CN106011173A - Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof - Google Patents
Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof Download PDFInfo
- Publication number
- CN106011173A CN106011173A CN201610034981.0A CN201610034981A CN106011173A CN 106011173 A CN106011173 A CN 106011173A CN 201610034981 A CN201610034981 A CN 201610034981A CN 106011173 A CN106011173 A CN 106011173A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- people
- ggf
- gap
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
-
- 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
- C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention provides a preparation method of a human oligodendrocyte precursor cell inhibiting nerve secondary. Specifically, the method includes: constructing a virus expression vector of two glial growth factors, conducting cotransfection of a human oligodendrocyte precursor cell to prepare the human oligodendrocyte precursor cell, which can achieve high expression of the two glial growth factors, i.e. a growth related factor 43 and a glial growth factor 2, give play to the combined action of the two to promote oligodendrocyte repair, and inhibit nerve secondary injury caused by proliferative response and glial scar formation after oligodendrocyte injury. Also, the proliferation and differentiation ability of oligodendrocyte precursor cell itself is also utilized to participate in nerve function repair, and no tumorigenesis exists. The invention also provides a kit for preparation of the human oligodendrocyte precursor cell inhibiting nerve secondary. The human oligodendrocyte precursor cell provided by the invention has very good therapeutic effect on nervous system diseases.
Description
Technical field
The present invention relates to a kind of people's Oligodendrocyte precursor cells suppressing neural secondary damage preparation method and
Obtained people's Oligodendrocyte precursor cells of the neural secondary damage of suppression by described method, especially, the present invention relates to
And building the virus expression carrier containing two glial growth factor so that people's Oligodendrocyte precursor cells can be steady
Surely this two glial growth factor are expressed, common both biological functions of performance；And provide one to prepare
The test kit of people's Oligodendrocyte precursor cells of this suppression nerve secondary damage.People's oligodendroglia that the present invention provides
CFU-GM keeps good multiplication capacity, participates in the reparation of function of nervous system, and without Tumor formation, it is expected to it is used for facing
Bed treatment nervous system disease, promotes the reparation of damaged nerve cell.
Background technology
Nervous system disease, when ischemia, anoxia and damage, causes calcium after astrocyte injury
Ion concentration significant change, after in active cell, Calcium Signal conducts and participates in astrocyte injury
Proliferation response, astrocyte abnormality proliferation causes the formation of glial scar, hinders injured nerve axle
Prominent effective regeneration and growth, predominantly central nervous system injury disease, i.e. traumatic brain injury, cerebral hemorrhage,
Cerebral ischemia and spinal cord injury etc..
Nerve injury astrocyte in early days is in ripe early stage, can maintain with secrete cytokines and promote
Neuronal function plays, and plays the effect of nervous tissue's support；But after its maturation, have many in early days
Plant beneficial functions to fade away, secrete injurious factor on the contrary, form chemical colloid barrier, affect nerve again
Raw, hinder Neurite elongation.The astrocyte being positioned at damage interface ripe shows suppression axon growth
Dan Baiduotang proteoglycan PG express, and in the formation of scar tissue, play main effect.Glial cell hyperplasia
Formed with glial scar, cause mechanicalness obstacle, affect the regeneration of aixs cylinder, extend and merge, and permissible
Forming blood capillary set, oppress blood capillary, the blood supply of impact local, neurocyte is all caused by these
Secondary damage effect.
Present invention applicant has been surprisingly found that, the glial growth factor of adult stem cell secretion is at neural secondary
Played an important role in damage suppression, it can improve body microenvironment, promotes stem cell and endogenous
Stem cell repair, but the glial growth factor of stem cell secretion is very limited amount of, grinds
Study carefully and think actually cellular replacement therapy, really play a role be its secretion Glial growth because of
Son.
The present invention by build containing two glial growth factor fragments, recombinate on viral vector, altogether
Two glial growth factor, the common biology playing both is expressed after transfected with human Oligodendrocyte precursor cells
Function, and without Tumor formation, there is good synergy.People's Oligodendrocyte precursor cells convenient sources, mainly
Including umbilical blood, placental blood, bone marrow and abortus brain etc., cell is cultivated and is obtained simply, can accomplish individuation,
The variant cell that immunogenicity is low can also be used.Thus, the present invention expresses two glial growth factor
The preparation of people's Oligodendrocyte precursor cells simple, two glial growth factor of high expressed, suppress astroglia
Abnormal cell proliferation and glial scar are formed, the common microenvironment being conducive to neuranagenesis of creating, suppression nerve
Secondary damage, be expected to maincenter injury disease is produced more preferably therapeutic effect.
Summary of the invention
The clinical efficacy of stem-cell therapy nervous system disease is approved widely in recent years, stem cell transplantation
Promote neurocyte reparation and neuranagenesis, improve neural axon and behavioral function recovers.Nervus centralis damages
Wound property end-stage disease has caused astrocyte ripe and abnormality proliferation because of microglial activation, causes glue
Matter cicatrization and Neuroinflammation, cause secondary damage to neurocyte.
The present invention based on our previous research work basis, utilize people's Oligodendrocyte precursor cells proliferation and differentiation and
The characteristic " gone back to the nest ", is carried out the recombinant viral vector of two the glial growth factor genetic fragments built
Transfection, can high expressed the two glial growth factor, the collaborative biology playing glial growth factor
Effect and the function of Oligodendrocyte precursor cells itself, can fill the interface of damage, suppression glial scar shape
Become, rearrange receptor tissue and postpone the expression of neuritegrowth inhibitor Dan Baiduotang proteoglycan PG, suppression neural two
Secondary damage.
Wherein, described glial growth factor is that growth phase closes the factor 43 (growth-associated
Protein-43, GAP-43) and glial growth factor 2 (glial growth factor 2, GGF-2)；
GAP-43 up-regulated promotes that neure growth, suppression glial scar are formed；GGF-2 expresses rising and helps
In neurological functional recovery, and improve Neuroinflammation, control the damaging action to neurocyte.Both associations
Same-action, can fill the interface of damage, suppression glial scar formation, rearrange receptor tissue and delay
The expression of neuritegrowth inhibitor Dan Baiduotang proteoglycan PG, the neural secondary damage of suppression.
The present invention is found that the people that glial growth factor slow virus carrier transfects in our R&D work
Oligodendrocyte precursor cells (Oligodendrocyte precursor cells, OPCs), its stable nerve expressed
Glia growth factor can suppress neural secondary damage, and without becoming tumor activity.This result of study has pointed out me
Can build slow virus carrier transfected with human Oligodendrocyte precursor cells by these two glial growth factor,
Make people's Oligodendrocyte precursor cells the most stably express after repeatedly Secondary Culture these two Glial growth because of
Son, has the ability playing its respective biological function.Ensure that cell quantity that clinical treatment uses
Meanwhile, also maintain the strongest propagation of stem cell, differentiation, homing activity, may be clinical treatment maincenter god
Play in damage disease and more preferably act on.
The invention provides the preparation method of a kind of people's Oligodendrocyte precursor cells suppressing neural secondary damage, its
Being characterised by, described method includes building on the virus expression carrier of two glial growth factor, corotation
Dye people's Oligodendrocyte precursor cells, being prepared as can the oligodendroglia ancestral of these two glial growth factor of high expressed
Cell, suppression glial cells hyperplasia causes neural secondary damage to promote repairing of neural injury；Wherein, described god
It is that growth phase closes the factor 43 (GAP-43) and glial growth factor 2 (GGF-2) through glia growth factor.
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfecting restructuring GAP-43
After the OPCs Secondary Culture of GGF-2 viral vector to the 10th generation, it expresses GAP-43 and GGF-2
Gene is above 1st generation OPCs of untransfected, respectively higher than at least 45 and 67 times；
In rats with spinal cord injury model, the OPCs of GAP43/GGF2 virus cotransfection transplants latter 28 days BBB
(Basso Beattie Bresnahan) motor function scores improved values is 20.00 ± 5.00, and cortex body-sensing induces
Current potential CSEP wave amplitude improved values 6.00 ± 1.00；Described GAP43/GGF2-OPCs is in treatment spinal cord injury
In rat, its suppression astrocytes and glial scar are formed.
People's Oligodendrocyte precursor cells convenient sources of the present invention, it is simple that cell cultivates acquisition, can be from patient
Bone marrow individuation obtains, it is also possible to as allosome large-scale production, can accomplish that individuation is applied.People dashes forward glue less
Mesenchymal progenitor cell people from source discards Cord blood, placental blood, autologous bone marrow and abortus brain etc., it is preferable that source
In people's autologous bone marrow.
People's Oligodendrocyte precursor cells preparation method of the present invention, i.e. separates through lymphocyte separation medium from bone marrow
The mononuclearcell that purification obtains, with serum-free oligodendrocyte culture medium (i.e. DMEM/F12 (1: 1)
Become containing 1%N2,2mM L-glutaminate, 5-100ng/ml basic fibroblast growth factor, 1-50ng/ml
Fibroblast growth factor 4,1-100ng/ml stem cell factor and 1-100ng/ml FMS sample tyrosine kinase
3 parts) at the 10-200 μ coated 75cm of g/ml poly-D-lysine2In Tissue Culture Flask primary and pass on training
Support, it is thus achieved that people's Oligodendrocyte precursor cells；Preferably, serum-free oligodendrocyte culture medium is
DMEM/F12 (1: 1) containing 1%N2,2mM L-glutaminate, 30ng/ml basic fibroblast growth factor,
5ng/ml fibroblast growth factor 4,10ng/ml stem cell factor and 5ng/ml FMS sample tyrosine
Kinases 3 part；Use the 50 μ coated 75cm of g/ml poly-D-lysine2Tissue Culture Flask.
People's Oligodendrocyte precursor cells detects through Flow Cytometry, OPCs mark A2B5, O4 and Sox10
All express more than 95%.
Viral vector of the present invention, can be at cell inner stablity propagation and expression genes of interest, predominantly
Slow virus, adenovirus, retrovirus etc., it is preferable that select slow virus expression system.
The present invention provides the preparation method of a kind of people's Oligodendrocyte precursor cells suppressing neural secondary damage, and it is special
Levy and be, people GAP-43 and GGF-2 sequence are expanded by PCR from normal human peripheral blood's genomic DNA
Increasing is got off, and has obtained the fragment of GAP-43 and GGF-2 mesh.These two target DNA fragments are connected to table
Reach after being built into recombiant plasmid on plasmid, after positive colony PCR identifies and order-checking is identified, the matter built
Grain can be saved in-80 DEG C of ultra cold storage freezers.Again by GAP-43 and GGF-2 gene fragment clone to slow sick
On carrier, it is packaged into restructuring GAP-43 and GGF-2 slow virus carrier, both cotransfection people oligodendroglia ancestrals
Cell, is prepared as people's Oligodendrocyte precursor cells of GAP43/GGF2 slow-virus transfection.
Transfect latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins (Green fluorescent protein,
GFP) luminous situation, Oligodendrocyte precursor cells GFP develops the color more than more than 90%, shows cotransfection
GAP-43 and GGF-2 slow-virus transfection people's Oligodendrocyte precursor cells success, it is thus achieved that continuous expression GAP-43
People's Oligodendrocyte precursor cells with GGF-2；
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfected GAP-43 and GGF-2
Slow-virus transfection people's Oligodendrocyte precursor cells Secondary Culture is to after the 10th generation, and it expresses GAP-43 and GGF-2
Gene is respectively higher than the OPCs 45.89 of untransfected and 67.98 times.
People's Oligodendrocyte precursor cells of the neural secondary damage of suppression of the present invention, can train with subculture in vitro separately
Supporting, the OPCs i.e. having transfected the transfection of GAP-43 and GGF-2 slow virus carrier passes through complete culture solution
DMEM/F12 subculture in vitro separately cultivate, when cell is cultivated the 5-6 days, cell growth fusion reach 90% with
On, need the trypsinization using mass volume ratio to be 0.25% to pass on；
Slow virus double with untransfected GAP-43 and GGF-2 carries people's Oligodendrocyte precursor cells of the present invention
The OPCs of body compares, and has higher growth rate, and Secondary Culture reaches to the 10th its proliferation times of generation
More than 500 times, and there is the ability being divided into oligodendrocyte.
After people's Oligodendrocyte precursor cells Secondary Culture 10 generation of the present invention, still high expressed growth relevant because of
Son 43 and glial growth factor 2, the OPCs of slow virus carriers double with untransfected GAP-43 and GGF-2
Compare, be respectively provided with similar spider cell form, oligodendrocyte can be broken up.
In rats with spinal cord injury model, the OPCs of GAP43/GGF2 slow virus cotransfection of the present invention
After transplanting, BBB motor function scores and cortical somatosensory evoked potential improve the OPCs apparently higher than untransfected,
Promote the neurological functional recovery of rats with spinal cord injury；Glial fibrillary acidic protein immunization groupization shows
Compared with the OPCs and the OPCs of untransfected of GAP43/GGF2 slow virus cotransfection transplants, positive cell table
Reaching quantity to substantially reduce, glial scar scope the most substantially diminishes.
One-tenth tumor internal, external experiment does not finds into tumor phenomenon, it is ensured that its safety.
The present invention also provides for the reagent of people's Oligodendrocyte precursor cells of a kind of neural secondary damage of the suppression prepared
Box, it is characterised in that described test kit includes:
1) people's Oligodendrocyte precursor cells basal medium；
2) packaged growth correlation factor 43 and glial growth factor 2 high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include above-mentioned method.
The present invention also provides for the application of people's Oligodendrocyte precursor cells of the neural secondary damage of described suppression, it is ensured that
In vitro culture obtains sufficient amount of people's Oligodendrocyte precursor cells, and it is raw to have two neuroglia of stable expression
The physiological function that the long factor, common its biological function that plays, promotion neural axon regeneration and protection are neural,
And can effectively induce the linear arrangement of astrocyte of new life, carry for neural axon regeneration and growth
For preferable passage, the hyper-proliferative of regulation and control astrocyte and the formation of glial scar, thus inhibit
To neural secondary damage effect.
Accompanying drawing explanation
Fig. 1 is expressed as people's Oligodendrocyte precursor cells of embodiment 2 preparation through FCM analysis streaming figure
Fig. 2 is expressed as GAP-43 and GGF-2 slow virus immunofluorescence figure after transfection OPCs of recombinating
Fig. 3 is expressed as gene table after the 10th generation restructuring GAP-43 and GGF-2 slow virus cotransfection OPCs
Reach level view
Fig. 4 is expressed as the OPCs of the 10th generation restructuring GAP43/GGF2 slow virus cotransfection and is divided into and dashes forward less
Glial cell immunofluorescence figure
Fig. 5 is expressed as GFAP after rats with spinal cord injury model GAP43/GGF2-OPCs transplants and expresses and glue
Matter cicatrix improves result figure
Detailed description of the invention
The invention provides the preparation method of a kind of people's Oligodendrocyte precursor cells suppressing neural secondary damage, its
Being characterised by, described method includes building on the virus expression carrier of two glial growth factor, corotation
Dye people's Oligodendrocyte precursor cells, being prepared as can the oligodendroglia ancestral of these two glial growth factor of high expressed
Cell, suppression glial cells hyperplasia causes neural secondary damage to promote repairing of neural injury.
People's Oligodendrocyte precursor cells convenient sources of the present invention, can from people discard Cord blood, placental blood,
Autologous bone marrow and abortus brain etc., it is preferable that derive from people's autologous bone marrow.
People's Oligodendrocyte precursor cells preparation method of the present invention, it may be assumed that take from 20ml bone marrow through lymphocyte
Separate the isolated and purified mononuclearcell obtained of liquid, by serum-free oligodendrocyte culture medium (i.e.
DMEM/F12 (1: 1) containing 1%N2,2mM L-glutaminate, 5-100ng/ml basic fibroblast growth because of
Son, 1-50ng/ml fibroblast growth factor 4,1-100ng/ml stem cell factor and 1-100ng/ml
FLT3L) it is resuspended to 2-5 × 106/ ml, draws 10ml and joins 10-200 μ g/ml
The coated 75cm of poly-D-lysine2In Tissue Culture Flask, in 37 DEG C, 5%CO2Cell culture incubator is cultivated
48 hours.The serum-free oligodendrocyte culture medium changing fresh factor-containing is cultivated the most continuously, until thin
Intracellular growth fusion reaches about 90%, uses 0.25% (mass volume ratio) pancreatin (containing mass volume ratio
0.02%EDTA) digesting, carry out Secondary Culture, i.e. 1 bottle passes on 2 bottles, supplements fresh containing cell
The serum-free oligodendrocyte culture medium of the factor continues to cultivate, it is thus achieved that people's Oligodendrocyte precursor cells.
Preferably, serum-free oligodendrocyte culture medium is DMEM/F12 (1: the 1) L-Han 1%N2,2mM
Glutamine, 30ng/ml basic fibroblast growth factor, 5ng/ml alkali fibroblast growth factor 4,
10ng/ml stem cell factor and 5ng/ml FLT3L；Use 50 μ g/ml
The coated 75cm of poly-D-lysine2Tissue Culture Flask.
People's Oligodendrocyte precursor cells detects through Flow Cytometry, OPCs mark A2B5, O4 and Sox10
All express more than 95%.
The genetic fragment of two glial growth factor of the present invention is to be expanded also by round pcr
Recombinate on expression plasmid, then recombinate on virus expression carrier, then pack 293T cell and be prepared as height
Express the virus of genes of interest.
The glial growth factor of the neural secondary damage of suppression of the present invention, closes the factor for growth phase
43 (GAP-43) and glial growth factor 2 (GGF-2).
Viral vector of the present invention, can be at cell inner stablity propagation and expression genes of interest, predominantly
Slow virus, adenovirus, retrovirus etc., it is preferable that select slow virus expression system, be commercialization
Product, is commercially available from commercial company.
People GAP-43 and GGF-2 sequence are expanded by PCR from normal human peripheral blood's genomic DNA and obtains
, the fragment of GAP-43 and GGF-2 mesh respectively with the pET28a carrier segments of XhoI/Nde I double digestion,
Under T4DNA ligase effect, in 4 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, convert large intestine
After bacillus competent cell DH5a, carry out positive colony PCR qualification and order-checking is identified；PCR primer gel
After electrophoresis detection and order-checking qualification meet GAP-43 and GGF-2 size and sequence, by bacterium solution correct for order-checking
Transfer in 10ml contains the LB fluid medium of corresponding antibiotic, 37 DEG C of overnight incubation, raw with sky, Beijing root
Thing carries middle amount test kit carry out plasmid extraction without endotoxin plasmid is little, extracts qualified recombiant plasmid and places
-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer；Described GAP-43 and GGF-2 size respectively may be about 706bp and
1268bp；
Respectively GAP-43, GGF-2DNA fragment and GV358 carrier are carried out XhoI/Nde I double digestion,
Under T4DNA ligase effect, by GAP-43 fragment and enzyme action GV358 carrier and GGF-2 fragment and
Enzyme action GV358 carrier, respectively at 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, converts escherichia coli
Carry out positive colony PCR qualification after competent cell DH5a and order-checking is identified；
Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to each
The culture dish 6 × 10 of 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in cultivate
Overnight；Remove culture fluid before transfection in second day, change 5ml Opti-MEM culture fluid；Take 9 μ g and pack mixed liquor
Add in 1.5ml Opti-MEM with 3 μ g slow virus expression plasmids, mix gently, take 36 μ l lipofectamine
2000 add in 1.5ml Opti-MEM, mix gently, and room temperature places 5min；Mixing plasmid solution and
Lipofectamine 2000 diluent, puts room temperature 20min；Mixed liquor is slowly added dropwise the cultivation to 293T cell
In liquid, mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；Discard containing transfection mixed after cultivating 6h
With the culture medium of thing, the PBS liquid adding 10ml cleans once, softly rocks culture dish with remaining the turning of washing
Abandon after dye mixture；It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2
Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10
Min, removes cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Respectively
Trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant
In, arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C；Centrifugal
After end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus and preserve liquid, the most instead
Blow and beat resuspended again；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence
Method measures titre, and packaged GAP-43 and GGF-2 slow virus, according to 50 μ l 2E+8TU/ml subpackages, protects
It is stored in-80 DEG C of ultra cold storage freezers；
Each to restructuring GAP-43 and GGF-2 slow virus 10 μ l lE+7TU/ml are joined people oligodendroglia ancestral
In 10-200 coated six well culture plates of μ g/ml poly-D-lysine of cell, system is 2ml, mixing, 37 DEG C,
5%CO2Incubator in hatch 8-12 hour after, change complete culture solution DMEM/F12 (1: 1),
Described complete culture solution containing 1%N2,2mM L-glutaminate, 5-100ng/ml basic fibroblast growth because of
Son, 1-50ng/ml fibroblast growth factor 4,1-100ng/ml stem cell factor and 1-100ng/ml
FLT3L；When cell growth fusion reaches 90%, use 0.25% trypsinization,
Proceed to the 10-200 μ coated 25cm of g/ml poly-D-lysine2Culture bottle grows, corresponding 1 cultivation in 1 hole
Bottle, at 25cm2Culture bottle merges and when reaching 90%, continues had digestive transfer culture cultivation；Growth transfection is divided into 3
Group: untransfected matched group, blank slow-virus transfection group, cotransfection GAP-43 and GGF-2 slow virus
Transfection group；
Transfect the luminous situation of latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins, oligodendroglia ancestral
Cell GFP develops the color more than more than 90%, shows cotransfection GAP-43 and GGF-2 slow-virus transfection people
Oligodendrocyte precursor cells success, it is thus achieved that people's Oligodendrocyte precursor cells of continuous expression GAP-43 and GGF-2；
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfected GAP-43 and GGF-2
Slow-virus transfection people's Oligodendrocyte precursor cells Secondary Culture is to after the 10th generation, and it expresses GAP-43 and GGF-2
Gene is respectively higher than the OPCs 45.89 of untransfected and 67.98 times.
People's Oligodendrocyte precursor cells of the neural secondary damage of suppression of the present invention, can train with subculture in vitro separately
Supporting, the OPCs i.e. having transfected the transfection of GAP-43 and GGF-2 slow virus carrier passes through complete culture solution
DMEM/F12 subculture in vitro separately cultivate, when cell is cultivated the 5-6 days, cell growth fusion reach 90% with
On, need the trypsinization using mass volume ratio to be 0.25% to pass on；
Slow virus double with untransfected GAP-43 and GGF-2 carries people's Oligodendrocyte precursor cells of the present invention
The OPCs of body compares, and has higher growth rate, and Secondary Culture reaches to the 10th its proliferation times of generation
More than 500 times, and there is the ability being divided into oligodendrocyte.
After people's Oligodendrocyte precursor cells Secondary Culture 10 generation of the present invention, still high expressed growth relevant because of
Son 43 and glial growth factor 2, the OPCs of slow virus carriers double with untransfected GAP-43 and GGF-2
Compare, be respectively provided with similar spider cell form, oligodendrocyte can be broken up.
In rats with spinal cord injury model, the OPCs of GAP43/GGF2 slow virus cotransfection of the present invention
After transplanting, BBB motor function scores and cortical somatosensory evoked potential improve the OPCs apparently higher than untransfected,
Promote the neurological functional recovery of rats with spinal cord injury；Glial fibrillary acidic protein immunization groupization shows
Compared with the OPCs and the OPCs of untransfected of GAP43/GGF2 slow virus cotransfection transplants, positive cell table
Reaching quantity to substantially reduce, glial scar scope the most substantially diminishes.
People's Oligodendrocyte precursor cells of the neural secondary damage of suppression of the present invention, subcutaneous injection in nude mouse
After, do not find into tumor phenomenon, it is ensured that its safety, and the breast cancer cell line MCF-7 inoculated is positive
Comparison occurs in that into tumor.
The present invention also provides for the reagent of people's Oligodendrocyte precursor cells of a kind of neural secondary damage of the suppression prepared
Box, it is characterised in that described test kit includes:
1) people's Oligodendrocyte precursor cells basal medium；
2) packaged growth correlation factor 43 and glial growth factor 2 high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include above-mentioned method.
The present invention also provides for suppressing the application of people's Oligodendrocyte precursor cells of neural secondary damage, high expressed
People's Oligodendrocyte precursor cells of GAP-43 and GGF-2 can fill the interface of damage, suppresses glial scar shape
Become, rearrange receptor tissue and postpone the expression of neuritegrowth inhibitor Dan Baiduotang proteoglycan PG；It is thus possible to it is non-
Often effectively improve the microenvironment of axon regeneration, the growth of aixs cylinder after promotion acute spinal cord injury；Suppression nerve
Secondary damage, i.e. suppression facial neurons after neuron atrophy and promote behavioral competence recover significantly.
Hereinafter, the specific embodiment of the present invention is illustrated, but the technical scope of the present invention is not limited to these
Example.
Embodiment 1 is recombinated the structure of GAP-43 and GGF-2 slow virus carrier and packaging
Under people GAP-43 and GGF-2 sequence are expanded from normal human peripheral blood's genomic DNA by PCR
Come, the fragment of GAP-43 and GGF-2 mesh respectively with XhoI/Nde I (Japan TAKARA company) double digestion
PET28a carrier (American I nvitrogen company) fragment, at T4DNA ligase, (Japan TAKARA is public
Department) effect under, in 4 DEG C, coupled reaction in 12 hours preparation clone connect liquid, convert E. coli competent thin
Carry out positive colony PCR qualification after born of the same parents DH5a (American I nvitrogen company) and order-checking is identified；Identify
GAP-43 and GGF-2 genetic fragment size respectively may be about 706bp and 1268bp, meets GAP-43 and GGF-2
Size and sequence.The correct bacterium solution that checks order is transferred in 10ml contains the LB fluid medium of ammonia benzyl antibiotic,
37 DEG C of overnight incubation, carry middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid are little,
Extract qualified recombiant plasmid and place-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer.
Respectively GAP-43, GGF-2DNA fragment and GV358 carrier (Shanghai Ji is triumphant) are carried out XhoI/Nde I
Double digestion, under T4DNA ligase effect, by GAP-43 fragment and enzyme action GV358 carrier and
GGF-2 fragment and enzyme action GV358 carrier respectively at 37 DEG C, coupled reaction in 12 hours preparation clone connect liquid,
After converting competent escherichia coli cell DH5a, carry out positive colony PCR qualification and order-checking is identified.
Take cell state good, be in the 293T cell (American I nvitrogen company) of exponential phase, carefully
After born of the same parents' counting, according to the culture dish 6 × 10 of each 10cm6Individual cell number is inoculated in culture dish, 37 DEG C,
5%CO2Incubator in overnight incubation；Remove culture fluid before transfection in second day, change 5ml Opti-MEM training
Nutrient solution；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add in 1.5ml Opti-MEM, gently
Mixing, takes 36 μ l lipofectamine2000 (American I nvitrogen company) and adds in 1.5ml Opti-MEM,
Mixing gently, room temperature places 5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature
20min；Mixed liquor is slowly added dropwise to the culture fluid of 293T cell, and mixing, in 37 DEG C, 5%CO2Cell
Incubator is cultivated；Discard the culture medium containing transfection mixture after cultivating 6h, add the PBS liquid of 10ml
Clean once, abandon after softly rocking the culture dish transfection mixture with washing remnants；It is slowly added to containing 10%
The cell culture medium 20ml of serum, in 37 DEG C, containing 5%CO2Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10
Min, removes cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Respectively
Trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant
In, arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C；Centrifugal
After end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus and preserve liquid, the most instead
Blow and beat resuspended again；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence
Method measures titre, and packaged GAP-43 and GGF-2 slow virus, according to 50 μ l 2E+8TU/ml subpackages, protects
It is stored in-80 DEG C of ultra cold storage freezers；
The preparation of embodiment 2 people's Oligodendrocyte precursor cells
Gather and contribute volunteer's 20ml bone marrow, thin through the isolated and purified single core obtained of lymphocyte separation medium
Born of the same parents, tongue expects that orchid is counted as 3 × 107Individual, with serum-free oligodendrocyte culture medium (i.e. DMEM/F12 (1: 1)
(Gibco company of the U.S.) contains 1%N2,2mM L-glutaminate (Gibco company of the U.S.), 30ng/ml
Basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4,10ng/ml stem cell factor and
5ng/ml FLT3L (PeproTech company of the U.S.)) it is resuspended to 3 × 106/ ml,
Draw 10ml and join the 50 μ coated 75cm of g/ml poly-D-lysine2In Tissue Culture Flask, in 37 DEG C,
5%CO2Cell culture incubator is cultivated 48 hours.
The serum-free oligodendrocyte culture medium changing fresh factor-containing after cultivating 48 hours is trained the most continuously
Supporting, hereafter the serum-free oligodendrocyte culture medium of fresh factor-containing is changed at interval for 2 days, until cell is raw
Long fusion reaches about 90%, uses 0.25% (mass volume ratio) pancreatin (containing mass volume ratio
0.02%EDTA) (Gibco company of the U.S.) digests, and carries out Secondary Culture, and i.e. 1 bottle passes on 2 bottles,
The serum-free oligodendrocyte culture medium supplementing fresh factor-containing continues to cultivate, it is thus achieved that people's oligodendroglia
CFU-GM.
Take tongue and expect 0.6 × 10 after blue dyeing counting6OPC cell, divides three groups, and first group has been respectively added to
20 μ L mouse-anti people's A2B5 monoclonal antibody (Millipore company of the U.S.), the two anti-sheep anti mouse IgM-FITC (U.S.
Invitrogen company)；20 μ L mouse-anti people's O4 monoclonal antibody (Millipore company of the U.S.), two anti-sheep anti mouses
IgM-PE (American I nvitrogen company)；It is (beautiful that second component does not add 20 μ L mouse-anti people's Sox10 monoclonal antibodies
Millipore company of state), two anti-sheep anti mouse IgM-FITC (American I nvitrogen company)；20 μ L rabbits resist
People's GFAP monoclonal antibody (Millipore company of the U.S.), (American I nvitrogen is public for two anti-sheep anti-mouse igg-PE
Department)；3rd group is Isotype control, has been respectively added to 20 μ L FITC labelling Mus IgM and 20 μ L PE mark
Note Mus IgM.It is placed in 4 DEG C of refrigerators to dye 30 minutes, then with the 1 × phosphate buffer (PBS) of 1mL
Washing three times, finally with the cell after the resuspended washing of 1 × PBS of 0.5mL, the cell after gained washing is used
FACS Calibur flow cytometer (U.S. company BD) detects.Fig. 1 result shows, people's oligodendroglia
CFU-GM detects through Flow Cytometry, and OPCs mark A2B5, O4 and Sox10 express and be respectively
99.08%, 96.40% and 96.90%, GFAP is expressed as 9.62%.
Embodiment 3 is recombinated the preparation of GAP-43 and GGF-2 slow-virus transfection people's Oligodendrocyte precursor cells
Each to restructuring GAP-43 and GGF-2 slow virus 10 μ l 1E+7TU/ml are joined people oligodendroglia ancestral
In 50 coated six well culture plates of μ g/ml poly-D-lysine of cell, system is 2ml, mixing, 37 DEG C,
5%CO2Incubator in hatch 8-12 hour after, change complete culture solution DMEM/F12 (1: 1),
Described complete culture solution containing 1%N2,2mM L-glutaminate, 30ng/ml basic fibroblast growth factor,
5ng/ml fibroblast growth factor 4,10ng/ml stem cell factor and 5ng/ml FMS sample tyrosine
Kinases 3 part；When cell growth fusion reaches 90%, use 0.25% trypsinization, proceed to 50 μ g/ml
The coated 25cm of poly-D-lysine2Growing in culture bottle, corresponding 1 culture bottle in 1 hole, at 25cm2Cultivate
Merge in Ping and when reaching 90%, continue had digestive transfer culture cultivation；Growth transfection is divided into 3 groups: untransfected matched group,
Blank slow-virus transfection group, cotransfection GAP-43 and GGF-2 slow-virus transfection group.
Fig. 2 is restructuring GAP-43 and GGF-2 slow virus immunofluorescence picture after transfection OPCs.Restructuring
GAP-43 and GGF-2 slow virus carrier Carrying Green Fluorescent Protein (GFP), transfected restructuring GAP-43 and
In green under the OPCs fluorescence inverted microscope of GGF-2 slow virus, show that restructuring GAP-43 and GGF-2 is slow
Virus transfection OPCs successfully constructs.
Fig. 3 is that GAP-43 and GGF-2 is in the 10th generation transfecting restructuring GAP-43 and GGF-2 slow virus
Expression in OPCs.By PCR kit for fluorescence quantitative according to the operation instructions (U.S.
Invitrogen company) detection, the OPCs having transfected restructuring GAP-43 and GGF-2 slow virus carrier passes on training
After supporting for the 10th generation, it is expressed GAP-43 and GGF-2 gene and is above 1st generation OPCs of untransfected, respectively
Higher than 45.89 and 67.98 times.
The test kit of people's Oligodendrocyte precursor cells of the neural secondary damage of suppression of embodiment 4 preparation
People's Oligodendrocyte precursor cells test kit of the neural secondary damage of suppression includes:
1) people's Oligodendrocyte precursor cells basal medium, i.e. DMEM/F12 (1: 1)；
2) packaged growth correlation factor 43 and glial growth factor 2 high expressed virus；
3) enzyme of peptic cell, 0.25% (mass volume ratio) pancreatin (containing mass volume ratio 0.02%EDTA)；
4) cytokine, i.e. 1%N2,2mM L-glutaminate, 5-100ng/ml basic fibroblast growth
The factor, 1-50ng/ml fibroblast growth factor 4,1-100ng/ml stem cell factor and 1-100ng/ml
FLT3L；
5) operation instructions；
Wherein, described operation instructions include the method described in embodiment 1-3.
Recombinate people's Oligodendrocyte precursor cells of GAP43/GGF2 slow-virus transfection of embodiment 5 becomes oligodendroglia
Cell breaks up
The OPCs taking the 10th generation restructuring GAP43/GGF2 slow-virus transfection is inoculated into and is equipped with 50 μ g/ml in advance
6 well culture plates of poly-D-lysine coating coverslips, induce liquid after cell grows to merge completely
(DMEM/F12 (1: 1) contains 10ng/ml NT3 (PeproTech company of the U.S.) and 50ng/ml
Beta-mercaptoethanol (American I nvitrogen company)), at 37 DEG C, 5%CO2Incubator is cultivated 24h, goes
Except induction liquid, add differentiation complete culture solution DMEM/F12 (1: 1) (containing 2mM L-glutaminate, 30ng/ml
Basic fibroblast growth factor, 5ng/ml fibroblast growth factor 4,10ng/ml stem cell factor and
5ng/ml FLT3L and 10% hyclone), at 37 DEG C, 5%CO2Incubator
Middle cultivation, changes liquid 1 time every 72h, cultivates 7 day time.Will be thin with 4% paraformaldehyde after cultivating 7 days
Born of the same parents, with fixing, carry out glial fibrillary acidic albumen (glial fibrillary acidic protein, GFAP) and exempt from
Epidemic disease cytochemistry checks.
GAP43/GGF2-OPCs after 4% cold paraformaldehyde fixes 15 minutes, lucifuge.Suck poly
After formaldehyde, wash three times with ice PBS, each 5 minutes.0.5%Triton X-100 covers cell 10 minutes,
Ice PBS washes three times, each 5 minutes.The import fetal lamb serum room temperature of identical host is resisted to close 30 points with two
Clock.Preparation one resists: the anti-human GFAP of rabbit (Millipore company of the U.S.) fetal lamb serum-dilution 200 times,
Adding an anti-covering cell, tinfoil 4 degree of lucifuges of parcel are overnight.Next day: take out cell and warm to room temperature again about
1h.Ice 1 ‰ Tween washes twice, each 5 minutes, in shaking table.Ice PBS washes once, 5 minutes, in
Shaking table.Preparation fluorescent labeling two resists: goat anti-rabbit immunoglobulin G (the Goat anti-rabbit of PE labelling
IgG (H&L) TRITC, Abcam company of the U.S., Ab50598), prepare with PBS or FBS, concentration
1∶200.Add two to resist, incubated at room 1 hour (lucifuge).Ice 1 ‰ Tween washes twice, each 5 minutes,
In shaking table.Ice PBS washes once, 5 minutes, in shaking table.DAPI contaminates core, 1, every ware, is completely covered
Live cell.Ice 1 ‰ Tween washes twice, each 5 minutes, in shaking table.Ice PBS washes once, and 5
Minute, in shaking table.Add anti-fluorescent quenching mountant, lucifuge.Upper machine copolymerization Jiao Mianyiyingguangxianweijingguan
Survey, take pictures.
Fig. 4 is that to be divided into oligodendroglia thin for the OPCs of the 10th generation restructuring GAP43/GGF2 slow-virus transfection
Born of the same parents' immunofluorescence figure.Figure is the oligodendrocyte immunofluorescence figure that 4 times of object lens are observed, and can observe in figure
To the oligodendrocyte of green starlike GFAP dyeing, show that the 10th generation restructuring GAP43/GGF2 is slow
The OPCs of virus transfection can be divided into oligodendrocyte.
Embodiment 6 rats with spinal cord injury model GAP43/GGF2-OPCs transplants experiment in vivo
Purchased from 20 experiment SD rats of Military Medical Science Institute's animal center, with 3% pentobarbital sodium
After (30mg/kg body weight) intraperitoneal injection of anesthesia rat, ventricumbent position is fixed on operating-table, depilation, sterilization,
Drape, positions T12 with rat back highest point and hits exactly way of escape longitudinal incision, being about 1.5cm, to both sides
Retract paraspinal muscle, appear T12 spinous process and vertebral plate, sting except T12 spinous process and vertebral plate, form rear diameter 4.0mm
Bone window, fully expose canalis spinalis and spinal dura mater, as deep as canalis spinalis spinal dura mater, with a heavy 10.0g circular metal
Impact rod (metal impact rod lower end diameter about 2.5mm, almost coincide with spinal cord diameter), by internal diameter be
The hollow pipe of 4.0mm, away from spinal cord 7cm eminence, makes metallic rod specify position, Vertical Free at hollow pipe
Falling, cause spinal cord injury, locally spinal cord hematoma shows and hits successfully, and observing whether the double hind leg of Mus have stress
Reflection, and row somatosensory evoked potential detection after surgery, assess the credibility of spinal cord bump test further, hit
Hit unsuccessfully animal to be rejected.
The rats with spinal cord injury model set up is randomly divided into A, B and C tri-groups, often group 6, and A group is real
Execute the OPCs transplantation group of the restructuring GAP43/GGF2 slow-virus transfection of example 3 preparation, B group embodiment 2
The OPCs transplantation group of preparation, C group is normal saline transplantation group.
Rats with spinal cord injury model sub-cage rearing is after 72 hours, and A group carries out tail vein transplantation
GAP43/GGF2-OPCs 2×106/ kg, 100 μ l；B group carries out tail vein transplantation OPCs 2 × 106/ kg,
100μl；C group carries out tail vein injection 100 μ l normal saline；Transplant the same day, 1d, 3d, 5d, 7d, 14d,
28d carries out Basso Beattie Bresnahan (BBB) motor function scores, result such as following table to all rats
Shown in 1.
Table 1
Illustrate: BBB motor function scores 0-7 divides the judge each joint motion of animal hind leg；8-13 divides judge
The gait of hind leg and coordination function；14-21 divides the fine movement passing judgment on motion median claw, and three full marks are 21
Point, the highest damage of score value is the least.
Can find out from table 1, transplant latter 3 days start C group compared with A or B group * p value and#P value is equal
Much smaller than 0.05, and transplant latter 14 days start A group compared with B group p value be much smaller than 0.05；A and B
The BBB scoring of group is apparently higher than C group, and wherein the BBB of A group marks apparently higher than B group, is i.e. used for
The OPCs of the GAP43/GGF2 slow-virus transfection of the present invention damaged that grafts spinal cord has the treatment of excellence
Effect, and immunologic rejection is the least, the OPCs of GAP43/GGF2 slow-virus transfection and the OPCs of untransfected
The neurological functional recovery of rats with spinal cord injury, the wherein OPCs of GAP43/GGF2 slow-virus transfection are promoted
Effect is more notable.
Embodiment 7 rats with spinal cord injury model GAP43/GGF2-OPCs transplants cortical somatosensory evoked potential
Analyze
The rats with spinal cord injury model of Example 5, through the restructuring GAP43/GGF2 of embodiment 3 preparation
After the OPCs of the OPCs of slow-virus transfection and embodiment 2 preparation transplants, apply evoked potentuial measuring system (Shanghai
Sea God medical electronics company limited) detection, stimulating electrode is placed in the middle part of tibialis anterior, and reference electrode is placed in
At the 1cm of distally, stimulus intensity is 1.5～4mA, ripple width 0.2mA, and the continuous square wave of frequency 1.9Hz stings
Swash superposition 20～40 times.Recording electrode is placed under head center line and coronal suture point of intersection scalp, with reference to electricity
Pole is placed at the 0.5cm of its rear.The animal afterbody that normal saline soaks is close to by ground wire.With rat hindlimb
Occur that slight twitch is as the criterion, before observing rat wound respectively, transplant the same day, transplant after 14d and 28d after transplanting
Time cortical somatosensory evoked potential (cortical somatosensory evoked potentials, CSEP) P1-N1 ripple
The change of width.
Table 2
From table 2 it appeared that after Spinal Cord Injury in Rats, CSEP wave amplitude substantially reduces the same day.14d after transplanting,
The wave amplitude of A group substantially rises, and compared with B group and C group, difference has statistical significance (* p ＜ 0.05).
28d after transplanting, C group CSEP wave amplitude substantially rises, and statistically significant with comparing difference during 14d
(&p ＜ 0.05)；A group CSEP wave amplitude is significantly raised compared with during 14d simultaneously, and difference is statistically significant
(P ＜ 0.05), the most statistically significant compared with the B group same period and C group (* p ＜ 0.05)；And B group is moved
Plant rear 14d and 28d CSEP wave amplitude significantly raised, the most statistically significant compared with same period C group
(#p ＜ 0.05).Show that the GAP43/GGF2-OPCs injury rats that grafts spinal cord has neurological functional recovery
Effect, and significantly better than simple OPCs.
Embodiment 8 rats with spinal cord injury model GAP43/GGF2-OPCs transplants glial scar analysis
In embodiment 5, rats with spinal cord injury model is after OPCs transplants 28 days, at the neck that broken by rat after death
Take out 1cm myeloid tissue immediately, 4% paraformaldehyde is fixed 48 hours.Paraffin embedding, myeloid tissue
Section (5 μm), carries out SABC detection GFAP expression.Paraffin section sticks to poly-D-lysine
On the slide processed, place at room temperature before dewaxing 60 minutes.Paraffin section dimethylbenzene I successively, 10
Minute；Dimethylbenzene II, 10 minutes；Dehydrated alcohol, 5 minutes；95% ethanol, five minutes；75% ethanol,
Five minutes；50% ethanol, five minutes；Deionized water, five minutes.PBS wash 3 times each 5 minutes.Sample
Digest 30 minutes in 37 DEG C of wet boxes with 0.1% pancreatin and carry out antigen retrieval；3 are washed with PBS after abandoning enzyme liquid
Secondary each 5 minutes；3%H2O2 deionized water processes 10 minutes, makes endogenous peroxidase inactivate.PBS washes
3 times each 5 minutes, drips an anti-rabbit anti-Mus GFAP (1: 500) (Millipore company of the U.S.) 50 μ l, 4 DEG C
Overnight after PBS wash 3 times each 5 minutes；The anti-mouse two of dropping alkali phosphatase enzyme mark resists
(1: 1000) 50 μ l (buys Beijing Zhong Shan company), and 37 DEG C stand 1 hour；PBS wash 3 times each 5 minutes, drip
Add fresh configuration DAB to develop the color 10 minutes, grasp dye levels under the microscope；PBS rinses 10 minutes；
Haematoxylin redyeing 2 minutes, hydrochloride alcohol breaks up；Tap water rinses 10 minutes；Dehydration, transparent, neutral
Resin mounting, microscope is observed and is taken pictures.
Sections and A and B group GFAP is damaged from Fig. 5 it may be seen that at C group 28d spinal cord injury
Positive cell is expressed more, and positive cell cell space is bigger, and projection is the most longer, and mutually tangling is woven into
Netted, form fine and close glial scar；In A group, GFAP positive cell expresses quantity when 28d and B
Group and C group more all significantly reduce, and GFAP positive cell volume diminishes, and projection tails off, and dyeing becomes
Light, glial scar scope diminishes, and shows that having transplanted GAP43/GGF2-OPCs treatment rats with spinal cord injury presses down
Astrocytes processed and glial scar are formed.
Claims (10)
1. suppress a preparation method for people's Oligodendrocyte precursor cells (OPCs) of neural secondary damage, its
Being characterised by, described method includes the virus expression carrier building two glial growth factor, is packaged into
After virus, cotransfection people's Oligodendrocyte precursor cells, being prepared as can these two glial growth factor of high expressed
Oligodendrocyte precursor cells, its without become tumor activity；Wherein, described glial growth factor is that growth phase is closed
The factor 43 (GAP-43) and glial growth factor 2 (GGF-2)；
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfecting restructuring GAP-43
After the OPCs Secondary Culture of GGF-2 viral vector to the 10th generation, it expresses GAP-43 and GGF-2
Gene is above 1st generation OPCs of untransfected, the highest 45 times and 67 times；
In rats with spinal cord injury model, the OPCs of GAP43/GGF2 virus cotransfection transplants latter 28 days BBB
(Basso Beattie Bresnahan) motor function scores improved values is 20.00 ± 5.00, and cortex body-sensing induces
Current potential CSEP wave amplitude improved values 6.00 ± 1.00；
Described GAP43/GGF2-OPCs is in treatment rats with spinal cord injury, and its suppression astrocyte increases
Grow and formed with glial scar.
Method the most according to claim 1, it is characterised in that by polymerase chain reaction (PCR)
Technology amplification growth correlation factor 43 and glial growth factor 2cDNA sequence, restructuring is to table respectively
Reach on plasmid, be connected on virus expression carrier through restriction enzyme site, two gene recombined virus carrier cotransfections
People's Oligodendrocyte precursor cells, it is thus achieved that multiplication capacity strengthens and people's Oligodendrocyte precursor cells of differentiation capability.
3. according to the method described in any one of claim 1-2, it is characterised in that described oligodendroglia ancestral
Cell derived people discards what Cord blood, placental blood, umbilical cord or autologous bone marrow In vitro culture obtained, it is preferable that
Derive from people's autologous bone marrow.
4. according to the method described in any one of claim 1-3, it is characterised in that described oligodendroglia ancestral is thin
Born of the same parents' preparation method is as follows:
Take from 20ml bone marrow through the isolated and purified mononuclearcell obtained of lymphocyte separation medium, use serum-free
Oligodendrocyte culture medium is resuspended to 2-5 × 106/ ml, draws 10ml and joins 10-200 μ g/ml poly
The coated 75cm of lysine2In Tissue Culture Flask, in 37 DEG C, 5%CO2Cell culture incubator is cultivated 48
Hour；The serum-free oligodendrocyte culture medium changing fresh factor-containing is cultivated the most continuously, until cell is raw
Long fusion reaches 90%, and using mass volume ratio is 0.25% to be that pancreatin digests, and carries out Secondary Culture,
I.e. 1 bottle passes on 2 bottles, and the serum-free oligodendrocyte culture medium supplementing fresh factor-containing continues to cultivate, and obtains
Obtain people's Oligodendrocyte precursor cells；Wherein, described cell culture medium is preferably DMEM/F12, its containing 1%N2,
2mM L-glutaminate, 5-100ng/ml basic fibroblast growth factor, 1-50ng/ml become fiber growth because of
Son 4,1-100ng/ml stem cell factor and 1-100ng/ml FLT3L；Institute
State pancreatin preferably containing mass volume ratio is the EDTA of 0.02%；
People's Oligodendrocyte precursor cells detects through Flow Cytometry, OPCs mark A285, O4 and Sox10
All express more than 95%.
5. the preparation method of the people's Oligodendrocyte precursor cells suppressing neural secondary damage, it is characterised in that
Under people GAP-43 and GGF-2 sequence are expanded from normal human peripheral blood's genomic DNA by PCR
Come, by the fragment of GAP-43 and GGF-2 mesh respectively with the pET28a carrier segments of XhoI/Nde I double digestion,
Under T4DNA ligase effect, in 4 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, convert large intestine
Carry out positive colony PCR qualification after bacillus competent cell DH5a and order-checking is identified；PCR primer gel electricity
After swimming detection and order-checking qualification meet GAP-43 and GGF-2 size and sequence, by checking order, correct bacterium solution turns
Being connected in the 10ml LB fluid medium containing corresponding antibiotic, 37 DEG C of overnight incubation, with without endotoxin plasmid
Little carry middle amount test kit and carry out plasmid extraction, extract qualified recombiant plasmid and place in-80 DEG C of ultra cold storage freezers
Long-term preservation；Described GAP-43 and GGF-2 genetic fragment size is respectively 706bp and 1268bp；
Respectively GAP-43, GGF-2DNA fragment and GV358 carrier are carried out XhoI/Nde I double digestion,
Under T4DNA ligase effect, by GAP-43 fragment and enzyme action GV358 carrier and GGF-2 fragment and
Enzyme action GV358 carrier, respectively at 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, converts escherichia coli
After competent cell DH5a, carry out positive colony PCR qualification and order-checking is identified；
Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to each
The culture dish 6 × 10 of 10cm6Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in cultivate
Overnight；Remove culture fluid before transfection in second day, change 5ml Opti-MEM culture fluid；Take 9 μ g and pack mixed liquor
Add in 1.5ml Opti-MEM with 3 μ g slow virus expression plasmids, mix gently, take 36 μ l lipofectamine
2000 add in 1.5ml Opti-MEM, mix gently, and room temperature places 5min；Mixing plasmid solution and
Lipofectamine 2000 diluent, puts room temperature 20min；Mixed liquor is slowly added dropwise the cultivation to 293T cell
In liquid, mixing, in 37 DEG C, 5%CO2Cell culture incubator is cultivated；Discard containing transfection mixed after cultivating 6h
With the culture medium of thing, the PBS liquid adding 10ml cleans once, softly rocks culture dish with remaining the turning of washing
Abandon after dye mixture；It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO2
Continue in incubator to cultivate 48-72h；
According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged
10min, removes cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Point
Other trim sample, puts into Beckman supercentrifuge one by one by the ultracentrifugation pipe with vial supernatant
In, arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature controls at 4 DEG C；Centrifugal
After end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus and preserve liquid, the most instead
Blow and beat resuspended again；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence
Method measures titre, and packaged GAP-43 and GGF-2 slow virus, according to 50 μ l 2E+8TU/ml subpackages, protects
It is stored in-80 DEG C of ultra cold storage freezers；
Each to restructuring GAP-43 and GGF-2 slow virus 10 μ l 1E+7TU/ml are joined people oligodendroglia ancestral
In 10-200 coated six well culture plates of μ g/ml poly-D-lysine of cell, system is 2ml, mixing, 37 DEG C,
5%CO2Incubator in hatch 8-12 hour after, change complete culture solution DMEM/F12, described
Complete culture solution DMEM/F12 preferably becomes containing 1%N2,2mM L-glutaminate, 5-100ng/ml alkalescence
Fibroblast growth factor, 1-50ng/ml fibroblast growth factor 4,1-100ng/ml stem cell factor and
1-100ng/ml FLT3L；When cell growth fusion reaches 90%, with 0.25%
Trypsinization, proceeds to the 10-200 μ coated 25cm of g/ml poly-D-lysine2Culture bottle grows, 1 hole pair
Answer 1 culture bottle, at 25cm2Culture bottle merges and when reaching 90%, continues had digestive transfer culture cultivation；Growth turns
Dye is divided into 3 groups: untransfected matched group, blank slow-virus transfection group, cotransfection GAP-43 and GGF-2
Slow-virus transfection group；
Infect latter 72 hours fluorescence microscopy Microscopic observation green fluorescent proteins (Green fluorescent protein,
GFP) luminous situation, GFP develops the color more than more than 90%, shows to have transfected GAP-43 and GGF-2
Slow-virus transfection people's Oligodendrocyte precursor cells success, it is thus achieved that the people of continuous expression GAP-43 and GGF-2 is few
Prominent glial progenitor cell；
Detected according to operation instructions by PCR kit for fluorescence quantitative, transfected GAP-43 and GGF-2
Slow-virus transfection people's Oligodendrocyte precursor cells Secondary Culture is to after the 10th generation, and it expresses GAP-43 and GGF-2
Gene is respectively higher than the OPCs 45.89 times and 67.98 times of untransfected；
Wherein, the preparation method of described Oligodendrocyte precursor cells is as claimed in claim 4.
6. the preparation method as described in any one of claim 1-5, it is characterised in that
The OPCs having transfected the transfection of GAP-43 and GGF-2 slow virus carrier passes through complete culture solution
DMEM/F12 subculture in vitro separately cultivate, when cell is cultivated the 5-6 days, cell growth fusion reach 90% with
On, need the trypsinization using mass volume ratio to be 0.25% to pass on；
Take Secondary Culture to the cellular morphology of observation, untransfected under 3 generations and 10 generation OPCs inverted microscopes
OPCs to OPCs of other transfection genes of interest, there is similar spider cell form, can break up few
Prominent glial cell.
7. according to the method described in any one of claim 1-6, it is characterised in that described people's oligodendroglia
CFU-GM high expressed growth correlation factor 43 and glial growth factor 2, fall after Secondary Culture to 10 generations
Put basis of microscopic observation and still there is stem cell morphology；Secondary Culture reaches 500 to the 10th its proliferation times of generation
More than Bei.
8. people's Oligodendrocyte precursor cells of a GAP43/GGF2 slow-virus transfection of recombinating becomes oligodendroglia thin
The method of born of the same parents' differentiation, it is characterised in that
The OPCs taking the 10th generation restructuring GAP43/GGF2 slow-virus transfection is inoculated into and is equipped with 50 μ g/ml in advance
6 well culture plates of poly-D-lysine coating coverslips, induce liquid after cell grows to merge completely
DMEM/F12, at 37 DEG C, 5%CO2Incubator is cultivated 24h, removes induction liquid, add and broken up
Full culture fluid DMEM/F12, at 37 DEG C, 5%CO2Incubator is cultivated, changes liquid 1 time every 72h,
Cultivate 7 day time；Cell is used fixing with 4% paraformaldehyde after cultivating 7 days, carry out neuroglia fibres
Acidic protein (glial fibrillary acidic protein, GFAP) immunocytochemistry inspection；
GAP43/GGF2-OPCs after 4% cold paraformaldehyde fixes 15 minutes, lucifuge；Suck poly
After formaldehyde, wash three times with ice PBS, each 5 minutes；0.5%Triton X-100 covers cell 10 minutes,
Ice PBS washes three times, each 5 minutes；The import fetal lamb serum room temperature of identical host is resisted to close 30 points with two
Clock；Preparation one resists: rabbit anti-human GFAP fetal lamb serum-dilution 200 times, adds an anti-covering cell, stannum
Paper bag wraps up in 4 DEG C of lucifuges overnight；Next day: take out cell and warm to room temperature about 1h again；Ice 1 ‰ Tween washes twice,
Each 5 minutes, in shaking table；Ice PBS washes once, 5 minutes, in shaking table；Preparation fluorescent labeling two resists:
The goat anti-rabbit immunoglobulin G of PE labelling, prepares with PBS or FBS, concentration 1: 200；Add two to resist,
Incubated at room 1 hour, lucifuge；Ice 1 ‰ Tween washes twice, each 5 minutes, in shaking table；Ice PBS
Wash once, 5 minutes, in shaking table；DAPI contaminates core, 1, every ware, covers all cell；Ice
1 ‰ Tween wash twice, each 5 minutes, in shaking table；Ice PBS washes once, 5 minutes, in shaking table；
Add anti-fluorescent quenching mountant, lucifuge；Upper machine copolymerization Jiao's immunofluorescence microscopy is observed, is taken pictures；Result
Display, it can be observed that the oligodendrocyte of green starlike GFAP dyeing, shows the restructuring of the 10th generation
The OPCs of GAP43/GGF2 slow-virus transfection can be divided into oligodendrocyte；
Wherein, described GAP43/GGF2-OPCs uses claim 1-Prepared by method described in 7 any one.
9. the people of the neural secondary damage of suppression that a kind is prepared as described in any one of claim 1-8 dashes forward glue less
The test kit of mesenchymal progenitor cell, it is characterised in that described test kit includes:
1) people's Oligodendrocyte precursor cells basal medium；
2) packaged growth correlation factor 43 and glial growth factor 2 high expressed virus；
3) enzyme of peptic cell；
4) cytokine and；
5) operation instructions；
Wherein, described operation instructions include the method described in any one of claim 1-8.
10. the people's Oligodendrocyte precursor cells promoting CO2 laser weld as described in any one of claim 1-9 and
Its application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610034981.0A CN106011173B (en) | 2016-01-20 | 2016-01-20 | Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610034981.0A CN106011173B (en) | 2016-01-20 | 2016-01-20 | Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106011173A true CN106011173A (en) | 2016-10-12 |
| CN106011173B CN106011173B (en) | 2020-08-11 |
Family
ID=57082711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610034981.0A Active CN106011173B (en) | 2016-01-20 | 2016-01-20 | Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106011173B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109694882A (en) * | 2018-12-27 | 2019-04-30 | 吉林大学 | The schwann cell of application, the improvement of miR comprising 5 ' end specific seed base sequences and its application |
| CN110951734A (en) * | 2019-12-16 | 2020-04-03 | 南通大学 | RNA inhibitor and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20230159890A1 (en) * | 2021-10-20 | 2023-05-25 | University Of Rochester | Treatment with genetically modified cells, and genetically modified cells per se, with increased competitive advantage and/or decreased competitive disadvantage |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995032724A1 (en) * | 1994-05-26 | 1995-12-07 | Ludwig Institute For Cancer Research | Glial mitogenic factors, their preparation and use |
| CN103215225A (en) * | 2012-01-18 | 2013-07-24 | 孙勇 | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell |
| CN105920617A (en) * | 2016-06-13 | 2016-09-07 | 南通大学 | Application of transcription factor Fo x O3 in preparing cerebral ischemia reperfusion nerve injury drug |
-
2016
- 2016-01-20 CN CN201610034981.0A patent/CN106011173B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995032724A1 (en) * | 1994-05-26 | 1995-12-07 | Ludwig Institute For Cancer Research | Glial mitogenic factors, their preparation and use |
| CN103215225A (en) * | 2012-01-18 | 2013-07-24 | 孙勇 | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell |
| CN105920617A (en) * | 2016-06-13 | 2016-09-07 | 南通大学 | Application of transcription factor Fo x O3 in preparing cerebral ischemia reperfusion nerve injury drug |
Non-Patent Citations (7)
| Title |
|---|
| L.C.PLANTINGA ET AL.: ""The expression of B-50/GAP-43 in Schwann cells is upregulated in degenerating peripheral nerve stumps following nerve injury"", 《BRAIN RESEARCH》 * |
| MICHAE¨L HOCQUEMILLER ET AL.: ""GAP43 Overexpression and Enhanced Neurite Outgrowth in Mucopolysaccharidosis Type IIIB Cortical Neuron Cultures"", 《JOURNAL OF NEUROSCIENCE RESEARCH》 * |
| P.-N. MOHANNA ET AL.: ""A composite poly-hydroxybutyrate–glial growth factor conduit for long nerve gap repairs"", 《J. ANAT.》 * |
| 俞志明等: ""神经胶质生长因子促进受损周围神经功能恢复"", 《现代康复》 * |
| 刘玲等: ""神经生长相关蛋白在脑损伤修复中的作用"", 《中国病理生理杂志》 * |
| 杨娟等: ""生长相关蛋白 43( GAP43) 基因修饰的骨髓间充质干细胞移植对大鼠视网膜变性疾病的治疗作用"", 《细胞与分子免疫学杂志》 * |
| 胡建国等: ""大鼠GAP-43基因的克隆及其在COS-7细胞的表达"", 《蚌埠医学院学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109694882A (en) * | 2018-12-27 | 2019-04-30 | 吉林大学 | The schwann cell of application, the improvement of miR comprising 5 ' end specific seed base sequences and its application |
| CN110951734A (en) * | 2019-12-16 | 2020-04-03 | 南通大学 | RNA inhibitor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106011173B (en) | 2020-08-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018103406A1 (en) | Neural stem cell injection for treating brain damage diseases and preparation method and use method thereof | |
| CN103702674B (en) | Composition, purposes and the preparation of platelet cracking content | |
| US8697048B2 (en) | Peptide amphiphile suspension to prevent or reduce tumor formation from administered embryonic stem cells | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| EP2076588B1 (en) | Expansion method for adult stem cells from blood, particularly peripheral blood, and relative application in medical field | |
| CN100516200C (en) | Adipose tissue-derived stromal cells for the repair of corneal and intra-orbital defects and uses thereof | |
| RU2005105065A (en) | STEM ROOF CELLS AND METHODS FOR TREATING THEM WITH THE USE OF EYE DISEASES ASSOCIATED WITH VASCULAR NEW FORMATION | |
| CN105154407B (en) | Preparation method and application of human adipose-derived stem cells with improved skin repair function | |
| CN101801396A (en) | methods and compositions for treating neural degeneration | |
| CN103865956B (en) | Utilize the method that recombinant slow virus induced nerve stem cells breaks up to dopaminergic neuron | |
| CN106163572A (en) | For compositions that the direct transdifferentiation of inducing somatic is vascular progenitor and application thereof | |
| EP2530144A2 (en) | Composition comprising cell and biocompatible polymer | |
| CN103191445A (en) | Use of mesenchymal stem cell and preparation method thereof | |
| JP2019532636A (en) | Photogenetically transformed photoreceptor precursor cells for use in the treatment of retinal degenerative diseases | |
| CN106011173A (en) | Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof | |
| CN106459904A (en) | Method for generating endothelial colony forming cell-like cells | |
| Hu et al. | Transplantation site affects the outcomes of adipose-derived stem cell-based therapy for retinal degeneration | |
| CN105255836A (en) | Method for preparing person stem cells with improved neural restoration function and application of person stem cells | |
| KR20180122288A (en) | 3D bioprinting construct using human nasal inferior turbinate derived mesenchymal stem cell and uses thereof | |
| CN114209814B (en) | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion | |
| CN108753730A (en) | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application | |
| CN108144120B (en) | Bifunctional collagen scaffold material, preparation method thereof and application thereof in spinal cord injury repair | |
| CN106399318A (en) | Method for modifying cells by virtue of oxygen-regulated basic fibroblast growth factor | |
| Hu et al. | The impact of bone marrow-derived mesenchymal stem cells on neovascularisation in rats with brain injury | |
| CN110172442A (en) | A kind of people's induced pluripotent stem cells, construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huang Qiming Inventor after: Wang Xu Inventor after: Wang Yudong Inventor before: Li Yimin Inventor before: Li Yihan Inventor before: Chen Zongming |
|
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170703 Address after: 330008, Jiangxi Nanchang Nanchang hi tech Industrial Development Zone, AI Sihu North Road No. 3 workshop No. 688, west section of the 2 Applicant after: Jiangxi Austria Biological Technology Co. Ltd. Address before: 100070 Beijing city Fengtai District East No. 249 Building 2 Room 301 Applicant before: Beijing Xipusheng International Biotechnology Co., Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |