CN102174578A - Lentiviral vector and preparation method of lentiviral vector-mediated bone mesenchymal stem cell line for secreting brain derived neurotrophic factors - Google Patents
Lentiviral vector and preparation method of lentiviral vector-mediated bone mesenchymal stem cell line for secreting brain derived neurotrophic factors Download PDFInfo
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Abstract
The invention discloses a lentiviral vector and a preparation method of a lentiviral vector-mediated bone mesenchymal stem cell line for secreting brain derived neurotrophic factors (BDNF). The method comprises the following steps of: (1) constructing the lentiviral expression vector, namely amplifying a cDNA sequence of the human BDNF by using a polymerase chain reaction (PCR) method, cloning the amplified human BDNF to the lentiviral expression vector, and naming the recombinant lentiviral expression vector as lenti-BDNF; (2) packing lentiviruses by using a calcium phosphate transfection method of optimized parameters, concentrating the lentivirus granules by using an ultracentrifugation method, and measuring the titer of the packed viruses; and (3) transferring the packed lentivirus granules to the human bone mesenchymal stem cells by using a reverse transfection method. By optimizing the cell density before transfection, pH value of 2X transfection buffer solution, solution changing time after transfection and CaCl2 concentration, an efficient method for low-cost calcium phosphate transfection and virus packing is provided. The transduction human bone mesenchymal stem cells can efficiently and constantly secrete the BDNF.
Description
Technical field
The present invention relates to the preparation method of the mesenchymal stem cells MSCs system of secreting Brain Derived Neurotrophic Factor efficiently, lastingly of a kind of lentiviral vectors and mediation thereof, belong to medical biotechnology field.
Background technology
Comprise two types stem cell in people's marrow, a kind of is hemopoietic stem cell, for the regeneration of various hemocytes provides the source; Another kind is a mescenchymal stem cell, can provide the microenvironment that is suitable for growing for hemopoietic stem cell, has hematopoiesis support in marrow, promotes the function that hemocyte grows.In addition, all right (1) self of mescenchymal stem cell, the mescenchymal stem cell of just isolated or frozen back recovery has stronger multiplication capacity from marrow, can increase rapidly in a short time, to reach the sufficient amount for the treatment of or studying.(2) multidirectional differentiation potential, mescenchymal stem cell can be divided into the cell in dissimilar mesoderm source such as osteocyte, chondrocyte and adipocyte under external different inductive condition; Under physiology and the pathological conditions, mescenchymal stem cell also can be realized interdepartmental differentiation in vivo, is divided into the glandular epithelium that neurocyte (comprising neurone and spongiocyte) and internal organ have secreting function.(3) immunity modulation, mescenchymal stem cell be for allergy states such as graft versus host disease (GVH disease), transplantation immunity, various autoimmune diseases, and immune deficiency waited low immunizing power all to have regulating effect, and it is normal to be that functions of immune system is tending towards.(4) as previously mentioned; mescenchymal stem cell can provide microenvironment for hemopoietic stem cell in medullary space; if heterotopic transplantation is to other tissue; then can secrete various nutritional factor; and by with the contact action of local organization cell; form favourable local cells survival, suppress local cells apoptosis microenvironment, thereby have the cytoprotective function.Because mesenchymal stem cells MSCs has above characteristic and function, and can derive from from body, the adverse consequences of avoiding the potential immunological rejection to cause, to the basis of bone marrow transplantation, the rich experiences of clinical study, autologous bone marrow mesenchymal stem cells or the genetic engineering modified mesenchymal stem cells MSCs of process ex vivo have become the new tool of treatment disease gradually in recent years in addition.
(brain derived neurotrophic factor's Neurotrophic Factor BDNF) not only extensively distributes in central nervous system, also is distributed in retina, peripheral motoneurons, kidney and prostata tissue.It is in the central nervous system growth course, to neuronic differentiation with grow and play an important role; In adult, be neurone survive and normal physiological function institute essential.The more important thing is that BDNF also has the neuronal damage of preventing and hinders death, improves neuronic pathological state, promotes biological effects such as damaged neuron regeneration and differentiation.
Patent of the present invention is secreted the human marrow mesenchymal stem cell system of people BDNF efficiently, lastingly with the lentiviral vectors preparation; be intended to utilize BDNF protection, trophism to nerve under pathological conditions; and the effect of keeping of the immunity modulation of mescenchymal stem cell or microenvironment; building up of this kind clone; the eye retrograde affection that will cause a variety of causes, neural system degeneration, nerve injury and autoimmunity nervous system disorders play the effect of improvement.
Summary of the invention
The technical problem to be solved in the present invention provides the preparation method of the mesenchymal stem cells MSCs system of secreting Brain Derived Neurotrophic Factor efficiently, lastingly of a kind of lentiviral vectors and mediation thereof, makes the human marrow mesenchymal stem cell stability and high efficiency expressing human Brain Derived Neurotrophic Factor (BDNF) after the transduction.
The present invention is achieved by the following technical solutions:
The slow virus expression vector of a kind of expression-secretion type Neurotrophic Factor (BDNF).
The preparation method of the mesenchymal stem cells MSCs system of a kind of secretion BDNF of lentiviral vectors mediation may further comprise the steps:
(1) structure of slow virus expression vector: utilize the method for PCR, amplification people BDNF.People's BDNF gene structure complexity has tens kinds of spliceosomes, but the variation of most spliceosomes all is at noncoding promoter region, does not influence its coded protein sequence.So when the design primer, taking the BDNF encoding sequence that the frequency of occurrences is maximum in spliceosome is template (GenBank accession number BC029795, full name Homo sapiens brain-derived neurotrophic factor).Upstream primer: 5 '-GC
ACCATGACCATCCTTTTCCTTACTA-3 ' (SEQ IDNO.2); top GC is the protection base; underscore is the restriction enzyme site NheI for adding partly; shadow zone GCCACC is the Kozak sequence; the shared GC of latter two base of this sequence and restriction enzyme site GCTAGC; this design has effectively utilized sequence, has reduced the tediously long of primer.It again cDNA initiator codon ATG one section sequence afterwards of BDNF.Downstream primer: 5 '-GC
CTCGAGCTATCTCCCCTTTTAATG-3 ' (SEQ ID NO.3); in like manner; GC is the protection base; underscore is the XhoI restriction enzyme site for adding partly; CTA is the complementary sequence of terminator codon TAG; people BDNF after the amplification is cloned into the slow virus expression vector, and with recombinant slow virus expression plasmid called after lenti-BDNF.
(2) packing of slow virus: pack slow virus with the calcium phosphate transfection method of parameters optimization, and with ultracentrifugation concentrating virus particle, the titre of mensuration packaging virus; The calcium phosphate transfection method of described parameters optimization is meant and utilizes calcium phosphate method transfection 293T cell, the pH value 7.07-7.13 of 2X transfection damping fluid, changed liquid in 4-7 hour after the transfection, change 48-60 hour collecting cell supernatant liquor behind the liquid, again with cell conditioned medium liquid transduction human mesenchymal stem cells MSCs system.
(3) with the slow virus particle after packing, with contrary infection protocol transduction human mesenchymal stem cells MSCs, transduction back cell can efficiently be secreted people BDNF, is 336 times of transduction human mesenchymal stem cells MSCs not, and along with the growth of cell with go down to posterity, the function of emiocytosis BDNF is able to long term maintenance.
Beneficial effect of the present invention is: (1) the present invention utilizes the signal peptide of people BDNF itself, and to adopt the sequence of the highest spliceosome of the frequency of occurrences be template, and adding Kozak sequence, the cDNA of coding people BDNF is cloned into the slow virus expression vector, follow-up test proves that this strategy can be realized stable the efficiently expressing of BDNF fully; (2) the present invention is by optimizing the preceding cell density of transfection, and the pH value of 2X transfection damping fluid is changed the liquid time after the transfection, and CaCl
2Concentration is found out the method for efficient, low-cost calcium phosphate transfection and packaging virus, and virus titer reaches 10
9More than the IU/ml.(3) the present invention's method of dying by reverse with the slow virus particle after pack, transduction human mesenchymal stem cells MSCs, can reach fast (saving 1 day time than normal transfection) like this and transduce.And the human marrow mesenchymal stem cell after the transduction can efficiently (exceed more than 330 times than the stem cell of not transduceing), continuous release (until back 72 days of transduction, secretion showed downtrending yet) BDNF.
Description of drawings
Fig. 1 be human marrow mesenchymal stem cell in vitro culture 1.5 days (A), 3 days (B), 7 days (C), the situation of 10 days (D).
Fig. 2 is the slow virus expression vector establishment of expression-secretion type people BDNF.Fig. 2-A is the pcr amplification product of people BDNF, adds restriction enzyme site and Kozak sequence, and length is 760bp (SEQ ID NO.1).Fig. 2-B is the positive strain that PCR identifier BDNF is cloned into the A-T carrier, and the bacterial strain of visible screening is all positive.Fig. 2-C is that the A-T positive strain NheI and the XhoI double digestion that sift out identify that wherein small segment is the BDNF fragment that cuts out.Fig. 2-D is the lentiviral vectors (big fragment) of purifying and the BDNF fragment of inserting (small segment).Fig. 2-E is that PCR identifies that BDNF is cloned into the positive strain (3 positive colonies) of slow virus.
Fig. 3 is after the lentiviruses transduction 64 hours, non-situation about concentrating with the human marrow mesenchymal stem cell secretion BDNF of concentrated slow virus particle transduction that ELISA detects.
Fig. 4 is back 25 days of transduction, and 34 days, 39 days, 72 days, western blot detected the secretion situation of BDNF in the cell culture supernatant.
Embodiment
The present invention will be further described below in conjunction with drawings and Examples, limits this invention never in any form, and all being equal to of any this area of carrying out according to the disclosure of invention replace or conspicuous variation or change, all belong to protection scope of the present invention.
Embodiment 1: the cultivation of human marrow mesenchymal stem cell
One, method:
1. the recovery of human marrow mesenchymal stem cell (this step purpose is to remove dead cell and not adherent cell)
(1) perfect medium of human marrow mesenchymal stem cell comprises α-MEM (Invitrogen/GIBCO, USA), 16.5% foetal calf serum (Atlanta Biologicals, USA), glutamine (the Invitrogen/GIBCO of 2-4mM, USA) and green grass or young crops/Streptomycin sulphate (final concentration is for being respectively 100 units/ml and 100 μ g/ml, Invitrogen/GIBCO).37 ℃ of preheatings and put into the Tissue Culture Dish that diameter is 15cm (Corning, USA).When human marrow mesenchymal stem cell is taken out back 37 ℃ of water-bath quick-thawings to 95% and melts from dry ice,, be added with the Tissue Culture Dish of substratum evenly, lentamente with the aseptic pipettor of 5ml with its rapid taking-up.
(2) at 37 ℃, the 5%CO2 cell culture incubator was cultivated after 24 hours, drew supernatant not remove adherent or dead cell, and PBS rinsing 2 times is to remove the residual serum in the substratum.37 ℃ of trypsin digestion cells 2-7 minute, increase serum or perfect medium termination reaction.
(3) draw cell in aseptic 50ml centrifuge tube, the centrifugal 450-500g of room temperature, 10 minutes, re-suspended cell precipitation, counting.
2. the amplification of human marrow mesenchymal stem cell
(1) be the Tissue Culture Plate of 15cm with the plantation of the density of 60 cell/cm2 with diameter with human marrow mesenchymal stem cell, promptly every plate comprises about 10
4Individual cell.According to Cytometric result, cell concn is adjusted into 10
4Cell/ml, that is each culture plate adds the 1ml cell suspension.
(2) in the 15ml Tissue Culture Plate, add earlier the perfect medium 24ml of 37 ℃ of preheatings, evenly, slowly add the 1ml cell suspension again, and be 8 fonts and move horizontally Tissue Culture Plate, make the cell uniform distribution.
(3) renew bright substratum in per 3 days, and in time under phase microscope, monitor cell growth condition.
Two, result: cultivate after 1.5 days (as Fig. 1-A), the human marrow mesenchymal stem cell adherent growth, cell space is less, becomes oval; Cultivate after 3 days, human marrow mesenchymal stem cell is elongated fusiformis, and stretches out elongated projection, and individual cells is flat sheet, be with the peripheral cell convergence (as Fig. 1-B); Cultivate after 7 days, cell continues to grow up, and most cells are flat sheet, but cell merges as yet (as Fig. 1-C); Cultivated the 10th day, cell merges gradually, is the growth of typical corrugated (as Fig. 1-D).
Embodiment 2: the slow virus expression vector establishment of coding people Brain Derived Neurotrophic Factor cDNA
One, method:
1.PCR amplification contains the cDNA sequence of restriction enzyme site coding people BDNF
After the design of primers, the gene template of people BDNF is available from U.S. Openbiosystem company (Catlog#MHS1010-7430336), with hi-fi archaeal dna polymerase pfu (Stratagene, USA), with following method performing PCR:
Component volume (μ l)
Nuclease?free?H
2O 78.2
dNTP(25mM?each) 0.8
Template (0.1 μ g/ μ l) 1
Upstream primer (10 μ M) 4
Downstream primer (10 μ M) 4
Cloned?pfu(2.5u/μl) 2
Cumulative volume 100
2. get PCR product 5 μ l and see special, a clear band through agarose gel electrophoresis.Satisfy with Qiagen PCR cleanup purification kit, the by specification requirement, purified pcr product is with 80 μ l wash-outs.Get 80 μ l purified products, add dNTP (25mM each) 0.8 μ l, 10XPCR damping fluid 10 μ l, the TaqDNA polysaccharase (Promega, USA) 1 μ l, and add water and supply 100 μ l volumes, 72 ℃ 10 minutes, the product end is added base A.And then utilize Qiagen PCR cleanup purification kit will have the PCR product purification of base A, same 80 μ l water elutions.
3. get 1 μ l purified product, (Invitrogen USA), goes into the A-T carrier by reagent and specification sheets with the PCR product cloning with TOPO TA clone test kit.
4.PCR the positive strain of identifying send order-checking, order-checking identifies and meets fully not have sudden change, called after BDNF-AT plasmid, with lentiviral vectors 1 μ g and contain BDNF A-T plasmid 2 μ g use simultaneously XhoI and NheI (New England Biolab, USA) double digestion as follows:
Component volume (μ l)
10XNEB?bufffer2 4
100XBSA 0.4
Lentiviral vectors (1 μ g) or BDNF in
Add water to 34.4
A-T carrier (2 μ g)
NheI(10u/μl) 0.6
XhoI(20u/μl) 0.6
Cumulative volume 40
Above reaction mixture, 37 ℃ are spent the night
5. capable 1% agarose electrophoresis of product that will spend the night and cut after BDNF-AT carrier enzyme is cut, proves that institute's DCRP is and contains the segmental positive colony of BDNF.(Qiagen USA), reclaims BDNF and lentiviral vectors fragment by the requirement of test kit specification sheets, and with the capable electrophoresis of the product behind the purifying, the result detects purification effect and estimates relative concentration shown in Fig. 2 D with Qiaquick gel extraction Kit.With 3-10: 1 mol ratio with BDNF fragment and lentiviral vectors with T4DNAligase (Invitrogen USA) connects as follows:
Component volume (μ l)
5Xligase?buffer 4
T4ligase(1u/μl) 1
The BDNF fragment: lentiviral vectors rubs and adds water to 15
You are than (3-10: 1)
Reaction mixture is placed 14 ℃ of water-baths connection (16-18 hour) of spending the night.
6. the ligation mixture is diluted with water to 100 μ l, get then 10 μ l by 42 ℃ of heat-shockeds of test kit specification sheets requirement transform TOP 10 chemoreception attitude cells (Invitrogen, USA).
7. after transforming, reaction mixture is divided into two parts, every part of about 150 μ l evenly coat on the agar plate that contains penbritin, and 37 ℃, CO
2Cultivated 16-20 hour in the incubator.
8. the single bacterium colony of picking contains in the conventional liq LB substratum of ammonia benzyl in 3ml, and 37 ℃ jolt cultivation in 14-16 hour.
9. get after jolting bacterium liquid, as on ice.Utilize GoTaq Green Master Mix (Promega, USA), performing PCR screening positive strain by the following method:
Component volume (μ l)
GoTaq?Green?Master?Mix(2X) 10
Upstream primer (10 μ M) 4
Downstream primer (10 μ M) 4
Bacterium liquid 1
Nuclease?free?H
2O 1
10.PCR after reaction finished, row 1% agarose gel electrophoresis was incited somebody to action, and filters out positive strain, with the slow virus expression vector called after lenti-BDNF of reorganization.Positive bacteria liquid 800 μ l bacterium liquid are mixed fully mixing of back with 200 μ l glycerine, be stored in-80 ℃ standby.
Two, result:
PCR reacts back row 1% agarose gel electrophoresis, gets the cDNA fragment of homogeneous 760bp, consistent with expected results (as Fig. 2 A).The cDNA fragment cloning of people's Brain Derived Neurotrophic Factor is gone into after the A-T carrier, and PCR identifies positive strain (as Fig. 2 B), shows among the figure to be positive strain.The positive A-T recombinant vectors that the PCR screening goes out by NheI and XhoI double digestion after, must expect 760bp small segment (as Fig. 2 C), confirm that positive bacterium colony that PCR sifts out comprises people BDNF really and inserts fragment.Behind positive strain and the lentiviral vectors while double digestion, purifying respective segments, electrophoresis purification Identification result are also estimated carrier and are inserted fragment relative concentration (as Fig. 2 D).After people's Brain Derived Neurotrophic Factor neurotrophic factor was cloned into lentiviral vectors, PCR identified positive strain (see Fig. 2 E, the positive strain that is of amplification is arranged).
Embodiment 3: the packing of slow virus
One, method:
1. transfection is preceding 24 hours, with the 293T cell with 8X10
6The density of/plate is planted in (Crorning on the Tissue Culture Dish of 150mm, USA), and adding 25ml substratum (DMEM+10% foetal calf serum+1% glutamine+100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates, all reagent are all available from Invitrogen/GIBCO), make the preceding cell of transfection reach the fusion of 30%-40%.
2. every plate adds the chloroquine of 1/1000 volume, and (Sigma USA), gently shakes culture plate, makes it mixing.
3. in the 50ml centrifuge tube, prepare transfection mixture, add 0.5ml water, add following composition then:
Amount of reagent (μ g)
Helper?plasmid 14
Envelop?plasmid 7
Lenti-BDNF 21
2M?CaCl
2 190-370μl
Continue to add water and supply volume to 3ml/ culture dish (if transfectional cell culture dish number increases the then proportional increase of amount of reagent).CaCl
2Available from U.S. Sigma company.
4. stretch into the centrifuge tube bottom with the 5ml transfer pipet, constantly blow and beat transfection mixture, with dripping 2X transfection damping fluid 3ml.The 2X damping fluid comprise HEPES 50mM (Gibco, USA), Na
2HPO
41.5mM (Fisher Scientific, USA), sucrose 12mM (Fisher Scientific, USA), NaCl 280mM (Sigma, USA), KCl 10mM (Sigma USA), regulates the pH value to 7.07-7.13).Then the 6ml mixing solutions is added drop-wise to Tissue Culture Plate and mixing gently, cell cultures is spent the night for 37 ℃.
5. after transfection 4-7 hour, the silt sample of visible black color deposition accumulates in cell peripheral and substratum is concentrated under the light microscopic.With 37 ℃ of warm aseptic PBS solution rinsing cells 2-3 time.To alleviate the toxicity that calcium phosphate precipitation causes to cell.Every then plate adds the 17ml fresh culture.
6. change 48-60 hour collecting cell supernatant liquor behind the liquid, supernatant liquor is transferred in the 50ml centrifuge tube centrifugal 5 minutes of 1500rpm room temperature, sedimentation cell fragment and impurity.(Corning, Cat#430768 USA) filter centrifuged supernatant, and filtered solution is non-concentrating virus (a viral rough liquid) with 0.45 micron filter membrane.The non-concentrating virus of portioning section, and be stored in-80 ℃ standby.
7. remaining non-concentrating virus is moved on to the ultracentrifugation pipe (Beckmen, USA) in, manage sucrose solution that the end slowly adds 4ml 20% certainly as cusion, 25000rpm then, 4 ℃ centrifugal 2-3 hour.
8. carefully draw supernatant, the visible virion precipitation in the pipe end.(Invitrogen/Gibco USA), is hatched half an hour for 4 ℃ with PBS to add the cold cell cultures of 100 μ l on precipitation.Packing concentrating virus then, and be stored in-80 ℃ standby.
Two, result:
With spissated virion transfection human myeloma cell, and to measure virus titer by PCR in real time be 3.68X10
9IU/ml.Embodiment 4: slow virus particle transduction human mesenchymal stem cells MSCs, and measure the BDNF secretion
One, method:
1. the human marrow mesenchymal stem cell of cultivating with trysinization, cell counting, human marrow mesenchymal stem cell concentration is adjusted into 2X10
4/ ml, with the 0.5ml cell suspension, final concentration is the polybrene (polybrene is available from U.S. Sigma company) of 8 μ g/ml, mixes mutually with non-concentrating virus of 100 μ l and 1 μ l concentrating virus respectively, plants in 6 orifice plates.
2. transduceed back 16 hours, and changed the substratum of 0.5ml into 2ml, nutrient media components is mescenchymal stem cell perfect medium component as above.
3. changing liquid after 48 hours, to human marrow mesenchymal stem cell concentrated, non-concentrating virus transduction and that do not transduce, getting substratum 2ml respectively, (Millipore, USA) explanation detects the secretion situation of people BDNF to press the ELISA test kit.
4. cell continue to be cultivated, and after 6 orifice plates cover with, change over to and continue in the T150 Tissue Culture Dish to cultivate, and the different time points of cultivating after transduction gets substratum, detects the wherein content of BDNF with western blot.
Two, result:
1. the amount of the human marrow mesenchymal stem cell secretion BDNF after transduceing 64 hours with non-concentrated and spissated slow virus particle is much larger than the stem cell of not transduceing, and its secretory volume is respectively 9.8ng/ml/10
4Individual cell, 14.1ng/ml/10
4Individual cell, and 4.2pg/ml/10
4Individual cell (see figure 3).
2. after the transduction in the long-term cultivation process the 25th day, 34 days, 39 days and 72 days, measure BDNF content in the substratum, visible human marrow mesenchymal stem cell continuous expression justacrine BDNF (see figure 4) after transduction with western blot.
Claims (3)
1. the slow virus expression vector of an expression-secretion type Neurotrophic Factor.
2. the preparation method of the mesenchymal stem cells MSCs system of the secretion BDNF of a lentiviral vectors mediation is characterized in that, may further comprise the steps:
(1) structure of slow virus expression vector: utilize the method for PCR, the cDNA sequence of amplification people BDNF, the people BDNF after the amplification is cloned into the slow virus expression vector, recombinant slow virus expression vector called after lenti-BDNF;
(2) pack slow virus with the calcium phosphate transfection method of parameters optimization, and with ultracentrifugation concentrating virus particle, the titre of mensuration packaging virus;
(3) with the slow virus particle after packing, with contrary infection protocol transduction human mesenchymal stem cells MSCs.
3. according to the preparation method of the mesenchymal stem cells MSCs system of the secretion Brain Derived Neurotrophic Factor of the described lentiviral vectors of claim 2 mediation, it is characterized in that, described step (2) is packed slow virus with the calcium phosphate transfection method of parameters optimization and is meant: utilize calcium phosphate method transfection 293T cell, the pH value 7.07-7.13 of 2X transfection damping fluid, changed liquid after the transfection in 4-7 hour, change 48-60 hour collecting cell supernatant liquor behind the liquid, again with cell conditioned medium liquid transduction human mesenchymal stem cells MSCs system.
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| CN103031278A (en) * | 2012-12-11 | 2013-04-10 | 中国人民解放军第二军医大学 | Method of modifying mesenchymal stem cell by JAM1 gene and application thereof |
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| CN111139266A (en) * | 2019-12-19 | 2020-05-12 | 吉林大学 | Transfection method for BDNF (brain-derived neurotrophic factor) to transfect mesenchymal stem cells |
| CN111989404A (en) * | 2018-01-24 | 2020-11-24 | 斯比根公司 | Mesenchymal stem cell for expressing brain-derived neurotrophic factor and application thereof |
| CN114729324A (en) * | 2019-07-24 | 2022-07-08 | 斯比根公司 | Preparation method and application of immortalized stem cell strain |
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| CN103045540A (en) * | 2011-10-14 | 2013-04-17 | 北京清美联创干细胞科技有限公司 | Novel method for promoting bone mesenchymal stem cells to be differentiated into neurons in rat brain |
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| CN104195160A (en) * | 2014-08-08 | 2014-12-10 | 厦门北大之路生物工程有限公司 | Method for preparing human brain-derived neurotrophic factor in form of Pro-BDNF |
| CN111989404A (en) * | 2018-01-24 | 2020-11-24 | 斯比根公司 | Mesenchymal stem cell for expressing brain-derived neurotrophic factor and application thereof |
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| JP7016556B2 (en) | 2018-01-24 | 2022-02-07 | エスエルバイジェン インコ―ポレイテッド | Mesenchymal stem cells expressing brain-derived neurotrophic factor and their uses |
| CN114729324A (en) * | 2019-07-24 | 2022-07-08 | 斯比根公司 | Preparation method and application of immortalized stem cell strain |
| CN111139266A (en) * | 2019-12-19 | 2020-05-12 | 吉林大学 | Transfection method for BDNF (brain-derived neurotrophic factor) to transfect mesenchymal stem cells |
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Application publication date: 20110907 |