CN114854688B - Method for improving expression level of mast cell exosomes and membrane surface Fc epsilon RI and application thereof - Google Patents
Method for improving expression level of mast cell exosomes and membrane surface Fc epsilon RI and application thereof Download PDFInfo
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Abstract
The application discloses a method for improving the expression quantity of mast cell exosomes and Fc epsilon RI on the membrane surface and application thereof, and relates to the technical field of biological cells. The application provides a method for improving the expression quantity of mast cell exosomes and the membrane surface Fc epsilon RI thereof, which comprises the following steps: (1) Mast cells were inoculated in petri dishes and placed at 37℃in 5% CO 2 Culturing in the complete culture medium, and changing the culture medium when the cell density reaches 75-85%; (2) Culturing the cells in the step (1) at 29-41 ℃; (3) Collecting the cell supernatant in the step (2) and carrying out gradient centrifugation to extract exosomes. According to the application, the temperature is changed to stimulate the mast cells, so that the expression level of the Fc epsilon RI on the exosomes of the mast cells and the membrane surface of the mast cells can be effectively improved; the application adopts temperature to stimulate the mast cells, has easy realization of experimental conditions and simple operation, and is suitable for mass extraction of exosomes.
Description
Technical Field
The application relates to the technical field of biological cells, in particular to a method for improving the expression level of mast cell exosomes and their membrane surface Fc epsilon RI and application thereof.
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) severely affect the daily life and work of patients. At present, the clinical popular medicines for treating asthma are mainly anti-inflammatory medicines and bronchodilators, but the curative effects of the medicines can only relieve symptoms at present and can not solve the occurrence and development of inflammation in asthma patients from the source, so that the research on allergic asthma is urgent and important.
The development and progression of asthma depends on numerous factors, igE being the primary mediator in allergic reactions, and despite the low IgE content in the blood, the engagement of innate and adaptive immunity plays a key role in the sustained state of allergic asthma and participates in the pathogenesis of asthma at various stages. IgE is mainly achieved by sensitization by binding to IgE high affinity receptors (fceri) on the surface of hypertrophied cell membranes. The sensitized mast cells can be activated by encountering specific antigens in the outside again, release chemical medium and trigger anaphylactic reaction. The treatment of anti-inflammatory and antihistamines can only suppress clinical symptoms at the time of asthma attack; the desensitization treatment period is long, the cost is high, and the effective rate is only 40%; anti-IgE antibodies are expensive, are used in large doses, may further elicit allergic reactions, and are limited to treatment of moderately severe allergic patients. Studies report that the presence of the mast cell-specific expressed protein fceri in the mast cell exosomes, fceri in the mouse bone marrow cell-derived mast cell exosomes can bind free IgE, competitively inhibit mast cell binding to IgE, thereby causing mast cell degranulation, reduced histamine secretion, reduced serum IgE levels, and an anti-IgE effect of the mast cell exosomes. Therefore, it is important to find a way to promote secretion of mast cell exosomes.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provides a method for improving the expression quantity of mast cell exosomes and the membrane surface Fc epsilon RI thereof and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows: a method for increasing the expression level of mast cell exosomes and their membrane surface fceri, the method comprising the steps of:
(1) Mast cells were inoculated in petri dishes and placed at 37℃in 5% CO 2 Culturing in the complete culture medium, and changing the culture medium when the cell density reaches 75-85%;
(2) Culturing the cells in the step (1) at 29-41 ℃;
(3) Collecting the cell supernatant in the step (2) and carrying out gradient centrifugation to extract exosomes.
The inventor of the present application has found through a great deal of experiments that, on the basis of secretion of exosomes by mast cells, by changing the culture temperature, mast cells can be stimulated, and expression of the exosomes of mast cells and their membrane surfaces fceri can be improved.
In a preferred embodiment of the method for increasing the expression level of fceri on the surface of a mast cell exosome or a membrane thereof according to the present application, the culture temperature in step (2) is 41 ℃.
The present inventors have found that the expression level of the mast cell exosomes and their membrane surfaces, fceri, is higher when they are continuously cultured at 41 ℃.
In a preferred embodiment of the method for increasing the expression level of fceri on the surface of a mast cell exosome or a membrane thereof according to the present application, the culture time in step (2) is 2 hours.
The present inventors have found that the method of the present application is used for culturing mast cells, and further culturing the mast cells at 41℃for 2 hours on the basis of secretion of exosomes by the mast cells, and that the expression level of fceri on the surface of the mast cells exosomes and membranes thereof is higher than the expression level for culturing times of less than and more than 2 hours.
In a preferred embodiment of the method for increasing the expression level of fcer on the surface of a mast cell exosome or a membrane thereof according to the present application, the medium to be replaced in the step (1) is a complete medium without exosomes.
As a preferred embodiment of the method for increasing the expression level of fceri on the surface of a mast cell exosome or a membrane thereof according to the present application, the gradient centrifugation in step (3) is: 300g for 10min,2000g for 10min,10000g for 30min,100000g for 70min and 110000g for 70min.
The application also provides the mast cell exosome prepared by the method for improving the expression quantity of the Fc epsilon RI on the surface of the mast cell exosome and the mast cell exosome.
The application also provides application of the mast cell exosome in preparing medicines for treating allergic asthma.
As a preferred embodiment of the use according to the application, the medicament prepared is administered by intravenous injection.
The application has the beneficial effects that: the application provides a method for improving the expression quantity of mast cell exosomes and the membrane surface Fc epsilon RI thereof. The inventor of the present application has found that, based on the secretion of the exosomes by the mast cells, the expression level of the exosomes and the fceri on the surface of the membrane can be effectively increased by stimulating the mast cells by changing the temperature, and when the temperature is 41 ℃ and the stimulation time is 2 hours, the expression level of the exosomes and the fceri on the surface of the membrane of the exosomes is the highest; the application adopts temperature to stimulate the mast cells, has easy realization of experimental conditions and simple operation, and is suitable for 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 hyperresponse index of mice after treatment of different treatment groups; c is IgE levels in mice after treatment in different treatment groups.
Fig. 2: lung tissue section HE staining pattern after treatment by different treatment groups.
Detailed Description
The above-described aspects of the present application will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present application is limited to the following examples only. All techniques implemented based on the above description of the application are within the scope of the application.
Example 1
The embodiment provides a method for improving the expression level of mast cell exosomes and their membrane surface fceri, which comprises the following specific experimental methods:
mast cells grown to 90% density in T175 flasks were grown at 1:3, after being plated, the mixture is placed at 37 ℃ and 5 percent CO 2 Culturing under the condition for 24h, when the cells grow to 80%, changing the cell culture medium into a complete culture medium without exosomes, dividing the cells into three groups, and culturing at 29 ℃,37 ℃ and 41 ℃ for 2h respectively. After the end of the culture, the culture was performed by ultracentrifugation: 300g for 10min,2000g for 10min,10000g for 30min,100000g for 70min and 110000g for 70min. After centrifugation, the supernatant was carefully aspirated and the pellet was transferred to a sterile EP tube for storage using 200. Mu.l of sterile PBS.
Example 2
In this example, allergic asthma mice were infused with exosomes prepared in example 1, and mice were tested for Airway Hyperresponsiveness (AHR) by Penh, and serum IgE levels were tested by the Elisa method. The pathological morphological changes of the alveolar tissues in the alveolar lavage fluid of the mice are observed by adopting HE staining, and the infiltration degree of inflammatory cells and the proportion of inflammatory cells in the alveoli are observed.
The specific experimental method comprises the following steps:
(1) Allergic asthma models of mice were established using Ovalbumin (OVA) -sensitized mice, which were randomly divided into 4 groups, respectively (B) OVA group: allergic asthma group; (C) OVA+29-exo group: treating the allergic asthma group with exosomes secreted under stimulation at 29 ℃; (D) OVA+37% -exo group: treating the allergic asthma group with exosomes secreted under stimulation at 37 ℃; (E) OVA+41-exo group: treating allergic asthma groups with exosomes secreted under 41 ℃ stimulation; set up (a) Ctrl group: negative control group. Mice were sacrificed 24h after airway hyperresponsiveness detection following treatment and alveolar lavage fluid, serum, and lung tissue were collected.
(2) Penh assay mice were tested for Airway Hyperresponse (AHR) assay: at 24 hours post-treatment, mice were weighed and examined for lung function after the instrument was run normally. After placing the mice in the scan box, the detection device is connected and the program is run. After filling the mice with body weight, the program was set to record the basal value of airway resistance of the mice for 1 minute, nebulized for 1 minute, and recorded for 3 minutes. Nebulization challenge was performed with PBS, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml, 64mg/ml acetylcholine, and the average Penh value for each experiment was recorded.
(3) Serum IgE content was measured by Elisa method: after 100 μl of each standard of the sample was added to the 96-well plate in the kit, the samples were incubated at 37℃for 120 minutes; the liquid from each well was poured out and incubated at 37℃for 60 minutes after the direct addition of 100. Mu.l of biotin antibody (1X); after sucking away the liquid from each well, washing with 200 μl of washing liquid, repeating for 2 minutes 3 times; after subsequent addition of HRP-avidin (1X) per well, incubation was carried out for 60 min at 37 ℃; repeating the above washing step 5 times; mu.l TMB substrate was added to each well. Incubation at 37 ℃ for 20 minutes; after 50. Mu.l of stop solution was then added to each well, the absorbance was rapidly read at a wavelength of 450nm using a microplate reader and calculated.
(4) HE staining: after the lung tissue of each experimental group was sectioned, the slide was placed in an oven at 65 ℃ and heated for 2 hours to completely evaporate the water on the slide, and then placed in xylene for 10 minutes to repeat dewaxing 3 times. And then sequentially hydrating the mixture by 100%, 90%, 80% and 70% ethanol concentration, wherein the time of each concentration is 2 minutes. Flushing under running water. The method comprises the steps of dripping hematoxylin dye on a slide for 10 minutes, washing under tap water for 3 minutes, dripping 1% ethanol hydrochloride for 5 seconds, washing under tap water for 3 minutes, dripping eosin dye solution for 2 minutes, and washing under tap water for 3 minutes. After the neutral resin sealing sheet is used, automatic sheet scanning is carried out in a full-automatic sheet scanning instrument.
The experimental results are shown in fig. 1 and 2. As can be seen from FIG. 1b, the airway hyperresponsiveness index of mice was decreased by injecting the exosomes produced by the stimulation at 41℃compared with the exosomes produced by the stimulation at 29℃and the exosomes produced by the stimulation at 37 ℃. As can be seen from FIG. 1c, igE levels in asthmatic mice were also significantly reduced by injecting exosomes produced by stimulation at 41 ℃. As can be seen from FIG. 2, the degree of infiltration of inflammatory cells in alveoli of mice was significantly reduced by injecting the exosomes produced by the stimulation at 41℃compared with the exosomes produced by the stimulation at 29℃and the exosomes produced by the stimulation at 37 ℃.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been 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 to the technical solution of the present application without departing from the spirit and scope of the technical solution of the present application.
Claims (7)
1. A method for preparing a fat large cell exosome, comprising the steps of:
(1) Inoculating mast cells in a culture dish, culturing in a complete culture medium with CO2 concentration of 5% at 37 ℃, and replacing the culture medium when the cell density reaches 75-85%;
(2) Culturing the cells of step (1) at a temperature of 41 ℃;
(3) Collecting the cell supernatant in the step (2) and carrying out gradient centrifugation to extract exosomes.
2. The method according to claim 1, wherein the incubation time in step (2) is 2 hours.
3. The method of claim 1, wherein the medium exchanged in step (1) is a complete medium without exosomes.
4. The method of claim 1, wherein the gradient centrifugation of step (3) is: 300g for 10min,2000g for 10min,10000g for 30min,100000g for 70min and 110000g for 70min.
5. The mast cell exosome produced by the production method according to any one of claims 1 to 4.
6. Use of a mast cell exosome according to claim 5 in the manufacture of a medicament for treating allergic asthma.
7. The use according to claim 6, wherein the prepared medicament is administered by intravenous injection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
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| Mast cell exosomes can suppress allergic reactions by binding to IgE;Xie Guogang等;J ALLERGY CLIN IMMUNOL;第141卷(第2期);第788-791页 * |
| Temperature regulates Rab3a and mast cell-derived exosomal FcεRI to inhibit mast cell activation;Liao Wenjing 等;Allergy;第78卷(第6期);第1707-1710页 * |
| 外泌体在支气管哮喘发病机制中的作用;李竹英;王婷;李寒梅;;中华全科医学(02);第291-294页 * |
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