WO2023179000A1 - 骨髓间充质干细胞外泌体在修复关节软骨缺损中的应用 - Google Patents

骨髓间充质干细胞外泌体在修复关节软骨缺损中的应用 Download PDF

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WO2023179000A1
WO2023179000A1 PCT/CN2022/126040 CN2022126040W WO2023179000A1 WO 2023179000 A1 WO2023179000 A1 WO 2023179000A1 CN 2022126040 W CN2022126040 W CN 2022126040W WO 2023179000 A1 WO2023179000 A1 WO 2023179000A1
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bone marrow
mesenchymal stem
marrow mesenchymal
exosomes
stem cell
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PCT/CN2022/126040
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English (en)
French (fr)
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孙诚
顾晓松
龚蕾蕾
从猛
杨洪伟
张愉
李枚原
王晓敏
徐来
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南通大学
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Priority to EP22933051.9A priority Critical patent/EP4324469A1/en
Priority to JP2023551722A priority patent/JP2024515005A/ja
Publication of WO2023179000A1 publication Critical patent/WO2023179000A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the invention belongs to the field of biomedicine and relates to a method for preparing bone marrow mesenchymal stem cell exosomes and its application in promoting chondrocyte vitality, increasing chondrocyte proliferation, enhancing chondrocyte migration, and repairing cartilage defects.
  • Cartilage is composed of collagen, proteoglycans, glycosaminoglycans, glycoproteins, a few cells, and 60-80% water. Chondrocytes and the surrounding extracellular matrix (ECM) together constitute the cartilage unit, which is regarded as the main structural, functional and metabolic unit of cartilage. In normal cartilage, chondrocytes synthesize collagen, proteoglycans, glycosaminoglycans and other substances to maintain cartilage homeostasis or replace damaged cartilage. Cartilage plays important roles in the human body such as bearing load and reducing bone friction between joints. However, adult cartilage tissue lacks blood vessels or nerves, so the ability of injured cartilage tissue to repair itself is very limited.
  • Cartilage dysfunction is often closely related to the occurrence of degenerative joint diseases, such as degenerative arthritis, senile arthritis, hypertrophic arthritis, etc.
  • Clinical methods for treating degenerative joint disease include non-surgical treatment and surgical treatment.
  • Non-surgical treatments mainly include oral non-steroidal anti-inflammatory drugs, intra-articular injection of drugs and rehabilitation exercises. Although they can relieve short-term pain and improve symptoms, the long-term effects are not good.
  • Surgical treatments include microfracture, drilling, joint replacement surgery, autologous chondrocyte implantation, juvenile allograft cartilage transplantation, autologous cartilage transplantation, etc.
  • the purpose of the present invention is to provide a bone marrow mesenchymal stem cell exosome for use in promoting chondrocyte vitality, increasing chondrocyte proliferation, enhancing chondrocyte migration, and repairing cartilage defects.
  • the exosomes can improve chondrocyte vitality, promote cell proliferation, and enhance cell migration ability.
  • Exosome treatment can significantly promote the repair of defective cartilage and significantly improve the regeneration of defective cartilage.
  • the application of bone marrow mesenchymal stem cell exosomes in the preparation and treatment of articular cartilage diseases are produced by stimulating bone marrow mesenchymal stem cells in the culture medium. After passage, the bone marrow mesenchymal stem cells are extracted from the culture medium. Stem cell exosomes, the bone marrow mesenchymal stem cells are derived from the bone marrow of the ilium or femur.
  • applications include any of the following:
  • the culture medium is a complete culture medium.
  • the culture conditions of bone marrow mesenchymal stem cells were 37°C and 5% CO 2 .
  • the complete medium to the bone marrow, mix by pipetting, and place it in an incubator for culture. Half the medium is replaced on the 3rd day, and all the medium is replaced on the 6th day. Subsequently, the medium is replaced every 2 days until the cells grow to 80-90%. Passage to the P1 generation, and then pass three times to obtain primary bone marrow mesenchymal stem cells.
  • the cells when culturing bone marrow mesenchymal stem cells, the cells are grown to the logarithmic growth phase, the medium is discarded, washed with PBS and replaced with 2% vesicle-free serum for culture until the cells grow to 80-90%, and the cells are collected. The clear liquid and filtrate were used for exosome extraction.
  • membrane affinity spin column filtration was used to collect bone marrow mesenchymal stem cell exosomes.
  • the filtered supernatant was added to an equal volume of XBP buffer, mixed, and then added to a membrane affinity spin column for processing.
  • the exosomes adsorbed on the column were eluted with XE buffer.
  • the filtration method is 0.2 ⁇ m membrane filtration.
  • the present invention provides the application of bone marrow mesenchymal stem cell exosomes in the preparation and treatment of articular cartilage diseases.
  • the exosomes can improve chondrocyte vitality, promote cell proliferation, and enhance cell migration ability.
  • Exosome treatment can significantly promote defective cartilage repair and significantly improve defective cartilage regeneration.
  • Figure 1 shows the extraction and identification of exosomes from bone marrow mesenchymal stem cells.
  • Figure 2 shows that bone marrow mesenchymal stem cell exosomes promote chondrocyte viability and cell proliferation.
  • Bone marrow mesenchymal stem cell exosomes improve chondrocyte viability;
  • Bone marrow mesenchymal stem cell exosomes promote chondrocyte proliferation. *p ⁇ 0.05,***p ⁇ 0.001, Student’s t-test analysis.
  • Figure 3 shows that bone marrow mesenchymal stem cell exosomes promote chondrocyte migration.
  • Figure 4 is an analysis of the internalization kinetics of bone marrow mesenchymal stem cell exosomes in chondrocytes.
  • the expression of bone marrow mesenchymal stem cell exosomes in chondrocytes is different. Quantification of PHK26 intensity at time points.
  • FITC-Phalloidin fluorescein isothiocyanate-phalloidin, used to label cytoskeleton
  • PKH-26 red fluorescein, used to label exosomes
  • DAPI 4',6-diamidino-2- Phenylindole, used to label cell nuclei
  • Merge is a composite image. ***p ⁇ 0.001, one-way ANOVA analysis.
  • Figure 5 shows bone marrow mesenchymal stem cell exosomes promote cartilage repair.
  • Figure 6 shows bone marrow mesenchymal stem cell exosomes promoting cartilage regeneration.
  • Human bone marrow specimens were obtained from the ilium or femur of 6 normal adults (average age 35 years, range 30-40 years; 3 males, 3 females). Place 1 ml of extracted human bone marrow into a 60 mm petri dish, add 8 ml of complete culture medium and mix evenly by pipetting.
  • the complete culture medium is DMEM cell culture medium, supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. Afterwards, they were cultured in an incubator containing 5% CO2 at 37°C. Half the medium was changed on the 3rd day, and all the medium was changed on the 6th day. Subsequently, the medium was changed every 2 days until the cells grew to 80-90% and were passaged to the P1 generation. . In this way, relatively pure primary bone marrow mesenchymal stem cells were obtained after three passages, and the P3-P5 generation cells were used for subsequent experiments.
  • the cells were grown to the logarithmic growth phase.
  • the medium was discarded, washed once with PBS, and replaced with a medium of 2% vesicle-free serum.
  • the medium of 2% vesicle-free serum was: ultracentrifuged fetal bovine serum ( 100000g, 16 hours, 4°C) to remove the vesicles and then use it to prepare cell culture medium.
  • the culture medium consists of: DMEM cell culture medium containing 2% vesicle-free fetal bovine serum, 1% penicillin and streptomycin. Until the cells grow to 80-90%, the supernatant is collected and filtered with a 0.2 ⁇ M filter, and the filtrate is used for exosome extraction.
  • NTA Nanoparticle tracking analyzes
  • Chondrocyte extraction (1) Cartilage slices were taken from the knee joints of 4-week-old New Zealand rabbits; (2) Cut into small pieces. After washing three times with PBS; (3) Digest with 0.25% trypsin for 30 minutes; (4) Discard the supernatant, add 0.2% type 2 collagenase, and place it in a 37°C 5% CO2 incubator for digestion overnight; (5) Filter with 200 mesh Filter through a mesh, and centrifuge the filtrate at 500g for 5 minutes; (6) Resuspend the pellet in complete chondrocyte culture medium and culture it in an incubator.
  • chondrocytes were seeded at a density of 5 ⁇ 10 3 /ml. 4 hours after inoculation, the medium was changed to medium containing 2% vesicle-free serum, and different concentrations of exosomes (5.0 ⁇ 10 8 /ml, 1.0 ⁇ 10 9 /ml, 2.0 ⁇ 10 9 /ml) were added and cultured for 24 hours. Add 1/10 of the total medium volume of CCK-8 solution. Place the culture plate in a 37°C incubator and incubate for 2-4 hours. Measure the absorbance value at a wavelength of 450nm.
  • chondrocytes were seeded at a density of 5 ⁇ 10 3 /ml. 4 hours after inoculation, the medium was changed to medium containing 2% vesicle-free serum, and prepared exosomes (1.0 ⁇ 10 9 /ml) were added and cultured for 24 hours. Aspirate the culture medium from each well, add 50 ⁇ l EdU culture medium to each well, and incubate for 2 hours.
  • Discard the culture medium add 100 ⁇ l of fixative after washing with PBS, and fix at room temperature for 15 minutes; add 100 ⁇ l of 2 mg/ml glycine solution, and neutralize for 5 minutes; add 100 ⁇ l of PBS, and wash for 5 minutes; add 100 ⁇ l of 0.5% TritonX-100 PBS for reaction for 10 minutes, and wash with PBS for 5 minutes. .
  • ibidi device Use the ibidi device to simulate scratch experiments: (1) Insert a 2-hole device into a pre-coated small dish to conduct a wound healing test. (2) Prepare the pretreated cell suspension, that is, resuspend the cells in serum-free medium. Add cell suspension at a concentration of 2 ⁇ 10 5 /ml (100 ⁇ l) per well. (3) After 12 hours, use sterile tweezers to gently pull out the 2 devices and wash them once with PBS. (4) The control group was replaced with 2% vesicle-free serum medium, and the treatment group was replaced with 2% vesicle-free serum medium containing exosomes (1.0 ⁇ 10 9 /ml), and culture was continued for 24h and 48h. (5) Observe the cell migration process under a microscope and take pictures.
  • PKH26 to label exosomes.
  • the process is as follows: (1) Prepare an exosome solution (100 ⁇ l) with a concentration of 1 ⁇ 10 11 /ml. (2) Add exosomes to 250 ⁇ l Diluent C and mix gently. (3) Take a new EP tube and add 2 ⁇ l PKH26 to 250 ⁇ l Diluent C. (4) Add (2) to (3) and mix well. (5) Leave at room temperature for 4 minutes, and mix with a pipette every 1 minute. (6) Add an equal volume of 1% BSA to terminate staining. (7) Concentrate the solution with Amicon Ultra Centrifugal Filters and set aside.
  • New Zealand rabbits were provided by the Animal Center of Nantong University. They were divided into 4 groups: control group, surgery group, surgery + low-dose exosomes group, surgery + high-dose exosomes group, with 6 animals in each group.
  • Penicillin (50,000 units/kg) was intramuscularly injected for 3 consecutive days to prevent infection, and tramadol 2 mg/kg was administered for analgesia.
  • 300 ⁇ l of exosomes of different concentrations (low dose: 1.0 ⁇ 10 10 /ml; high dose: 5.0 ⁇ 10 10 /ml) were injected into the joint cavity once a week for a total of 4 treatments.
  • the rabbit was euthanized, the complete knee joint was removed, fixed in paraformaldehyde for 24 hours, decalcified with 10% EDTA (pH 7.4) for 2-3 months, embedded in paraffin, and cut into 5 ⁇ m thick slices.
  • Dewax the paraffin sections to water Place the sections in sequence in xylene (I) for 20 min, xylene (II) for 20 min, absolute ethanol (I) for 5 min, absolute ethanol (II) for 5 min, and 75% ethanol for 5 min, and wash with tap water.
  • Stain hematoxylin semen (60°C) for 30 to 60 seconds, wash away the hematoxylin semen with running water for 5 to 10 seconds, 1% hydrochloric acid ethanol for 1 to 3 seconds, slightly wash with water for 1 to 2 seconds, return the blue solution to blue for 5 to 10 seconds, and rinse with running water for 15 to 30 seconds , stain with 0.5% eosin solution for 30 to 60 seconds, rinse slightly with distilled water for 1 to 2 seconds, 80% ethanol for 1 to 2 seconds, 95% ethanol for 1 to 2 seconds, absolute ethanol for 1 to 2 seconds, xylene (I) for 2 to 3 seconds, xylene ( II) 2 ⁇ 3s, seal with neutral gum.
  • Fast green staining soak the sections in fast green staining solution for 5-10 minutes, wash away excess dyeing solution with water until the cartilage becomes colorless, soak slightly in differentiation solution, and rinse with distilled water.
  • Safranin staining Place the sections in safranin staining solution for 15-30 seconds and quickly dehydrate with absolute ethanol.
  • Transparent sealing clear in clean xylene for 5 minutes, seal with neutral gum. Microscopic examination, image acquisition and analysis.
  • Cartilage damage is graded according to the International Cartilage Repair Society (ICRS).
  • ICRS International Cartilage Repair Society
  • the culture supernatant of bone marrow mesenchymal stem cells was collected for exosome extraction.
  • cell debris in the supernatant will be removed by centrifugation, and the resulting supernatant will be filtered and then extracted with exosomes using a commercial exosome extraction kit ( Figure 1a). Perform relevant identification on the obtained secretosomes.

Abstract

提供了一种骨髓间充质干细胞外泌体在制备治疗关节软骨疾病的药物中的应用。所述骨髓间充质干细胞外泌体由培养基刺激骨髓间充质干细胞经传代后于培养液中提取获得，所述骨髓间充质干细胞来源于髂骨或股骨的骨髓。所述骨髓间充质干细胞外泌体可提高软骨细胞活力，促进软骨细胞增殖和迁移的活性，促进软骨修复。

Description

骨髓间充质干细胞外泌体在修复关节软骨缺损中的应用 技术领域
本发明属于生物医药领域，涉及一种骨髓间充质干细胞外泌体的制备方法及其在促进软骨细胞活力、提高软骨细胞增殖、增强软骨细胞迁移、修复软骨缺损中的应用。
背景技术
软骨是由胶原蛋白、蛋白聚糖、糖胺聚糖、糖蛋白、少许细胞、以及60-80％的水分等成份所构成。软骨细胞与周围的胞外基质(ECM)共同构成软骨单位，被视为软骨的主要结构、功能及代谢单位。在正常软骨中，软骨细胞通过合成胶原蛋白、蛋白聚糖及糖胺聚糖等物质，用以维持软骨稳态或置换损坏软骨。软骨在人体中具有承重负荷，减少关节间骨骼摩擦等重要作用。然而，成人软骨组织中缺乏血管或神经，因此受伤软骨组织自行修复能力十分有限。软骨功能缺损常与退行关节病发生密切相关，如退行性关节炎、老年性关节炎、肥大性关节炎等。临床上治疗退行关节病的方法包括非手术治疗和手术治疗两类。非手术治疗以口服非甾体类抗炎药、关节腔内注射药物和康复锻炼为主，虽能缓解近期疼痛改善症状，但远期效果不佳。手术治疗包括微骨折、钻孔、关节置换手术、自体软骨细胞植入、幼年异体软骨移植、自体软骨移植等。然而，由于这些不同的外科手术的局限性，包括纤维软骨下位形成，有限的组织可用性，原发性软骨细胞在扩张过程中脱分化导致功能丧失，以及可能出现的供体软骨周围炎症等，使得软骨再生的新型替代治疗策略成为迫切需要。基于干细胞分化潜能治疗软骨损伤已经被临床证实了巨大的发展潜力，同时为避免干细胞致瘤性等方面的制约，干细释放的细胞外囊泡，特别是外泌体产生的旁分泌作用修复软骨损伤越发受到重视。
发明内容
本发明的目的在于提供一种骨髓间充质干细胞外泌体，在促进软骨细胞活力、提高软骨细胞增殖、增强软骨细胞迁移、修复软骨缺损中的应用。所述外泌体可以提高软骨细胞活力，促进细胞增殖，并增强细胞迁移能力。外泌体处 理可显著促进缺损软骨修复，并显著改善缺损软骨的再生。
骨髓间充质干细胞外泌体在制备治疗关节软骨疾病中的应用，所述骨髓间充质干细胞外泌体由培养基刺激骨髓间充质干细胞产生，传代后于培养液中提取骨髓间充质干细胞外泌体，所述骨髓间充质干细胞来源于髂骨或股骨的骨髓。
进一步的，所述应用包括以下任一项：
在制备促进软骨细胞活力药物中的应用；
在制备促进软骨细胞增殖药物中的应用；
在制备促进软骨细胞迁移药物中的应用；
在制备修复软骨缺损药物中的应用。
进一步的，所述培养基为完全培养基。
进一步的，骨髓间充质干细胞的培养条件为37℃，5％CO 2。
进一步的，将骨髓加入完全培养基吹打混匀，置于培养箱中培养，第3天半换液，第6天全换液，后续每2天换液，直至细胞长至80-90％，传代至P1代，再经过3次传代得到原代骨髓间充质干细胞。
进一步的，培养骨髓间充质干细胞时，细胞生长至对数生长期，弃培养基，PBS清洗后换成2％无囊泡血清的培养基培养，直至细胞长至80-90％，收集上清液，滤液用于外泌体提取。
进一步的，采用膜亲和离心柱过滤收集骨髓间充质干细胞外泌体。
进一步的，将过滤后的上清液加入等体积XBP缓冲液，混匀后加到膜亲和离心柱处理，吸附在柱上的外泌体用XE缓冲液进行洗脱。
进一步的，所述过滤方法为0.2μm滤膜过滤。
进一步的，混匀后加到膜亲和离心柱处理，500g离心1分钟。
有益效果
本发明提供骨髓间充质干细胞外泌体在制备治疗关节软骨疾病中的应用，所述外泌体可以提高软骨细胞活力，促进细胞增殖，并增强细胞迁移能力。外泌体处理可显著促进缺损软骨修复，并显著改善缺损软骨的再生。
附图说明
图1为骨髓间充质干细胞外泌体提取与鉴定。(a)骨髓间充质干细胞外泌 体提取流程；(b)骨髓间充质干细胞外泌体电镜照片；(c)纳米颗粒跟踪仪分析骨髓间充质干细胞外泌体；(d)骨髓间充质干细胞外泌体蛋白质鉴定；1：骨髓间充质干细胞总蛋白样品；2：骨髓间充质干细胞外泌体蛋白样品。蛋白表达采用western blot方法进行分析；(e)骨髓间充质干细胞外泌体内化鉴定。FITC-Phalloidin：异硫氰酸荧光素-鬼笔环肽，用于标记细胞骨架；PKH-26：红色荧光素，用于标记外泌体；DAPI：4',6-二脒基-2-苯基吲哚，用于标记细胞核；Merge为合图。
图2为骨髓间充质干细胞外泌体促进软骨细胞活力及细胞增殖。(a)骨髓间充质干细胞外泌体提高软骨细胞活力；(b)骨髓间充质干细胞外泌体促进软骨细胞增殖。*p<0.05,***p<0.001,Student’s t-test分析。
图3为骨髓间充质干细胞外泌体促进软骨细胞迁移。(a)用骨髓间充质干细胞外泌体处理软骨细胞，用伤口愈合实验检测细胞迁移能力；(b)对伤口愈合实验结果进行量化分析。***p<0.001,one-way ANOVA分析。
图4为骨髓间充质干细胞外泌体在软骨细胞中内化动力学分析。(a)骨髓间充质干细胞外泌体内化。将骨髓间充质干细胞外泌体进行PKH26标记，用标记的外泌体处理软骨细胞，不同时间点检测细胞内PKH26的信号；(b)对骨髓间充质干细胞外泌体在软骨细胞中不同时间点PHK26强度的量化分析。FITC-Phalloidin：异硫氰酸荧光素-鬼笔环肽，用于标记细胞骨架；PKH-26：红色荧光素，用于标记外泌体；DAPI：4',6-二脒基-2-苯基吲哚，用于标记细胞核；Merge为合图。***p<0.001,one-way ANOVA分析。
图5为骨髓间充质干细胞外泌体促进软骨修复。(a)实验流程；(b)兔膝关节软骨外观图；(c)兔膝关节软骨苏木精-伊红染色(hematoxylin-eosin staining)。
图6为骨髓间充质干细胞外泌体促进软骨再生。(a)番红-固绿染色(Saf-O/Fast Green staining)。(b)根据国际软骨学会评分标准对软骨再生进行评分。*p<0.05,**p<0.01,***p<0.001,one-way ANOVA分析。
具体实施方式
一、实验步骤
1.人骨髓间充质干细胞外泌体的提取、培养、鉴定
人骨髓标本来源于6名正常成年人的髂骨或者股骨(平均年龄35岁，范围30-40岁；男：3名，女：3名)。将提取的人的骨髓1ml置60mm培养皿中，加入8ml完全培养基吹打混匀。所述完全培养基为DMEM细胞培养液，并补加10％的胎牛血清，1％的盘尼西林及链霉素。之后置于37℃含5％CO 2的培养箱中培养，第3天半换液，第6天全换液，后续每2天换液，直至细胞长至80-90％，传代至P1代。这样经过3次传代后即获得了较纯的原代骨髓间充质干细胞,P3-P5代细胞用于后续实验。
2.骨髓间充质干细胞外泌体提取
细胞生长至对数生长期，弃培养基，PBS洗一次，换成2％无囊泡血清的培养基培养，所述2％无囊泡血清的培养基为：将胎牛血清经超速离心(100000g,16小时，4℃)去除囊泡后用于细胞培养基配制。培养基组成为：DMEM细胞培养液内含2％无囊泡胎牛血清，1％盘尼西林及链霉素。直至细胞长至80-90％，收集上清，0.2μM滤器过滤，滤液用于外泌体提取。将0.2μM滤器过滤后的滤液转移到一个新的管中，加入等体积XBP缓冲液，将试管轻轻翻转5次，混匀溶液。将混合物加到外泌体试剂盒exoEasy Kit柱，500g离心1分钟。丢弃流出的溶液，再次加样直至混合物全部过滤。将柱子置于同一收集管中，加入10ml XWP缓冲液，5000g离心5分钟，去除柱中的残余液体。弃去下层液体。将柱子移至新的收集管，吸附在柱上的外泌体用XE缓冲液进行洗脱。将得到的外泌体保存于-80℃冰箱，用于后续实验。
3.外泌体鉴定
(1)用PBS重悬外泌体，Nanoparticle tracking analyses(NTA)测定外泌体粒径大小及浓度。
(2)将外泌体溶液沉积在聚醋酸甲基乙烯脂和碳包覆的300目铜网格上，在室温下放置3min，然后用1.5％的乙酸铀酰染色。透射电子显微镜对网格进行成像、观察、拍照。
(3)Western Blot用于检测骨髓间充质干细胞外泌体相关蛋白包括CD81,Flotillion-1,TSG101的表达情况。
4.软骨细胞的提取与培养
软骨细胞提取：(1)软骨切片取自4周大的新西兰兔的膝关节；(2)将其切成小块。PBS洗涤三次后；(3)0.25％胰蛋白酶消化30min；(4)弃上清，0.2％的2型胶原酶，置于37℃含5％CO2培养箱中消化过夜；(5)200目滤网过滤，滤液以500g离心5分钟；(6)沉淀用软骨细胞完全培养基重悬后置培养箱培养。
软骨细胞传代：(1)每2d用完培换液，直至细胞长至80-90％；(2)PBS清洗2遍后，加入适当体积0.25％胰酶消化，完培终止消化收集细胞，离心后重悬种板；(3)这样经过1次传代后即获得了较纯的原代软骨细胞,P1-P3代的软骨细胞用于后续的实验。
5.软骨细胞活力检测
在96孔板中，按照5×10 3/ml的密度接种软骨细胞。接种4h后，换成含2％无囊泡血清的培养基，并加入不同浓度的外泌体(5.0×10 8/ml,1.0×10 9/ml，2.0×10 9/ml)培养24h。加入1/10培养基总体积的CCK-8溶液。将培养板置于37℃培养箱内孵育2-4h。测定450nm波长处的吸光值。
6.软骨细胞增值检测
在96孔板中，按照5×10 3/ml的密度接种软骨细胞。接种4h后，换成含2％无囊泡血清的培养基，并加入制备好的外泌体(1.0×10 9/ml)培养24h。吸去每孔中的培养基，每孔加入50μl EdU培养基孵育2h。弃培养基，PBS清洗后加入100μl固定液室温固定15min；加入100μl 2mg/ml甘氨酸溶液，中和反应5min；加入100μl PBS，清洗5min；加入100μl 0.5％TritonX-100的PBS反应10min，PBS清洗5min。加入100μl染色反应液，避光室温孵育30min；加入100μl 0.5％TritonX-100的PBS清洗2次，每次10min；加入100μl Hoechst染色液，避光室温孵育15min；PBS脱色摇床清洗3次，每次5-10min。细胞图像用荧光显微镜EVOS FL Auto拍摄。
7.细胞划痕实验
采用ibidi装置模拟划痕实验：(1)在预包被的小皿中插入2孔装置，进行伤口愈合试验。(2)准备预处理的细胞悬液，即用无血清培养基重悬细胞。按 每孔2×10 5/ml浓度(100μl)加入细胞悬液。(3)12h后用无菌镊轻轻拔起2装置，PBS洗1遍。(4)对照组换成2％无囊泡血清培养基，处理组换成含外泌体(1.0×10 9/ml)的2％无囊泡血清培养基，继续培养24h和48h。(5)显微镜下观察细胞迁移过程并拍照。
8.外泌体内化实验
使用PKH26对外泌体进行标记，过程如下：(1)准备浓度为1×10 11/ml的外泌体溶液(100μl)。(2)将外泌体加入250μl Diluent C中，轻轻混匀。(3)取新的EP管，将2μl PKH26加入250μl Diluent C中。(4)将(2)加入(3)中，混匀。(5)室温放置4min，每隔1min用移液枪混匀。(6)加入等体积1％BSA终止染色。(7)将溶液用Amicon Ultra Centrifugal Filters浓缩后备用。(8)用无血清培养基稀释浓缩后的外泌体，最终配成含有1.0×10 9/ml的外泌体的溶液。(9)待软骨细胞接种4h后，加入PKH26标记的外泌体，共同培养3,6,12,24,48,72h。(10)处理结合后用4％PFA固定细胞。(11)PBS清洗2次，每次10min；(12)加入100μl DAPI染色液，避光室温孵育15min。(13)共聚焦显微镜观察、拍照。
9.软骨缺损模型构建
新西兰兔由南通大学动物中心提供。将其分为4组：对照组、手术组、手术+低剂量外泌体组、手术+高剂量外泌体组，每组6只。首先给予肌注美托咪定(20μg/kg)，氯胺酮(5mg/kg)、丁丙诺啡(0.03mg/kg)联合用药。使用异氟烷进行麻醉，在兔左后肢用钻头在膝关节股骨滑车凹槽处打出一个直径为4mm、深度为3mm的骨软骨缺损。关闭切口，逐层缝合。连续3天肌肉注射青霉素(5万单位/kg)预防感染，曲马多2mg/kg镇痛。将300μl不同浓度(低剂量：1.0×10 10/ml；高剂量：5.0×10 10/ml)外泌体注射至关节腔内，每周注射1次，共处理4次。
10.石蜡切片
实验结束，对兔子实施安乐死，取出完整膝关节，多聚甲醛固定24h，10％EDTA(pH 7.4)脱钙2-3个月，石蜡包埋膝关节，切成5μm厚的薄片。石蜡切片脱蜡至水：依次将切片放入二甲苯(Ⅰ)20min-二甲苯(Ⅱ)20min-无水乙醇 (Ⅰ)5min-无水乙醇(Ⅱ)5min-75％乙醇5min，自来水洗。
11.苏木精和伊红(H&E)染色
苏木精液染色(60℃)30～60s，流水洗去苏木精液5～10s，1％盐酸乙醇1～3s，稍水洗1～2s，促蓝液返蓝5～10s，流水冲洗15～30s，0.5％伊红液染色30～60s蒸馏水稍洗1～2s，80％乙醇1～2s，95％乙醇1～2s，无水乙醇1～2s，二甲苯(Ⅰ)2～3s，二甲苯(Ⅱ)2～3s，中性树胶封固。
12.翻红-固绿染色(Safranin O/Fast Green)染色
固绿染色：切片入固绿染液5-10min，水洗去多余染液，至软骨呈无色，分化液稍浸泡，三蒸水清洗。番红染色：切片入番红染液15-30s，无水乙醇快速脱水。透明封片：干净的二甲苯透明5min，中性树胶封片。显微镜镜检，图像采集分析。
13.软骨再生评分
根据国际软骨修复协会(International Cartilage Repair Society，ICRS)对软骨损伤分级评分。
将骨髓间充质干细胞培养上清收集起来用于外泌体提取。首先将用离心去除上清中的细胞碎片，所得上清经过滤后用商品化外泌体提取试剂盒进行外泌体提取(图1a)。对所得的分泌体进行相关鉴定。首先，用电子显微镜对所提取的外泌体进行观察，发现所提外泌体显现典型外泌体外形，即具有双层膜的杯状囊泡(图1b)；随后用纳米颗粒跟踪仪进行分析，发现所提外泌体平均直径为131.20nm，浓度为1.2x 10 11颗粒/毫升(图1c)；蛋白质检测发现，所提外泌体表达外泌体的典型蛋白包括CD81，TSG10，Flotillion 1，不表达Actin(图1d)；最后，我们检测了所提外泌体的内化能力。结果表明，将外泌体与软骨细胞共培养，发现外泌体可内化(图1e)。上述结果显示从骨髓间充质干细胞上清所提的胞外囊泡为外泌体。
功能实验表明，所提外泌体可以提高软骨细胞活力(图2a)，促进细胞增殖(图2b)，并增强细胞迁移能力(图3a,b)。为确定体内处理方式，进行了外泌体内化动力学检测。结果表明，外泌体与软骨细胞共培养3小时即可在细胞内观察到外泌体，随处理时间延长，细胞内的外泌体逐渐增多，内化高峰出 现在处理后72小时(图4a,b)。体内实验采用兔后肢膝关节软骨缺损模型，将制备的外泌体注射至关节腔内，共注射4次，随后进行形态学检测分析软骨修复情况(图5a)。对兔膝关节大体形态观察发现，手术组兔膝关节软骨缺损依然清晰可见；经过外泌体处理后，软骨缺损则明显恢复，并且高剂量组恢复更好(图5b)。苏木精-伊红染色结果也表明，外泌体处理可显著促进缺损软骨修复(图5c)。番红-固绿染色结果发现，手术组兔膝关节软骨缺损没有得到改善，但外泌体处理可显著改善缺损软骨的再生(图6a)；用国际软骨学会制定的标准进行评分，外泌体处理可明显增加该项评分，高剂量外泌体组评分优于低剂量组(图6b)。

Claims (10)

  1. 骨髓间充质干细胞外泌体在制备治疗关节软骨疾病中的应用，其特征在于，所述骨髓间充质干细胞外泌体由培养基刺激骨髓间充质干细胞产生，传代后于培养液中提取骨髓间充质干细胞外泌体，所述骨髓间充质干细胞来源于髂骨或股骨的骨髓。
  2. 根据权利要求1所述的应用，其特征在于，所述应用包括以下任一项：
    在制备促进软骨细胞活力药物中的应用；
    在制备促进软骨细胞增殖药物中的应用；
    在制备促进软骨细胞迁移药物中的应用；
    在制备修复软骨缺损药物中的应用。
  3. 根据权利要求1所述的应用，其特征在于，所述培养基为完全培养基。
  4. 根据权利要求1所述的应用，其特征在于，骨髓间充质干细胞的培养条件为37℃，5％CO 2。
  5. 根据权利要求1所述的应用，其特征在于，将骨髓加入完全培养基吹打混匀，置于培养箱中培养，第3天半换液，第6天全换液，后续每2天换液，直至细胞长至80-90％，传代至P1代，再经过3次传代得到原代骨髓间充质干细胞。
  6. 根据权利要求1所述的应用，其特征在于，培养骨髓间充质干细胞时，细胞生长至对数生长期，弃培养基，PBS清洗后换成2％无囊泡血清的培养基培养，直至细胞长至80-90％，收集上清液，滤液用于外泌体提取。
  7. 根据权利要求1所述的应用，其特征在于，采用膜亲和离心柱过滤收集骨髓间充质干细胞外泌体。
  8. 根据权利要求6所述的应用，其特征在于，将过滤后的上清液加入等体积XBP缓冲液，混匀后加到膜亲和离心柱处理，吸附在柱上的外泌体用XE缓冲液进行洗脱。
  9. 根据权利要求8所述的应用，其特征在于，所述过滤方法为0.2μm滤膜过滤。
  10. 根据权利要求8所述的应用，其特征在于，混匀后加到膜亲和离心柱处理，500g离心1分钟。
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