CN109266610A - A method of promotion Derived from Mesenchymal Stem Cells is neuron - Google Patents

A method of promotion Derived from Mesenchymal Stem Cells is neuron Download PDF

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CN109266610A
CN109266610A CN201811095661.1A CN201811095661A CN109266610A CN 109266610 A CN109266610 A CN 109266610A CN 201811095661 A CN201811095661 A CN 201811095661A CN 109266610 A CN109266610 A CN 109266610A
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mesenchymal stem
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neuron
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stem cell
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CN109266610B (en
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陈金虎
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Ningbo Jin Wei Biotechnology Co Ltd
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    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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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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Abstract

The invention discloses a kind of promotion Derived from Mesenchymal Stem Cells into the method for neuron, comprising the following steps: 1) prepares human mesenchymal stem cell and secondary culture；2) human mesenchymal stem cell after making secondary culture is divided into human mesenchymal stem cell-nerve ball in differential medium；3) human mesenchymal stem cell-nerve ball is made to be divided into neuron in induced medium.Promote neuronotropic differentiation by adding neurotrophic factor in induced medium, the results showed that the cell for expressing mature neural markers object (MAP2ab) and immature neural markers object ('beta '-tubulin III) is significantly increased relative to blank control.Method of the invention provides certain direction for the new treatment of promotion spinal cord injury reparation.

Description

A method of promotion Derived from Mesenchymal Stem Cells is neuron
Technical field
It is neuron the present invention relates to technical field of cell culture more particularly to a kind of promotion Derived from Mesenchymal Stem Cells Method.
Background technique
Spinal cord injury (spinal cord injury, SCI) is a kind of serious cental system illness, after most serious Fruit is to lead to damage segment with the serious dysfunction of lower limb body, caused by paraplegia patient can be made to lose self-care ability, this is not Serious body and menticide only are brought to patient, also causes heavy burden to family.
At present in the treatment of patients with spinal cord injury, use generally by means of some drugs and mutually tie with physiotherapy It closes to slow down the degree of paralysis.But by such treatment, the quality of life of patient is still unable to get good promotion.
How to make the reparation of nervous centralis and reconstruction after spinal cord injury, is currently still a problem.Although refreshing Discovery through growth factor brings hope to the clinical drug therapy of central nervous system injury, also achieved the effect that it is certain, but It is that neurotrophy molecular weight of material is too big, blood-brain barrier can not be penetrated and enter central nervous tissue to play its activity.
In spinal cord injury, transplanting may be a kind of practical method for carrying out central nervous system reparation.But it is real The transplantation treatment of existing cell, it is necessary first to cell differentiation be studied and be assessed at the ability of neuron, and it is thin to develop promotion The method of born of the same parents' differentiation.
Therefore, those skilled in the art is dedicated to developing a kind of method that promotion stem cell is divided into neuron.
Summary of the invention
To achieve the above object, the present invention provides a kind of promotion Derived from Mesenchymal Stem Cells into the method for neuron.
In a better embodiment of the invention, this method, comprising the following steps:
1) human mesenchymal stem cell and secondary culture are prepared；
2) human mesenchymal stem cell after making secondary culture is divided into human mesenchymal stem cell-in differential medium Nerve ball；
3) human mesenchymal stem cell-nerve ball is made to be divided into neuron in induced medium.
Preferably, step 3) is specially and smashes human mesenchymal stem cell-nerve ball in step 2), is inoculated into poly- In L-lysine and the double coated culture vessel containing the induced medium of laminin, in 37 DEG C, 5%CO2Culture Differentiation 7-10 days.
Further, induced medium is added with 0.5umol/L all-trans retinoic acid, 1%FBS, 5% horse serum, 1% The NB culture medium of N2 supplement, 1% penicillin/streptomycin and 10ng/mL recombinant human B DNF.
In another preferred embodiment, step 3) is specially by human mesenchymal stem cell-nerve in step 2) Ball is smashed, and is inoculated into poly-l-lysine and the double coated culture vessel of laminin, is first used first containing BDNF Induced medium carries out induction differentiation 5-6 days, and the second induced medium containing TGF-β 1 is then used to carry out induction differentiation 4-5 It, cell is in 37 DEG C, 5%CO2Culture.
Further, then after adding the second induced medium containing TGF-β 1, first by cell at 40 DEG C, 5%CO2Middle training It supports 3-4 hours, is then then transferred to 37 DEG C, 5%CO2Continue culture differentiation 4-5 days.
Further, the first induced medium is added with 0.5umol/L all-trans retinoic acid, 1%FBS, 5% horse blood Clearly, the NB culture medium of 1%N2 supplement, 1% penicillin/streptomycin and 10ng/mL recombinant human B DNF；Second induced medium For added with 0.5umol/L all-trans retinoic acid, 1%FBS, 5% horse serum, 1%N2 supplement, 1% penicillin/streptomycin with And the NB culture medium of 1-5ng/L TGF-β 1.
Further, step 1) is specially to carry out alcohol disinfecting to umbilical cord, and blood vessel is removed in balanced salt solution, and China is logical Mescenchymal tissue in family name's glue is cut into 0.4-0.6cm3Size, centrifugation obtain mescenchymal tissue part, use serum-free DMEM/ The washing of F12 culture medium；The sediment fraction obtained after washing centrifugation carries out enzymic digestion；Cytometer is carried out to the suspension after enzymic digestion Number, then carries out inoculated and cultured, i.e. acquisition human mesenchymal stem cell；It is passed when cell grows to 70%-80% fusion rate Generation.
Preferably, step 2) is carried out using the human mesenchymal stem cell of step 1) forth generation to the 6th generation.
Further, step 2) includes: enzyme process separating step 1) in cultivate human mesenchymal stem cell, and access containing point Change and is cultivated in the tissue cultures low-adhesion plastic flask of culture medium；Wherein, differential medium is to contain 20ng/ml epidermis Growth factor, 20ng/mL alkalinity at Porcine HGF and the diluted B27 of 1:50 NB culture medium；Cell is at 37 DEG C, 5%CO2 Lower culture, the fresh epidermal growth factor of addition in every 3-4 days, alkalinity replace once a week differentiation training at Porcine HGF and B27 Support base.
Another aspect of the present invention additionally provide it is a kind of using above-mentioned promotion Derived from Mesenchymal Stem Cells at neuron Method is studying the application in the neural restoration after spinal cord injury.
Using the method in the present invention, the ratio that Derived from Mesenchymal Stem Cells is neuron can be effectively improved.Using containing The induced medium of BDNF carries out in the cell after induction differentiation, expresses mature neural markers object (MAP2ab) and immature nerve The cell proportion of marker ('beta '-tubulin III) has significant raising compared with blank control group, it can respectively reach 8 ± 1.9% and 38.6 ± 2.9%.And use the first induced medium containing BDNF and the second induced medium containing TGF-β 1 In cell after carrying out induction differentiation, mature neural markers object (MAP2ab) and immature neural markers object (β-micro-pipe egg are expressed White III) cell proportion compared with blank control group, also significantly improve, also, relative to only use containing BDNF induction training After feeding base carries out induction differentiation, two kinds of positive cell ratios are also further increased.Method of the invention is to promote spinal cord injury The new treatment of reparation provides certain direction.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific steps and generation, with It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is that two kinds of cells occur in the Initial culture of the human mesenchymal stem cell of a preferred embodiment of the invention Form, Fig. 1 a are strips, and Fig. 1 b is circular.
Fig. 2 is that the human mesenchymal stem cell third generation passage cell culture of a preferred embodiment of the invention is melted to 80% Cellular morphology when conjunction rate；
Fig. 3 is the human mesenchymal stem cell renewed vaccination of a preferred embodiment of the invention to nerve ball differential medium Cell after 1-2 days starts the form of aggregation.
Fig. 4 is many drifts of appearance in 3-4 days of the human mesenchymal stem cell of a preferred embodiment of the invention in post-conversion The sphere of floating cell.
Fig. 5 is the CD44 coloration result in the 3rd generation of the human mesenchymal stem cell of a preferred embodiment of the invention.
Fig. 6 is the nesting for the nerve ball that the human mesenchymal stem cell of a preferred embodiment of the invention is derived Positive mark's figure.
It is (immature that Fig. 7 is that the human mesenchymal stem cell of a preferred embodiment of the invention is divided into 'beta '-tubulin III Neural markers object) positive cell Fluorescent immunohistochemistry method result figure.
Fig. 8 is that the human mesenchymal stem cell of a preferred embodiment of the invention is divided into GFAP (astroglia mark Remember object) positive cell Fluorescent immunohistochemistry method result figure.
Fig. 9 is that the human mesenchymal stem cell of a preferred embodiment of the invention is divided into CalC (oligodendroglia mark Remember object) positive cell Fluorescent immunohistochemistry method result figure.
Figure 10 is that the human mesenchymal stem cell of a preferred embodiment of the invention is divided into MAP2ab (mature neural markers Object) positive cell Fluorescent immunohistochemistry method result figure.
Figure 11 is that the human mesenchymal stem cell of a preferred embodiment of the invention is divided into the different neural markers objects positives The Western blot testing result figure of the GAP-associated protein GAP level of cell.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits The embodiment that Yu Wenzhong is mentioned.
Experimental method used in the specific embodiment of the invention, if being the method for this field routine without specified otherwise. Reagent used in the specific embodiment of the invention, if can be obtained by purchase without specified otherwise.
Human mesenchymal stem cell has many advantages of embryonic stem cell and adult stem cell simultaneously, and relative to other Stem cell, it is advantageous that: plasticity is strong；It is easy to obtain by way of low invasion, and being capable of fast breeding；It is immune Compatible, the human mesenchymal stem cell of patient oneself can be used for autotransplantation.Therefore, the present invention is using human mesenchymal stem cell Beginning object carries out differentiation research.
In nerve to occur, microenvironment factor pair proliferation and differentiation have highly important influence.Wherein, BDNF widely divides Cloth plays the role of in the development, survival and reparation of nerve cell highly important in development and mature nervous system.
This research is by the combination of different trophic factors and different conditions, and observer's Derived from Mesenchymal Stem Cells is at mind Effect through member.
The preparation of 1 human mesenchymal stem cell of embodiment
Neonatal umbilical cord is obtained, is placed in hanks' balanced salt solution (HBSS, Gibco, USA), 4 DEG C of preservations.Umbilical cord With alcohol disinfecting 30 seconds of 75%.In HBSS, cord vessels is removed.The mescenchymal tissue being present in magnificent Tong Shi glue is cut It is cut into about 0.5cm3, and be centrifuged 5 minutes at 1,200rpm.Supernatant, the sediment fraction of mescenchymal tissue are removed after centrifugation It is washed with serum-free DMEM/F12 culture medium (Gibco, USA), is centrifuged 5 minutes at 1,200rpm later.Sediment fraction uses 0.075%II Collagenase Type (Sigma) at 37 DEG C enzyme process separate 18 hours, then further use 0.125% trypsase/ EDTA (Gibco) digests 30 minutes at 37 DEG C.Suspension uses in the DMEM/F12 progress containing 10% (v/v) fetal calf serum With the cell in suspension is counted under the microscope.It adjusts to 5-7 × 103A/cm2It is seeded in tissue culture flasks, In 37 DEG C, 5%CO2Under cultivated, and be maintained at subconfluent state.When cell grows to 70% fusion rate, with the ratio of 1:3 Example carries out secondary culture.Follow-up study is carried out using the cell of forth generation to the 6th generation.
As seen in figure la and lb, in the Initial culture of human mesenchymal stem cell, there are two kinds of cellular morphologies, one is strips Shape, one is circular.By the passage in three to five generations, the pinacocyte form of single layer is presented in human mesenchymal stem cell, and And the form of all cells tends to be similar.Fig. 2 shows cellular morphologies when third generation passage cell culture to 80% fusion rate.
2 human mesenchymal stem cell of embodiment forms human mesenchymal stem cell-nerve ball
With 0.125% trypsase/0.02%EDTA separation forth generation human mesenchymal stem cell, the human world after separation is filled Matter stem cell is with 2 × 105A/cm2Density access tissue cultures low-adhesion plastic flask in, in flask containing differentiation culture Base.Differential medium be containing 20ng/ml epidermal growth factor (EGF), 20ng/mL alkalinity at Porcine HGF (bFGF) and The NB culture medium (Invitrogen) of the diluted B27 of 1:50 (Gibco).
Cell is at 37 DEG C, 5%CO2Lower culture.The fresh growth factor of addition in every 3-4 days, replaces once a week culture medium. It is observed that spherical formed within 4-5 days after trigger differentiation.Before passage, cell is cultivated in differential medium always.
Fig. 3 shows the cellular morphology after human mesenchymal stem cell renewed vaccination to nerve ball differential medium 1-2 days, carefully Born of the same parents start to assemble, and form human mesenchymal stem cell-nerve ball.The sphere of many floating cells in post-conversion 3-4 days is (i.e. again 7 days or so after inoculation) occur, as shown in Figure 4.
It is passed on enzyme process within every 10-12 days, i.e., adds 0.6% glucose to carry out enzyme process biography using 0.025% trypsase Generation.Before neuron terminal differentiation, these nerve ball spline structures continue to expand 4-6 week (about 3-4 generation).
The human mesenchymal stem cell in nearly all 3rd generation all shows very strong CD44 dyeing (Fig. 5).It is dry from human mesenchyme Nerve ball that is cell-derived and coming is confirmed (Fig. 6) by nestin antibody.
The further differentiation (BDNF induction) of embodiment three-occupant apartment mesenchymal stem cells-nerve ball
Collect cultivated in embodiment 2 by human mesenchymal stem cell-nerve ball, and smashed, renewed vaccination to poly- L-lysine and laminin it is double coated containing induced medium six hole chamber slides (chamber slides, NUNC in).Cell is in 37 DEG C, 5%CO2Culture differentiation 7-10 days.
Wherein, induced medium be added with 0.5umol/L all-trans retinoic acid (Sigma), 1%FBS, 5% horse serum, 1%N2 supplement, 1% penicillin/streptomycin (being all purchased from Gibco) and 10ng/mL recombinant human B DNF (rhBDNF, R&D Systems NB culture medium).
In control group, as hereinbefore, only induced medium replaces with the induction training of the control without BDNF to experiment condition Support base, i.e., it is green added with 0.5umol/L all-trans retinoic acid (Sigma), 1%FBS, 5% horse serum, 1%N2 supplement and 1% The NB culture medium of mycin/streptomysin (being all purchased from Gibco).
Cell after collecting differentiation is analyzed:
Human mesenchymal stem cell can be divided into that 'beta '-tubulin III (immature neural markers object), (astroglia is thin by GFAP Born of the same parents' marker), the cell of CalC (oligodendroglia marker) and MAP2ab (mature neural markers object) positive, using immune The positive cell of different markers is observed by fluorescence histochemical method, as a result as is seen in figs 7-10.Pass through Hoechst 33342+Dyeing, quantifies the cell of the various positives after differentiation, quantitative result calculation method are as follows: corresponding positive cell number Amount/Hoechst 33342+Quantity × 100% of staining cell, the results are shown in Table 1.
Table 1: the quantitative percentage of positive cell
Induced medium 'beta '-tubulin III GFAP CalC MAP2ab
Control 19.2 ± 3.0% 42.8 ± 3.8% 27 ± 2% 4.4 ± 1.8%
BDNF induction 38.6 ± 2.9% 15.8 ± 4.5% 20.6 ± 4.6% 8 ± 1.9%
The result shows that the 'beta '-tubulin III positive cell and MAP2ab sun that are generated using nerve-specific abductive approach Property cell be apparently higher than using conventional abductive approach.
Western blot is further verified:
Western blot is conventional method in that art, in this research with for 'beta '-tubulin III (1:500), A process resistant of MAP2ab (1:500), two process resistant then being connect with peroxidase.Pass through the chemiluminescence method of enhancing Develop.Use β-Actin (β-Actin) as internal reference.Antibody is all from Chemicon.
The result of Western blot verifying is as shown in figure 11, and result is consistent with ImmunohistochemistryResults Results.
The result shows that expressing mature neural markers object (MAP2ab) and immature neural markers object (β-after adding BDNF Tubulin III) cell proportion compared with blank control group, have significant raising, that is to say, that be added brain source nerve Growth factor group significantly improves the ratio that umbilical cord source nerve stem cell transdifferentiation is neuron.
The further differentiation (1 combined induction of BDNF+TGF- β) of 4 human mesenchymal stem cells of embodiment-nerve ball
Collect cultivated in embodiment 2 by human mesenchymal stem cell-nerve ball, and ground, renewed vaccination to poly- L-lysine and the double coated six hole chamber slide (chamber containing BDNF induced medium of laminin Slides, NUNC) in.Cell is in 37 DEG C, 5%CO2Culture differentiation 5-6 days.Later, BDNF induced medium is replaced with into TGF- 1 induced medium of β, first at 40 DEG C, 5%CO2Middle culture 3-4 hours, is then then transferred to 37 DEG C, 5%CO2Continue culture differentiation 4-5 days.
Wherein, BDNF induced medium is added with 0.5umol/L all-trans retinoic acid (Sigma), 1%FBS, 5% horse Serum, 1%N2 supplement, 1% penicillin/streptomycin (being all purchased from Gibco) and 10ng/mL recombinant human B DNF (rhBDNF, R& D Systems) NB culture medium；
1 induced medium of TGF-β is added with 0.5umol/L all-trans retinoic acid (Sigma), 1%FBS, 5% horse blood Clearly, the NB culture of 1%N2 supplement, 1% penicillin/streptomycin (being all purchased from Gibco) and 1-5ng/L TGF-β 1 (Sigma) Base.
In control group, experiment condition as hereinbefore, only all replace by BDNF induced medium and 1 induced medium of TGF-β It is changed to the control induced medium without BDNF, that is, is added with 0.5umol/L all-trans retinoic acid (Sigma), 1%FBS, 5% The NB culture medium of horse serum, 1%N2 supplement and 1% penicillin/streptomycin (being all purchased from Gibco).
Cell after collecting differentiation is analyzed:
It is observed, and passed through using positive cell of the Fluorescent immunohistochemistry method to different markers Hoechst33342+Dyeing, quantifies the cell of the various positives after differentiation, quantitative result calculation method are as follows: corresponding sun Property cell quantity/Hoechst 33342+Quantity × 100% of staining cell, the results are shown in Table 2.
Table 2: the quantitative percentage of positive cell
The result shows that the 'beta '-tubulin III positive cell and MAP2ab sun that are generated using nerve-specific abductive approach Property cell be apparently higher than using conventional abductive approach.Also, the method with the independent BDNF induction in embodiment 3, expression are mature There has also been a degree of for neural markers object (MAP2ab) and the cell proportion of immature neural markers object ('beta '-tubulin III) It improves.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea Scheme, all should be within the scope of protection determined by the claims.

Claims (10)

1. a kind of method for promoting Derived from Mesenchymal Stem Cells into neuron, which comprises the following steps:
1) human mesenchymal stem cell and secondary culture are prepared；
2) human mesenchymal stem cell after making secondary culture is divided into human mesenchymal stem cell-nerve in differential medium Ball；
3) human mesenchymal stem cell-nerve ball is made to be divided into neuron in induced medium.
2. promoting Derived from Mesenchymal Stem Cells at the method for neuron as described in claim 1, which is characterized in that step 3) tool Body is to smash human mesenchymal stem cell-nerve ball in step 2), is inoculated into poly-l-lysine and laminin double-contracting In the culture vessel containing the induced medium of quilt, in 37 DEG C, 5%CO2Culture differentiation 7-10 days.
3. promoting Derived from Mesenchymal Stem Cells at the method for neuron as claimed in claim 2, which is characterized in that the induction Culture medium is added with 0.5umol/L all-trans retinoic acid, 1%FBS, 5% horse serum, 1%N2 supplement, 1% penicillin/chain The NB culture medium of mycin and 10ng/mL recombinant human B DNF.
4. promoting Derived from Mesenchymal Stem Cells at the method for neuron as described in claim 1, which is characterized in that step 3) tool Body is to smash human mesenchymal stem cell-nerve ball in step 2), is inoculated into poly-l-lysine and laminin double-contracting In the culture vessel of quilt, the first induced medium containing BDNF is first used to carry out induction differentiation 5-6 days, then using containing Second induced medium of TGF-β 1 carries out induction differentiation 4-5 days, and cell is in 37 DEG C, 5%CO2Culture.
5. promoting Derived from Mesenchymal Stem Cells at the method for neuron as claimed in claim 4, which is characterized in that contain in addition After having the second induced medium of TGF-β 1, first by cell at 40 DEG C, 5%CO2Middle culture 3-4 hours, is then then transferred to 37 DEG C, 5%CO2Continue culture differentiation 4-5 days.
6. promoting Derived from Mesenchymal Stem Cells at the method for neuron as claimed in claim 5, which is characterized in that described first Induced medium is added with 0.5umol/L all-trans retinoic acid, 1%FBS, 5% horse serum, 1%N2 supplement, 1% mould The NB culture medium of element/streptomysin and 10ng/mL recombinant human B DNF；Second induced medium is added with 0.5umol/L All-trans retinoic acid, 1%FBS, 5% horse serum, 1%N2 supplement, 1% penicillin/streptomycin and 1-5ng/L TGF-β 1 NB culture medium.
7. promoting Derived from Mesenchymal Stem Cells at the method for neuron as described in claim 1, which is characterized in that step 1) tool Body is to carry out alcohol disinfecting to umbilical cord, and blood vessel is removed in balanced salt solution, the mescenchymal tissue in magnificent Tong Shi glue is cut into 0.4-0.6cm3Size, centrifugation obtain mescenchymal tissue part, are washed using serum-free DMEM/F12 culture medium；After washing centrifugation The sediment fraction of acquisition carries out enzymic digestion；Cell count is carried out to the suspension after enzymic digestion, inoculated and cultured is then carried out, that is, obtains Obtain human mesenchymal stem cell；It is passed on when cell grows to 70%-80% fusion rate.
8. promoting Derived from Mesenchymal Stem Cells at the method for neuron as described in claim 1, which is characterized in that step 2) is adopted It is carried out with the human mesenchymal stem cell in step 1) forth generation to the 6th generation.
9. promoting Derived from Mesenchymal Stem Cells at the method for neuron as claimed in claim 8, which is characterized in that step 2) packet Include: enzyme process separating step 1) in the human mesenchymal stem cell cultivated, and access the low adherency of the tissue cultures containing differential medium It is cultivated in property plastic flask；Wherein, differential medium is containing 20ng/ml epidermal growth factor, 20ng/mL alkalinity at thin The NB culture medium of the intracellular growth factor and the diluted B27 of 1:50；Cell is at 37 DEG C, 5%CO2Lower culture, addition in every 3-4 days are fresh Epidermal growth factor, alkalinity replace once a week differential medium at Porcine HGF and B27.
10. promoting Derived from Mesenchymal Stem Cells at the method for neuron after studying spinal cord injury as described in claim 1 Application in neural restoration.
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