WO2022242189A1 - 干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用 - Google Patents

干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用 Download PDF

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WO2022242189A1
WO2022242189A1 PCT/CN2021/143667 CN2021143667W WO2022242189A1 WO 2022242189 A1 WO2022242189 A1 WO 2022242189A1 CN 2021143667 W CN2021143667 W CN 2021143667W WO 2022242189 A1 WO2022242189 A1 WO 2022242189A1
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exosomes
application according
stem cells
obstructive pulmonary
chronic obstructive
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PCT/CN2021/143667
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French (fr)
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孙瑞婷
冉丕鑫
吴祖泽
周玉民
杨月峰
王�华
白鸽
孔攀月
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广州医科大学附属第一医院（广州呼吸中心）
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Publication of WO2022242189A1 publication Critical patent/WO2022242189A1/zh
Priority to ZA2023/07972A priority Critical patent/ZA202307972B/en

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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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  • the present invention relates to the technical field of lung disease treatment, in particular to the application of exosomes derived from stem cells in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.
  • Chronic obstructive pulmonary disease characterized by persistent respiratory symptoms and airflow limitation, is a common preventable and treatable chronic respiratory disease.
  • Smoking is one of the main causes of COPD, and its pathogenesis is very complex.
  • pathogenesis is very complex.
  • lung transplantation there is still no effective cure for COPD clinically.
  • stem cell transplantation and the application of cytokines and other biotherapeutic methods have become the treatment methods for COPD because there is no transplant rejection, and they can be enriched to the damaged site to exert anti-inflammatory, immunosuppressive, and anti-apoptotic effects.
  • One of the most promising candidates for the lung Although it has been developed for a long time, only 11 stem cell therapy products of 11 companies in the world have been approved. In my country's stem cell therapy market, especially the treatment of lung diseases has great potential.
  • the problems brought about by its application have gradually become prominent.
  • the application of stem cells has its limitations. After transplantation, the survival time of stem cells in the body is short, and the effect of colonization treatment after transplantation cannot be guaranteed. In addition, we cannot ignore its inevitable side effects, especially tumorigenicity and immunogenicity.
  • the purpose of the present invention is to provide an application of exosomes derived from stem cells in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.
  • the present invention seeks an alternative drug that not only inherits the function of MSC, but also is easier to be ingested: exosomes.
  • exosomes are vesicles with a lipid bilayer membrane structure, and their small structure is easier to be taken up by recipient cells , and then reach the injury site to play the role of immunotherapy, which provides a new idea and strategy for the treatment of chronic obstructive pulmonary disease.
  • exosomes derived from stem cells in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.
  • exosomes with small structure are easier to be taken up by recipient cells, which have similar therapeutic effects to stem cells in the treatment of chronic obstructive pulmonary disease, and are more likely to be absorbed by recipient cells because they do not have immune rejection. Ingested, so it can be used as a substitute for stem cells.
  • the exosomes are extracted from stem cell culture supernatant.
  • stem cell-derived exosomes are prepared by the following method:
  • the culture supernatant obtained by culturing the mesenchymal stem cells is obtained by gradient ultracentrifugation.
  • the exosomes in the culture supernatant obtained from culturing mesenchymal stem cells are gradually extracted by a kit.
  • exosomes isolated in the present invention are identified by mass spectrometry to identify the functional proteins wrapped in exosomes, which are rich in more than 300 proteins. Enrichment analysis found that these functional proteins are indeed mainly involved in response to injury, cell activation, tissue repair, and factor immunity Modulation and other biological processes.
  • the mesenchymal stem cells are derived from fat, bone marrow, dental pulp or umbilical cord.
  • the drug has any one or more functions in the following options:
  • the research of the present invention shows that for chronic obstructive pulmonary disease, stem cell-derived exosomes have the functions of inhibiting bronchoalveolar lavage fluid inflammation, improving lung structure, improving lung function and inhibiting the apoptosis of alveolar epithelial cells, through which function to realize the treatment of chronic obstructive pulmonary disease.
  • the inhibition of bronchoalveolar lavage fluid inflammation includes: reducing the percentage of neutrophils, increasing the percentage of macrophages and/or reducing the content of inflammatory factors.
  • exosomes derived from stem cells have the functions of reducing the percentage of neutrophils, increasing the percentage of macrophages and reducing the content of inflammatory factors, which play a role To suppress the effect of bronchoalveolar lavage fluid inflammation.
  • the inflammatory factors include at least one of IL-1 ⁇ , IL-6 and IL-10.
  • lung structure includes: inhibiting the thickening of alveolar septa.
  • the research of the present invention shows that, on a more specific functional level, for chronic obstructive pulmonary disease, the stem cell-derived exosomes of the present invention can significantly inhibit the thickening of alveolar septa and improve lung structure.
  • the improving lung function includes: reducing functional residual capacity, increasing Cfvc50 and/or increasing FEV100/FVC.
  • the stem cell-derived exosomes of the present invention can reduce pulmonary functional residual capacity, increase Cfvc50 and/or increase FEV100/FVC to improve lung function. Function.
  • the inhibition of alveolar epithelial cell apoptosis includes: restoring the viability of alveolar epithelial cells, promoting cell proliferation, and inhibiting disease-induced apoptosis.
  • the research of the present invention shows that, on a more specific functional level, for chronic obstructive pulmonary disease, the stem cell-derived exosomes of the present invention can restore the proliferation ability of alveolar epithelial cells and inhibit cell apoptosis.
  • Chronic obstructive pulmonary disease in the present invention is not limited to humans, but may be other mammals.
  • a method for treating chronic obstructive pulmonary disease comprising administering to a subject an effective amount of stem cell-derived exosomes or a preparation containing stem cell-derived exosomes.
  • Stem cell-derived exosomes or preparations containing stem cell-derived exosomes are used in the treatment of chronic obstructive pulmonary disease.
  • the effective amount refers to the amount of the preparation that treats, alleviates or prevents the target disease or condition, or exhibits a detectable therapeutic or preventive effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of symptoms, and the therapeutic agents and/or combination of therapeutic agents chosen for administration.
  • the method of administration can be injection, in vivo administration or transdermal penetration.
  • the drug is applied to the patient in need of treatment, and the specific dosage and method of administration can be determined by the physician according to the condition of the above-mentioned patient.
  • the medicine also includes pharmaceutically acceptable auxiliary materials.
  • Such as auxiliary materials can include any one or more of wetting agents, emulsifiers, diluents, excipients, fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, stabilizers, etc. kind.
  • the dosage form of the drug includes injection, nasal spray or nasal drops.
  • the pharmaceutical dosage form of the present invention is not limited thereto, and those skilled in the art can choose reasonably according to actual needs, and no matter what the dosage form is, it all falls within the protection scope of the present invention.
  • the medicine includes any one or more of the following: preparations for inhibiting bronchoalveolar lavage fluid inflammation, preparations for improving lung structure and preparations for improving lung function.
  • the stem cell-derived exosomes in the present invention can be made into different dosage forms according to the situation, and can also be made into different dosage forms according to the difference in use, such as preparations for inhibiting bronchoalveolar lavage fluid inflammation, preparations for improving lung structure, Preparations for improving lung function, etc.
  • the pharmaceutical dosage forms provided by the present invention can be prepared according to conventional methods in the art.
  • the present invention has at least the following beneficial effects:
  • the present invention uses exosomes derived from stem cells to treat chronic obstructive pulmonary disease. Since immune rejection is prevented, and because of their small structure, they are easier to be taken up by recipient cells, and then reach the damaged site to play the role of immunotherapy , providing a new idea and strategy for the treatment of chronic obstructive pulmonary disease.
  • exosomes derived from stem cells have the functions of inhibiting bronchoalveolar lavage fluid inflammation, improving lung structure, improving lung function, and inhibiting the apoptosis of alveolar epithelial cells. Through the exertion of these functions, the treatment of chronic Treatment of obstructive lung disease.
  • the stem cell-derived exosomes of the present invention can be made into different dosage forms or preparations according to needs, so as to better treat chronic obstructive pulmonary disease.
  • FIG. 1 is a diagram of the Western Blot reaction (WB) detection results of exosomes purified in Example 1 of the present invention
  • Example 2 is an electron microscope (EM) observation diagram of exosomes purified in Example 1 of the present invention
  • Figure 3 and Figure 4 are the results of the NTA method for the detection of purified exosomes in Example 1 of the present invention.
  • Fig. 5 is a map of protein types detected and identified by mass spectrometry in stem cell-derived exosomes in Example 1 of the present invention
  • Example 6 is an enrichment analysis diagram of protein types of stem cell-derived exosomes in Example 1 of the present invention.
  • Figure 7 is a diagram of the in vivo and in vitro experiments in Example 1 of the present invention to verify the uptake of exosomes by recipient cells;
  • Fig. 8 is the comparative figure of the phenotype of lung tissue and lung function in the treatment group and the control group in Example 2 of the present invention.
  • Fig. 9 is the comparison chart of the results of lung tissue staining in the treatment group and the control group in Example 2 of the present invention.
  • Fig. 10 is the airway thickening lesion situation diagram of lung tissue in the treatment group and the control group in Example 2 of the present invention.
  • Fig. 11 is the inflammatory changes in the treatment group and the control group in Example 2 of the present invention.
  • Figure 12 is a graph showing the expression of pro-inflammatory factors in the treatment group and the control group in Example 2 of the present invention.
  • Figure 13 is a graph showing the slowing down of proliferation caused by CSE (cigarette smoke extract, Cigarette Smoke Extract) stimulation in the treatment group and control group in Example 2 of the present invention
  • Fig. 14 is a diagram of CSE-induced apoptosis in the treatment group and the control group in Example 2 of the present invention.
  • the obtained P0 mesenchymal stem cells were isolated, and the original cell bank and the working cell bank were established in the P3, P5 and P6 generations, respectively.
  • Cell phenotype detection and identification of P6 generation cells After the cell density is greater than 70%, the medium is replaced, and the cell culture supernatant of the P5-P6 generation cell expansion culture is collected after 48 hours.
  • the precipitate was resuspended in 100 ⁇ l PBS, and the purified MSC-exo drug was obtained.
  • the purified MSC-exo drug was detected and identified by WB, EM, and NAT techniques.
  • Stem cell-derived exosomes are rich in about 300 proteins, as shown in Figure 5.
  • FIG. 6 Through enrichment analysis (as shown in Figure 6), it is found that these functional proteins are indeed mainly involved in biological processes such as response to injury, cell activation, tissue repair, and factor immune regulation, which may be the way they exert their therapeutic effects.
  • C57BL/6J mice were randomly divided into normal control group (CTL), model group, MSCs group, and MSCs-derived exosomes group.
  • CTL normal control group
  • model group MSCs group
  • MSCs-derived exosomes group MSCs-derived exosomes group.
  • smoke exposure for 6 months establish a mouse COPD animal model, specifically: 4-6 weeks old mice, after a week of adaptation, use domestic cheap cigarettes, conduct whole-body exposure in a smoke box, and give mice passive smoking , exposed to cigarette smoke 6 days a week for 6 months.
  • Cell therapy was started after 3 months of smoke exposure, administered once a month at a dose of 200 ⁇ g/200 ⁇ l/mouse, and treated for 3 times in total, and samples were taken to evaluate the therapeutic effect after 6 months of smoke exposure.
  • the functional residual capacity (FRC) of the mice increased (P ⁇ 0.05), the forced expiratory peak flow rate in one second and the compliance of 50% vital capacity (Cfvc50, FEVpef) all increased, and the bronchoalveolar lavage fluid inflammation
  • the total number of cells and neutrophils were significantly increased (P ⁇ 0.01), and the mean linear intercept (MLI) of the alveoli was thickened (P ⁇ 0.05).
  • MMI mean linear intercept
  • H&E staining of the lung tissue showed that, as shown in Figure 9, after CS exposure, the alveolar structure of the mice was obviously destroyed, and the alveolar cavity expanded and fused, showing the characteristics of emphysema; while the exosome and MSC intervention group were all inhibited, The alveolar structure was relatively complete, the number of damaged alveoli was reduced, and the average linear intercept of the alveoli decreased significantly.
  • exosomes On airway thickening lesions, as shown in Figure 10, exosomes also showed a good inhibitory effect.
  • alveolar walls became thinner, partial compensatory emphysema was formed, blood vessels were dilated and congested, and large airway thickening was suppressed, indicating that the lung structure was improved.
  • COPD chronic inflammatory response process.
  • BALF a chronic inflammatory response process.
  • the total number of inflammatory cells and neutrophil count in bronchoalveolar lavage fluid were significantly lower Compared with the model group (P ⁇ 0.05), it indicated that the inflammatory response was effectively suppressed. That is, exosome therapy can effectively reduce the abnormal increase of inflammatory cells in the alveoli, especially neutrophils and lymphocytes.
  • each pro-inflammatory factor is in the order of Contral, CS, CS+MSC, CS+MSC-exosome from left to right.
  • stem cell-derived exosomes can be made into different dosage forms according to the situation, and can also be made into different dosage forms according to the difference in use, such as preparations for inhibiting bronchoalveolar lavage fluid inflammation, and for improving lung structure. preparations, preparations for improving lung function, etc.

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Abstract

提供干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用。结构小更容易被受体细胞摄取的外泌体在治疗慢性阻塞性肺疾病具有和干细胞相似的治疗效果，而其本身因没有免疫排斥而更容易被受体细胞所摄取，所以可以作为干细胞的替代物，为治疗慢性阻塞性肺疾病提供了一种新的思路和策略。

Description

干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用
相关申请的交叉引用
本申请要求于2021年05月20日提交中国专利局的申请号为CN202110551341.8、名称为“干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用”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
本发明涉及肺疾病治疗技术领域，具体而言，涉及干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用。
背景技术
慢阻肺以持续的呼吸道症状和气流受限为特征，是一种常见的可预防、可治疗的慢性呼吸系统疾病。抽烟是慢阻肺发病的主要原因之一，而且其发病机制非常复杂。目前临床上除了肺移植，慢阻肺尚缺乏行之有效的根治措施。
因为无移植排斥反应、且能够向损伤部位富集发挥抗炎、免疫抑制、抗凋亡等作用，针对慢阻肺的发病机制，干细胞移植以及其细胞因子应用等生物治疗手段已经成为治疗慢阻肺最有潜力的备选方案之一。虽然发展已久，但目前全球仅有11家企业的11个干细胞治疗产品获批。在我国干细胞治疗市场特别是肺部疾病的治疗更是潜力巨大。但是，随着对干细胞研究的深入，其应用所带来的问题也逐渐凸显。干细胞的应用有他的局限性，移植后的干 细胞在体内存活时间短，并不能保证移植后的定植治疗效果。另外，我们也不能忽视其难以避免的副作用，尤其是致瘤性和免疫原性。
针对上述问题，可以寻求更好、更安全地应用干细胞从而向临床转化应用的途径和方法。
发明内容
本发明的目的在于提供一种干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用。本发明寻找一种既遗传MSC功能、又更容易被摄取的替代药物：外泌体。研究发现，采用外泌体对慢性阻塞性肺疾病的治疗和干细胞一样具有很好的效果，同时外泌体是具有脂质双层膜结构的囊泡，它们结构小更容易被受体细胞摄取，进而到达损伤部位从而发挥免疫治疗的作用，为治疗慢性阻塞性肺疾病提供了一种新的思路和策略。
为了实现本发明的上述目的，特采用以下技术方案：
干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用。
本发明的研究显示，结构小更容易被受体细胞摄取的外泌体，其在治疗慢性阻塞性肺疾病具有和干细胞相似的治疗效果，而其本身因没有免疫排斥而更容易被受体细胞所摄取，所以可以作为干细胞的替代物。
可选地，所述外泌体从干细胞培养上清液中提取得到。
可选地，所述干细胞来源外泌体通过如下方法制备得到：
将间充质干细胞培养所得的培养上清通过梯度超速离心所得。
可选地，在本发明的一些实施方式中，通过试剂盒逐步提取间充质干细胞培养所得的培养上清中的外泌体。
需要说明的是，在本发明所公开的内容基础上，本领域技术人员可以采用本领域其他熟知的试剂盒提取技术，提取上清中的外泌体，这对本来领域 技术人员来说是容易实现，无论其采用何种方法得到外泌体并用于慢性阻塞性肺疾病的治疗，其均是属于本发明的保护范围。
本发明分离的外泌体，通过质谱鉴定外泌体包裹的功能蛋白，大约富含三百多个蛋白，富集分析发现这些功能蛋白确实是主要参与应对损伤、细胞活化、组织修复、因子免疫调剂等生物学过程。
可选地，在本发明的一些实施方式中，所述所述间充质干细胞来源于脂肪、骨髓、牙髓或脐带。
进一步地，所述药物具有以下选项中的任一种或多种功能：
(a)抑制支气管肺泡灌洗液炎症；
(b)改善肺脏结构；
(c)改善肺脏功能；
(d)抑制肺泡上皮细胞的凋亡。
本发明的研究显示，对于慢性阻塞性肺疾病来说，干细胞来源的外泌体具有抑制支气管肺泡灌洗液炎症、改善肺脏结构、改善肺脏功能以及抑制肺泡上皮细胞的凋亡的功能，通过这些功能的发挥，实现对慢性阻塞性肺疾病的治疗。
进一步地，所述抑制支气管肺泡灌洗液炎症包括：减少中性粒细胞百分比、增加巨噬细胞百分比和/或减少炎症因子含量。
本发明的研究显示，在更为具体的功能层面上，对于慢性阻塞性肺疾病，干细胞来源的外泌体具有减少中性粒细胞百分比、增加巨噬细胞百分比以及减少炎症因子含量的功能，起到抑制支气管肺泡灌洗液炎症的效果。
进一步地，所述炎症因子包括IL-1β、IL-6和IL-10中的至少一种。
进一步地，所述改善肺脏结构包括：抑制肺泡间隔增厚。
本发明的研究显示，在更为具体的功能层面上，对于慢性阻塞性肺疾病，本发明的干细胞来源的外泌体能够明显抑制肺泡间隔增厚，改善肺脏结构。
可选地，所述改善肺脏功能包括：降低功能残气量、提升Cfvc50和/或提升FEV100/FVC。
本发明的研究显示，在更为具体的功能层面上，对于慢性阻塞性肺疾病，本发明的干细胞来源的外泌体能够降低肺功能残气量、提升Cfvc50和/或提升FEV100/FVC以改善肺脏功能。
可选地，所述抑制肺泡上皮细胞的凋亡包括：恢复肺泡上皮细胞的活力，促进细胞增殖，抑制疾病诱导的细胞凋亡。
本发明的研究显示，在更为具体的功能层面上，对于慢性阻塞性肺疾病，本发明的干细胞来源的外泌体能够恢复肺泡上皮细胞的增殖能力，抑制细胞的凋亡。
本发明中的慢性阻塞性肺疾病并不限于人，还可以为其他哺乳动物。
慢性阻塞性肺疾病的治疗方法，包括给予受试者有效量的干细胞来源的外泌体或含有干细胞来源的外泌体的制剂。
干细胞来源的外泌体或含有干细胞来源的外泌体的制剂应用于治疗慢性阻塞性肺疾病。
其中，有效量是指制剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、症状的性质和程度、以及选择给与的治疗剂和/或治疗剂的组合。给药方式可以为注射、体内给药或者是透皮渗透方式，药物施加 于需要治疗的患者，具体给药剂量以及给药方式可根据上述病人的情况由医师决定。
进一步地，所述药物还包括药学上可接受的辅料。
如辅料可以包括润湿剂、乳化剂、稀释剂、赋形剂、填充剂、崩解剂、粘合剂、润滑剂、表面活化剂、矫味剂、稳定剂等中的任一种或多种。
进一步地，所述药物的剂型包括注射剂、鼻腔喷雾剂或滴鼻剂。
本发明的药物剂型并不限于此，本领域技术人员可以根据实际需要合理选择，无论何种剂型，其均属于本发明的保护范围内。
进一步地，所述药物包括以下中的任一种或多种：用于抑制支气管肺泡灌洗液炎症的制剂、用于改善肺脏结构的制剂和用于改善肺脏功能的制剂。
本发明中的干细胞来源外泌体可根据情况制成不同剂型，也可以根据用途的差异制成不同剂型，如用于抑制支气管肺泡灌洗液炎症的制剂、用于改善肺脏结构的制剂、用于改善肺脏功能的制剂等。
本发明提供的药物剂型按照本领域的常规方法制备即可。
与现有技术相比，本发明至少具有以下有益效果：
(1)本发明采用干细胞来源的外泌体对慢性阻塞性肺疾病的治疗，既然防止了免疫排斥，又由于它们结构小更容易被受体细胞摄取，进而到达损伤部位从而发挥免疫治疗的作用，为治疗慢性阻塞性肺疾病提供了一种新的思路和策略。
(2)本发明经研究发现干细胞来源的外泌体具有抑制支气管肺泡灌洗液炎症、改善肺脏结构、改善肺脏功能、抑制肺泡上皮细胞的凋亡等作用，通过这些功能的发挥，实现对慢性阻塞性肺疾病的治疗。
(3)本发明的干细胞来源的外泌体可根据需要制成不同的剂型或制剂，以更好的治疗慢性阻塞性肺疾病。
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，以下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为本发明实施例1中纯化的外泌体的蛋白免疫印迹反应(WB)检测结果图；
图2为本发明实施例1中纯化的外泌体的电镜(EM)观察图；
图3和图4为本发明实施例1中NTA方法对纯化的外泌体检测的结果图；
图5为本发明实施例1中干细胞来源外泌体经质谱技术检测鉴定到的蛋白种类图谱；
图6为本发明实施例1中干细胞来源外泌体的蛋白种类的富集分析图；
图7为本发明实施例1中体内和体外实验验证受体细胞对外泌体的摄取情况图；
图8为本发明实施例2治疗组和对照组中肺组织的表型以及肺功能的对比图；
图9为本发明实施例2治疗组和对照组中肺脏组织染色的结果对照图；
图10为本发明实施例2治疗组和对照组中肺脏组织的气道增厚病变情况图；
图11为本发明实施例2治疗组和对照组中炎症变化情况；
图12为本发明实施例2治疗组和对照组中促炎因子的表达情况图；
图13为本发明实施例2治疗组和对照组中恢复CSE(香烟烟雾提取物，Cigarette Smoke Extract)刺激导致的增殖减缓的情况图；
图14为本发明实施例2治疗组和对照组中CSE诱导的细胞凋亡情况图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
1、间充质干细胞的制备及扩增
分离获得的P0代间充质干细胞，分别在P3代、P5代和P6代建立原始细胞库和工作细胞库。P6代细胞进行细胞表型检测鉴定。细胞密度大于70％后，更换培养基，48h后收集P5-P6代细胞扩增培养的细胞培养上清。
2、外泌体的提取及鉴定
将收集得到的培养上清液A mL 4℃离心机3000g离心30min去除死细胞，取上清继续10000g离心30min去除细胞碎片，将上清转移至超滤管中浓缩至B mL(A:B约为20:1)。使用超速离心机分离及近一步纯化外泌体，超速离心机110000g离心70min两次。沉淀用100μl PBS重悬，得到的即为纯化的MSC-exo药剂，通过WB、EM、NAT的技术对纯化的MSC-exo药剂检测鉴定。
(1)纯化外泌体后，裂解制备蛋白样品，BCA检测蛋白浓度后通过WB的方法进行外泌体标志蛋白的检测鉴定，结果如图1所示。
(2)透射电镜观察外泌体形态：取10μl MSC-exo滴于铜网，静置2min，滴加10μl乙酸双氧铀，染色1min，烤干后透射电镜观察，结果如图2所示。
(3)NTA测外泌体粒径大小分布和外泌体浓度：使用马尔文公司NanoSight Nanoparticle Tracking Analysis仪器检测外泌体的粒径大小分布和浓度，结果如图3、4所示。
3、外泌体成分的分析
分离三个人供体来源的干细胞后，分别提取他们的外泌体，通过质谱鉴定外泌体包裹的功能蛋白，进一步解释外泌体发挥作用的方式。干细胞来源外泌体大约富含三百多个蛋白，如图5所示。通过富集分析(如图6)，发现这些功能蛋白确实是主要参与应对损伤、细胞活化、组织修复、因子免疫调剂等生物学过程，这些可能是它们发挥治疗作用的方式。
4、验证受体细胞对外泌体的摄取情况
通过体内和体外实验验证受体细胞对外泌体的摄取情况，结果如图7所示。说明受体细胞可以摄入外泌体。
实施例2
1、干细胞来源外泌体治疗CODP模型鼠的疗效评价
(1)COPD模型鼠的构建及给药设计
C57BL/6J小鼠经检疫合格后，随机分为正常对照组(CTL)、模型组、MSCs组、MSCs来源外泌体组。利用烟雾暴露6个月，建立小鼠类慢阻肺动物模型，具体为：4-6周龄小鼠，适应一周后，采用国产平价香烟，于熏烟箱中进行全身暴露给予小鼠被动吸烟，每周香烟烟雾暴露6天，持续6个 月。在烟雾暴露3个月开始细胞治疗，每月给药一次，给药剂量为200μg/200μl/小鼠，共治疗3次，烟雾暴露6个月后取材评估治疗效果。
(2)外泌体治疗COPD的效价评估
模型组小鼠肺功能残气量(FRC)增大(P＜0.05)，用力呼气峰流速的一秒量及50％肺活量的顺应性(Cfvc50，FEVpef)均升高，支气管肺泡灌洗液炎症细胞总数、中性粒细胞显著升高(P<0.01)，肺泡平均内衬间隔(mean linear intercept，MLI)，增厚(P<0.05)。上述结果表明，已经成功建立了COPD小鼠模型。
如图8所示，经过治疗后取材发现，MSC或其外泌体处理后，均可看到肺组织颜色一定程度减轻，颗粒物的沉积出现不同程度的改善；采用小动物肺功能仪(PFT系统)评估大鼠的肺功能，通过肺功能的结果可以看到，CS暴露后，小鼠肺功能指标功能残气量和用力肺活量均显著增加，治疗后FRC、Cfvc50和FEVpef均下降，提示肺功能得到有效改善。提示了烟草烟雾暴露导致小鼠肺功能改变；与模型组相比，外泌体及MSC均可不同程度的改善烟雾暴露导致的肺功能异常。
肺脏组织进行H&E染色发现，如图9所示，CS暴露后，小鼠肺泡结构明显被破坏，肺泡腔扩大、融合，出肺气肿特征；而外泌体及MSC干预组均有所抑制，肺泡结构相对完整，被破坏的肺泡数量减少，肺泡平均线性截距下降MLI值明显降低。
在气道增厚病变上，如图10所示，外泌体也显示出了良好的抑制效果。外泌体及MSC干预组，肺泡壁变薄，部分代偿性肺气肿形成，血管扩张充血，大气道增厚病变得到抑制，说明肺脏结构改善。
慢阻肺病变是慢性的炎症反应过程，通过检查BALF中细胞成分发现(如图11所示)，干细胞和外泌体治疗后，支气管肺泡灌洗液炎症细胞总数、中性粒细胞计数显著低于模型组(P<0.05)，表明炎症反应得到有效抑制。即外泌体治疗可以有效减少肺泡中细胞炎症细胞的异常增多，特别是中性粒细胞和淋巴细胞。
图12中，每个促炎因子的图中，均是按照Contral、CS、CS+MSC、CS+MSC-exosome依次从左到右的顺序。如图12所示，检测肺部微环境中IL1\IL6和IL10的这些促炎因子的表达，外泌体及MSC的治疗，明显减少了他们的表达，而且这一抗炎影响不仅在局部，在全身都有展现，血清检测得到了相似的结果。
外泌体的作用机制不仅是免疫调节。所以我们利用CSE提取物在体外刺激原代培养的肺泡上皮细胞中，在72h的培养观察中，发现外泌体的加入可以有效恢复CSE刺激导致的增殖减缓，在12h就开始出现明显效果(见图13)。其中，图13中的细胞计数和时间的关系曲线中，最上边的为Control组，中间的线条为CSE+MSC-exo组，最下边的线条为CSE组。同样流式结果显示外泌体可以抑制CSE诱导的细胞凋亡(见图14)。
尽管已用具体实施例来说明和描述了本发明，然而应意识到，在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
以上所描述的实施例是本发明一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领 域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
工业实用性
通过应用本申请的技术方案，干细胞来源外泌体可根据情况制成不同剂型，也可以根据用途的差异制成不同剂型，如用于抑制支气管肺泡灌洗液炎症的制剂、用于改善肺脏结构的制剂、用于改善肺脏功能的制剂等。

Claims (14)

  1. 干细胞来源的外泌体在制备治疗慢性阻塞性肺疾病药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述外泌体从干细胞培养上清液中提取得到。
  3. 根据权利要求2所述的应用，其特征在于，所述干细胞来源外泌体通过如下方法制备得到：
    将间充质干细胞培养所得的培养上清通过梯度超速离心所得。
  4. 根据权利要求2所述的应用，其特征在于，通过试剂盒逐步提取间充质干细胞培养所得的培养上清中的外泌体。
  5. 根据权利要求3所述的应用，其特征在于，所述间充质干细胞来源于脂肪、骨髓、牙髓或脐带。
  6. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物具有以下选项中的任一种或多种功能：
    (a)抑制支气管肺泡灌洗液炎症；
    (b)改善肺脏结构；
    (c)改善肺脏功能；
    (d)抑制肺泡上皮细胞的凋亡。
  7. 根据权利要求6所述的应用，其特征在于，所述抑制支气管肺泡灌洗液炎症包括：减少中性粒细胞百分比、增加巨噬细胞百分比和/或减少炎症因子含量。
  8. 根据权利要求7所述的应用，其特征在于，所述炎症因子包括IL-1β、IL-6和IL-10中的至少一种。
  9. 根据权利要求6所述的应用，其特征在于，所述改善肺脏结构包括：抑制肺泡间隔增厚。
  10. 根据权利要求9所述的应用，其特征在于，所述改善肺脏功能包括：降低功能残气量、提升Cfvc50和/或提升FEV100/FVC。
  11. 根据权利要求9所述的应用，其特征在于，所述抑制肺泡上皮细胞的凋亡包括：恢复肺泡上皮细胞的活力，促进细胞增殖，抑制疾病诱导的细胞凋亡。
  12. 根据权利要求1-11任一项所述的应用，其特征在于，所述药物还包括药学上可接受的辅料。
  13. 根据权利要求12所述的应用，其特征在于，所述药物的剂型包括注射剂、鼻腔喷雾剂或滴鼻剂。
  14. 根据权利要求12所述的应用，其特征在于，所述药物包括以下中的任一种或多种：用于抑制支气管肺泡灌洗液炎症的制剂、用于改善肺脏结构的制剂和用于改善肺脏功能的制剂。
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