CN101397573A - Lentiviral vectors expressing glial derived neurotrophic factor and uses thereof - Google Patents
Lentiviral vectors expressing glial derived neurotrophic factor and uses thereof Download PDFInfo
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Abstract
The invention discloses a lentiviral vector for expressing glial cell line-derived neurotrophic factor and application thereof, belonging to the technical field of medical biology. The lentivirus vector for expressing the glial cell line-derived neurotrophic factor has a preservation number of CGMCCNo.2086. The invention has the advantages that: the slow virus vector constructed by the invention contains two promoter structures, can express GDNF gene and hygromycin resistance gene at the same time, and the slow virus vector supernatant packaged by a transient transfection method can efficiently transfect and divide relatively slow neural stem cells, so that the GDNF gene is integrated into the target cell genome and stably expressed for a long time; the neural stem cells after transfection can remove the neural stem cells which are not successfully transfected by hygromycin screening to obtain a uniform cell system for over-expressing GDNF; the nerve stem cell modified by the lentivirus vector gene can still keep the original biological characteristics.
Description
Technical field
The present invention relates to a kind of lentiviral vectors of expressing glial cell line-derived neurotrophic factor and uses thereof, belong to medical biotechnology field.
Background technology
Glial cell line-derived neurotrophic factor (Glial cell derived neurotrophic factor; GDNF) be a kind of neurotrophic factor, confirm in the test of various clinical The Animal Model Study that GDNF can promote the survival of dopaminergic neuron, motor neuron and the reconstruction of damage back nerve synapse with neurotrophy and neuroprotective.But because GDNF is a kind of macro-molecular protein, be difficult to arrive the local patholoic change cerebral tissue, become the major cause that hinders its clinical application by hemato encephalic barrier.
Neural stem cell has self duplication and the multidirectional ability that is divided into neurone and spongiocyte, for the cell therapy of nerve degenerative diseases has brought hope.The neural stem cells transplantation experimental study confirms that the neural stem cell that is implanted in the animal model brain can be integrated into local brain tissue, and moving within the specific limits and breaking up becomes neurone and spongiocyte.Therefore neural stem cell also is used as a kind of cell carrier that carries GDNF.Studies confirm that, go into the diseased region of Parkinson's animal model of primates and muroid with crossing the neural stem cells transplantation of expressing GDNF, obviously improved former generation survival of dopaminergic neurons efficient of co-transplantation, it is a kind of carrier of cell therapy preferably that explanation can be crossed the neural stem cell of expressing gdnf gene.But the neural stem cell division growth is slow relatively, and particularly through after the secular cultivation of going down to posterity, the cell proportion that is in the division growth state can progressively descend.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of lentiviral vectors of stably express glial cell line-derived neurotrophic factor.
Another technical problem that the present invention will solve provides the method for the lentiviral vectors of expression glial cell line-derived neurotrophic factor in external efficient transfection neural stem cell.
For achieving the above object, the present invention is by the following technical solutions:
Express the lentiviral vectors of glial cell line-derived neurotrophic factor, its preserving number is CGMCC No.2086.
Express the lentiviral vectors in-vitro transfection neural stem cell of glial cell line-derived neurotrophic factor and the method for expression, neural ball is dispersed as single cell suspension with pancreatin, with 2.5 * 10
5The density of/ml is inoculated in the culture dish with fibronectin bag quilt, cultivate after at least six hours, the supernatant that in the 250ul nerve stem cell culture medium, adds the lentiviral vectors CGMCC No.2086 of MOI=0.5-10 (preferred 1.0), adding concentration is the polybrene (cohesion amine) of 2ug/ml, transfection 20 hours.
The lentiviral vectors CGMCC No.2086 that expresses glial cell line-derived neurotrophic factor is used for preparation treatment nerve degenerative diseases such as Parkinsonian purposes.
The host bacterium of the lentiviral vectors that the present invention relates to preserved at China Committee for Culture Collection of Microorganisms common micro-organisms center on June 14th, 2007, deposit number is CGMCC No.2086, the microbial strain of ginseng certificate is DUET101-GDNF, the classification called after colon bacillus of suggestion.
The polygene expression vector that we have expressed GDNF and hygromycin gene when having made up by house-keeping gene promotor EF1-a and PGK driving under study for action.Confirm in the experiment that the lentiviral vectors that carries GDNF and GFP (green fluorescent protein) gene can continue high expression level GDNF and GFP in external successful transfection neural stem cell.The neural stem cell of transfection success is expressed hygromycin gene simultaneously, screens through external adding Totomycin, and we can obtain the neural stem cell system that homogeneous is expressed GDNF and GFP.Use this purity higher, the character more neural stem cell of the high expression level GDNF of homogeneous is transplanted and will further be improved the security and the validity of transplantation treatment.Compare with the carrier of other type, lentiviral vectors has higher transfection efficiency for the relative neural stem cell slowly of propagation, and can make foreign gene GDNF obtain more continual and steady expression.We have carried out continuous monitoring to the GDNF secretion level of the neural stem cell of transfection GDNF, find that GDNF can be in external continuous expression secretion, and secretion level does not take place significantly to change.In the long-term cultivation after hygromycin selection, the neural stem cell after the transfection can keep original biological characteristics.
Advantage of the present invention is: the lentiviral vectors that the present invention makes up contains two promoter structures, can express gdnf gene and hygromycin gene simultaneously, the lentiviral vectors supernatant that can pack out by the method for transient transfection is transfection division neural stem cell relatively slowly efficiently, gdnf gene is integrated in the target cell genome, the steady in a long-term expression; Neural stem cell after the transfection can be removed successfully neural stem cell of untransfected through hygromycin selection, and the mistake that obtains homogeneous is expressed the cell system of GDNF; The neural stem cell of modifying through lentiviral vector genome still can keep its original biological characteristics.
The invention will be further described below in conjunction with drawings and Examples, do not limit the present invention in any way, all any this areas of carrying out according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the lentiviral vectors design of graphics that contains gdnf gene; Fig. 1-A wherein: isolate original segment after lentiviral vectors DUET101 enzyme is cut; Fig. 1-B:PCR amplifies the gdnf gene total length that contains restriction enzyme site; Fig. 1-C: plasmid enzyme restriction is identified after the successful connection.
Lentiviral vectors ring junction composition and structure back mode chart: Fig. 2-A of Fig. 2 for expressing glial cell line-derived neurotrophic factor: ring junction composition: as figure plasmid total length 11430bp, the disconnected gdnf gene segment for being connected into of figure medium green color chips is between restriction enzyme site Xba-I and BsrG1; Fig. 2-B: structure iron: as scheme wherein that EF1-a and PGK are promotor, drive the plain resistant gene of gdnf gene and damp enzyme respectively.LTR is the long terminal repeat of lentiviral vectors.
Fig. 3 is the lentiviral vectors transfection neural stem cell observations under light microscopic and the fluorescent fiber mirror after 10 days that contains GFP: wherein, and Fig. 3-A: neural ball under the light microscopic (400 times); Fig. 3-B: fluorescent microscope is observed down, the neural ball GFP positive; Fig. 3-C: neural ball is dispersed as light microscopic figure behind the single cell suspension; Fig. 3-D: fluorescent microscope is observed down, the 95% cell journey GFP positive.
Fig. 4 is transfection conditions optimization experiment figure as a result: Fig. 4-A: transfection efficiency relatively shows the neural ball transfection efficiency of direct transfection average out to 7.08% ± 0.80% under the different growth forms; Neural ball is dispersed as transfection behind the single cell suspension, transfection efficiency average out to 22.31% ± 0.28%; The ANOVA check, p＜0.01, the n=10 group difference has the significance meaning.Fig. 4-B: the GDNF transfection efficiency is measured under the different adherent conditions: normal control group and poly-lysine bag are organized a transfection efficiency there was no significant difference (ANOVA, n=8, p〉0.05); The fibronectin bag organized and other two groups of transfection efficiencies between significant difference (p＜0.05) is arranged.Fig. 4-C: different transfection time point transfection efficiency measurement results (n=10): under the identical transfection conditions, with the transfection time lengthening, transfection efficiency improves gradually, arrives plateau after 20-20 hours.
Fig. 5 is GDNF secretion situation ELISA measurement result figure under the different copy number conditions: the GDNF secretory volume all has significant difference (p＜0.05) between ANOVA analysis of control group and transfection group, wherein, MOI=5 group and MOI=10 group and MOI=0.5,1,2.5 10 group differences have significance (p＜0.01).
Fig. 6 is different times GDNF secretory volume measurement result figure after the hygromycin selection: the sustainable expression of GDNF after the transfection, secretory volume prolong in time not have obviously and change; The GDNF secretory volume is relevant with the transfection copy number, and copy number is high more, and the GDNF secretory volume is high more.
Embodiment
Embodiment 1: Culture of neural stem cells
One. method: former generation neural stem cell take from people embryo spontaneous abortion fetus (8-16w) (Xuan Wu obstetrics and gynecology hospital provides, through Ethics Committee's approval).Make single cell suspension after drawing materials and cross 400 mesh filter screens and remove and to organize residue, trypan blue counting back is with 2.5 * 10
5The density inoculation of/ml.Substratum is DMEM-F12 (U.S. GIBICO company), N
2Tonic (U.S. GIBICO company), 20ng/mlbFGF (U.S. R﹠amp; D company), 20ng/ml EGF (U.S. R﹠amp; D company), mycillin (U.S. GIBICO company).5%CO
2Cultivate in the incubator.Visible little cell colony forms about 3 days, all visible neural balls.Change liquid every three and half, when treating that neural ball grows to the 50um left and right sides, shear and go down to posterity.Treat that neural ball increases after the some amount, neural ball is dispersed as single cell suspension with pancreatin after, again with 2 * 10
5The density inoculation of/ml, this method can make neural stem cell be increased in a large number at short notice.
Two. the result: after former generation, the people embryo cortex neural stem cell of drawing materials was cultivated a week, visible neural ball formed.Combination is tight between each cell space of neural ball at this moment, the character homogeneous, and refractivity is stronger, when treating that neural ball grows to the 0.5mm left and right sides, begins to shear and goes down to posterity.Go down to posterity through 3-4 shearings, neural ball is expanded to some amount, and we adopt the digestion succusion that neural ball is dispersed as single cell suspension, with 2 * 10
5The density inoculation is gone down to posterity, and obviously shortened by the unicellular cycle that forms neural ball this moment, can obtain 2-3 times cell after the week of going down to posterity.After the passage 10 times, vitro culture was carried out the differentiation and proliferation ability and has been identified about 2 months, was used for next step experiment.
The lentiviral vectors that embodiment 2. contains gdnf gene makes up
One. material source: the plasmid PLNCX-2 that comprises people GDNF receives the Neuroscience Research institute in the Capital University of Medical Sciences, and wherein gdnf gene sequence total length is 558bp, is sequence shown in the sequence table SEQ ID No.3.Lentiviral vectors plasmid DUET101 receives in the U.S. Cheng Linzhao of Johns Hopkins University and teaches.
Two. method:
1.PCR method amplifies the gdnf gene sequence that contains corresponding restriction enzyme site
According to lentiviral vectors plasmid structure, designed the primer of the amplification GDNF that comprises XbaI and two restriction enzyme sites of BsrGI, upstream primer: 5 '-T
TCTAGACCACCATGAAGTTATGGGATGTCGTG-3 ' (SEQ IDNO.1); underscore is the restriction enzyme site XbaI for adding partly; the restriction enzyme site front adds protection base T, and ccacc thereafter is the Kozark sequence, is thereafter the one section sequence that begins from GDNF beginning password ATG again.Downstream primer 5 '-CA
TGTACATCAGATACATCCACACCTTTTAGCG-3 ' (SEQ ID NO.2), underscore be the restriction enzyme site BsrG I for adding partly, and the front adds the protection base equally, thereafter for comprising one section sequence of termination codon in the gdnf gene sequence.By PCR method, be template with plasmid PLNCX-2, amplify gdnf gene sequence total length with the archaeal dna polymerase of high-fidelity.The PCR reaction conditions is as follows.
Reaction system:
Reaction conditions:
Fs: 98 ℃ of 1 circulations in 30 seconds
Phase III: 72 ℃ of 1 circulations in 7 fens
2. lentiviral vectors DUET101 and GDNF PCR product use restriction enzyme Xba I and BsrG I double digestion.
The transferring plasmid DUET101 of gained PCR product and lentiviral vectors is carried out double digestion with XbaI and two enzymes of BsrGI (U.S. NEB company), and reaction system and condition are as follows.
The endonuclease reaction system: be total to 50ul, concrete composition is as follows:
Reaction conditions is that 37 degree water-baths were hatched 2 hours.It is standby that enzyme is cut product recovery back.
Carrier DUET101 enzyme is cut product 1% agarose gel electrophoresis, make the carrier segment separate segment with original green fluorescent protein (GFP) gene fragment and separate.
3. carrier segment gel reclaims: this process utilization glass milk gel reclaims test kit (Japanese Takara company) to carry out, and concrete grammar is with reference to the specification sheets of product, and step is as follows:
(1) ultraviolet lamp incision steamed sandwich contains the pulsating gel of carrier, removes the unnecessary gel tissue around the DNA during cutting as far as possible, and the gel of well cutting is put in the 1.5mlEP pipe of weighing in advance.
(2) use scales/electronic balance weighing, add the NaI solution of 3 times of volume gel weight, effect treated that gel melted fully in 15 minutes in the 55 degree water-baths.
(3) add 20ul glass milk solution, abundant mixing, room temperature effect 20 minutes repeats mixing several times in the mechanism.
(4) 10000rpm is centrifugal 1 minute, removes supernatant, remaines in earlier in another 1.5mlEP pipe, if organic efficiency is lower, can repeat (3) once.Glass milk precipitation remaines in the pipe end.
(5) with the resuspended glass milk of lavation buffer solution precipitation, centrifugal 1 minute of 10000rpm abandons supernatant.Repeat once.
(6) add and the isopyknic TE damping fluid of glass milk solution, 55 degree effects 10 minutes are fully dissolved DNA.Centrifugal 1 minute of 10000rpm draws supernatant and is stored in the 1.5ml EP pipe.Get 1ul and carry out 1:40 dilution its concentration of spectrophotometric determination.
4. ligation: utilization T4 dna ligase (U.S. NEB company) carries out ligation with the gel recovery segment that the PCR enzyme of GDNF cuts back to close product and carrier DUET101, and gdnf gene is connected among the carrier DUET101.
(1) the PCR enzyme is cut back to close product and carrier gel and reclaim in the segment 10:1 adding in molar ratio reaction system, whether enzyme cuts entirely to set up empty carrier connection control tube to see carrier simultaneously.
Reaction system:
Reaction conditions: 16 degree overnight incubation.
(2) connect product transformed competence colibacillus bacterium
Melt competent cell Stbl3 (American I nvitrogen company) on ice, get 5ul (10pg-100ngDNA) and connect in the product adding competent cell, stir gently, fully placed on ice 30 minutes behind the mixing with the rifle head; Change the EP pipe in 42 degree water-baths thermal shock 45 seconds, take out immediately, placed on ice 2 minutes; The SOC substratum that adds the 250ul preheating, the tight pipe lid of lid is sealed with sealing film, is put in the 37 degree shaking tables 225rpm shaking culture 1 hour; Get at last on the microbial culture plate that 25-100ul coats preheating, the dull and stereotyped inversion is put in the incubator, 37 degree overnight incubation.
(3) bacterium liquid PCR screening positive clone: a plurality of mono-clonal bacterium colonies of picking are put in respectively and fill the shaking in the tube of 3mlLB liquid nutrient medium, 230rpm, 37 degree shaking culture 8 hours.Getting 1ul bacterium liquid is template, carries out pcr amplification with the GDNF primer.2% agarose gel electrophoresis is chosen positive colony.Preserve bacterial classification.
(4) bacterium liquid PCR positive colony is carried out plasmid and carry for a short time, further identify with BsrG I double digestion with Xba I to be connected the result.The evaluation of checking order of positive colony plasmid.
(5) the correct plasmid DUET101-GDNF of order-checking preserves bacterium liquid in-80 ℃ of refrigerators, and standby with transfection level plasmid extraction kit extraction preservation.
Three. the result:
With the plasmid PLNCX2 that comprises the gdnf gene sequence is template, and behind pcr amplification, the trip of 2% sepharose electricity shows the band that conforms to purpose segment size, as Fig. 1-B.Lentiviral vectors is behind the xbaI/BsrGI double digestion, and 1.2% agarose gel electrophoresis shows that the original segment that is positioned between two restriction enzyme sites in the carrier is separated, and carrier is cut into linearity, as Fig. 1-A.The PCR product is selected positive colony after being connected back transformed competence colibacillus bacterium with the carrier segment T4DNA ligase enzyme that reclaims through gel, and it is separated that the visible segment that conforms to GDNF segment size of plasmid enzyme restriction evaluation is extracted in the amplification back, as Fig. 1-C.Insert segment and Genebank (this section sequence numbering through order-checking
AY052832, sequence is listed in file sequence tabulation) in length be that the sequence of 558bp conforms to.Illustrate that gdnf gene successfully is cloned into lentiviral vectors.Carrier structure of the present invention as shown in Figure 2.
Plasmid DUET101-GDNF preserved at China Committee for Culture Collection of Microorganisms common micro-organisms center on June 14th, 2007, deposit number is CGMCC No.2086, the microbial strain of ginseng certificate is DUET101-GDNF, the classification called after colon bacillus of suggestion.
The condition and the optimization of embodiment 3. transfection neural stem cell
One. method:
1. transfection: slow virus particulate packing adopts liposome-mediated transient transfection method to carry out, and transfection (is received the 293T cell life science institute in Peking University, seen document: Xu Xingang, Hu Jianhe, Zhang Yanming, Deng Hongkui the day before yesterday; The HCV Envelope Protein Gene Expression of retroviral vector mediation and animal immune test; " Chinese virusology "; 200419 (6): 563-567.) go down to posterity in the 100mm culture dish, culture plate is used 100ug/ml before inoculation poly-lysine bag was washed twice with DPBS before using by 1 hour.Inoculating cell adds up to 5 * 10
6, 5%CO
2Cultivate 24h in the incubator, treat to carry out transfection behind cell 70%-80% remittance sheet.Coding GDNF that will build respectively and the transferring plasmid DUET101 of GFP, coding lentiviral vectors gag, the plasmid CMV △ 8.91 of pol structure (receives in U.S. Johns Hopkins University, see document: Zhou BY, Ye Z, Chen G, Gao ZP, zhang YA, Cheng L.Inducible and reversible transgene expression in human stem cells afterefficient and stable gene transfer.Stem Cells.2007 Mar; 25 (3): 779-89.), and the plasmid PMD.G of coding VSV-G structure (receives in U.S. Johns Hopkins University, see document: zhou BY, Ye Z, Chen G, Gao ZP, Zhang YA, Cheng L.Inducible and reversible transgene expressionin human stem cells after efficient and stable gene transfer.Stem Cells.2007), ratio in 3:4:1 is carried out transfection, total amount 16ug, put into the polystyrene tube that contains 800ul OPTI-MEM (U.S. GIBICO company), the liposome of DNA:Lipofectamine (American I nvitrogen company)=1:2.5 is dissolved among isopyknic OPTI-MEM in addition, and room temperature is placed after five minutes DNA and liposome are mixed, and room temperature left standstill 20 minutes behind the soft mixing.Discard original 293T cell culture medium, add 8ml OPTI-MEM substratum, add DNA and liposome mixed solution, discard transfection liquid behind cultivation 8-12h, change and collect substratum ITS (DMEM+Insulin+transferin).Respectively at collecting supernatant behind 24h and the 48h, the 0.45um filter filters removes cell debris, and Centricon plus-20 whizzer concentrates back transfection 293T cell and carries out the titre evaluation, and-80 degree are preserved.
2. condition optimizing: be to improve transfection efficiency, seek the condition of the most suitable neural stem cell transfection, we adopt poly-lysine respectively, and the fibronectin bag is made the method for the temporary transient adherent growth of neural stem cell by culture dish.Transfection the day before yesterday, neural ball is dispersed as single cell suspension with pancreatin, with 2.5 * 10
5The density of/ml is inoculated in respectively with poly-lysine or fibronectin (Fibronectin U.S. SIGMA company) and wraps in 24 orifice plates of quilt, and bag is control group by group.At least after cultivating six hours, be 0.5,1 with MOI respectively, 2.5,5,10 add the lentiviral vectors supernatant of GDNF and GFP in the 250ul nerve stem cell culture medium, respectively with 2ug/ml, 6ug/ml, 8ug/ml concentration adds cohesion amine (Polybrene, U.S. SIGMA company), cultivates 20 hours, renew bright substratum and cell is blown afloat gently with the blue electron gun head, make it recover the suspension growth state.A week after the transfection is dispersed as single cell suspension with neural ball, and fluorescent microscope is observed counting GFP positive cell percentage down, further measures the per-cent of GFP positive neurons stem cell with the method for flow cytometer, analyzes transfection efficiency.Simultaneously difference cohesion amine concentration group is carried out trypan blue counting cells survival rate.To MOI=1, cohesion amine concentration is that the 2ug/ml group has been carried out different transfection time point transfection efficiencies mensuration.Do not express fluorescently-labeled supernatant for only containing GDNF, we adopt the Totomycin (hygromycin) that transforms back adding in the 5th day 100ug/ml to screen, and screen back 72 hours, and failing, it is dead to transform the neural stem cell meeting.With GFP-transfected neural stem cell in contrast, Analysis and Screening efficient is used for next step experiment after transforming the amplification of successful neural stem cell.
Two. the result:
1. three of slow virus carrier system plasmid DUET101, CMV8.91, PMD.G cotransfection 293T cell were collected after the transfection 24 hours, after 48 hours supernatant concentrates, and transfection 293T cell, counting GFP positive cell percentage carries out titre and identifies T=5.2 * 10
6TU/ml.The 293T cell GDNF antibody staining counting positive cell ratio of transfection GDNF, titre is 4.6 * 10
6TU/ml.
2. after neural ball is dispersed as single cell suspension, the concentrated supernatant of LV-GDNF and LV-GFP transforms with different MOI values respectively, and experimental result shows, lentiviral vectors can successful transfection neural stem cell, make its expressing green fluorescent protein and GDNF, as shown in Figure 3.It is about 40% that GFP-transfected neural stem cell is dispersed as single cell suspension FACS mensuration transformation efficiency.After the 100ug/ml hygromycin selection 3 days, cells were tested by flow cytometry GFP positive cell ratio illustrates through antibiotic-screening all more than 90%, can obtain higher expression GFP of purity and the neural stem cell of GDNF.Experimental result shows under the same terms neural ball is dispersed as the unicellular transfection of carrying out, and can significantly improve transfection efficiency, shown in Fig. 4-A.The fibronectin bag is organized with control group and is compared by group with the PDL bag in addition, and transfection efficiency has significant difference, illustrates with the fibronectin bag to be improved transfection efficiency, shown in Fig. 4-B.With the prolongation of transfection time, transfection efficiency progressively improves simultaneously, arrives plateau in the time of 20 hours, shown in Fig. 4-C.But along with the prolongation of transfection time, the also corresponding enhancing of toxic action of cohesion amine pair cell, concentration is big more, and cell mortality is high more.In order to obtain best transfection efficiency and minimum cell mortality, we have selected with the fibronectin bag by culture dish, and neural ball is dispersed as single cell suspension, the cohesion amine of 2ug/ml, transfection 20 hours.
Embodiment 4.GDNF expression-secretion is measured
One. method: the cortex neural stem cell of different MOI values was increased after with the plain screening of damp enzyme after transfection finished, and neural ball is dispersed as single cell suspension, with 2 * 10
5The density of/ml is inoculated in six orifice plates, adds the 2ml nutrient solution, and it is in vegetative state to cultivate 3 angels, gets 24 hours the frozen mensuration GDNF concentration of preparing in-80 degree of supernatant 1ml after 48 hours behind the adding fresh culture respectively.Every kind of copy number is inoculated 3 holes, has measured inoculation 3 days equally respectively, and 7 days, the 10th day, 21 days, in the time of 40 days per 1 * 10
5The GDNF secretory volume of individual cell, GDNF measure to adopt the ELISA measuring method, and specifically the experimental procedure that provides of the GDNF enzyme-linked immuno sorbent assay kit that provides with reference to Promega company is carried out.The method of application cell immunochemistry and RT-PCR is identified simultaneously.
Two. the result: after three days, each organizes different times per 10 to the cortex neural stem cell of transfection GDNF through the hygromycin selection of 100ug/ml
5The individual cell per hour secretory volume of GDNF is respectively control group, MOI=0,339.92 ± 231.262; MOI=0.5, GDNF=1556.79 ± 450.087pg/m; MOI=1, GDNF=1879.62 ± 342.651, MOI=2.5,1927.45 ± 251.600pg/ml; MOI=5,4680.78 ± 1041.574pg/ml; MOI=10,4452.74 ± 628.975pg/ml.ANOVA analyzes, and all there is significant difference p＜0.05 between each group and control group between control group and the transfection group.Between each transfection group, MOI=0.5,1,2.5 and MOI=5, analyze P＜0.05 between 10 groups, difference has significance.As shown in Figure 5.Prolongation in time in addition, GDNF can continuous expression more than January, and the GDNF secretory volume does not obviously change.As shown in Figure 6.
Sequence table
＜110〉Xuanwu Hospital of Capital University of Medical Science
＜120〉lentiviral vectors of expression glial cell line-derived neurotrophic factor and uses thereof
<130>
<160>3
<170>PatentIn?version?3.3
<210>1
<211>33
<212>DNA
＜213〉synthetic
<400>1
<210>2
<211>33
<212>DNA
＜213〉synthetic
<400>2
<210>3
<211>558
<212>DNA
＜213〉people's gdnf gene sequence
<400>3
Claims (5)
1. express the lentiviral vectors of glial cell line-derived neurotrophic factor, its preserving number is CGMCC No.2086.
2. express the lentiviral vectors CGMCC No.2086 in-vitro transfection neural stem cell of glial cell line-derived neurotrophic factor and the method for expression, it is characterized in that: neural ball is dispersed as single cell suspension with pancreatin, with 2.5 * 10
5The density of/ml is inoculated in the culture dish with fibronectin bag quilt, cultivate after at least six hours, add the supernatant of the lentiviral vectors CGMCC No.2086 of MOI=0.5-10 in the 250ul nerve stem cell culture medium, adding concentration is the polybrene (cohesion amine) of 2ug/ml, transfection 20 hours.
3. the lentiviral vectors CGMCC No.2086 in-vitro transfection neural stem cell of expression glial cell line-derived neurotrophic factor according to claim 2 and the method for expression is characterized in that: described MOI=1.0.
4. the lentiviral vectors CGMCC No.2086 that expresses glial cell line-derived neurotrophic factor is used to prepare the purposes for the treatment of nerve degenerative diseases.
5. the lentiviral vectors CGMCC No.2086 of expression glial cell line-derived neurotrophic factor according to claim 4 is used to prepare the purposes for the treatment of nerve degenerative diseases, it is characterized in that: described nerve degenerative diseases is Parkinson's disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215225A (en) * | 2012-01-18 | 2013-07-24 | 孙勇 | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell |
| CN103251957A (en) * | 2012-02-16 | 2013-08-21 | 孙勇 | Treatment of spinal cord injury by transplanting stem cells modified by neurotrophic factor gene |
| CN114990163A (en) * | 2022-03-31 | 2022-09-02 | 中海峡(福建)细胞生物科技有限公司 | Lentiviral vector for stem cell gene modification and construction method and application thereof |
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2007
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215225A (en) * | 2012-01-18 | 2013-07-24 | 孙勇 | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell |
| CN103251957A (en) * | 2012-02-16 | 2013-08-21 | 孙勇 | Treatment of spinal cord injury by transplanting stem cells modified by neurotrophic factor gene |
| CN114990163A (en) * | 2022-03-31 | 2022-09-02 | 中海峡(福建)细胞生物科技有限公司 | Lentiviral vector for stem cell gene modification and construction method and application thereof |
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