CN106399249A - Culture method for promoting proliferation and differentiation of neural stem cells - Google Patents
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Abstract
The invention discloses a culture method for promoting proliferation and differentiation of neural stem cells. According to the culture method, exogenous neurotrophic factors act to the cultured neural stem cells, so that the neural stem cells are induced to have proliferation and differentiation, and then neurons are formed, wherein the exogenous neurotrophic factors belong to a polypeptide with the sequence shown as SEQ ID NO.1; the exogenous neurotrophic factors and enhanced peptide act to the cultured neural stem cells together, so that the neural stem cells are induced to have proliferation and differentiation, and then neurons are formed, wherein the enhanced peptide is an oligopeptide with the sequence shown as SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. According to the method provided by the invention, by supplementing the exogenous neurotrophic factors, the proliferation of neural stem cells and the differentiation of the neural stem cells towards the neurons can be obviously promoted, the enhanced peptide can enhance the effects of promoting proliferation and inducing differentiation of the exogenous neurotrophic factors, and the effects are obvious.
Description
Technical field
The invention belongs to biological field, be related to the culture of stem cell and in particular to a kind of promote cell proliferation of nerve cord and
The cultural method of differentiation, for promoting propagation and the differentiation to neuron direction of neural stem cell.
Background technology
Neural stem cell (Neural Stem Cells, NSCs) is one kind of stem cell, is present in brain and spinal cord
Undifferentiated cell.As other stem cell, neural stem cell also has the list point of division growth and various differentiation, possesses self
Updating ability.It can be divided into polytype neural tissue cell such as neuron, neurogliocyte.Neural thousand cells
Various functions are completed jointly by neuron and neurogliocyte, when the numerous disease in central nervous system, such as：Brain
All there is different degrees of neural cell loss in damage, cerebral hemorrhage, parkinson disease etc. or deformation is dead, and thus causes neural work(
The infringement of energy, neural stem cell can differentiate new neuron to replace impaired neuron to repair brain.
NSCs has self renewal and differentiation capability, and this makes NSCs in the treatment of the diseases such as central nervous system injury
Provide new Therapeutic Method, but the ability of endogenouss NSCs its own amplification and differentiation be extremely limited, and the propagation of NSCs and
Break up and do not fix, it is subject to the dynamic regulation of cell micro-environment.Researcher attempts by supplemented with exogenous neurotrophy
The factor, improves cell micro-environment, thus adjusting propagation and the differentiation of NSCs, but effect is barely satisfactory.
Content of the invention
It is an object of the invention to provide a kind of cultural method promoting cell proliferation of nerve cord and differentiation, by supplemented with exogenous
Property neurotrophic factor promote the propagation of neural stem cell and the differentiation to neuron direction.
Above-mentioned purpose is achieved by the following technical solution：
A kind of cultural method promoting cell proliferation of nerve cord and differentiation：Exogenous neurotrophic factor is acted on culture
Neural stem cell, induce its propagation and be divided into neuron；Wherein, described exogenous neurotrophic factor is a kind of polypeptide,
Its sequence is as shown in SEQ ID NO.1.The facilitation to cell proliferation of nerve cord and differentiation for this exogenous neurotrophic factor
Substantially.
Preferably, the concentration of described exogenous neurotrophic factor is 300-900 μ g/ml.
Preferably, described exogenous neurotrophic factor and strengthening peptide are cooperated with the neural stem cell of culture, lure
Lead its propagation and be divided into neuron；Described strengthening peptide is a kind of oligopeptide, its sequence such as SEQ ID NO.2 or SEQ ID NO.3
Or shown in SEQ ID NO.4.Research finds, sequence is as shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4
When oligopeptide acts solely on neural stem cell, do not promote cell proliferation of nerve cord and differentiation effect, but with above-mentioned external source
After property neurotrophic factor is used in combination, exogenous neurotrophic factor can be significantly improved to cell proliferation of nerve cord and differentiation
Effect.
Preferably, the concentration of strengthening peptide shown in SEQ ID NO.2 is 120-240 μ g/ml.
Preferably, the concentration of strengthening peptide shown in SEQ ID NO.3 is 150-250 μ g/ml.
Preferably, the concentration of strengthening peptide shown in SEQ ID NO.4 is 80-200 μ g/ml.
Preferably, neural stem cell isolated culture method is：Take pregnant 14d mice, cervical dislocation is placed in 75% after putting to death
Ethanol in 5min, take out gravid uterus, pre-cooling D-hank ' s rinse 2 times, take out embryo after peel off cerebral cortex, be placed in pre-cooling
In D-hank ' s liquid, eye scissorss shred and repeatedly blow and beat, and make cell suspension, aseptic 200 mesh steel meshes filter and 1500r/min from
Heart 5min, with DMEM/F12 (1:1) culture medium re-suspended cell, blows and beats again, counts, with 2 × 105Individual/mL is inoculated in improvement
DMEM/F12(1:1) in culture medium, 37 DEG C, 5%CO2With culture 2-3d under 95% air conditionses, neural stem cell composition primary
Neural ball, blows and beats neural ball with 0.25% pancreatin cooperative mechanical after continuing culture 2-3d, makes single cell suspension, with 2 × 105
Individual/mL is re-seeded into culture medium, and 4-5d was passed on for 1 generation；Improvement DMEM/F12 (1:1) culture medium is every 100mL
DMEM/F12(1:1) 200 μ L B27,200ng bFGF, 10,000 U penicillins and 10mg streptomycin are added in culture medium.
Preferably, neural stem cell authentication method is：Take 3 generation neural stem cell suspension digestion piping and druming, monolayer is inoculated into interior
Put in 12 well culture plates of the coverslip that poly-D-lysine was processed, after culture 3d, cell climbing sheet is rinsed with PBS, 4% poly
Formaldehyde fixes 20min, Nestin immunofluorescence dyeing, an anti-dilution factor 1:200, Deca DAPI contaminates core 5min, mounting, microscopy.
Preferably, the method for detecting cell proliferation of nerve cord is：3 generation neural stem cell of Isolation and culture are disappeared
Changing piping and druming is in individual cells, and monolayer is inoculated in 12 well culture plates of the cell climbing sheet that built-in poly-D-lysine was processed, and will train
Foster neural stem cell is divided into matched group and dosing group, in 37 DEG C, 5%CO2Cultivate with 95% air conditionses, add nerve trunk
Cell non-serum culture medium, culture 48h adds BrdU, the final concentration of 20ng/L of BrdU, collects cell climbing sheet after continuing culture 2h,
Carry out BrdU dyeing, an anti-dilution factor 1:200, and Deca DAPI dye core, statistics BrdU staining positive cells number and total cellular score,
Calculate BrdU positive cell ratio；Wherein, neural stem cell serum-free medium is DMEM/F12 (1:1) culture medium.
Preferably, the method for detecting neural stem cell differentiating one-tenth neuron is：3 generation nerve trunk of Isolation and culture
Cell dissociation piping and druming is in individual cells, and monolayer is inoculated into 12 well culture plates of the cell climbing sheet that built-in poly-D-lysine was processed
In, the neural stem cell of culture is divided into matched group and dosing group, in 37 DEG C, 5%CO2Cultivate with 95% air conditionses, plus
Enter neural stem cell division culture medium, after culture 5d, collect cell climbing sheet, carry out MAP2 and GFAP immunity skilful light dyeing one anti-dilute
Degree of releasing 1:200, and add DAPI dye core, statistics MAP2 or GFAP positive cell number and total cellular score, calculate MAP2 and GFAP sun
Sexual cell ratio；Wherein, neural stem cell differentiating culture medium is the DMEM/F12 (1 containing 10% hyclone:1) culture medium.
Beneficial effects of the present invention：
The method that the present invention provides passes through supplemented with exogenous neurotrophic factor, can remarkably promote the increasing of neural stem cell
Grow and the differentiation to neuron direction.
Brief description
Fig. 1：Matched group and the contrast of dosing group BrdU staining positive cells；
Fig. 2：Matched group and the contrast of dosing group MAP2 positive cell；
Fig. 3：Matched group and the contrast of dosing group GFAP positive cell；
Fig. 4：Matched group and dosing group MAP2 and the contrast of GFAP expression.
Specific embodiment
Specifically introduce technical scheme below in conjunction with the accompanying drawings.It is normal that unspecified experimental technique is this area
Rule method, the experiment material not being discussed in detail is the commercially available conventional material that those skilled in the art can obtain.
Laboratory animal：2 monthly age of SPF level kunming mice, male Mus 6, raettin 12, weight 25-30g, by Qinghai University
Experimental Animal Center provides conventinal breeding.
Experiment reagent：DMEM/F12 culture medium is purchased from Hyclone company of the U.S.；B27Serum-Frrsupplement, many
Polylysine and MTS are purchased from Sigma Co., USA；Hyclone (FBS), pancreatin are purchased from Gibco company of the U.S.；BFGF is purchased from U.S.
R＆D company of state；Anti- MAP2 monoclonal antibody, pvdf membrane and Western blotting chemical luminescence reagent kit are purchased from the U.S.
Millipore company；Anti- GFAP monoclonal antibody is purchased from Chemicon company of the U.S.；One anti-diluent, lowlenthal serum confining liquid,
PBS, the two of FITC and TRITC labelling anti-it is purchased from bio tech ltd of Zhong Shan Golden Bridge of BeiJing, China；DAPI, penicillium sp
Element and streptomycin are purchased from Chinese green skies bio tech ltd；Protein concentration detection BCA test kit is purchased from U.S. Biorad
Company；Pre- dsred protein rainbow Marker is purchased from Fermentas company of Lithuania.
Embodiment 1：Exogenous neurotrophic factor and the preparation of strengthening peptide
The synthetic method of polypeptide is highly developed, can adopt Solid phase synthesis in laboratory oneself it is also possible to directly
Entrust to the synthesis of biological outsourcing company.The limited public affairs of the polypeptide being related to equal student on commission work biological engineering (Shanghai) share in the present invention
Department's synthesis, and through ESI Mass Spectrometric Identification, correct to guarantee peptide sequence.
Fmoc method solid phase synthesis technique：Synthetic reaction is carried out to N-terminal according to from C-terminal, and Rink medium has free amino group；Often
In one step connection procedure, amino acid residue will activate, and has the HBTU of free amino group on 4 times of media in activator mixture,
HOBt, DIEA and Fmoc- aminoacid；Every time after the coupled reaction of aminoacid, all use a pyridine/acetic acid/N- Methylimidazole.
(3:2:0.5) mixture is closing the free amino group being not connected with, capping 10 minutes；Every time the coupled reaction of aminoacid it
Afterwards, before next aminoacid connects, the Fmoc- group on medium will be removed, remove Fmoc- group using containing 20% piperidines
Dimethylformamide, need 15 minutes；Finally, after all amino acid residues are linked in sequence, it is situated between from Rink with 98% trifluoroacetic acid
Cut down in matter, cutting is carried out 2 hours at room temperature.
The exogenous neurotrophic factor of the present invention (shown in SEQ ID NO.1), strengthening peptide A (shown in SEQ ID NO.2), strong
Change peptide B (shown in SEQ ID NO.3), all through Mass Spectrometric Identification, structure is accurate for strengthening PEPC (shown in SEQ ID NO.4).
Embodiment 2：The separation and Culture of neural stem cell and identification
The isolated culture method of primary neural stem cell is：Take pregnant 14d mice, cervical dislocation is placed in 75% after putting to death
5min in ethanol, takes out gravid uterus, and pre-cooling D-hank ' s rinses 2 times, peels off cerebral cortex, be placed in pre-cooling D- after taking out embryo
In hank ' s liquid, eye scissorss shred and repeatedly blow and beat, and make cell suspension, and aseptic 200 mesh steel meshes filter and 1500r/min centrifugation
5min, with DMEM/F12 (1:1) culture medium re-suspended cell, blows and beats again, counts, with 2 × 105Individual/mL is inoculated in improvement DMEM/
F12(1:1) in culture medium, 37 DEG C, 5%CO2With culture 2-3d under 95% air conditionses, neural stem cell will form primary neural
Ball, blows and beats neural ball with 0.25% pancreatin cooperative mechanical after continuing culture 2-3d, makes single cell suspension, with 2 × 105Individual/mL
It is re-seeded into culture medium, 4-5d was passed on for 1 generation；Described improvement DMEM/F12 (1:1) culture medium is every 100mL
DMEM/F12(1:1) 200 μ L B27,200ng bFGF, 10,000 U penicillins and 10mg streptomycin are added in culture medium.
The authentication method of neural stem cell is：Take 3 generation neural stem cell suspension digestion piping and druming, monolayer is inoculated into built-in poly
In 12 well culture plates of the coverslip that lysine was processed, after culture 3d, cell climbing sheet is rinsed with PBS, 4% paraformaldehyde is solid
Determine 20min, carry out Nestin immunofluorescence dyeing, an anti-dilution factor 1:200, and Deca DAPI dye core 5min, mounting, microscopy.
Through the double dyeing of Specific marker Nestin and DAPI, more than 98% cell is in that Nestin and DAPI dyes as a result
Positive.This shows, the isolated culture method of above-mentioned primary neural stem cell is feasible, can be used for the separation and Culture of neural stem cell.
Embodiment 3：The impact that exogenous neurotrophic factor is bred to NSCs
3 generation neural stem cell digestion piping and druming of Isolation and culture are in individual cells, monolayer is inoculated into built-in poly and relies
In 12 well culture plates of the acid-treated cell climbing sheet of ammonia, the neural stem cell of culture is divided into matched group and dosing group, in 37
DEG C, 5%CO2Cultivate with 95% air conditionses, add neural stem cell serum-free medium, culture 48h adds BrdU, BrdU
Final concentration of 20ng/L, collects cell climbing sheet after continuing culture 2h, carries out BrdU dyeing, an anti-dilution factor 1:200, and Deca
DAPI contaminates core, statistics BrdU staining positive cells number and total cellular score, calculates BrdU positive cell ratio；Wherein, described nerve
Stem cell serum-free culture medium is DMEM/F12 (1:1) culture medium.Contain in the neural stem cell serum-free medium of dosing group
The exogenous neurotrophic factor (shown in SEQ ID NO.1) of 600 μ g/ml.
BrdU staining positive cells ratio=BrdU staining positive cells number/total cellular score × 100%
Result：Dosing group BrdU staining positive cells ratio is significantly higher than matched group, as shown in figure 1, difference has statistics
Learn meaning (P ＜ 0.05).With matched group ratio, the rate of increase of dosing group NSCs is 180 ± 15%.
When the concentration of exogenous neurotrophic factor is in the range of 300-900 μ g/ml, all there is significant promotion propagation and make
With.
Result shows, exogenous neurotrophic factor can promote the propagation of NSCs.
Embodiment 4：The impact that exogenous neurotrophic factor breaks up to NSCs
3 generation neural stem cell digestion piping and druming of Isolation and culture are in individual cells, and monolayer is inoculated into built-in poly and relies ammonia
In 12 well culture plates of acid-treated cell climbing sheet, the neural stem cell of culture is divided into matched group and dosing group, in 37 DEG C,
5%CO2Cultivate with 95% air conditionses, add neural stem cell differentiating culture medium, collect cell climbing sheet after culture 5d, carry out
The skilful light of MAP2 and GFAP immunity dyes an anti-dilution factor 1:200, and add DAPI dye core, count MAP2 or GFAP positive cell number
And total cellular score, calculate MAP2 and GFAP positive cell ratio；Wherein, described neural stem cell differentiating culture medium is containing 10% tire
The DMEM/F12 (1 of Ox blood serum:1) culture medium.Exogenous containing 600 μ g/ml in the neural stem cell differentiating culture medium of dosing group
Neurotrophic factor (shown in SEQ ID NO.1).
MAP2 positive cell ratio=MAP2 positive cell number/total cellular score × 100%
GFAP positive cell ratio=GFAP positive cell number/total cellular score × 100%
Result：Immunofluorescence dyeing shows, after culture 5d, dosing group is compared with matched group, and MAP2 positive cell (represents god
Through unit) ratio significantly increases, difference statistically significant (P ＜ 0.05), as shown in Figure 2；And GFAP positive cell (represents colloid
Cell) ratio no difference of science of statistics (P ＞ 0.05), as shown in Figure 3.With matched group ratio, dosing group NSCs Differentiating Into Neurons
Increment rate is 150 ± 10%；With matched group ratio, dosing group NSCs is broken up inconspicuous to glial cell.
Western blotting shows, after culture 5d, compared with matched group, MAP2 expression is significantly raised for dosing group,
And GFAP expression no difference of science of statistics, as shown in Figure 4.
When the concentration of exogenous neurotrophic factor is in the range of 300-900 μ g/ml, all there is significant promotion differentiation and make
With.
Result shows, exogenous neurotrophic factor can promote NSCs Differentiating Into Neurons.
Embodiment 6：The invigoration effect of strengthening peptide A (shown in SEQ ID NO.2) exogenous neurotrophic factor
Measure strengthening peptide A (shown in SEQ ID NO.2) exogenous neurotrophy according to the method for embodiment 3 and 4 respectively
The invigoration effect of the factor, result such as following table：
Result shows, when strengthening peptide A acts solely on neural stem cell, is no obviously promoted propagation and induction of differentiation；
But strengthening peptide A can strengthen promotion propagation and the induction of differentiation of exogenous neurotrophic factor, effect is significant.
Embodiment 7：The invigoration effect of strengthening peptide B (shown in SEQ ID NO.3) exogenous neurotrophic factor
Measure strengthening peptide B (shown in SEQ ID NO.3) exogenous neurotrophy according to the method for embodiment 3 and 4 respectively
The invigoration effect of the factor, result such as following table：
Result shows, when strengthening peptide B acts solely on neural stem cell, is no obviously promoted propagation and induction of differentiation；
But strengthening peptide B can strengthen promotion propagation and the induction of differentiation of exogenous neurotrophic factor, effect is significant.
Embodiment 8：The invigoration effect of strengthening PEPC (shown in SEQ ID NO.4) exogenous neurotrophic factor
Measure strengthening PEPC (shown in SEQ ID NO.4) exogenous neurotrophy according to the method for embodiment 3 and 4 respectively
The invigoration effect of the factor, result such as following table：
Result shows, when strengthening PEPC acts solely on neural stem cell, is no obviously promoted propagation and induction of differentiation；
But strengthening PEPC can strengthen promotion propagation and the induction of differentiation of exogenous neurotrophic factor, effect is significant.
In sum, the method that the present invention provides passes through supplemented with exogenous neurotrophic factor, can remarkably promote nerve
The propagation of stem cell and the differentiation to neuron direction；Strengthening peptide A, B, C can strengthen the promotion of exogenous neurotrophic factor
Propagation and induction of differentiation, effect is significant.
The effect of above-described embodiment is only that the essentiality content of the explanation present invention, but does not limit the guarantor of the present invention with this
Shield scope.It will be understood by those within the art that, technical scheme can be modified or be equal to replace
Change, the essence without deviating from technical solution of the present invention and protection domain.
Claims (10)
1. a kind of promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：By exogenous neurotrophic factor
Act on the neural stem cell of culture, induce its propagation and be divided into neuron；Wherein, described exogenous neurotrophic factor is
A kind of polypeptide, its sequence is as shown in SEQ ID NO.1.
2. according to claim 1 promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：Described outer
The concentration of derived neurotrophic factor is 300-900 μ g/ml.
3. according to claim 1 promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：Will be described
Exogenous neurotrophic factor cooperates with the neural stem cell of culture with strengthening peptide, induces its propagation and is divided into nerve
Unit；Described strengthening peptide is a kind of oligopeptide, and its sequence is as shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4.
4. according to claim 3 promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：SEQ ID
The concentration of strengthening peptide shown in NO.2 is 120-240 μ g/ml.
5. according to claim 3 promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：SEQ ID
The concentration of strengthening peptide shown in NO.3 is 150-250 μ g/ml.
6. according to claim 3 promote cell proliferation of nerve cord and differentiation cultural method it is characterised in that：SEQ ID
The concentration of strengthening peptide shown in NO.4 is 80-200 μ g/ml.
7. according to the arbitrary described cultural method promoting cell proliferation of nerve cord and differentiation of claim 1-6 it is characterised in that
The isolated culture method of neural stem cell is：Take pregnant 14d mice, cervical dislocation is placed in 5min in 75% ethanol after putting to death,
Take out gravid uterus, pre-cooling D-hank ' s rinses 2 times, peel off cerebral cortex after taking out embryo, be placed in pre-cooling D-hank ' s liquid,
Eye scissorss shred and repeatedly blow and beat, and make cell suspension, and aseptic 200 mesh steel meshes filter and 1500r/min centrifugation 5min, use
DMEM/F12(1:1) culture medium re-suspended cell, blows and beats again, counts, with 2 × 105Individual/mL is inoculated in improvement DMEM/F12 (1:
1) in culture medium, 37 DEG C, 5%CO2With cultivate 2-3d under 95% air conditionses, neural stem cell will form primary neural ball, continue
With the neural ball of 0.25% pancreatin cooperative mechanical piping and druming after continuous culture 2-3d, make single cell suspension, with 2 × 105Individual/mL connects again
Plant in culture medium, 4-5d was passed on for 1 generation；Described improvement DMEM/F12 (1:1) culture medium is the DMEM/F12 of every 100mL
(1:1) 200 μ LB27,200ng bFGF, 10,000 U penicillins and 10mg streptomycin are added in culture medium.
8. the cultural method promoting cell proliferation of nerve cord and differentiation according to claim 7 is it is characterised in that nerve trunk
The authentication method of cell is：Take 3 generation neural stem cell suspension digestion piping and druming, monolayer is inoculated into what built-in poly-D-lysine was processed
In 12 well culture plates of coverslip, after culture 3d, cell climbing sheet is rinsed with PBS, 4% paraformaldehyde fixes 20min, carries out
Nestin immunofluorescence dyeing, an anti-dilution factor 1:200, and Deca DAPI dye core 5min, mounting, microscopy.
9. the cultural method of promotion cell proliferation of nerve cord according to claim 7 and differentiation is it is characterised in that be used for examining
Survey cell proliferation of nerve cord method be：3 generation neural stem cell digestion piping and druming of Isolation and culture are in individual cells, monolayer
It is inoculated in 12 well culture plates of the cell climbing sheet that built-in poly-D-lysine was processed, the neural stem cell of culture is divided into comparison
Group and dosing group, in 37 DEG C, 5%CO2Cultivate with 95% air conditionses, add neural stem cell serum-free medium, culture
48h adds BrdU, the final concentration of 20ng/L of BrdU, collects cell climbing sheet, carry out BrdU dyeing, an anti-dilution after continuing culture 2h
Degree 1:200, and Deca DAPI dye core, statistics BrdU staining positive cells number and total cellular score, calculate BrdU positive cell ratio；
Wherein, described neural stem cell serum-free medium is DMEM/F12 (1:1) culture medium.
10. the cultural method of promotion cell proliferation of nerve cord according to claim 7 and differentiation is it is characterised in that be used for
Detection neural stem cell differentiating become neuron method be：3 generation neural stem cell digestion piping and druming of Isolation and culture are in single
Cell, monolayer is inoculated in 12 well culture plates of the cell climbing sheet that built-in poly-D-lysine was processed, will be thin for the nerve trunk of culture
Born of the same parents are divided into matched group and dosing group, in 37 DEG C, 5%CO2Cultivate with 95% air conditionses, add neural stem cell differentiating culture
Base, collects cell climbing sheet after culture 5d, carries out the skilful light of MAP2 and GFAP immunity and dyes an anti-dilution factor 1:200, and add DAPI
Dye core, statistics MAP2 or GFAP positive cell number and total cellular score, calculate MAP2 and GFAP positive cell ratio；Wherein, described
Neural stem cell differentiating culture medium is the DMEM/F12 (1 containing 10% hyclone:1) culture medium.
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| CN109172864A (en) * | 2018-10-10 | 2019-01-11 | 上海市第人民医院 | A kind of schwann cell combines LC-PLGA conduit and its application with neural stem cell |
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