CN114854688A - Method for improving expression quantity of mast cell exosome and Fc epsilon RI on membrane surface of mast cell exosome and application of mast cell exosome and Fc epsilon RI - Google Patents
Method for improving expression quantity of mast cell exosome and Fc epsilon RI on membrane surface of mast cell exosome and application of mast cell exosome and Fc epsilon RI Download PDFInfo
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Abstract
The invention discloses a method for improving the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes and application of the mast cell exosomes and the Fc epsilon RI, and relates to the technical field of biological cells. The invention provides a method for improving the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes, which comprises the following steps: (1) the culture dish was inoculated with mast cells and placed at 37 ℃ in 5% CO 2 Culturing in the complete culture medium, and replacing the culture medium when the cell density reaches 75-85%; (2) culturing the cells in the step (1) at the temperature of 29-41 ℃; (3) and (3) collecting the cell supernatant obtained in the step (2), and performing gradient centrifugation to extract exosomes. The invention can effectively improve the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes by changing the temperature to stimulate the mast cells; the invention adopts temperature to stimulate mast cells and experimental conditionsEasy realization, simple operation and suitability for the mass extraction of exosomes.
Description
Technical Field
The invention relates to the technical field of biological cells, in particular to a method for improving the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes and application of the mast cell exosomes.
Background
Allergic Asthma (AA) is the most common clinical phenotype in all types of asthma, and chronic inflammatory diseases of the nasal mucosa, mediated by serum Immunoglobulin E (IgE), seriously affect the daily life and work of patients. At present, the drugs for treating asthma which are clinically popular are mainly anti-inflammatory drugs and bronchodilators, but the curative effect of the drugs can only relieve symptoms at present, and the occurrence and development of inflammation in the body of an asthma patient cannot be solved from the source, so that the research on allergic asthma is urgent and important.
The development of asthma depends on a number of factors, IgE being the major mediator of the allergic reaction, and despite its low content in blood, the key role in linking innate and adaptive immunity in the persistent state of allergic asthma and being involved in the development of asthma in various stages. IgE is mainly sensitized by binding to the high affinity receptor for IgE (fceri) on the surface of mast cell membranes. The sensitized mast cells can be activated when meeting specific antigens in the outside again, release chemical mediators and trigger anaphylactic reaction. Treatment with anti-inflammatory and antihistamine drugs can only suppress the clinical symptoms at the time of asthma attack; the desensitization treatment has long period and high cost, and the effective rate is only 40 percent; anti-IgE antibodies are expensive, large in dosage, and further likely to cause allergic reactions, and are limited to treatment of moderately and severely allergic patients. The research reports that the protein Fc epsilon RI specifically expressed by mast cells exists in mast cell exosomes, and the Fc epsilon RI in the mast cell exosomes derived from mouse bone marrow cells can bind to free IgE and competitively inhibit the binding of the mast cells and the IgE, so that the mast cells are degranulated, the secretion histamine is reduced, the serum IgE level is reduced, and the anti-IgE effect of the mast cell exosomes is realized. Therefore, it appears to be crucial to find a way to promote secretion of mast cell exosomes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for improving the expression level of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes and application of the mast cell exosomes.
In order to achieve the purpose, the invention adopts the technical scheme that: a method of increasing the expression of mast cell exosomes and their membrane surface fceri, comprising the steps of:
(1) the culture dish was inoculated with mast cells and placed at 37 ℃ in 5% CO 2 Culturing in the complete culture medium, and replacing the culture medium when the cell density reaches 75-85%;
(2) culturing the cells in the step (1) at the temperature of 29-41 ℃;
(3) and (3) collecting the cell supernatant obtained in the step (2), and performing gradient centrifugation to extract exosomes.
The inventor of the application finds that the mast cells can be stimulated and the expression of the mast cell exosomes and the expression of the Fc epsilon RI on the membrane surface of the mast cell exosomes can be improved by changing the culture temperature on the basis of the secretion of the exosomes by the mast cells.
In a preferred embodiment of the method for increasing the expression level of mast cell exosomes and fceri on the membrane surface thereof according to the present invention, the culture temperature in the step (2) is 41 ℃.
The inventor of the application researches and discovers that on the basis that the mast cells secrete the exosomes, when the mast cells are continuously cultured at 41 ℃, the expression levels of the mast cell exosomes and the Fc epsilon RI on the membrane surface of the mast cell exosomes are higher.
In a preferred embodiment of the method for increasing the expression level of mast cell exosomes and fceri on the membrane surface thereof according to the present invention, the culture time in the step (2) is 2 hours.
The inventor of the present application has found that the mast cell is cultured by the method of the present invention, and the mast cell is cultured for 2 hours at 41 ℃ on the basis of secretion of exosome from mast cell, and the expression level of the mast cell exosome and its membrane surface fcepsilonri is higher than that of the mast cell exosome and its membrane surface fcepsilonri for less than and more than 2 hours in culture time.
In a preferred embodiment of the method for increasing the expression level of mast cell exosomes and fcepsilonr on the membrane surface thereof according to the present invention, the medium to be replaced in step (1) is a complete medium without exosomes.
As a preferred embodiment of the method for increasing the expression level of mast cell exosomes and fceri on the membrane surface thereof according to the present invention, the gradient centrifugation in step (3) is: centrifuging at 300g for 10min, 2000g for 10min, 10000g for 30min, 100000g for 70min, and 110000g for 70 min.
The invention also provides the mast cell exosome prepared by the method for improving the expression level of the mast cell exosome and the Fc epsilon RI on the membrane surface of the mast cell exosome.
The invention also provides application of the mast cell exosome in preparing a medicament for treating allergic asthma.
As a preferred embodiment of the use according to the invention, the mode of administration of the prepared medicament is intravenous injection.
The invention has the beneficial effects that: the invention provides a method for improving the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface of the mast cell exosomes. Through a large number of experimental screenings, the inventor of the application finds that the expression level of the mast cell exosome and the Fc epsilon RI on the membrane surface of the mast cell exosome can be effectively improved by stimulating the mast cell by changing the temperature on the basis of the secretion of the mast cell exosome, and the expression level of the mast cell exosome and the Fc epsilon RI on the membrane surface of the mast cell exosome is the highest when the temperature is 41 ℃ and the stimulation time is 2 hours; the invention adopts temperature to stimulate mast cells, has easy realization of experimental conditions and simple operation, and is suitable for the mass extraction of exosomes.
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FIG. 1: a is an experimental step of treating asthma mice by exosomes with different concentrations; b is the airway hyperresponsiveness index of mice treated by different treatment groups; c is the IgE level in mice after treatment by different treatment groups.
FIG. 2: HE staining of lung tissue sections after treatment in different treatment groups.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1
The present example provides a method for increasing the expression level of mast cell exosomes and Fc epsilon RI on the membrane surface thereof, and the specific experimental method is as follows:
mast cells grown to a density of 90% in T175 flasks were grown at 1: 3 after plating, the mixture is placed at 37 ℃ and 5 percent CO 2 Culturing for 24h, when the cell growth reaches 80%, replacing the cell culture medium with complete culture medium without exosome, dividing the cell into three groups, and culturing at 29 deg.C, 37 deg.C, and 41 deg.C for 2 h. After the completion of the culture, the culture was performed by ultracentrifugation: centrifuging at 300g for 10min, 2000g for 10min, 10000g for 30min, 100000g for 70min, and 110000g for 70 min. After centrifugation, the supernatant was carefully aspirated, and the pellet was resuspended using 200. mu.l sterile PBS and transferred to a sterile EP tube for storage.
Example 2
This example used the exosomes prepared in example 1 to infuse allergic asthma mice and tested the Airway Hyperreactivity (AHR) of the mice by Penh, and serum IgE content by Elisa. And (3) observing pathological morphology change of the mouse alveolus and alveolus tissues in the alveolar lavage fluid, the infiltration degree of inflammatory cells in the alveolus and the proportion of the inflammatory cells by adopting HE staining.
The specific experimental method comprises the following steps:
(1) an Ovalbumin (OVA) sensitized mouse is adopted to establish an allergic asthma model of the mouse, and the mouse is randomly divided into 4 groups which are respectively (B) an OVA group: allergic asthma group; (C) OVA +29 deg.C-exo group: treating allergic asthma group with exosome secreted under 29 deg.C stimulation; (D) OVA +37 ℃ -exo group: treating allergic asthma group with exosome secreted under 37 deg.C stimulation; (E) OVA +41 ℃ -exo group: treating allergic asthma group with exosome secreted at 41 deg.C; let also (A) Ctrl group: and (4) a negative control group. Mice were sacrificed 24h after airway hyperreactivity detection following treatment and alveolar lavage fluid, serum and lung tissue were collected.
(2) Penh assay Airway Hyperresponse (AHR) assay in mice: at 24 hours post-treatment, mice were weighed and tested for lung function after checking that the instrument was functioning properly. After the mouse is placed in the scanning box, the detection device is connected, and the program is operated. After filling the weight of the mice, the program was set to record the basal value of airway resistance of the mice for 1 minute, nebulization for 1 minute, and recording for 3 minutes. Nebulization challenge was performed in PBS, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml, 64mg/ml acetylcholine and the average Penh value for each experiment was recorded.
(3) The Elisa method for detecting the content of serum IgE: adding 100 mul of each standard sample into a 96-well plate in the kit, and incubating for 120 minutes at 37 ℃; the liquid from each well was decanted and after adding 100. mu.l biotin antibody (1X) directly, incubated for 60 minutes at 37 ℃; after the liquid in each well was aspirated, the wells were washed with 200. mu.l of washing solution and repeated 3 times for 2 minutes each; followed by addition of HRP-avidin (1X) per well and incubation at 37 ℃ for 60 min; repeating the washing step for 5 times; add 90. mu.l TMB substrate per well. Incubation at 37 ℃ for 20 min; subsequently, 50. mu.l of stop solution was added to each well, and the absorbance was rapidly read at a wavelength of 450nm in a microplate reader and calculated.
(4) HE staining: after the lung tissues of mice in each experimental group were sliced, the slides were placed in an oven at 65 ℃ and heated for 2 hours to completely evaporate the water of the slides, then placed in xylene and left for 10 minutes, and dewaxing was repeated 3 times. Then, hydration was performed sequentially by using 100%, 90%, 80%, and 70% ethanol concentrations for 2 minutes. And (5) flushing the water with tap water. The slide is dripped with hematoxylin dye for 10 minutes, washed under tap water for 3 minutes, dripped with 1% hydrochloric acid ethanol for differentiation for 5 seconds, washed under tap water for 3 minutes, dripped with eosin dye for 2 minutes, and washed under tap water for 3 minutes. After the neutral resin is used for sealing, the automatic piece sweeping is carried out in a full-automatic piece sweeping instrument.
The results of the experiment are shown in FIGS. 1 and 2. As can be seen from FIG. 1b, the mice showed a decreased airway hyperreactivity index when injected with exosomes stimulated at 41 ℃ as compared to exosomes stimulated at 29 ℃ and exosomes stimulated at 37 ℃. As can be seen from FIG. 1c, IgE levels were also significantly reduced in asthmatic mice injected with exosomes stimulated at 41 ℃. As shown in FIG. 2, when the exosome stimulated at 41 ℃ was injected, the degree of alveolar inflammatory cell infiltration of mice was significantly reduced compared to the exosome stimulated at 29 ℃ and the exosome stimulated at 37 ℃.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for increasing the expression level of mast cell exosomes and Fc epsilon RI on the membrane surface thereof, comprising the following steps:
(1) the culture dish was inoculated with mast cells and placed at 37 ℃ in 5% CO 2 Culturing in the complete culture medium, and replacing the culture medium when the cell density reaches 75-85%;
(2) culturing the cells in the step (1) at the temperature of 29-41 ℃;
(3) and (3) collecting the cell supernatant obtained in the step (2), and performing gradient centrifugation to extract exosomes.
2. The method according to claim 1, wherein the culture temperature in the step (2) is 41 ℃.
3. The method according to claim 1, wherein the culturing time in the step (2) is 2 hours.
4. The method according to claim 1, wherein the medium replaced in step (1) is a complete medium without exosomes.
5. The method of claim 1, wherein the gradient centrifugation of step (3) is: centrifuging at 300g for 10min, 2000g for 10min, 10000g for 30min, 100000g for 70min, and 110000g for 70 min.
6. Mast cell exosomes prepared according to the method of any one of claims 1 to 5.
7. Use of a mast cell exosome according to claim 6 in the preparation of a medicament for treating allergic asthma.
8. The use according to claim 7, wherein the medicament is prepared for administration by intravenous injection.
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CN108324735A (en) * | 2017-01-20 | 2018-07-27 | 李莉 | For the extracellular body preparation of disease treatment and its application |
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WO2008116889A1 (en) * | 2007-03-26 | 2008-10-02 | Institut Pasteur | A novel in vitro model of mature serosal-type mast cells for the analysis of autoimmune and allergic inflammation |
CN108324735A (en) * | 2017-01-20 | 2018-07-27 | 李莉 | For the extracellular body preparation of disease treatment and its application |
KR20190070227A (en) * | 2017-12-12 | 2019-06-20 | 주식회사 엑소코바이오 | A method to evaluate functional activity of exosome and/or extracellular vesicle |
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Patent Citations (4)
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WO2008116889A1 (en) * | 2007-03-26 | 2008-10-02 | Institut Pasteur | A novel in vitro model of mature serosal-type mast cells for the analysis of autoimmune and allergic inflammation |
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US20200060970A1 (en) * | 2017-01-20 | 2020-02-27 | Li Li | Exosome preparation for treating disease and application thereof |
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Cited By (2)
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CN108324735A (en) * | 2017-01-20 | 2018-07-27 | 李莉 | For the extracellular body preparation of disease treatment and its application |
CN108324735B (en) * | 2017-01-20 | 2024-02-09 | 李莉 | Exosome preparation for treating diseases and application thereof |
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