CN113521101A - Application of stem cell-derived exosomes in the preparation of drugs for the treatment of chronic obstructive pulmonary disease - Google Patents
Application of stem cell-derived exosomes in the preparation of drugs for the treatment of chronic obstructive pulmonary disease Download PDFInfo
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Abstract
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。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，为治疗慢性阻塞性肺疾病提供了一种新的思路和策略。
The invention relates to the technical field of pulmonary disease treatment, in particular to the application of stem cell-derived exosomes in the preparation of drugs for treating chronic obstructive pulmonary disease. The research of the present invention shows that exosomes with small structures are more easily taken up by recipient cells, which have similar therapeutic effects as stem cells in the treatment of chronic obstructive pulmonary disease, and are more easily absorbed by recipient cells because they have no immune rejection. Therefore, it can be used as a substitute for stem cells, which provides a new idea and strategy for the treatment of chronic obstructive pulmonary disease.
Description
Technical Field
The invention relates to the technical field of lung disease treatment, in particular to application of exosomes derived from stem cells in preparation of a medicament for treating chronic obstructive pulmonary disease.
Background
Chronic obstructive pulmonary disease is characterized by persistent respiratory symptoms and airflow limitation, and is a common preventable and treatable chronic respiratory disease. Smoking is one of the major causes of the onset of chronic obstructive pulmonary disease, and its pathogenesis is very complex. At present, effective radical treatment measures are not available clinically except for lung transplantation and chronic obstructive lung diseases.
Because there is no graft rejection reaction and the damaged part can be enriched to play the functions of anti-inflammation, immune suppression, anti-apoptosis, etc., aiming at the pathogenesis of the chronic obstructive pulmonary disease, biological treatment means such as stem cell transplantation and cytokine application thereof, etc. have become one of the most potential alternatives for treating the chronic obstructive pulmonary disease. Although it has been developed for a long time, only 11 stem cell therapy products are currently approved in 11 enterprises worldwide. The potential is huge in the stem cell treatment market in China, particularly in the treatment of lung diseases. However, as stem cells have been studied, problems associated with their use have become more prominent. The application of stem cells has other limitations, and the survival time of transplanted stem cells in vivo is short, so that the planting treatment effect after transplantation cannot be ensured. In addition, we cannot ignore the unavoidable side effects thereof, especially tumorigenicity and immunogenicity.
In response to the above problems, approaches and methods for better and safer application of stem cells to clinical transformation applications may be sought.
Disclosure of Invention
The invention aims to provide application of exosome derived from stem cells in preparation of a medicament for treating chronic obstructive pulmonary disease. The present invention seeks a substitute drug that inherits MSC function and is more easily ingested: an exosome. Researches show that the exosome has a good effect on treating the chronic obstructive pulmonary disease like stem cells, and meanwhile, the exosome is a vesicle with a lipid bilayer membrane structure, is small in structure and easy to be absorbed by receptor cells, and further reaches an injured part to play a role in immunotherapy, so that a new thought and strategy are provided for treating the chronic obstructive pulmonary disease.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
application of exosome derived from stem cells in preparation of drugs for treating chronic obstructive pulmonary disease.
The research of the invention shows that the exosome with a small structure and easy uptake by the receptor cells has the similar treatment effect with the stem cells in the treatment of the chronic obstructive pulmonary disease, and can be used as a substitute of the stem cells because the exosome is not subjected to immune rejection and is easy to be taken by the receptor cells.
Optionally, the exosomes are extracted from a stem cell culture supernatant.
Alternatively, the stem cell-derived exosome is prepared by the following method:
the culture supernatant obtained by culturing the mesenchymal stem cells is obtained by gradient ultracentrifugation.
Alternatively, in some embodiments of the present invention, exosomes in culture supernatant from mesenchymal stem cell culture are gradually extracted by a kit.
It should be noted that, based on the disclosure of the present invention, it is easy for those skilled in the art to extract exosomes in the supernatant by using other well-known kit extraction techniques, and it is within the scope of the present invention to use exosomes obtained by any method for treating chronic obstructive pulmonary disease.
The functional proteins encapsulated by the exosomes are identified by mass spectrometry, so that the exosomes are about more than three hundred proteins, and enrichment analysis finds that the functional proteins are really mainly involved in the biological processes of dealing with injury, cell activation, tissue repair, factor immune regulation and the like.
Optionally, in some embodiments of the invention, the mesenchymal stem cell is derived from adipose, bone marrow, dental pulp, or umbilical cord.
Further, the medicament has any one or more of the following options:
(a) inhibiting bronchoalveolar lavage fluid inflammation;
(b) improving lung structure;
(c) improving lung function;
(d) inhibiting apoptosis of alveolar epithelial cells.
The study of the present invention shows that, in the case of chronic obstructive pulmonary disease, exosomes derived from stem cells have functions of inhibiting bronchoalveolar lavage fluid inflammation, improving lung structure, improving lung function, and inhibiting apoptosis of alveolar epithelial cells, and by exerting these functions, treatment of chronic obstructive pulmonary disease is achieved.
Further, the inhibiting bronchoalveolar lavage fluid inflammation comprises: decreasing the percentage of neutrophils, increasing the percentage of macrophages, and/or decreasing the level of inflammatory factors.
The research of the invention shows that, on a more specific functional level, for chronic obstructive pulmonary disease, 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, and have the effect of inhibiting the inflammation of bronchoalveolar lavage fluid.
Further, the inflammatory factor includes at least one of IL-1 β, IL-6, and IL-10.
Further, the improving the lung structure includes: inhibit alveolar septal thickening.
The research of the invention shows that on a more specific functional level, for chronic obstructive pulmonary disease, the exosome derived from the stem cells can obviously inhibit alveolar septal thickening and improve lung structure.
Optionally, the improving lung function comprises: decreasing functional residual capacity, increasing Cfvc50, and/or increasing FEV 100/FVC.
The study of the invention shows that, on a more specific functional level, for chronic obstructive pulmonary disease, the stem cell-derived exosomes of the invention can reduce lung functional residual capacity, increase Cfvc50 and/or increase FEV100/FVC to improve lung function.
Optionally, the inhibiting apoptosis of alveolar epithelial cells comprises: restoring the activity of alveolar epithelial cells, promoting cell proliferation, and inhibiting apoptosis induced by diseases.
The research of the invention shows that, on a more specific functional level, for chronic obstructive pulmonary disease, the exosome derived from the stem cells can restore the proliferation capacity of alveolar epithelial cells and inhibit apoptosis of the cells.
The chronic obstructive pulmonary disease in the present invention is not limited to humans, and may be other mammals.
A method of treating chronic obstructive pulmonary disease comprising administering to a subject an effective amount of stem cell-derived exosomes or a formulation comprising stem cell-derived exosomes.
The exosome derived from the stem cells or the preparation containing the exosome derived from the stem cells is applied to the treatment of chronic obstructive pulmonary disease.
Wherein an effective amount is an amount of the formulation that treats, ameliorates, or prevents the target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the symptoms, and the therapeutic agent and/or combination of therapeutic agents selected for administration. The mode of administration may be injection, in vivo or transdermal, and the drug is administered to the patient in need of treatment, and the specific dose and mode of administration may be determined by a physician according to the patient's condition.
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.
For example, the adjuvant may include any one or more of wetting agent, emulsifier, diluent, excipient, filler, disintegrant, binder, lubricant, surfactant, flavoring agent, stabilizer, etc.
Further, the dosage form of the medicine comprises injection, nasal spray or nasal drops.
The dosage form of the present invention is not limited thereto, and those skilled in the art can select the dosage form according to the actual needs, and the dosage form is within the protection scope of the present invention.
Further, the medicament comprises any one or more of: an agent for inhibiting bronchoalveolar lavage fluid inflammation, an agent for improving lung structure, and an agent for improving lung function.
The stem cell-derived exosome of the present invention may be formulated into different dosage forms according to circumstances, or may be formulated into different dosage forms according to the difference in the use, such as a preparation for inhibiting bronchoalveolar lavage fluid inflammation, a preparation for improving lung structure, a preparation for improving lung function, and the like.
The pharmaceutical dosage form provided by the invention is prepared according to the conventional method in the field.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the invention adopts exosomes derived from stem cells to treat chronic obstructive pulmonary disease, prevents immune rejection, and plays a role in immunotherapy by being absorbed by receptor cells due to small structures and easy to be absorbed by the receptor cells, thereby providing a new idea and strategy for treating the chronic obstructive pulmonary disease.
(2) According to researches, the exosome derived from the stem cells has the effects of inhibiting bronchoalveolar lavage fluid inflammation, improving lung structure, improving lung function, inhibiting apoptosis of alveolar epithelial cells and the like, and the treatment of the chronic obstructive pulmonary disease is realized through the exertion of the functions.
(3) The exosome derived from the stem cells can be prepared into different dosage forms or preparations according to needs so as to better treat the chronic obstructive pulmonary disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph showing the result of Western blotting reaction (WB) assay of the exosomes purified in example 1 of the present invention;
FIG. 2 is an Electron Microscope (EM) observation image of the purified exosome in example 1 of the present invention;
FIGS. 3 and 4 are graphs showing the results of the NTA method for detecting purified exosomes in example 1 of the present invention;
FIG. 5 is a protein species map identified by mass spectrometry detection of the stem cell-derived exosome in example 1 of the present invention;
FIG. 6 is a graph showing the enrichment analysis of the protein species of the stem cell-derived exosomes in example 1 of the present invention;
FIG. 7 is a graph showing the experimental confirmation of the uptake of exosomes by recipient cells in vivo and in vitro in example 1 of the present invention;
FIG. 8 is a graph comparing the phenotype of lung tissue and lung function in the treated and control groups of example 2 of the present invention;
FIG. 9 is a graph showing a comparison of the results of staining lung tissues in the treatment group and the control group in example 2 of the present invention;
FIG. 10 is a graph showing the lesion of thickened airways in lung tissue in the treatment group and the control group in accordance with example 2 of the present invention;
FIG. 11 is a graph showing the changes in inflammation in the treatment group and the control group according to example 2 of the present invention;
FIG. 12 is a graph showing the expression of proinflammatory factors in the treatment group and the control group of example 2 of the present invention;
FIG. 13 is a graph showing the reduction of proliferation caused by the recovery of CSE (Cigarette Smoke Extract) stimulation in the treatment group and the control group according to example 2 of the present invention;
FIG. 14 is a graph of CSE-induced apoptosis in the treatment and control groups of example 2 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
1. Preparation and amplification of mesenchymal stem cells
And (3) separating the obtained mesenchymal stem cells of the P0 generation, and respectively establishing an original cell bank and a working cell bank in the P3 generation, the P5 generation and the P6 generation. The P6 generation cells were subjected to phenotypic cell assay. And (4) replacing the culture medium after the cell density is more than 70%, and collecting cell culture supernatant obtained by P5-P6 generation cell expansion culture after 48 hours.
2. Exosome extraction and identification
Centrifuging the collected culture supernatant A mL at 4 deg.C for 30min at 3000g for 30min to remove dead cells, centrifuging the supernatant for 30min at 10000g for removing cell debris, transferring the supernatant to an ultrafiltration tube, and concentrating to B mL (A: B is about 20: 1). The exosome was separated and further purified using an ultracentrifuge, which centrifuged 110000g twice for 70 min. And (3) resuspending the precipitate with 100 μ l of PBS to obtain the purified MSC-exo medicament, and detecting and identifying the purified MSC-exo medicament by WB, EM and NAT technologies.
(1) After purifying the exosomes, preparing a protein sample by lysis, detecting and identifying the exosome marker proteins by a WB method after detecting the protein concentration by BCA, and the result is shown in figure 1.
(2) The exosome morphology was observed by transmission electron microscopy: and dripping 10 mu l of MSC-exo into a copper net, standing for 2min, dripping 10 mu l of uranyl acetate, dyeing for 1min, drying and observing by a transmission electron microscope, wherein the result is shown in figure 2.
(3) NTA measures exosome particle size distribution and exosome concentration: the particle size distribution and concentration of exosomes were measured using a markov NanoSight Tracking Analysis instrument and the results are shown in fig. 3, 4.
3. Analysis of exosome composition
After the stem cells from the three human donors are separated, exosomes of the stem cells are respectively extracted, functional proteins encapsulated by the exosomes are identified through mass spectrometry, and the mode of functioning of the exosomes is further explained. Stem cell-derived exosomes are approximately more than three hundred proteins rich, as shown in fig. 5. By enrichment analysis (see fig. 6), it was found that these functional proteins are indeed mainly involved in biological processes of dealing with injury, cell activation, tissue repair, immune modulation of factors, etc., which may be the way they exert therapeutic effects.
4. Verification of exosome uptake by recipient cells
The uptake of exosomes by recipient cells was verified by in vivo and in vitro experiments, and the results are shown in figure 7. Indicating that the recipient cell can take up exosomes.
Example 2
1. Evaluation of therapeutic Effect of Stem cell-derived exosome on treatment of CODP model mouse
(1) Construction and administration design of COPD model mice
After the C57BL/6J mice are qualified by quarantine, the mice are randomly divided into a normal control group (CTL), a model group, an MSCs group and an MSCs source exosome group. Establishing a mouse slow lung blocking animal model by using smoke exposure for 6 months, which specifically comprises the following steps: 4-6 weeks old mice, after a week of adaptation, using domestic flat cigarettes, were exposed systemically in a smoking box and were given passive smoking for 6 days per week for 6 months. Cell therapy was started at 3 months of smoke exposure, once a month at a dose of 200 μ g/200 μ l/mouse for 3 total treatments, and treatment effect was assessed by drawing material after 6 months of smoke exposure.
(2) Potency assessment for exosome treatment of COPD
The lung Functional Residual Capacity (FRC) of the model group mice is increased (P <0.05), the compliance of one second amount of forced expiratory peak flow rate and 50% vital capacity is increased (Cfvc50, FEVpef), the total number of bronchoalveolar lavage fluid inflammatory cells and neutrophils are obviously increased (P <0.01), the alveolar Mean Lining Interval (MLI) is increased, and the thickness is increased (P < 0.05). The above results indicate that a mouse model of COPD has been successfully established.
As shown in fig. 8, after treatment, the material was obtained, and after treatment with MSC or exosome, the color of lung tissue was reduced to some extent, and the deposition of particulate matter was improved to some extent; the pulmonary function of the rat is evaluated by adopting a small animal pulmonary function instrument (PFT system), and the result of the pulmonary function shows that after CS exposure, the functional residual capacity and forced vital capacity of the pulmonary function indexes of the rat are obviously increased, and after treatment, FRC, Cfvc50 and FEVpef are all reduced, thereby prompting that the pulmonary function is effectively improved. Suggesting that tobacco smoke exposure results in altered lung function in mice; compared with the model group, the exosomes and the MSCs can improve the lung dysfunction caused by smoke exposure to different degrees.
H & E staining is carried out on lung tissues, and as shown in figure 9, after CS is exposed, the pulmonary alveolar structure of a mouse is obviously destroyed, the pulmonary alveolar spaces are enlarged and fused, and emphysema characteristics are generated; and the exosome and the MSC intervention group are inhibited, the alveolar structure is relatively complete, the number of damaged alveoli is reduced, and the MLI value of the decrease of the average linear intercept of the alveoli is obviously reduced.
On airway thickening lesions, exosomes also showed good inhibitory effect as shown in figure 10. Exosome and MSC intervention group, lung alveolar wall becomes thin, partial compensatory emphysema is formed, blood vessel dilatation and congestion are caused, and thickening lesion of large airway is inhibited, which indicates that the lung structure is improved.
Chronic obstructive pulmonary disease is a chronic inflammatory response process, and the total number of inflammatory cells and neutrophil counts of bronchoalveolar lavage fluid after treatment with stem cells and exosomes is significantly lower than that of a model group (P <0.05) by examining the cellular components in BALF (as shown in fig. 11), indicating that the inflammatory response is effectively inhibited. That is, exosome therapy may be effective in reducing the abnormal increase of cellular inflammatory cells, particularly neutrophils and lymphocytes, in the alveoli.
In FIG. 12, each proinflammatory factor is plotted from left to right in the order Contral, CS + MSC, and CS + MSC-exosome. As shown in fig. 12, the expression of these pro-inflammatory factors of IL1\ IL6 and IL10 in the lung microenvironment, treatment of exosomes and MSCs was examined, and their expression was significantly reduced, and this anti-inflammatory effect was demonstrated not only locally but also systemically, with similar results obtained in serum examinations.
The mechanism of action of exosomes is not only immune regulation. Therefore, we used CSE extract to stimulate primary cultured alveolar epithelial cells in vitro, and in 72h culture observation, it was found that the addition of exosomes was effective in restoring the proliferation retardation caused by CSE stimulation, and a significant effect began to appear at 12h (see FIG. 13). In the graph of FIG. 13 showing the relationship between cell count and time, the top line is the Control group, the middle line is the CSE + MSC-exo group, and the bottom line is the CSE group. The same flow results showed that exosomes could inhibit CSE-induced apoptosis (see fig. 14).
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
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| PCT/CN2021/143667 WO2022242189A1 (en) | 2021-05-20 | 2021-12-31 | Application of exosomes derived from stem cells in preparing medicine for treating chronic obstructive pulmonary diseases |
| ZA2023/07972A ZA202307972B (en) | 2021-05-20 | 2023-08-16 | Application of stem cell-derived exosomes in the preparation of drugs for the treatment of chronic obstructive pulmonary disease |
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| CN114480260A (en) * | 2022-01-24 | 2022-05-13 | 同济大学 | Adult lung stem cell exosome and preparation method and application thereof |
| WO2022242189A1 (en) * | 2021-05-20 | 2022-11-24 | 广州医科大学附属第一医院（广州呼吸中心） | Application of exosomes derived from stem cells in preparing medicine for treating chronic obstructive pulmonary diseases |
| CN116162597A (en) * | 2022-12-12 | 2023-05-26 | 中国科学技术大学 | Engineered extracellular vesicles loaded with Wnt proteins and uses thereof |
| WO2023123216A1 (en) * | 2021-12-30 | 2023-07-06 | 中山大学 | Use of vesicles in preparation of drug for treating lung diseases |
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| CN112121063B (en) * | 2020-09-01 | 2022-04-29 | 北京诺德观呈医疗科技有限公司 | Application of exosome in preparation of medicine for treating pulmonary fibrosis |
| CN113521101A (en) * | 2021-05-20 | 2021-10-22 | 广州医科大学附属第一医院（广州呼吸中心） | Application of stem cell-derived exosomes in the preparation of drugs for the treatment of chronic obstructive pulmonary disease |
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| CN114480260A (en) * | 2022-01-24 | 2022-05-13 | 同济大学 | Adult lung stem cell exosome and preparation method and application thereof |
| CN114480260B (en) * | 2022-01-24 | 2024-02-09 | 同济大学 | An adult lung stem cell exosome and its preparation method and application |
| CN116162597A (en) * | 2022-12-12 | 2023-05-26 | 中国科学技术大学 | Engineered extracellular vesicles loaded with Wnt proteins and uses thereof |
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