-3p的靶基因，并提出科学假设：miR-139-3p高表达会抑制Stat1的表达和激活，从而抑制巨噬细胞向M1极化、促进向M2极化。In order to comprehensively analyze the target genes related to miR-139-3p and M2 polarization, the present invention simultaneously uses four software, namely miRWalk, TargetScan, miRDB and miRTarBase, to predict its target genes. Among them, TargetScan predicted that Stat1 is a target gene of miR-139-3p. Since Stat1 can promote M1 polarization after activation, the present invention proposes Stat1 as the target gene of miR-139-3p, and proposes a scientific hypothesis: high expression of miR-139-3p will inhibit the expression and activation of Stat1, thereby inhibiting macrophages. Polarize to M1 and promote polarization to M2.
本实施例做了荧光素酶报告基因实验验证miR-139-3p与Stat1转录本3’UTR靶片段的配对结合。将构建好的pmiR-RB-Report TM双荧光素酶质粒载体和miR-139-3p mimic共同转染293T细胞，荧光素酶活性检测结果表明：miR-139-3p mimic降低了Stat1野生型WT 3’UTR组的荧光素酶活性，但对突变型Mut 3’-UTR组的荧光素酶活性无明显影响，表明miR-139-3p可与Stat1转录本3’UTR靶片段配对结合。 In this example, a luciferase reporter gene experiment was performed to verify the paired binding of miR-139-3p to the 3'UTR target fragment of the Stat1 transcript. The constructed pmiR-RB-Report TM dual-luciferase plasmid vector and miR-139-3p mimic were co-transfected into 293T cells. The luciferase activity detection results showed that: miR-139-3p mimic reduced Stat1 wild-type WT 3 The luciferase activity of the 'UTR group, but had no significant effect on the luciferase activity of the mutant Mut 3'-UTR group, indicating that miR-139-3p can pair with the 3'UTR target fragment of the Stat1 transcript.
接着，本发明用miR-139-3p mimic或inhibitor转染巨噬细胞，Western blot实验结果表明：miR-139-3p mimic可降低Stat1总蛋白的表达水平，而使用miR-139-3p inhibitor后Stat1总蛋白水平则不会降低(图5I、图5J)。LPS刺激巨噬细胞后再转染miR-139-3p mimic或inhibitor，发现过表达miR-139-3p可明显降低Stat1的磷酸化水平、促进Arg1蛋白表达、抑制iNOS蛋白表达；而敲低miR-139-3p表达后，LPS刺激后的巨噬细胞中Stat1的磷酸化水平和iNOS蛋白表达水平依旧较高，Arg1蛋白表达水平依旧较低(图5K、图5L)。Next, the present invention uses miR-139-3p mimic or inhibitor to transfect macrophages. Western blot experimental results show that: miR-139-3p mimic can reduce the expression level of Stat1 total protein, and using miR-139-3p inhibitor reduces Stat1 The total protein level did not decrease (Figure 5I, Figure 5J). Macrophages were stimulated with LPS and then transfected with miR-139-3p mimic or inhibitor. It was found that overexpression of miR-139-3p could significantly reduce the phosphorylation level of Stat1, promote Arg1 protein expression, and inhibit iNOS protein expression; while knockdown of miR-139-3p could significantly reduce the phosphorylation level of Stat1, promote Arg1 protein expression, and inhibit iNOS protein expression. After 139-3p expression, the phosphorylation level of Stat1 and the expression level of iNOS protein in macrophages stimulated by LPS were still high, while the expression level of Arg1 protein was still low (Figure 5K, Figure 5L).
综上，以上结果证明了miR-139-3p主要通过靶向抑制Stat1的表达和激活发挥促进M2、抑制M1的功能。In summary, the above results prove that miR-139-3p mainly promotes M2 and inhibits M1 by targeting the expression and activation of Stat1.
实施例2.miR-139-3p介导了MSC ATV-Exo的促心肌巨噬细胞M2极化功能 Example 2.miR-139-3p mediates the function of MSC ATV -Exo in promoting cardiac macrophage M2 polarization
本实施例在动物体内验证了miR-139-3p的作用。按照同样的方法给予急性心肌梗死模型大鼠心肌内注射miR-139-3p表达水平受干扰后的外泌体即MSC ATV-Exo(miR inhibitor)或MSC-Exo(miR mimic)，检测心梗急性期即第3天时心肌组织中巨噬细胞极化 的情况。免疫荧光结果表明，相对于MSC ATV -Exo组，MSC ATV-Exo(miR inhibitor)组心肌CD68+巨噬细胞数量增加，CD206+巨噬细胞数量降低；而MSC-Exo(miR mimic)组CD68+巨噬细胞数量降低水平、CD206+巨噬细胞数量升高水平则和MSC ATV-Exo组相当(图6A、图6D)。 This example verified the role of miR-139-3p in animals. According to the same method, acute myocardial infarction model rats were given intramyocardial injection of exosomes with disturbed expression levels of miR-139-3p, namely MSC ATV -Exo (miR inhibitor) or MSC-Exo (miR mimic), to detect acute myocardial infarction. The phase is the polarization of macrophages in myocardial tissue on day 3. Immunofluorescence results showed that compared with the MSC ATV - Exo group, the number of myocardial CD68+ macrophages increased and the number of CD206+ macrophages decreased in the MSC ATV -Exo(miR inhibitor) group; while the number of CD68+ macrophages in the MSC-Exo(miR mimic) group The level of reduction in number and increase in the number of CD206+ macrophages was comparable to that of the MSC ATV -Exo group (Figure 6A, Figure 6D).
小动物超声结果显示：在心梗后第28天，相对于MSC ATV-Exo组，MSC ATV-Exo-miR inhibitor组的LVEF值和LVFS值显著降低，而相对于MSC ATV-Exo-miR inhibitor组，MSC-Exo-miR mimic组LVEF与LVFS值又显著提高(图6B、图6E)。Masson染色结果表明，相对于MSC ATV-Exo组，MSC ATV-Exo-miR inhibitor组的心肌梗死面积显著升高；且MSC Exo-miR mimic组的心肌梗死面积较MSC ATV Exo-miR inhibitor组心梗面积显著减小(图6C、图6F)。以上结果表明，miR-139-3p在MSC ATV-Exo发挥治疗心肌梗死、缩小梗死面积的作用中起到重要作用。 Small animal ultrasound results showed that on the 28th day after myocardial infarction, the LVEF value and LVFS value of the MSC ATV -Exo-miR inhibitor group were significantly reduced compared with the MSC ATV -Exo group, and compared with the MSC ATV -Exo-miR inhibitor group , the LVEF and LVFS values of the MSC-Exo-miR mimic group were significantly increased (Figure 6B, Figure 6E). Masson staining results showed that compared with the MSC ATV -Exo group, the myocardial infarction area of the MSC ATV -Exo-miR inhibitor group was significantly increased; and the myocardial infarction area of the MSC Exo-miR mimic group was significantly higher than that of the MSC ATV Exo-miR inhibitor group. The area was significantly reduced (Figure 6C, Figure 6F). The above results indicate that miR-139-3p plays an important role in MSC ATV -Exo's ability to treat myocardial infarction and reduce infarct size.
Western blot和qPCR结果表明：相对于MSC ATV-Exo组，MSC ATV-Exo(miR inhibitor)组梗死周边区心肌组织中Arg1、IL-10、CD206的蛋白或mRNA表达水平降低，iNOS、IL-12、TNF-α的蛋白或mRNA表达水平升高，Stat1蛋白磷酸化水平升高，表明敲低miR-139-3p表达水平后的MSC ATV-Exo治疗组心肌组织中浸润的M2型巨噬细胞比例降低了；而MSC-Exo(miR mimic)组Arg1、IL-10、CD206、iNOS、IL-12、TNF-α及Stat1磷酸化水平则与MSC ATV-Exo组的相当，与MSC ATV-Exo(miR inhibitor)组的有明显差异(图6G、图6H、图6I)。 Western blot and qPCR results showed that: compared with the MSC ATV -Exo group, the MSC ATV -Exo (miR inhibitor) group had reduced protein or mRNA expression levels of Arg1, IL-10, and CD206 in the myocardial tissue in the peri-infarct area, while iNOS and IL-12 , TNF-α protein or mRNA expression levels increased, and Stat1 protein phosphorylation levels increased, indicating the proportion of M2 macrophages infiltrating in the myocardial tissue of the MSC ATV -Exo treatment group after knocking down the expression level of miR-139-3p. decreased; while the phosphorylation levels of Arg1, IL-10, CD206, iNOS, IL-12, TNF-α and Stat1 in the MSC-Exo (miR mimic) group were comparable to those in the MSC ATV -Exo group, and were similar to those in the MSC ATV -Exo ( There were significant differences in the miR inhibitor) group (Figure 6G, Figure 6H, Figure 6I).
以上结果综合说明，ATV预处理使MSC衍生的外泌体中高表达miR-139-3p，MSC ATV -Exo心肌内注射后通过向损伤心肌中传递miR-139-3p，抑制更多的巨噬细胞浸润心肌、促进已浸润的巨噬细胞向M2极化，发挥抑制炎症、促进心肌修复效应(图7)。 The above results comprehensively indicate that ATV pretreatment causes high expression of miR-139-3p in MSC-derived exosomes. After intramyocardial injection of MSC ATV - Exo, it inhibits more macrophages by delivering miR-139-3p to the injured myocardium. Infiltrate the myocardium and promote the polarization of infiltrated macrophages to M2, exerting the effect of inhibiting inflammation and promoting myocardial repair (Figure 7).

Claims (13)

  1. miR-139-3p在制备调控巨噬细胞极化的制剂中的应用。Application of miR-139-3p in the preparation of preparations for regulating macrophage polarization.
  2. 根据权利要求1所述的应用，该应用包括：在外泌体中过表达miR-139-3p的试剂材料在制备用于促进巨噬细胞向M2型极化的制剂中的应用。The application according to claim 1, which application includes: application of a reagent material that overexpresses miR-139-3p in exosomes in the preparation of a preparation for promoting the polarization of macrophages toward the M2 type.
  3. 根据权利要求2所述的应用，其中，用于促进巨噬细胞向M2型极化的制剂包括过表达miR-139-3p的外泌体。The application according to claim 2, wherein the preparation for promoting the polarization of macrophages toward the M2 type includes exosomes overexpressing miR-139-3p.
  4. 根据权利要求3所述的应用，其中，所述外泌体为骨髓间充质干细胞衍生的外泌体。The application according to claim 3, wherein the exosomes are exosomes derived from bone marrow mesenchymal stem cells.
  5. 根据权利要求2所述的应用，其中，在外泌体中过表达miR-139-3p的试剂材料包括：通过基因技术在外泌体中过表达miR-139-3p的试剂材料，和/或通过药物预处理干细胞而使得干细胞分泌高表达miR-139-3p的外泌体的试剂材料。The application according to claim 2, wherein the reagent material for overexpressing miR-139-3p in exosomes includes: a reagent material for overexpressing miR-139-3p in exosomes through genetic technology, and/or through drugs A reagent material that pretreats stem cells to cause them to secrete exosomes that highly express miR-139-3p.
  6. 根据权利要求5所述的应用，其中，所述药物预处理干细胞的药物为他汀类药物，例如阿托伐他汀。The application according to claim 5, wherein the drug used to pretreat stem cells is a statin drug, such as atorvastatin.
  7. 根据权利要求1所述的应用，该应用包括：抑制和/或敲低外泌体中miR-139-3p的表达的试剂材料在制备用于抑制巨噬细胞向M2型极化的制剂中的应用。The application according to claim 1, which application includes: the use of a reagent material that inhibits and/or knocks down the expression of miR-139-3p in exosomes in the preparation of a preparation for inhibiting the polarization of macrophages toward the M2 type. application.
  8. 根据权利要求7所述的应用，其中，抑制和/或敲低外泌体中miR-139-3p的表达的试剂材料包括：miR-139-3p抑制剂、通过基因技术敲低外泌体中miR-139-3p的表达的试剂材料中的一种或多种。The application according to claim 7, wherein the reagent materials for inhibiting and/or knocking down the expression of miR-139-3p in exosomes include: a miR-139-3p inhibitor, knocking down the expression of miR-139-3p in exosomes through genetic technology One or more of the reagent materials for the expression of miR-139-3p.
  9. miR-139-3p在制备调控信号转导与转录激活因子1(signal transducer and activator of transcription 1,Stat1)的表达的制剂中的应用。Application of miR-139-3p in the preparation of preparations that regulate the expression of signal transducer and activator of transcription 1 (Stat1).
  10. 根据权利要求9所述的应用，该应用包括：过表达miR-139-3p的试剂材料在制备降低巨噬细胞中总Stat1表达水平的制剂中的应用。The application according to claim 9, which application includes: application of a reagent material overexpressing miR-139-3p in preparing a preparation for reducing the total Stat1 expression level in macrophages.
  11. 根据权利要求9所述的应用，该应用包括：抑制和/或敲低外泌体中miR-139-3p的表达的试剂材料在制备用于增加巨噬细胞中总Stat1表达水平的制剂中的应用。The application according to claim 9, which application includes: the use of a reagent material that inhibits and/or knocks down the expression of miR-139-3p in exosomes in the preparation of a preparation for increasing the total Stat1 expression level in macrophages. application.
  12. 一种提高心肌组织M2型标志物表达水平的方法，该方法包括：向需要的受试者给予过表达miR-139-3p的外泌体。A method for increasing the expression level of M2-type markers in cardiac tissue, the method comprising: administering exosomes overexpressing miR-139-3p to a subject in need.
  13. 一种治疗急性心肌梗死的方法，该方法包括：向需要的受试者给予过表达miR-139-3p的外泌体，从而实现以下至少一种效果：A method for treating acute myocardial infarction, the method comprising: administering exosomes overexpressing miR-139-3p to a subject in need, thereby achieving at least one of the following effects:
    减少心梗急性期心肌中巨噬细胞浸润、控制炎症反应，并促进浸润的巨噬细胞从M1型向M2型转化，促进心肌修复，减小心梗面积，改善心功能；和/或Reduce the infiltration of macrophages in the myocardium in the acute phase of myocardial infarction, control the inflammatory response, and promote the transformation of infiltrating macrophages from M1 type to M2 type, promote myocardial repair, reduce the size of myocardial infarction, and improve cardiac function; and/or
    传递心脏保护性miR-139-3p到巨噬细胞中，通过抑制Stat1的表达与激活促进M2巨噬细胞极化。Delivering cardioprotective miR-139-3p to macrophages promotes M2 macrophage polarization by inhibiting Stat1 expression and activation.
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