CN108441476B - A kind of processing method of neural stem cell - Google Patents
A kind of processing method of neural stem cell Download PDFInfo
- Publication number
- CN108441476B CN108441476B CN201810022157.2A CN201810022157A CN108441476B CN 108441476 B CN108441476 B CN 108441476B CN 201810022157 A CN201810022157 A CN 201810022157A CN 108441476 B CN108441476 B CN 108441476B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- neural stem
- culture medium
- bfgf
- preculture
- 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.)
- Active
Links
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 66
- 238000003672 processing method Methods 0.000 title claims abstract description 18
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 46
- 241000700605 Viruses Species 0.000 claims abstract description 31
- 239000001963 growth medium Substances 0.000 claims abstract description 27
- 208000015181 infectious disease Diseases 0.000 claims abstract description 18
- NJSSICCENMLTKO-HRCBOCMUSA-N [(1r,2s,4r,5r)-3-hydroxy-4-(4-methylphenyl)sulfonyloxy-6,8-dioxabicyclo[3.2.1]octan-2-yl] 4-methylbenzenesulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)O[C@H]1C(O)[C@@H](OS(=O)(=O)C=2C=CC(C)=CC=2)[C@@H]2OC[C@H]1O2 NJSSICCENMLTKO-HRCBOCMUSA-N 0.000 claims abstract description 10
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims abstract description 7
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims abstract description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims abstract 5
- 238000000034 method Methods 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- -1 diallyl dimethyl ammoniumchlorides Chemical class 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims 1
- 210000001772 blood platelet Anatomy 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 6
- 238000003780 insertion Methods 0.000 abstract description 6
- 230000037431 insertion Effects 0.000 abstract description 6
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 38
- 210000004027 cell Anatomy 0.000 description 20
- 230000000052 comparative effect Effects 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 13
- 210000000130 stem cell Anatomy 0.000 description 12
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 6
- 210000005036 nerve Anatomy 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 206010008092 Cerebral artery thrombosis Diseases 0.000 description 4
- 210000005013 brain tissue Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000302 ischemic effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 206010008088 Cerebral artery embolism Diseases 0.000 description 2
- 208000022306 Cerebral injury Diseases 0.000 description 2
- 206010008132 Cerebral thrombosis Diseases 0.000 description 2
- 206010008190 Cerebrovascular accident Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 2
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
- 208000006011 Stroke Diseases 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 201000010849 intracranial embolism Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000007096 poisonous effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 101710101493 Viral myc transforming protein Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical compound COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000009251 neurologic dysfunction Effects 0.000 description 1
- 230000007971 neurological deficit Effects 0.000 description 1
- 208000015015 neurological dysfunction Diseases 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
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
- 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
-
- 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
- C07K14/50—Fibroblast growth factors [FGF]
- C07K14/503—Fibroblast growth factors [FGF] basic FGF [bFGF]
-
- 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
Abstract
The present invention provides a kind of processing methods of neural stem cell, comprising: 1) carries out preculture to neural stem cell with the culture medium containing platelet derived growth factor；2) by the neural stem cell bed board through preculture；3) it is pre-processed with the neural stem cell that diallyl dimethyl ammoniumchloride treats infection slow virus；4) it is infected with slow virus carrier through the pretreated neural stem cell of step 3).Processing method through the invention can make C17.2 neural stem cell be easier to be carried the slow virus carrier infection of exogenous bFGF, can also improve the C17.2 Neural Stem Cells ' Growth situation of insertion bFGF and the expression quantity of bFGF.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of processing method of neural stem cell.
Background technique
Cerebral arterial thrombosis is as caused by cerebral thrombosis or cerebral embolism, and cerebral thrombosis or cerebral embolism will lead to local brain tissue and lack
Blood, anoxic, and then cause brain tissue softening, death.Cerebral arterial thrombosis not only has the characteristics that morbidity and mortality are high, also
Have the characteristics that high disability rate and high relapse rate, is current difficult medical problem in the urgent need to address.
In recent years, Stem cell transplantation for ischemic stroke is increasingly valued by people.It is many different types of
Stem cell such as embryonic stem cell (ES), mesenchymal stem cell (BMSC), induction versatile stem cell (iPS) and nerve cord are thin
Born of the same parents (NSC) are used to treatment ischemia apoplexy, and transplant these cells and can reduce neurologic impairment.C17.2 nerve
Stem cell line is the cell of the immortalization obtained after v-myc genetic modification, is separated from the cerebellum of newborn mice earliest
Arrive.Transplanting C17.2 NSC system is widely used in research central nervous system disorder disease, such as Parkinson
Disease, Huntington disease, craniocerebral injury, hypoxic-ischemic cerebral injury, spinal cord injury etc..C17.2 NSC system is a kind of good
The cell origin for treating cerebral arterial thrombosis.
But toxicants, the formation such as a large amount of excitatory neurotransmitters of tissue release and oxygen radical are unfavorable for after cerebral injury
The local microenvironment of neural stem cells transplantation survival, most of transplanted cells pass through apoptosis pathway death.So improving transplanting
Stem cell becomes a critical issue for the resistivity of ischemic microenvironment.
The 18kD polypeptide that basic fibroblast growth factor (bFGF) is made of 155 amino acid, is distributed mainly on
The tissue such as hypophysis, brain and nerve fiber and retina, adrenal gland, placenta, can promote from mesoderm and neuroderm
Multiple types cell Proliferation, differentiation, have extensive biological effect.BFGF is in low-level in Adult Mammals brain tissue
Expression, is mainly generated by astroglia, but the significant up-regulation of bFGF expression after brain tissue impairment.BFGF promotes blood vessel raw
At, axon regeneration, neuroprotection, tissue repair, mobilize Endogenous neural stem cells, repaired in development of central nervous system and damage
Play a significant role in multiple.In focal cerebral ischemia injury rat model, bFGF can inhibit nerve cell apoptosis, increase brain
Ischemic region blood supply is reduced significantly cerebral infarct size, improves neurological dysfunction.
But bFGF half-life short in vivo, it degrades rapidly after intravenously administrable, it is difficult to keep its bioactivity；BFGF is raw
Object macromolecular, it is difficult to penetrate blood-brain barrier；It is widely distributed in FGF receptor body, it is present in many histoorgans of whole body, it is complete through vein
It is big that side effect is administered in body.
Both stem cell transplantation and bFGF are joined together, bFGF gene order is inserted into using Gene transfer techniques dry
In cell, enable the high expressed bFGF albumen of stem cell.On the one hand transplanting can be improved in the bFGF albumen of stem cell expression
Survival ability of the stem cell under ischemic microenvironment, and can be used as protein therapeutic and go to improve nervous function.And it is dry thin
On the one hand born of the same parents are used as the carrier of expressed bFGF albumen, avoid bFGF albumen as protein drug half-life period section, be difficult to penetrate blood
The defects of brain barrier, general reaction, on the other hand but also the survival rate of transplanting stem cell itself increases, while bFGF albumen
The function of promoting differentiation can promote the differentiation of stem cell, can preferably improve the neurological deficit after cerebral arterial thrombosis.
Slow virus (Lentivirus) carrier is grown up based on HIV-1 (human immune deficiency I type virus)
Gene therapy vector.Its advantage is that it can infect cell such as primary cell, stem cell of more difficult infection etc..Slow disease simultaneously
Objective gene sequence can be integrated into the genome of target cell by poisonous carrier, guarantee target gene expression steady in a long-term.Slowly
Viral vectors has been widely used in gene therapy, production of vaccine, transgenic animals as a kind of infection means of maturation
The various aspects such as preparation.
BFGF gene can be inserted into C17.2 NSC system using slow virus carrier, obtain high expressed bFGF
The C17.2 NSC system of albumen.Then by obtained C17.2 neural stem cell by way of intravenous injection until lack
In the rat body of hemorrhagic cerebral apoplexy, observe it whether can good improvement nervous function damage due to caused by hypoxic-ischemic and
Infarction volume is reduced, and further detects its migration, proliferation and differentiation capability.In the process, inventor's discovery pair
The processing mode of C17.2 neural stem cell is different, and the efficiency for infecting bFGF gene is different, but also will affect insertion bFGF's
The expression of C17.2 Neural Stem Cells ' Growth situation and bFGF.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of processing method of neural stem cell, through this process
Method can make C17.2 neural stem cell be easier to be carried the slow virus carrier infection of exogenous bFGF, can also improve insertion
The C17.2 Neural Stem Cells ' Growth situation of bFGF and the expression quantity of bFGF.
The present invention relates to a kind of processing methods of neural stem cell, comprising:
1) preculture: from infecting the 2-5 days before slow virus carrier, with (the Platelet containing platelet derived growth factor
Derived growth factor, PDGF) culture medium preculture C17.2 neural stem cell；
2) it bed board: by the C17.2 neural stem cell Jing Guo step 1) preculture in 24 orifice plate bed boards, is incubated overnight；
3) it pre-processes: diallyl dimethyl ammoniumchloride being added into the culture medium of C17.2 neural stem cell, and continues
Cultivate 2-3 hour；
4) with carrying the slow virus carrier infection of exogenous bFGF through the pretreated C17.2 neural stem cell of step 3), 37 DEG C
Incubator is incubated for 6 hours, is inhaled the culture medium that abandoning culture medium more renews and is continued to cultivate.
Preferably, the final concentration of 5-10ng/ml of the platelet derived growth factor in the step 1) in the medium.
Preferably, in the step 1), from infecting the 3-5 days before slow virus carrier, with containing platelet derived growth because
The pre- base culture C17.2 neural stem cell of culture of son.
Preferably, in the step 2), by the C17.2 neural stem cell Jing Guo step 1) preculture in 24 orifice plate bed boards
Concentration is 1 × 104-5×104A/hole.
Preferably, the final concentration of 3-5 μ g/ of the diallyl dimethyl ammoniumchloride in the step 3) in the medium
ml。
Preferably, in the step 3), diallyl dimethyl chlorine is added into the culture medium of C17.2 neural stem cell
Change ammonium, and continues to cultivate 2 hours.
In a preferred embodiment, the processing method of the neural stem cell includes:
1) preculture: from infecting the before slow virus carrier the 3rd day, with the culture of the platelet derived growth factor containing 5ng/ml
Base preculture C17.2 neural stem cell；
2) bed board: by the C17.2 neural stem cell Jing Guo step 1) preculture in 24 orifice plate bed boards, 1 × 104A/hole, mistake
Night culture；
3) it pre-processes: 3 μ g/ml diallyl dimethyl ammoniumchlorides being added into the culture medium of C17.2 neural stem cell,
And continue to cultivate 2 hours；
4) with carrying the slow virus carrier infection of exogenous bFGF through the pretreated C17.2 neural stem cell of step 3), 37 DEG C
Incubator is incubated for 6 hours, is inhaled the culture medium that abandoning culture medium more renews and is continued to cultivate.
By carrying out preculture to nerve cell in advance with the culture medium containing PDGF, and exogenous bFGF is carried being added
Slow virus before first be added diallyl dimethyl ammoniumchloride cell is pre-processed, it is thin to improve C17.2 nerve cord
Born of the same parents are carried the efficiency of the slow-virus infection of exogenous bFGF, and improve the C17.2 nerve of insertion bFGF to a certain extent
The expression quantity of stem cell growth situation and bFGF.
In method provided by the invention, although mechanism is not also very clear, PDGF can promote oneself of neural stem cell
I such as updates at the abilities, by preculture, neural stem cell is made to be in the state for being easier to infection slow virus；Add before virus infection
Enter diallyl dimethyl ammoniumchloride to pre-process cell, the diallyl dimethyl ammoniumchloride in culture medium can be with
Reduce the electrical charge rejection between cell and slow virus；By the way that above two reagent is creatively applied to C17.2 nerve simultaneously
In processing method in stem cell, and optimize the dosage and use condition of each reagent, it is thin to have obtained nerve cord of the invention
The processing method of born of the same parents.Compared with prior art, the present invention having the following advantages that.
Processing method through the invention can make C17.2 neural stem cell be easier to be carried the slow disease of exogenous bFGF
Poisonous carrier infection can also improve the C17.2 Neural Stem Cells ' Growth situation of insertion bFGF and the expression quantity of bFGF.
Detailed description of the invention
In Fig. 1 embodiment 1-2 and comparative example 1-3 infect slow virus after C17.2 neural stem cell expressed bFGF Western
Blot figure；
The efficiency of infection of Fig. 2 embodiment 1-2 and comparative example 1-3 statistics；
C17.2 neural stem cell is under fluorescence microscope after infecting slow virus in Fig. 3 embodiment 1-2 and comparative example 1-3
Photo；
Now in conjunction with drawings and examples, the invention will be further described:
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment 1-2 and comparative example 1-3
Embodiment 1-2 and comparative example 1-3 is carried out by following experimental procedure,
Experimental procedure:
1 is overexpressed the building of bFGF slow virus carrier
1.1 building pShuttle-5HRE-bFGF-IRES-hrGFP plasmids
The genetic fragment is synthesized according to the sequence (Gene ID:2247 in NCBI) of bFGF gene, in the gene when synthesis
Upstream and downstream introduces a SalI restriction enzyme site (Synesis Company is Jin Sirui Biotechnology Co., Ltd) respectively, according to operation instruction
Book distinguishes the digestion segment and pShuttle-IRES-hrGFP-1 plasmid (Agilent) with SalI restriction enzyme (NEB),
Fragment purification is carried out using DNA purification kit (Invotrigen) to get the bFGF piece of SalI restriction enzyme site is had to two sections
Section and there are the pShuttle-IRES-hrGFP-1 plasmid vectors of SalI restriction enzyme site.
According to operation instructions, the aforementioned bFGF segment with SalI restriction enzyme site is connected with T4 ligase (NEB) and is stayed
There is the pShuttle-IRES-hrGFP-1 plasmid vector of SalI restriction enzyme site, converts bed board.The sequencing of picking monoclonal, sequencing are drawn
Object is pShuttle-IRES-hrGFP-1-F:CTCACGGGGATTTCCAAGTC, by comparing sequencing result, is correctly inserted
Enter the plasmid pShuttle-5HRE-bFGF-IRES-hrGFP of bFGF segment.
1.2 amplification bFGF-IRES-hrGFP genes；
Using pShuttle-5HRE-bFGF-IRES-hrGFP plasmid as template, bFGF-IRES-hrGFP gene is expanded.Instead
Answer system as follows:
1.3bFGF-IRES-hrGFP PCR product is connect with pENTER/D-TOPO plasmid；
Using PENTR/D-TOPO CLONING KIT (Invotrigen), and according to its specification by bFGF-IRES-
HrGFP PCR product is attached with pENTER/D-TOPO plasmid:
It is incubated at room temperature 10min.BFGF-IRES-hrGFP PCR product and pENTER/D-TOPO plasmid connection product are turned
Change, amplification, extract plasmid and carry out digestion verification, digestion verification is correct.
1.4 building pLenti6.4/R4R2/V5-CMV-bFGF-IRES-hrGFP plasmids
Reaction system is as follows
1ul Proteinase K is added later and terminates reaction, converts bed board by 25 DEG C of reaction 16h, and picking monoclonal carries out digestion mirror
It is fixed, it selects digestion and identifies correct bacterium to get the successful pLenti6.4/R4R2/V5-CMV-bFGF-IRES-hrGFP of building is arrived
Plasmid.Using the plasmid as the slow virus carrier of this experiment, which can be overexpressed bFGF and mark with green fluorescence.
2. virus packaging
First day:
Bed board: cell number reaches 2*10 when transfecting T25 culture bottle6(90%-95% fusion), 5ml contains without antibiotic
Blood serum medium is incubated overnight.
Second day:
1. the virapower of 4.5ug (4.5ul is added in concentration 1ug/ul) is added in 1.5ml EP pipe
DMEM not serum-containing media of the middle upgrading grain of packaging mix and 1.5ug (concentration 450ng/ul, 7ul) to 700ul
In, softly suction mixes liquid-transfering gun up and down；
B, the DMEM that 12ul lipofectamine2000 to 700ul is added in another 1.5ml EP pipe is trained without serum
Base is supported, softly suction mixes liquid-transfering gun up and down, is incubated at room temperature 5 minutes；
C, after incubation, A and B is mixed, softly suction mixes liquid-transfering gun up and down；
D, it is incubated at room temperature 20min.(must be transfected in 6 hours)
2. discarding culture medium, the not antibiotic complete medium of 5ml is added.
3. the transfection mixture that front has been incubated for is added in the culture medium of 293T cell, front-rear direction softly rocks training
It supports bottle to mix, in incubator overnight.
Third day
1. removing culture medium, the not antibiotic complete medium of 5ml is added；
2. being incubated for 48-72h again.
5th or six day
1. by media transfer into 15ml centrifuge tube；
2.4 DEG C of centrifugation 2000g 15min；
3. gained supernatant is slow virus liquid, supernatant is divided into 1ml and is transferred in five 1.5 centrifuge tubes, wherein
A pipe (1ml) is taken to be divided into 200ul points five pipe again；- 80 DEG C of preservations
3.C17.2 neural stem cell preculture
To infection slow virus carrier from infecting the 2-8 days before slow virus carrier, C17.2 neural stem cell is carried out pre-
Culture, basal medium are as follows: DMEMF12+bFGF10ng/ml+EGF20ng/ml+B272ml/100ml is implemented on this basis
Example 1-2 and comparative example 1-3 used medium additive and starting cultivated days see the table below:
4. cell infection
First day: by the C17.2 neural stem cell Jing Guo preculture in 24 orifice plate bed boards, 1 × 104A/hole is trained overnight
It supports.
Second day: before virus liquid is added, the C17.2 neural stem cell to be transformed of embodiment 1-2 and comparative example 1-3 being carried out
Different pretreatments, processing mode are that reagent is added into cell culture medium, and handling agents useful for same and handling duration see the table below:
| Handle agents useful for same | Handling duration | |
| Embodiment 1 | Diallyl dimethyl ammoniumchloride (4 μ g/ml) | 2h |
| Embodiment 2 | Diallyl dimethyl ammoniumchloride (3 μ g/ml) | 2h |
| Comparative example 1 | Polybrene Polybrene (8 μ g/ml) | 2h |
| Comparative example 2 | Diallyl dimethyl ammoniumchloride (3 μ g/ml) | 2h |
| Comparative example 3 | Nothing | - |
After pretreatment, 200ul virus liquid is added into 24 orifice plates, 37 DEG C of incubators are incubated for 6 hours, are inhaled and are abandoned culture medium replacement
New culture medium, and continue to cultivate.
Detection efficiency of infection is shown in embodiment 4 within 6th day.
Embodiment 3 verifies bFGF protein expression
Detected by Western Blot in the cell that embodiment 1-2 and comparative example 1-3 are obtained whether successful expression
BFGF albumen, experimental procedure are as follows:
1. collecting protein
1.1 by the plating cells being proliferated after infection to 6 orifice plates, and 1 × 105A/hole.
1.2, which continue culture to degrees of fusion, reaches 90%-95%.It inhales and abandons culture medium, PBS is rinsed twice, and it is entirely thin that 80ul is added
Born of the same parents' protein extract.
1.3 are incubated for 10 minutes on ice, are gently scraped cell with cell scraper, collect to EP and manage.
2. determination of protein concentration
2.1 by 4 DEG C of centrifugation 10min of protein lysate 12000rp/min of collection.
2.2 are added to the diluted Coomassie Brillant Blue solution of 200ul 1:4 in 96 orifice plates.
2.3 take the protein lysate supernatant 2ul being centrifuged to be added in Coomassie brilliant blue (3 multiple holes)
2.4 concussion 5min are mixed, and choose the bubble in net 96 orifice plate.
2.5 measure the absorbance value (ODs) of 96 orifice plates at 595nm.
2.6 by calculating, spare by all sample trims to 40ug/20ul.
3.Western Blot
The protein sample prepared through step 2 is carried out SDS- polyacrylamide gel electrophoresis by 3.1.
3.2 transferring film
3.3 are immunoreacted and take pictures
3.3.1 it after the film to take a turn for the better being cleaned once with TBST, is put into 5% skim milk, is sealed as room temperature on shaking table
Close 1~1.5h.It is washed 3 times with TBST, cleans confining liquid.Primary antibody is added to be put into 4 DEG C later as 1h is incubated at room temperature on shaking table and shake
Bed is overnight.
3.3.2 next day, then primary antibody is recycled primary antibody, marked in being incubated at room temperature 1h on shaking table.It is washed film 3 times with TBST,
Each 7min.The corresponding secondary antibody of primary antibody is added and is incubated for 1h, secondary antibody is generally with secondary antibody: (TBS+TBST)=1:3000 is prepared.It discards
Secondary antibody washes film 3 times with TBST, each 7min.
3.3.3 Western Blotting chemical illuminating reagent ECL-A liquid, each 500uL mixed in equal amounts of B liquid are taken, then
Even add to is incubated for 2min on film, be exposed, preservation of taking pictures.
Experimental result in the cell that embodiment 1-2 and comparative example 1-3 are obtained as shown in Figure 1, as shown in Figure 1, detect
The expression of bFGF is arrived.
The detection of 4 efficiency of infection of embodiment
In this experiment, because the bFGF infected is with hrGFP (green fluorescent protein), it is possible to by aobvious with being inverted
Micro mirror observes fluorescence, and with the efficiency of infection of this Statistics Implementation example 1-2 and comparative example 1-3, efficiency of infection statistics is shown in Fig. 2, in fluorescence
Microscopically observation to Part of photos taken see Fig. 3.
As shown in Figure 2, the method provided through the invention can make C17.2 neural stem cell be easier to be carried
The slow virus carrier of exogenous bFGF infects.From the figure 3, it may be seen that under same operation and conditions of exposure, the cell green of embodiment 1-2
Fluorescent brightness is higher than the cell green fluorescence brightness in comparative example 1-3, illustrates the method provided through the invention, can also change
The C17.2 Neural Stem Cells ' Growth situation of kind insertion bFGF and the expression quantity of bFGF.
The technical means disclosed in the embodiments of the present invention is not limited to the technical means disclosed in the above technical means, and further includes
Technical solution consisting of any combination of the above technical features.The foregoing is a specific embodiment of the present invention, should refer to
Out, for those skilled in the art, without departing from the principle of the present invention, can also make several
Improvements and modifications, these modifications and embellishments are also considered to be within the scope of the present invention.
Claims (7)
1. a kind of processing method of neural stem cell, which is characterized in that comprise the steps of:
1) preculture: from infecting the 2-5 days before slow virus carrier, with the culture medium preculture containing platelet derived growth factor
C17.2 neural stem cell；
2) it bed board: by the C17.2 neural stem cell Jing Guo step 1) preculture in 24 orifice plate bed boards, is incubated overnight；
3) it pre-processes: diallyl dimethyl ammoniumchloride being added into the culture medium of C17.2 neural stem cell, and continues to cultivate
2-3 hour；
4) it is infected with the slow virus carrier for carrying exogenous bFGF through the pretreated C17.2 neural stem cell of step 3), 37 DEG C of cultures
Case is incubated for 6 hours, is inhaled the culture medium that abandoning culture medium more renews and is continued to cultivate.
2. the processing method of neural stem cell described in accordance with the claim 1, which is characterized in that the blood platelet in the step 1)
The final concentration of 5-10ng/ml of source growth factor in the medium.
3. the processing method of neural stem cell described in accordance with the claim 1, which is characterized in that in the step 1), from infection
Before slow virus carrier from 3-5 days, with the culture medium preculture C17.2 neural stem cell containing platelet derived growth factor.
4. the processing method of neural stem cell described in accordance with the claim 1, in the step 2, step 1) preculture will be passed through
C17.2 neural stem cell in 24 orifice plate bed boards concentration be 1 × 104-5×104A/hole.
5. the processing method of neural stem cell described in accordance with the claim 1, which is characterized in that the polydiene in the step 3)
The final concentration of 3-5 μ g/ml of diallyidimethylammonium chloride in the medium.
6. the processing method of neural stem cell described in accordance with the claim 1, which is characterized in that in the step 3), to C17.2
Diallyl dimethyl ammoniumchloride is added in the culture medium of neural stem cell, and continues to cultivate 2 hours.
7. the processing method of neural stem cell described in accordance with the claim 1, which comprises the following steps:
1) preculture: pre- with the culture medium of the platelet derived growth factor containing 5ng/ml from infecting the before slow virus carrier the 3rd day
Cultivate C17.2 neural stem cell；
2) bed board: by the C17.2 neural stem cell Jing Guo step 1) preculture in 24 orifice plate bed boards, 1 × 104A/hole is trained overnight
It supports；
3) it pre-processes: being added 3 μ g/ml diallyl dimethyl ammoniumchlorides into the culture medium of C17.2 neural stem cell, and after
2 hours of continuous culture；
4) it is infected with the slow virus carrier for carrying exogenous bFGF through the pretreated C17.2 neural stem cell of step 3), 37 DEG C of cultures
Case is incubated for 6 hours, is inhaled the culture medium that abandoning culture medium more renews and is continued to cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810022157.2A CN108441476B (en) | 2018-01-10 | 2018-01-10 | A kind of processing method of neural stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810022157.2A CN108441476B (en) | 2018-01-10 | 2018-01-10 | A kind of processing method of neural stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108441476A CN108441476A (en) | 2018-08-24 |
| CN108441476B true CN108441476B (en) | 2019-01-15 |
Family
ID=63190820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810022157.2A Active CN108441476B (en) | 2018-01-10 | 2018-01-10 | A kind of processing method of neural stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108441476B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830778A (en) * | 2012-11-23 | 2014-06-04 | 上海微创医疗器械（集团）有限公司 | Polyelectrolyte-containing drug coating and preparation method thereof |
| CN104958786A (en) * | 2015-07-27 | 2015-10-07 | 广东医学院 | PF-127-LV-NFcocktail compound loaded with portable neural stem cells as well as preparation method and application of PF-127-LV-NFcocktail compound |
-
2018
- 2018-01-10 CN CN201810022157.2A patent/CN108441476B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830778A (en) * | 2012-11-23 | 2014-06-04 | 上海微创医疗器械（集团）有限公司 | Polyelectrolyte-containing drug coating and preparation method thereof |
| CN104958786A (en) * | 2015-07-27 | 2015-10-07 | 广东医学院 | PF-127-LV-NFcocktail compound loaded with portable neural stem cells as well as preparation method and application of PF-127-LV-NFcocktail compound |
Non-Patent Citations (2)
| Title |
|---|
| Transplantation of bFGF-expressing neural stem cells promotes cell migration and functional recovery in rat brain after transient ischemic stroke;Jin-Jing Zhang et al;《Oncotarget》;20171027;第8卷(第60期);摘要，第102074页Materials and Methods部分，图1-5 |
| 阳离子聚合物在非病毒基因转染中的研究进展;任先越等;《生物工程学报》;20130525;第29卷(第5期);第568-577页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108441476A (en) | 2018-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gagliardi et al. | Characterization and transplantation of CD73-positive photoreceptors isolated from human iPSC-derived retinal organoids | |
| JP6396988B2 (en) | Methods and compositions for double glycan binding AAV vectors | |
| Zhang et al. | CRISPR/Cas9‐mediated sheep MSTN gene knockout and promote sSMSCs differentiation | |
| JP6387350B2 (en) | Adeno-associated virus vector targeting oligodendrocytes | |
| JP2022188202A (en) | Cells and methods of using and making the same | |
| CN108713059A (en) | VCN enhancer combinations object and its application method | |
| CN103958667A (en) | Compounds for improved viral transduction | |
| Liu et al. | Human neural progenitors derived from integration-free iPSCs for SCI therapy | |
| US20220401491A1 (en) | Mesenchymal stem cell expressing trail and cd, and use thereof | |
| WO2001046384A2 (en) | A method for isolating and purifying multipotential neural progenitor cells and multipotential neural progenitor cells | |
| CN109097402A (en) | A kind of preparation method of recombinant vector CAR-CD244-antiCD19 | |
| Du et al. | Induced pluripotent stem cell therapies for geographic atrophy of age-related macular degeneration | |
| WO2023011248A1 (en) | Gene therapy for wet age-related macular degeneration using ipsc-derived cells as vectors | |
| McClements et al. | Tropism of AAV vectors in photoreceptor-like cells of human iPSC-derived retinal organoids | |
| CN109790521A (en) | Generate the method for the optimization of parvovirus H-1 on a large scale in the culture medium of substantially serum-free | |
| EP3413896B1 (en) | Vcn enhancer compositions and methods of using the same | |
| CN113621611B (en) | Marrow specific promoter and application thereof | |
| CN108441476B (en) | A kind of processing method of neural stem cell | |
| CN110229790A (en) | Immortal human derived neural stem cell line, preparation method, recombinant viral vector and application | |
| CN105255836A (en) | Method for preparing person stem cells with improved neural restoration function and application of person stem cells | |
| JP2022532535A (en) | How to select nutritional requirements | |
| CN106399318A (en) | Method for modifying cells by virtue of oxygen-regulated basic fibroblast growth factor | |
| CN113717929B (en) | Pharmaceutical composition containing gene-edited adipose-derived stem cells | |
| KR101269125B1 (en) | Method for Proliferating Stem Cells Using Genes Activating Notch Signaling | |
| JP7144829B2 (en) | Mesenchymal stem cells with enhanced safety and anti-inflammatory effects |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190925 Address after: 100029 B-2-303, 3rd floor, No. 1 Building, 11 Huixin East Street, Chaoyang District, Beijing Patentee after: Beijing Rebos Cell Biotechnology Co.,Ltd. Address before: 510000 D2-101 building twenty-eighth, 61 Daling mountain road, Tianhe District, Guangzhou, Guangdong. Patentee before: GCH REGENERATIVE MEDICINE TECHNOLOGY CO.,LTD. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 100029 B-2-303, 3rd floor, No. 1 Building, 11 Huixin East Street, Chaoyang District, Beijing Patentee after: Beijing Darwin Cell Biotechnology Co.,Ltd. Address before: 100029 B-2-303, 3rd floor, No. 1 Building, 11 Huixin East Street, Chaoyang District, Beijing Patentee before: Beijing Rebos Cell Biotechnology Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200228 Address after: 510000 D2-101 building twenty-eighth, 61 Daling mountain road, Tianhe District, Guangzhou, Guangdong. Patentee after: GCH REGENERATIVE MEDICINE TECHNOLOGY CO.,LTD. Address before: 100029 B-2-303, 3rd floor, No. 1 Building, 11 Huixin East Street, Chaoyang District, Beijing Patentee before: Beijing Darwin Cell Biotechnology Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510000 room d2-101, building 28, No.61 Dalingshan Road, Tianhe District, Guangzhou City, Guangdong Province Patentee after: Jisai international Regenerative Medicine Technology Co.,Ltd. Address before: 510000 room d2-101, building 28, No.61 Dalingshan Road, Tianhe District, Guangzhou City, Guangdong Province Patentee before: GCH REGENERATIVE MEDICINE TECHNOLOGY CO.,LTD. |
|
| CP01 | Change in the name or title of a patent holder |