CN107904258A - A kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property - Google Patents

A kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property Download PDF

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CN107904258A
CN107904258A CN201711085893.4A CN201711085893A CN107904258A CN 107904258 A CN107904258 A CN 107904258A CN 201711085893 A CN201711085893 A CN 201711085893A CN 107904258 A CN107904258 A CN 107904258A
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nsc
cell
cerebral infarction
genes
rat
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高山
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SHANGHAI FENGXIAN CENTRL HOSPITAL
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    • 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
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention belongs to medical field, disclose a kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property, NSC is separated from neonate rat forebrain tissue, utilize flow cytometer and single cell clone technology, the characteristic of its self-renewing and pluripotent differentiation is shown by cultured and amplified in vitro, and by rAAV mediation EPO genes and LacZ gene transfers, establish the NSC transfection systems for stablizing expression EPO genes and LacZ genes and analyze NSC transfection systems in vitro, internal express transgenic protein status；NSC transfection systems through EPO genes and LacZ genetic modifications are implanted into rat cerebral infarction ischemic focus, analyze the repair of EPO genes and LacZ gene pairs rat cerebral infarction ischemic focuses.EPO modifications NSC transplanting of the present invention effectively promotes the recovery of cerebral ischemia convalescence nervous function, improves therapeutic effect.

Description

A kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property
Technical field
The invention belongs to medical field, more particularly to a kind of rat model structure for verifying treatment cerebral infarction ischemia drugs pharmacological property Construction method, it is particularly applicable to hematopoietin and modifies neural stem cells transplantation to experimental rat cerebral infarction ischemic focus reparation The analysis of effect.
Background technology
Cerebral arterial thrombosis refers to feeding artery (arteria carotis and vertebral artery) narrow or inaccessible, cerebral insufficiency due to brain The general name of caused brain tissue necrosis.There is the cerebral ischemia of four types：Transient ischemic attack (TIA)；Reversible nerve work( Can obstacle (RIND)；Advancing stroke (SIE)；Complete stroke (CS).TIA exists without cerebral infarction, and RIND, SIE and CS have Different degrees of cerebral infarction exists, and positive medical treatment should be given to the patients with cerebral ischemic of early stage, it is therefore intended that prevents The further development of cerebral ischemia.Mitigate cerebral lesion.Should be according to patient's different pathogeny, pathogenesis, Clinical types, disease time etc. Situation determines therapeutic scheme, gives individualized treatment.However, the existing treatment to cerebral arterial thrombosis, it is impossible to be effectively facilitated The recovery of cerebral ischemia convalescence nervous function, causes therapeutic effect poor.
In conclusion problem existing in the prior art is：The existing treatment to cerebral arterial thrombosis, it is impossible to be effectively facilitated The recovery of cerebral ischemia convalescence nervous function, causes therapeutic effect poor；The prior art is by being subject to theory to be limited, target gene table The albumen receipts technology reached limits the performance that cannot have certain abundance, thus improve and be very necessary to significantly express.
The content of the invention
In view of the problems of the existing technology, the big for the treatment of cerebral infarction ischemia drugs pharmacological property is verified the present invention provides a kind of Mouse model construction method.The present invention can effectively show the abundance of target gene albumen by transgenic virus carrier.Purpose Gene includes EPO genes and LacZ genes.
The present invention is achieved in that the present invention follows gene expression theory, and dual gene expression abundance is better than single base Because of expression, expressed substrate can more preferably play biological effects.
The present invention provides a kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property, including：
Step 1, NSC is separated from neonate rat forebrain tissue, using flow cytometer and single cell clone technology, is led to The characteristic that cultured and amplified in vitro shows its self-renewing and pluripotent differentiation is crossed, and by rAAV mediation EPO genes and LacZ Gene transfer, establish stablize expression EPO genes and LacZ genes NSC transfection systems and analyze NSC transfection systems in vitro, body Interior express transgenic protein status；
Step 2, rat cerebral infarction ischemic focus is implanted into by the NSC transfection systems through EPO genes and LacZ genetic modifications, Analyze the repair of EPO genes and LacZ gene pairs rat cerebral infarction ischemic focuses；And further analyze through EPO genes and LacZ Therapy mechanism of the stem cell of the genetic modifications such as gene to rat cerebral infarction.
Further, NSC is separated in the forebrain tissue from neonate rat includes：
The original cuiture of NSC：
(1) draw materials：Newborn SD suckling mouse in 24h, cervical dislocation are put to death, and 75% alcohol soaking disinfection, divest skull and meninx, Forebrain tissue is isolated under disecting microscope；
(2) digest：Using mechanical digestion method, by brain tissue, sharp property is cut on culture dish, is transferred to the centrifugation of 10ml cells Pipe, adds PBS to 3ml, stands 2min after careful piping and druming, suction out and collect supernatant, in the centrifuge tube containing precipitate The PBS liquid of same volume is added, blows and beats again, so repeatedly, is gradually reduced tissue volume；
(3) centrifuge：75g centrifuges 10min or 900 turn/min centrifugations 5min；Abandon supernatant；NSC training liquid is added, piping and druming is equal Even, concentration is simultaneously adjusted to 1*10 by cell count6/ml；
(4) cultivate：Cell suspension is transferred in 24ml Tissue Culture Flasks, is placed in 37 DEG C, 5%CO2Cultivated in incubator.
Further, NSC is separated in the forebrain tissue from neonate rat to further include：
Passage：
When 50% cell mass>Passed on during 30 cells；Cell suspension is transferred to 10ml centrifuge tubes, with 110g from Heart 10min, abandons supernatant.Add 2ml nutrient solutions；5min, transfer supernatant to blake bottle are stood after soft piping and druming；In centrifuge tube 2ml training liquid is added, repetitive operation to cell mass disappears；
Concentration is simultaneously adjusted to 1*10 by postdigestive cell suspension cell count6/ ml, is placed in 37 DEG C, 5%CO2In incubator Culture；
Continuous passage is more than 18 times, until removing all dead cell, cell fragment and irregular cell agglomerates, formation rule Nerve ball.
Further, the screening of flow cytometer includes：
Heochst33342 dye liquors：The storing liquid concentration of 10ug/ml is made into PBS, 4 DEG C are kept in dark place, suspension growth Cell adds Heochst33342, final concentration of 1ug/ml in the state of culture；After 37 DEG C are incubated 1h, low temperature 500-1000r/ Min centrifugations 5min discards dye liquor, is stored in after washing 2 times with PBS in ice；Then using fluidic cell separation SP cells.
Further, the single cell clone includes：
Using being transferred to after limiting dilution assay diluting cells suspension in 96 orifice plates；
Analyze and select the culture hole containing individual cells；
37 DEG C are placed in, 5%CO2Cultivated in incubator, the cell for selecting active growth formation nerve ball is passed on, and is expanded Culture.
Further, the method for the rAAV mediation EPO genes and LacZ gene transfers includes：
The design of the primer of gene transfer and the expression of synthesis, amplification and rAAV-EPO and rAAV-LacZ.
Further, the NSC transfection systems for establishing stable expression EPO genes and LacZ genes include：
The NSC transfection systems of culture are cloned with 2*106/ ml density is placed in 10cm culture dishes, and adds 2*1010urAAV- EPO or rAAV-LacZ, new nutrient solution is converted to after transfecting 12h, continues to cultivate 72h.
Further, the analysis NSC transfection systems in vitro, internal express transgenic protein status using histochemistry and Westem blot verify the expression of transgene protein, including：
X-gal is dyed：
Nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO2Trained in incubator Foster 2h makes cell attachment；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min；
0.01%PBS develops a film 3 times and dries, and adds 0.1%Triton ruptures of membranes 10min；
0.01%PBS develops a film 3 times and dries, and adds X-gal analysis cellular colours；
1*10 is transplanted in rat brain respectively11NSC, the NSC for transfecting LacZ genes and the NSC for transfecting EPO genes, after 2 days Implantation site brain tissue and the NSC supernatants of culture is taken to carry out the expression of Westernblot detection EPO genes and LacZ genes.
Further, the rat model construction method of the verification treatment cerebral infarction ischemia drugs pharmacological property further includes：NSC reflects It is fixed：Including：
Nestin is dyed：
Nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO2Trained in incubator Foster 2h makes cell attachment；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min；
0.01%PBS develops a film 3 times and dries；
Add 0.1%Triton ruptures of membranes 10min；
0.01%PBS develops a film 3 times and dries；
Add 2%BSA and close 30min at room temperature；
Add the anti-nestin antibody of mouse, in 4 DEG C；
0.01%PBS develops a film 3 times and dries up, and adds fluorescence secondary antibody sheep mouse IgG.CY3；
Lucifuge 2h and dry, analyzed under fluorescence microscope at room temperature.
Further, cell differentiation is carried out after NSC identifications：
Nerve ball is suctioned out and is transferred in 24 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, is trained in 5%CO2 incubators Foster 2h makes cell attachment；
Cell culture fluid is absorbed, is added per hole containing 20% hyclone nutrient solution 1ml, 37 DEG C, is trained in 5%CO2 incubators Support 7 days；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min, 0.01%PBS develops a film 3 times and dries in the air It is dry；
0.1%Triton ruptures of membranes 10min is added, 0.01%PBS develops a film 3 times and dries；
Add 2%BSA and close 30min at room temperature；
The anti-b-tubilinIII antibody of primary antibody mouse is added in 1,2 holes and exempts from anti-MAP2 antibody, in 4 DEG C overnight.0.01% PBS is rinsed 3 times and dried up, and adds fluorescence secondary antibody sheep anti-mouse igg-CY3 and goat anti-rabbit igg-FITC.Lucifuge 2h and dry at room temperature, Analyzed under fluorescence microscope；
GFAP antibody is added in 3 holes, is placed in 4 DEG C of refrigerator overnights.0.01%PBS is rinsed 3 times and dried up, and adds fluorescence secondary antibody Sheep anti-mouse igg-CY3, is analyzed under fluorescence microscope；
CNPase oligodendroglia mark antigen-antibodies are added in 3 holes, in 4 DEG C of refrigerator overnights.0.01%PBS is rinsed 3 times and dry up, add fluorescence secondary antibody sheep anti-mouse igg-CY3, analyzed under fluorescence microscope.
Advantages of the present invention and good effect are：The present invention is using adeno-associated virus (AAV) rotaring dyeing technology mediation EPO genes With Lacz gene transfers, NSC express transgenic albumen situations in position are analyzed, and the NSC of the genetic modification through purpose is moved Rat cerebral infarction ischemic focus is implanted into, further analyzes therapeutic effect of the stem cell transplantation to rat cerebral infarction, Primary Study is done carefully The mechanism of action of born of the same parents' transplantation treatment cerebral infarction, lays the foundation for the experimental analysis of further nervous system disease.EPO is repaiied Decorations NSC transplanting is effectively facilitated the recovery of cerebral ischemia convalescence nervous function, improves therapeutic effect.
The present invention initially sets up rat cerebral infarction ischemia model, then using transgenic technology that EPO modification nerve cords is thin Born of the same parents are simultaneously implanted into the big intracerebral of animal model, the nervous function of animal model from Point of View of Clinical, while pass through experimental method pair Bcl-2 protein expressions are detected, the potential mechanism of action having found that it is likely that, for cerebral ischemia clinical treatment new way is provided Footpath.
Brief description of the drawings
Fig. 1 is the rat model construction method flow that cerebral infarction ischemia drugs pharmacological property is treated in the verification that the present invention implements to provide Figure.
Fig. 2 is the separation, culture and transfection EPO gene expression analysis technologies for the neonate rat NSC that the present invention implements offer Route flow chart.
Fig. 3 is that the EPO joints NSC that the present invention implements to provide is transplanted to rat cerebral infarction ischemic focus repairing analysis technology path Flow chart.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
The present invention follows gene expression theory, and dual gene expression abundance is better than term single gene expression, and expressed substrate can More preferably to play biological effects.
The present invention is using adeno-associated virus (AAV) rotaring dyeing technology mediation EPO genes and Lacz gene transfers, and NSC is in body for analysis Express transgenic albumen situation in position, and the NSC of the genetic modification through purpose is implanted into rat cerebral infarction ischemic focus, further Analyze therapeutic effect of the stem cell transplantation to rat cerebral infarction, the mechanism of action of Primary Study cellular replacement therapy cerebral infarction.
Below in conjunction with the accompanying drawings and specific embodiment is further described the application principle of the present invention.
As shown in Figure 1, the present invention implements the rat model structure side of the verification treatment cerebral infarction ischemia drugs pharmacological property provided Method comprises the following steps：
S101：NSC is separated from neonate rat forebrain tissue, using flow cytometer and single cell clone technology, is passed through Cultured and amplified in vitro shows the characteristic of its self-renewing and pluripotent differentiation, and by rAAV mediation EPO genes and LacZ bases Because of transfer, establish the NSC transfection systems for stablizing expression EPO genes and LacZ genes and analyze NSC transfection systems in vitro, in vivo Express transgenic protein status.
S102：NSC transfection systems through EPO genes and LacZ genetic modifications are implanted into rat cerebral infarction ischemic focus, point Analyse the repair of EPO genes and LacZ gene pairs rat cerebral infarction ischemic focuses；And further analyze through EPO genes and LacZ bases Because waiting the stem cell of genetic modification to the therapy mechanism of rat cerebral infarction.
With reference to specific embodiment, the invention will be further described.
Embodiment：
First, the separation, culture and transfection EPO gene expression analysis of neonate rat NSC is carried out.
1st, the culture of NSC
1.1 original cuiture：
(1) draw materials：Newborn SD suckling mouse in 24h, cervical dislocation are put to death, and 75% alcohol soaking disinfection, divest skull and meninx, Disecting microscope bearing subdivision separates out forebrain tissue.
(2) digest：Using mechanical digestion method.By brain tissue, the sharp property cutting on culture dish, is transferred to the centrifugation of 10ml cells Pipe, adds PBS to 3ml, stands 2min after careful piping and druming, suction out and collect supernatant, in the centrifuge tube containing precipitate The PBS liquid of same volume is added, blows and beats again, so repeatedly, is gradually reduced tissue volume.
(3) centrifuge：75g centrifugations 10min (or 900 turns/min centrifugations 5min), abandons supernatant.NSC training liquid is added, piping and druming is equal Even, concentration is simultaneously adjusted to 1*10 by cell count6/ml。
(4) cultivate：Cell suspension is transferred in 24ml Tissue Culture Flasks, is placed in 37 DEG C, 5%CO2Cultivated in incubator.
1.2 passage
(1) when 50% cell mass>Passed on during 30 cells.Cell suspension is transferred to 10ml centrifuge tubes, with 110g centrifuges 10min, abandons supernatant.Add 2ml nutrient solutions；5min, transfer supernatant to blake bottle are stood after soft piping and druming；From 2ml training liquid is added in heart pipe, aforesaid operations to cell mass is repeated and disappears.
(2) postdigestive cell suspension cell count and concentration is adjusted to 1*106/ ml, is placed in 37 DEG C, 5%CO2Culture Cultivated in case.
(3) continuous passage more than 18 times, until removing all dead cell, cell fragment and irregular cell agglomerates, form The nerve ball of rule.
1.3 flow cytometers screen
Heochst33342 dye liquors：The storing liquid concentration of 10ug/ml is made into PBS, 4 DEG C are kept in dark place, suspension growth Cell adds Heochst33342, final concentration of 1ug/ml in the state of culture；After 37 DEG C are incubated 1h, low temperature 500-1000r/ Min centrifugations 5min discards dye liquor, is stored in after washing 2 times with PBS in ice.Then using fluidic cell separation SP cells.
1.4 single cell clone
(1) it is transferred to after limiting dilution assay diluting cells suspension in 96 orifice plates.
(2) observe and select the culture hole containing individual cells.
(3) 37 DEG C are placed in, 5%CO2To be cultivated in incubator, the cell for selecting active growth formation nerve ball is passed on, Amplification cultivation.
2nd, NSC is identified
2.1nestin dyeing
(1) nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO2In incubator Culture 2h makes cell attachment.
(2) cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min.
(3) 0.01%PBS develops a film 3 times and dries.
(4) 0.1%Triton ruptures of membranes 10min is added.
(5) 0.01%PBS develops a film 3 times and dries.
(6) add 2%BSA and close 30min at room temperature.
(7) the anti-nestin antibody of mouse is added, in 4 DEG C.
(8) 0.01%PBS develops a film 3 times and dries up, and adds fluorescence secondary antibody sheep mouse IgG.CY3.
(9) lucifuge 2h and dry, fluorescence microscopy Microscopic observation at room temperature.
2.2 cell differentiation
(1) nerve ball is suctioned out and is transferred in 24 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, in 5%CO2 incubators Culture 2h makes cell attachment.
(2) cell culture fluid is absorbed, is added per hole containing 20% hyclone nutrient solution 1ml, 37 DEG C, in 5%CO2 incubators Culture 7 days.
(3) cell culture fluid is absorbed, the fixation of 4% paraformaldehyde attached cell 30min, 0.01%PBS is added and develops a film 3 times simultaneously Dry.
(4) addition 0.1%Triton ruptures of membranes 10min, 0.01%PBS develop a film 3 times and dry.
(5) add 2%BSA and close 30min at room temperature.
(6) the anti-b-tubilinIII antibody of primary antibody mouse is added in 1,2 holes and exempts from anti-MAP2 antibody (neuronal marker resists It is former) antibody, in 4 DEG C overnight.0.01%PBS is rinsed and 3 times and dried up, add fluorescence secondary antibody sheep anti-mouse igg-CY3 and goat anti-rabbit igg- FITC.Lucifuge 2h and dry, fluorescence microscopy Microscopic observation at room temperature.
(7) GFAP (astroglia mark antigen) antibody is added in 3 holes, is placed in 4 DEG C of refrigerator overnights.0.01% PBS is rinsed 3 times and dried up, and adds fluorescence secondary antibody sheep anti-mouse igg-CY3, fluorescence microscopy Microscopic observation.
(8) CNPase (oligodendroglia mark antigen) antibody is added in 3 holes, in 4 DEG C of refrigerator overnights.0.01% PBS is rinsed 3 times and dried up, and adds fluorescence secondary antibody sheep anti-mouse igg-CY3, fluorescence microscopy Microscopic observation.
3rd, the gene transfer of virus related to rocombinant adenovirus rAAV mediations
(1) design of the primer of gene transfer and synthesis, amplification and rAAV-EPO and rAAV-LacZ or rAAV-LacZ Expression.
(2) NSC of clone's culture is with 2*106/ ml density is placed in 10cm culture dishes, and adds 2*1010uRAAV-EPO, New nutrient solution is converted to after transfection 12h, continues to cultivate 72h.
4th, histochemistry and Westem blot confirm the expression of transgenosis
4.1X-gal dyeing
(1) nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO2In incubator Culture 2h makes cell attachment.
(2) cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min.
(3) 0.01%PBS develops a film 3 times and dries, and adds 0.1%Triton ruptures of membranes 10min.
(4) 0.01%PBS develops a film 3 times and dries, and adds X-gal observation cellular colours.
1*10 is transplanted respectively in 4.2 rat brains11NSC, the NSC for transfecting LacZ genes and the NSC for transfecting EPO genes, 2 days After take implantation site brain tissue, and the NSC supernatants of culture carry out the expression of Westernblot detections EPO.
With reference to experimental rat model foundation and packet the invention will be further described.
Under 20-25 DEG C of room temperature, and the monitoring of vim and vigour, blood glucose, by SD rats through 10% chloraldurate 3.2ml/kg abdominal cavities After injecting anesthetic, make neck veutro center skin incision, exposing right side successively, neck is total, neck is outer and internal carotid.Ligature outside neck After arterial distal, the blunt nosed nylon wires of diameter 0.5mm are inserted into its near-end, and reached through internal carotid and initial part is moved in brain.Resistance After disconnected right side arteria cerebri media 2h, nylon rope yarn is pulled out, ligation of external carotid artery, skin suture notch.In 20- after rat recovery 25 DEG C are raised, and give gentamicin 20KU intraperitoneal injections after model foundation in 3d.After middle cerebral artery occlusion model in rats is established 3d, to being randomly divided into 4 groups after the progress nerve function lesion scoring of all animals with digits table：Control group, NSC transplantation groups, LacZ-NSC transplantation groups, EPO-NSC transplantation groups, every group 15.
Fig. 2 is the separation, culture and transfection EPO gene expression technique routes for the neonate rat NSC that the present invention implements offer Flow chart.
Fig. 3 is that the EPO joints NSC that the present invention implements to provide is transplanted to rat cerebral infarction ischemic focus recovery technique route flow Figure.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of rat model construction method for verifying treatment cerebral infarction ischemia drugs pharmacological property, it is characterised in that the verification is controlled Treating the rat model construction method of cerebral infarction ischemia drugs pharmacological property includes：
Step 1, NSC is separated from neonate rat forebrain tissue, using flow cytometer and single cell clone technology, passes through body Outer culture amplification shows the characteristic of its self-renewing and pluripotent differentiation, and by rAAV mediation EPO genes and LacZ genes Transfer, establish stablize expression EPO genes and LacZ genes NSC transfection systems and analyze NSC transfection systems in vitro, internal table Up to transgene protein situation；
Step 2, rat cerebral infarction ischemic focus is implanted into by the NSC transfection systems through EPO genes and LacZ genetic modifications, analysis The repair of EPO genes and LacZ gene pairs rat cerebral infarction ischemic focuses；And further analyze through EPO genes and LacZ genes Stem cell Deng genetic modification is to the therapy mechanism of rat cerebral infarction.
2. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In NSC is separated in the forebrain tissue from neonate rat to be included：
The original cuiture of NSC：
(1) draw materials：Newborn SD suckling mouse in 24h, cervical dislocation are put to death, and 75% alcohol soaking disinfection, divest skull and meninx, is dissecting Forebrain tissue is isolated under microscope；
(2) digest：Using mechanical digestion method, by brain tissue, sharp property is cut on culture dish, is transferred to 10ml cell centrifuge tubes, is added Enter PBS to 3ml, stand 2min after careful piping and druming, suction out and collect supernatant, phase is added in the centrifuge tube containing precipitate The PBS liquid of same volume, blows and beats again, so repeatedly, is gradually reduced tissue volume；
(3) centrifuge：75g centrifuges 10min or 900 turn/min centrifugations 5min；Abandon supernatant；NSC training liquid is added, piping and druming is uniform, carefully Born of the same parents count and concentration are adjusted to 1*106/ml；
(4) cultivate：Cell suspension is transferred in 24ml Tissue Culture Flasks, is placed in 37 DEG C, 5%CO2Cultivated in incubator.
3. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In separation NSC is further included in the forebrain tissue from neonate rat：
Passage：
When 50% cell mass>Passed on during 30 cells；Cell suspension is transferred to 10ml centrifuge tubes, is centrifuged with 110g 10min, abandons supernatant.Add 2ml nutrient solutions；5min, transfer supernatant to blake bottle are stood after soft piping and druming；In centrifuge tube again 2ml training liquid is added, repetitive operation to cell mass disappears；
Concentration is simultaneously adjusted to 1*10 by postdigestive cell suspension cell count6/ ml, is placed in 37 DEG C, 5%CO2Trained in incubator Support；
Continuous passage is more than 18 times, until removing all dead cell, cell fragment and irregular cell agglomerates, the god of formation rule Through ball.
4. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In the screening of flow cytometer includes：
Heochst33342 dye liquors：The storing liquid concentration of 10ug/ml is made into PBS, 4 DEG C are kept in dark place, the cell of suspension growth Heochst33342, final concentration of 1ug/ml are added in the state of culture；After 37 DEG C are incubated 1h, low temperature 500-1000r/min Centrifugation 5min discards dye liquor, is stored in after washing 2 times with PBS in ice；Then using fluidic cell separation SP cells.
5. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In the single cell clone includes：
Using being transferred to after limiting dilution assay diluting cells suspension in 96 orifice plates；
Analyze and select the culture hole containing individual cells；
37 DEG C are placed in, 5%CO2Cultivated in incubator, the cell for selecting active growth formation nerve ball is passed on, amplification cultivation.
6. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In the method for the rAAV mediation EPO genes and LacZ gene transfers includes the design and synthesis, amplification of the primer of gene transfer And the expression of rAAV-EPO and rAAV-LacZ.
7. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In the NSC transfection systems for establishing stable expression EPO genes and LacZ genes include：
The NSC transfection systems of culture are cloned with 2*106/ ml density is placed in 10cm culture dishes, and adds 2*1010uRAAV-EPO or RAAV-LacZ, new nutrient solution is converted to after transfecting 12h, continues to cultivate 72h.
8. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In, analysis NSC transfection systems in vitro, internal express transgenic protein status use histochemistry and Westem blot Verify the expression of transgene protein, including：
X-gal is dyed：
Nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO22h is cultivated in incubator to be made Cell attachment；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min；
0.01%PBS develops a film 3 times and dries, and adds 0.1%Triton ruptures of membranes 10min；
0.01%PBS develops a film 3 times and dries, and adds X-gal analysis cellular colours；
1*10 is transplanted in rat brain respectively11NSC, the NSC for transfecting LacZ genes and the NSC for transfecting EPO genes, take shifting after 2 days Plant the NSC supernatants progress Westernblot detection EPO genes for selecting brain tissue and culture and the expression of LacZ genes.
9. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 1, its feature exist In the rat model construction method of the verification treatment cerebral infarction ischemia drugs pharmacological property further includes：
NSC is identified：Including：
Nestin is dyed：
Nerve ball is suctioned out and is transferred in 96 orifice plates for scribbling 0.1%Gelatin, 37 DEG C, 5%CO22h is cultivated in incubator to be made Cell attachment；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min；
0.01%PBS develops a film 3 times and dries；
Add 0.1%Triton ruptures of membranes 10min；
0.01%PBS develops a film 3 times and dries；
Add 2%BSA and close 30min at room temperature；
Add the anti-nestin antibody of mouse, in 4 DEG C；
0.01%PBS develops a film 3 times and dries up, and adds fluorescence secondary antibody sheep mouse IgG.CY3；
Lucifuge 2h and dry, analyzed under fluorescence microscope at room temperature.
10. the rat model construction method of verification treatment cerebral infarction ischemia drugs pharmacological property as claimed in claim 9, its feature exist In NSC carries out cell differentiation after identifying：
Nerve ball is suctioned out and is transferred in 24 orifice plates for scribbling 0.1%Gelatin, 370C, cultivates 2h in 5%CO2 incubators Make cell attachment；
Cell culture fluid is absorbed, adds to contain in 20% hyclone nutrient solution 1ml, 370C, 5%CO2 incubator per hole and cultivates 7 My god；
Cell culture fluid is absorbed, 4% paraformaldehyde is added and fixes attached cell 30min, 0.01%PBS develops a film 3 times and dries；
0.1%Triton ruptures of membranes 10min is added, 0.01%PBS develops a film 3 times and dries；
Add 2%BSA and close 30min at room temperature；
The anti-b-tubilinIII antibody of primary antibody mouse is added in 1,2 holes and exempts from anti-MAP2 antibody, in 4 DEG C overnight.0.01%PBS is rushed Wash 3 times and dry up, add fluorescence secondary antibody sheep anti-mouse igg-CY3 and goat anti-rabbit igg-FITC.Lucifuge 2h and dry at room temperature, fluorescence is shown Analyzed under micro mirror；
GFAP antibody is added in 3 holes, is placed in 4 DEG C of refrigerator overnights.0.01%PBS is rinsed 3 times and dried up, and adds fluorescence secondary antibody goat-anti Mouse IgG-CY3, is analyzed under fluorescence microscope；
CNPase oligodendroglia mark antigen-antibodies are added in 3 holes, in 4 DEG C of refrigerator overnights.0.01%PBS is rinsed 3 times And dry up, add fluorescence secondary antibody sheep anti-mouse igg-CY3, analyzed under fluorescence microscope.
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