WO2023098088A1 - 外泌体在胸主动脉夹层/动脉瘤中的作用 - Google Patents

外泌体在胸主动脉夹层/动脉瘤中的作用 Download PDF

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WO2023098088A1
WO2023098088A1 PCT/CN2022/105071 CN2022105071W WO2023098088A1 WO 2023098088 A1 WO2023098088 A1 WO 2023098088A1 CN 2022105071 W CN2022105071 W CN 2022105071W WO 2023098088 A1 WO2023098088 A1 WO 2023098088A1
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exosomes
aneurysm
aortic dissection
week
bapn
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李杨欣
刘冯
沈振亚
张瑜
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苏州大学
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    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0668—Mesenchymal stem cells from other natural sources
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  • the present invention relates to the field of biomedicine, in particular to the role of exosomes in thoracic aortic dissection/aneurysm.
  • Thoracic aortic dissection/aneurysm is an aggressive vascular disease requiring early diagnosis and effective treatment.
  • TAAD refers to the permanent expansion of the entire thickness of the aortic wall, and the diameter of the lumen exceeds 50% of the normal to form an aortic aneurysm.
  • the annual incidence of thoracic aortic dissection/aneurysm is about 7-9 cases/100,000.
  • the mortality rate will be as high as 50%-68% within 48 hours, and 90% within 3 months.
  • the annual case fatality rate is still 20%.
  • acute TAAD is mainly treated with surgical thoracotomy or endovascular repair, but the mortality rate during the operation is high, and the postoperative complications and recurrence rate are high. Therefore, for the pathogenesis of TAAD, it is particularly important to find a drug that can precisely target the pathological changes in the early stage of the disease.
  • the present invention provides exosomes that can delay the occurrence of arterial dissection/aneurysm.
  • Exosomes can delay the occurrence of thoracic aortic dissection/aneurysm in a minimally invasive way without thoracotomy, and improve the survival rate.
  • the present invention provides the application of exosomes in the preparation of drugs or preparations for preventing, delaying and/or treating thoracic aortic dissection/aneurysm.
  • the exosomes are administered via the inner canthal vein, each administration dose is 100 ⁇ L, and injected 3 times a week.
  • the inducer of the thoracic aortic dissection/aneurysm animal model includes ⁇ -aminopropionitrile (BAPN).
  • BAPN ⁇ -aminopropionitrile
  • the concentration of BAPN in the first week and the second week is 6 mg/mL, and the concentration of BAPN in the third week and the fourth week increases. Up to 8mg/mL.
  • the method for constructing exosomes includes the following steps:
  • Step 1 UMSC cells in logarithmic growth phase and in good growth state were mixed with fetal bovine serum and centrifuged to remove exosomes previously present in the serum, and ⁇ -Cultivate in MEM medium for 48 hours, collect the supernatant of the medium when the cell density reaches 90%, and pass passage;
  • Step 2 take the supernatant collected in step 1, centrifuge, remove dead cells and debris, and collect the supernatant;
  • Step 3 Take the supernatant collected in step 2, mix it with the exosome isolation reagent, extract at 4°C for 24 hours, and obtain the extract;
  • Step 4 Centrifuge the extract obtained in step 3, collect the precipitate, resuspend in PBS, and measure the concentration of exosomes; store the exosomes with the measured concentration at -80°C in ultra-low temperature.
  • the centrifugation described in step 1 is specifically: centrifugation at a speed of 110000g for 7h;
  • the centrifugation described in step 2 is specifically: centrifuging at a speed of 2000g for 30min;
  • the centrifugation described in step 4 is specifically: centrifuging at 13000g for 1h at 4°C.
  • the exosome isolation reagent in step 3 includes PEG4000; the volume ratio of the exosome isolation reagent to the supernatant is 1:2.
  • the present invention also provides drugs or preparations for preventing, delaying and/or treating thoracic aortic dissection/aneurysm, including exosomes and pharmaceutically acceptable excipients;
  • the exosomes were prepared according to the construction method described.
  • the construction method of the exosomes includes the following steps:
  • Step 1 UMSC cells in logarithmic growth phase and in good growth state were mixed with fetal bovine serum and centrifuged to remove exosomes previously present in the serum, and ⁇ -Cultivate in MEM medium for 48 hours, collect the supernatant of the medium when the cell density reaches 90%, and pass passage;
  • Step 2 take the supernatant collected in step 1, centrifuge, remove dead cells and debris, and collect the supernatant;
  • Step 3 Take the supernatant collected in step 2, mix it with the exosome isolation reagent, extract at 4°C for 24 hours, and obtain the extract;
  • Step 4 Centrifuge the extract obtained in step 3, collect the precipitate, resuspend in PBS, and measure the concentration of exosomes; store the exosomes with the measured concentration at -80°C in ultra-low temperature.
  • the centrifugation described in step 1 is specifically: centrifugation at a speed of 110000g for 7h;
  • the centrifugation described in step 2 is specifically: centrifuging at a speed of 2000g for 30min;
  • the centrifugation described in step 4 is specifically: centrifuging at 13000g for 1h at 4°C.
  • the exosome isolation reagent in step 3 includes PEG4000; the volume ratio of the exosome isolation reagent to the supernatant is 1:2.
  • the present invention also provides a drug combination for preventing, delaying and/or treating thoracic aortic dissection/aneurysm, including exosomes, any other active ingredients and pharmaceutically acceptable excipients.
  • the experimental results of the present invention show that exosomes can delay the occurrence of thoracic aortic dissection/aneurysm in a minimally invasive manner without thoracotomy, and improve the survival rate.
  • the survival rate of the normal mouse group is 100% within 30 days, and the survival rate of the mice induced with BAPN drugs to establish aortic dissection/aneurysm began to decrease at the beginning of the third week, until the experiment At the 4th week of the cycle, the overall survival rate was maintained at 50%.
  • the survival rate of the mice decreased slightly after the third week.
  • the survival rate maintained at 70%. From the above data, we can conclude that the injection of exosomes significantly delayed the occurrence of thoracic aortic dissection/aneurysm.
  • Figure 1 shows the identification results of exosomes derived from human umbilical cord mesenchymal stem cells; wherein, the present invention uses an inverted microscope to observe the morphology of human umbilical cord mesenchymal stem cells derived from exosomes, and it can be seen that it is in a long spindle shape, such as As shown in Figure 1A, the supernatant of the cell culture medium was then collected, and impurities such as cell debris were gradually removed by differential centrifugation.
  • the exosomes were extracted and separated using PEG4000 and an exosome extraction kit, and the collected human umbilical cord
  • the exosomes derived from mesenchymal stem cells were identified; the scanning electron microscope was used for morphological observation, and the results showed that the exosomes had a diameter of about 60 nm and were round particles with a typical cup-shaped shape, as shown in Figure 1B;
  • the results of the nanoparticle size analyzer showed that the diameter of exosomes was distributed between 30-100 nm, as shown in Figure 1C; flow cytometry further detected the expression of the exosome marker protein CD63, as shown in Figure 1D It was found that the CD63+ content in exosomes was as high as 99.9%;
  • Western Blot detected the expression of exosome marker protein TSG101, and found that exosomes had the characteristics of high expression of TSG101, as shown in Figure 1E; the above identification results showed that we isolated The exosomes derived from human umbilical cord mesenchymal
  • Size size (diameter nanometers); Number (Percent): number (percentage); CD63: exosome marker protein; Count: number; UMSC-Exo: human umbilical cord mesenchymal stem cells Exosomes; UMSC: human umbilical cord mesenchymal stem cells; TSG101: exosome marker protein; GAPDH: internal reference;
  • Fig. 2 shows the construction result of aortic dissection/aneurysm model
  • the present invention has set up mouse thoracic aortic dissection/aneurysm model by the mode of BAPN administration, we can find that with the advancement of administration time, small The diameter of the aortic arch of the mice gradually increased, and even obvious swelling appeared (Fig. 2A); EVG elastic fibers showed that the normal structure of the aortic wall was destroyed, and the arrangement of elastic and stress fibers on the blood vessels was gradually disordered (Fig. 2B); HE staining showed The arrangement of smooth muscle cells on the aortic arch was disordered and the number decreased (Figure 2C); Figure D shows the difference in the aorta before and after administration;
  • BAPN 1W BPAN administration for 1 week
  • Control control
  • TAAD thoracic aortic dissection/aneurysm
  • Figure 3 shows that exosomes can slow down the occurrence of aortic dissection/aneurysm; among them, in order to explore the role of exosomes, we constructed a mouse model of thoracic aortic dissection/aneurysm according to the above method, and injected exosomes intravenously, Then a series of tests were carried out; the experiment was divided into 3 groups, namely the normal group, the BAPN administration group and the BAPN administration + exosomes group; by detecting the weight change within 4 weeks, it was found that there was no significant weight difference among the three groups. The difference between BAPN administration group + exosomes group (Fig.
  • POST after treatment, here refers to after administration; 1W: 1 week; BodyWeight: body weight; Control: control; Exo: exosomes; BAPN+Exo: administration of BAPN, while injecting exosomes; Percent survial (%): survival rate; MMP9PlasmaInterleukin: the expression level of matrix metalloproteinase 9; Diameter (mm): diameter (millimeter); Maximal diameter (mm): maximum diameter (millimeter); Sinus: sinus portion; AscendingAorta: rising main; Bow: bow.
  • the present invention discloses the role of exosomes in thoracic aortic dissection/aneurysm. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • exosomes play a certain role as the carrier of biological information in physiological and pathological processes such as immune response, apoptosis, angiogenesis, and inflammatory response.
  • the safety has been confirmed by research, so exosomes derived from various stem cells have great therapeutic prospects.
  • Exosomes are secreted by a variety of cells and are lipid membrane vesicles with a diameter of 30-100 nm. Exosomes contain a variety of DNA, ncRNA, proteins, etc., which play an indispensable role in many physiological and pathological processes.
  • Exosomes are mainly derived from a variety of mesenchymal stem cells, and exosomes derived from mesenchymal stem cells have been considered as new drug candidates for cell-free therapy of various diseases.
  • human umbilical cord mesenchymal stem cells have the advantages of a wide range of sources, easy access, and no ethical and moral issues. They are expected to become seed cells in the process of clinical research, treatment and application.
  • exosomes are regarded as important mediators of intercellular communication in many physiological and pathological processes.
  • the experimental results of the present invention show that: the survival rate of the normal mouse group within 30 days is 100%, and the survival rate of the aortic dissection/aneurysm group induced by BAPN drugs begins to decrease at the beginning of the third week, until the experimental period is 4 weeks At this time, the overall survival rate was maintained at 50%, and on this basis, in the exosome injection group, we can see that the survival rate of the mice decreased slightly after the third week, and the survival rate was maintained at 70% by the 4th week of the experimental cycle. %. From the above data, we can conclude that the injection of exosomes significantly delayed the occurrence of thoracic aortic dissection/aneurysm.
  • the raw materials and reagents used can be purchased from the market.
  • Embodiment 2 Construction of mouse thoracic aortic dissection/aneurysm model
  • BAPN ⁇ -aminopropionitrile
  • mice were anesthetized by respiratory inhalation with 2% isoflurane gas. After successful anesthesia, the skin was prepared on the chest and abdomen of the mice, and the hair on the body surface was removed to reduce the impact on the ultrasound images. The mice were placed in the supine position on the echocardiography platform, the limbs were fixed with adhesive tape on the flat-panel electrocardiogram monitoring position, the ultrasonic coupling agent was applied to the extremities, and the heated ultrasonic coupling agent was evenly applied to the chest and abdomen of the mouse.
  • the MS400C ultrasonic probe fix the ultrasonic probe above the echocardiography platform and adjust it down to a suitable position to avoid exerting too much pressure on the thoracic and abdominal cavity of the mouse while maintaining a clear image in the thoracic and abdominal cavity.
  • Find the long-axis section of the heart along the median direction adjust the probe to the direction of the left ventricular outflow tract until the ascending aorta of the mouse can be completely observed, keep the image, then move the probe to the abdomen, and press down the probe to give a little pressure to the abdominal cavity, find The blood vessel that is still full under pressure is the abdominal aorta.
  • the largest transverse diameter of the thoracoabdominal aorta was measured for three times and the data was recorded. Pay attention to observe the vital signs such as the heart rate and respiratory rate of the mice, and the operation time of each mouse is about 5-10 minutes.
  • BAPN is an inhibitor of lysyl oxidase, and the latter participates in the cross-linking of elastin fibers and collagen fibers. Plays an important role in maintaining the homeostasis of the elastic plate. Lysyl oxidase activity decreases with age, so mice should be selected for BAPN treatment during the juvenile fast-growing period rather than in adulthood.

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Abstract

涉及生物医学领域，特别涉及外泌体在胸主动脉夹层/动脉瘤中的作用。正常小鼠组在30天内的生存率是100％，使用BAPN药物诱导小鼠建立主动脉夹层/动脉瘤组的生存率在第三周初时开始降低，到实验周期4周时，总体生存率维持在50％，而在此基础上，注射外泌体组，可以看到小鼠生存率在第三周后略微降低，到实验周期4周时，生存率维持在70％。由以上数据，可以得出注射外泌体后，显著延缓了胸主动脉夹层/动脉瘤的发生。

Description

外泌体在胸主动脉夹层/动脉瘤中的作用
本申请要求于2021年11月30日提交中国专利局、申请号为202111444763.1、发明名称为“外泌体在胸主动脉夹层/动脉瘤中的作用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医学领域，特别涉及外泌体在胸主动脉夹层/动脉瘤中的作用。
背景技术
胸主动脉夹层/动脉瘤(TAAD)是一种侵袭性血管疾病，需要早期诊断和有效治疗。但是，由于特定的血管结构和病变部位狭窄，目前尚无有效的治疗TAAD的方法。TAAD是指主动脉壁全层发生永久性扩张，管腔直径超过正常50％形成主动脉瘤，随着血流的冲击，主动脉内壁发生损伤，高速血流通过撕裂内膜形成夹层。胸主动脉夹层/动脉瘤的年发病率约为7～9例/10万，破裂后若未及时救治，48h时内病死率高达50％～68％，3个月内可达90％，10年病死率仍为20％。目前临床上急性TAAD主要采用外科开胸手术或血管内修复治疗，但手术期病死率高，术后并发症多、复发率高。因此，针对TAAD发病机制，寻找一种可在疾病早期针对病理学改变进行精准靶向治疗的药物尤为重要。
发明内容
有鉴于此，本发明提供外泌体可以延缓动脉夹层/动脉瘤的发生。外泌体可以通过不开胸，以微创的方式延缓胸主动脉夹层/动脉瘤的发生，提高了存活率。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了外泌体在制备预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物或制剂中的应用。
在本发明的一些具体实施方案中，所述外泌体的施用方式为内眦静脉 施用，每次施用剂量100μL，每周注射3次。
在本发明的一些具体实施方案中，所述胸主动脉夹层/动脉瘤动物模型的诱导剂包括β-氨基丙腈(BAPN)。
在本发明的一些具体实施方案中，所述胸主动脉夹层/动脉瘤动物模型的构建方法中，第一周和第二周BAPN浓度为6mg/mL，第三周和第四周BAPN浓度增大至8mg/mL。
在本发明的一些具体实施方案中，所述外泌体的构建方法包括如下步骤：
步骤1：将处于对数生长期、生长状态良好的UMSC细胞，与胎牛血清混合后离心，去除先前存在于血清中的外泌体，使用含有10％不含外泌体胎牛血清的α-MEM培养基培养48h，待细胞密度达到90％时收集培养基的上清，传代；
步骤2：取步骤1收集的所述上清，离心，除去死的细胞和碎片，收集上清；
步骤3：取步骤2收集的所述上清，与外泌体分离试剂混匀后4℃条件下抽提24h，获得抽提液；
步骤4：取步骤3获得的抽提液离心，收集沉淀，经PBS重悬，测定外泌体的浓度；将测好浓度的外泌体置于-80℃超低温保存。
在本发明的一些具体实施方案中，步骤1中所述离心具体为：以110000g的速度离心7h；
步骤2中所述离心具体为：以2000g的速度离心30min；
步骤4中所述离心具体为：4℃，以13000g的速度离心1h。
在本发明的一些具体实施方案中，步骤3中所述外泌体分离试剂包括PEG4000；所述外泌体分离试剂与所述上清的体积比为1：2。
更重要的是，本发明还提供了预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物或制剂，包括外泌体和药学上可接受的辅料；
所述外泌体如所述构建方法制得。
具体的，所述外泌体的构建方法包括如下步骤：
步骤1：将处于对数生长期、生长状态良好的UMSC细胞，与胎牛 血清混合后离心，去除先前存在于血清中的外泌体，使用含有10％不含外泌体胎牛血清的α-MEM培养基培养48h，待细胞密度达到90％时收集培养基的上清，传代；
步骤2：取步骤1收集的所述上清，离心，除去死的细胞和碎片，收集上清；
步骤3：取步骤2收集的所述上清，与外泌体分离试剂混匀后4℃条件下抽提24h，获得抽提液；
步骤4：取步骤3获得的抽提液离心，收集沉淀，经PBS重悬，测定外泌体的浓度；将测好浓度的外泌体置于-80℃超低温保存。
在本发明的一些具体实施方案中，步骤1中所述离心具体为：以110000g的速度离心7h；
步骤2中所述离心具体为：以2000g的速度离心30min；
步骤4中所述离心具体为：4℃，以13000g的速度离心1h。
在本发明的一些具体实施方案中，步骤3中所述外泌体分离试剂包括PEG4000；所述外泌体分离试剂与所述上清的体积比为1：2。
此外，本发明还提供了预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物组合，包括外泌体、任意其他有效成分和药学上可接受的辅料。
本发明实验结果表明：外泌体可以通过不开胸，以微创的方式延缓胸主动脉夹层/动脉瘤的发生，提高了存活率。如3B所示，我们可以看到正常小鼠组在30天内的生存率是100％，使用BAPN药物诱导小鼠建立主动脉夹层/动脉瘤组的生存率在第三周初时开始降低，到实验周期4周时，总体生存率维持在50％，而在此基础上，注射外泌体组，我们可以看到小鼠生存率在第三周后略微降低，到实验周期4周时，生存率维持在70％。由以上数据，我们可以得出注射外泌体后，显著延缓了胸主动脉夹层/动脉瘤的发生。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示人脐带间充质干细胞来源外泌体的鉴定结果；其中，本发明使用倒置显微镜观察了外泌体来源的人脐带间充质干细胞的形态，可以看到呈长梭形的状态，如图1A所示，随后通过收集细胞培养基上清，采用差速离心法逐步去除细胞碎片等杂质，使用PEG4000和外泌体抽提试剂盒将外泌体抽提分离，将收集到的人脐带间充质干细胞来源的外泌体进行鉴定；使用扫描电子显微镜进行形态学观察，结果显示，外泌体的直径在60nm左右，为具有典型杯状形态的圆形颗粒，如图1B所示；与此同时，纳米粒度分析仪结果显示，外泌体的直径分布在30-100nm之间，如图1C所示；流式细胞术进一步检测了外泌体标志蛋白CD63的表达，如图1D所示，发现外泌体中CD63+含量高达99.9％；Western Blot检测了外泌体标志蛋白TSG101的表达，发现外泌体具有高表达TSG101的特性，如图1E所示；以上鉴定结果表明我们分离得到的人脐带间充质干细胞来源的外泌体符合检测要求，可应用于后续实验；
图例解释：Size(d.nm):尺寸(直径纳米)；Numeber(Percent):数量(百分比)；CD63:外泌体标志蛋白；Count:数量；UMSC-Exo:人脐带间充质干细胞来源的外泌体；UMSC:人脐带间充质干细胞；TSG101:外泌体标志蛋白；GAPDH:内参；
图2示主动脉夹层/动脉瘤模型的构建结果；其中，本发明通过BAPN给药的方式，建立了小鼠胸主动脉夹层/动脉瘤模型，我们可以发现随着给药时间的推进，小鼠的主动脉弓直径逐渐变大，甚至出现明显的膨起(图2A)；EVG弹力纤维显示主动脉管壁正常结构破坏，血管上的弹力及应力纤维排布逐渐紊乱(图2B)；HE染色显示主动脉弓上平滑肌细胞排布紊乱，数量减少(图2C)；图D展示了给药前后主动脉的差异；
图例解释：BAPN 1W:BPAN给药1周；Control:对照；TAAD:胸主动脉夹层/动脉瘤；
图3示外泌体可以减缓主动脉夹层/动脉瘤的发生；其中，为了探究外泌体的作用，我们按照上述方法构建小鼠胸主动脉夹层/动脉瘤模型，通过静脉注射外泌体，随即进行了一系列的检测；实验分为3组，分别为正常组、BAPN给药组和BAPN给药+外泌体组；通过检测4周内的体重变化， 发现3组之间体重无明显的差异性(图3A)；BAPN给药组+外泌体组的生存率高于BAPN组(图3B)；Elisa检测不同组小鼠血液中MMP9的表达量，结果显示BAPN给药+外泌体组的表达量高于另外2组(图3C)；通过心超测量小鼠主动脉弓的直径，发现随着时间的推进，窦部、升主及弓部直径均增大(图3D、图3F、图3H)，我们又统计了给药4周后的直径差异，结果显示每组之间窦部及升主的直径无差异性(图E、G)，弓部直径具有差异性，且BAPN给药+外泌体组的直径小于BAPN组(图3I)；以上实验结果均表明外泌体可以减缓胸主动脉夹层/动脉瘤的发生；
图例解释：POST:处理后，在这里指给药后；1W:1周；BodyWeight:身体体重；Control:对照；Exo:外泌体；BAPN+Exo:给药BAPN，同时注射外泌体；Percent survial(％):生存率；MMP9PlasmaInterleukin:基质金属蛋白酶9的表达量；Diameter(mm):直径(毫米)；Maximal diameter(mm):最大直径(毫米)；Sinus:窦部；AscendingAorta:升主；Bow:弓部。
具体实施方式
本发明公开了外泌体在胸主动脉夹层/动脉瘤中的作用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
近年来，科研工作者提出“无细胞的干细胞治疗”，在功能上替代了干细胞，是一种新型的治疗方法。外泌体作为“无细胞的干细胞治疗”的主要方式，主要作为生物信息的载体在免疫应答、细胞凋亡、血管生成、炎性反应等生理病理过程中均发挥一定的作用，其治疗效果和安全性均已经得到研究的证实，因此多种干细胞来源的外泌体具有巨大的治疗前景。外泌体由多种细胞分泌产生，是直径在30-100nm之间的脂质膜性囊泡。外泌体内含有多种DNA、ncRNA、蛋白质等，在许多生理和病理过程中均发挥着不可或缺的作用。外泌体主要来源于多种间充质干细胞，间充质干细胞来源的外泌体已经被认为是各种疾病无细胞治疗的新候选药物。人脐带 间充质干细胞作为外泌体的主要来源，具有来源广泛、较易获取、无伦理道德问题等优点，有望成为临床研究治疗及应用过程中的种子细胞。外泌体作为旁分泌的主要形式，在许多生理和病理过程中被看作细胞间通讯的重要介质。
本发明实验结果表明：正常小鼠组在30天内的生存率是100％，使用BAPN药物诱导小鼠建立主动脉夹层/动脉瘤组的生存率在第三周初时开始降低，到实验周期4周时，总体生存率维持在50％，而在此基础上，注射外泌体组，我们可以看到小鼠生存率在第三周后略微降低，到实验周期4周时，生存率维持在70％。由以上数据，我们可以得出注射外泌体后，显著延缓了胸主动脉夹层/动脉瘤的发生。
本发明提供的外泌体在胸主动脉夹层/动脉瘤中的作用中，所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 外泌体的获取
①将处于对数生长期、生长状态良好的UMSC细胞均匀接种于10cm大皿内，将胎牛血清以110000g的速度离心7h，去除先前存在于血清中的外泌体，使用含有10％不含外泌体胎牛血清的α-MEM培养基培养。继续培养48h，待细胞密度达到90％时收集旧的培养基上清，细胞进行传代处理。
②将上述收集到的上清，以2000g的速度离心30min，目的是除去死的细胞和碎片。
③用吸管吸取新的上清，转移至新的50mL离心管中，加入1/2体积上清的PEG4000外泌体分离试剂，上下晃动，充分混匀后4℃条件下抽提过夜。
④24h后，设置离心机的参数温度为4℃，转速为13000g，时间1h，离心结束后可见离心管内壁及底部有沉淀产生，即为分离得到的外泌体。
⑤弃上清，根据沉淀的量用PBS重悬，采用BCA蛋白浓度定量 试剂盒测定外泌体的浓度。
⑥将测好浓度的外泌体置于-80℃超低温冰箱保存待用。
实施例2 小鼠胸主动脉夹层/动脉瘤模型的构建
采用β-氨基丙腈(BAPN)饮水的方式处理3周、C57BL/6J雄性小鼠，称取BAPN粉末，溶于无菌的双蒸水中，第一周和第二周BAPN浓度为6mg/mL，第三周和第四周BAPN浓度增大至8mg/mL。
实施例3 超声测量直径
对小鼠使用2％的异氟烷气体呼吸道吸入麻醉。麻醉成功后，小鼠胸腹部备皮，去除体表毛发以减少对超声图像的影响。将小鼠以仰卧位放置在心超机平台上，胶布固定四肢于平板心电图监测位置，涂抹超声耦合剂于四肢末端，在小鼠胸腹部均匀涂抹加温的超声耦合剂。采用MS400C超声探头，将超声探头固定于心超平台上方，向下调至合适的位置，避免给小鼠胸腹腔施加太大压力，同时保持胸腹腔内图像清晰。沿正中方向找到心脏长轴切面，将探头向左室流出道方向调整，直至能够完整观察出小鼠升主动脉，保留图像，再向腹部移动探头，并下压探头，给予腹腔少许压力，找到压力状态下依然充盈的血管，即为腹主动脉，取胸腹主动脉最大横径，测量三次，记录数据。注意观察小鼠心率、呼吸频率等生命体征，每只小鼠操作时间约5-10min。
效果例1 人脐带间充质干细胞来源外泌体的鉴定
实验目的：
检测我们获得的外泌体是否可应用于后续实验
实验方法和结果：
首先我们使用倒置显微镜观察了外泌体来源的人脐带间充质干细胞的形态，可以看到呈长梭形的状态，如图1A所示，随后通过收集细胞培养基上清，采用差速离心法逐步去除细胞碎片等杂质，使用PEG4000和外泌体抽提试剂盒将外泌体抽提分离。将收集到的人脐带间充质干细胞来 源的外泌体进行鉴定。首先，使用扫描电子显微镜进行形态学观察，结果显示，外泌体的直径在60nm左右，为具有典型杯状形态的圆形颗粒，如图1B所示。与此同时，纳米粒度分析仪结果显示，外泌体的直径分布在30-100nm之间，如图1C所示。流式细胞术进一步检测了外泌体标志蛋白CD63的表达，如图1D所示，发现外泌体中CD63+含量高达99.9％。Western Blot检测了外泌体标志蛋白TSG101的表达，发现外泌体具有高表达TSG101的特性，如图1E所示。以上鉴定结果表明，我们成功分离得到人脐带间充质干细胞来源的外泌体，可应用于后续实验。
效果例2 主动脉夹层/动脉瘤模型的构建
实验目的：
观测BAPN给药过程中主动脉的变化，以此来确定建模的成功
实验方法和结果：
在本发明中，我们采用BAPN饮水的方式构建小鼠主动脉夹层/动脉瘤模型，其原理主要是BAPN是赖氨酰氧化酶的抑制剂，后者参与弹性蛋白纤维和胶原蛋白纤维交联，在维持弹力板的稳态中起重要作用。随着年龄的增长，赖氨酰氧化酶活性下降，因此应选择在幼年快速生长期的小鼠给予BAPN处理，而不是在成年期。此外，在人群中男性主动脉夹层/动脉瘤发生率高于女性，并且C57BL/6J小鼠为心血管疾病研究常用小鼠，所以本研究最终选择3周龄雄性C57BL/6J小鼠作为实验对象。首先，我们通过BAPN给药的方式，建立了小鼠胸主动脉夹层/动脉瘤模型，我们可以发现随着给药时间的推进，小鼠的主动脉弓直径逐渐变大，甚至出现明显的膨起(图2A)；EVG弹力纤维显示主动脉管壁正常结构破坏，血管上的弹力及应力纤维排布逐渐紊乱(图2B)；HE染色显示主动脉弓上平滑肌细胞排布紊乱，数量减少(图2C)；图2D展示了给药前后主动脉的差异。
效果例3 外泌体可以减缓主动脉夹层/动脉瘤的发生
实验目的：
探究外泌体在胸主动脉夹层/动脉瘤中的作用。
实验方法和结果：
为了探究外泌体的作用，我们按照上述方法构建小鼠胸主动脉夹层/动脉瘤模型，通过内眦静脉注射外泌体，随即进行了一系列的检测。实验分为3组，分别为正常组、BAPN给药组和BAPN给药+外泌体组。
实验结果如下所示：
表1.图3A的数据
Control 3W 4W 5W 6W 7W
B21 6.7 10.8 14.4 17.4 22.6
B22 5.7 7.5 11.3 15.6 16.1
B23 7.7 11.9 15.1 16.1 20.8
BAPN          
B3 7.1 9.7 12.3 14.7 17.8
B7 6.1 9.2 13.3 16.4 19.8
B11 8.2 12.4 13.7 17.5 23.8
B17 7.3 11.4 14 17.3  
B14 6.5 11.1 15.1 18.1  
B19 6.9 8.9 12.5    
B24 5.9 7.2 11.3 16.7  
BAPN+Exo          
B1 6.3 8.9 12.8 17.9 21.1
B4 5.4 9.9 15.2 17.4 24.6
B5 6.9 12.3 13.7 16.1  
B6 7.9 11.7 14.3 18.6  
B8 7.3 11.6 13.6 16.7  
B9 6.5 8.8 12.7 16.1 18.1
B10 7.1 13.1 14.9 18.2 21.7
备注：B加数字无实际意义，仅代表不同只小鼠的编号 表2.图3B的数据
周数：W Control死亡数 BAPN死亡数 BAPN+Exo死亡数
0 0 0 0
1 0 0 0
2 0 0 0
3 0 1 0
4 0 4 3
表3.图3C的数据
Figure PCTCN2022105071-appb-000001
Figure PCTCN2022105071-appb-000002
表4.图3D、图3E的数据
  3W 4W 5W 6W 7W
Control          
B21 1.316 1.31 1.435 1.407 1.519
B22 1.077 1.137 1.449 1.413 1.387
B23 0.927 1.38 0.978 1.328 1.49
BAPN          
B3 1.109 1.115 1.2 1.334 1.612
B7 1.085 1.23 1.077 1.368 1.664
B11 1.078 1.215 1.403 1.416 1.347
B17 0.918 1.182 1.304 1.351  
B14 1.161 1.158 1.212 1.328  
B19 1.121 1.344 1.389    
B24 1.180 1.158 1.520 1.558  
BAPN+Exo          
B1 0.987 1.205 1.266 1.475 1.343
B4 0.884 1.354 1.371 1.329 1.406
B5 0.969 1.304 1.433 1.464  
B6 1.057 1.353 1.304 1.54  
B8 1.152 0.917 1.099 1.118  
B9 0.987 1.205 1.080 1.469 1.392
B10 1.081 1.291 1.379 1.341 1.298
备注：B加数字无实际意义，仅代表不同只小鼠的编号；单位：mm。
表5.图3F、图3G的数据
  3W 4W 5W 6W 7W
Control          
B21 1.25 1.294 1.18 1.095 1.372
B22 1.094 1.001 1.173 1.446 1.392
B23 0.921 1.217 1.153 1.406 1.525
BAPN          
B3 1.076 1.234 1.192 1.348 1.486
B7 0.938 1.26 1.224 1.35 1.926
B11 1.003 1.25 1.376 1.439 1.342
B17 1.031 1.233 1.13 1.407  
B14 0.919 1.137 1.272 1.445  
B19 0.992 1.312 1.329    
B24 1.154 1.025 1.398 1.446  
BAPN+Exo          
B1 1.118 1.141 1.275 1.267 1.447
B4 0.978 1.18 1.27 1.41 1.426
B5 1.055 1.34 1.311 1.527  
B6 0.96 1.038 1.083 1.523  
B8 1.133 0.725 0.899 1.27  
B9 0.999 1.211 0.962 1.31 1.387
B10 1.095 1.098 1.173 1.27 1.29
备注：B加数字无实际意义，仅代表不同只小鼠的编号；单位：mm。
表6.图3H、图3I的数据
  3W 4W 5W 6W 7W
Control          
B21 1.314 1.133 1.152 1.41 1.356
B22 1.161 1.266 1.186 1.48 1.328
B23 1.076 1.368 1.179 1.479 1.41
BAPN          
B3 1.056 1.359 1.223 1.482 1.609
B7 0.926 1.26 1.693 1.754 1.605
B11 1.208 1.27 1.575 1.47 1.625
B17 1.061 0.96 1.384 1.466  
B14 1.099 1.192 1.362 1.615  
B19 1.040 1.175 1.440    
B24 1.066 1.058 1.291 1.483  
BAPN+Exo          
B1 1.115 1.415 1.272 1.267 1.349
B4 1.189 1.143 1.355 1.211 1.563
B5 1.076 1.217 1.289 1.408  
B6 1.038 1.182 1.328 1.612  
B8 1.124 0.976 1.240 1.402  
B9 1.149 1.228 1.085 1.364 1.506
B10 1.061 1.298 1.299 1.371 1.488
备注：B加数字无实际意义，仅代表不同只小鼠的编号；单位：mm。
通过检测4周内的体重变化，发现3组之间体重无明显的差异性(图3A)；BAPN给药组+外泌体组的生存率高于BAPN组(图3B)；Elisa检测不同组小鼠血液中MMP9的表达量，结果显示BAPN给药+外泌体组的表达量高于另外两组(图3C)；通过心超测量小鼠主动脉弓的直径，发现随着时间的推进，窦部、升主及弓部直径均增大(图3D、3F、3H)，我们又统计了给药4周后的直径差异，结果显示每组之间窦部及升主的直 径无差异性(图3E、3G)，弓部直径具有差异性，且BAPN给药+外泌体组的直径小于BAPN组(图3I)。
综上，实验结果均表明外泌体可以减缓胸主动脉夹层/动脉瘤的发生。
以上对本发明所提供的外泌体在胸主动脉夹层/动脉瘤中的作用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 外泌体在制备预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物或制剂中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述外泌体的施用方式为内眦静脉施用，每次施用剂量100μL，每周注射3次。
  3. 如权利要求2所述的应用，其特征在于，所述胸主动脉夹层/动脉瘤动物模型的诱导剂包括β-氨基丙腈(BAPN)。
  4. 如权利要求3所述的应用，其特征在于，所述胸主动脉夹层/动脉瘤动物模型的构建方法中，第一周和第二周BAPN浓度为6mg/mL，第三周和第四周BAPN浓度增大至8mg/mL。
  5. 如如权利要求1至4任一项所述的应用，其特征在于，所述外泌体的构建方法包括如下步骤：
    步骤1：将处于对数生长期、生长状态良好的UMSC细胞，与胎牛血清混合后离心，去除先前存在于血清中的外泌体，使用含有10％不含外泌体胎牛血清的α-MEM培养基培养48h，待细胞密度达到90％时收集培养基的上清，传代；
    步骤2：取步骤1收集的所述上清，离心，除去死的细胞和碎片，收集上清；
    步骤3：取步骤2收集的所述上清，与外泌体分离试剂混匀后4℃条件下抽提24h，获得抽提液；
    步骤4：取步骤3获得的抽提液离心，收集沉淀，经PBS重悬，测定外泌体的浓度；将测好浓度的外泌体置于-80℃超低温保存。
  6. 如权利要求5所述的应用，其特征在于，步骤1中所述离心具体为：以110000g的速度离心7h；
    步骤2中所述离心具体为：以2000g的速度离心30min；
    步骤4中所述离心具体为：4℃，以13000g的速度离心1h。
  7. 如权利要求5或6所述的应用，其特征在于，步骤3中所述外泌体分离试剂包括PEG4000；所述外泌体分离试剂与所述上清的体积比为 1：2。
  8. 预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物或制剂，其特征在于，包括外泌体和药学上可接受的辅料；
    所述外泌体如权利要求5～7任一项所述应用中所述构建方法制得。
  9. 预防、延缓和/或治疗胸主动脉夹层/动脉瘤的药物组合，其特征在于，包括外泌体、任意其他有效成分和药学上可接受的辅料。
  10. 预防、延缓和/或治疗胸主动脉夹层/动脉瘤的方法，其特征在于，施用外泌体或如权利要求8所述的药物或制剂，或如权利要求9所述的药物组合。
PCT/CN2022/105071 2021-11-30 2022-07-12 外泌体在胸主动脉夹层/动脉瘤中的作用 WO2023098088A1 (zh)

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