CN109370989A - A method of promoting nerve stem cell proliferation - Google Patents
A method of promoting nerve stem cell proliferation Download PDFInfo
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- CN109370989A CN109370989A CN201811354436.5A CN201811354436A CN109370989A CN 109370989 A CN109370989 A CN 109370989A CN 201811354436 A CN201811354436 A CN 201811354436A CN 109370989 A CN109370989 A CN 109370989A
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Abstract
The invention belongs to field of biotechnology, are related to the method for stem cell culture, disclose and are applied to VX-702 to prepare nerve stem cell culture medium, for promoting nerve stem cell proliferation or maintaining the stemness of neural stem cell.VX-702 can effectively improve the proliferation of neural stem cell, and promotion forms Neural Stem Cells Neural ball, can also significantly improve the expression of neural stem cell marker, such as NESTIN and SOX2.The present invention also provides a kind of methods for promoting nerve stem cell proliferation, and step includes, and 35 DEG C~39 DEG C, under the conditions of 3%~8%CO2, with the nerve stem cell culture medium containing VX-702, adhere-wall culture neural stem cell.The present invention provides nerve stem cell culture medium and promotes the method for nerve stem cell proliferation using VX-702 as additive, helps to establish stable, reliable neural stem cell in-vitro culture model.
Description
Technical field
The invention belongs to field of biotechnology, are related to the method for stem cell culture, specially promotion nerve stem cell proliferation
Method.
Background technique
Neural stem cell is to be present in mammalian central nervous system to have self-renewal capacity and Multidirectional Differentiation latent
The cell mass of energy.During brain development, neural stem cell can be divided into new neuron, astroglia and dash forward less
Spongiocyte can construct brain structure and functional unit；After brain development is mature, neural stem cell still have it is limited again
Raw ability, provides possibility for brain damage reparation.
The proliferation of neural stem cell and differentiation are always the difficult point and hot spot of research, effectively improve the proliferation of neural stem cell
Have great importance.Since neural stem cell number itself is less in body, caused body is only stimulated by environmental damage
The feedback of itself is still not enough to achieve the purpose that realize neural self-regeneration and reconstruction, and neural stem cells transplantation is deposited
Motility rate is not high, directed differentiation is difficult, easy tumor formation, is difficult to the problems such as penetrating scar tissue.So how to pass through regulation endogenous mind
Proliferation through stem cell is of great significance for obtaining neural stem cell.
The method for finding nerve stem cell proliferation promotes the original new drug of differentiation and proliferation of neural stem cells to exploitation and attacks
It gram is difficult to the nervous system injury cured and retrogression pathological changes is significant.
The reagent used at present for promoting nerve stem cell proliferation is more single, and the most commonly used is ginseng saponin(e Rg1, this reagent promotees
Effect into cell Proliferation is weaker.
Therefore, it is necessary to be improved the prior art, screening can more effectively regulate and control the additive of nerve stem cell proliferation,
And it is used for promoting nerve stem cell proliferation and maintains the stemness of neural stem cell.
Shown in VX-702 (p38 mitogen activated protein kinase inhibitor) structure such as formula (I).
It has been investigated that VX-702 can promote the proliferation of neural stem cell, and effectively maintain the stemness of neural stem cell.
Summary of the invention
For the deficiency in the prior art, the present invention is intended to provide a kind of culture medium for promoting nerve stem cell proliferation and new
Method, and VX-702 is used to promote nerve stem cell proliferation and be maintained the stemness of neural stem cell.
To achieve the above object, the present invention solve the problems, such as itself the technical solution adopted is that:
VX-702 can be used in terms of promoting nerve stem cell proliferation or maintain the stemness of neural stem cell.
When containing 1~10 μm of ol/L VX-702 in culture medium, the proliferation of neural stem cell is significantly improved.Preferably,
When content is 5~8 μm of ol/L, when especially 7 μm of ol/L, the raising of the proliferation rate of neural stem cell is especially pronounced.Also, it cultivates
It when containing VX-702 in base, can effectively facilitate to form Neural Stem Cells Neural ball, can also significantly improve and maintain nerve cord thin
The expression of born of the same parents' marker, such as NESTIN and SOX2.Thus provable, VX-702 can be used in promoting nerve stem cell proliferation side
Face or the stemness for maintaining neural stem cell.
VX-702 can be used for preparing nerve stem cell culture medium.
The present invention also provides a kind of nerve stem cell culture mediums, including basal medium and VX-702.
Preferably, in the nerve stem cell culture medium, the concentration of VX-702 is 1~10 μm of ol/L.It is furthermore preferred that VX-
702 concentration is 5~8 μm of ol/L.
Preferably, the basal medium is DMEM/F12 culture solution.
Preferably, it is raw also to contain epithelical cell growth factor, basic fibroblast for the nerve stem cell culture medium
The long factor, B27 cell culture additive and antibiotic.
It is furthermore preferred that wherein the content of epithelical cell growth factor be 15~25ng/mL, basic fibroblast growth because
The content of son is 15~25ng/mL, antibiotic be 80~120U/mL penicillin, 0.05~0.15mg/mL streptomysin and 200~
300ng/mL anphotericin.
Above-mentioned nerve stem cell culture medium can be used for culture of neural stem cells neural, or promote the proliferation of neural stem cell, or
The stemness of person's maintenance neural stem cell.
The present invention also provides a kind of methods for promoting nerve stem cell proliferation, and step includes, 35 DEG C~39 DEG C, 3%~8%
Under the conditions of CO2, with above-mentioned nerve stem cell culture medium, adhere-wall culture neural stem cell.
Preferably, incubation time is 1~12 day；If incubation time is more than 1 day, every 1~3 day replacement Culture of neural stem cells
Base.
Inoculum concentration and the ratio of culture medium are 5000~30000 cell/mL, preferably 8000~15000 cells/
mL。
A preferred embodiment of the present invention is that nerve stem cell culture medium is cultivated based on DMEM/F12 culture solution
Base, wherein the content of VOX-2 is 7 μm of ol/L, and contains mycillin (penicillin 100U/mL, streptomysin 0.1mg/mL), both sexes
Mycin 250ng/mL, epidermal growth factor 20ng/mL, basic fibroblast growth factor 2 0ng/mL and 2% (volume ratio)
B27 cell culture additive.The condition of culture of neural stem cells neural are as follows: the neural stem cell of logarithmic growth phase, according to above-mentioned
Nerve stem cell culture medium, in 37 DEG C, 5%CO2Under the conditions of carry out adhere-wall culture；Incubation time is 1~12 day, and every 1~2 day more
Change culture medium.
The proliferation of neural stem cell and differentiation are always the difficult point and hot spot of research, effectively improve the proliferation of neural stem cell
Have great importance.The present invention provides nerve stem cell culture medium and promotes nerve cord thin using VX-702 as additive
The method of born of the same parents' proliferation helps to establish stable, reliable neural stem cell in-vitro culture model, and then is neural stem cell conduct
The cell origin of cell replacement therapy or the carrier of gene therapy are for treating central lesion, degenerative disease and brain
The offers reference such as tumour can finally provide fundamental basis for the research of the molecule mechanism of nervous system development.
Detailed description of the invention
Fig. 1 is influence (culture 2 day) of the 1 various concentration VOX-702 of embodiment to nerve stem cell proliferation situation
After Fig. 2 is the VX-702 that embodiment 2 is added 7 μM, nerve stem cell proliferation rate changes with time
After Fig. 3 is 3 control group of embodiment (A) and 7 μM of VX-702 (B) is added, the phase contrast microscope of neural molecular biology
Picture
After Fig. 4 is 3 control group of embodiment and 7 μM of VX-702 is added, the size and number of neural molecular biology are distributed.
Fig. 5 be embodiment 4 in, VX-702 promote neural stem cell marker expression, figure A, B be respectively control group and
The fluorescence detection result of experimental group.
Specific embodiment
Basic culture solution (culture medium): DMEM/F12 culture solution+2%B27 (50 ×)+epidermal growth factor EGF (20ng/
ML)+basic fibroblast growth factor bFGF (20ng/mL)+blueness/streptomysin (penicillin 100U/mL, streptomysin 0.1mg/
ML)+amphotericin B (250ng/mL).
Experimental group culture solution (culture medium): the VX-702 of various concentration is added in basic culture solution.
The proliferative conditions of 1 various concentration VX-702 of embodiment intervention neural stem cell
Neural stem cell control group culture solution uses basic culture solution: DMEM/F12 culture solution+2%B27 (50 ×)+epidermis
Growth factor EGF (20ng/mL)+basic fibroblast growth factor bFGF (20ng/mL)+blueness/streptomysin (penicillin
100U/mL, streptomysin 0.1mg/mL)+amphotericin B (250ng/mL)；Experimental group culture solution adds respectively on the basis of the control group
Add 1 μM, 3 μM, 5 μM, 7 μM, 10 μM of VX-702.
Steps are as follows:
(1) it cultivates to the neural stem cell of logarithmic growth phase after counting, every hole presses 3 × 103It is a to be inoculated with neural stem cell
Into 96 orifice plates, and 5 multiple holes of every group of setting.
(2) cell is divided into blank well (containing only culture medium), control wells (containing cell, contain culture medium, without VX-702), reality
It verifies and (adds 1 μM, 3 μM, 5 μM, 7 μM, 10 μM of VX-702 respectively on the basis of the control group), be separately added into corresponding culture solution
Or pharmaceutical culture medium mixed liquor, volume 0.1mL are placed in incubator and are incubated for for 24 hours, make cell adherent growth.
(3) it replaces a subculture: culture medium in 96 orifice bores being blotted with liquid-transfering gun, takes and 96 orifice plates is tipped upside down on into high pressure
On the paper handkerchief to sterilize, culture plate bottom is gently patted, culture medium in hole is thoroughly fallen dry；It is separately added into corresponding culture solution again
Or pharmaceutical culture medium mixed liquor, volume 0.1mL, it is placed in and continues to be incubated for for 24 hours in incubator.
(4) 10 μ L CCK8 (5mg/mL) are added in each hole, are placed in after being incubated for 30min-1h in incubator, are surveyed with microplate reader
Wavelength is the OD value of 450nm.
As a result it as shown in Figure 1, when the concentration of VX-702 is 1 μM, 3 μM, 5 μM, 7 μM, 10 μM, after culture 2 days, and compares
Group is compared, and experimental group nerve stem cell proliferation degree increases, and when VX-702 concentration is 7 μM, nerve stem cell proliferation degree
Maximum shows that VX-702 can promote nerve stem cell proliferation, and optimum concentration is 7 μM.
The VX-702 that embodiment 2 detects 7 μM intervenes the proliferative conditions of neural stem cell
Neural stem cell is divided into control group and experimental group, control group culture solution uses DMEM/F12 culture solution+2%B27
(50 ×)+EGF (20ng/mL)+bFGF (20ng/mL)+blueness/streptomysin (penicillin 100U/mL, streptomysin 0.1mg/mL)+two
Property mycin B (250ng/mL), experimental group culture solution add 7 μM of VX-702 on the basis of the control group.
Steps are as follows:
(1) being in logarithmic growth phase after tally counts to neural stem cell takes 1000/hole cell kind in 96 orifice plates
In, each group repeats 5 holes, and the ordinary culture medium of 100 μ L is added in every hole.It keeps all hole cell concentrations equal, is weighed if gap is excessive
Novel species plate.
(2) it to avoid culture solution in culture plate from being cultured case drying, at the end of bed board, is added in outermost collar aperture a small amount of
PBS solution is placed in 37 DEG C, 5%CO2Incubator in be incubated for 6h wait for that cell is adherent.
(3) after cell is completely adherent, culture medium in 96 orifice bores is blotted with liquid-transfering gun, 96 orifice plates are tipped upside down on into high pressure
On the paper handkerchief to sterilize, culture plate bottom is gently patted, culture medium in hole is thoroughly fallen dry；It is general that 100 μ L are added in cellular control unit
The culture medium that 100 μ L contain 7 μM of VX-702 is added in logical culture medium, experimental group cell, is placed in incubator and is incubated for 12h.
(4) 10 μ L MTT solution (5mg/mL) are added in every hole, are uniformly mixed and put back to incubator culture 4h.
(5) supernatant is sucked out, and 100 μ L DMSO (dimethyl sulfoxide), low-speed oscillation 10min object to be crystallized is added in every hole
After completely dissolution, when the use of microplate reader Detection wavelength being 490nm each hole OD value.Detection control group uses Nostoc commune Vanch respectively again
The OD value that the culture medium containing 7 μM of VX-702 is incubated for the 2nd, 4,6,8,10 day in incubator is added in base and experimental group.
As a result as shown in Fig. 2, nerve cord is thin after 4,6,8,10 days of the VX-702 of 7 μM of addition (every 2 days replacement culture mediums)
The proliferation rate of born of the same parents is much higher than control group, this shows that 7 μM of VX-702 can promote the proliferation of neural stem cell.
The VX-702 that 37 μM of embodiment intervenes Neural Stem Cells Neural ball and is formed
According to 2 step of embodiment (1)~(3) method, control group and experimental group, experimental group 7 μM of VX- of addition are set
702 culture medium culture of neural stem cells neural, every 2 days one subcultures of replacement are cultivated 3 days.
As shown in figure 3, observing under phase contrast microscope after having cultivated 3 days, the visible neural molecular biology of experimental group gradually increases
(Fig. 3 B) greatly.
The size and number of neural molecular biology are as shown in Figure 4.Whether diameter is at 20-50 μm, 51-80 μm or 81-
The neural molecular biology of 110 μm of sizes, the quantity of experimental group (VX-702) far more than control group (Ctrl), show VX-702
(7 μM) can promote nerve stem cell proliferation.
Embodiment 4 detects the marker expression that VX-702 promotes neural stem cell
Research generally believes NESTIN and SOX2Have expression in the neural stem cell of entire manhood, thus NESTIN and
SOX2The stem cell labeling object occurred for adult neural.If VX-702 can promote the expression of above-mentioned neural stem cell marker,
Then illustrate that it has the function of maintaining neural stem cell stemness.
(1) neural stem cell is divided into control group and experimental group, control group culture solution uses DMEM/F12 culture solution+2%
B27+EGF (20ng/mL)+bFGF (20ng/mL)+blueness/streptomysin (100U/mL)+amphotericin B (250ng/mL), experimental group
Culture solution adds 7 μM of VX-702,37 DEG C, 5%CO on the basis of the control group2Under the conditions of co-culture 12 days, replacement in every 2 days is primary
Culture medium.
(2) cell climbing sheet of sterilizing is clipped in 24 orifice plates with tweezers.Two groups of cells that digestion step (1) obtains, cell
Taken after counting in 20000 cell kinds to the cell climbing sheet of 24 orifice plates, be separately added into corresponding culture medium (1mL), be placed in 37 DEG C,
5%CO2Continue culture in incubator 3~6 hours.
(3) observation cell when sprawl completely, it is in the best state when be quickly sucked out culture medium, 1mL PBS buffer solution is added, is placed in
200 turns of 5min on shaking table rinse 3 times, wash away cell fragment and extra culture medium.
(4) net PBS buffer solution is blotted, every hole is added 200 μ L immunostaining fixers (4% paraformaldehyde), rocks up and down
Uniformly, 10min is stood.
(5) immunostaining fixer is abandoned, PBS buffer solution is added, is placed in 200 turns of 5min on shaking table, is rinsed 3 times.
(6) net PBS buffer solution is blotted after, and 200 μ L 0.5%Triton are added (with PBS by 100% Triton in every hole
It is diluted to 0.5%, dilution ratio 1:200), it rocks up and down uniformly, stands 20min.
(7) 0.5%Triton is abandoned, 1mL PBS buffer solution is added, is placed in 200 turns of 5min on shaking table, is rinsed 3 times.
(8) net PBS buffer solution is blotted, every hole is added 200 μ L 5%BSA confining liquids and (weighs BSA solid and be dissolved in TBST buffering
In liquid), stand 1h.
(9) 5%BSA confining liquid is recycled, 200 μ L SOX are added in every hole2Primary antibody and NESTIN primary antibody rock uniformly up and down,
It is put in 4 DEG C (or being at least incubated for 6-8h) overnight.
(10) second days recycling primary antibodies are added PBST buffer (being prepared by PBS+ polysorbas20), are placed on shaking table 200 turns
5min, rinsing is three times.
(11) secondary antibody that 200 μ L carry fluorescence is added in every hole, and masking foil wraps up 24 orifice plates, is rocked uniformly up and down, is put in and keeps away
90-105min is incubated at room temperature at light.
(12) secondary antibody is abandoned, 1mL PBST buffer is added, is placed in 200 turns of 5min on shaking table, rinsing is three times.
(13) net PBST is blotted, the DAPI dyeing liquor that 200 μ L are used for nuclear targeting is added in every hole, it rocks up and down uniformly,
Stand 4min.
(14) DAPI dyeing liquor is abandoned, 1mL TBST buffer is added, is placed in 200 turns of 5min on shaking table, rinsing is three times.
(15) glass slide is placed on the masking foil torn, fluorescence extract liquor is dripped on glass slide, and carry out cell kind
Creep plate is taken out with hook and tweezers, has the one side of cell to contact with fluorescence extract liquor and (climb by the label of class by TBST wash clean
Piece tips upside down on glass slide), outermost masking foil is wrapped immediately, and is marked in masking foil outer layer.
The fluorescence of DAPI, SOX2 and NESTIN of control group and experimental group are observed in detection respectively under fluorescence microscope, with
And three kinds of fluorescence (being indicated with MERGE) are detected simultaneously, as a result such as Fig. 5.
As shown in figure 5, compared after (7 μM) of VX-702 processing 12 days (Fig. 5 B, VX-702) with control group (Fig. 5 A, Ctrl),
The marker NESTIN and SOX of experimental group neural stem cell2Fluorescence intensity is higher, and the DAPI fluorescence intensity of two groups of cells connects
Closely, show that VX-702 (7 μM) can effectively maintain the stemness of neural stem cell.
Claims (10)
- Application of the 1.VX-702 in terms of promoting nerve stem cell proliferation or in terms of the stemness of maintenance neural stem cell.
- 2.VX-702 the application in terms of preparing nerve stem cell culture medium.
- 3. a kind of nerve stem cell culture medium, which is characterized in that including basal medium and VX-702.
- 4. nerve stem cell culture medium as claimed in claim 3, which is characterized in that wherein the concentration of VX-702 is 1~10 μm of ol/ L。
- 5. nerve stem cell culture medium as claimed in claim 3, which is characterized in that the basal medium is DMEM/F12 training Nutrient solution.
- 6. nerve stem cell culture medium described in claim 3 or 5, which is characterized in that also contain epithelical cell growth factor, alkali Property fibroblast growth factor, B27 cell culture additive and antibiotic.
- 7. nerve stem cell culture medium as claimed in claim 6, which is characterized in that the content of epithelical cell growth factor be 15~ 25ng/mL, the content of basic fibroblast growth factor are 15~25ng/mL, antibiotic be 80~120U/mL penicillin, 0.05~0.15mg/mL streptomysin and 200~300ng/mL anphotericin.
- 8. a kind of method for promoting nerve stem cell proliferation, which is characterized in that 35 DEG C~39 DEG C, 3%~8%CO2Under the conditions of, it uses The described in any item nerve stem cell culture mediums of claim 3~7, adhere-wall culture neural stem cell.
- 9. promoting the method for nerve stem cell proliferation described in claim 8, which is characterized in that incubation time is 1~12 day；Culture When time is more than 1 day, every 1~3 day replacement nerve stem cell culture medium.
- 10. any one of claim 4~8 nerve stem cell culture medium is used for culture of neural stem cells neural, or promotes nerve The proliferation of stem cell, or maintain the stemness of neural stem cell.
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Cited By (1)
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| CN109652374A (en) * | 2019-02-27 | 2019-04-19 | 赛璟生物医药科技(上海)有限公司 | It is a kind of for maintaining the serum free medium of neural stem cell in vitro culture |
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| WO2016141084A1 (en) * | 2015-03-03 | 2016-09-09 | The Board Of Trustees Of The Leland Stanford Junior University | Producing mesodermal cell types and methods of using the same |
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