CN109554341B - Application of non-invasive ultrasonic treatment cells in preparation of exosomes, exosomes and preparation method and application of exosomes - Google Patents
Application of non-invasive ultrasonic treatment cells in preparation of exosomes, exosomes and preparation method and application of exosomes Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly provides application of non-invasive ultrasonic treatment cells in preparation of exosomes, and a preparation method and application of exosomes. The preparation method of the exosome provided by the invention adopts noninvasive ultrasonic treatment of cells, regulates and controls the expression abundance of microRNA in the extracellular exosome by stimulating the cells to a certain extent, and/or potentially improves the expression of brain-derived neurotrophic factors, and/or promotes harmful proteins to be removed from the brain, and/or reduces the oxidative damage of harmful substances to brain tissues. The method is suitable for all cells, has good universality and high exosome yield, is simple to operate, and can obtain the target exosome in a short time. In addition, the method can realize the simultaneous promotion of the expression abundance of various microRNAs, and more exosomes with changed microRNA expression can be obtained instead of a single exosome with changed microRNA expression.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to application of noninvasive ultrasonic cell treatment in preparation of exosomes, and a preparation method and application of exosomes.
Background
The exosome is a multivesicular body existing outside cells, and can form a multivesicular endosome through inwards sinking of a cell endocytic vesicle membrane, and the endosome is fused with a cell membrane to release vesicles in the endosome. The exosome has the diameter of 30-150nm, contains multiple components such as RNA, protein, microRNA, DNA fragments and the like, and is distributed in various body fluids such as blood, saliva, urine, cerebrospinal fluid, breast milk and the like. The microRNA is a non-coding RNA with an endogenous regulation function, and can be combined with a 3 'untranslated region of a target mRNA to cause different expressions of target genes to different degrees, so that exosomes can regulate the expression of the target genes by transferring the microRNA into the cytoplasm of a target cell and then specifically combining the microRNA with a 3' end non-coding region of the corresponding mRNA. The cell selectively generates exosome containing different contents under different pathological and physiological conditions. Furthermore, exosomes secreted by different cells have different components and have different regulating effects on target cells.
In the current research on exosomes, the research on microRNA is the most extensive, and usually, cells are subjected to gene transfection, genes are transfected into a cell line through electrotransformation, time-consuming and labor-consuming screening is performed to construct a cell line expressing the target microRNA, then the cell line is cultured, and exosomes in cell culture supernatant are collected, so that the exosomes expressing the target microRNA are obtained. Such methods have the disadvantage that it may take several months of experimental time to complete the construction of the cell line and usually only allow high expression of a single microRNA. In addition, each construction approach has poor universality due to differentiation of different cells.
Therefore, it is of great significance to develop an exosome preparation method which can simultaneously and efficiently improve the abundance of a plurality of target substances, has good universality and is simple to operate and low in cost.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of exosome and exosome, so as to alleviate the technical problems that the preparation method of exosome in the prior art is high in cost, complex in operation and difficult to realize expression regulation of a plurality of target substances.
The second purpose of the invention is to provide the application of the non-invasive ultrasonic treatment of cells in the preparation of exosomes.
The third purpose of the invention is to provide the application of the exosome in the preparation of the medicine.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a preparation method of exosome comprises the following steps: and (3) adopting noninvasive ultrasonic treatment to the cells, and extracting to obtain the exosome.
Further, the method comprises the following steps: culturing the nerve cells subjected to non-invasive ultrasonic treatment for 36-60 hours, and extracting exosomes to obtain exosomes.
Further, the non-invasive ultrasound processing comprises: non-invasive ultrasonic cell treatment is carried out for 1-30min under the conditions that the frequency of a probe is 0.5-1.5MHz and the amplitude is 10-200mV, wherein the amplitude is preferably 50-10mV, and is further preferably 90-110 mV; the ultrasonic treatment time is preferably 1 to 20min, more preferably 1 to 10min, and still more preferably 3 to 7 min.
Further, the nerve cells include SH-SY5Y cells, IMR-32 cells, TGW cells, LAN-1 cells, HA cells or N2A cells, preferably SH-SY5Y cells.
Further, the method for extracting exosomes comprises the following steps: concentrating the culture medium without nerve cells and apoptotic bodies to obtain a concentrated solution, obtaining a precipitate of the concentrated solution and a precipitating agent, and separating the precipitate to obtain the exosome.
Further, the precipitating agent comprises polyethylene glycol 4000 or polyethylene glycol 6000, preferably polyethylene glycol 6000, and further preferably 14% -18% (w/v) polyethylene glycol 6000;
preferably, the volume ratio of the concentrated solution to the precipitating agent is 1: 0.5-1.5;
preferably, the action conditions of the concentrated solution and the precipitating agent are as follows: precipitating at 2-8 deg.C for 20-28 hr;
preferably, the step of separating the precipitate is: 90000-110000 Xg was centrifuged for 1.5-2.5 hours to precipitate as exosomes.
The application of non-invasive ultrasonic treatment of cells in preparing exosomes.
The exosome prepared by the preparation method.
Further, the expression abundance of miR-27a-3p, miR-27b-3p and miR-7-5p in the exosome is improved.
The preparation method or the application of the exosome in any one of the following A) to D):
A) preparing a medicament for preventing and/or treating Alzheimer disease;
B) preparing a medicament for preventing and/or treating Parkinson's disease;
C) preparing a medicament for preventing and/or treating spinal injuries;
D) preparing the medicine for preventing and/or treating the brain injury after stroke.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the exosome provided by the invention adopts noninvasive ultrasonic treatment of cells, regulates and controls the expression abundance of microRNA in the extracellular exosome by stimulating the cells to a certain extent, and/or potentially improves the expression of brain-derived neurotrophic factors, and/or promotes harmful proteins to be removed from the brain, and/or reduces the oxidative damage of harmful substances to brain tissues. The method is suitable for all cells, such as tumor cells, stem cells, nerve cells, osteoarthritis cells and the like, has good universality and high exosome yield, is simple to operate, avoids complicated molecular biological operation, can obtain target exosomes in a short time, and greatly shortens the preparation time of the exosomes. In addition, the method can realize the simultaneous improvement of the expression abundance of various microRNAs, and compared with a gene transfection method, more exosomes with changed microRNA expression can be obtained instead of a single exosome with changed microRNA.
The exosome provided by the invention comprises the change of the expression abundance of various microRNAs, and/or the potential improvement of the expression of brain-derived neurotrophic factors, and/or the promotion of the elimination of harmful proteins from the brain, and/or the reduction of the oxidative damage of the brain tissues by the harmful substances, and can be used as a carrier to realize the simultaneous diagnosis of various diseases or prepare medicines for treating various diseases.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a method of non-invasive ultrasound treatment in an embodiment of the present invention;
FIG. 2 is a flow chart of exosome extraction in example 2 of the present invention;
FIG. 3 is a graph showing the results of particle size measurements performed by an exosome Malvern particle size analyzer in example 3 of the present invention;
FIG. 4 is a transmission electron microscope observation result chart of exosomes in example 3 of the present invention;
FIG. 5 is a diagram showing the result of miR-27a-3p content detection of exosomes in example 4 of the present invention;
FIG. 6 is a diagram showing the result of miR-27b-3p content detection of exosomes in example 4 of the present invention;
FIG. 7 is a miR-7-5p content detection result diagram of exosomes in example 4 of the present invention;
FIG. 8 shows the results of the test for the exosome uptake in the Parkinson's cell model in example 5 of the present invention,
wherein, 1-Parkinson cell model + exosomes; 2-parkinson cell model; 3-SH-SY5Y cells + exosomes; 4-SH-SY5Y cells can find that a Parkinson cell model takes a large amount of exosomes;
FIG. 9 shows the result of apoptosis in normal culture of the Parkinson's cell model in example 5 of the present invention;
FIG. 10 shows the result of apoptosis of the Parkinson's cell model treated with exosome in example 5 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.
In the present invention, all the embodiments and preferred methods mentioned herein can be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the components referred to or the preferred components thereof may be combined with each other to form a novel embodiment, if not specifically stated.
In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "6 to 22" means that all real numbers between "6 to 22" have been listed herein, and "6 to 22" is simply a shorthand representation of the combination of these values.
The "ranges" disclosed herein may have one or more lower limits and one or more upper limits, respectively, in the form of lower limits and upper limits.
In the present invention, unless otherwise specified, the individual reactions or operation steps may or may not be performed in sequence. Preferably, the reaction processes herein are carried out sequentially.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A preparation method of exosome comprises the following steps: and (3) adopting noninvasive ultrasonic treatment to the cells, and extracting to obtain the exosome.
The preparation method of the exosome provided by the invention adopts noninvasive ultrasonic treatment of cells, regulates and controls the expression abundance of microRNA in the extracellular exosome by stimulating the cells to a certain extent, and/or potentially improves the expression of brain-derived neurotrophic factors, and/or promotes harmful proteins to be removed from the brain, and/or reduces the oxidative damage of harmful substances to brain tissues. The method is suitable for all cells, such as tumor cells, stem cells, nerve cells, osteoarthritis cells and the like, has good universality and high exosome yield, is simple to operate, avoids complicated molecular biological operation, can obtain target exosomes in a short time, and greatly shortens the preparation time of the exosomes. In addition, the method can realize the simultaneous improvement of the expression abundance of various microRNAs, and compared with a gene transfection method, more exosomes with changed microRNA expression can be obtained instead of a single exosome with changed microRNA.
Non-invasive ultrasound in the present invention refers to medical ultrasound, typically sound waves with a frequency in the interval of 0.1-50 MHz. Ultrasound, a mechanical wave, is generated by the vibration of an object (acoustic source) and caused to propagate through a medium that compresses and expands.
Based on the mechanical effect of ultrasound, ultrasound nerve regulation is a new technology of noninvasive brain stimulation and regulation appearing in recent years, and the central nerve of a stimulation part generates stimulation or inhibition effect through different intensity, frequency, pulse repetition frequency, pulse width and duration, so that reversible change of bidirectional regulation is generated on nerve function. Therefore, the cells are treated by non-invasive ultrasound, the cells are stimulated to change the composition of exosomes of the cells, the exosomes with high-abundance expression of target products can be obtained by adjusting different ultrasound conditions and selecting different cells, and meanwhile, a specific cell line can generate specific exosomes under specific ultrasound treatment conditions.
In some embodiments, the non-invasive ultrasound may be, but is not limited to, the method shown in fig. 1, the non-invasive ultrasound is used to stimulate the cells in the culture medium by processing the culture medium containing the cells, and since the exosomes are present in the culture medium, in order to ensure the purity of the exosomes to be extracted subsequently, the cells are suspended in a serum-free culture medium before the non-invasive ultrasound processing, so as to avoid the contamination of the product purity by impurities such as the exosomes in the serum.
In a preferred embodiment, the method comprises the following steps: culturing the nerve cells subjected to non-invasive ultrasonic treatment for 36-60 hours, and extracting exosomes to obtain exosomes. Experiments show that the abundance of exosome components secreted by cells in 36-60 hours of continuous culture of the cells after non-invasive ultrasonic treatment is changed greatly, and the target microRNA is expressed in high abundance and has high content. The culture time of the treated neural cells is typically, but not limited to, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours.
In a preferred embodiment, the non-invasive ultrasound treatment comprises: non-invasive ultrasonic cell treatment is carried out for 1-30min under the conditions that the probe frequency is 0.5-1.5MHz and the amplitude is 10-200 mV. The probe frequency is typically, but not limited to, 0.5MHz, 0.7MHz, 1MHz, 1.3MHz, or 1.5 MHz; the probe amplitude is typically, but not limited to, 10mV, 20mV, 30mV, 40mV, 50mV, 60mV, 70mV, 80mV, 90mV, 100mV, 110mV, 120mV, 130mV, 140mV, 150mV, 160mV, 170mV, 180mV, 190mV, or 200 mV; the ultrasound time is typically, but not limited to, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 9min, 10min, 15min, 20min, 25min, or 30 min. Wherein the nerve cells include SH-SY5Y cells, IMR-32 cells, TGW cells, LAN-1 cells, HA cells or N2A cells, preferably SH-SY5Y cells. The HA cell is an astrocyte, and the N2A cell is a mouse brain neuroma cell; SH-SY5Y cells, IMR-32 cells, TGW cells and LAN-1 cells are all human neuroblastoma cells, tests show that noninvasive ultrasonic treatment under the conditions is carried out on nerve cells, exosomes in a culture medium is extracted after the nerve cells are cultured for 36-60 hours, the expression abundance of miR-27a-3p, miR-27b-3p and miR-7-5p is multiplied to different degrees, and the method has important significance for obtaining high-expression miR-27a-3p, miR-27b-3p and miR-7-5p exosomes simultaneously, greatly shortens the production time, avoids genetic engineering operation and reduces the cost.
In a preferred embodiment, the method for extracting exosomes includes, but is not limited to, concentrating the culture medium from which the nerve cells and apoptotic bodies are removed to obtain a concentrate, obtaining a precipitate of the concentrate and a precipitating agent, and separating the precipitate to obtain exosomes. Other methods of density gradient centrifugation, differential centrifugation, size exclusion, immune separation and polymer precipitation
In a preferred embodiment, the precipitating agent comprises polyethylene glycol 4000 or polyethylene glycol 6000, preferably polyethylene glycol 6000, more preferably 14% to 18% (w/v) polyethylene glycol 6000, still more preferably 16% (w/v) polyethylene glycol. Wherein w/v is the mass number of polyethylene glycol contained in a unit volume.
In a preferred embodiment, the volume ratio of the concentrate to the precipitant is 1:0.5 to 1.5.
In a preferred embodiment, the conditions under which the concentrate and precipitant act are: precipitating at 2-8 deg.C for 20-28 hr.
In a preferred embodiment, the step of separating the precipitate is: 90000-110000 Xg was centrifuged for 1.5-2.5 hours to precipitate as exosomes.
In a preferred embodiment, the method for extracting exosomes is specifically:
the cell culture supernatant was subjected to a series of centrifugation treatments to remove cell debris, apoptotic bodies, and the like. And concentrating by using an ultrafiltration tube with the molecular weight of 3KDa, mixing the concentrated culture medium with 16% (w/v) polyethylene glycol according to the proportion of 1:1, placing the mixed liquid at 2-8 ℃ for precipitation, centrifuging after 24 hours, continuously centrifuging the obtained precipitate for 2 hours at 100,000g, and obtaining the final precipitate, namely the exosome.
The application of non-invasive ultrasonic treatment of cells in preparing exosomes. The non-invasive ultrasonic treatment is carried out on the cells, the component content in the exosomes of the cells can be stimulated to be greatly changed, and the target exosomes can be obtained through screening and detection.
The invention provides an exosome prepared by the preparation method. The expression abundance of multiple microRNAs in the exosome provided by the invention is increased simultaneously, and the exosome can be used as a carrier to realize simultaneous diagnosis of multiple diseases or prepare medicines for treating multiple diseases.
In a preferred embodiment, the research shows that the expression abundance of miR-27a-3p, miR-27b-3p and miR-7-5p in exosomes is improved by treating nerve cells for 3-7min under the conditions that the probe frequency is 0.5-1.5MHz and the amplitude is 90-110mV, and extracting exosomes after culturing for 36-60 hours. It is understood that the increase in expression abundance is relative to cells that have not been non-invasively sonicated.
The expression of miR-27a-3p in cerebrospinal fluid of Alzheimer's Disease (AD) patients is reduced, and the miR-27a-3p can be used as a target point for treating AD. miR-23a can relieve neuronal apoptosis after brain injury, and the expression of miR-23a has potential application in protecting neuronal activity. miR-7 can be combined with alpha-synuclein to down-regulate the mRNA and protein expression level of the alpha-synuclein. Alpha-synuclein is a 144 amino acid protein of three types and is a potential protein that may cause Parkinson (PD). Meanwhile, miR-7 controls the development of cerebral cortex through a P53 pathway.
The preparation method or the application of the exosome in any one of the following A) to D):
A) preparing a medicament for preventing and/or treating Alzheimer disease;
B) preparing a medicament for preventing and/or treating Parkinson's disease;
C) preparing a medicament for preventing and/or treating spinal injuries;
D) preparing the medicine for preventing and/or treating the brain injury after stroke.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1 serum-free Medium culture of cells and ultrasound non-invasive stimulation of cells
SH-SY5Y cells are adopted in the embodiment, and after SH-SY5Y cells are cultured to the fusion degree of 70% -80%, the cells are replaced by serum-free high-sugar DMEM medium.
The cell culture dish was stimulated by an ultrasound device for a total ultrasound stimulation time of 5 minutes, the specific ultrasound stimulation method being shown in fig. 1. Specific ultrasonic parameters, probe frequency: 1MHz, amplitude: 100mV, 5 minutes of ultrasonic time.
After the stimulation is finished, the cell morphology is observed under a microscope, and the result shows that the cell morphology after the ultrasonic treatment is good and has no difference with the unstimulated SH-SY5Y cell.
The SH-SY5Y cells after ultrasonic treatment are continuously placed in a 37 ℃ incubator for continuous culture, and after 24 hours and 48 hours of culture, cell culture supernatants are collected to prepare for extracting exosomes.
Example 2 exosome collection after ultrasound stimulation
The extraction flow chart of exosomes is shown in fig. 2, and specifically includes:
1) the cell culture supernatant collected in example 1 was centrifuged at 300 Xg for 5 minutes to remove cells;
2) centrifuging the solution supernatant obtained in the step 1) at 2000 Xg for 10 minutes to remove apoptotic bodies;
3) concentrating the solution in the step 2) by adopting a 3kDa ultrafiltration tube, centrifuging for 30 minutes at 3000 Xg, and collecting trapped fluid;
4) mixing the concentrated culture medium obtained in the step 3) with 16% (w/v) polyethylene glycol according to the volume ratio of 1:1, and precipitating the mixed liquid at 4 ℃ for 24 hours to obtain a precipitate;
5) centrifuging the precipitate in the step 4) at 100,000 Xg for 2 hours to obtain the exosome.
Example 3 validation of exosomes
The exosomes collected in example 2 were measured by a malvern particle size analyzer, and as a result, as shown in fig. 3, the particle size of the exosomes was substantially about 100 nm.
The exosomes collected in example 2 were observed by transmission electron microscopy, and as a result, a tea-cup-like classical exosome morphology was observed as shown in fig. 4.
Example 4MicroRNA assay
Exosomes obtained in example 2 after the cells were ultrasonically stimulated for 24 hours and 48 hours were cultured, total RNA was extracted, and fluorescence quantitative qRT-PCR was performed. Designing primers, carrying out quantitative analysis on miRNA, selecting U6 as an internal reference, wherein specific sequences of the primers are shown in the following table 1:
TABLE 1
The results are shown in FIGS. 5 to 7. The expression of three microRNAs of SH-SY5Y cells is greatly improved after 48 hours of ultrasonic stimulation, specifically comprising miR-27a-3p, miR-27b-3p and miR-7-5p, and the contents of the three microRNAs in exosomes after 48 hours of ultrasonic stimulation are respectively increased by 9.79 times, 10.19 times and 3.6 times.
The experiment is repeated for three times, and the result shows that the technical scheme provided by the invention can really realize high-abundance expression of miR-27a-3p, miR-27b-3p and miR-7-5p in the exosome secreted by the SH-SY5Y cell, and the experiment has good reproducibility.
Example 5 uptake of exosomes and inhibition of apoptosis
SH-SY5Y cells are treated by 1mM MPP, and a Parkinson cell model is established. Exosomes secreted by cells cultured for 48 hours after the ultrasonic stimulation in example 2 were co-cultured with parkinson cells, where the exosomes were labeled with exosome-specific fluorescent dye pkh26 and the fluorescence intensity was examined using flow cytometry. The results are shown in FIG. 8, where 1: parkinson cell model + exosomes; 2: a model of Parkinson's cells; 3: SH-SY5Y cell + exosome; 4: SH-SY5Y cells can find that the Parkinson cell model takes up a large amount of exosomes.
After the Parkinson cell model is incubated for 72 hours, exosomes secreted by cells cultured for 48 hours after ultrasonic stimulation and serum-free DMEM medium in the embodiment 2 are added and cultured for 72 hours, and meanwhile, the cell apoptosis condition is detected by a flow cytometer by adopting an Annexin V/PI double staining method by taking the serum-free DMEM medium as a control group. The results are shown in fig. 9 and fig. 10, where the proportion of apoptotic cells in the control group was Q3+ Q2-31.94% and the proportion of apoptotic cells in the exosome-treated group was Q3+ Q2-15.78%, indicating that exosomes extracted from the sonicated cells can reverse apoptosis of SH-SY5Y cells.
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.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute
Application of non-invasive ultrasonic treatment cells in preparation of exosomes, exosomes and preparation method and application of exosomes
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Claims (7)
1. A preparation method of exosome is characterized by comprising the following steps: non-invasive ultrasonic treatment is adopted to treat cells, and exosomes are extracted;
the method comprises the following steps: culturing the nerve cells subjected to non-invasive ultrasonic treatment for 36-60 hours, and extracting exosomes to obtain exosomes;
the nerve cells comprise SH-SY5Y cells;
the ultrasonic device stimulates the cell culture dish, the total ultrasonic stimulation time lasts for 5 minutes, and the specific ultrasonic parameters, the probe frequency: 1MHz, amplitude: 100mV, 5 minutes of ultrasonic time.
2. The method of preparation of claim 1, wherein the non-invasive ultrasound treatment comprises: non-invasive ultrasonic cell treatment is carried out for 1-30min under the conditions that the probe frequency is 0.5-1.5MHz and the amplitude is 10-200 mV.
3. The method for preparing according to claim 1, wherein the method for extracting exosomes comprises: concentrating the culture medium without nerve cells and apoptotic bodies to obtain a concentrated solution, obtaining a precipitate of the concentrated solution and a precipitating agent, and separating the precipitate to obtain the exosome.
4. The method of claim 3, wherein the precipitating agent comprises polyethylene glycol 4000 or polyethylene glycol 6000,
the volume ratio of the concentrated solution to the precipitator is 1: 0.5-1.5;
the action conditions of the concentrated solution and the precipitating agent are as follows: precipitating at 2-8 deg.C for 20-28 hr;
the step of separating the precipitate is: 90000-110000 Xg was centrifuged for 1.5-2.5 hours to precipitate as exosomes.
5. An exosome produced by the production method according to any one of claims 1 to 4.
6. The exosome according to claim 5, characterized in that miR-27a-3p, miR-27b-3p and miR-7-5p expression abundance is increased in the exosome.
7. Use of the preparation method according to any one of claims 1 to 4 or the exosomes according to claim 5 or 6 in any one of the following A) -D):
A) preparing a medicament for preventing and/or treating Alzheimer disease;
B) preparing a medicament for preventing and/or treating Parkinson's disease;
C) preparing a medicament for preventing and/or treating spinal injuries;
D) preparing the medicine for preventing and/or treating the brain injury after stroke.
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| PCT/CN2019/123568 WO2020125447A1 (en) | 2018-12-18 | 2019-12-06 | Application of non-invasive ultrasonic cell in preparing exosome, exosome, and preparation method therefor and application thereof |
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| CN110777142A (en) * | 2019-06-27 | 2020-02-11 | 上海交通大学 | Method for promoting secretion of extracellular fluid by low-intensity pulse ultrasonic stimulation |
| CN110772483B (en) * | 2019-11-13 | 2021-03-26 | 山东大学 | Application of hydrogen sulfide modified mesenchymal stem cell outer vesicle serving as miRNA delivery vector in hypoxic-ischemic brain injury |
| CN110951669A (en) * | 2019-12-09 | 2020-04-03 | 益善生物技术股份有限公司 | Coprecipitator, reagent group, kit and extraction method for extracting exosome |
| CN113061579B (en) * | 2019-12-12 | 2023-04-18 | 中国科学院深圳先进技术研究院 | Exosome and preparation method and application thereof |
| WO2021114164A1 (en) * | 2019-12-12 | 2021-06-17 | 中国科学院深圳先进技术研究院 | Exosome, preparation method therefor and use thereof |
| CN112980791B (en) * | 2019-12-12 | 2023-05-16 | 中国科学院深圳先进技术研究院 | Method for producing microvesicles, microvesicles obtained based on the same and use thereof |
| WO2021114160A1 (en) * | 2019-12-12 | 2021-06-17 | 中国科学院深圳先进技术研究院 | Method for producing microvesicles, and microvesicles obtained based on the method for producing microvesicles and application thereof |
| AU2021325575A1 (en) * | 2020-08-11 | 2023-03-02 | Royal Melbourne Institute Of Technology | Stimulating cellular production of exosomes |
| CN111876390B (en) * | 2020-08-12 | 2021-04-09 | 湖南南华爱世普林生物技术有限公司 | Application of compound-loaded transgenic stem cell exosome in preparation of medicines or whitening cosmetics |
| CN118236401A (en) * | 2021-04-09 | 2024-06-25 | 上海市同仁医院 | New application of exosomes derived from cerebral cortex neuron cells |
| CN113684130B (en) * | 2021-08-17 | 2024-04-26 | 深圳高性能医疗器械国家研究院有限公司 | Device and method for stimulating secretion of cell exosomes, exosomes obtained by method and application of exosomes |
| CN114107205B (en) * | 2021-11-29 | 2023-08-22 | 哈尔滨医科大学 | Method for stimulating cells to secrete exosomes rapidly and application of method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN116731958A (en) * | 2014-10-09 | 2023-09-12 | 细胞结构公司 | Placenta-derived adherent cell exosomes and uses thereof |
| CN108865983A (en) * | 2017-09-11 | 2018-11-23 | 江苏国立生物研究院有限公司 | A kind of extracting method of cell excretion body |
| CN108624557A (en) * | 2018-05-31 | 2018-10-09 | 章毅 | The preparation method and applications of mescenchymal stem cell excretion body |
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-
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Non-Patent Citations (5)
| Title |
|---|
| Isolation of exosomes from whole blood by integrating acoustics and microfluidics.;Wu M et al;《Proc Natl Acad Sci U S A》;20171003;第114卷(第40期);第10584-10589页 * |
| 叶冬熳等.肿瘤外泌体的提取与鉴定.《现代肿瘤医学》.2018,第26卷(第 20 期), * |
| 外泌体内microRNA-135a跨血脑屏障转运的初步研究;刘辰庚等;《中国医药导报》;20171125(第33期);摘要 * |
| 肿瘤外泌体的提取与鉴定;叶冬熳等;《现代肿瘤医学》;20181030;第26卷(第 20 期);第3342页右栏"3.2.5"，第3341页右栏第2段，第3342页右栏"3.2.2" * |
| 辐射对神经相关细胞外泌体的生物效应影响研究;李福涛;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20161015;摘要 * |
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