CN108148809A - A kind of method that excretion body is detached in the supernatant from tumour cell - Google Patents
A kind of method that excretion body is detached in the supernatant from tumour cell Download PDFInfo
- Publication number
- CN108148809A CN108148809A CN201710885748.8A CN201710885748A CN108148809A CN 108148809 A CN108148809 A CN 108148809A CN 201710885748 A CN201710885748 A CN 201710885748A CN 108148809 A CN108148809 A CN 108148809A
- Authority
- CN
- China
- Prior art keywords
- supernatant
- excretion body
- cell
- tumour cell
- detached
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of methods that excretion body is detached in supernatant from tumour cell, include the following steps：When growth of tumour cell converges rate up to 85% 95%, collect culture medium, obtain cell culture supernatant, after carrying out membrane filtration, it collects filtrate again to centrifuge filtrate ultrafiltration, collects centrifugal ultrafiltration liquid, 10w/v% 12w/v% polyethanol solution is added in 1: 1 ratio of ultrafiltrate volume, and 4 DEG C of centrifugations fully after mixing, last gained precipitation is excretion body.Convenient material drawing of the present invention is easy to Enrichment Amplification culture；The enrichment of excretion body is effectively increased using means such as membrane filtration, ultrafiltration centrifugation and addition polyglycol solutions in separation process, it is time-consuming short, it is at low cost.
Description
Technical field
The present invention relates to a kind of for detaching the method for excretion body from biofluid, and in particular to a kind of from tumour cell
The method that excretion body is detached in supernatant.
Background technology
Excretion body (exosomes) is a kind of subcellular vesica knot as secreted by various kinds of cell, a diameter of 30-140nm
Structure is in spherical or cup-shaped under electron microscope.It is derived from intracellular early stage endosome (early endosome), endosome to
Endogenous budding forms more vesica endosomes (multivesicular endosome, MVE), and the latter is with cell membrane fusion thus will be small
Vesica is discharged into extracellularly, forms excretion body.The component analysis of excretion body shows in excretion body comprising a large amount of protein, core
Acid and lipid etc., a portion is the generality component common to all excretion bodies, remaining is then because of excretion body derived tissues
Different and distinctive component.Either which kind of ingredient rises during the intercellular substance mediated in excretion body, information interchange
To key effect.The study found that contain protein relevant with cell derived, RNA and microRNA in excretion body, and excretion
Body can be by biological barrier, in intercellular trafficking functional nucleic acid, so as to play various biological functions, as tumour is thin
Born of the same parents source excretion body can participate in interacting between tumour cell and tumor microenvironment in tumor microenvironment through a variety of ways, and then
Promote the growth and transfer of tumour cell.Therefore excretion body is expected to become a kind of novel medicine feeding approach and gene therapy vector.
Tumour is the big killer for being only second to cardiovascular and cerebrovascular disease at present and influencing human health, local infiltration and long distance
From transfer be the most important feature of malignant tumour, and the main reason for this is also tumour causing death.Numerous studies discovery,
Compared with normal cell, tumour cell discharges more excretion bodies to adjust tumor microenvironment, escapes the killing of immune system, increases
The drug resistance of strong tumour cell.In recent years, it with going deep into excretion body function research, is found in the excretion body in tumour source
A series of protein and miRNAs that key effect is played in the infiltration of tumour and transfer process is promoted.Meanwhile this
It was found that also important theoretical foundation is provided for exploitation diagnosing tumor and the new method for the treatment of.
At present, the extraction of excretion body mainly using ultrahigh speed gradient centrifugation, Density ultracentrifugation, hypervelocity from
The heart-immunomagnetic beads are affine method and kit centrifugal column method etc..Ultracentrifugation method uses Ultracentrifuge, and price is held high
Expensive, generally use is not less than 100, and 000 × g centrifugation 5-8h, the requirement to consumptive material is high, and takes very long.Immunomagnetic beads are affine
Method can carry out the excretion body of extraction the purifying of specific marker, but due at present for the mark of the excretion body of separate sources
Remember that albumen is also indefinite, so the excretion body yield obtained is far below ultracentrifugal yield, and generally require a variety of labels
Immunomagnetic beads extract, and improve reagent cost.Kit centrifugal column is usually applicable only to the acquisition of small amount excretion body, and
It is and expensive.Therefore, the excretion body of isolated reliable in quality and high degree of enrichment is still a current weight of the research field
Big challenge.
Invention content
The purpose of the present invention is to provide a kind of more efficient, simple, inexpensive excretion body extracting methods, big absolutely so as to overcome
Most researchers can not extract the situation of all good excretion body of acquisition amount matter due to experiment condition limits, which can be thin from tumour
The excretion body of high-purity, high yield is obtained in born of the same parents' supernatant, popularization of the excretion body in scientific research and clinical detection is played
Important role.
To achieve the above object, the technical solution taken of the present invention is：
A kind of method that excretion body is detached in supernatant from tumour cell, includes the following steps：
S1：With complete medium culture tumour cell, when cell density reaches 50%-90%, no excretion is used instead
The culture medium of body serum, culture is for 24 hours after -48h, collects cell supernatant, under the conditions of 0 DEG C -4 DEG C, successively 500-1000 × g and
1500-2500 × g respectively centrifuges 15min to remove cell or cell fragment, collects supernatant；
S2：The supernatant of gained with sterilizing filter is filtered, filtrate is collected, under the conditions of 0 DEG C -4 DEG C, by gained filtrate
2000-4000 × g centrifuges 15min, collects supernatant；
S3：The obtained supernatants of step S2 are transferred to ultra-filtration centrifuge tube, under the conditions of 0 DEG C -4 DEG C, 3000-5000 × g
30min is centrifuged, collects ultrafiltrate；
S4：Gather in the ultrafiltrate obtained to step S3 in the ratio of ultrafiltrate volume 1: 1 addition 5w/v%-20w/v%
Ethylene glycol solution, turn upside down centrifuge tube, mixing, 4 DEG C of overnight incubations；
S5：By the liquid after incubation in room temperature or 4 DEG C, 1500 × g centrifugation 30min, the visible rice white of centrifugation bottom of the tube sinks
It forms sediment；Supernatant is removed, 1500 × g continues to centrifuge 5min, removes the liquid on upper strata, obtained precipitation is excretion body.
Preferably, the specification of the sterilizing filter is 0.22 μm or 0.45 μm.
Preferably, the specification of the ultra-filtration centrifuge tube in the step S3 is 10KD, 30KD or 100KD.
Preferably, the time being incubated in the step S4 is no less than 12h.
The invention has the advantages that：
The methods of present invention is by integrated use membrane filter method, ultra-filtration centrifuge tube simplifies entire preparation process, shortens system
The time is taken, increases substantially the amount to obtain of excretion body, and then effectively reduce and produce cost, it is easy to utilize.
Description of the drawings
Fig. 1 is the droplet measurement schematic diagram of glioma U251 cell source excretion bodies in the embodiment of the present invention；
In figure：Breadth coefficient (PDI) is a nondimensional value according to the breadth coefficient that accumulation is obtained away from method, represents grain
The distribution of sub- size)：0.08~0.7 represents moderate dispersion degree system, is the best scope of application of algorithm.
Fig. 2 is glioma U251 cell source excretion vivo flow cytometry surface antigens CD63 and CD81 in the embodiment of the present invention
Expression.
Fig. 3 is that protein concentration detects in glioma U251 cell source excretion bodies in the embodiment of the present invention.
Fig. 4 is the coomassie brilliant blue staining result of albumen in glioma U251 cell source excretion bodies in the embodiment of the present invention.
Fig. 5 is protein immunoblot figure in glioma U251 cell source excretion bodies in the embodiment of the present invention.
Specific embodiment
In order to which objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
It is described in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
Embodiment 1
The separation and extraction of people's glioma U251 cell supernatant excretion bodies
Human glioma cells U251 (being purchased from Cyagen companies of China) is incubated at 75cm by step 124 culture bottles in,
And add in containing 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomysins DMEM/F12 culture mediums, 37 DEG C, 5%
CO2It is cultivated in incubator, when cell density reaches 60%~70%, sucks original cell supernatant, addition contains
The 10% fetal calf serum culture medium without excretion body, every bottle of 15mL culture medium obtain cell supernatant 50mL, then after cultivating 48h
Supernatant a is obtained after 800 × g and 2000 × g centrifugations 15min successively under the conditions of 4 DEG C；
Step 2 filters 0.22 μm of (Millex-GP, Millipore) sterilizing filter of supernatant a, collects filtrate,
Then 4 DEG C, 3000 × g centrifuges 15min to remove cell or cell fragment, obtains supernatant b；
Supernatant b is transferred to ultra-filtration centrifuge tube (30KD, Millipore Amicon Ultra 15) by step 3) in,
4 DEG C, 4000 × g, centrifugation makes the volume of the concentrated liquid ultrafiltrate c be drawn, according to 1: 1 ratio of volume, to ultrafiltrate to 800-1000 μ L
The solution (being dissolved in PBS) of 10w/v%-12w/v% polyethylene glycol is added in c, turn upside down centrifuge tube, mixing, 4 DEG C of incubations
Overnight (at least 12h).Keep centrifuge tube upright during overnight incubation.
Step 4, will be incubated after filtrate c in room temperature or 4 DEG C, 1500 × g centrifugation 30min, the visible rice white of centrifugation bottom of the tube
Precipitation；Supernatant is sucked, 1500 × g continues to centrifuge 5min, and the careful liquid component for removing upper strata, obtained precipitation is excretion
Body.The excretion body of extraction precipitation is directly frozen in -80 DEG C of refrigerator, in a short time in case the identification of subsequent excretion body with
Other experiments.
Embodiment 2
The granularmetric analysis of people's glioma U251 cell source excretion bodies and marker identification
1) excretion body droplet measurement：Through the isolated excretion body precipitation of the above method excretion is redissolved using the filtered PBS of 1mL
Body, sealing preserve on ice；The disposable clean sample cell of selection ensures without particle to adhere in light path to light using dust-free paper wiping
Outer tube wall；Be slowly injected into excretion liquid solution and avoid bubble, can support with favored policy sample cell, sample cell is sealed using lid；By sample
Product pond is put into instrument, is detected according to standard practice instructions operating instrument；According to machine testing in instrumentation regulation：ZETASIZER
Nano series-Nano-ZS detect the grain size of excretion body.
Testing result as shown in figure 1 and table 1, uses ZETASIZER Nano series-Nano-ZS Instrumental Analysis samples
1, obtained average grain diameter and grain size main peak is in the particle size range of Exosome, the particle distribution coefficient that detects
(PDI) between 0.08-0.7, it was demonstrated that system dispersion degree is moderate, and testing result confidence level is high.The particle diameter distribution of sample is wherein
20nm-200nm ranges account for 60% or so, coincide with the particle diameter distribution of Exosome.
Table 1
| Testing index | As a result |
| Average grain diameter (nm) | 20.3 |
| Breadth coefficient (PDI) | 0.356 |
| Grain size main peak (nm) | 30.24 |
| Grain size 20nm-200nm percentages (%) | 59 |
2) excretion vivo flow cytometry surface markers analyte detection：Through the isolated excretion body precipitation of the above method, use
The filtered PBS of 100ul answer even excretion body, and sealing preserves on ice；The 20 straight labeling antibodies of μ L BD fluorescence (CD63 or CD81) are added in incubate
It educates；Sample 2 respectively using do not dye Exosome as negative control, labeled as NC；Sample 2 carries out CD63 and CD81 respectively
Two groups of antibody dyeing, labeled as CD63 and CD81；According to machine testing in instrumentation regulation：BD accuri C6 flow
cytomenter。
Testing result uses BD accuri C6 flow cytomenter Instrumental Analysis sample 2 as shown in Fig. 2 and table 2
The expression of two specific antibodies of middle CD63 and CD81, obtained dyed rear Exosome are positive, and positive rate exists
Between 60-80%.Two antibody test positive rates of same sample are slightly different, may with the specific binding capacity of antibody or
The distribution of two antigens is related.
Table 2
| Grouping | Negative ratio (%) | Positive ratio (%) |
| Negative control | 97.7 | 2.3 |
| CD63 | 39.8 | 60.2 |
| CD81 | 14.7 | 85.3 |
Embodiment 3
People's glioma U251 cell sources excretion body protein content and excretion body specific proteins CD9, HSP70 and Tsg101's
Expression detects
The efficient abundant freezing-thawing and cracking of RIPA lysates for the excretion body precipitation 1mL that embodiment 1 is obtained, extracts total egg
In vain.
According to the method for Bradfod determination of protein concentration, BSA standard curves are made, add in testing protein sample, are added in
The Coomassie Brillant Blue solution of 285 μ L detects absorbance (OD values) in microplate reader, and calculates protein content.
The electrophoresis of 60-90min is carried out in voltage 80-100V.Electrophoresis has just been run out of to bromophenol blue can terminate electrophoresis, rear to carry out
Coomassie brilliant blue protein staining and transferring film.
After transferring film, protein film is placed into preprepared TBST cleaning solutions immediately, 5min is rinsed, to wash away
Electricity on film turns liquid.After add in confining liquid, slowly shaken on shaking table, room temperature closing 60min.
After closing, the primary antibody (CD9, HSP70, Tsg101) diluted is added in immediately.4 DEG C are slowly shaken overnight incubation
Afterwards, primary antibody is recycled, TBST cleaning solutions wash 3 times, each 15min.
The secondary antibody that horseradish peroxidase (HRP) marks is diluted according to proper proportion secondary antibody diluent, room temperature or 4 DEG C exist
It is slowly shaken on shaking table and is incubated 60min, TBST cleaning solutions wash 3 times, each 15min.
Protein powder adds in ECL luminescent solutions and develops on film.
Testing result is as shown in Fig. 3 and table 3, and albumen is dense in the isolated excretion body of 50mL cell supernatants as stated above
Degree can reach 1.8 μ g/ μ L, and coomassie brilliant blue staining shows protein band in different even molecular weight distributions, with concentration
Increase, the color of band is gradually deepened, and Western Blot detection displays, separated excretion body can express significant albumen point
Sub- CD9, HSP70 and Tsg101, and with the increase of concentration, the expression quantity of albumen gradually raises.
Table 3
| Excretion body protein | As a result |
| OD values | 0.12 |
| Concentration (μ g/ μ L) | 1.8 |
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, several improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (4)
1. the method for excretion body is detached in a kind of supernatant from tumour cell, which is characterized in that include the following steps：
S1：With complete medium culture tumour cell, when cell density reaches 50%-90%, no excretion body blood is used instead
Clear culture medium, culture is for 24 hours after -48h, collects cell supernatant, under the conditions of 0 DEG C -4 DEG C, 500-1000 × g and 1500- successively
2500 × g respectively centrifuges 15min to remove cell or cell fragment, collects supernatant；
S2：The supernatant of gained with sterilizing filter is filtered, filtrate is collected, under the conditions of 0 DEG C -4 DEG C, by gained filtrate
2000-4000 × g centrifuges 15min, collects supernatant；
S3：The obtained supernatants of step S2 are transferred to ultra-filtration centrifuge tube, under the conditions of 0 DEG C -4 DEG C, 3000-5000 × g centrifugations
30min collects ultrafiltrate；
S4：In the ultrafiltrate obtained to step S3 the poly- second two of 5w/v%-20w/v% is added in the ratio of ultrafiltrate volume 1: 1
Alcoholic solution, turn upside down centrifuge tube, mixing, 4 DEG C of overnight incubations；
S5：By the liquid after incubation in room temperature or 4 DEG C, 1500 × g centrifugation 30min, the visible rice white precipitation of centrifugation bottom of the tube；It goes
Except supernatant, 1500 × g continues to centrifuge 5min, removes the liquid on upper strata, obtained precipitation is excretion body.
2. the method for excretion body is detached in a kind of supernatant from tumour cell as described in claim 1, which is characterized in that described
The specification of sterilizing filter is 0.22 μm or 0.45 μm.
3. the method for excretion body is detached in a kind of supernatant from tumour cell as described in claim 1, which is characterized in that described
The specification of ultra-filtration centrifuge tube in step S3 is 10KD, 30KD or 100KD.
4. the method for excretion body is detached in a kind of supernatant from tumour cell as described in claim 1, which is characterized in that described
The time being incubated in step S4 is no less than 12h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710885748.8A CN108148809A (en) | 2017-09-18 | 2017-09-18 | A kind of method that excretion body is detached in the supernatant from tumour cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710885748.8A CN108148809A (en) | 2017-09-18 | 2017-09-18 | A kind of method that excretion body is detached in the supernatant from tumour cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108148809A true CN108148809A (en) | 2018-06-12 |
Family
ID=62468122
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710885748.8A Pending CN108148809A (en) | 2017-09-18 | 2017-09-18 | A kind of method that excretion body is detached in the supernatant from tumour cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148809A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523536A (en) * | 2017-10-20 | 2017-12-29 | 上海市同济医院 | A kind of extracting method of tissue-derived excretion body and application |
| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
| CN109609458A (en) * | 2018-12-29 | 2019-04-12 | 暨南大学 | A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body |
| CN110747158A (en) * | 2019-11-14 | 2020-02-04 | 赵凯 | Cell supernatant exosome extraction process based on precipitation reagent method |
| CN111434769A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院分子细胞科学卓越创新中心 | Preparation method and application of exosome |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
| CN106222126A (en) * | 2016-07-27 | 2016-12-14 | 郑州点石生物技术有限公司 | The extracting method of body is secreted by human body fluid China and foreign countries |
| CN106282107A (en) * | 2016-08-30 | 2017-01-04 | 章毅 | Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body |
| CN108225873A (en) * | 2016-12-14 | 2018-06-29 | 成都中医药大学 | A kind of composition for extracting urine excretion body and its preparation method and application |
-
2017
- 2017-09-18 CN CN201710885748.8A patent/CN108148809A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
| CN106222126A (en) * | 2016-07-27 | 2016-12-14 | 郑州点石生物技术有限公司 | The extracting method of body is secreted by human body fluid China and foreign countries |
| CN106282107A (en) * | 2016-08-30 | 2017-01-04 | 章毅 | Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body |
| CN108225873A (en) * | 2016-12-14 | 2018-06-29 | 成都中医药大学 | A kind of composition for extracting urine excretion body and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 杨诚 等: "基于PEG6000富集精液来源外泌体的提取与鉴定", 《J SOUTH MED UNIV》 * |
| 胡国文 等: "旋转超滤：一种提取细胞外泌体的新方法", 《第二军医大学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523536A (en) * | 2017-10-20 | 2017-12-29 | 上海市同济医院 | A kind of extracting method of tissue-derived excretion body and application |
| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
| CN109468265B (en) * | 2018-11-06 | 2021-06-22 | 广州市创唯曦旺生物科技有限公司 | Method for extracting milk exosomes |
| CN109609458A (en) * | 2018-12-29 | 2019-04-12 | 暨南大学 | A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body |
| CN111434769A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院分子细胞科学卓越创新中心 | Preparation method and application of exosome |
| CN110747158A (en) * | 2019-11-14 | 2020-02-04 | 赵凯 | Cell supernatant exosome extraction process based on precipitation reagent method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108148809A (en) | A kind of method that excretion body is detached in the supernatant from tumour cell | |
| CN112352047A (en) | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells | |
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN102298055B (en) | Human blood H-FABP nano-gold labeled test paper and preparation method thereof | |
| CN107893050A (en) | A kind of extracellular vesica and its production and use | |
| CN106574927A (en) | Method for screening cancer prevention agent or anticancer agent using morphological characteristics of luterial | |
| CN109355259A (en) | A kind of umbilical cord mesenchymal stem cells excretion body culture and separation method | |
| CN107669697A (en) | Application of the DNA tetrahedrons in alzheimer Apoptosis is suppressed | |
| CN109609458A (en) | A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body | |
| CN110777142A (en) | Method for promoting secretion of extracellular fluid by low-intensity pulse ultrasonic stimulation | |
| CN114854676B (en) | Construction method and application of grass carp skeletal muscle myoblast cell line | |
| CN110241071A (en) | A kind of normal renal tubule primary cell of people and its Isolation and culture and application | |
| CN107663539A (en) | Circular rna circ PTGR1 purposes | |
| CN109679897A (en) | To mesenchymal stem cells differentiation cardiac-like muscle cell induced drug and detection method | |
| CN104974977B (en) | A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method | |
| CN106906271A (en) | The application of the outer body secretion in mesenchymal stem cells MSCs source and its test method | |
| CN113616674A (en) | Application of extracellular vesicle with anti-inflammatory effect | |
| CN109395096A (en) | Mark the method for excretion body and excretion body and its application of AIE fluorescent molecule label | |
| CN116286376A (en) | Separation method of haematococcus pluvialis extracellular vesicles and application thereof | |
| CN104894217A (en) | Method for detecting human peripheral blood lymphocyte genomic damage | |
| CN108410822A (en) | A kind of the CIK cell preparation and its cultural method of high cell toxicity | |
| CN108192925A (en) | Applications of the SIRT3 in old human adipose mesenchymal stem cells rejuvenation is promoted | |
| CN106566802A (en) | Preparation method for preparing culture kit of umbilical cord blood mesenchemal stem cells | |
| CN110452948A (en) | Deer heart protein hydrolysate and its preparation method and application | |
| CN109929804B (en) | Human ovarian cancer cell line and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180612 |
|
| RJ01 | Rejection of invention patent application after publication |