CN103656677A - Medicine composition for treating neuron degeneration disease - Google Patents
Medicine composition for treating neuron degeneration disease Download PDFInfo
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- CN103656677A CN103656677A CN201210352867.4A CN201210352867A CN103656677A CN 103656677 A CN103656677 A CN 103656677A CN 201210352867 A CN201210352867 A CN 201210352867A CN 103656677 A CN103656677 A CN 103656677A
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Abstract
The invention discloses a medicine composition for treating neuron degeneration disease. The invention provides a method for forming neurons by specifically inducing spongiocyte differentiation. The method disclosed by the invention specifically comprises the following steps: firstly establishing a Myt1L (Myelin transcription factor 1-like) gene on a virus vector and packaging to obtain pseudovirus particles with Myt1L; and infecting spongiocyte by the pseudovirus particles with the Myt1L, and culturing for a period of time, thus differentiating the spongiocyte into neuron cells. The neuron cells and the composition thereof have a potential value in treatment of neuron degeneration diseases, such as Parkinson disease.
Description
Technical field
The present invention relates to biological technical field, relate to particularly the method and composition that a species specificity induction glial cell changes into neuronal cell.
Background technology
Stem cell has very wide application prospect in biomedical sector, and embryonic stem cell has the potential of comprehensive differentiation, becomes the focus of research.The people such as British scientist Wilmut in 1997 proceed to the somatic nucleus of sheep in non-nucleus egg mother cell and develop into ripe individuality---famous sheep Dolly, thus confirmed that in practice somatic cell reverses the probability that becomes myeloid-lymphoid stem cell.2006, Japan scientist Takahashi and Yamanaka find after 4 transcription factor (Oct-4, Sox-2, Klf4 and c-Myc) combined effect mouse skin cell, can successfully be induced and reverse that be divided into can be to the pluripotent stem cell of three differentiation of germinal layers, Takahashi in 2007 and Yamanaka utilize these four transcription factor successfully people's epidermis cell induction to be differentiated to form to versatile stem cell.Although the research of Takahashi and Yamanaka becomes a milestone in stem-cell research field, but must experience obtain versatile stem cell from somatic induction in practical operation, from versatile stem cell basis, directed differentiation is object target cell again, whole process steps is many, the cycle is long, mortality is high, the cell breaking up also has swollen neoplastic potential danger, and these problems are unfavorable for the promotion and application of this technology.
Wernig laboratory in 2010 has reported by Brn2, Ascl1 and tri-factors of Myt1l, the somatic cell of mice directly to be induced to obtain to have functional similar neuronal cell first on Nature, has opened the new page of the research in neuroscience field.Wernig laboratory in 2011, by Brn2, Ascl1, Myt1l and tetra-transcription factor of NeuroD1, forms functional nerve unit cell by people's fibroblast induction.These researchs have been simplified and from somatic induction to versatile stem cell reorientation, have been divided into neuronic loaded down with trivial details approach, for neurobiological study provides more efficiently instrument.
Parkinson disease are mainly because of the neuronal cell generation pathological changes in the district of midbrain position, position " black substance ", the function reduction of the synthetic minimizing of dopamine, inhibition acetylcholine causes; after pathological changes; neuronal quantity reduces, function reduction; this region will " be captured by glial cell institute " if can allow these glial cells become again again as neuronal cell, so perhaps have certain help for Parkinsonian treatment.
To sum up, this area forms neuronic method and composition in the urgent need to developing simple and effective induction glial cell.
Summary of the invention
The object of the invention is for providing a kind of simple and effective induction to form neuronic method and composition.
In a first aspect of the present invention, the purposes of a kind of Myt1L gene or Myt1L albumen is provided, for the preparation of induction glial cell, form neuronic compositions; Or the compositions of preparation treatment neurodegenerative diseases.
In another preference, described compositions comprises pharmaceutical composition.
In a second aspect of the present invention, a kind of pseudovirion is provided, described pseudovirion has following characteristics:
(a) carry the coded sequence of the Myt1L of external source;
(b) can infect glial cell, and in glial cell, express the Myt1L transcription factor of external source.
In another preference, described Myt1L derives from people.
In another preference, described pseudovirion is selected from lower group of slow virus, adenovirus, herpesvirus or poxvirus.
In another preference, described pseudovirion is prepared as follows:
The carrier that carries the coded sequence of Myt1L is imported in the incasing cells of pseudovirion, thereby form pseudovirion.
In a third aspect of the present invention, a kind of expression cassette is provided, described expression cassette from 5 ' to 3 ' comprises following element successively:
The specific promoter of glial cell, the coded sequence of Myt1L, termination codon.
In another preference, the specific promoter of described glial cell comprises: GFAP promoter (GFAP, glial fibrillary acidic protein) and HRE-GFAP hybrid promoter (HRE, hypoxia response element).
In another preference, Myt1L derives from mammal, preferably people.
In a fourth aspect of the present invention, a kind of expression vector is provided, described expression vector contains the expression cassette described in third aspect present invention.
In another preference, described expression vector comprises plasmid, viral vector.
In another preference, described viral vector comprises slow virus carrier, adenovirus vector, herpesvirus vector, poxvirus vector or gland relevant viral vector.
In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, described compositions comprises (i) pharmaceutically acceptable carrier and (ii) expression vector described in fourth aspect present invention or the pseudovirion described in second aspect present invention.
In a sixth aspect of the present invention, the purposes of the pseudovirion described in expression vector described in a kind of fourth aspect present invention or second aspect present invention is provided, they are used to preparation induction glial cell and form neuronic compositions; Or the compositions of preparation treatment neurodegenerative diseases.
In a seventh aspect of the present invention, a kind of method of inducing glial cell to form neuronal cell is provided, comprise step:
Under external source Myt1L albumen exists, cultivate glial cell, thereby induction glial cell forms neuronal cell.
In another preference, described external source Myt1L albumen is included in the Myt1L albumen adding in cultivating system, or in described glial cell, expresses external source Myt1L coded sequence and the external source Myt1L albumen that produces.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, at this, tire out and state no longer one by one.
Accompanying drawing explanation
Fig. 1 has shown for building the structure chart of the carrier FUGW of Myt1L slow virus carrier.
Fig. 2 has shown by the bright field figure of the U251 cell after Myt1L slow virus infection.
Fig. 3 has shown that the infected cell of presentation of results has been expressed Tuj1 albumen by the wild figure of the fluorescence of the U251 cell after Myt1L slow virus infection.
Fig. 4 has shown by the DAPI colored graph of the U251 cell after Myt1L slow virus infection, illustrates that nucleus place is colored.
The demonstration of Fig. 2-Fig. 4 result, infected cell has been expressed nerve-specific albumen Tuj1.
The specific embodiment
The long-term deep research of inventor's process, is surprised to find that, Myt1L single-gene can induce glial cell to form neuronal cell effectively first.The inventor is gene constructed on viral vector by Myt1L, and packing obtains the pseudovirion with Myt1L, and infects after glial cell by Myt1L pseudovirion, through cultivation after a while, glial cell can be induced to differentiate into neuronal cell.On this basis, completed the present invention.
promoter
In the present invention, available promoter is not particularly limited, and is one section of DNA sequence that is positioned at structural gene 5 ' end upstream, can activate RNA polymerase, has the specificity that combines exactly with Myt1L template DNA and have transcription initiation.Can be selected from the composition factor, the inductivity factor and organizing specific sex factor.Be more preferably tissue-specific promoter, as glial cell specificity promoter, can in glial cell, start transcribing of hereditary material by specificity.
myt1L gene and albumen
Myt1L, myelin transcription factor sample albumen, as a transcription factor, participates in the differentiation of neurocyte; In central nervous system, in the growth course of neuron and oligodendroglia, play a key effect.
Gene title: MYT1L species: people source; The Genbank numbering of transcript: NM_015025.
The accession number of the aminoacid sequence of MYT1L is NP_055840, and concrete sequence is as follows:
MEVDTEEKRH RTRSKGVRVP VEPAIQELFS CPTPGCDGSG HVSGKYARHR SVYGCPLAKK 60
RKTQDKQPQE PAPKRKPFAV KADSSSVDEC DDSDGTEDMD EKEEDEGEEY SEDNDEPGDE 120
DEEDEEGDRE EEEEIEEEDE DDDEDGEDVE DEEEEEEEEE EEEEEEENED HQMNCHNTRI 180
MQDTEKDDNN NDEYDNYDEL VAKSLLNLGK IAEDAAYRAR TESEMNSNTS NSLEDDSDKN 240
ENLGRKSELS LDLDSDVVRE TVDSLKLLAQ GHGVVLSENM NDRNYADSMS QQDSRNMNYV 300
MLGKPMNNGL MEKMVEESDE EVCLSSLECL RNQCFDLARK LSETNPQERN PQQNMNIRQH 360
VRPEEDFPGR TPDRNYSDML NLMRLEEQLS PRSRVFASCA KEDGCHERDD DTTSVNSDRS 420
EEVFDMTKGN LTLLEKAIAL ETERAKAMRE KMAMEAGRRD NMRSYEDQSP RQLPGEDRKP 480
KSSDSHVKKP YYDPSRTEKK ESKCPTPGCD GTGHVTGLYP HHRSLSGCPH KDRVPPEILA 540
MHESVLKCPT PGCTGRGHVN SNRNSHRSLS GCPIAAAEKL AKAQEKHQSC DVSKSSQASD 600
RVLRPMCFVK QLEIPQYGYR NNVPTTTPRS NLAKELEKYS KTSFEYNSYD NHTYGKRAIA 660
PKVQTRDISP KGYDDAKRYC KDPSPSSSST SSYAPSSSSN LSCGGGSSAS STCSKSSFDY 720
THDMEAAHMA ATAILNLSTR CREMPQNLST KPQDLCATRN PDMEVDENGT LDLSMNKQRP 780
RDSCCPILTP LEPMSPQQQA VMNNRCFQLG EGDCWDLPVD YTKMKPRRID EDESKDITPE 840
DLDPFQEALE ERRYPGEVTI PSPKPKYPQC KESKKDLITL SGCPLADKSI RSMLATSSQE 900
LKCPTPGCDG SGHITGNYAS HRSLSGCPRA KKSGIRIAQS KEDKEDQEPI RCPVPGCDGQ 960
GHITGKYASH RSASGCPLAA KRQKDGYLNG SQFSWKSVKT EGMSCPTPGC DGSGHVSGSF 1020
LTHRSLSGCP RATSAMKKAK LSGEQMLTIK QRASNGIEND EEIKQLDEEI KELNESNSQM 1080
EADMIKLRTQ ITTMESNLKT IEEENKVIEQ QNESLLHELA NLSQSLIHSL ANIQLPHMDP 1140
INEQNFDAYV TTLTEMYTNQ DRYQSPENKA LLENIKQAVR GIQV 1184
(SEQ ID NO.: 1)
Should understand, in the present invention, term " Myt1L gene and albumen " not only comprises Myt1L gene and the albumen of wild type and saltant type, the active fragment or the active derived protein (and corresponding coded sequence) that also comprise Myt1L albumen, for example, as long as described active fragment or active derived protein still have the basic function (retaining >=50%, >=70% above wild type Myt1L protein active) of wild type Myt1L albumen.
Myt1L gene can use conventional method (as PCR or complete synthesis) to obtain, and then imports carrier, proceeds to host cell, thereby express, obtains Myt1L albumen.In addition, Myt1L gene or clone also can buy acquisition, for example, can buy purchased from OriGene company (article No. SC304207).
viral vector
In the present invention, a kind of preferred carrier is viral vector.Viral vector of the present invention can directly be induced formation neuron by glial cell.
For viral vector of the present invention, being not particularly limited, can be that any virus of can utilizing has its genomic feature of transmission, brings hereditary material into other cell, the viral vector infecting.Can betide in complete live body or cell culture.Comprise slow virus carrier, adenovirus vector, herpesvirus vector, poxvirus vector, gland relevant carriers.
A kind of preferred viral vector is slow virus carrier.In an example of the present invention, by round pcr, Myt1L is gene constructed to FUGW slow virus carrier, thus form FUGW-MYT1L slow virus carrier.
A kind of particularly preferred viral vector is pseudovirion.In the art, the method for preparing pseudovirion is well known to those skilled in the art, for example, adopt incasing cells maternity leave in next life virion.
In an example of the present invention, a kind of packing method of Myt1L slow virus carrier comprises: by after the FUGW-MYT1L slow virus carrier in carrier system, pCMV-dR8.2 dvpr carrier and the common transfection host cell of pCMV-VSV-G carrier, can obtain MYT1L slow virus granule at host cell intermediate package.Host cell is 293T cell.
external evoked method
What the present invention also provided a kind of external, non-therapeutic directly induces into neuronic method by glial cell.Method by Myt1L slow virus granule induction glial cell comprises step: by a Myt1L slow virus infection glial cell, metainfective cell maintain is more than 20 days, thus formation neuron.
Through inducing formed neuronal cell, can detect by immunofluorescence, for example, with the antibody of neuronal specificity albumen Tuj1, detect, thereby identify neuronal cell.
pharmaceutical composition and administering mode
The present invention also provides a kind of can be used for that non-neuronal cell (as glial cell) induction is formed to neuronic compositions.Pharmaceutical composition of the present invention also can be treated or prevention of neurodegenerative diseases.
Pharmaceutical composition of the present invention comprises expression vector or pseudovirion and the pharmaceutically acceptable carrier that the present invention is above-mentioned.
At pharmaceutical composition of the present invention, conventionally contain 10
6-10
12the pseudovirion of PFU/ml, preferably 10
7-10
12the pseudovirion of PFU/ml, more preferably 10
9-10
11the pseudovirion of PFU/ml.
" pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.
Term refers to some medicament carriers like this: they itself are not necessary active component, and after using, there is no undue toxicity.Suitable carrier is well known to those of ordinary skill in the art.On combination of Chinese medicine is learned, acceptable carrier can contain liquid, as water, saline, buffer.In addition, in these carriers, also may there is complementary material, as filler, lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance etc.In described carrier, can also contain cell transfecting reagent.
Conventionally, after described expression vector or pseudovirion and pharmaceutically acceptable carrier are mixed, i.e. obtainable pharmaceutical composition of the present invention.
The administering mode of compositions of the present invention is not particularly limited, and representational example comprises (but being not limited to): intravenous injection, subcutaneous injection, brain injection etc.
beneficial effect of the present invention comprises:
1. the present invention only needs to pack transcription factor of Myt1L to viral vector, glial cell induction can be divided into neuronal cell, has solved to only have a plurality of transcription factor are imported to the step that carrier could obtain functional nerve unit.
2. to have simplified somatic induction be versatile stem cell in the present invention, and reorientation is divided into neuronic loaded down with trivial details approach.
3. promoter is glial cell identification specificity promoter, and only transcribing of hereditary material carried out in glial cell, avoided infecting the possibility of other cells.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber are percentage by weight and parts by weight.
experiment material
1. carrier:
Container name: FUGW (purchased from Addgene company, article No. 14883)
Element order: Ubiquitin promoter-MCS-GFP-WRE
Cloning site: BamHI/AgeI
Carrier collection of illustrative plates: as shown in Figure 1.
2. amplimer:
Forward:
aggtcgactc tagaggatcc cgccaccatg gaggtggaca ccgaggagaa gcggcat (SEQ ID NO.: 2)
Reverse:
agtccatggt ggcgaccggg acctgaattc ctctcacagc ctgcttta (SEQ ID NO.: 3)
3. cell
3.1. 293T cell:
Purchased from U.S. ATCC.The 293T cell of trypsinization exponential phase for cell transfecting, cell density is 1.2 * 10
7cell/20ml (0.6 * 10
9/ L) time, be re-seeded into the Tissue Culture Dish of 15cm, 37 ℃, 5%CO
2in incubator, cultivate transfection when cell density reaches 70%～80%.Before transfection 24h, cell culture medium is replaced by serum-free medium.
glial cell U251:
Purchased from U.S. ATCC.With the U251 cell of trypsinization exponential phase, cell density is 1.2 * 10
7cell/20ml (0.6 * 10
9/ L) time, be re-seeded into the Tissue Culture Dish in 24 holes, 37 ℃, 5% CO
2in incubator, cultivate.
embodiment 1
the structure of Myt1L slow virus carrier:
(1) take by MYT1L cDNA clone is template, by amplimer, carries out PCR reaction, and amplification obtains the PCR product of big or small 3.5 Kb left and right.
(2) PCR product and viral vector are carried out to the double digestion of BamHI and AgeI.
(3) cross after T4 DNA ligase (purchased from Takara company) connects and carry out conversion reaction.
(4) by the evaluation to transformant, select positive colony and send survey, the sequencing sequence of usining consistent with expected sequence as correctly cloning, called after FUGW-MYT1L slow virus carrier.
the packing of Myt1L slow virus carrier:
(1) prepare DNA solution (the FUGW-MYT1L slow virus carrier 20 μ g of 3 kinds of plasmids in slow virus packaging system, pCMV-dR8.2 dvpr carrier (purchased from Addgene) 15 μ g, pCMV-VSV-G carrier (purchased from Addgene) 10 μ g, mix homogeneously dilution with the Opti-MEM of respective volume, adjustment cumulative volume is 2.5ml, and at room temperature incubation is 5 minutes.
(2) get 100 μ lLipofectamine2000 (purchased from invitrogen) reagent at another Guan Zhongyu 2.4ml Opti-MEM (purchased from Invitrogen) mixed diluting, at room temperature incubation is 5 minutes.
(3) DNA after dilution described in (1) is mixed with the Lipofectamine2000 after dilution described in (2), in 5 minutes, put upside down and mix lightly.Incubation 20min under room temperature.
(4) DNA and Lipofectamine 2000 mixed liquors are transferred in the culture fluid of 293T cell, mix, in 37 ℃, 5%CO
2in cell culture incubator, cultivate.Cultivate and go the culture medium that contains transfection mixture after 8 h, every bottle of cell adds the PBS liquid of 20ml, gently double swerve once culture bottle to wash remaining transfection mixture, then go.
(5) in every bottle of cell, add the cell culture medium 25ml containing 10% serum, in 37 ℃, 5%CO
2in incubator, continue to cultivate 48 hours.
(6) collect the 293T cell conditioned medium liquid of transfection after 48 hours.In 4 ℃, the centrifugal 10min of 4000g, removes cell debris.With 0.45 μ m filter filtering supernatant in 40ml ultra-filtration centrifuge tube (purchased from Millipore).Viral crude extract sample is joined in filter cup and cover lid, filter cup is inserted in filtered solution collecting pipe.After combining, carry out balance, be placed in rotary head.At the centrifugal about 10-15 minute of 4000g.After centrifugal end, take out centrifugal device, filter cup and filtered solution collection cups are below separated.In sample collection cup, be viral concentrated solution.
(7) viral concentrated solution is shifted out, be kept in viral pipe after subpackage ,-80 degree are long-term to be preserved.The viral nomenclature obtaining is MYT1L slow virus granule.
myt1L slow virus induction glial cell;
(1) when glial cell U251 density reaches 50%～60%, carry out viral infection, before infecting, cell culture medium is replaced by serum-free medium.
(2), according to the MOI value (MOI=5) of U251 cell, add the MYT1L slow virus (adding viral number=cell number x MOI value) of Sq.Observation of cell state after 12h: if there is no obvious cytotoxic effect, continue to change culture medium after cultivation 24h; If there is obvious cytotoxic effect, change immediately culture medium.
(3) culture medium is changed: after U251 cell is by MYT1L slow virus infection three days time, use is with the N27 factor (Invitrogen, 17504-044) Neurobasal culture medium (Invitrogen, 12348-017) progressively replace original culture medium.Replacement method retains original culture medium of 1/2 at every turn, adds 1/2 the fresh Neurobasal culture medium with the N27 factor, within every three days, changes a subculture, guarantees that cell growth state is good.
(4) cell maintains: the U251 cell after viral infection need to continue to cultivate more than 20 days, needs during this period cell regularly to change liquid.
embodiment 4
the immunofluorescence of induced neuronal cell is detected;
U251 cell after continuing to cultivate above infection in 20 days is carried out to immunofluorescence experiment, and experimental procedure is as follows:
(1) washing: clean gently cell with TBS, wash three times, to remove the impurity in cell;
(2) fixing step: fresh 4% paraformaldehyde preparing is room temperature 20 minutes fixedly;
(3) washing: the TBS of pre-cooling washes three times, every all over 5 minutes;
(4) permeable membrane: blot liquid, by 0.2% Triton-X room temperature 10 minutes.
(5) washing: TBS washes three times, every all over 5 minutes;
(6) sealing: add 200 μ l 2% BSA, room temperature 1 hour.
(7) primary antibodie is hatched: β 3-Tubulin (TU-20) Mouse mAb (Cell Signaling Technology, #4466), add 100 μ l primary antibodies (1:200), and with 1%BSA, join antibody, 4 spend night, or 37 spend 2 hours.
(8) washing: wash primary antibodie, each 10 minutes of TBS, washes three times, notes: must cover surface of glass slide, as far as possible wash clean.
following steps are all at dark room operation
(9) two anti-hatching: (dark room operation) Alexa Fluor 546 Donkey Anti-Mouse IgG (Invitrogen, A10036), add 100 μ l bis-anti-(1:500), black out, incubated at room 1 hour.
(10) washing: (dark room operation) washed two and resisted, and each 10 minutes of TBS, washes three times, notes: must cover surface of glass slide, the wash clean of trying one's best, lucifuge.
(11) mounting: (dark room operation) uses VECTASHIELD
dAPI stain (4 ', 6-diamidino-2-phenylindone 4 ', 6-diamidino-2-phenylindole, be a kind of can with the fluorescent dye of the powerful combination of DNA) (H-1200) (3 ng/mL), room temperature 10 minutes; Attention: the bubble of having tried not; Slide just can not move after putting again.
(12) take pictures: use fluorescence microscope to take pictures:
U251 cell after MYT1L slow virus infection, while maintaining again 20 days left and right, by the immunofluorescence experiment of neuronal specificity albumen Tuj1, result shows that U251 cell expressed Tuj1 albumen, and Tuj1 positive cell has more than 80%, illustrate that U251 cell now has possessed neuronic characteristic, as Fig. 2-4.The U251 cell that does not infect MYT1L slow virus cannot detect the expression of Tuj1.
This shows, after MYT1L slow virus infection glial cell, can induce glial cell to express neuronal specificity albumen, makes induced glial cell possess neuron behavior.
embodiment 5
build specific expressed expression cassette and carrier in glial cell
GFAP promoter and HRE-GFAP hybrid promoter are known promoteres that can be specific expressed in glial cell.In the present embodiment, with the FUGW-MYT1L based lentiviral vector building, build the slow virus carrier of glial cell specifically expressing.Concrete method is as follows:
In FUGW-MYT1L carrier, for starting the ubiquitin promoter of MYT1L, from carrier, by the method for enzyme action, excise, insert colloid specificity promoter-GFAP promoter or HRE-GFAP promoter that pcr amplification obtains, thereby obtain the MYT1L slow virus carrier that contains following expression cassette:
(a) coded sequence-termination codon of GFAP promoter-Myt1L;
(b) coded sequence-termination codon of HRE-GFAP hybrid promoter-Myt1L.
Described expression cassette can be in glial cell specific expressed Myt1L albumen.
Test result shows, after the slow virus carrier infection glial cell containing this tissue specific expression box, can induce equally glial cell to express neuronal specificity albumen (Tuj1 albumen), makes induced glial cell possess neuron behavior.In addition, should containing the slow virus carrier of this tissue specific expression box, infect after epidermis cell the Myt1L albumen of not expressing external source.
discuss:
The invention provides a species specificity induction glial cell and be differentiated to form neuronic method, particularly, first Myt1L is gene constructed on viral vector, packing obtains the pseudovirion with Myt1L, and infect after glial cell by Myt1L pseudovirion, through cultivation after a while, glial cell can be divided into neuronal cell.Neurodegenerative diseases is because glial cell after neuron senescence and death has occupied leading position, thereby has blocked the electrophysiologic response between neuron.MYT1L slow virus can form neuronal cell by glial cell induction, MYT1L viral vector has potential value in the treatment of neurodegenerative diseases (such as parkinson disease etc.), by glial cell is induced as after neuron the neutral net of interruption is repaired, thereby alleviate patient's misery.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Sequence table
<110> Shanghai JiKai Gene Chemical Technology Co., Ltd
<120> pharmaceutical composition that can be used for treating neuron degeneration
<130> P2012-0911
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1184
<212> PRT
<213> homo sapiens (homo sapiens)
<400> 1
MEVDTEEKRH RTRSKGVRVP VEPAIQELFS CPTPGCDGSG HVSGKYARHR SVYGCPLAKK 60
RKTQDKQPQE PAPKRKPFAV KADSSSVDEC DDSDGTEDMD EKEEDEGEEY SEDNDEPGDE 120
DEEDEEGDRE EEEEIEEEDE DDDEDGEDVE DEEEEEEEEE EEEEEEENED HQMNCHNTRI 180
MQDTEKDDNN NDEYDNYDEL VAKSLLNLGK IAEDAAYRAR TESEMNSNTS NSLEDDSDKN 240
ENLGRKSELS LDLDSDVVRE TVDSLKLLAQ GHGVVLSENM NDRNYADSMS QQDSRNMNYV 300
MLGKPMNNGL MEKMVEESDE EVCLSSLECL RNQCFDLARK LSETNPQERN PQQNMNIRQH 360
VRPEEDFPGR TPDRNYSDML NLMRLEEQLS PRSRVFASCA KEDGCHERDD DTTSVNSDRS 420
EEVFDMTKGN LTLLEKAIAL ETERAKAMRE KMAMEAGRRD NMRSYEDQSP RQLPGEDRKP 480
KSSDSHVKKP YYDPSRTEKK ESKCPTPGCD GTGHVTGLYP HHRSLSGCPH KDRVPPEILA 540
MHESVLKCPT PGCTGRGHVN SNRNSHRSLS GCPIAAAEKL AKAQEKHQSC DVSKSSQASD 600
RVLRPMCFVK QLEIPQYGYR NNVPTTTPRS NLAKELEKYS KTSFEYNSYD NHTYGKRAIA 660
PKVQTRDISP KGYDDAKRYC KDPSPSSSST SSYAPSSSSN LSCGGGSSAS STCSKSSFDY 720
THDMEAAHMA ATAILNLSTR CREMPQNLST KPQDLCATRN PDMEVDENGT LDLSMNKQRP 780
RDSCCPILTP LEPMSPQQQA VMNNRCFQLG EGDCWDLPVD YTKMKPRRID EDESKDITPE 840
DLDPFQEALE ERRYPGEVTI PSPKPKYPQC KESKKDLITL SGCPLADKSI RSMLATSSQE 900
LKCPTPGCDG SGHITGNYAS HRSLSGCPRA KKSGIRIAQS KEDKEDQEPI RCPVPGCDGQ 960
GHITGKYASH RSASGCPLAA KRQKDGYLNG SQFSWKSVKT EGMSCPTPGC DGSGHVSGSF 1020
LTHRSLSGCP RATSAMKKAK LSGEQMLTIK QRASNGIEND EEIKQLDEEI KELNESNSQM 1080
EADMIKLRTQ ITTMESNLKT IEEENKVIEQ QNESLLHELA NLSQSLIHSL ANIQLPHMDP 1140
INEQNFDAYV TTLTEMYTNQ DRYQSPENKA LLENIKQAVR GIQV 1184
<210> 2
<211> 57
<212> DNA
<213> artificial sequence
<400> 2
aggtcgactc tagaggatcc cgccaccatg gaggtggaca ccgaggagaa gcggcat 57
<210> 3
<211> 48
<212> DNA
<213> artificial sequence
<400> 3
agtccatggt ggcgaccggg acctgaattc ctctcacagc ctgcttta 48
Claims (10)
1. a purposes for Myt1L gene or Myt1L albumen, is characterized in that, for the preparation of induction glial cell, forms neuronic compositions; Or the compositions of preparation treatment neurodegenerative diseases.
2. a pseudovirion, is characterized in that, described pseudovirion has following characteristics:
(a) carry the coded sequence of the Myt1L of external source;
(b) can infect glial cell, and in glial cell, express the Myt1L transcription factor of external source.
3. pseudovirion as claimed in claim 2, is characterized in that, described pseudovirion is selected from lower group of slow virus, adenovirus, herpesvirus, adeno-associated virus or poxvirus.
4. an expression cassette, is characterized in that, described expression cassette from 5 ' to 3 ' comprises following element successively:
The specific promoter of glial cell, the coded sequence of Myt1L, termination codon.
5. expression cassette as claimed in claim 4, is characterized in that, the specific promoter of described glial cell comprises: GFAP promoter and HRE-GFAP hybrid promoter; And/or
Myt1L derives from mammal, preferably people.
6. an expression vector, is characterized in that, described expression vector contains expression cassette claimed in claim 4.
7. expression vector as claimed in claim 6, is characterized in that, described expression vector comprises plasmid, viral vector;
More preferably, described viral vector comprises slow virus carrier, adenovirus vector, herpesvirus vector, poxvirus vector or gland relevant viral vector.
8. a pharmaceutical composition, is characterized in that, described compositions comprises (i) pharmaceutically acceptable carrier and (ii) expression vector claimed in claim 6 or pseudovirion claimed in claim 2.
9. a purposes for expression vector claimed in claim 6 or pseudovirion claimed in claim 2, is characterized in that, for the preparation of induction glial cell, forms neuronic compositions; Or the compositions of preparation treatment neurodegenerative diseases.
10. induce glial cell to form a method for neuronal cell, it is characterized in that, comprise step:
Under external source Myt1L albumen exists, cultivate glial cell, thereby induction glial cell forms neuronal cell.
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