CN109554341A - Noninvasive ultrasonic treatment cell is preparing the application in excretion body, excretion body and its preparation method and application - Google Patents
Noninvasive ultrasonic treatment cell is preparing the application in excretion body, excretion body and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically, providing the noninvasive ultrasonic treatment cell of one kind is preparing the application in excretion body, excretion body and its preparation method and application.Excretion preparation provided by the invention, using noninvasive ultrasonic treatment cell, by carrying out certain stimulation to cell, the gene expression abundance of microRNA in regulating cell excretion body, and/or the potential expression for improving brain derived neurotrophic factor, and/or deleterious protein is promoted to remove from intracerebral, and/or mitigate harmful substance to the oxidative damage of brain tissue.This method is all suitable for all cells, and universality is good and excretion body yield easy to operate is big, and target excretion body can be obtained in a short time.In addition, promotion while a variety of microRNA gene expression abundances may be implemented in this method, available more microRNA express the excretion body changed, rather than the individually excretion body of microRNA change.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of noninvasive ultrasonic treatment cell in preparation excretion body
In application, excretion body and its preparation method and application.
Background technique
Excretion body is that one kind is present in extracellular more vesica bodies, can be recessed inwardly to form more bubbles by cell endocytic vacuolar membrane
Vesicles therein are discharged after endosome, endosome and cell membrane fusion.The diameter of excretion body is 30-150nm, includes RNA, egg
The Multiple components such as white matter, microRNA, DNA fragmentation have in a variety of body fluid such as blood, saliva, urine, cerebrospinal fluid and breast milk
Distribution.MicroRNA is the non-coding RNA that a kind of endogenous has adjusting function, can be with 3 ' non-translational region knots of said target mrna
It closes, so as to cause the different degrees of differential expression of target gene, therefore, excretion body can be thin by the way that microRNA is transmitted to target
In born of the same parents' cytoplasm, then it is specifically bound to the end the 3' noncoding region of corresponding mRNA, to regulate and control expression of target gene.Cell is in different diseases
The excretion body containing different contents is selectively produced under reason, physiological condition.And the excretion body of different cell secretions has not
Same component is not also identical to target cell adjustment effect.
At present in the research of excretion body, the research of microRNA is the most extensive, generallys use and carries out gene turn to cell
Dye is turned by electricity, gene is transfected to cell line, then carry out the screening of time and effort consuming, and building expression target microRNA's is thin
Then the excretion body in cells and supernatant is collected, to obtain expression target by cultivating cell line again by born of the same parents system
The excretion body of microRNA.Such method drawback is that the experimental period that possible need some months could complete the structure of cell line
It builds, and is typically only capable to carry out high expression to single microRNA.Further, since the differentiation of different cells, every kind of building side
The universality of formula is very poor.
Therefore, develop one kind and can while efficiently improve multiple target substance abundance, universality it is good and it is easy to operate at
This low excretion preparation has great importance.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide the preparation method and excretion body of a kind of excretion body, to alleviate in the prior art
The preparation method of excretion body is at high cost, complicated for operation and the technical issues of be difficult to realize multiple target substance expression regulations.
The second object of the present invention is to provide application of the noninvasive ultrasonic treatment cell in preparation excretion body.
The third object of the present invention is to provide the application of excretion body in medicine preparation.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of preparation method of excretion body, comprising the following steps: use noninvasive ultrasonic treatment cell, extraction obtains excretion
Body.
Further, comprising the following steps: outer by being extracted after the noninvasive neuronal cell cultures being ultrasonically treated 36-60 hours
Body is secreted, excretion body is obtained.
Further, the noninvasive ultrasonic treatment includes: in frequency probe 0.5-1.5MHz and amplitude 10-200mV condition
Under noninvasive ultrasonic cell 1-30min, amplitude is preferably 50-10mV, further preferably 90-110mV；Ultrasonic time is preferably 1-
20min, further preferably 1-10min are still more preferably 3-7min.
Further, the nerve cell includes SH-SY5Y cell, IMR-32 cell, TGW cell, LAN-1 cell, HA
Cell or N2A cell, preferably SH-SY5Y cell.
Further, the method for extracting excretion body include: will remove the culture medium of nerve cell and apoptotic body into
Row is concentrated to get concentrate, obtains the sediment of concentrate and precipitating reagent effect, and sediment separate out obtains excretion body.
Further, the precipitating reagent includes Macrogol 4000 or Macrogol 6000, preferably Macrogol 6000,
Further preferably 14%-18% (w/v) Macrogol 6000；
Preferably, the volume ratio of the concentrate and the precipitating reagent is 1:0.5-1.5；
Preferably, the action condition of the concentrate and the precipitating reagent are as follows: 2-8 DEG C precipitating 20-28 hours；
Preferably, the step of sediment separate out are as follows: 90000-110000 × g is centrifuged 1.5-2.5 hours, is precipitated as outer
Secrete body.
Application of the noninvasive ultrasonic treatment cell in preparation excretion body.
The excretion body that above-mentioned preparation method is prepared.
Further, miR-27a-3p, miR-27b-3p and miR-7-5p gene expression abundance improve in the excretion body.
Above-mentioned preparation method or excretion body are in following A)-D) in any one application:
A) preparation prevention and/or treatment Alzheimer disease drugs；
B) preparation prevention and/or treatment anti-parkinson drug；
C) preparation prevention and/or treatment spinal injury drug；
D) preparation prevention and/or apoplexy Brain Injury After drug.
Compared with prior art, the invention has the benefit that
Excretion preparation provided by the invention, using noninvasive ultrasonic treatment cell, by being carried out centainly to cell
It stimulates, the gene expression abundance of the microRNA in regulating cell excretion body, and/or the potential table for improving brain derived neurotrophic factor
It reaches, and/or deleterious protein is promoted to remove from intracerebral, and/or mitigate harmful substance to the oxidative damage of brain tissue.This method is to institute
Some cells are all suitable for, such as tumour cell, stem cell, nerve cell and osteoarthritic cells etc., and universality is good and grasps
It is big to make simple excretion body yield, avoids cumbersome molecular biology manipulations, target excretion body, pole can be obtained in a short time
The earth shortens the preparation time of excretion body.In addition, promotion while a variety of microRNA gene expression abundances may be implemented in this method,
Compared with gene transfection method, the excretion body that available more microRNA expression change, rather than single microRNA
The excretion body of change.
Neurological disease is all the origin cause of formation with Various Complex, and a kind of change of substance is difficult to meet actual need in excretion body
It asks, includes the change of a variety of microRNA gene expression abundances, and/or potential raising brain source nerve in excretion body provided by the invention
The expression of trophic factors, and/or promote deleterious protein to remove from intracerebral, and/or mitigate harmful substance and damage to the oxidation of brain tissue
Wound can be used as while carrier realizes a variety of diseases and diagnose or prepare the drug for treating a variety of diseases.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the method schematic diagram of noninvasive ultrasonic treatment in embodiment of the present invention；
Fig. 2 is that excretion body extracts flow chart in the embodiment of the present invention 2；
Fig. 3 is excretion body Malvern particle size analyzer droplet measurement result figure in the embodiment of the present invention 3；
Fig. 4 is excretion body transmission electron microscope observing result figure in the embodiment of the present invention 3；
Fig. 5 is the miR-27a-3p content detection result figure of excretion body in the embodiment of the present invention 4；
Fig. 6 is the miR-27b-3p content detection result figure of excretion body in the embodiment of the present invention 4；
Fig. 7 is the miR-7-5p content detection result figure of excretion body in the embodiment of the present invention 4；
Fig. 8 is that Parkinson's cell model absorbs excretion body test result in the embodiment of the present invention 5,
Wherein, 1- Parkinson cell model+excretion body；2- Parkinson's cell model；3-SH-SY5Y cell+excretion body；4-
SH-SY5Y cell, it is possible to find Parkinson's cell model huge uptake excretion body；
Fig. 9 is the Apoptosis result that Parkinson's cell model is normally cultivated in the embodiment of the present invention 5；
Figure 10 is the Apoptosis result that Parkinson's cell model is treated through excretion body in the embodiment of the present invention 5.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method
It can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with
Intercombination forms new technical solution.
In the present invention, if related each component or its preferred ingredient can be combined with each other shape without particularly illustrating
The technical solution of Cheng Xin.
In the present invention, unless otherwise indicated, numberical range " a~b " indicates the contracting of any real combinings between a to b
Sketch form shows that wherein a and b is real number.Such as numberical range " 6~22 " indicate herein all listed " 6~22 " it
Between whole real numbers, " 6~22 " be these combinations of values breviary indicate.
" range " disclosed in this invention can be respectively one or more lower limits and one in the form of lower and upper limit
A or multiple upper limits.
In the present invention, unless otherwise indicated, each reaction or operating procedure can be carried out sequentially, can not also be in sequence
It carries out.Preferably, reaction method herein is that sequence carries out.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
A kind of preparation method of excretion body, comprising the following steps: use noninvasive ultrasonic treatment cell, extraction obtains excretion
Body.
Excretion preparation provided by the invention, using noninvasive ultrasonic treatment cell, by being carried out centainly to cell
It stimulates, the gene expression abundance of the microRNA in regulating cell excretion body, and/or the potential table for improving brain derived neurotrophic factor
It reaches, and/or deleterious protein is promoted to remove from intracerebral, and/or mitigate harmful substance to the oxidative damage of brain tissue.This method is to institute
Some cells are all suitable for, such as tumour cell, stem cell, nerve cell and osteoarthritic cells etc., and universality is good and grasps
It is big to make simple excretion body yield, avoids cumbersome molecular biology manipulations, target excretion body, pole can be obtained in a short time
The earth shortens the preparation time of excretion body.In addition, promotion while a variety of microRNA gene expression abundances may be implemented in this method,
Compared with gene transfection method, the excretion body that available more microRNA expression change, rather than single microRNA
The excretion body of change.
Noninvasive ultrasound in the present invention refers to medical ultrasonic, and typically referring to frequency is the sound wave in the section 0.1-50MHz.
Ultrasound is used as a kind of mechanical wave, is to be vibrated to generate by object (sound source), and lead to its propagation by compression and expansion medium.
Based on the mechanics effect of ultrasound, ultrasonic neuromodulation is the non-invasive brain stimulation occurred in recent years and the new skill of regulation
Art generates the nervous centralis of stimulation location by different intensity, frequency, pulse recurrence frequency, pulse width, duration
Stimulation or depression effect, the invertibity for generating bidirectional modulation to nervous function change.Therefore, using noninvasive ultrasonic treatment cell,
Stimulation cell makes the excretion body ingredient of cell change, can by adjusting the different ultrasound conditions cell different with selection
To obtain the excretion body of target product high abundance expression, simultaneously because under the conditions of specific ultrasonic treatment, specific cell line
Can produce specific excretion body, this method has good repeatability, and will not damaging cells, institute in this way can be big
Amount quickly produces target excretion body.
In some embodiments, noninvasive ultrasound can be, but not limited to by the way of as shown in Figure 1, using noninvasive ultrasound
Stimulation of the culture medium realization containing cell to the cell in culture medium is handled, since excretion body is present in culture medium, in order to
Cell, is suspended in serum free medium before noninvasive ultrasonic treatment, avoids serum by the purity for guaranteeing subsequent extracted excretion body
In the pollution of the impurity to product purity such as excretion body.
In a preferred embodiment, comprising the following steps: by the noninvasive neuronal cell cultures 36- being ultrasonically treated
Excretion body is extracted after 60 hours, obtains excretion body.It for nerve cell, is found by experiment that, cell is after noninvasive ultrasonic treatment
The excretion body Composition Abundance for continuing to cultivate the secretion of 36-60 hours time inner cells changes greatly, at this time target microRNA Gao Feng
Degree expression, content are higher.It is 36 hours that the processed neuronal cell cultures time is typical but non-limiting, 42 hours, it is 48 small
When, 54 hours or 60 hours.
In being preferably carried out mode, noninvasive ultrasonic treatment includes: in frequency probe 0.5-1.5MHz and amplitude 10-
Noninvasive ultrasonic cell 1-30min under the conditions of 200mV.Frequency probe it is typical but non-limiting for 0.5MHz, 0.7MHz, 1MHz,
1.3MHz or 1.5MHz；Pop one's head in amplitude it is typical but non-limiting for 10mV, 20mV, 30mV, 40mV, 50mV, 60mV, 70mV,
80mV, 90mV, 100mV, 110mV, 120mV, 130mV, 140mV, 150mV, 160mV, 170mV, 180mV, 190mV or 200mV；
Ultrasonic time it is typical but non-limiting for 1min, 2min, 3min, 4min, 5min, 6min, 7min, 9min, 10min,
15min, 20min, 25min or 30min.Wherein, nerve cell includes SH-SY5Y cell, IMR-32 cell, TGW cell, LAN-
1 cell, HA cell or N2A cell, preferably SH-SY5Y cell.HA cell is star spongiocyte, and N2A cell is mouse brain
Neural oncocyte；SH-SY5Y cell, IMR-32 cell, TGW cell and LAN-1 cell are human neuroblastoma cells, are led to
Overtesting discovery, the noninvasive ultrasonic treatment of above-mentioned condition is carried out to nerve cell, extracts culture medium after cultivating 36-60 hours time
In excretion body, the gene expression abundance of miR-27a-3p, miR-27b-3p and miR-7-5p all obtains different degrees of multiple and increases,
For being of great significance for high expression miR-27a-3p, miR-27b-3p and miR-7-5p excretion body simultaneously, greatly
The production time is shortened, genetic engineering operation is avoided, reduces costs.
In being preferably carried out mode, the method for extracting excretion body includes but is not limited to that removal nerve cell and apoptosis is small
The culture medium of body is concentrated to give concentrate, obtains the sediment of concentrate and precipitating reagent effect, and sediment separate out obtains outer
Secrete body.The methods of other density gradient centrifugations, differential centrifugation, volume exclusion, immune separation and polymer precipitating
In being preferably carried out mode, precipitating reagent includes Macrogol 4000 or Macrogol 6000, preferably poly- second two
Alcohol 6000, further preferably 14%-18% (w/v) Macrogol 6000 is still more preferably the poly- second two of 16% (w/v)
Alcohol.Wherein, w/v is the polyethylene glycol mass number contained in unit volume.
In being preferably carried out mode, the volume ratio of concentrate and precipitating reagent is 1:0.5-1.5.
In being preferably carried out mode, the action condition of concentrate and precipitating reagent are as follows: 2-8 DEG C precipitating 20-28 hours.
In being preferably carried out mode, the step of sediment separate out are as follows: 90000-110000 × g is centrifuged 1.5-2.5 hours,
It is precipitated as excretion body.
In being preferably carried out mode, the method for extraction excretion body specifically:
A series of centrifugal treatings are carried out to cells and supernatant, remove cell fragment, apoptotic body etc..Recycle molecular weight
It is concentrated for the super filter tube of 3KDa, the culture medium after concentration is mixed according to the ratio of 1:1 with 16% (w/v) polyethylene glycol
It closes, mixed liquid is placed in 2-8 DEG C of precipitating, is centrifuged after 24 hours, and the precipitating of acquisition continues to be centrifuged 2 hours in 100,000g
Afterwards, the last precipitating of acquisition is excretion body.
Application of the noninvasive ultrasonic treatment cell in preparation excretion body.Noninvasive ultrasonic treatment is carried out to cell, can be stimulated
Constituent content generation significantly changes in the excretion body of cell, by screening and detecting available target excretion body.
The present invention provides the excretion body that above-mentioned preparation method is prepared.It is a variety of in excretion body provided by the invention
Increase while microRNA gene expression abundance, can be used as to diagnose or prepare while carrier realizes a variety of diseases and treat a variety of diseases
The drug of disease.
In being preferably carried out mode, by the study found that using frequency probe 0.5-1.5MHz and width to nerve cell
Value 90-110mV condition handles 3-7min, and excretion body, miR-27a-3p, miR- in excretion body are extracted after being further cultured for 36-60 hours
27b-3p and miR-7-5p gene expression abundance improves.It is understood that the raising of gene expression abundance is not relative to carrying out noninvasive ultrasound
For the cell of processing.
MiR-27a-3p expresses reduction, table in Alzheimer disease (Alzheimer disease, AD) patient's cerebrospinal fluid
Bright miR-27a-3p can be used as the target spot of AD treatment.MiR-23a can mitigate the Neuron Apoptosis situation after cerebral injury,
The expression of miR-23a has potential purposes for protection neuronal activity.MiR-7 can be by tying with α-synuclein
It closes, to lower the mRNA and protein expression level of a-synuclein.And α-synuclein is a kind of containing there are three types of type
The albumen of 144 amino acid is the potential albumen that may cause Parkinson (PD).Meanwhile miR-7 is big by the control of P53 access
The development of cortex.
Above-mentioned preparation method or excretion body are in following A)-D) in any one application:
A) preparation prevention and/or treatment Alzheimer disease drugs；
B) preparation prevention and/or treatment anti-parkinson drug；
C) preparation prevention and/or treatment spinal injury drug；
D) preparation prevention and/or apoplexy Brain Injury After drug.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for being described in more detail.
1 serum free medium culture cell of embodiment and ultrasound non-invasive stimulate cell
The present embodiment changes nothing into after SH-SY5Y cell culture to 70%-80% fusion degree using SH-SY5Y cell
Serum DMEM in high glucose culture medium.
Vltrasonic device stimulates Tissue Culture Dish, and total ultrasound stimulation time continues 5 minutes, specific ultrasound stimulation side
Method is as shown in Figure 1.Specific ultrasound parameter, frequency probe: 1MHz, amplitude: 100mV, ultrasonic time: 5 minutes.
After stimulation, cellular morphology is observed under the microscope, the results showed that the cellular morphology after ultrasonic treatment is good, with
SH-SY5Y cell is not stimulated not have difference.
Continue the SH-SY5Y cell being ultrasonically treated to be placed in 37 ° of incubators and continue to cultivate, and is small in culture 24 respectively
When and after 48 hours, collect cells and supernatant, prepare extract excretion body.
Excretion body of the embodiment 2 after ultrasound stimulation is collected
The extraction flow chart of excretion body as shown in Fig. 2, specifically:
1) 300 × g of cells and supernatant collected in embodiment 1 is centrifuged 5 minutes, removes cell；
2) 2000 × g of solution supernatant in step 1) is centrifuged 10 minutes, removes apoptotic body；
3) solution in step 2) is concentrated using the super filter tube of 3kDa, 3000 × g is centrifuged 30 minutes, collects retention
Liquid；
4) enrichment medium in step 3) is mixed with 16% (w/v) polyethylene glycol according to volume ratio 1:1, will be mixed
Liquid after conjunction precipitates 24 hours under the conditions of being placed in 4 DEG C, obtains precipitating；
5) 100 will be deposited in step 4), 000 × g is centrifuged 2 hours, and precipitating is excretion body.
The verifying of 3 excretion body of embodiment
The excretion body being collected into embodiment 2 is measured using Malvern particle size analyzer, as a result as shown in figure 3,
The partial size of excretion body is obtained substantially in 100nm or so.
The excretion body being collected into embodiment 2 is utilized into transmission electron microscope observing, as a result as shown in figure 4, observable obtains tea
The classical excretion volume morphing of cup-shaped.
Embodiment 4microRNA detection
24 hours and 48 hours resulting excretion bodies will be cultivated after ultrasound stimulation cell in embodiment 2 carries out total serum IgE progress
It extracts, carries out fluorescent quantitation qRT-PCR.Design primer carries out quantitative analysis to wherein miRNA, and internal reference selects U6, the tool of primer
Body sequence is as shown in table 1 below:
Table 1
As a result as shown in Figure 5-Figure 7.It can be found that after ultrasound stimulation 48 hours, three kinds of microRNA's of SH-SY5Y cell
Expression greatly improves, and specifically includes miR-27a-3p, miR-27b-3p and miR-7-5p, the excretion after ultrasound stimulation 48 hours
In body, content increases 9.79 times, 10.19 times and 3.6 times respectively.
In triplicate by above-mentioned experiment, the results showed that technical solution provided by the invention really can be by SH-SY5Y cell
MiR-27a-3p, miR-27b-3p and miR-7-5p in the excretion body of secretion obtain high abundance expression, and experiment has good
Reproducibility.
The intake of 5 excretion body of embodiment and inhibition Apoptosis
1mM MPP handles SH-SY5Y cell, establishes Parkinson's cell model.Using being trained after the ultrasound stimulation in embodiment 2
The excretion body and Parkinson's cell for supporting the secretion of 48 hour cells co-culture, wherein excretion body excretion body specific fluorescence dye
Pkh26 label checks fluorescence intensity using flow cytometer.As a result as shown in Figure 8, wherein 1: Parkinson's cell model+excretion
Body；2: Parkinson's cell model；3:SH-SY5Y cell+excretion body；4:SH-SY5Y cell, it is possible to find Parkinson's cell model is big
Amount intake excretion body.
The secretion of 48 hour cells is cultivated after the ultrasound stimulation being added in embodiment 2 after being incubated for Parkinson's cell model 72 hours
Excretion body and plasma-free DMEM medium culture 72 hours, while plasma-free DMEM medium is only added as a control group,
Using Annexin V/PI double-staining, with flow cytomery Apoptosis situation.As a result as shown in Figure 9 and Figure 10, right
It is Q3+Q2=31.94% according to apoptotic cell ratio in group, apoptotic cell ratio is Q3+Q2=in excretion body treatment group
15.78%, illustrate that the excretion body for the cell extraction being ultrasonically treated can reverse the apoptosis of SH-SY5Y cell.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen Xianjin Technology Academe
<120>noninvasive ultrasonic treatment cell is preparing the application in excretion body, excretion body and its preparation method and application
<160> 8
<170> PatentIn version 3.5
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<213>artificial sequence
<400> 6
ctcaactggt gtcgtggagt cggcaattca gttgagaaca acaa 44
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<400> 7
aacgcttcac gaatttgcgt 20
<210> 8
<211> 17
<212> DNA
<213>artificial sequence
<400> 8
ctcgcttcgg cagcaca 17
Claims (10)
1. a kind of preparation method of excretion body, which comprises the following steps: use noninvasive ultrasonic treatment cell, extract
Obtain excretion body.
2. preparation method according to claim 1, which comprises the following steps: be ultrasonically treated noninvasive
Excretion body is extracted after neuronal cell cultures 36-60 hours, obtains excretion body.
3. preparation method according to claim 1 or 2, which is characterized in that the noninvasive ultrasonic treatment includes: in probe frequency
Noninvasive ultrasonic cell 1-30min, amplitude are preferably 50-10mV under the conditions of rate 0.5-1.5MHz and amplitude 10-200mV, further
Preferably 90-110mV；Ultrasonic time is preferably 1-20min, further preferably 1-10min, is still more preferably 3-
7min。
4. according to claim want 2 described in preparation method, which is characterized in that the nerve cell include SH-SY5Y cell,
IMR-32 cell, TGW cell, LAN-1 cell, HA cell or N2A cell, preferably SH-SY5Y cell.
5. preparation method according to claim 2, which is characterized in that the method for extracting excretion body includes: that will remove
The culture medium of nerve cell and apoptotic body is concentrated to give concentrate, obtains the sediment of concentrate and precipitating reagent effect,
Sediment separate out obtains excretion body.
6. preparation method according to claim 5, which is characterized in that the precipitating reagent includes Macrogol 4000 or poly- second
Glycol 6000, preferably Macrogol 6000, further preferably 14%-18% (w/v) Macrogol 6000；
Preferably, the volume ratio of the concentrate and the precipitating reagent is 1:0.5-1.5；
Preferably, the action condition of the concentrate and the precipitating reagent are as follows: 2-8 DEG C precipitating 20-28 hours；
Preferably, the step of sediment separate out are as follows: 90000-110000 × g is centrifuged 1.5-2.5 hours, is precipitated as excretion
Body.
7. application of the noninvasive ultrasonic treatment cell in preparation excretion body.
8. the excretion body that preparation method described in any one of claims 1-6 is prepared.
9. excretion body according to claim 8, which is characterized in that miR-27a-3p, miR-27b-3p in the excretion body
It is improved with miR-7-5p gene expression abundance.
10. excretion body is in following A described in preparation method described in any one of claims 1-6 or claim 8 or 9)-D) in
The application of any one:
A) preparation prevention and/or treatment Alzheimer disease drugs；
B) preparation prevention and/or treatment anti-parkinson drug；
C) preparation prevention and/or treatment spinal injury drug；
D) preparation prevention and/or apoplexy Brain Injury After drug.
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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 |
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| WO2020125447A1 (en) * | 2018-12-18 | 2020-06-25 | 深圳先进技术研究院 | Application of non-invasive ultrasonic cell in preparing exosome, exosome, and preparation method therefor and application thereof |
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| WO2020125447A1 (en) | 2020-06-25 |
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