CN109136187A - A kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell - Google Patents

A kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell Download PDF

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Publication number
CN109136187A
CN109136187A CN201811148056.6A CN201811148056A CN109136187A CN 109136187 A CN109136187 A CN 109136187A CN 201811148056 A CN201811148056 A CN 201811148056A CN 109136187 A CN109136187 A CN 109136187A
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cell
nerve cell
induction
differentiation
crinial bone
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麻育源
卢刚
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Zhejiang Provincial Peoples Hospital
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Zhejiang Provincial Peoples Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • 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
    • C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources

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  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Neurology (AREA)
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  • General Engineering & Computer Science (AREA)
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Abstract

The present invention relates to a kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell, include the following steps: to take crinial bone mescenchymal stem cell, with 104~106A/cm2Density be seeded in induction liquid, at 37 DEG C, 5%CO2Fiber differentiation 21 days in incubator obtain mature nerve cell；Liquid composition: 1 × N2,1 × B27,30~50ng/ml b-FGF, 10~30 μ g/ml VB12 is induced, solvent is DMEM/F12 basic culture solution.The beneficial effects of the present invention are: it is nerve cell that the method for the invention, which can effectively induce crinial bone Derived from Mesenchymal Stem Cells, differentiation gained cell not only expresses nerve cell marker protein, more importantly differentiation and maturation cell has a neuron ultra microstructure.

Description

A kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell
Technical field
The present invention relates to cell inductive technology fields, and in particular to crinial bone mesenchyma stem cell differentiation induction nerve cell Method.
Background technique
The neurodegenerative diseases such as Parkinson's disease, alzheimer's disease and amyotrophic lateral sclerosis have become clinical common And refractory disease.Cellular transplantation therapy provides new direction for above-mentioned disease treatment, but nerve cell is difficult to obtain and expand, It finds alternative cell origin and has become key point of the cell therapy technology for neurodegenerative disease treatment.
The mescenchymal stem cell (Mesenchymal stem cells, MSCs) for being derived from the tissues such as umbilical cord and ilium is middle embryo The one kind in layer source has the adult stem cell of height self-renewal capacity and multi-lineage potential.Research shows that MSCs is in addition to energy It is enough to break up to the mesoderms such as skeletonization, fat and cartilage direction, it under certain conditions can also be thin across liver cell, cardiac muscle is divided into Other germinal layer cell types such as born of the same parents, islet cells, that is, show the plasticity of height.Therefore, research foundation belongs to ectodermic Skull MSCs breaks up the induction system of nerve cell, not only has theoretical basis, but also will be for the rear something lost of cranial vascular disease in the future The offers such as disease, spinal cord injury and neural restoration treatment are direct, effective cell origin, are of great significance.
Summary of the invention
The purpose of the present invention is to overcome the above shortcomings and to provide a kind of neurons of crinial bone source for mesenchymal stem cells The suspension culture method of cell.
A kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell, includes the following steps:
Crinial bone mescenchymal stem cell is taken, with 104~106A/cm2Density be seeded to induction liquid in, at 37 DEG C, 5% CO2Fiber differentiation 21 days in incubator obtain mature nerve cell；
Induce liquid composition: 1 × N2,1 × B27,30~50ng/ml b-FGF, 10~30 μ g/ml VB12, solvent are DMEM/F12 basic culture solution.
As preferred: the induction liquid final concentration composition: 1 × N2,1 × B27,50ng/ml b-FGF, 10 μ g/ml VB12, solvent are DMEM/F12 basic culture solution.
As preferred: the cell density being seeded in induction liquid is 105A/cm2。
The beneficial effects of the present invention are: the method for the invention can effectively induce the crinial bone Derived from Mesenchymal Stem Cells to be Nerve cell, differentiation gained cell not only expresses nerve cell marker protein, more importantly differentiation and maturation cell has one Neuron ultra microstructure.
Detailed description of the invention
Fig. 1 is the cellular morphology micrograph for inducing liquid to induce the 21st day；A figure is that skull MSCs, the B figure for not starting to induce is Induction liquid induces the 21st day cell.40 times of amplification of A figure, 100 times of amplification of B figure.
Fig. 2 is the Immunofluorescence test figure of cell expression nerve cell marker protein after inducing liquid induction；A is Tuj-1 table It reaches；B is that NeuN and NSE is expressed；C is Syn expression.Amplify 100 times.
Fig. 3 is cellular neural cell ultrastructure (synaptic vesicle) detection figure after induction liquid induction.
Specific embodiment
The present invention is described further below with reference to embodiment.The explanation of following embodiments is merely used to help understand this Invention.It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, also Can be with several improvements and modifications are made to the present invention, these improvement and modification also fall into the protection scope of the claims in the present invention It is interior.
Embodiment 1: the separation of crinial bone mescenchymal stem cell
By 10cm3Skull osteocomma with medical electric drill sawing it is broken after, be transferred in 50mL centrifuge tube, be added 25mL physiology salt Water after mixing, is placed in low temperature shaking table, in 8 DEG C, under the conditions of revolving speed is 150 revs/min, is shaken 15 minutes, 200 mesh net filtrations, Filtrate 1000rpm is centrifuged 6min, and after removing supernatant, precipitating is suspended with cell culture fluid, and a small amount of suspension is taken to pass through fully automatic blood After analyzer is counted, with 2 × 105/cm2Density is seeded to 25cm2In plastic cell culture bottle, 37 degree are placed in, contains 5%CO2 It is cultivated in incubator, topples over after 48h and remove non-attached cell, change fresh medium, change liquid within then every 2 days, it is long extremely to cell 90% when converging, had digestive transfer culture, until in P3 generation, obtain the single fiber-like crinial bone mescenchymal stem cell (MSCs) of form.
Embodiment 2: the induction of crinial bone derived mesenchymal stem cells in vitro differentiation nerve cell
The P3 that 1 method of Example obtains is seeded to induction liquid for crinial bone MSCs, and 37 DEG C, 5%CO2It is lured in incubator Culture 21 days is led, wherein changing within every 2 days liquid, inducing cell is collected and is identified, obtain mature nerve cell.
Induce I final concentration of liquid composition: 1 × N2,1 × B27,50ng/ml b-FGF, 10 μ g/ml VB12, solvent DMEM/ F12 basic culture solution (is purchased from Corning company).
N2, B27 are purchased from Gibco company, and b-FGF is purchased from Peprotech company, and VB12 is purchased from Sigma company.
Embodiment 3: the identification of noble cells
(1) morphological observation: the 21st day cell is induced to utilize micro- sem observation without the MSCs and induction liquid of induction Cellular morphology, and photograph to record, shown in the result is shown in Figure 1, figure A amplifies 40 times, and figure B amplifies 100 times.
(2) nerve cell marker protein Immunofluorescence test: the cell after induction liquid induction is through 4% paraformaldehyde room temperature After (25 DEG C) fixed 15min, is rinsed with PBS (pH value 7.4) and remove paraformaldehyde, then (purchased with 0.05%Triton X-100 From Sigma company) soaking at room temperature processing 10 minutes, 1h then is closed in room temperature with 5% lowlenthal serum (purchased from raw work biology), PBS is cleaned twice, and cell is divided into 4 groups, is separately added into primary antibody, and group 1 is anti-Tuj-1, and group 2 is anti-NeuN and anti-NSE, and group 3 is anti- Syn, primary antibody are purchased from Abcam company, and 4 DEG C of wet box are incubated overnight；PBS embathes three times, each 10min；Sequentially add fluorescence mark Remember secondary antibody, group 1 is Alexa 488, group 2 is Alexa 488 and Alexa 647 marks secondary antibody, and (secondary antibody is equal for Alexa 647 for group 3 Purchased from Lian Ke biotech firm), 37 DEG C of incubation 1h, PBS embathe three times；3 groups of cell DAPI (being purchased from Sigma company) dye cores, laser Laser Scanning Confocal Microscope (Carl Zeiss) observation is taken pictures.As a result see Fig. 2 (amplifying 100 times).
(3) nerve cell micro structure testing: collecting and induce 21 days cells in centrifuge tube, 1000rpm/min centrifugation 10min abandons supernatant, and cell precipitation is fixed with 2.5% glutaraldehyde, and 4 DEG C of fixed 30min, PBS are rinsed 3 times, the fixed 2h of 1% osmic acid, PBS rinsing, acetone dehydration, embedding, 90nm slice, transmission electron microscope observing are taken pictures.As a result as shown in Figure 3.
The result shows that induction gained noble cells has nerve cell sample form, nerve cell marker protein is expressed, more It is important that differentiation and maturation cell has certain nerve cell ultra microstructure (synaptic vesicle) feature, show this abductive approach energy Enough effective induced calvarial Derived from Mesenchymal Stem Cells nerve cells.

Claims (3)

1. a kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell, which comprises the steps of:
Crinial bone mescenchymal stem cell is taken, with 104~106A/cm2Density be seeded in induction liquid, at 37 DEG C, 5%CO2Culture Fiber differentiation 21 days in case obtain mature nerve cell；
Induce liquid composition: 1 × N2,1 × B27,30~50ng/ml b-FGF, 10~30 μ g/ml VB12, solvent DMEM/F12 Basic culture solution.
2. the method for crinial bone mesenchyma stem cell differentiation induction nerve cell according to claim 1, it is characterised in that: The induction liquid final concentration composition: 1 × N2,1 × B27,50ng/ml b-FGF, 10 μ g/ml VB12, solvent are DMEM/F12 base Plinth culture solution.
3. the method for crinial bone mesenchyma stem cell differentiation induction nerve cell according to claim 1, it is characterised in that: The cell density being seeded in induction liquid is 105A/cm2。
CN201811148056.6A 2018-09-29 2018-09-29 A kind of method of crinial bone mesenchyma stem cell differentiation induction nerve cell Pending CN109136187A (en)

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EP3636286A1 (en) * 2011-05-18 2020-04-15 The Regents of The University of California Compositions and methods for treating retinal diseases
EP3169771B1 (en) * 2014-07-14 2020-04-01 Neelamkrishnan Venkataramanaa A method for the regeneration and differentiation of human perinephric fat derived mesenchymal stromal cells into astroglial, renal, neuronal and pancreatic progenitor cells
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Application publication date: 20190104