WO2023273428A1 - 基于通心络预处理的间充质干细胞来源外泌体及其制备方法 - Google Patents

基于通心络预处理的间充质干细胞来源外泌体及其制备方法 Download PDF

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WO2023273428A1
WO2023273428A1 PCT/CN2022/082234 CN2022082234W WO2023273428A1 WO 2023273428 A1 WO2023273428 A1 WO 2023273428A1 CN 2022082234 W CN2022082234 W CN 2022082234W WO 2023273428 A1 WO2023273428 A1 WO 2023273428A1
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mesenchymal stem
stem cells
exosomes
tongxinluo
centrifuge
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杨跃进
熊语嫣
汤瑞杰
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中国医学科学院阜外医院
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  • the present invention relates to a mesenchymal stem cell-derived exosome based on drug pretreatment and its preparation method, in particular to a high-efficiency mesenchymal stem cell-derived exosome based on drug Tongxinluo pretreatment and its preparation method.
  • AMI can lead to ischemia and necrosis of a large number of myocardium in a short period of time, which is then replaced by scar tissue, leading to heart failure and death.
  • Existing treatments cannot effectively regenerate and repair heart muscle.
  • stem cell transplantation therapy especially mesenchymal stem cells (MSCs, mesenchymal stem cells), such as bone marrow mesenchymal stem cells (bone marrow-mesenchymal stem cells, BM-MSCs), has high hopes for transplantation treatment of AMI (Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM and others.
  • MSCs mesenchymal stem cells
  • BM-MSCs bone marrow mesenchymal stem cells
  • Bone marrow cells regenerate infarcted myocardium. Nature2001; 410(6829):701-705; Fisher SA ,Doree C, Mathur A, Martin-Rendon E. Meta-Analysis of Cell Therapy Trials for Patients With Heart Failure. Circulation Research 2015; 116(8):1361-1377; Afzal MR, Samanta A, Shah ZI, Jeevanantham V, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult Bone Marrow Cell Therapy for Ischemic Heart Disease: Evidence and Insights From Randomized Controlled Trials. Circ Res 2015;117(6):558-575).
  • MSCs The paracrine function of MSCs is an important way for them to function (Ranganath, S.H., et al., Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease. Cell Stem Cell, 2012.10(3): p.244-58.)
  • mesenchymal stem cells can improve cardiac function after infarction through paracrine protection to a certain extent, the effect is not significant.
  • exosomes an extracellular vesicle structure secreted by MSCs in a paracrine manner, have been widely reported to be involved in improving the microenvironment and repairing the myocardium after AMI (Cheng, H., et al., Hypoxia-challenged MSC-derived exosomes deliver miR-210 to attenuate post-infarction cardiac apoptosis.Stem Cell Res Ther,2020.11(1):p.224; Tan,S.J.O.,et al.,Novel Applications of Mesenchymal Exosomes Stem Cell-derived for Myocardial Infarction Therapeutics.Biomolecules,2020.10(5); Xiao,C.,et al.,Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125b.Circ Res,20158.123( 564-578.).
  • Exosomes can carry a wide range of bioactive molecules, including RNA, protein, etc., among which microRNA has been widely reported to participate in the heart repair process (Gebert, L.F.R. and I.J.MacRae, Regulation of microRNA function in animals. Nat Rev Mol Cell Biol, 2019.20( 1): p.21-37; Zhou, S.S., et al., miRNAS in cardiovascular diseases: potential biomarkers, therapeutic targets and challenges. Acta Pharmacol Sin, 2018.39 (7): p.1073-1084.), and miRNA- 146a has been widely reported to have functions such as cardiac repair and anti-fibrosis (Boon RA, Dimmeler S. MicroRNAs in myocardial infarction. Nat Rev Cardiol.
  • exosomes derived from BM-MSCs can improve the microenvironment of myocardial infarction and promote heart repair. Therefore, it is expected to become a new generation of myocardial repair products and provide new ideas for clinical treatment of acute myocardial infarction (Lamichhane TN, Sokic S, Schardt JS, Raiker RS, Lin JW, Jay SM. Emerging Roles for Extracellular Vesicles in Tissue Engineering and Regenerative Medicine. Tissue Engineering Part B: Reviews 2015;21(1):45-54).
  • One object of the present invention is to provide a preparation method for improving the performance of exosomes derived from mesenchymal stem cells based on Tongxinluo drug pretreatment.
  • Another object of the present invention is to provide the prepared exosomes derived from mesenchymal stem cells.
  • pretreatment of mesenchymal stem cells by Tongxinluo can prepare mesenchymal stem cells with the ability to significantly improve cardiac function after myocardial infarction, reduce myocardial infarction size, reduce myocardial cell apoptosis, and reduce inflammation.
  • TXL Tongxinluo
  • the present invention provides a method for preparing exosomes derived from mesenchymal stem cells that upregulates the expression level of miR-146a, the method comprising: pretreating mesenchymal stem cells with Tongxinluo, and culturing mesenchymal stem cells to collect exosomes secreted by them.
  • Tongxinluo (TXL) used in the present invention is a kind of medicine, which should meet the requirements of relevant quality standards.
  • the method for preparing exosomes derived from mesenchymal stem cells that up-regulates the expression level of miR-146a of the present invention includes:
  • the time for pretreating mesenchymal stem cells with Tongxinluo is 24 hours.
  • the present invention found that miR-146a expressed in exosomes in the collected cell supernatant was significantly up-regulated when the treatment time was 24 hours.
  • the ultracentrifugation method includes:
  • centrifuge After collecting the conditioned medium, centrifuge to remove cells, centrifuge to remove cell debris, and centrifuge to remove large vesicles, then centrifuge to collect the pellet and resuspend, then centrifuge again to obtain exosomes.
  • the centrifugation method includes: after collecting the conditioned medium, centrifuge at 300g for 10min to remove cells; centrifuge at 2000g for 20min to remove cell debris; 16500g high-speed centrifugation for 30min to remove large vesicles; 120000g ultracentrifugation for 70min to collect the precipitate and resuspend, and then 120000g ultracentrifugation for 70min to obtain exosomes.
  • the mesenchymal stem cells include bone marrow mesenchymal stem cells or fat mesenchymal stem cells .
  • the present invention also provides exosomes prepared according to the method of the present invention.
  • the exosomes prepared by the present invention highly express miR-146a; the miR-146a can reduce post-myocardial infarction reaction inflammation, cell apoptosis, and reduce the size of myocardial infarction.
  • the present invention also provides the preparation of mesenchymal stem cells that secrete exosomes that highly express miR-146a and have the ability to improve cardiac function after myocardial infarction, reduce infarct size, reduce myocardial cell apoptosis, and reduce inflammation. application in formulations.
  • the mesenchymal stem cells are pretreated with 400 ⁇ g/mL Tongxinluo for 12-24 hours, preferably 24 hours.
  • the mesenchymal stem cells include bone marrow mesenchymal stem cells or fat mesenchymal stem cells.
  • exosomes with higher miR-146a content can be obtained by pretreating BM-MSCs with 400 ⁇ g/mL TXL for 24 hours, which can improve cardiac function after myocardial infarction, reduce infarct size, and reduce myocardial infarction. Apoptosis and reduce inflammation.
  • Figure 1A- Figure 1B shows the morphology and structure of exosomes derived from mesenchymal stem cells in an embodiment of the present invention under an electron microscope and the results of particle size analysis (NTA) of the exosomes.
  • NTA particle size analysis
  • Figure 2 shows that exosomes derived from mesenchymal stem cells in the examples of the present invention highly express exosome marker proteins such as Alix, TSG101, and CD63.
  • Figure 3A- Figure 3B shows that Tongxinluo pretreatment of mesenchymal stem cell-derived exosomes can significantly improve the test results of cardiac function after myocardial infarction in rats.
  • Figure 3C- Figure 3D shows the detection results of Tongxinluo pretreatment of mesenchymal stem cell-derived exosomes can reduce the size of myocardial infarction.
  • Figure 4 shows that Tongxinluo pretreatment of mesenchymal stem cell-derived exosomes can effectively reduce cardiomyocyte apoptosis.
  • Figure 5 shows that Tongxinluo pretreatment of mesenchymal stem cell-derived exosomes can significantly reduce the release of related inflammatory factors.
  • Figure 6 shows the volcano plot of the difference between exosomes derived from mesenchymal stem cells pretreated with Tongxinluo and exosomes derived from ordinary mesenchymal stem cells.
  • Figure 7 shows that miRNA-146a related to cardioprotection in exosomes derived from mesenchymal stem cells pretreated with Tongxinluo was nearly 10 times higher than that of exosomes derived from ordinary mesenchymal stem cells.
  • each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.
  • TXL used in the examples was purchased from Shijiazhuang Yiling Pharmaceutical Co., Ltd., and its main components are as follows:
  • BM-MSCs The preparation method of exosomes derived from mesenchymal stem cells: the primary rat (Sprague-Dawley rat, 60-80g) BM-MSCs were isolated by the differential attachment method, and the BM-MSCs were subcultured and expanded to the 3rd-4th generation for later use.
  • BM-MSCs complete medium IMDM, Gibco, USA
  • IMDM IMDM
  • the conditioned medium was harvested and the exosomes (MSC TXL -Exo) secreted by BM-MSCs pretreated with Tongxinluo were isolated by ultracentrifugation.
  • the specific steps of the ultracentrifugation method include: after collecting the conditioned medium, centrifuge at 300g for 10min to remove cells; centrifuge at 2000g for 20min to remove cell debris; Obtain exosomes.
  • MSC TXL -Exo prepared by pretreatment of Tongxinluo was identified, and then functional evaluation was carried out, including proof that intramyocardial injection improved rat heart function, reduced myocardial infarction size, reduced myocardial cell apoptosis and improved microenvironment; MSC- Exo and MSC TXL -Exo high-throughput sequencing to find out significantly changed miRNA, and further detection of miRNA-146a RT-PCR level in MSC TXL -Exo, which proves that the protection of MSC TXL -Exo on the heart is mainly caused by miRNA- 146a-mediated.
  • the MSC TXL -Exo separated by ultracentrifugation is spherical or disc-shaped under the electron microscope, with a size of about 100nm; NTA analysis shows that the particle size distribution is in the range of 30-150nm. There was no significant difference in the shape and particle size distribution of exosomes secreted by BM-MSCs after Tongxinluo pretreatment and without pretreatment.
  • Figure 1A- Figure 1B The specific results can be seen in Figure 1A- Figure 1B, in which, Figure 1A: Observation of the morphology and structure of mesenchymal stem cell-derived exosomes (MSC-Exo) under an electron microscope, which is spherical or disc-shaped, with a size of about 100nm, passed through the heart After Tongxinluo pretreatment, the morphology remained unchanged; Figure 1B: The particle size distribution of MSC-Exo was analyzed by NTA, and the particle size distribution of Tongxinluo pretreated and untreated MSC-Exo were both in the range of 30-150nm; Figure 2, western The blot showed that exosomes highly expressed Alix, TSG101, CD63 and other exosome marker proteins.
  • MSC-Exo mesenchymal stem cell-derived exosomes
  • intramyocardial injection of MSC TXL -Exo can significantly improve cardiac function after myocardial infarction, reduce infarct size, reduce myocardial cell apoptosis, and reduce inflammatory factors release.
  • Figure 3A- Figure 5 Transplantation of mesenchymal stem cell-derived exosomes (MSC TXL -Exo) pretreated by Tongxinluo significantly improved cardiac function in rats with myocardial infarction;
  • Figure 3C- Figure 3D Masson staining showed that MSC TXL -Exo transplantation significantly reduced the size of myocardial infarction in rats;
  • Figure 4 Apoptotic cell staining suggested that MSC TXL -Exo transplantation could reduce cardiomyocyte apoptosis;
  • Figure 5 ELISA detection showed that MSC TXL -Exo Transplantation can effectively reduce the release of inflammatory factors, thereby improving the microenvironment of transplantation.
  • Pretreatment of mesenchymal stem cells with 400 ⁇ g/mL TXL for 24 hours can obtain exosomes that can improve cardiac function, reduce myocardial infarction size, and promote angiogenesis, and the mechanism is related to up-regulation of miR-146a levels in exosomes .

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Abstract

提供了一种基于通心络预处理的间充质干细胞来源外泌体及其制备方法。所提供的一种上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法，包括：采用通心络预处理间充质干细胞，并培养处理后的间充质干细胞以收集其分泌的外泌体。该方法可制备出具有促进血管新生和/或心肌修复能力的间充质干细胞来源外泌体。

Description

基于通心络预处理的间充质干细胞来源外泌体及其制备方法 技术领域
本发明是关于一种基于药物预处理的间充质干细胞来源外泌体及其制备方法，具体是关于一种基于药物通心络预处理的间充质干细胞来源的高效外泌体及其制备方法。
背景技术
WHO统计数据表明：2016年心血管疾病是全球第一大死因(约1760万人，32.2％)尽管目前医疗水平已大大提升，但急性心肌梗死(acute myocardial infarction,AMI)仍然是目前全球致死致残的主要原因之一(GBD 2016 Mortality Collaborators.Global,regional,and national under-5 mortality,adult mortality,age-specific mortality,and life expectancy,1970-2016:a systematic analysis for the Global Burden of Disease Study 2016.Lancet.2017 Sep 16；390(10100):1084-1150；Roth,G.A.,et al.,Global,Regional,and National Burden of Cardiovascular Diseases for 10 Causes,1990 to 2015.J Am Coll Cardiol,2017.70(1):p.1-25；Virani,S.S.,et al.,Heart Disease and Stroke Statistics-2020 Update:A Report From the American Heart Association.Circulation,2020.141(9):p.e139-e596.)。AMI可导致大量心肌在短时间内缺血坏死，继而被瘢痕组织替代引发心衰和死亡。现有治疗手段无法有效再生和修复心肌。近年来干细胞移植治疗，尤其是间充质干细胞(MSCs,mesenchymal stem cells)，例如骨髓间充质干细胞(bone marrow-mesenchymal stem cells,BM-MSCs)，移植治疗AMI被寄予厚望(Orlic D,Kajstura J,Chimenti S,Jakoniuk I,Anderson SM,Li B,Pickel J,McKay R,Nadal-Ginard B,Bodine DM and others.Bone marrow cells regenerate infarcted myocardium.Nature2001；410(6829):701-705；Fisher SA,Doree C,Mathur A,Martin-Rendon E.Meta-Analysis of Cell Therapy Trials for Patients With Heart Failure.Circulation Research 2015；116(8):1361-1377；Afzal MR,Samanta A,Shah ZI,Jeevanantham V,Abdel-Latif A,Zuba-Surma EK,Dawn B.Adult Bone Marrow Cell Therapy for Ischemic Heart Disease:Evidence and Insights From Randomized Controlled Trials.Circ Res 2015；117(6):558-575)。MSCs的旁分泌功能是他们发挥作用的重要方式(Ranganath,S.H.,et al.,Harnessing the mesenchymal stem cell secretome for the treatment of  cardiovascular disease.Cell Stem Cell,2012.10(3):p.244-58.)，然而临床研究表明，间充质干细胞虽能一定程度通过旁分泌保护作用改善梗死后心功能，但效果并不显著。进一步研究表明，MSCs通过旁分泌的方式分泌的一种细胞外囊泡结构——外泌体(exosome)被广泛报道可参与AMI后改善微环境以及修复心肌(Cheng,H.,et al.,Hypoxia-challenged MSC-derived exosomes deliver miR-210 to attenuate post-infarction cardiac apoptosis.Stem Cell Res Ther,2020.11(1):p.224；Tan,S.J.O.,et al.,Novel Applications of Mesenchymal Stem Cell-derived Exosomes for Myocardial Infarction Therapeutics.Biomolecules,2020.10(5)；Xiao,C.,et al.,Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125b.Circ Res,2018.123(5):p.564-578.)。外泌体可以广泛携带生物活性分子，包括RNA、蛋白质等，其中microRNA被广泛报道可以参与心脏修复过程中(Gebert,L.F.R.and I.J.MacRae,Regulation of microRNA function in animals.Nat Rev Mol Cell Biol,2019.20(1):p.21-37；Zhou,S.S.,et al.,miRNAS in cardiovascular diseases:potential biomarkers,therapeutic targets and challenges.Acta Pharmacol Sin,2018.39(7):p.1073-1084.)，而miRNA-146a被广泛报道具有心脏修复、抗纤维化等功能(Boon RA,Dimmeler S.MicroRNAs in myocardial infarction.Nat Rev Cardiol.2015 Mar；12(3):135-42；Wang X,Ha T,Liu L,Zou J,Zhang X,Kalbfleisch J,Gao X,Williams D,Li C.Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury.Cardiovasc Res.2013 Mar 1；97(3):432-42；Barile L,Moccetti T,Marbán E,Vassalli G.Roles of exosomes in cardioprotection.Eur Heart J.2017 May 7；38(18):1372-1379)。同时外泌体具有来源广、稳定、无免疫原性等优点。另一方面，BM-MSCs来源的外泌体(MSC-Exo)可改善心梗微环境及促进心脏修复，因此，有望成为新一代的心肌修复产品，为临床治疗急性心肌梗死提供新思路(Lamichhane TN,Sokic S,Schardt JS,Raiker RS,Lin JW,Jay SM.Emerging Roles for Extracellular Vesicles in Tissue Engineering and Regenerative Medicine.Tissue Engineering Part B:Reviews 2015；21(1):45-54)。
发明内容
本发明的一个目的在于提供一种基于通心络药物预处理提高间充质干细胞来源外泌体效能的制备方法。
本发明的另一目的在于提供所制备的间充质干细胞来源外泌体。
本发明的研究发现，通心络(Tongxinluo,TXL)预处理间充质干细胞，可制备出具有显著改善心梗后心功能、减少心梗面积、减少心肌细胞凋亡及减轻炎症能力的间充质干细胞来源外泌体。
从而，一方面，本发明提供了一种上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法，该方法包括：采用通心络预处理间充质干细胞，并培养处理后的间充质干细胞以收集其分泌的外泌体。
本发明中所用通心络(Tongxinluo,TXL)为一种药物，其应符合相关质量标准要求。
根据本发明的具体实施方案，本发明的上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法包括：
在间充质干细胞的培养基中加入通心络预处理间充质干细胞12-24小时后，更换细胞培养基为无外泌体的完全培养基继续培养；48小时后收集条件培养基并利用超速离心法分离得到经通心络预处理间充质干细胞分泌的外泌体。
根据本发明的具体实施方案，优选地，采用通心络预处理间充质干细胞的时间为24h。本发明发现，处理时间为24h时，收集到的细胞上清里面的外泌体表达的miR-146a显著上调。
根据本发明的具体实施方案，本发明的上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法中，超速离心法包括：
收集条件培养基后，依次离心去除细胞、离心去除细胞碎片、离心去除大囊泡，然后离心收取沉淀并重悬后，再次离心获得外泌体。
具体地，本发明的上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法中，离心法包括：收集条件培养基后，300g离心10min去除细胞；2000g离心20min去除细胞碎片；16500g高速离心30min去除大囊泡；120000g超速离心70min收取沉淀并重悬后，再次120000g超速离心70min获得外泌体。
根据本发明的具体实施方案，本发明的上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法中，所述间充质干细胞包括骨髓间充质干细胞或脂肪间充质干细胞。
另一方面，本发明还提供了按照本发明所述方法制备得到的外泌体。
与现有技术得到的外泌体相比，本发明制备得到的外泌体高表达miR-146a；所述miR-146a可减轻心梗后反应炎症、细胞凋亡、缩小心梗面积。
另一方面，本发明还提供了通心络在制备间充质干细胞分泌高表达miR-146a并且具有改善心梗后心功能、减少梗死面积、减少心肌细胞凋亡及减轻炎症的外泌体的制剂中的应用。
根据本发明的一些具体实施方案，本发明中，利用400μg/mL通心络预处理间充质干细胞12-24小时，优选为24h。
根据本发明的具体实施方案，本发明中，所述间充质干细胞包括骨髓间充质干细胞或脂肪间充质干细胞。
在本发明的一个具体实施方案中，利用400μg/mL TXL预处理BM-MSC 24小时可获得miR-146a含量更高的外泌体，可改善心梗后心功能、减小梗死面积、减少心肌细胞凋亡及减轻炎症。
附图说明
图1A-图1B显示本发明实施例中间充质干细胞来源外泌体在电镜下的形态结构以及外泌体粒径分析(NTA)的结果。
图2显示本发明实施例中间充质干细胞来源外泌体高表达Alix,TSG101,CD63等外泌体标记蛋白。
图3A-图3B显示通心络预处理间充质干细胞来源外泌体可显著改善大鼠心梗后心功能的检测结果。
图3C-图3D显示通心络预处理间充质干细胞来源外泌体可减少心梗面积的检测结果。
图4显示通心络预处理间充质干细胞来源外泌体可减少心肌细胞凋亡有效。
图5显示通心络预处理间充质干细胞来源外泌体可显著减轻相关炎症因子的释放。
图6显示通心络预处理间充质干细胞来源外泌体与普通间充质干细胞来源外泌体差异的火山图。
图7显示通心络预处理间充质干细胞来源外泌体中与心脏保护作用相关的miRNA-146a比普通间充质干细胞来源外泌体升高了近10倍。
具体实施方式
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。
实施例中所用TXL购自石家庄以岭药业股份有限公司，其主要成分如下：
通心络主要成分 药材部位 比例(％)
人参 根和地下茎 1.7
赤芍 根 1.6
酸枣仁 种 1.2
檀香提取物 树干心材 0.4
降香 树干心材和根 4.0
乳香 树脂 6.0
冰片 C 10H 18O 3.6
水蛭 干体 23.6
全蝎 干体 18.1
土蟞虫 雌性干体 18.1
蜈蚣 干体 3.6
蝉蜕 皮 18.1
实施例1
间充质干细胞来源外泌体的制备方法：
间充质干细胞来源外泌体的制备方法：利用差速贴壁法分离原代大鼠(Sprague-Dawley大鼠，60-80g)BM-MSCs并传代扩增至第3-4代备用。在BM-MSCs完全培养基(IMDM,Gibco,美国)中加入400μg/mL通心络预处理24小时后，更换细胞培养基为IMDM部分培养基(不加入胎牛血清)继续培养。48小时后收取条件培养基并利用超速离心法分离得到经通心络预处理BM-MSCs分泌的外泌体(MSC TXL-Exo)。超速离心法具体步骤包括：收取条件培养基后，300g离心10min 去除细胞；2000g离心20min去除细胞碎片；16500g高速离心30min去除大囊泡；120000g超速离心70min收取沉淀并重悬后，再次120000g超速离心70min获得外泌体。
对制备好的MSC TXL-Exo进行鉴定：包括电镜(FEI，Tecnai G2 Spirit BioTwin)分析观察形态结构，NTA(PARTICLE METRIXs,ZetaVIEW S/N 17-310)分析外泌体的粒径分布。
对通心络预处理制备的MSC TXL-Exo进行鉴定，然后进行功能学评价，包括证明心肌内注射后改善大鼠心功能、减少心梗面积、减轻心肌细胞凋亡及改善微环境；MSC-Exo和MSC TXL-Exo高通量测序，找出显著改变的miRNA，并进一步进行MSC TXL-Exo中miRNA-146a RT-PCR水平检测，即证明MSC TXL-Exo对心脏的保护主要是由miRNA-146a介导。
评价指标(研究结果)
利用超速离心法分离得到的MSC TXL-Exo在电镜下呈现球形或圆盘形，大小在100nm左右；NTA分析其粒径分布在30-150nm范围内。通心络预处理后与无预处理的BM-MSC分泌的外泌体在形态、粒径分布上没有显著差异。具体结果可参见图1A-图1B，其中，图1A：电镜下观察间充质干细胞来源外泌体(MSC-Exo)的形态结构，呈球形或圆盘形，大小在100nm左右，经通心络预处理后形态不变；图1B：用NTA分析MSC-Exo的粒径分布，通心络预处理和未处理的MSC-Exo的粒径分布均在30-150nm范围内；图2，western blot显示外泌体高表达Alix,TSG101,CD63等外泌体标记蛋白。
与未经通心络预处理的MSC-Exo相比，MSC TXL-Exo心肌内注射后可显著改善大鼠心梗后心功能、减小梗死面积，并能减少心肌细胞凋亡、减少炎症因子的释放。具体可参见图3A-图5，其中，图3A-图3B：经通心络预处理间充质干细胞来源外泌体(MSC TXL-Exo)移植显著改善心梗大鼠心功能；图3C-图3D：Masson染色显示MSC TXL-Exo移植显著减小大鼠心梗面积；图4：凋亡细胞染色提示MSC TXL-Exo移植可以减少心肌细胞凋亡；图5:ELISA检测提示MSC TXL-Exo移植可以有效减轻炎症因子的释放，从而改善移植微环境。*：P<0.05；**：P<0.01；***：P<0.001。
与未经通心络预处理的MSC-Exo相比，高通量测序的结果显示MSC TXL-Exo中miRNA-146a显著增加，升高了近4倍。图6:高通量测序的结果显示，MSC TXL-Exo 中miRNA-146a显著增加；图7：RT-PCR结果显示MSC TXL-Exo中miRNA-146a较MSC-Exo升高了约10倍。而miR-146a在减轻心肌纤维化、减少细胞凋亡、减轻炎症反应等方面发挥着重要作用。
结论：利用400μg/mL TXL预处理间充质干细胞24小时后可获得具有改善心功能、减少心梗面积、促进血管新生的外泌体，其机制与上调外泌体中的miR-146a水平有关。

Claims (11)

  1. 一种上调miR-146a的表达水平的间充质干细胞来源外泌体的制备方法，该方法包括：采用通心络预处理间充质干细胞，并培养处理后的间充质干细胞以收集其分泌的外泌体。
  2. 根据权利要求1所述的方法，该方法包括：
    在间充质干细胞的培养基中加入通心络预处理间充质干细胞12-24小时后，更换细胞培养基为无外泌体的完全培养基继续培养；48小时后收集条件培养基并利用超速离心法分离得到经通心络预处理间充质干细胞分泌的外泌体。
  3. 根据权利要求2所述的方法，其中，通心络预处理间充质干细胞的时间为24h。
  4. 根据权利要求2所述的方法，其中，超速离心法包括：
    收集条件培养基后，依次离心去除细胞、离心去除细胞碎片、离心去除大囊泡，然后离心收取沉淀并重悬后，再次离心获得外泌体。
  5. 根据权利要求4所述的方法，其中，收集条件培养基后，300g离心10min去除细胞；2000g离心20min去除细胞碎片；16500g高速离心30min去除大囊泡；120000g超速离心70min收取沉淀并重悬后，再次120000g超速离心70min获得外泌体。
  6. 根据权利要求1-5中任一项所述的方法，其中，所述间充质干细胞包括骨髓间充质干细胞或脂肪间充质干细胞。
  7. 按照权利要求1-6中任一项所述方法制备得到的外泌体。
  8. 根据权利要求7所述的外泌体，其中，所述外泌体高表达miRNA-146a。
  9. 通心络在制备促进间充质干细胞分泌高表达miR-146a并且具有改善心梗后心功能、减少梗死面积、减少心肌细胞凋亡及减轻炎症的外泌体的制剂中的应用。
  10. 根据权利要求9所述的应用，其中，利用400μg/mL通心络加入间充质干细胞的IMDM培养基中预处理12-24小时。
  11. 根据权利要求9或10所述的应用，其中，所述间充质干细胞包括骨髓间充质干细胞或脂肪间充质干细胞。
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