CN101389761A - De-differentiation of astrocytes into neural stem cell using bmi-1 - Google Patents

De-differentiation of astrocytes into neural stem cell using bmi-1 Download PDF

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CN101389761A
CN101389761A CNA2006800535005A CN200680053500A CN101389761A CN 101389761 A CN101389761 A CN 101389761A CN A2006800535005 A CNA2006800535005 A CN A2006800535005A CN 200680053500 A CN200680053500 A CN 200680053500A CN 101389761 A CN101389761 A CN 101389761A
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cell
bmi
neural stem
stem cell
astroglia
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刘承权
文哉喜
尹炳善
金淇东
朴圭万
刘承骏
全恩暻
金甫那
郭成植
孟萨克
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Eun Tak KK
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Abstract

Disclosed is a composition and a method for inducing the de-differentiation of astrocytes into neural stem cells using Bmi-1. The de-differentiated neural stem cells have the ability to differentiate into astrocytes, neurons, and oligodendrocytes.

Description

Use BMI-1 that astroglia cell is dedifferentiated into and be neural stem cell
Technical field
Neural stem cell (NSCs) is the hypotype of neural progenitor cell, and it has the astroglia cell of being divided into, oligodendrocyte and neuronic ability.From central nervous system (CNS) and peripheral nervous system (PNS), neural stem cell forms the neural ball of many cells, and they are divided into colloid system and nervous system cell people such as (, 2001) SallyTemple under background condition separately.These neural stem cell are used for the treatment of incurable disease, and study its potential method as cell therapy.Because neural stem cell is the adult stem cell that can produce ethics problem hardly, so it has been carried out broad research.Recently the hot research about dedifferenting that carries out has been strengthened the importance of adult stem cell.Adult stem cell still still has many difficulties than the easier acquisition of embryonic stem cell in its practical application.In addition, when using other people adult stem cell, immunological rejection also is a problem.Therefore, use patient's self cell induction to dedifferente and to solve a question at the front.For this purpose, must induce the cell that has broken up to dedifferente.Now, carrying out broad research so that induce with the method for for example cytogamy and nuclear transplantation and to dedifferente.In another study group that uses different methods, Alexis J. has reported the characteristic (Lancet2004) that has neural stem cell under the Culture of neural stem cells condition when isolated cells is cultivated from skin.In addition, Toru K. successfully makes oligodendroglia precursor be dedifferentiated into to be neural stem cell (Genes ﹠amp; Development2004).The article that this study group delivered about this problem from 2000, and in one piece of article in 2004, reported that the genetic expression in each stage is relevant with Chromatin Remodeling and histone modification.
Background technology
In the present invention, propose a kind of approach that solves the problem of aforementioned institute introduction method, and set up a kind of suitable new using method.In the present invention, select and cross transcription factor research of having expressed a kind of known adjusting NSCs performance and be divided into neural stem cell.The selecteed factor is accredited as " Bmi-1 ", is to be used to one of protein of regulating histone modification and Cycle Regulation (people such as Jan W., 1999).It is reported that the Cdkn2a/INK4A locus is the target spot of Bmi-1.Bmi-1 is known as transcribing of this target gene and suppresses son.Recently, the Bmi-1 gene has been reported in the neural stem cell express and play an important role (people such as Anna V.M., 2003,2005, people such as In-Kyoung Park, 2004) in the self of neural stem cell.
The present inventor has carried out under this background deeply and research completely, thereby found crossing expression and can inducing the astroglia cell that has broken up to be dedifferentiated into to can self and be divided into the cell of the neural stem cell sample of astroglia cell, neurone and oligodendrocyte of Bmi-1 wherein, thereby caused the present invention.
Summary of the invention
The purpose of this invention is to provide and induce astroglia cell to be dedifferentiated into composition, comprise Bmi-1 protein or contain the nucleic acid material of the proteinic nucleotide sequence of coding Bmi-1 into neural stem cell.
Another object of the present invention has provided a kind of astroglia cell of inducing and has been dedifferentiated into method into neural stem cell, comprises with Bmi-1 protein or contains the nucleic acid material processing astroglia cell of the proteinic nucleotide sequence of coding Bmi-1.
Another object of the present invention provides the neural stem cell of using this method preparation.
It is astroglia cell, oligodendrocyte and neuronic method with the cell differentiation of nerve cord of dedifferenting that another object of the present invention provides a kind of.
The accompanying drawing summary
Above and other objects of the present invention, feature and other advantage will be from the following detailed description and more being expressly understood with reference to the accompanying drawings, wherein:
Fig. 1 has shown the Bmi-1 target p16 with immunocytochemical assay Ink4aAnd p19 ArfCross to express;
Fig. 2 has shown that inductive dedifferentes, wherein shown ball formation (A) and direct kick form (B) (when cultivate under the condition that is being suitable for neural stem cell form direct kick when unicellular and only be suitable for attaching under the culture condition of astroglia cell the back be suitable for neural stem cell condition under cultivate and form ball when unicellular, and stablized 12 hours);
Fig. 3 has shown (a) expression with the neural stem cell marker of immunocytochemical assay, wherein is divided into the cell (D to F) of neural stem cell (A to C) and neural stem cell sample;
Fig. 4 has shown the expression (Ast: astroglia cell, NSC: neural stem cell, Ast-Bmi-1: astroglia cell+Bmi-1NSCLC: the cell of neural stem cell sample) of the neural stem cell specific marker thing that detects with RT-PCR; With
Fig. 5 has shown that cell (D-G) vitro differentiation of neural stem cell (A-C) and neural stem cell sample is astroglia cell, oligodendrocyte and neurone.
Realize optimised form of the present invention
One aspect of the present invention relates to can induce astroglia cell to be dedifferentiated into composition into neural stem cell, and it comprises Bmi-1 protein or contains the nucleic acid material of the proteinic nucleotide sequence of coding Bmi-1.
Term " cell of neural stem cell sample " expression multipotential stem cell dedifferentes by being divided into astroglia cell, oligodendrocyte and neuronic somatocyte as used herein.In the present invention, the cell of neural stem cell sample also is described as neural stem cell.
Recently openly when Bmi-1 crosses expression, even break up, astroglia cell also can be dedifferentiated into the cell into the multipotent neural stem cell sample in the present invention.
In the present invention, Bmi-1 provides with the form of protein or the proteinic nucleic acid of coding Bmi-1.
So long as derive from Mammals for example people, horse, sheep, pig goat, camel, antelope and dog, any Bmi-1 can be used in the composition of the present invention.It is in addition, of the present invention that to be used for being dedifferentiated into for the Bmi-1 protein of neurocyte can be its wild-type or varient.
Term " Bmi-1 protein variants " relates to nature or the artificial Bmi-1 protein that produces, wherein Bmi-1 protein on aminoacid sequence because amino acid whose disappearance, insertion, non-conservative substitution or conservative substitution or its combination and different with wild-type.Varient is to have with natural protein to have identical bioactive function coordinator, although their physics and/or chemical property change.Preferably, varient has strengthened structural stability or the physiologically active to physics and chemical environment.
In a preferred embodiment of the invention, Bmi-1 is the form that comprises the nucleic acid material of the proteinic nucleotide sequence of coding Bmi-1.
Coding Bmi-1 proteinic nucleotide sequence is its wild-type or varient, and the varient of wherein natural generation or synthetic is because being combined on the aminoacid sequence of disappearance, insertion, non-conservative substitution or the conservative substitution of base or they is different with wild-type.
Coding Bmi-1 proteinic nucleotide sequence can be comprise dna molecular (genome, cDNA) or the strand of RNA molecule or two strands.
In the preferred embodiment of the present invention, the nucleic acid material that contains the proteinic nucleotide sequence of coding Bmi-1 is the carrier that allows the Bmi-1 protein expression.
Term " carrier " relates to the DNA construct that contains dna sequence dna as used herein, and being connected in of described dna sequence dna operability can influence on the regulating and controlling sequence that described DNA expresses in the host cell that is fit to.
As used herein term " connection of operability " thus relate to the expression of nucleic acid regulating and controlling sequence and the coding target protein second nucleotide sequence between functional the connection can finish general function.With can use gene recombination technology preparation well-known in the art being connected of the operability of recombinant vectors, and site-specific dna break can use enzyme well known in the art to carry out with being connected.
The carrier that is suitable for the present invention's use comprises signal sequence or is used for membrane orienting or excretory homing sequence and expression regulation element, for example promotor, operon, initiator codon, terminator codon, polyadenylation signal and enhanser, and can be built into various ways according to their purpose.The promotor of carrier can be composing type or induction type.In addition, expression vector comprises optionally marker, and described marker allows to select carrier-containing host cell, and reproducible expression vector comprises replication origin.This carrier can be self-replacation, perhaps can be to be integrated on the DNA of host cell.
The carrier that can use in the present invention can be plasmid vector, cosmid vector or virus vector, the preferred virus carrier.But the example of virus vector comprises being not restricted to and derives from for example carrier of HIV (human immunodeficiency virus), MLV (murine leukemia virus), ASLV (avian sarcomata virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (mouse mammary tumour virus) or the like, adenovirus, adeno associated virus and hsv of retrovirus.In a practical embodiments of the present invention, use pBabe puro carrier, it derives from the virus vector based on moloney leukemia virus, carries the selected marker of tetracycline.
The nucleic acid material that contains coding Bmi-1 proteinic nucleotide sequence can be with the form of carrier as naked DNA (people such as Wolff, Science, 247:1465-8,1990; People such as Wolff, J.Cell Sci.103:1249-59,1992), or under the help of liposome or cationic polymers, introduce cell.In order to use in gene transfection, liposome is for example immobilized artificial membrane made of DOTMA and DOTAP of cationic phospholipid.When mixing with certain proportion with negative charge Nucleotide, cationic-liposome forms nucleic acid-liposome complex.
In another preferred embodiment of the present invention, the nucleic acid material that contains the proteinic nucleotide sequence of coding Bmi-1 can be wherein to express the proteinic virus of Bmi-1.
Term " virus " relates to the virus vector that carries coding Bmi-1 proteinic nucleotide sequence and transforming or transfection packing cell and the Bmi-1 for preparing expresses virus as used herein.
Include, but are not limited to retrovirus, adenovirus, adeno associated virus and hsv according to employed viral example in the preparation of Bmi-1 expression virus of the present invention.Preferred retrovirus.In following examples, the reorganization pBabepuro carrier that Bmi-1 expresses the nucleotide sequence of virus by will carrying coding Bmi-1 protein (pBabe puro Bmi-1) is converted in the PT67 packing cell and prepares.
Another aspect of the present invention relates to astroglia cell and is dedifferentiated into method into neural stem cell, comprises with Bmi-1 protein or contains the nucleic acid material processing of the proteinic nucleotide sequence of coding Bmi-1.
In more detail, this method comprises step (i) cultivate astroglia cell in substratum; (ii) with Bmi-1 protein or contain the nucleic acid material processing substratum of coding Bmi-1 proteinic nucleotide sequence; (iii) induce astroglia cell to be dedifferentiated into and be neural stem cell.
Any conventional substratum that is used for culture of neural stem cells neural can be as the substratum at step (i) astroglia cell.Substratum contains carbon source, nitrogenous source and micro-component usually.In addition, substratum can comprise microbiotic, for example penicillin, Streptomycin sulphate and gentamicin.Substratum preferably contains bFGF.
The nucleic acid material that step is used to handle the Bmi-1 protein of cell in (ii) or contains the proteinic nucleotide sequence of coding Bmi-1 as mentioned above.
Another aspect of the present invention relates to the neural stem cell with preceding described method preparation.
Confirmed to express neural stem cell specific marker thing nidogen, CD133 and the Sox2 of same level, and had and the general identical differentiation capability of neural stem cell by neural stem cell of dedifferenting preparation according to the present invention.Also shown the self feature by neural stem cell of dedifferenting preparation according to the present invention.
Another aspect of the present invention also relates to the cell differentiation of nerve cord that obtains according to dedifferenting of preceding method becomes astroglia cell, oligodendrocyte and neuronic method.
When the neural stem cell of using the compositions and methods of the invention to dedifferente is placed in the following time of condition of differentiation separately, is divided into astroglia cell, neurone and oligodendrocyte and can be specific to separately the marker representation of cell by detection and monitors.
Better understanding of the present invention can obtain by following examples, and they are used for understanding and are not used in restriction the present invention.
Embodiment 1: experimental technique
1. the cultivation of mouse astroglia cell and mouse neural stem cell
Separation is cultivated under suitable condition from the mouse astroglia cell of E13.5CNS.They (handle (Gibco0.05%) with Regular Insulin before cultivating among the DMEM (high sugar, HyClone)) at Eagle ' the s substratum of the improvement that replenishes 10%FBS (HyClone), 1% penicillin/streptomycin and 1%L-glutamine (Cambrex).From second day, changed a subculture with fresh culture every day.Use the cell between a generation and three generations.Neural stem cell is separated from mouse (E13.5) and in contrast at additional B27 serum replacement (Gibco), people's recombinant basic FGF and the people EGF (R﹠amp that recombinates; D), Eagle ' s substratum/F12 (DMEM/F12, Gibco) the middle cultivation of containing the Dulbecco ' improvement of Regular Insulin (Sigma), Transferrins,iron complexes (Sigma), selenium (Sigma), progesterone (Sigma) and penicillin/streptomycin (Cambrex).
2. the infection of retrovirus-mediated method
PBabe puro Bmi-1 (uses Lipofectamine (Invitrogen) transfection to select to PT67 packaging cell line (Clontech) and in the presence of tetracycline (3 μ g/ml) (BD science) with insert people Bmi-1 (NCBI registration number: L13689NM_024865) prepare) and pBabe puro to pBabe puro carrier.The transformant strain grow to 90% or higher coverage (confluency) after, supernatant liquor by filter (0.45 μ m) (Millipore) to remove cell debris, make supernatant liquor superinfection astroglia cell then, (polybrene (Sigma) separates in the time of 10 hours at the interval to use cohesion amine.In the presence of tetracycline (0.5 μ g/ml), select subsequently.
3. western blot analysis and sxemiquantitative PCR
Use the RIPA damping fluid that whole cell extracts is separated from cell, use the definite protein concn wherein of Branford detection method (Bio-rad) subsequently.The cell extract of per 50 μ g carries out 10% or 4～12% prefabricated SDS-PAGE (Invitrogen).Thus separated protein transduction being moved to pvdf membrane (Millipore) goes up and seals with 3～5% skimming milk TBST.With anti-Bmi-1 (Upstate), anti-p16 INK4A(Santacruz), anti-p19 ARF(Novous) and alpha-tubulin (sigma) 4 ℃ of vibration incubated overnight, the anti-mouse IgG that puts together with HRP, anti-rabbit igg (Zymed) are at room temperature reaction subsequently.Supersignal West Pico test kit (Pierce) is used for detecting.
Use Trizol (Invitrogen) that whole RNA separation from each cell strain is come.In the presence of oligomerization d (T) 12-18-mer (Invitrogen), use Superscriptase II reversed transcriptive enzyme (Invitrogen) to synthesize cDNA total RNA from 500ng.In the presence of each primer that is fit to of 10pmol, be pre-mixed liquid (1U Tag archaeal dna polymerase, 250 μ M dNTPs, 10mM Tris-HCl, 40mM KCl and 1.5mM MgCl with 1 μ l cDNA with PCR 2Bioneer Korea) carries out RT-PCR.The primer that is used for RT-PCR be suitable for increasing mouse marker GAPDH, nidogen and Sox2, the following demonstration of their base sequence.
The primer that is used for mouse GAPDH
Forward: 5 '-GATGACATCAAGAAGGTGGTGAAG-3 ' (SEQ ID NO.:1)
Oppositely: 5 '-GTTGCTGTAGCCGTATTCATTGTC-3 ' (SEQ ID NO.:2)
The primer that is used for the mouse nidogen
Forward: 5 '-GGCATCCCTGAATTACCCAA-3 ' (SEQ ID NO.:3)
Oppositely: 5 '-AGCTCATGGGCATCTGTCAA-3 ' (SEQ ID NO.:4)
The primer that is used for mouse sox2
Forward: 5 '-AGTGGTACGTTAGGCGCTTC-3 ' (SEQ ID NO.:5)
Oppositely: 5 '-TGCCTTAAACAAGACCACGA-3 ' (SEQ ID NO.6)
4. induce and dedifferente
With the identical culture condition that is used for neural stem cell under induce and dedifferente.Cell cultures uses two kinds of diverse ways to carry out.Cell is with 1 * 10 5The density of individual cells/well is inoculated on 6 orifice plates and cultivated 12 hours, substratum is changed to replenish B27 serum-free (Gibco), people's recombinant basic FGF and the people EGF (R﹠amp that recombinates subsequently; D), contain Regular Insulin (Sigma), Transferrins,iron complexes (Sigma), selenium (Sigma), progesterone (Sigma) and penicillin/streptomycin (Cambrex) fresh Dulbecco ' s modified Eagle ' s substratum/F12 (N2) (DMEM/F12, Gibco).Cell is handled with bFGF every day, and substratum every other day once is changed to fresh culture.Perhaps, under proper condition cultured cells with trypsin acting and with 3 * 10 5The density of individual cell is seeded on the 60mm microbial culture flat board.Replenish B27 serum-free (Gibco), people's recombinant basic FGF and the people EGF (R﹠amp that recombinates; D), (DMEM/F12 is Gibco) as substratum to contain fresh Dulbecco ' the s modified Eagle ' s substratum/F12 (N2) of Regular Insulin (Sigma), Transferrins,iron complexes (Sigma), selenium (Sigma), progesterone (Sigma) and penicillin/streptomycin (Cambrex).
5. cross the marker of expressing by immunocytochemical determination with separately
With 4% Paraformaldehyde 96 (EMS) 4 ℃ fix 1 hour after, neural ball vibrates overnight incubation in 20% sucrose.Then, neural ball uses OCT compound very low temperature to refrigerate in 8 hole cell slide glasss (Nunc) and to be cut into 8～10 μ m before dyeing thick.After the PBS sealing that contains 10% normal sheep serum (Jackson ImmunoResearch)+0.1%BSA (Sigma)+0.3%Triton X-100 (Sigma), anti--the nidogen (Chemicon) of section, anti--CD133 (MACS) and anti--Sox2 (Sigma) then with resisting-mouse-cy3 (Jackson ImmunoResearch), anti--rabbit-FITC (molecular probe Molecular probe) and anti--goat-cy3 (Zymed) incubated at room temperature, use DAPI (Sigma) nuclear staining 4 ℃ of overnight incubation at last.Inspection after the confocal lens of Zeiss (confocal lens) (Ka Er Zeiss Carl Zeiss) are used to dye.The cell of differentiation dyes in the same manner as described above.In this respect, anti--GFAP (Dako), anti--S100 β (Zymed), anti--'beta '-tubulin III (Covance), anti--Map2a (Sigma), anti--TH (Sigma), anti--O4 (R﹠amp; D) and anti--CNPase (Chemicon) as antibody.
6. vitro differentiation
With PLO (Polyornithine) (Sigma) and ln or Zeta protein (Sigma) bag by after, cultivate under the differentiation condition that cell provides below.
Become astroglia cell in order to break up, cell cultures is being replenished among the DMEM of 10%FBS (HyClone) (HyClone, high sugar) at people's recombinant bfgf and EGF (R﹠amp; D) or CNTF (recombinant rat ciliary neurotrophic factor) (Upstate) carried out 5～7 days under existing.
By contain N2 and B27 serum-free, replenish the people recombinate in the substratum of FGF culturing cell 4 days then in no FGF substratum cultivation induced it to be divided into neurone in 8 days.Perhaps, (Sigma) there are cultivation down 7～14 days in vitamin A acid as differentiating inducer to cell at 1～10 μ m RA.As other selection, (valproic acid, Sigma) (vitamin A acid is cultivated 7～14 days to induce differentiation to become neurone to cell under the co-existing in Sigma) with 1～10 μ m RA at 1～10mM VPA.
By in replenishing the N2 substratum of B27 serum-free at PDGF-AA (Thr6 PDGF BB-AA, R﹠amp; D), T3 (3,3,5-three iodo-L-thyronine, Sigma), people's recombinant basic FGF and EGF (R﹠amp; D) existence is cultivated down and is induced to differentiate into oligodendrocyte.
When cultivating under aforementioned differentiation condition, whether the morphology of monitoring cell and use is specific to separately the antibody of differentiation marker thing carries out immunocytochemistry and correctly carries out to detect differentiation.
Embodiment 2: the result
The Bmi-1 gene uses the retroviral vector transduction system to cross in the mouse astroglia cell that has broken up and expresses.As Bmi-1 target gene p16 Ink4aAnd p19 ArtCrossing of Bmi-1 gene expressed by western blot analysis confirmation (Fig. 1) when not expressing.
Then, the condition that is used for culture of neural stem cells neural is induced and is dedifferented.Astroglia cell is with 1 * 10 5The density of individual cells/well is inoculated on 6 orifice plates, and after 12 hours, cell is cultivated under the condition of neural stem cell being suitable for.Cultivate in 3-4 days, observe the form that cell has the neural stem cell sample.When cell with 3 * 10 5The density of individual cell/plate is inoculated in when cultivating on the 60mm bacterium flat board and under the condition identical with neural stem cell, also observes the form that they have neural stem cell.On the contrary, when cultivating, do not form neural ball (Fig. 2) with control vector pBabe puro EGFP cells transfected.At the 6th day, observing neural ball had and is transferred to new flat board when neural stem cell and identical form when cultivating thereon.Whether can self in order to detect the cell that dedifferentes with the Bmi-1 gene, carry out inferior ball (subsphere) and form and detect.The result has formed ball (data are unlisted) after one week in unicellular.
In order to find whether these cells have the characteristic identical with neural stem cell, use marker separately to carry out immunocytochemistry.In this respect, main great expression is also being observed in the cell of neural stem cell sample after the dyeing in marker nidogen, CD133 and the Sox2 of neural stem cell.In addition, the freezing microtome section of the neural ball that therefore forms has been proved conclusively the expression (Fig. 3) of nidogen, CD133 and Sox2.
In addition, analyze by RT-PCR, the nidogen and the Sox2 that all are expressed in neural stem cell find to be expressed in according in the cell that dedifferentes of the present invention.Therefore, judge that the cell that dedifferentes according to the present invention is the cell (Fig. 4) of neural stem cell sample.
For whether the cell that detects the neural stem cell sample can break up as neural stem cell, be induced to differentiate into astroglia cell, oligodendrocyte and neurone in the same manner as described above.By using the analysis at each self-marker's specific antibody, the cell of discovery neural stem cell sample is divided into three types of cell as neural stem cell.Differentiate with the expression of GFAP and S100 to be divided into astroglia cell, differentiate to be divided into neurone and to differentiate with the expression of 'beta '-tubulin III (Tuj1) and Map2a to be divided into oligodendrocyte (Fig. 5) with the expression of O4 and CNPOase.
In a word, the data declaration Bmi-1 gene that obtains by this research plays an important role in the cell that astroglia cell is dedifferented for the neural stem cell sample, and the cell of these neural stem cell samples that dedifferente can break up back astroglia cell, oligodendrocyte and neurone.
Commercial Application
As described before, Bmi-1 is useful inducing astroglia in being dedifferentiated into to NSC, and The NSC of dedifferenting can be used for the treatment of various diseases.
The bibliography of prior art
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3.Sally Temple.The development of neural stem cells Nature calendar year 2001; 414:112-117
4.Anna V.Molofsky, Ricardo Pardal, Toshihide Iwashita, In-Kyung Park, Michael F.Clarke ﹠amp; Sean J.Morrison.Bmi-1 dependence distinguishes neural stem cell self-renewalfrom progenitor proliferation Nature2003; 425:962-967
5.Jacqueline J.L.Jacobs, Blanca Scheijen, Jan-Willem Voncken, Karin Kieboom, Anton Berns, and Maarten van Lohuizen.Bmi-1 collaborates with c-Myc in tumorigenesis byinhibiting c-Myc-induced apoptosis via INK4a/ARF Genes ﹠amp; Development 1999; 13:2678-2690
6.S 1' lvia Bea, Frederic Tort, Magda Pinyol, Xavier Puig, Luis Herna ' ndez, SilviaHerna ' ndez, Pedro L.Ferna ' ndez, Maarten van Lohuizen, Dolors Colomer, and Elias Campo.BMI-1 Gene Amplification and Overexpression in Hematological Malignancies Occur Mainlyin Mantle Cell Lymphomas.2001; 61:2409-412
7.Dusan Zencak, Merel Lingbeek, Corinne Kostic, Meriem Tekaya, Ellen Tanger, DanaHornfeld, Muriel Jaquet, Francis L.Munier, Daniel F.Schorderet, Maarten van Lohuizen, andYvan Arsenijevic.Bmil Loss Produces an Increase in Astroglial Cells and a Decrease inNeural Stem Cell Population and Proliferation.The Journal of Neuroscience 2005; 25:5774-783
8.Carly Leung, Merel Lingbeek, Olga Shakhova, James Liu, Ellen Tanger, ParvinSaremaslani, Maarten van Lohuizen ﹠amp; Silvia Marino.Bmil is essential for cerebellardevelopment and is overexpressed in human medulloblastomas Nature 2004; 428:337-341
9.Atsushi Iwama, Hideyuki Oguro, Masamitsu Negishi, Yuko Kato, YouheiMorita, Hiroko Tsukui, Hideo Ema, Takehiko Kamijo, Yuko Katoh-Fukui, HaruhikoKoseki, Maarten van Lohuizen, and Hiromitsu Nakauchi.Enhanced Self-Renewal ofHematopoietic Stem Cells Mediated by the Polycomb Gene Product Bmi-1 of transcriptionalactivators 2004; 21:843-851
10.Katalin Schlett and Emilia Madara ' sz.Retinoic Acid Induced Neural Differentiationin a Neuroectodermal Cell Line Immortalized by p53 Deficiency.Journal of NeuroscienceResearch 1997; 47:405-15
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12.Tomoya Kitayama, Masanori Yoneyama, Keisuke Tamaki, and Yukio Yoneda.Regulation of Neuronal Differentiation by N-Methyl-D-Aspartate Receptors Expressed inNeural Progenitor Cells Isolated From Adult Mouse Hippocampus Journal of NeuroscienceResearch 2004: 76:599-12
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15.Anna V.Molofsky, Shenghui He, Mohammad Bydon, Sean J.Morrison, RicardoPardal.Bmi-1 promotes neural stem cell self-renewal and neural development but notmouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways.Genes ﹠amp; Development 2005; 19:1432-1437
16.Sopia W.M.bruggeman, Merel E.Valk-Lingbeek, Petra P.M.van der Stoop, Jacqueline J.L., Jacobs, Karin kieboom, Ellen Tanger, Danielle Hulsman, Carly Leung, YvanArsenijevic, Silvia Marino and Maarten van Lohuizen.Ink4a and Arf differentiallu affect cellproliferation and neural stem cell self-renewal in Bmi-l-deficient mice.Genes ﹠amp; Development 2005; 19:1438-1443
17.Kishi Y, Takahashi J, Koyanagi M, Morizane A, Okamoto Y, Horiguchi S, Tashiro K, Honjo T, Fujii S, Hashimoto N..Estrogen promotes differentiation and survival ofdopaminergic neurons derived from neural stem cells Journal of neuroscience research 2005; 79:279-286
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21.Hu X, Jin L, Feng L.Erk1/2 but not PI3K pathway is required for neurotrophin3-induced oligodendrocyte differentiation of post-natal neural stem cells.Journal ofNeurochemistry 2004; 90:1339-1347
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Sequence table
＜110〉Eun Tak Kabushiki Kaisha
＜120〉use Bmi-1 that astroglia cell is dedifferentiated into and be neural stem cell
<160>6
<170>KopatentIn?1.71
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the forward primer of GAPDH
<400>1
Figure A200680053500Q00151
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the reverse primer of GAPDH
<400>2
Figure A200680053500Q00152
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the forward primer of nidogen
<400>3
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the reverse primer of nidogen
<400>4
Figure A200680053500Q00162
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the forward primer of SOX2
<400>5
Figure A200680053500Q00163
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be specific to the reverse primer of SOX2
<400>6
Figure A200680053500Q00164

Claims (10)

1. induce astroglia cell to be dedifferentiated into composition, comprise Bmi-1 protein or contain the nucleic acid material of the proteinic nucleotide sequence of coding Bmi-1 into neural stem cell.
2. composition as claimed in claim 1, the wherein said nucleic acid material that contains the proteinic nucleotide sequence of coding Bmi-1 is the carrier that allows the Bmi-1 protein expression.
3. composition as claimed in claim 1, the wherein said nucleic acid material that contains the proteinic nucleotide sequence of coding Bmi-1 is to express the proteinic virus of Bmi-1.
4. composition as claimed in claim 1, wherein said neural stem cell of dedifferenting has the ability that is divided into astroglia cell, neurone and oligodendrocyte.
5. by induce astroglia cell to be dedifferentiated into method with Bmi-1 protein or the nucleic acid material processing astroglia cell that contains coding Bmi-1 proteinic nucleotide sequence into neural stem cell.
6. method as claimed in claim 5, wherein said neural stem cell of dedifferenting has the ability that is divided into astroglia cell, neurone and oligodendrocyte.
7. method as claimed in claim 5 comprises step:
(i) in substratum, cultivate astroglia cell;
(ii) use described Bmi-1 protein or the described astroglia cell of described nucleic acid material processing; With
(iii) induce described astroglia cell to be divided into neural stem cell.
8. substratum as claimed in claim 7 contains bFGF in the substratum in the wherein said step (i).
9. neural stem cell is used the described method preparation of claim 5.
10. use the method for the prepared cell differentiation of nerve cord of the described method of claim 5 as astroglia cell, neurone and oligodendrocyte.
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