CN109097328A - One species specific mescenchymal stem cell excretion body extracting method - Google Patents
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- 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
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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Abstract
The invention discloses a species specific mescenchymal stem cell excretion body extracting methods, the method includes the following steps: the collection of the supernatant of the mescenchymal stem cell of serum-free: carrying out the culture of mescenchymal stem cell first, replace culture medium culture, centrifugation, cell fragment is removed, initial supernatant liquid is obtained；It takes the initial supernatant liquid in further gradient centrifugation, the supernatant after the centrifugation is transferred in sterile centrifugation tube, be centrifuged, be collected into the supernatant containing microcapsule bubble；By the supernatant centrifugation containing microcapsule bubble, the precipitating containing excretion body is obtained, cleans, is then centrifuged again, obtains excretion body precipitating；Obtained excretion body is precipitated, sterile PBS is added, is filtered, the mescenchymal stem cell excretion body of the specificity is obtained.The excretion body that the present invention collects is filtered using filter membrane, it can be ensured that the excretion body of extraction is nanoscale；Revolving speed is reduced and optimized simultaneously, reduces the mechanical damage of high speed, separative efficiency is high, at low cost.
Description
Technical field
The invention belongs to cell technology fields, and in particular to a species specific mescenchymal stem cell excretion body extraction side
Method.
Background technique
Mescenchymal stem cell (Mesenchyaml stem cells, MSCs) is a kind of multipotential stem cell, it is derived from development
The mesoderm and ectoderm of early stage can separate in the Various Tissues such as marrow, fat, synovial membrane, bone, muscle and umbilical cord, warp
The Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, neural liver, cardiac muscle and endothelium can be divided into after induction.It crosses
It goes, directly directed differentiation is target cell to play a role after being often regarded as mescenchymal stem cell and can living on trees to damaged part.However,
Some researches show that, mescenchymal stem cell live on trees to damage location ratio and to be divided into the quantity of target cell be all very limited
, and this process for being divided into required target cell is very of short duration, conflicting in time with the therapeutic effect observed.
Therefore, more and more research trends are in thinking that mescenchymal stem cell mainly secretes cytokine profiles by paracrine mechanism
Other cells are acted on to play a role.
An important component of the excretion body (Exosome, Exo) as paracrine, it is by various active somatic cells point
The size secreted is about double-layer of lipoid membrane vesicle shape structure corpusculum of the density between 1.13~1.19g/ml between 30~100nm,
After plasma membrane fusion, it is discharged into extracellular environment in the form of exocytosis.After excretion body and target cell contact, excretion body and its take
The biomolecule (such as functional lipid, protein, the molecules such as mRNA s (mRNAs), microRNAs) of band is with the shape of endocytosis
Formula is received and plays a role.Therefore, largely research it has also been found that mescenchymal stem cell really can be by itself excretion body
The effects of playing the tissue repair similar with cell, immunosupress adjusting immune function.Such as early in 2007, Timmer etc. is rolled into a ball
Knot finds that the extracellular matrix of MSC can effectively mitigate ischemical reperfusion injury, passes through heart ischemia/Reperfu- sion of pig
Matter is handled when model gives the extracellular base of MSC, it is possible to reduce infarct size reaches nearly 60%.Pass through extracellular matrix
Intrinsic activity substance is researched and analysed, and Electronic Speculum observes the lipid bilayer vesica for being a group size between 50~200nm, and
The albumen such as specific expressed CD81, CD9, ALIX substantially conform to the fundamental characteristics of excretion body.In addition to this, in injury of kidney reparation
Aspect gives 30ug mescenchymal stem cell excretion body when someone cuts building murine chronic renal injury model by 5/6 kidney entirely respectively
With 1 × 106Mescenchymal stem cell is handled by tail vein injection, display is dissected after 7 days, with the blank control group phase for giving PBS
Than mouse blood urea nitrogen, the serum creatinine, uric acid ratio of mescenchymal stem cell processing group and mescenchymal stem cell excretion body processing group
It all reduces, improves tissue fibrosis and interstitial lymphocyte infiltration phenomenon.These confirm that mescenchymal stem cell is certain
Paracrine function can be played by secretion excretion body.And compared with cell therapy, excretion volume property is more stable, saves transport
It is convenient, risk is formed without transplanted cells bring immunological rejection and tumour, therefore this " cell-free excretion body " has
Hoping becomes a kind of new therapeutic strategy.How obtaining separation excretion body and obtaining the excretion body of high-purity is current researcher
And its problem of concern, it is most important to subsequent application study.Therefore, this patent invents a kind of collect in response to this problem
The excretion body of high concentration, specific source for mesenchymal stem cells.
Currently, the separating and extracting process of excretion body mainly has: ultracentrifugation, immunomagnetic beads, ultrafiltration, precipitating or kit
The methods of.These methods are specific as follows:
1, supercentrifugation (differential centrifugation): surpassing from method is most common excretion body means of purification, using low-speed centrifugal,
High speed centrifugation is alternately: i.e. cell culture supernatant is centrifuged in 300g, 10min, and precipitating takes cell supernatant；2000g centrifugation
10min removes dead cell precipitating, takes supernatant；It is separated followed by 1000g, 30min, precipitating removes pellet cell debris object, obtains
The supernatant obtained is at > 10000g, 70min separation condition, and acquisition is precipitated as excretion body precipitating and other interferencing proteins, most
These precipitatings are washed with PBS again afterwards, are centrifuged 70min in > 100000g, excretion body can be obtained in precipitating.Exceed the speed limit from method because
Easy to operate, the vesica quantity of acquisition is more and very popular, but process is relatively time-consuming, and the rate of recovery it is unstable (may with turn
Subtype is related), purity is also under suspicion；In addition, repeated centrifugation operation is it is also possible to damage vesica, to reduce it
Quality.
2, density gradient centrifugation: this method is that sample and different sucrose density materials are mixed centrifugation.It is exceeding the speed limit
Under centrifugal force effect, sucrose solution is set to form density stratum continuously distributed from low to high, to cause the different component in sample
It is deposited to respective isodensity area, forms a kind of method that zone separates in turn.Sucrose density gradient centrifugation is such as commonly used in experiment
Method is that two kinds of concentration sucrose solutions (such as 2.5M and 0.25M) are made into continuous gradient system in advance to be placed in ultracentrifugation pipe, sample
Originally it is layered on sucrose solution, 100000g is centrifuged 16h, and excretion cognition is deposited to isodensity area (1.10~1.18g/ml).With such
The excretion body purity is high that method is separated to, but preliminary preparation is many and diverse, time-consuming, yield is few.In the excretion of source of human stem cell
Body extracts related patents and has not been reported.
3, ultrafiltration is centrifuged: since excretion body is about tens nanometers of a size of cryptomere corpusculum, being greater than general protein, benefit
Selective Separation is carried out to sample with the ultrafiltration membrane of different retentions relative molecular mass (MWCO), excretion body can be obtained.Ultrafiltration
Centrifugal process is simple and efficient, and does not influence the bioactivity of excretion body, is a kind of new method for extracting cell excretion body.
Application No. is 201710447410.4 Chinese patents to disclose the separation method of stem cell excretion body, they pass through
The different size of concentration tube of conjunctive use can quickly collect stem cell excretion body.That is: by being adopted respectively to sterile excretion body
4 DEG C are carried out with 10KD super filter tube, it, again will be with new by the concentrate of acquisition after 3500~4500g is centrifuged 35~45min
10KD super filter tube is centrifuged again under normal temperature conditions, condition 150000g, 8~12min, collects concentrate；Last then room temperature item
(15~30 DEG C) are further centrifuged 1.5~2.5min using 900~1100g under part, and the liquid obtained in collecting pipe is then containing height
The solution of concentration stem cell excretion body.The partition method method centrifugation time that they show that they provide is shorter than the ultracentrifugation time, can
The mechanical damage of excretion body is effectively reduced, the activity of excretion body is preferably retained.
4, magnetic bead immunization: there is its specific marker object (such as CD63, CD9 albumen) in excretion body surface face, with the anti-label of coating
The magnetic bead of object antibody combines after being incubated for excretion somatocyst bubble, can excretion body be adsorbed and be separated.Paramagnetic particle method has special
Property is high, easy to operate, does not influence the advantages that excretion volume morphing is complete, but low efficiency, and excretion body bioactivity is vulnerable to pH and salt
Concentration influences, and is unfavorable for downstream experiment, it is difficult to widely available.
5, the PEG-base precipitation method: polyethylene glycol (PEG) can be co-precipitated in conjunction with hydrophobic proteins and lipid molecular, previously
Applied to virus is collected from serum equal samples, it is also used to precipitating excretion body now, principle may be swum with competitive binding
It is related from hydrone.Using PEG precipitating excretion body, there are many problems: such as purity and the rate of recovery are low, more (the false sun of foreign protein
Property), granular size is inhomogenous, generates the polymer for being difficult to remove, and the chemical additives such as mechanical force or Tween-20 will destroy
Excretion body etc., therefore vulnerable to query when publishing an article.
6, kit extracts: in recent years, having occurred various commercialized excretion body extracts kits in the market, has had plenty of
Impurity component is filtered out by the filter of special designing, some is then isolated and purified using spatial exclusion chromatography (SEC),
Also method excretion body is then precipitated out using compound precipitation by some.
Although these methods have their own characteristics, magnetic bead immunization, the PEG-base precipitation method and kit extracting method
The excretion body for being only applicable in small sample extracts, and is not particularly suited for the collection of extensive excretion body.And the method for sucrose gradient centrifugation
Relatively time consuming, ultrafiltration centrifugation needs to constantly change filter membrane.In addition, these schemes do not carry out specific excretion for all kinds of samples
The separation of body optimizes.Therefore this programme is mainly for excretion body most common, suitable for separating, extracting source for mesenchymal stem cells
Supercentrifugation optimize, inventing a kind of suitable for mescenchymal stem cell and can isolate the separation of specific excretion body
Extracting method.
As described above, supercentrifugation in simple terms by cell culture fluid or body fluid equal samples successively 300g, 2000g,
10000g centrifugation removal cell fragment and macro-molecular protein, last 100000g are centrifuged to obtain excretion body.
Application No. is 201610279473.9 Chinese patent disclose a kind of excretion body, excretion body preparation method and its
Application in preparation treatment medication for treating pyemia or preparation, the umbilical cord mesenchyma for disclosing Interleukin -1β (IL-1 β) optimization are dry
Cell origin excretion body is being treated in pyemia in application, excretion body is collected and the method for separation and Extraction is: 1. taking 3-7
The mescenchymal stem cell (MSCs) in generation is inoculated in 10cm culture dish, and when cell fusion reaches 60-70%, recombined human is added
IL-1 β, so that final concentration of 10ng/m1, is handled 12-24 hours；Cell is cleaned twice with PBS, and replacement is containing 10% without excretion body
The DMEM/F12 culture medium of FBS continues culture 48-72 hours.FBS wherein without excretion body is by 100,00g ultracentrifugation 18
The FBS of precipitating is removed after hour；2. collection step 1. in medium supernatant, collect excretion body with the method for differential centrifugation:
Sterile collection step 1. in the resulting medium supernatant of final step in 500m1 sterile centrifuge bottles or 50ml polypropylene centrifuge tube
In, it is centrifuged 10 minutes in 4 DEG C, 2000g, to remove dead cell and big fragment；Carefully by supernatant be transferred to it is new it is sterile from
In heart pipe, in 4 DEG C, 10,000g are centrifuged 30 minutes, to remove organelle and little particle；Carefully supernatant is transferred to sterile super
It in fast centrifuge tube, in 4 DEG C, 110,000g ultracentrifugation 70 minutes, carefully discards supernatant, then plus injection physiological saline cleaning one
Secondary, in 4 DEG C, 110000g ultracentrifugation 70 minutes, obtained precipitating was that the human umbilical cord mesenchymal stem cells of IL-1 β optimization come
The excretion body in source.Excretion body is resuspended with physiological saline, -80 DEG C of packing save after measuring protein concentration.
Application No. is 201711104768.3 Chinese patent, to disclose a kind of human umbilical cord mesenchymal stem cells excretion external
With creams and preparation method thereof, it is also supercentrifugation that the excretion body in invention formula, which collects the method extracted, it may be assumed that take P2~
The human umbilical cord mesenchymal stem cells in P5 generation are cultivated with DMEM/F12 culture solution, when cell is merged up to 70%, use nothing instead
The hungry culture solution of serum cultivates 12h, the hungry culture solution of serum-free is changed to containing 10ng/ml epithelical cell growth factor
DMEM/F12 culture solution, culture to cell be in logarithmic growth phase, collection cell culture supernatant 300g~10000g gradient
It is centrifuged 60-120min, centrifugation gained supernatant 0.22um filter is filtered, human umbilical cord mesenchymal stem cells excretion body is obtained and mentions
Take liquid.
Application No. is 201510779110.7 Chinese patents to disclose miR-145-5p modification umbilical cord mesenchymal stem cells
The excretion body of secretion and its preparation and application, the invention is in the UMSC excretion body for extracting highly expressed microR-145-5p modification
When, the method for use is also supercentrifugation, it may be assumed that is replaced with to the UCMSC for the miR-145-5p modification that will be extracted
Exosome-free FBS culture medium collects supernatant after cultivating 48-72h, and then by it in 4 DEG C, 300 × g, 10min centrifugation are removed
Remove cell fragment；4 DEG C, the impurity such as 2000 × g, 10min centrifugation removal dead cell；0.22um sterilised membrane filter filtering further removal
Impurity；4 DEG C, 100000 × g, 120min ultracentrifugation obtains the exosome precipitating being overexpressed containing microRNA145-5p, adds
Enter about 200ul PBS to wash one time；4 DEG C again, 100000 × g, 120min ultracentrifugation, can be obtained purer concentration by
The excretion body in the umbilical cord mesenchymal stem cells source of microRNA145-5p modification.80 DEG C are stored in, it can long-term preservation.
The excretion body method in separation and Extraction mesenchymal stem cells in the prior art source is although relatively common, but exist with
Lower problem:
(1) although the excretion body that is collected into of supercentrifugation is relatively more, since microvesicle and excretion body are not united very much now
One standard of perfection, the excretion body purity being collected into is bad, so the aggregation of discovery excretion body is blocking when Electronic Speculum is identified, quality is not
It is good；
(2) the existing cell-free specificity of excretion body extracting method, such as supercentrifugation, different size and type it is thin
Born of the same parents are same Ultracentrifugation conditions, i.e., 300g, 2000g, 10000g, 100000g are centrifuged, and such ultracentrifugal method is certain
It is difficult to meet the centrifugal condition of different cell types, it is therefore desirable to carry out the excretion body centrifugal condition of different cell types excellent
Change；
(3) although ultrafiltration centrifugal process is simple, 1h is only needed using super filter tube centrifugally operated, opposite ultracentrifugation pipe time-consuming is close
2.5h can save 1.5h, but entire centrifugal process needs replacing at least 2 filter membranes, expensive.And simple ultrafiltration side
The excretion bulk concentration that method is collected cannot reach very high, and it is also one that detection and the treatment etc. that need higher concentration excretion body, which are applied,
Kind limitation.
For these reasons, the present invention is specifically proposed.
Summary of the invention
In order to solve problem above of the existing technology, the present invention provides outside a species specific mescenchymal stem cell
Body extracting method is secreted, this method mainly on the basis of traditional ultracentrifugation, optimizes eccentricity, has investigated one kind
Mescenchymal stem cell excretion body process for separation and purification, the mesenchymal stem cells excretion body that this method is extracted are extracted suitable for large-scale separation
Not only with high purity, the excretion body protein content of acquisition is also high, includes active material and enriches, between also there is Specific marker to assess
The quality and potential curative effect of the excretion body of mesenchymal stem cells.
To achieve the goals above, the present invention adopts the following technical scheme:
One species specific mescenchymal stem cell excretion body extracting method, the method include the following steps:
(1) collection of the supernatant of the mescenchymal stem cell of serum-free: carrying out the culture of mescenchymal stem cell first, will be thin
Born of the same parents' degrees of fusion reaches the mescenchymal stem cell of 60-70%, is cleaned with PBS, and the culture medium for being replaced with serum-free Related Component carries out
Culture, flat, collection culture medium, centrifugation are covered with to cell, and removal cell fragment obtains initial supernatant liquid；
(2) it takes the initial supernatant liquid to be centrifuged 10min in further gradient centrifugation 2000g, removes dead cell and greatly broken
Piece, the supernatant after being centrifuged；
(3) supernatant after the centrifugation is transferred in centrifuge tube, is centrifuged 30min in 10000g, is collected into containing micro-
The supernatant of vesica；
(4) supernatant containing microcapsule bubble is centrifuged 60min in 70000g, obtains the precipitating containing excretion body, cleans, so
70000g is centrifuged 70min again afterwards, obtains excretion body precipitating；
(5) obtained excretion body is precipitated and PBS is added, used membrane filtration, the mesenchyma for obtaining the specificity is done
Cell excretion body.
Further, the ɑ-MEM culture that the culture medium of culture mescenchymal stem cell is the PLA that concentration is 5% in step (1)
ɑ-MEM the culture medium of base or the FBS for being 15% for concentration.
Further, the culture medium is placed on 37 DEG C, CO2It is cultivated in the incubator that concentration is 5%.Further,
The culture medium that step (1) is replaced with serum-free Related Component is ɑ-MEM or DMEM/F12 culture medium, in the culture medium PLA or
The concentration of FBS is 0.
The culture medium that serum-free Related Component is replaced in the present invention is in order to avoid PLA or FBS (platelet cracking content)
Remain the influence extracted to subsequent excretion body.
Further, replacing the incubation time carried out after culture medium is 48-72 hours.
Further, the condition being centrifuged in step (1) is that 300-500g is centrifuged 5-10min.
Further, the temperature being centrifuged in step (2)-(4) is 4 DEG C.
Further, the sterile PBS of 180-220ul being added in step (5).
Further, the aperture of filter membrane is 0.1um in step (5).
Aperture is used to filter in the present invention for the filter membrane of 0.1um, it is ensured that obtain real nanoscale excretion body.
Further, each step that excretion body extracts is operated under sterile conditions.
Compared with prior art, beneficial effects of the present invention are as follows:
(1) the excretion body that the present invention collects is filtered using filter membrane, it can be ensured that the excretion body of extraction is nanoscale；This hair
Ultracentrifugation use 70000g, active material abundant is contained in the mescenchymal stem cell excretion body extracted, reduces height
The mechanical damage of speed, separative efficiency is high, at low cost；
(2) extracting method of the invention is optimized, obtains to eccentricity on the basis of traditional ultracentrifugation
Mescenchymal stem cell excretion body extracting method is extracted in a kind of suitable large-scale separation, outside the mescenchymal stem cell that this method is extracted
Secrete that body is not only with high purity, the excretion body protein total amount of acquisition is also high, and it is abundant to include active material, also comments with Specific marker
Estimate the quality and potential curative effect of the excretion body of mescenchymal stem cell；
(3) the excretion body that method of the invention is extracted contains small-molecule substance and has to promotion vascular repair and anti-fibrosis
Preferable effect.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1: the CD29 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 2: the CD90 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 3: the CD44 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 4: the CD73 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 5: the CD105 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 6: the CD14 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 7: the CD34 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 8: the CD45 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Fig. 9: the CD19 streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Figure 10: the HLA-DR streaming Phenotypic examination of the third generation mescenchymal stem cell in umbilical cord source；
Figure 11: aspect graph of the excretion body that control group extracting method is extracted under Electronic Speculum observation；
Figure 12: aspect graph of the excretion body that test group extracting method is extracted under Electronic Speculum observation；
Figure 13: CD63 the and CD81 egg of Western blotting qualification test group and the excretion of control group difference separating and extracting process
White expression.
Relative expression quantity of Figure 14: the qRT-PCR detection miR-486-5p in the excretion body in umbilical cord mesenchymal stem cells source
Figure.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
Mescenchymal stem cell of the invention is human umbilical cord mesenchymal stem cells.
Embodiment 1
The species specific mescenchymal stem cell excretion body extracting method of the one of the present embodiment, the method include following step
It is rapid:
(1) collection of the supernatant of the mescenchymal stem cell of serum-free: carrying out the culture of mescenchymal stem cell first, described
ɑ-MEM the culture medium that culture medium is the PLA that concentration is 5%, is put into 37 DEG C, CO2It is cultivated in the incubator that concentration is 5%；When thin
Born of the same parents' degrees of fusion reaches 60%, is cleaned with PBS and is replaced with the culture medium of serum-free Related Component twice and is cultivated, long to cell
It is full flat, collect culture medium, the culture medium for being replaced with serum-free Related Component is ɑ-MEM, and in the culture medium after replacing PLA or
The concentration of FBS is 0, carries out culture 48 hours；300g is centrifuged 10min centrifugation, removes cell fragment, obtains initial supernatant liquid；
(2) take the initial supernatant liquid in further gradient centrifugation, at 4 DEG C, 2000g is centrifuged 10min, remove dead cell and
Big fragment, the supernatant after being centrifuged；
(3) supernatant after the centrifugation is transferred in centrifuge tube, at 4 DEG C, 10000g is centrifuged 30min, is collected into and contains
There is the supernatant of microcapsule bubble；
(4) by the supernatant containing microcapsule bubble at 4 DEG C, 70000g is centrifuged 60min, obtains the precipitating containing excretion body, PBS
Cleaning one time, then 70000g is centrifuged 70min again, obtains excretion body precipitating；
(5) obtained excretion body is precipitated and 180ulPBS is added, filtered with 0.1um filter membrane, obtain the specificity
Mescenchymal stem cell excretion body；
Wherein, each step that excretion body extracts is operated under sterile conditions.
Embodiment 2
(1) collection of the supernatant of the mescenchymal stem cell of serum-free: carrying out the culture of mescenchymal stem cell first, described
ɑ-MEM the culture medium that culture medium is the FBS that concentration is 15%, is put into 37 DEG C, CO2It is cultivated in the incubator that concentration is 5%；When thin
Born of the same parents' degrees of fusion reaches 65%, is cleaned with PBS and is replaced with the culture medium of serum-free Related Component twice and is cultivated, long to cell
It is full flat, culture medium is collected, the culture medium for being replaced with serum-free Related Component is DMEM/F12, and in the culture medium after replacement
The concentration of PLA or FBS is 0, carries out culture 60 hours；400g is centrifuged 8min centrifugation, removes cell fragment, obtains initial supernatant
Liquid；
(2) take the initial supernatant liquid in further gradient centrifugation, at 4 DEG C, 2000g is centrifuged 10min, remove dead cell and
Big fragment, the supernatant after being centrifuged；
(3) supernatant after the centrifugation is transferred in centrifuge tube, at 4 DEG C, 10000g is centrifuged 30min, is collected into and contains
There is the supernatant of microcapsule bubble；
(4) by the supernatant containing microcapsule bubble at 4 DEG C, 70000g is centrifuged 60min, obtains the precipitating containing excretion body, PBS
Cleaning one time, then 70000g is centrifuged 70min again, obtains excretion body precipitating；
(5) obtained excretion body is precipitated and 200ulPBS is added, filtered with 0.1um filter membrane, obtain the specificity
Mescenchymal stem cell excretion body；
Wherein, each step that excretion body extracts is operated under sterile conditions.
Embodiment 3
(1) collection of the supernatant of the mescenchymal stem cell of serum-free: carrying out the culture of mescenchymal stem cell first, described
ɑ-MEM the culture medium that culture medium is the FBS that concentration is 15%, is put into 37 DEG C, CO2It is cultivated in the incubator that concentration is 5%；When thin
Born of the same parents' degrees of fusion reaches 70%, is cleaned with PBS and is replaced with the culture medium of serum-free Related Component twice and is cultivated, long to cell
It is full flat, collect culture medium, the culture medium for being replaced with serum-free Related Component is ɑ-MEM, and in the culture medium after replacing PLA or
The concentration of FBS is 0, carries out culture 60 hours；500g is centrifuged 5min centrifugation, removes cell fragment, obtains initial supernatant liquid；
(2) take the initial supernatant liquid in further gradient centrifugation, at 4 DEG C, 2000g is centrifuged 10min, remove dead cell and
Big fragment, the supernatant after being centrifuged；
(3) supernatant after the centrifugation is transferred in centrifuge tube, at 4 DEG C, 10000g is centrifuged 30min, is collected into and contains
There is the supernatant of microcapsule bubble；
(4) by the supernatant containing microcapsule bubble at 4 DEG C, 70000g is centrifuged 60min, obtains the precipitating containing excretion body, PBS
Cleaning one time, then 70000g is centrifuged 70min again, obtains excretion body precipitating；
(5) obtained excretion body is precipitated and 220ulPBS is added, filtered with 0.1um filter membrane, obtain the specificity
Mescenchymal stem cell excretion body；
Wherein, each step that excretion body extracts is operated under sterile conditions.
Influence of the different centrifugal conditions of test example 1 to mescenchymal stem cell excretion body separation and Extraction
Test method
Test group: excretion body is extracted using the method for the present invention；
Control group: using traditional Ultracentrifugation Method, i.e. 300g, 2000g, 10000g, 100000g；
1 test method
The influence of evaluation test group and excretion body the extracting total amount and phenotype to umbilical cord mesenchymal stem cells of control group, is commented
The effect and two kinds of extracting methods that valence difference Ultracentrifugation Method extracts mescenchymal stem cell are to neuronal cell growth
It influences, the specific method is as follows:
1.1 cell culture processes
Respectively by the umbilical cord mesenchymal stem cells being collected into (UC-MSCs) according to 2000-5000/cm2Density carry out
Then suitable stem cell complete medium is added in inoculation, culture medium is the 15%FBS or 5%PLA of aMEM, is placed in 37 DEG C,
CO2It is cultivated in the incubator that concentration is 5%, (the adherent cell growth to 70%- after the adherent adhesion area of born of the same parents is up to 70%~90%
When 90% fusion), it can be needed then to carry out subsequent experimental according to experiment.
The flow cytometer detection of 1.2 umbilical cord mesenchymal stem cells
After MSC cell recovery culture to 80-90% degrees of fusion when carry out flow cytometer detection.Prepare to have digested fills between umbilical cord
Matter stem cell, PBS washing after to cell carry out antibody label, labelled antibody be respectively CD29, CD73, CD105, CD90, CD44,
CD14,CD34,FCD45,HLA-DR,CD19.After being incubated for washing, upper machine testing.
1.3 excretion bodies extract
(1) traditional supercentrifugation: the MSC that be extracted and cell fusion degree reaches 70% is washed 2 times with PBS, is changed
At the a-MEM of no FBS or PLA, cultivate 48 hours.Then receive supernatant, be centrifuged by several times: 1. 300g, 10 minutes, remove cell and its
Fragment；2. 2000g 10 minutes, removes the cell and its fragment die；3. 10000g 30 minutes, removes cell fragment；4. will
Supernatant connects 0.22 μm of membrane filtration with 50 syringes；5. 100000g, 1 hour, it is seen that precipitating (excretion body and protein ingredient)；
6. discarding supernatant, retain precipitating, PBS is added；7. 100000g, 1 hour, it is seen that precipitating is purer concentration excretion body；Above from
Heart step is completed at 4 DEG C, and whole process is sterile working.
(2) MSC that be extracted and cell fusion degree reaches 70% supercentrifugation of the invention: is washed 2 with PBS
Time, it changes the a-MEM of no FBS or PLA into, cultivates 48 hours.Then receive supernatant, be centrifuged by several times: 1. 500g, 10 minutes, removal was thin
Born of the same parents and its fragment；2. 2000g 10 minutes, removes the cell and its fragment die；3. 10000g, 30 minutes, removal cell was broken
Piece；4. supernatant is connect 0.22 μm of membrane filtration with 50 syringes；5. 70000g, 1 hour, it is seen that precipitating (excretion body and albumen
Ingredient)；6. discarding supernatant, retain precipitating, PBS is added；7. 70000g, 1 hour, it is seen that precipitating is purer concentration excretion body；
8. obtained excretion body precipitating, is added the sterile PBS of 200ul, using the membrane filtration of 0.1um, it is ensured that obtain real nanoscale
Excretion body.The above centrifugation step is completed at 4 DEG C.Whole process is sterile working.
1.4BCA method measures excretion body protein concentration
This test uses the protein concentration of the BCA determination of protein concentration kit measurement excretion body of U.S. Thermo company.
It takes BSA to be dissolved in 1%SDS and is made into the protein standard that concentration is 0.5mg/ml.Every hole needs BCA working solution 200ul, counts as follows
The total volume of working solution needed for calculating: (number of standard items+testing protein quality sample number) × (experiment number of repetition, this reality
It tests as 2) × 200ul.It is BCA working solution needed for 50:1 mixes well configuration by A:B volume ratio.By 0.5mg/ml protein standard
It is added in 96 orifice plates with 0,1,2,4,8,12,16,20ul and 4ul albumen sample, 1%SDS is added to complement to 20ul.Every hole is added
200ul BCA working solution, 37 DEG C of incubation 30min.Each hole is measured in the absorption value of 562nm wavelength with microplate reader.And according to each mark
The concentration and absorbance value in quasi- hole draw standard concentration curve, and further according to the absorbance value of each sample aperture, to acquire it practical dense
Degree.It detects, is then needed according to above-mentioned surveyed concentration dilution concentration of specimens if you need to further progress Western Blot Western blotting,
It is close consistent to make its concentration, and appropriate albumen sample-loading buffer is added.Sample is placed in boiling water and boils 15min and makes albuminous degeneration,
Cooling stand-by or -20 DEG C of storages.
1.5Nanosight identifies excretion body volume and excretion volume density
This experiment uses the Nanosight detector of Britain Malvern company NS300 model.It will freeze in outside -80 DEG C
Body is secreted in 4 DEG C of refrigerator defrostings, and PBS rinses instrument connecting tube, extracts 100 μ l excretion liquid solutions and is slowly injected into connecting tube, instrument
Detection 1 minute, can be imaged by the Brownian movement of nanoparticle and detect its density.
2 test results
UC-MSC the 3rd generation MSC cell of the method culture of 2.1 pairs of this tests is tested, discovery discovery positive indication
CD29, CD90, CD44, CD73 and CD105 positive rate are respectively 99.3%, 98.8%, 98.5%, 99.6%, 99.3%, are owned
The equal > 95% of positive indication；And CD14, CD34, CD45, CD19 and HLA-DR positive rate are respectively less than 2%, are negative, and see Fig. 1-
10, illustrate the available purer umbilical cord mesenchymal stem cells of tissue stationary culture culture.
The collection and purifying of excretion body in 2.2MSC culture supernatant
This experiment can be obtained successfully super in MSC culture supernatant by using two different supercentrifugations
Fast centrifugal sediment, by carrying out protein quantification to ultracentrifugation precipitation object, it is found by the applicant that in this 200ml culture supernatant,
Traditional Ultracentrifugation Method can isolated about 0.31g total protein, therefore concentration is about 0.159 μ g/ml culture supernatant；And
The total protein that supercentrifugation of the invention extracts is 0.428ug, concentration 0.214ug/ml, higher than traditional separation side
Method is shown in Table 1.
The total protein concentration that the different excretion body centrifugal process of table 1 are extracted
| Number | Grouping | Protein concentration (ug/ml) |
| 1 | Blank cultures | 0 |
| 2 | Traditional supercentrifugation | 0.185 |
| 3 | Traditional supercentrifugation | 0.134 |
| 4 | Traditional supercentrifugation | 0.157 |
| 5 | Supercentrifugation of the invention | 0.223 |
| 6 | Supercentrifugation of the invention | 0.207 |
| 7 | Supercentrifugation of the invention | 0.213 |
The Property Identification of the source 2.3MSC excretion body
Collect whether obtained supernatant sediment is MSC source excretion body to study, size for excretion body and its
Show that distinctive marker is detected.First with the excretion body that Electronic Speculum observation both methods is collected into, discovery is
100nm grades or so of vesica is shown in Figure 11-13, and CD63 the and CD81 antigen of WB method detection excretion body characteristics, tradition side
Method and hypervelocity separation method of the invention are expressed, and illustrate that both methods can effectively extract mescenchymal stem cell
exosome.In addition, being also grouped the identification of size to the excretion body of both method for extracting, Nanosight detection discovery is outer
The uniform component of body is secreted, excretion body particle size is respectively less than 120nm.The excretion body for illustrating this new extracting is also nanoscale excretion
Body.
So in summary, the nanoscale excretion body total amount that the hypervelocity separation method collection of this invention is purified to is higher,
It is a kind of method suitable for the extracting of mescenchymal stem cell excretion body.
The mescenchymal stem cell excretion body rna expression spectrum analysis and functional verification that the method for the invention of test example 2 is extracted
1 test objective: rna expression composes situation in identification mescenchymal stem cell excretion body, finds Specific marker.
2, test method: 2 kinds of mescenchymal stem cell serum excretion bodies are separated using the method for the present invention, RNA is then extracted, answers
The transcript profile sequencing of 1 sample is carried out with two generation sequencing technologies, and is tested in another excretion body using real-time fluorescence quantitative PCR
Demonstrate,prove sequencing result.It is specific as follows:
The separation and Extraction of 2.1 excretion bodies:
The culture of mescenchymal stem cell is identical as cell culture processes in test example 1.
The MSC that be extracted and cell fusion degree reaches 70% is washed 2 times with PBS, changes the a- of no FBS or PLA into
MEM is cultivated 48 hours.Then receive supernatant, be centrifuged by several times: 1. 500g 10 minutes, removes cell and its fragment；2. 2000g, 10
Minute, remove the cell and its fragment die；3. 10000g 30 minutes, removes cell fragment；4. by supernatant with 50 syringes
Connect 0.22 μm of membrane filtration；5. 70000g, 1 hour, it is seen that precipitating (excretion body and protein ingredient)；6. discarding supernatant, it is heavy to retain
It forms sediment, PBS is added；7. 70000g, 1 hour, it is seen that precipitating is purer concentration excretion body；8. obtained excretion body precipitating, is added
The sterile PBS of 200ul, using the membrane filtration of 0.1um, it is ensured that obtain real nanoscale excretion body.The above centrifugation step is in 4
It is completed at DEG C.Whole process is sterile working.
The miRNA all expressed in 2.2 high-flux sequence MSC and its exo:
Excretion body total serum IgE is extracted referring to QIAzol kit operating procedure, after purity and Concentration Testing comply with standard, by
Guangzhou Rui Bo company using transcript profile sequencing technologies (RNAseq) detection 1 exception secretes body transcript profile expression data, by with
Major gene sequence is compared, and analysis obtains mRNA (message RNA, mRNA) and non-coding RNA (1non-coding
RNA, ncRNA) expression data.The every of exon is navigated to readings of every 1,000,000 bases on the position in chromosome
Segment number (fragment per kilobase of exon model per million mapped in 1000 bases
Reads, FPKM) > 1 think there is gene expression.
2.3qRT-PCR verifies the expression of miRNA in Exo, wherein miR-486-5p RT sequence are as follows: 5'-GTCGTA
TCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG-3', miR-486-5p mature sequence are as follows: F 5'-
CAGTCCTGTACTGAGCTGC-3', internal reference U6F sequence are as follows: 5'-CTCGCTTCGGCAGCACA-3'；U6R sequence are as follows: 5'-
AACGCTTCACGAATTTGCGT-3' primer sequence by the design of Ji Ma company, synthesizes.The reverse transcription of RNA and quantitative PCR according to
TOYOBO brand specification carries out, i.e., RNA reverse transcription synthesizes cDNA, acts on 30 minutes in 42 DEG C, acts on 5 minutes and inactivating through 95 DEG C
Template as Q-PCR reaction afterwards, is quantified using SYBR Green Realtime PCR Master Mix (TOYOBO)
PCR amplification (LightCycler 2.0Instrument, Roche Applied Science).The reverse transcription of miRNA and quantitative
PCR using miRCURY LN Universal RT microRNA PCR kit (203300and 203450, EXIGON Inc.,
MA, USA), it is operated according to kit specification.
3 test results
3.1 by detecting miRNA express spectra, the inventors discovered that in the excretion body of MSC source, MiR-486-
The high expression of the enrichment such as 5p, MiR-10a-5p, MiR-10b-5p, MiR-191-5p, MiR-222-3p, and excretion body these height expression
MiR-486-5p in cell low expression.It is found in conjunction with newest document, the miR-486-5p that excretion body surface reaches not only has
Pulmonary fibrosis resistant effect, can also can promote the reparation of renal ischaemia damage.Simultaneously, the BM-MSC under hypoxia condition, miR-
486 high expression, can be enhanced BM-MSCs secretion HGF, and the factors such as VEGF promote hematopoiesis.Therefore speculate, miR-486-5p can be used as
The specific marker of MSC-exosome, the excretion body for being alternatively arranged as evaluation MSC source repair the potential of curative effect in injury of blood vessel
Index the results are shown in Table 2.
The excretion body and the highly expressed miRNAs of cell in 2 source MSCs of table
3.2miR-486-5p the verifying in the excretion body of MSC source.
By carrying out testing for miR-486-5p qRT-PCR expression to the exosome for not carrying out being sequenced another MSC source
Card.Inventor's discovery is compared with internal reference, the really high expression of the exosome of excretion body.Prove that miR-486-5p can be used as mesenchyma
The specific maker of the excretion body in source, is shown in Figure 14.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a species specific mescenchymal stem cell excretion body extracting method, which is characterized in that the method includes following step
It is rapid:
(1) collection of the supernatant of the mescenchymal stem cell of serum-free: the culture of mescenchymal stem cell is carried out first, cell is melted
The right mescenchymal stem cell for reaching 60-70%, is cleaned with PBS, and the culture medium for being replaced with serum-free Related Component is cultivated,
Flat, collection culture medium, centrifugation are covered with to cell, removal cell fragment obtains initial supernatant liquid；
(2) it takes the initial supernatant liquid to be centrifuged 10min in further gradient centrifugation 2000g, removes dead cell and big fragment, obtain
Supernatant after to centrifugation；
(3) supernatant after the centrifugation is transferred in centrifuge tube, is centrifuged 30min in 10000g, is collected into containing microcapsule bubble
Supernatant；
(4) supernatant containing microcapsule bubble is centrifuged 60min in 70000g, obtains the precipitating containing excretion body, cleans, then again
70000g is centrifuged 70min, obtains excretion body precipitating；
(5) obtained excretion body is precipitated and PBS is added, used membrane filtration obtains the mescenchymal stem cell of the specificity
Excretion body.
2. a species specific mescenchymal stem cell excretion body extracting method according to claim 1, which is characterized in that step
Suddenly culture medium of culture mescenchymal stem cell is the PLA that concentration is 5% in (1) ɑ-MEM culture medium be concentration is 15%
ɑ-MEM the culture medium of FBS.
3. a species specific mescenchymal stem cell excretion body extracting method according to claim 2, which is characterized in that institute
The culture medium stated is placed on 37 DEG C, CO2It is cultivated in the incubator that concentration is 5%.
4. the species specific mescenchymal stem cell excretion body extracting method of one according to claim 1 to 3,
It is characterized in that, the culture medium that step (1) is replaced with serum-free Related Component is ɑ-MEM or DMEM/F12 culture medium, the culture
The concentration of PLA or FBS is 0 in base.
5. a species specific mescenchymal stem cell excretion body extracting method according to claim 4, which is characterized in that more
Changing the incubation time carried out after culture medium is 48-72 hours.
6. a species specific mescenchymal stem cell excretion body extracting method according to claim 1, which is characterized in that step
Suddenly the condition being centrifuged in (1) is that 300-500g is centrifuged 5-10min.
7. the mescenchymal stem cell excretion body extracting method of specificity described in -6 any one according to claim 1, feature
It is, the temperature being centrifuged in step (2)-(4) is 4 DEG C.
8. a species specific mescenchymal stem cell excretion body extracting method according to claim 1, which is characterized in that step
Suddenly the sterile PBS of 180-220ul being added in (5).
9. a species specific mescenchymal stem cell excretion body extracting method according to claim 6, which is characterized in that step
Suddenly the aperture of filter membrane is 0.1um in (5).
10. the mescenchymal stem cell excretion body extracting method of specificity described in -9 any one according to claim 1, feature
It is, each step that excretion body extracts is operated under sterile conditions.
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