CN106566810A - Composition, inducer containing composition and induction method - Google Patents
Composition, inducer containing composition and induction method Download PDFInfo
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- CN106566810A CN106566810A CN201610910757.3A CN201610910757A CN106566810A CN 106566810 A CN106566810 A CN 106566810A CN 201610910757 A CN201610910757 A CN 201610910757A CN 106566810 A CN106566810 A CN 106566810A
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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
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
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Abstract
The invention relates to the field of cells, especially to a composition, an inducer containing the composition and an induction method. The composition comprises B27, a basic fibroblast growth factor, an epidermal growth factor and a nerve growth factor. According to the induction method, the B27, the basic fibroblast growth factor (bFGF), the epidermal growth factor (EGF) and the nerve growth factor (NGF) are mainly utilized to induce gingiva mesenchymal stem cell differentiation into neuron-like cells which have a typical morphology of nerve cells and express neuronal marker nestin, neuronspecific enolase and neurogliocyte marker glial fibrillary acidic protein. It shows that the gingiva mesenchymal stem cells retain the capability of transdifferentiation of germinal layers into non-mesenchymal cells, can transdifferentiate germinal layers into neuron-like cells and are expected to become seed cells for nerve cell replacement therapy.
Description
Technical field
The present invention relates to cell field, more particularly to a kind of compositionss, the induction preparation containing said composition and induction side
Method.
Background technology
The recovery of function of nervous system is extremely difficult after central nervous system injury, and main cause is the Regenerated energy of intracerebral neuron
Power extreme difference.Research at present has shown that intracerebral although with the presence of neural stem cell, and can be divided into neuron and neurogliocyte, but
It is that quantity is also considerably less, therefore uses it for neuronal damage reparation also right and wrong because the position that neural stem cell is present is limited to very much
Often difficult.As the research of stem cell field deepens continuously, finding the stem cell at other positions can also be divided into nerve carefully
Born of the same parents.By embryonic neural or Umbilical Cord Blood Transplant to the Mus intracerebral being damaged, can moderately improve its function of nervous system, but
Because the factors such as ethics, immunologic rejection limit the extensive application clinically of these stem cell.Medulla mesenchyma is dry thin
Born of the same parents can obtain and can in vitro stablize amplification autologous, and can induce differentiation into neurocyte in vitro, be expected to become nerveous system
The seed cell of system disease transplantation treatment.But induce the neural-like cells for differentiating effectively can't stably express at present
Whether neurocyte characteristic, such neural-like cells possess the function of real neuron, however it remains dispute, and from normal human
Interior bone marrow extraction, also can cause a certain degree of actual bodily harm to donor.
Tooth in the research in odontology field, in the first-class adult jaw covering weave of dental pulp, periodontal membrane, jawbone, tip of a root cream
Property mescenchymal stem cell in source has powerful tissue regeneration ability, and the good of defect repair regeneration is presented in a series of researchs
Application prospect.Such as periodontal ligament stem cell shows the adjustment effect to immunologic cellular activity.Screen of the gingiva as periodontal tissue
Barrier, it is very sensitive to fuselage state, oral environment change, many disease phenotypes such as hypertrophy are easily produced, and also gingiva has in itself
Powerful healing ability, there may be in these phenomenons hint gingiva tissues tissue homeostasis and injury repairing are had it is important
The undifferentiated mescenchymal stem cell of effect.
Gingiva belongs to periodontal tissue, is attached to the mucosa screen of periodontal separation lower section periodontal tissue and space outerpace
Barrier.Gingiva includes epithelium and lamina propria two parts, from development source, epithelium from primitive oral cavity epithelium, on oral mucosa
Skin-deep continuity；Lamina propria is then developed from early stage neural crest cell and migrates the mesenchyme to be formed, epithelium and the continuous phase interaction of mesenchyme
With ultimately forming gingiva tissue.One typical feature of gingiva tissue is that have stronger wound healing ability, shows as cutting
Except, damage after can heal without scar, recovery gingival contour.Gingiva mescenchymal stem cell (gingival mesenchymal stem
Cells, GMSCs) it is the main mesenchymal cell for constituting gingival connective tissue, from mesoblastic fibroblast, not only
Ability with active self renewal, and also the function with synthesis and the extracellular matrixs such as collagen of degrading：I types and III type
Collagen, fibronectin splicing variants etc..
Gingiva mescenchymal stem cell draw materials conveniently, cell content is big in gingiva tissue, value-added speed is fast, external using different
Derivant can be neural-like cells by gingiva mesenchyma stem cell differentiation induction, disclose its and have under suitable environmental condition
There is the potential for being divided into neurocyte.This functional characteristic of gingiva mescenchymal stem cell greatly attracted from preclinical medicine and
Clinical medical researcher, to the researchers of basic science a kind of pattern of research and development biology is provided, and is filled between gingiva
Reparation, replacement and the regeneration capacity that matter stem cell has also provides the chance of a development new therapy for clinical medicine.
The content of the invention
In view of this, the present invention provides a kind of compositionss, the induction preparation containing said composition and abductive approach.The present invention
Purpose be the abductive approach for providing a kind of gingiva Derived from Mesenchymal Stem Cells for neuron cell, it is mainly used
B27, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF) induction gingiva
Derived from Mesenchymal Stem Cells is neuron cell, is not only provided with the representative configuration of neurocyte, and expresses neuronal marker
Thing nestin (Nestin), neuronspecific enolase (neuron specific enolase, NSE), neuroglia
Cell plastid mark glial fibrillary acidic protein (glial fibrilamentacidic protein, GFAP)；Show between gingiva
Mesenchymal stem cells are remained across the ability that differentiation of germinal layers is non-mesenchymal cell, can be neuron cell across differentiation of germinal layers, are had
Prestige becomes the seed cell of neurocyte replacement therapy.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of compositionss, including B27, basic fibroblast growth factor, epidermal growth factor and
Nerve growth factor.
Present invention also offers during described compositionss are neuron cell in induction gingiva Derived from Mesenchymal Stem Cells
Effect.
Present invention also offers a kind of induction preparation, including the compositionss that the present invention is provided.
In some specific embodiments of the present invention, basic fibroblast growth factor described in the induction preparation
Final concentration of 15～25ng/mL.
In some specific embodiments of the present invention, epidermal growth factor is final concentration of described in the induction preparation
5～15ng/mL.
In some specific embodiments of the present invention, nerve growth factor is final concentration of described in the induction preparation
20～30ng/mL.
In some specific embodiments of the present invention, the volumetric concentration of B27 described in the induction preparation is 2%.
In some specific embodiments of the present invention, the induction preparation also includes cell culture medium.
In some specific embodiments of the present invention, serum is also included in the induction preparation.The serum can be
Hyclone.
The invention provides a kind of gingiva Derived from Mesenchymal Stem Cells is the abductive approach of neuron cell, take between gingiva
The induction preparation that mesenchymal stem cells are provided with the present invention is cultivated after mixing.
In some specific embodiments of the present invention, the preparation method of gingiva mescenchymal stem cell comprises the steps：
1st, the primary separation and Culture of GMSCs：
1) raw material sources：Taking gingiva that clinically orthodontic uprighting art cuts or impaction exodontia needs and cuts off and surround perseverance
Periodontal gingiva tissue.It is required that patient does not have gingival hyperplasia, inflammation and using the medicine for causing gingival hyperplasia excessively.The tooth for cutting
In quick PBS of the immersion containing 3 times of dual anti-4 DEG C pre-coolings of gum tissue.
Wherein, 3 times of dual anti-concentration are 300u/mL penicillins, 300u/mL streptomycins.
2) rinse：Rinsed 3 times with 3 times of dual anti-PBS are contained, then gingiva is placed in mesenchymal stem cell serum-free culture medium
Immersion 5min；
Wherein, 3 times of dual anti-concentration are 300u/mL penicillins, 300u/mL streptomycins.
Wherein, 100u/mL penicillins, 100u/mL streptomycins are contained in mesenchymal stem cell serum-free culture medium.
3) digest for the first time：Gingiva tissue is cut into into 1cm3Size, adds appropriate NTx enzyme-Dispase enzymes mixing to disappear
Change liquid, put 37 DEG C, in 200R constant-temperature tables 45min is digested.After digestion terminates, 1000r/min centrifugation 5min abandon supernatant.Add
Appropriate PBS is resuspended, and 100um cell strainers filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons supernatant.
Wherein, the ratio of NTx enzyme-Dispase enzymes is 1:1, collagenase concentration be collagenase 3g/L, Dispase enzymes
Concentration is Dispase enzyme 4g/L.
Wherein, digestive environments are 37 DEG C, in 200R constant-temperature tables, and digestion time is 45min；
Wherein, cell strainer aperture used is 100um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
4) digest for second：Step 3) in filter under get off it is indigested tissue add isopyknic 0.125% pancreas
Protease, puts 37 DEG C, continues to digest 15min in 200R constant-temperature tables.After digestion terminates, 1000r/min centrifugation 5min are abandoned
Clearly.Add appropriate PBS resuspended, 70um cell strainers filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration are abandoned
Supernatant.
Wherein, the ratio of trypsin and gingiva tissue is volume ratio 1:1, tryptic concentration is 0.125%；
Wherein, cell strainer aperture used is 70um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
5) it is inoculated with：Step 3) and step 4) in the cell collected with filling between EGF containing 10ng/mL (epidermal growth factor)
Matter stem cell serum-free culture medium re-suspended cell, is inoculated in six orifice plates, 5%CO237 DEG C of cultures of incubator.
Wherein, basal medium is mesenchymal stem cell serum-free culture medium, can market purchase or self-control.
Wherein, the concentration of EGF (epidermal growth factor) is 10ng/mL in complete medium；
6) purification：Treat that cell covers with 80-90%, cell is collected, with magnetic bead sorting method purifying cells.By collect cell with
4 DEG C of incubation 1h of STRO-1 monoclonal antibodies, with 30min4 DEG C of incubation of magnetic bead, under magnetic force devices effect STRO-1 are filtered out+GMSCs, connects
Plant in six new orifice plates, 5%CO237 DEG C of cultures of incubator.
2nd, GMSCs surface markers identification：
Treat that cell covers with 80-90%, collect cell (about 1 × 106It is individual), add 3g/L Triton immersion 20min, PBS to wash
3 times, 10% lowlenthal serum room temperature closing 2h, difference Deca 1:CD146, CD105, CD90, CD34, CD45, HLA- of 200 dilutions
The each 50 μ L of DR antibody, while the simple μ L of PBS 50 of matched group Deca, 4 DEG C of process 16h.Next day room temperature places 30min, PBS rinsings 3
Time, each group difference Deca 1:The anti-igg of FITC labellings two of 500 dilutions, room temperature lucifuge incubation 1h, PBS is rinsed 2 times, 10% Fu Er
Malin fixes cell, and flow cytometer determines the positive rate of antigen.Testing result shows, clone cell surface Antigens CD14 6,
CD105, CD90 positive expression, and CD34, CD45, HLA-DR radiolucent table reaches, and illustrates that the GMSCs obtained in present invention category is derived from
Mesoblastic mescenchymal stem cell.
3rd, GMSCs is passed on：
Treat that cell covers with 80～90%, inhaled with suction pipe and abandon old culture fluid, add appropriate 0.25% trypsin, digest 1-3
Minute, Microscopic observation cellular contraction becomes bowlder, adds appropriate mesenchymal stem cell serum-free culture medium to terminate digestion immediately, collects
Cell, 1000r/min centrifugation 5min, abandons supernatant.Add the appropriate mesenchymal stem cell serum-free culture containing 10ng/mL EGF
Base, carries out cell counting, by 5 × 104Cells/mL density is inoculated in culture dish carries out Secondary Culture, 5%CO2Incubator 37
DEG C culture, liquid was changed every 2-3 days once.
Observation of cell form during supporting, as a result as shown in Figure 2.Cell inoculation about 4h after start it is adherent, 3～4d enter it is right
Number trophophase, 6～8d enters plateau.Vitro growth rates are steady, in monolayer adherence growth.Most cells are in spindle shape,
Form is irregular.
In some specific embodiments of the present invention, gingiva Derived from Mesenchymal Stem Cells is the induction of neuron cell
Method is specially：The 3rd generation GMSCs is chosen, by 1 × 104Cells/mL density is inoculated in and is placed with the aseptic of poly-D-lysine process
In six orifice plates of coverslip, DMEM/F12 culture medium of the 2ml containing 10%FBS is added per hole.Luring for present invention offer is provided after 24h
Preparation is led, in 37 DEG C, 5%CO2, saturated humidity quiescent culture, the culture fluid more renewed every 2～3 days.
The invention provides a kind of abductive approach of gingiva Derived from Mesenchymal Stem Cells for neuron cell, it is mainly
Using B27, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF) induction
Gingiva Derived from Mesenchymal Stem Cells is neuron cell, is not only provided with the representative configuration of neurocyte, and expresses neuron
Mark nestin (Nestin), neuronspecific enolase (neuron specific enolase, NSE), god
Jing glial cell mark glial fibrillary acidic protein (glial fibrilamentacidic protein, GFAP)；Show tooth
Gum mescenchymal stem cell is remained across the ability that differentiation of germinal layers is non-mesenchymal cell, can be that neuron is thin across differentiation of germinal layers
Born of the same parents, are expected to become the seed cell of neurocyte replacement therapy.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 shows GMSCs cell surface marker flow cytometer detection result figures；
Fig. 2 shows GMSCs aspect graphs；
Fig. 3 shows that each group induction differentiation rate compares.
Specific embodiment
The invention discloses a kind of compositionss, the induction preparation containing said composition and abductive approach, people in the art
Member can use for reference present disclosure, be suitably modified technological parameter realization.Specifically, all similar replacements and change
Apparent to those skilled in the art, they are considered as being included in the present invention.The method of the present invention and application
It is described by preferred embodiment, related personnel substantially can be right in without departing from present invention, spirit and scope
Method described herein and application are modified or suitably the technology of the present invention is realized and applied to change with combining.
The invention provides a kind of GMSCs is divided into the abductive approach of neuron cell, it mainly uses B27, alkali
Property fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF) induction GMSCs be divided into
Neuron cell, is not only provided with the representative configuration of neurocyte, and expresses neuronal marker nestin
(Nestin), neuronspecific enolase (neuron specific enolase, NSE), neurogliocyte mark
Glial fibrillary acidic protein (glial fibrilamentacidic protein, GFAP)；Show that GMSCs is remained across germinal layer
The ability of non-mesenchymal cell is divided into, can be neuron cell across differentiation of germinal layers, be expected to become neurocyte replacement therapy
Seed cell.
GMSCs is autologous draw materials conveniently, be easy to amplification in vitro, can be used for autotransplantation, it is anti-so as to avoid immunologic rejection
Should.
The present invention provides one kind and acts on GMSCs by various inducible factor use in conjunction, can effectively induce GMSCs Godwards
The like cell differentiation of Jing units.Show that GMSCs is remained across the ability that differentiation of germinal layers is non-mesenchymal cell, can be god across differentiation of germinal layers
Jing units like cell, is expected to become the seed cell of neurocyte replacement therapy.
Raw materials used and reagent is equal in compositionss, the induction preparation containing said composition and abductive approach that the present invention is provided
Can be buied by market.
With reference to embodiment, the present invention is expanded on further：
The primary separation and Culture of the GMSCs of embodiment 1
4) raw material sources：Taking gingiva that clinically orthodontic uprighting art cuts or impaction exodontia needs and cuts off and surround perseverance
Periodontal gingiva tissue.It is required that patient does not have gingival hyperplasia, inflammation and using the medicine for causing gingival hyperplasia excessively.The tooth for cutting
In quick PBS of the immersion containing 3 times of dual anti-4 DEG C pre-coolings of gum tissue.
Wherein, 3 times of dual anti-concentration are 300u/mL penicillins, 300u/mL streptomycins.
5) rinse：Rinsed 3 times with 3 times of dual anti-PBS are contained, then gingiva is placed in mesenchymal stem cell serum-free culture medium
Immersion 5min；
Wherein, 3 times of dual anti-concentration are 300u/mL penicillins, 300u/mL streptomycins.
Wherein, 100u/mL penicillins, 100u/mL streptomycins are contained in mesenchymal stem cell serum-free culture medium.
6) digest for the first time：Gingiva tissue is cut into into 1cm3Size, adds appropriate NTx enzyme-Dispase enzymes mixing to disappear
Change liquid, put 37 DEG C, in 200R constant-temperature tables 45min is digested.After digestion terminates, 1000r/min centrifugation 5min abandon supernatant.Add
Appropriate PBS is resuspended, and 100um cell strainers filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons supernatant.
Wherein, the ratio of NTx enzyme-Dispase enzymes is 1:1, collagenase concentration is collagenase 3g/L Dispase enzymes
Concentration is Dispase enzyme 4g/L.
Wherein, digestive environments are 37 DEG C, in 200R constant-temperature tables, and digestion time is 45min；
Wherein, cell strainer aperture used is 100um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
4) digest for second：Step 3) in filter under get off it is indigested tissue add isopyknic 0.125% pancreas
Protease, puts 37 DEG C, continues to digest 15min in 200R constant-temperature tables.After digestion terminates, 1000r/min centrifugation 5min are abandoned
Clearly.Add appropriate PBS resuspended, 70um cell strainers filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration are abandoned
Supernatant.
Wherein, the ratio of trypsin and gingiva tissue is volume ratio 1:1, tryptic concentration is 0.125%；
Wherein, cell strainer aperture used is 70um；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
5) it is inoculated with：Step 3) and step 4) middle mesenchyme of the cell collected containing 10ng/mLEGF (epidermal growth factor)
Stem cell serum-free culture medium re-suspended cell, is inoculated in six orifice plates, the 37 DEG C of cultures of 5%CO2 incubators.
Wherein, basal medium is mesenchymal stem cell serum-free culture medium, can market purchase or self-control.
Wherein, the concentration of EGF (epidermal growth factor) is 10ng/mL in complete medium；
6) purification：Treat that cell covers with 80-90%, cell is collected, with magnetic bead sorting method purifying cells.By collect cell with
4 DEG C of incubation 1h of STRO-1 monoclonal antibodies, with 30min4 DEG C of incubation of magnetic bead, under magnetic force devices effect STRO-1 are filtered out+GMSCs, connects
Plant in six new orifice plates, 5%CO237 DEG C of cultures of incubator.
GMSCs surface markers are identified
Treat that cell covers with 80-90%, collect cell (about 1 × 106It is individual), add 3g/L Triton immersion 20min, PBS to wash
3 times, 10% lowlenthal serum room temperature closing 2h, difference Deca 1:CD146, CD105, CD90, CD34, CD45, HLA- of 200 dilutions
The each 50 μ L of DR antibody, while the simple μ L of PBS 50 of matched group Deca, 4 DEG C of process 16h.Next day room temperature places 30min, PBS rinsings 3
Time, each group difference Deca 1:The anti-igg of FITC labellings two of 500 dilutions, room temperature lucifuge incubation 1h, PBS is rinsed 2 times, 10% Fu Er
Malin fixes cell, and flow cytometer determines the positive rate of antigen.Testing result shows, clone cell surface Antigens CD14 6,
CD105, CD90 positive expression, and CD34, CD45, HLA-DR radiolucent table reaches, and illustrates that the GMSCs obtained in present invention category is derived from
Mesoblastic mescenchymal stem cell.
The GMSCs cell surface marker expression rate results of table 1
| Cell phenotype | CD146 | CD105 | CD90 | HLA-DR | CD34 | CD45 |
| Positive expression rate (%) | 100.00 | 99.90 | 96.70 | 0.30 | 0.00 | 0.10 |
GMSCs is passed on
Treat that cell covers with 80～90%, inhaled with suction pipe and abandon old culture fluid, add appropriate 0.25% trypsin, digestion 1～3
Minute, Microscopic observation cellular contraction becomes bowlder, adds appropriate mesenchymal stem cell serum-free culture medium to terminate digestion immediately, collects
Cell, 1000r/min centrifugation 5min, abandons supernatant.The appropriate mesenchymal stem cell serum-free culture medium containing 10ng/mLEGF is added,
Cell counting is carried out, by 5 × 104Cells/mL density is inoculated in culture dish carries out Secondary Culture, 5%CO237 DEG C of trainings of incubator
Support, liquid was changed once every 2～3 days.
Observation of cell form in incubation, as a result as shown in Figure 2.Start adherent, 3～4d entrance after cell inoculation about 4h
Exponential phase, 6～8d enters plateau.Vitro growth rates are steady, in monolayer adherence growth.Most cells are in long shuttle
Shape, form is irregular.
The GMSCs of embodiment 2 breaks up
In order to verify the effect of induction of the present invention, while arranging 4 groups of matched groups and 3 groups of experimental grouies, chose for the 3rd generation
GMSCs, by 1 × 104Cells/mL density is inoculated in and is placed with six orifice plates of the sterile cover slips of poly-D-lysine process, per hole
Add DMEM/F12 culture medium of the 2mL containing 10%FBS.1～matched group of matched group 4 and 1～experimental group of experimental group are changed after 24h
The corresponding culture fluid of each group in 3, in 37 DEG C, 5%CO2, saturated humidity quiescent culture, the culture fluid more renewed every 2～3 days.
Matched group 1 is negative control group, is cellar culture group, and the culture fluid for using is：Containing volume fraction 10%FBS
DMEM/F12 culture fluid.
Matched group 2 is positive controls, and the induction liquid for using is：The DMEM/ of 10%FBS, 2%B27,10ng/mL EGF
F12 culture fluid.
Matched group 3 is positive controls, and the induction liquid for using is：The DMEM/ of 10%FBS, 2%B27,20ng/mL bFGF
F12 culture fluid.
Matched group 4 is positive controls, and the induction liquid for using is：The DMEM/ of 10%FBS, 2%B27,25ng/mL NGF
F12 culture fluid.
Induction liquid used in experimental group 1 is：10%FBS, 2%B27,5ng/mL EGF, 15ng/mL bFGF, 20ng/
The DMEM/F12 culture fluid of mL NGF.
Induction liquid used in experimental group 2 is：10%FBS, 2%B27,10ng/mL EGF, 20ng/mL bFGF, 25ng/
The DMEM/F12 culture fluid of mL NGF.
Induction liquid used in experimental group 3 is：10%FBS, 2%B27,15ng/mL EGF, 25ng/mL bFGF, 30ng/
The DMEM/F12 culture fluid of mL NGF.
The formula of each group culture fluid such as table 2：
The each group induction formula of liquid of table 2
| Group | Basal medium | FBS | B27 | EGF | bFGF | NGF |
| Experimental group 1 | DMEM/F12 | 10% | 2% | 5ng/mL | 15ng/mL | 20ng/mL |
| Experimental group 2 | DMEM/F12 | 10% | 2% | 10ng/mL | 20ng/mL | 25ng/mL |
| Experimental group 3 | DMEM/F12 | 10% | 2% | 15ng/mL | 25ng/mL | 30ng/mL |
| Matched group 1 | DMEM/F12 | 10% | 2% | -- | -- | -- |
| Matched group 2 | DMEM/F12 | 10% | 2% | 10ng/mL | -- | -- |
| Matched group 3 | DMEM/F12 | 10% | 2% | -- | 20ng/mL | -- |
| Matched group 4 | DMEM/F12 | 10% | 2% | -- | -- | 25ng/mL |
GMSCs breaks up morphologic observation：
It is visible part cell attachment and have short and small projection to stretch out after 12 hours, the adherent life of visible most cells after 3 days
Long, irregularly, projection constantly increases thick and elongation to form, form differs, the sheet of many projections of dispersion is divided into after 1 week starlike thin
Born of the same parents and neuron cell, projection is interleaved with each other and reticulates.
SABC:
Culture 7 days after take out cell climbing sheet, according to the description of immunohistochemical staining test kit carry out Nestin, NSE,
The immunohistochemical staining of GFAP, DAB colour developings.
Culture fluid in the well culture plate of coverslip 6 will be placed with to suction out, PBS rinsing cell tile 5 minutes is repeated 3 times.
15 minutes are fixed with 4% paraformaldehyde, is dried after PBS rinsing, neutral gum is adhered on microscope slide, 4 DEG C of refrigerator mistakes
Night.The hydrogen peroxide of Deca 3% is incubated 15 minutes, eliminates the activity of endogenous peroxydase, and PBS is rinsed 5 minutes, weight
It is multiple 3 times.Respectively appropriate one anti-(Nestin, NSE, the GFAP) of Deca, is incubated 1 hour in 37 DEG C of wet box, and 4 DEG C overnight.Next day takes out
Afterwards PBS is rinsed 5 minutes, and Deca two resists, and incubation 40 minutes in 37 DEG C of wet box, PBS is rinsed 5 minutes, repeats 3
Secondary, the freshly prepared DAB solution of Deca develops the color 5-10 minutes, in light Microscopic observation, with tap water color development stopping is rinsed.Will
After drying slice, thin piece, haematoxylin is redyed 1 minute, and indigo plant is returned in differentiation, and gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting.
To each group gained cell Jing after SABC detection, formula is pressed to the cell of Nestin, NSE, GFAP dyeing：
Induction differentiation rate=staining positive cells number/total cellular score × 100%
Counted, the differentiation rate for calculating each group is compared.Per group samples 3 times, statistical data after detecting respectively.System
Meter result such as table 3.
(* represents p to each group cell differentiation rate of table 3<0.05, * * represents p<0.01)
Table 3, Fig. 3 results show that cellular control unit Nestin, NSE, GFAP are showed no coloring, and differentiation rate is zero.Experimental group
Cell Nestin, NSE, GFAP coloring is most deep in 2, positive expression rate extremely significantly (p<0.01) higher than other each groups, the He of experimental group 1
Cell Nestin, NSE, GFAP coloring is relatively deep in experimental group 3, positive expression rate significantly (p<0.05) higher than each matched group.Experiment
(the p of cell differentiation rate 63.15 ± 0.85% in group 2<0.01), illustrate that division culture medium used of the invention can be induced effectively
GMSCs Differentiation into Neuron-like Cells.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of compositionss, it is characterised in that including B27, basic fibroblast growth factor, epidermal growth factor and nerve
Somatomedin.
2. work of the compositionss according to claim 1 in induction gingiva Derived from Mesenchymal Stem Cells is neuron cell
With.
3. a kind of induction preparation, it is characterised in that including compositionss as claimed in claim 1.
4. induction preparation according to claim 3, it is characterised in that the end of the basic fibroblast growth factor is dense
Spend for 15～25ng/mL.
5. the induction preparation according to claim 3 or 4, it is characterised in that final concentration of the 5 of the epidermal growth factor～
15ng/mL。
6. the induction preparation according to any one of claim 3 to 5, it is characterised in that the end of the nerve growth factor is dense
Spend for 20～30ng/mL.
7. the induction preparation according to any one of claim 3 to 6, it is characterised in that the volumetric concentration of the B27 is 2%.
8. the induction preparation according to any one of claim 3 to 7, it is characterised in that also including cell culture medium.
9. the induction preparation according to any one of claim 3 to 8, it is characterised in that also including serum.
10. a kind of gingiva Derived from Mesenchymal Stem Cells is the abductive approach of neuron cell, it is characterised in that takes and filled between gingiva
Matter stem cell is cultivated after mixing with the induction preparation as described in any one of claim 3 to 9.
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| CN109266609A (en) * | 2018-09-30 | 2019-01-25 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application in induction amnion mesenchymal stem cell into neural-like cells differentiation |
| CN109628405A (en) * | 2018-12-21 | 2019-04-16 | 金华职业技术学院 | Nerve growth factor is promoting the application in umbilical cord mesenchymal stem cells Differentiation into Neuron-like Cells |
| CN112094804A (en) * | 2019-06-18 | 2020-12-18 | 中国医学科学院基础医学研究所 | A kind of heterogeneous stem cell population, its preparation method and use |
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| CN105408470A (en) * | 2013-06-24 | 2016-03-16 | 南加利福尼亚大学 | A composition of mesenchymal stem cells |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266609A (en) * | 2018-09-30 | 2019-01-25 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application in induction amnion mesenchymal stem cell into neural-like cells differentiation |
| CN109628405A (en) * | 2018-12-21 | 2019-04-16 | 金华职业技术学院 | Nerve growth factor is promoting the application in umbilical cord mesenchymal stem cells Differentiation into Neuron-like Cells |
| CN112094804A (en) * | 2019-06-18 | 2020-12-18 | 中国医学科学院基础医学研究所 | A kind of heterogeneous stem cell population, its preparation method and use |
| CN113646425A (en) * | 2019-06-18 | 2021-11-12 | 中国医学科学院基础医学研究所 | Heterogeneous stem cell population, preparation method and application thereof |
| CN113646425B (en) * | 2019-06-18 | 2024-03-19 | 中国医学科学院基础医学研究所 | A heterogeneous stem cell population, its preparation method and use |
| CN112094804B (en) * | 2019-06-18 | 2024-05-14 | 中国医学科学院基础医学研究所 | Heterogeneous stem cell population, preparation method and application thereof |
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