CN107151653A - A kind of efficient amplification human marrow mesenchymal stem cell and the culture medium and method for keeping its dryness - Google Patents
A kind of efficient amplification human marrow mesenchymal stem cell and the culture medium and method for keeping its dryness Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
Abstract
The present invention provides a kind of efficient amplification human marrow mesenchymal stem cell and keeps the culture medium and method of its dryness, the culture medium contains at least one of Si ions and Sr ions, preferably contain Si ions and Sr ions, wherein, the concentration of Si ions is 1~10ppm, and the concentration of Sr ions is 2~50ppm.During using medium culture human marrow mesenchymal stem cell of the invention, the amplification efficiency of mesenchymal stem cells MSCs can be improved, can be that scientific research and clinical practice provide substantial amounts of mesenchymal stem cells MSCs while keeping good stem cell dryness.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of efficient amplification human marrow mesenchymal stem cell simultaneously keeps it
The culture medium and method of dryness, can significantly improve expansion of stem cells speed and good stem cell can be kept in breeding
Dryness.
Background technology
Mesenchymal stem cells MSCs (BMSCs) is as a kind of new widely used seed cell, with relatively strong propagation energy
Power and multi-lineage potential, can be under specific inductive condition to Gegenbaur's cell, fibroblast, desmacyte, fat cell
Break up with endothelial cell etc., autotransplantation can be carried out and in the absence of tissue matching and immunological rejection, controlled in cell transplantation, gene
Treat, the field such as cell therapy and organizational project is with a wide range of applications [Tissue Eng Regen Med.2016；13:
465-74]。
Mesenchymal stem cells MSCs needs a large amount of cells in applied to clinical treatment and organizational project, but is obtained from marrow
The stem cell population taken is limited, it is impossible to meet the cell concentration needed for practical application, while stem cell cultivates amplification procedure in vitro
In, easily differentiation, aging are so as to lose the dryness of multi-lineage potential, i.e. stem cell, therefore this area is at present in the urgent need to grinding
Mesenchymal stem cells MSCs rapid amplifying and the method for keeping dryness are studied carefully, the need for meeting scientific research and clinical practice.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide a kind of efficient amplification human marrow mesenchymal stem cell
(hBMSC) and the culture medium and method of its dryness are kept.
On the one hand, the present invention, which is provided, a kind of efficient amplification human marrow mesenchymal stem cell and can keep the culture of its dryness
Base, it contains at least one of Si ions and Sr ions, preferably containing Si ions and Sr ions, wherein, Si ions it is dense
Spend for 1~10ppm, the concentration of Sr ions is 2~50ppm.
During using medium culture human marrow mesenchymal stem cell of the invention, the expansion of mesenchymal stem cells MSCs can be improved
Increasing Efficiency, can be that scientific research and clinical practice provide substantial amounts of medulla mesenchyma and done while keeping good stem cell dryness
Cell.For example, the culture medium of the present invention promotes hBMSC propagation percentage to be 20%~50%.
It is preferred that the concentration of Si ions is 1.5~5ppm.
It is preferred that the concentration of Sr ions is 10.5~40ppm.
It is preferred that the culture medium is the special complete medium of human marrow mesenchymal stem cell to CaSiO3The extraction of powder
The special complete medium of liquid, human marrow mesenchymal stem cell is special complete to leaching liquor, the human marrow mesenchymal stem cell of SrO powders
Full culture medium is to SrSiO3The leaching liquor of powder or wherein any two or more mixing.
On the other hand, the present invention provides a kind of efficient amplification human marrow mesenchymal stem cell and the method for keeping its dryness,
With any one above-mentioned medium culture human marrow mesenchymal stem cell.
According to the above method, with efficient amplification human marrow mesenchymal stem cell and its dryness can be kept, can be with the short time
Obtaining largely has good xeric mesenchymal stem cells MSCs, is that scientific experiment research, clinical treatment and bone tissue engineer are fast
Fast expanding stem cells provide feasible method and foundation.
Brief description of the drawings
Fig. 1 is the Experimental Research flow chart of steps of an embodiment of the present invention.As seen from the figure, it is prepared for that there is difference first
Concentration Si, Sr and the extract culture medium of Si-Sr combinations, its rapid amplifying is promoted most by cultivating hBMSC and having filtered out
Good Si-Sr combined concentrations, and further probed into influence of this concentration to stem cell dryness；
Fig. 2 is influence of the single Si culture mediums containing various concentrations to Proliferation of Human Mesenchymal Stem Cells.As seen from the figure, single Si
Between 1/16-1/64 is diluted to, the table of comparisons 1, single Si concentration can be obviously promoted hBMSC propagation in 1.259-5.18ppm,
And single Si has extremely obvious promotion cultivation effect at 2.59ppm；
Fig. 3 is that Si concentration is 2.59ppm, and Si-Sr combinations and the correspondence single Sr of same concentrations are to people after addition various concentrations Sr
The influence of Proliferation of Bone Mesenchymal Stem Cells.As seen from the figure, single Sr concentration has in 0.32-40.39ppm to hBMSC propagation
Facilitation, after Si (2.59pm) is combined with Sr, Si (2.59ppm)-Sr (2.52-40.39ppm) can more obviously promote carefully
Born of the same parents breed, and wherein Si (2.59ppm)-Sr (20.20ppm) facilitation effect is most notable.Single Si (2.59 ppm) promotes thin simultaneously
Born of the same parents' proliferation rate is 11.10%, and single Sr (20.20ppm) promotes cell proliferation rate to be 16.89%, both stack up still less than
Actual Si (2.59ppm)-Sr (20.20ppm) combination promotes cell proliferation rate 47.48%, illustrates Si-Sr combinations for promoting
There is cooperative effect in hBMSC propagation；
Fig. 4 is that single Si (2.59ppm), single Sr (20.20ppm) and Si (2.59ppm)-Sr (20.20ppm) combinations are promoting
To the influence of stem cell dryness during stem cells hyperplasia.As can be seen, single Si (2.59ppm) and single Sr
(20.20ppm) has effect in terms of stem cell dryness is maintained, but Si (2.59ppm)-Sr (20.20ppm) combinations have pole
Its obvious effect, for four dryness related genes, single Si (2.59ppm) improves dryness rate and single Sr compared to control group
(20.20ppm), which improves both dryness rates and stacked up, is still significantly less than actual Si (2.59ppm)-Sr (20.20 ppm) combination phases
Dryness rate is improved than control group, illustrates Si-Sr combinations for maintaining hBMSC dryness to there is cooperative effect.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following embodiments, it should be appreciated that accompanying drawing and following embodiments
The present invention is merely to illustrate, is not intended to limit the present invention.
An embodiment of the present invention provide one kind can in vitro efficient amplification human marrow mesenchymal stem cell and keep its do
The culture medium of property.The culture medium contains at least one of Si ions and Sr ions.In the culture medium, the existence form of Si ions
Can be SiO4 4-.The existence form of Sr ions can be Sr2+。
In one example, the culture medium contains single Si ions, and the wherein concentration of Si ions can be 1~10ppm, preferably
For 1.259~5.18ppm, more preferably more preferably 1.5~5ppm, 2.59ppm.
In another example, the culture medium contains single Sr ions, and the wherein concentration of Sr ions can be 2~50ppm, preferably
For 2.52~40.39ppm, more preferably more preferably 10.5~40ppm, 20.20ppm.
In another example, the culture medium contains Si ions and Sr ions simultaneously, wherein the concentration of Si ions can for 1~
10ppm, preferably 1.259~5.18ppm, more preferably 1.5~5ppm, more preferably 2.59ppm, the concentration of Sr ions
Can be 2~50ppm, more preferably preferably 2.52~40.39ppm, more preferably 10.5~40ppm, 20.20ppm.
When containing Si ions and Sr ions simultaneously in culture medium, Si ions and Sr ion pairs promote hBMSC propagation and keep its dryness to deposit
In cooperative effect, i.e., its facilitation effect be more than the superposition of single Si ions and single Sr ions.Si (2.59ppm)-Sr
(20.20ppm) can be obviously promoted stem cells hyperplasia in this concentration, and collaboration keeps dry during stem cell rapid amplifying is promoted
Cell dryness.In the culture medium, if the concentration of Si ions and/or Sr ions is excessive, certain cytotoxicity is had；Such as
The concentration of fruit Si ions and/or Sr ions is too small, then is difficult to play efficient amplification human marrow mesenchymal stem cell and keeps it to do
The effect of property.
The basic components (component in addition to above-mentioned Si ions and/or Sr ions) of the culture medium can be known in the art
HBMSC culture mediums, the special complete mediums of such as hBMSC.The special complete mediums of hBMSC are purchased from commercialization, for example
Purchased from Sai Ye bio tech ltd, model HUXMA-03011-440.
The culture medium can be the ion leaching liquor combined containing single Si, Sr or Si-Sr.In one embodiment,
Using the special complete mediums of hBMSC as Extraction solvent, above-mentioned ion leaching liquor is prepared.The ion containing single Si is prepared to extract
, can be to CaSiO during liquid3Powder is extracted, and causes Ca ion concentrations in extraction stoste due to mineralising occurring in leaching process
It is very low, and Ca ion concentrations are higher in the special complete mediums of hBMSC, extraction stoste is dilute by the special complete mediums of hBMSC
Release that rear Ca ion concentrations are consistent with Ca concentration in the special complete mediums of hBMSC, therefore the influence of i.e. negligible Ca ions.
CaSiO3Can be by Ca (NO3)2·4H2O (such as purchased from traditional Chinese medicines) and NaSiO3·9H2O (such as purchased from traditional Chinese medicines) is by precipitation legal system
It is standby, through 800 DEG C~1500 DEG C calcinings.The CaSiO of gained3Purity be more than 99%.CaSiO3Ceramics can be 1 μm of -10mm
Particle or ceramic block, particle diameter are preferably 30 μm -150 μm, the CaSiO in this particle size range3It is ceramic easily to obtain, extract more convenient
And extraction stoste ion concentration it is moderate be easy to later stage stoste dilute., can be to SrO when preparing the ion leaching liquor containing single Sr
Powder (such as purchased from Aladdin) is extracted.The purity of SrO powders is preferably more than 99.7%, and particle diameter is preferably 30 μm -150
μm, the SrO powders in this particle size range are easily obtained, and extracting more convenient and extraction, stoste ion concentration is moderate is easy to later stage stoste dilute
Release., can be to SrSiO when preparing the ion leaching liquor containing Si-Sr combinations3Powder is extracted, can also be by above-mentioned CaSiO3
Leaching liquor, SrO leaching liquors, SrSiO3Any two or more mixing in leaching liquor.SrSiO3Can be by Sr (NO3)2(for example purchased from
Traditional Chinese medicines) and NaSiO3·9H2O (such as purchased from traditional Chinese medicines) is prepared by the precipitation method, through 800 DEG C~1500 DEG C calcinings.Gained
SrSiO3The purity of ceramics is more than 99%.SrSiO3Ceramics can be 1 μm of -10mm particle or ceramic block, and particle diameter is preferred
For 30 μm -150 μm, the SrSiO in this particle size range3It is ceramic easily to obtain, extract more convenient and extraction stoste ion concentration is moderate
It is easy to later stage stoste to dilute.Obtained extraction stoste can also be diluted to obtain by the special complete mediums of hBMSC
Need concentration.
In one example, the preparation method of single Si, Sr and Si-Sr combined ionic leaching liquor containing various concentrations is：According to
ISO 10993-5 standards prepare powder leaching liquor, and used medium is hBMSC special culture medias, pure CaSiO3Powder is used to make
Standby leaching liquor containing Si, pure SrO powders are used to prepare leaching liquor containing Sr, pure SrSiO3Powder is used to prepare leaching liquor containing Si-Sr, most
Extraction stoste is diluted by the special complete medium gradient proportionings of hBMSC afterwards.
In the case where not influenceing the purpose of the present invention, other components can also be contained in the culture medium.
Hereinafter, the concentration of the step of illustrating to obtain the optimal activity of Si, Sr ion and Si, Sr ion is to stem cell
The influence of dryness.
Here, by the method containing Si, Sr ion leaching liquor culture cell, having probed into Si-Sr ion populations and having promoted marrow
The optimal concentration combination of mescenchymal stem cell propagation, and find that Si-Sr ion populations can not only promote stem cell at this concentration
Rapid amplifying, moreover it is possible to keep the dryness of stem cell.The amplification in vitro stem cell methods are simple and easy to apply, inexpensive, can scale obtain
Obtaining largely has good xeric mesenchymal stem cells MSCs.
In one embodiment, as shown in figure 1, the step of obtaining the optimal activity of Si, Sr ion and Si, Sr
Influence of the concentration of ion to stem cell dryness comprises the following steps：(1) single Si, Sr and Si-Sr containing various concentrations combine from
The preparation of sub- leaching liquor；(2) it is the ion leaching liquor hBMSC containing single Si, Sr and Si-Sr combination obtained in step (1) is special
Detect thin using CCK8 methods as blank control group with complete medium culture hBMSC, and using the special complete mediums of hBMSC
Born of the same parents breed performance, and screening promotes the optimal Si-Sr combined concentrations of Proliferation of Human Mesenchymal Stem Cells；(3) by step (2)
Acquisition promotion Proliferation of Human Mesenchymal Stem Cells optimal Si-Sr combined concentrations and its corresponding same concentrations it is single
The special complete medium culture human marrow mesenchymal stem cells of Si, Sr ion leaching liquor hBMSC, and by the special complete trainings of hBMSC
Base is supported as blank control group, stem cell dryness correlation factor mRNA is detected using SYBR Green fluorescence real-time quantitative PCRs
Expression.
(1), the preparation of the leaching liquor of various concentrations Si, Sr and Si-Sr combinations：
Pass through CaSiO3, SrO and SrSiO3Powder is prepared containing single Si, single Sr and Si-Sr combination leaching liquors, with powder and
HBMSC special culture medias mass volume ratio is 200mg mL-1Addition, sealing in 37 DEG C of constant-temperature tables as extracting 24h.Then will
Extract stoste and matched by special complete medium gradient and diluted, obtain the extraction combined with various concentrations Si, Sr and Si-Sr
Liquid, and ion concentration is measured by inductive coupling plasma emission spectrograph (ICP-OES).
(2) screening of the optimal Si-Sr combined concentrations of Proliferation of Human Mesenchymal Stem Cells, is promoted：
A) it will be filled between the hBMSC complete medium culture people's marrow combined containing different Si, Sr and Si-Sr obtained in step (1)
Matter stem cell, and it regard the special complete mediums of hBMSC as blank control group.Wherein, it is dry thin for cultivating human bone marrow mesenchymal
Single Si concentration ranges are 0.162~41.44ppm in the nutrient solution of born of the same parents' propagation, and single Sr concentration ranges are 0.32~40.39
Ppm, Si-Sr combined concentration scope are Si (2.59ppm)-Sr (0.32~40.39ppm).
B) hBMSC is inoculated into 96 porocyte culture plates with the density of 800 cells/wells, each concentration sets 6 to put down
Row hole is tested, after cell attachment 24h, culture medium is changed into 100 μ L/ holes various concentrations ion leaching liquor complete medium after
Continuous culture, and liquid is changed every other day, reach after default time 1,3 and 7 days, cell proliferative energy is detected using CCK8 methods.
C) the optimal activity that single Si promotes Proliferation of Human Mesenchymal Stem Cells is filtered out first, on this basis
The Sr ions of various concentrations are added, further screening promotes the optimal Si-Sr combined concentrations of Proliferation of Human Mesenchymal Stem Cells.
The optimal Si-Sr combinations of the promotion Proliferation of Human Mesenchymal Stem Cells filtered out, Si (2.59ppm)-Sr (20.20ppm) exists
This concentration can be obviously promoted stem cells hyperplasia.
(3), influence of the optimal Si-Sr combined concentrations to stem cell dryness：
A) by the optimal Si-Sr combined concentrations of the promotion Proliferation of Human Mesenchymal Stem Cells obtained in step (2) and its correspondingly
Same concentrations the single special complete medium culture human marrow mesenchymal stem cells of Si, Sr ion hBMSC, and by hBMSC
Special complete medium is used as blank control group.
B) forth generation hBMSC is inoculated into 6 porocyte culture plates with the density of 2.0*10^5 cells/well, treats 12h
After cell attachment, culture medium change into 2ml/ holes contain optimal Si-Sr combined concentrations and its correspondence same concentrations single Si, Sr from
When cell reaches 80%-90% degree of converging after the culture medium and blank control group culture medium of son, 48h, part cell is collected simultaneously
With 1:2 ratios are passed on.
C) eighth generation is passed on always according to above method, the 4th generation, the 6th generation and the 8th generation cell are collected respectively.
D) expression of stem cell dryness correlation factor is detected using SYBR Green fluorescence real-time quantitative PCRs.Described
Promote Proliferation of Human Mesenchymal Stem Cells optimal Si-Sr combinations, Si (2.59ppm)-Sr (20.20ppm) can be in this concentration
Collaboration keeps stem cell dryness during promoting stem cell rapid amplifying.
The present invention is compounded using hBMSC complete mediums with Si, Sr ion population, have found the splendid ring of cell growth
Border, finally realizes rapid amplifying mesenchymal stem cells MSCs and ensures the purpose of its dryness.By experimental verification repeatedly, culture
Excellent.Specifically, preparing has different Si, the leaching liquor of Sr concentration, in different Si, Sr and Si-Sr combined concentrations
Lower culture hBMSC, detection cell propagation and dryness index.Experiment shows that Si, Sr is imitated to promoting stem cells hyperplasia to there is collaboration
Should, Si-Sr combinations can be obviously promoted stem cells hyperplasia in suitable concentration, and keep stem cell dryness.
Beneficial effects of the present invention：
(1) preparation is simple；(2) it is with low cost and be easy to promote；(3) higher amplification in vitro speed；(4) solve
The problem of Marrow Mesenchymal Stem Cells In Vitro easily loses multi-lineage potential.
In summary, it can in a short time be obtained in vitro using the inventive method and largely keep filling between good xeric marrow
Matter stem cell, can be used for scientific research, clinical treatment and organizational project etc..
Below by embodiments of the invention are introduced, so that the present invention is furture elucidated, substantive distinguishing features improve with significant.
It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.Unless otherwise defined or described herein,
All specialties used herein are identical with meaning known to those skilled in the art with scientific words.In addition it is any
Similar or impartial method and material all can be applied in the inventive method to described content.
In following examples, CaSiO3To analyze pure Ca (NO3)2·4H2O (being purchased from traditional Chinese medicines) and Na2SiO3·9H2O (is purchased from
Traditional Chinese medicines) it is raw material, CaSiO is prepared using chemical precipitation method3Powder.First by Na2SiO3·9H2O, Ca (NO3)2·4H2O points
It is not dissolved in deionized water, it is 0.5molL to be configured to concentration-1The aqueous solution, then by Ca (NO3)2Solution is slowly dropped into by force
The Na of strong stirring2SiO3In solution, until completion of dropping.Continue to stir 24 hours, reaction gained precipitation is filtered, deionization is used
Water and absolute ethyl alcohol are placed in 60 DEG C of thermostatic drying chambers after respectively washing 3 times and dried 48 hours.Finally by the presoma powder of gained
Calcined 2 hours at 900 DEG C, then by ground 200 mesh sieve, obtain purity more than 99%, particle diameter is less than 70 μm of CaSiO3Powder
Body.SrSiO3To analyze pure Sr (NO3)2(being purchased from traditional Chinese medicines) and Na2SiO3·9H2O (being purchased from traditional Chinese medicines) is raw material, heavy using chemistry
Shallow lake method prepares SrSiO3Powder.First by Na2SiO3·9H2O, Sr (NO3)2It is dissolved in respectively in deionized water, is configured to concentration equal
For 0.5molL-1The aqueous solution, then by Sr (NO3)2Solution is slowly dropped into intensively stirred Na2SiO3In solution, until being added dropwise
Finish.Continue to stir 24 hours, gained precipitation will be reacted and filtered, 60 are placed in after respectively being washed 3 times with deionized water and absolute ethyl alcohol
Dried 48 hours in DEG C thermostatic drying chamber.Finally the presoma powder of gained is calcined 2 hours at 900 DEG C, then by ground
200 mesh sieves, obtain purity more than 99%, particle diameter is less than 70 μm of SrSiO3Powder.SrO is purchased from Shanghai Aladdin biochemical technology stock
Part Co., Ltd, model 1314-11-10, hBMSC special culture media is purchased from Sai Ye bio tech ltd, model
HUXMA-03011-440。
Embodiment 1：
Influences of the single Si to Proliferation of Human Mesenchymal Stem Cells：
(1) CaSiO is prepared according to ISO 10993-5 standards3Leaching liquor.With powder and hBMSC special culture media mass volume ratios
200mg mL-1Powder is added in culture medium, sealing, which is placed in 37 DEG C of constant-temperature tables, extracts 24h, 4000rpm centrifugations
10min, takes supernatant and 200mg mL is obtained after 0.22 μm of bacteriological filtration membrane filtration-1CaSiO3Extract stoste.By 200 mg
mL-1CaSiO3Extraction stoste is diluted to 1/2,1/4,1/8,1/16,1/32,1/64,1/ with the special complete mediums of hBMSC
128 and 1/256, it is stand-by to be placed in 4 DEG C of refrigerators, and by inductive coupling plasma emission spectrograph (ICP- OES) measure from
Sub- concentration (being shown in Table 1)；
(2) hBMSC is inoculated into 96 porocyte culture plates with the density of 800 cells/wells, each concentration setting 6 is parallel
Hole is tested, and the control group using hBMSC complete medium cultures is used as blank control (CONT), CaSiO3Leaching liquor is used as experiment
Group.After cell attachment 24h into, culture medium is changed to the CaSiO of the gradient dilution in 100 μ l/ holes3Leaching liquor complete medium and blank
Control group culture medium continues to cultivate, and changes liquid every other day；
(3) reach the 1 of preset time, after 3 and 7 days, cell-proliferation activity is detected using CCK8 methods.
Si concentration (μ g mL in the single Si extract culture mediums of the gradient dilution of table 1-1)
Fig. 2 is shown, compared with blank control group, CaSiO3Leaching liquor complete medium can in suitable Si concentration ranges
It is obviously promoted hBMSC propagation.Dilute CaSiO3Stoste is extracted to 1/16-1/64, correspondence Si concentration is 5.18-1.295ppm energy
Promote hBMSC propagation, wherein being diluted to 1/32, correspondence Si concentration has for 2.59ppm more obviously promotes cultivation effect.
Embodiment 2：
The influence of Si-Sr combinations and the correspondence single Sr of same concentrations to Proliferation of Human Mesenchymal Stem Cells：
(1) CaSiO is prepared according to ISO 10993-5 standards3、SrSiO3With SrO leaching liquors.With powder and the special cultures of hBMSC
Base mass volume ratio 200mg mL-1Powder is added in culture medium, sealing, which is placed in 37 DEG C of constant-temperature tables, extracts 24h,
4000rpm centrifuges 10min, takes supernatant and 200mg mL are obtained after 0.22 μm of bacteriological filtration membrane filtration-1CaSiO3、 SrSiO3
Stoste is extracted with SrO.Stoste ion concentration is measured by inductive coupling plasma emission spectrograph (ICP-OES)；
(2) by CaSiO3It is 2.59ppm that extraction stoste is diluted to Si concentration with the special complete mediums of hBMSC.Pass through CaSiO3
Extract stoste and SrSiO3Stoste mixing is extracted, it is 2.59 ppm to dilute Si concentration with the special complete mediums of hBMSC, while Sr
Concentration is respectively 40.39ppm, 20.20ppm, 10.10ppm and halved successively, until Sr concentration is 0.32ppm (being shown in Table 2).Will
SrO extractions stoste is diluted to Sr concentration during Sr concentration is combined with Si-Sr with the special complete mediums of hBMSC and distinguishes identical (be shown in Table
3) 4 DEG C of refrigerators, are placed in stand-by；
(3) hBMSC is inoculated into 96 porocyte culture plates with the density of 800 cells/wells, each concentration setting 6 is parallel
Hole is tested, and using the control group of hBMSC complete medium cultures as blank control (CONT), single Sr leaching liquors are used as experiment
Group control, Si-Sr combines leaching liquor as experimental group, after after cell attachment 24h into, culture medium is changed to the Si-Sr in 100 μ L/ holes
Combine leaching liquor, single Sr leaching liquors complete medium and blank control group culture medium to continue to cultivate, and change liquid every other day；
(4) reach the 1 of preset time, after 3 and 7 days, cell-proliferation activity is detected using CCK8 methods.
Sr concentration (μ g mL in the single Sr extract culture mediums of the gradient dilution of table 2-1)
Si, Sr concentration (μ g mL in the various concentrations Si-Sr of table 3 combination extract culture mediums-1)
Fig. 3 is shown, compared with blank control group, and single Sr leaching liquors complete medium is 0.32-40.39 in Sr concentration
Ppm can promote hBMSC to breed, and the fixed Si concentration of Si-Sr combinations is 2.59ppm, and gradient adds to be found after various concentrations Sr, Si
(2.59ppm)-Sr (2.52-40.39ppm) can promote cell to breed, and its facilitation effect is higher than the single Si of correspondence and single
Sr.Wherein Si (2.59ppm)-Sr (20.20ppm) combines promotion hBMSC propagation that can be more obvious.Simultaneously in this concentration list
One Si promotes propagation percentage (11.10%) to promote to breed after percentage (16.89%) is superimposed still less than Si-Sr with single Sr
Combination promotes propagation percentage (47.48%), illustrates that Si-Sr combinations have cooperative effect in propagation is promoted.
Embodiment 3：
(1) it is experimental group, single Si to choose optimal Si (2.59ppm)-Sr (20.20ppm) combination leaching liquor complete medium
(2.59ppm) and single Sr (20.20ppm) leaching liquors complete medium are experimental comparison group, and hBMSC complete mediums are blank
Control group；
(2) forth generation hBMSC is inoculated into 6 porocyte culture plates with the density of 2.0*10^5 cells/well, treats 12h cells
After adherent, culture medium changes single Si, Sr ion that optimal Si-Sr combined concentrations and its correspondence same concentrations are contained in 2ml/ holes into
When cell reaches 80%-90% degree of converging after leaching liquor complete medium and blank control group culture medium, 48h, part is collected
Cell and with 1:2 ratios are passed on；
(3) eighth generation is passed on always according to above method, the 4th generation, the 6th generation and the 8th generation cell are collected respectively；
(4) detected using SYBR Green fluorescence real-time quantitative PCRs stem cell dryness correlation factor (Nanog, OCT4,
SOX2, TERT) expression.
Fig. 4 is shown, compared with blank control group, and single Si (2.59ppm) and single Sr (20.20ppm) leaching liquor are complete
Culture medium can stimulate hBMSC to maintain part dryness in breeding, but Si (2.59ppm)-Sr (20.20 ppm) is combined
More obvious effect is served in stem cell dryness is maintained, it maintains dryness effect to be higher than the single Si of correspondence and single
Sr Overlay, illustrates that Si (2.59ppm)-Sr (20.20ppm) combinations can stimulate stem cell rapid amplifying process in collaboration
In, further collaboration maintains stem cell dryness.
Claims (5)
1. a kind of efficient amplification human marrow mesenchymal stem cell and can keep the culture medium of its dryness, it is characterised in that contain
At least one of Si ions and Sr ions, preferably containing Si ions and Sr ions, wherein, the concentration of Si ions for 1~
The concentration of 10ppm, Sr ion is 2~50ppm.
2. culture medium according to claim 1, it is characterised in that the concentration of Si ions is 1.5~5ppm.
3. culture medium according to claim 1 or 2, it is characterised in that the concentration of Sr ions is 10.5~40ppm.
4. culture medium according to any one of claim 1 to 3, it is characterised in that the culture medium is filled between people's marrow
The special complete medium of matter stem cell is to CaSiO3The special complete medium pair of leaching liquor, human marrow mesenchymal stem cell of powder
The special complete medium of leaching liquor, human marrow mesenchymal stem cell of SrO powders is to SrSiO3The leaching liquor of powder is wherein appointed
The two or more mixing of meaning.
5. a kind of efficient amplification human marrow mesenchymal stem cell and the method for keeping its dryness, it is characterised in that use claim 1
To the medium culture human marrow mesenchymal stem cell any one of 4.
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| US20060198863A1 (en) * | 2005-03-03 | 2006-09-07 | Musculoskeletal Transplant Foundation | Ceramic composition for filling bone defects |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
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| US20060198863A1 (en) * | 2005-03-03 | 2006-09-07 | Musculoskeletal Transplant Foundation | Ceramic composition for filling bone defects |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
| CN105695407A (en) * | 2016-03-15 | 2016-06-22 | 佰通生物技术(苏州)有限公司 | Microelement composition having activating function on stem cells and application thereof |
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