CN101787359A - Recombinant lentivirus for carrying out targeted induction on osteogenic differentiated cell apoptosis as well as preparation method and application thereof - Google Patents
Recombinant lentivirus for carrying out targeted induction on osteogenic differentiated cell apoptosis as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses recombinant lentivirus for carrying out targeted induction on the osteogenic differentiated cell apoptosis as well as a preparation method and application thereof. A recombinant lentivirus genome contains an Apoptin gene expression box which contains an Osterix gene promoter, an Apoptin gene and a terminator, and the Apoptin gene expression is regulated and controlled by the Osterix gene promoter; the recombinant lentivirus takes a pan-osteogenic cell peculiar expression gene Osterix promoter as a promoting element, expresses Apoptin for inducing the osteogenic differentiated cell apoptosis in osteogenic differentiated cells, does not play a role on chondrogenic differentiated cells and normal cartilage cells, can be prepared into medicines for inducing the osteogenic differentiated cell apoptosis, carries out the treatment on residual osteogenic differentiated cells in seed cells after the osteogenic induced differentiation treatment and ensures a single chondrogenic differentiation path of the seed cells, thereby providing a favorable seed cell for the establishment of tissue engineering cartilage; and the preparation method of the recombinant lentivirus is simple, convenient, rapid and low in cost.
Description
Technical field
The present invention relates to a kind of recombinant virus, particularly a kind of recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis also relates to the preparation method and the application of this recombinant slow virus.
Background technology
Cartilage injury or disappearance are FAQs clinically, because the histology of cartilage and the repair ability that biological characteristics has limited himself still lack therapy measure at present.Method of Tissue Engineering provides a kind of brand-new means for the reparation of cartilage defect, utilizes timbering material for the propagation and the matrix secretion of seed cell provide the space framework, forms the structure of similar normal articular cartilage, and functionating.Seed cell comprises mescenchymal stem cell such as mesenchymal stem cells MSCs, peripheral blood mescenchymal stem cell, umbilical cord blood mesenchymal stem cells, fat mesenchymal stem cell and the umbilical cord mesenchymal stem cells etc. in embryonic stem cell and multiple source.Owing to more easily obtain to induce into the cartilage differentiation potential with having, (mesenchymal stem cells MSCs) is considered to present cartilage tissue engineered ideal seed cell to mesenchymal stem cells MSCs.But with MSCs and the compound structure cell-composite body of absorbability timbering material and carry out external cartilage induction when cultivating, having alkaline phosphatase and Bone Gla protein positive cell in tissue engineering bone/cartilage occurs, and, tissue engineering bone/cartilage is behind subcutaneous plantation of nude mice or flesh bag plantation some months, in typical cartilage cavities spline structure, be dispersed in the ossified kitchen range of appearance, show that the part seed cell enters the endochondral ossification program.The ossified phenomenon that this because seed cell heterogeneity causes is one of important bottleneck problem that restricts at present the tissue engineering bone/cartilage constructing technology, certainly will have influence on the structure of cartilage and at body damaged reparation fully and reconstruction.Therefore, how to regulate and control MSCs directed differentiation chondroblast and to suppress it be major issue in present cartilage tissue engineered to osteoblast differentiation.For obtaining efficient special cartilage seed cell, must partly handle the Osteoblast Differentiation of tissue engineering bone/cartilage seed cell.
In osteoblastic transcriptional regulatory, Osterix (Osx is also referred to as SP7) gene comes into one's own day by day as osteoblast differentiation and the essential transcription factor of bone forming.The Osterix gene belongs to Sp/XKLF family, it is a kind of transcription factor with zinc-finger motif structural domain, be positioned at the downstream of core binding factor Cbfa1/Runx2 and regulated and control by it, the Osterix gene promoter area (713～-700bp) have the identification regulatory site of Cbfa1.Discover that the Osterix knock out mice does not have bone and forms but have cartilage bone structure, and Cbfa1 is expressed as the positive, and the Cbfa1 deficient mice is not expressed Osterix.In view of the above, the part scholar has proposed the pattern that osteoblast differentiation forms: MSCs and is divided into the cartilage precursor cell → Cbfa1 gene promoter → cell that can secrete the II Collagen Type VI and is converted into scleroblast → scleroblast to the skeletonization precursor cell that can secrete type i collagen or loose type chondrocyte conversions → Osterix gene promoter → skeletonization precursor cell and secretes extracellular matrixs such as Bone Gla protein, bone sialoprotein and osteonectin.According to above-mentioned pattern, the cell of expressing Cbfa1 still has potential two-way differentiation capability, both can be divided into scleroblast, can be divided into the chondrocyte again, and the cell of expression Osterix has direct osteogenic ability.
Apoptosis element (Apoptin) be a kind ofly derive from chicken anaemia virus, by the small protein that 121 amino acid are formed, can induce chick hemocytoblast and T lymphocyte precursor apoptosis, it is low to cause chick serious anaemia and immunity function to occur.Apoptin can also induce human tumor cells and transformant apoptosis by the mode that is independent of the p53 action pathway and do not suppressed by the Bcl-2 overexpression, and normal cell is not played a role.Therefore, Apoptin is considered to first truly alternative inducing tumor cell and transformant apoptosis and the apoptotic proteins of injuring normal cell not.Discover that Apoptin activity inducing apoptosis and its Subcellular Localization are closely related.In normal cell, Apoptin is positioned tenuigenin, can not cell death inducing; And in tumour cell, Apoptin is positioned in the nucleus, can inducing apoptosis of tumour cell.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis, can handle passing through into remaining Osteoblast Differentiation cell in the seed cell of chondrocyte induction differentiation processing, induced osteogenesis noble cells apoptosis and to becoming cartilage noble cells and normal cartilage cell not to play a role, guarantee that seed cell becomes the single-pathway of cartilage differentiation, thereby provide a kind of good seed cell for the structure of tissue engineering bone/cartilage; Two of purpose is to provide the preparation method of the recombinant slow virus of described carrying out targeted induction on osteogenic differentiated cell apoptosis; Three of purpose is to provide the application of the recombinant slow virus of described carrying out targeted induction on osteogenic differentiated cell apoptosis.
For achieving the above object, the present invention adopts following technical scheme:
1, the recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis, comprise an Apoptin expression casette in the described recombinant slow virus genome, described Apoptin expression casette comprises Osterix gene promoter, Apoptin gene and terminator, and described Apoptin expression of gene is regulated and control by the Osterix gene promoter.
Further, described Apoptin expression casette also comprises V5 epitope gene sequence, and described V5 epitope gene sequence is between Apoptin gene and terminator;
Further, described recombinant slow virus is the Apoptin expression casette is inserted the HIV-1 viral genome and to obtain.
2, the preparation method of the recombinant slow virus of described carrying out targeted induction on osteogenic differentiated cell apoptosis may further comprise the steps:
A, the segmental clone of Osterix gene promoter: the design of primers according to segmental nucleotide sequence of Osterix gene promoter and lentiviral vectors pLenti6/V5-D-TOPO requires to design and synthesize primer, genomic dna with mice embryonic scleroblast MC3T3-E1 is a template, pcr amplification Osterix gene promoter fragment, be cloned into the pGEM-T carrier, get recombinant vectors pGEM-T/Osxp;
The clone of b, Apoptin gene fragment: the nucleotide sequence according to the Apoptin gene designs and synthesizes primer, genomic dna with extraction in the chicken bursa tissue is a template, pcr amplification Apoptin gene fragment is cloned into the pGEM-T carrier, gets recombinant vectors pGEM-T/Apo; In pGEM-T/Apo, go out the Apoptin gene fragment, under the effect of T4DNA ligase enzyme, be connected, get recombinant vectors pcDNA3.1-Apo with same pcDNA3.1 (+) carrier through EcoRV and XbaI double digestion with EcoRV and XbaI double digestion;
The structure of c, Osxp-Apoptin recombined lentivirus vector: in pGEM-T/Osxp, go out the Osxp fragment with KpnI and EcoRI double digestion, with be connected under the effect of T4DNA ligase enzyme through the pcDNA3.1-Apo of KpnI and EcoRI double digestion equally, recombinant vectors pcDNA3.1-Osxp-Apo; In pcDNA3.1-Osxp-Apo, go out the Osxp-Apoptin fragment again, be connected, get recombinant vectors pLenti6/V5-Osxp-Apo with the pLenti6/V5-D-TOPO carrier with KpnI and XbaI double digestion;
The packing of d, Osxp-Apoptin recombinant slow virus: adopt Lipofectamine 2000 reagent with pLenti6/V5-Osxp-Apo and slow virus packaging plasmid mixture cotransfection 293FT cell; after treating that obvious pathology appears in cell; collection contains the culture supernatant of virus, promptly gets the Osxp-Apoptin recombinant slow virus.
Further, primer sequence is described in the step a:
Upstream primer: 5 '-ggggtacccccacccagggaagcaagatgat-3 ';
Downstream primer: 5 '-cggaattccgggtcccaaggagtcaggcagat-3 ';
Further, the condition of pcr amplification described in the step a is: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 59 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 2 minutes, totally 35 circulations, last 72 ℃ were extended 10 minutes;
Further, primer sequence is described in the step b:
Upstream primer: 5 '-cggatatccgagtaatttcaaatgaacgct-3 ';
Downstream primer: 5 '-gctctagagccagtcttatacgccttcttgc-3 ';
Further, the condition of pcr amplification described in the step b is: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 60 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 5 minutes.
3, the application of the recombinant slow virus of described carrying out targeted induction on osteogenic differentiated cell apoptosis in the medicine of preparation induced osteogenesis noble cells apoptosis.
The recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis of the present invention mainly is made up of three parts: 1. with the promotor of general scleroblast specific expression gene Osterix as starting element; 2. at the gene A poptin of the cell inner expression and the Osteoblast Differentiation cell of committing suiside; 3. the lentiviral vectors that high infection rate and stable integration are taken into account.The selection of each several part is according to as follows: the 1. selection of starting element: utilizing specificity promoter regulation and control goal gene to express in target cell is the important means that realizes targeting gene therapy.Osterix is specific expressed in scleroblast, and on the one hand, it is scleroblast that Osterix can make the skeletonization precursor cell directed differentiation of expressing Cbfa1, has the effect that is separated into osteocyte system and chondrocyte system in the entochondrostosis process; On the other hand, Osterix is the negative regulatory factor of Sox9 (having vital role in chondrocyte and cartilaginous tissue differentiation) expression and chondrocyte's phenotype; These all make the Osterix gene promoter become and drive the desirable starting element that suicide gene is expressed; 2. the selection of suicide gene: the Apoptin gene is a preferable suicide gene, after exogenous Apoptin expresses, nuclear translocation takes place in target cell gradually, in normal cell, then be trapped in the tenuigenin, even Apoptin is expressed in the Normocellular nucleus, can cell death inducing yet.Under the guiding of Osterix gene promoter, Apoptin can kill and wound the Osteoblast Differentiation cell specifically, and can not injure into cartilage noble cells and normal cartilage cell.3. foreign gene imports the selection of carrier: non-virological gene transfer method efficient is lower, the gene therapy scheme overwhelming majority who has been used for human trial carries out transgenosis with virological method, and is wherein ripe with retroviral vector and adenovirus carrier.The ideal virus vector should be able to provide the genetic expression and the biological safety of transgenosis efficiently, long-term stability simultaneously.Though retroviral vector can make goal gene be integrated into the target cell genome and realize stably express, the division stage of can only transduceing cell, the non-division stage of can not transduceing cell.Though adenovirus carrier can be transduceed division stage cell and stationary phase cell, transduction efficiency is also higher, the goal gene unconformability is to the target cell genome, only can transient expression, and adenovirus protein can cause human immunity reaction and inflammatory reaction.Lentiviral vectors can infect non-division stage cell, goal gene and is integrated into advantages such as target cell genome long-term expression, immune response be little because of it has, and is expected to become the ideal gene transfer vector.Commonly used be that the lentiviral vectors of fundamental construction is that cis-acting elements relevant with virus packing, reverse transcription and integration in the HIV-1 gene is separated with the proteic sequence of trans-acting of encoding with 1 type human immunodeficiency virus (HIV-1), be transformed into carrier components and packing composition respectively, will pack composition usually and independently be placed on (packaging plasmid and coating plasmid) on 2 plasmids respectively.The lentiviral vectors that uses is four pUC pUCs mostly now, comprise vector plasmid, packaging plasmid, coating plasmid and pVSV-G plasmid, with the Env gene of VSV-G (herpes stomatitis virus glycoprotein G gene) replacement HIV, the false HIV virion of generation is safe, host cell wide, stability is strong, titre is high.
Beneficial effect of the present invention is: at a difficult problem that causes ossified kitchen range formation at present in tissue engineering bone/cartilage makes up owing to remaining Osteoblast Differentiation cell in the seed cell, the present invention has set up a kind of recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis, with the promotor of general scleroblast specific expression gene Osterix as starting element, the apoptotic Apoptin of induced expression Osteoblast Differentiation in the Osteoblast Differentiation cell, and to becoming cartilage noble cells and normal cartilage cell not to play a role, can make the medicine of induced osteogenesis noble cells apoptosis, to passing through seed cell (mescenchymal stem cell such as the mesenchymal stem cells MSCs of handling into the chondrocyte induction differentiation that comprise embryonic stem cell and multiple source, the peripheral blood mescenchymal stem cell, umbilical cord blood mesenchymal stem cells, fat mesenchymal stem cell and umbilical cord mesenchymal stem cells etc.) in remaining Osteoblast Differentiation cell handle, guarantee that seed cell becomes the single-pathway of cartilage differentiation, thereby provide a kind of good seed cell for the structure of tissue engineering bone/cartilage.The preparation method of this recombinant slow virus is easy, quick, cost is low.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is the structure synoptic diagram of Osterix gene promoter regulation and control Apoptin gene (Osxp-Apoptin) recombinant slow virus;
Fig. 2 is the segmental agarose gel electrophoresis result of pcr amplification Osterix gene promoter (Osxp), and wherein swimming lane 1 is a dna molecular amount standard, and swimming lane 2 and 3 is the segmental PCR product of Osxp;
Fig. 3 is through the blue coloration result of the A Erxin of the epiphyseal cartilage cell of Osxp-Apoptin recombinant slow virus processing;
Fig. 4 is the epiphyseal cartilage cell proliferation experiment result who handles through the Osxp-Apoptin recombinant slow virus;
Fig. 5 is the II Collagen Type VI immunofluorescence dyeing result through the MSCs of Osxp-Apoptin recombinant slow virus processing;
Fig. 6 is the blue coloration result of A Erxin that becomes the cartilage differentiation through the MSCs that the Osxp-Apoptin recombinant slow virus is handled;
Fig. 7 is the alkaline phosphatase staining result who becomes the cartilage differentiation through the MSCs that the Osxp-Apoptin recombinant slow virus is handled;
Fig. 8 observes the Osxp-Apoptin recombinant slow virus to chondrogenetic influence at body.
Embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.Unless otherwise indicated, various restriction enzymes, archaeal dna polymerase, dna ligase and the test kit etc. in the preferred embodiment are all available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing.
One, the preparation of Osxp-Apoptin recombinant slow virus
The structure synoptic diagram of Osxp-Apoptin recombinant slow virus as shown in Figure 1, its preparation method may further comprise the steps:
1, the segmental clone of Osxp
Design of primers requirement according to segmental nucleotide sequence of Osxp and lentiviral vectors pLenti6/V5-D-TOPO among the GenBank NT_039621.7 designs and synthesizes following primer:
F1:5 '-gggg
Tacccc
Cagggaagcaagatgat-3 ' (SEQ ID No.1), underscore partly is a restriction enzyme KpnI restriction enzyme site, bold Italic partly is topoisomerase (TOPO) connection site;
R1:5 '-cgg
AattcCgggtcccaaggagtcaggcagat-3 ' (SEQ ID No.2), underscore partly is a restriction enzyme EcoRI restriction enzyme site.
Adopt genome DNA extracting reagent kit from mice embryonic scleroblast strain MC3T3-E1, to extract genomic dna, operate according to the test kit specification sheets.
With the MC3T3-E1 cell genomic dna is template, is the upstream and downstream primer with F1 and R1, adopts PCR test kit amplification Osxp fragment, operates according to the test kit specification sheets.The pcr amplification system is: 10 * PCR damping fluid, 5 μ L, dNTPs 5 μ L, each 2 μ L of upstream and downstream primer, template 4 μ L, MgCl
24 μ L, Taq archaeal dna polymerase 1 μ L is diluted to cumulative volume 50 μ L with no DNA enzyme water.The pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 59 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 2 minutes, totally 35 circulations, last 72 ℃ were extended 10 minutes.The PCR product adopts agarose gel electrophoresis to identify, visible PCR product specific band (as shown in Figure 2) occurs at about 2.1kb place, and is consistent with the molecular weight size of target fragment.Adopt the PCR product to reclaim test kit and cut glue recovery purified pcr product, operate according to the test kit specification sheets.
PCR product behind the purifying is connected under the effect of T4DNA ligase enzyme with cloning vector pGEM-T (Promega company), connect product transformed into escherichia coli JM109 competent cell, blue hickie screening positive clone, the extracting plasmid, the evaluation of checking order, the result shows that it is consistent with the Osxp fragments sequence to insert fragments sequence (SEQ ID No.3) in the gained positive colony plasmid, with its called after pGEM-T/Osxp.
2, the clone of Apoptin gene fragment
Nucleotide sequence according to Apoptin gene among the GenBank AF313470 designs and synthesizes following primer:
F2:5 '-cg
GatatcCgagtaatttcaaatgaacgct-3 ' (SEQ ID No.4), underscore partly is a restriction enzyme EcoRV restriction enzyme site;
R2:5 '-gc
TctagaGccagtcttatacgccttcttgc-3 ' (SEQ ID No.5), underscore partly is restriction enzyme XbaI enzyme cutting position; In order to make the amalgamation and expression of Apoptin, deleted terminator at 3 ' end with the V5 epi-position.
With chicken jugular vein sacrificed by exsanguination, separate the fabricius bursa immediately, adopt genome DNA extracting reagent kit to extract genomic dna, operate according to the test kit specification sheets.
Genomic dna with extraction in the chicken bursa tissue is a template, is the upstream and downstream primer with F2 and R2, adopts PCR test kit amplification Apoptin gene fragment, operates according to the test kit specification sheets.The pcr amplification system is: 10 * PCR damping fluid, 5 μ L, dNTPs 5 μ L, each 2 μ L of upstream and downstream primer, template 4 μ L, MgCl
24 μ L, Taq archaeal dna polymerase 1 μ L is diluted to cumulative volume 50 μ L with no DNA enzyme water.The pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 60 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 5 minutes.The PCR product adopts agarose gel electrophoresis to identify, visible PCR product specific band occurs at about 0.4kb place, and is consistent with the molecular weight size of target fragment.Adopt the PCR product to reclaim test kit and cut glue recovery purified pcr product, operate according to the test kit specification sheets.
PCR product behind the purifying is connected under the effect of T4DNA ligase enzyme with cloning vector pGEM-T (Promega company), connect product transformed into escherichia coli JM109 competent cell, blue hickie screening positive clone, the extracting plasmid, the evaluation of checking order, the result shows that it is consistent with the sequence of Apoptin gene fragment to insert fragments sequence (SEQ ID No.6) in the gained positive colony plasmid, with its called after pGEM-T/Apo.
In positive colony plasmid pGEM-T/Apo, go out the Apoptin gene fragment with EcoRV and XbaI double digestion, with be connected under the effect of T4DNA ligase enzyme through the carrier for expression of eukaryon pcDNA3.1 (+) of EcoRV and XbaI double digestion (Invitrogen company) equally, connect product transformed into escherichia coli JM109 competent cell, blue hickie screening positive clone, the extracting plasmid, carrying out evaluation of EcoRV and XbaI double digestion and order-checking identifies, sequencing result shows, it is consistent with the sequence of Apoptin gene fragment to insert fragments sequence in the gained positive colony plasmid, with its called after pcDNA3.1-Apo.
3, the structure of Osxp-Apoptin recombined lentivirus vector
In positive colony plasmid pGEM-T/Osxp, go out the Osxp fragment with KpnI and EcoRI double digestion, be connected under the effect of T4DNA ligase enzyme through the positive colony plasmid pcDNA3.1-Apo of KpnI and EcoRI double digestion again with equally, connect product transformed into escherichia coli DH5 α competent cell, blue hickie screening positive clone, the extracting plasmid, carrying out evaluation of KpnI and EcoRI double digestion and order-checking identifies, sequencing result shows, Osxp fragment orientation is inserted into Apoptin gene fragment upstream in the gained positive colony plasmid, with its called after pcDNA3.1-Osxp-Apo.
In positive colony plasmid pcDNA3.1-Osxp-Apo, go out the Osxp-Apoptin fragment with KpnI and XbaI double digestion, with lentiviral vectors pLenti6/V5-D-TOPO (Invitrogen company) is to mix at 1: 1 by volume, add 6 * connection damping fluid (Invitrogen company), 1 μ L, be supplemented to cumulative volume 6 μ L with sterile purified water, temperature was hatched 5 minutes for 22 ℃, and the Osxp-Apoptin fragment is connected with lentiviral vectors; Connect product transformed into escherichia coli Stbl3 competent cell, with the LB plate screening positive colony that contains penbritin, the amplification of picking list bacterium colony, get bacterium liquid and carry out the PCR evaluation, the extracting plasmid evaluation of checking order simultaneously, sequencing result shows, inserts fragments sequence in the gained positive colony plasmid and conforms to fully with expected sequence, with its called after pLenti6/V5-Osxp-Apo.
4, the packing of Osxp-Apoptin recombinant slow virus
3 μ g positive colony plasmid pLenti6/V5-Osxp-Apo and 9 μ g slow virus packaging plasmid mixtures are dissolved in the 1.5mL serum-free Opti-MEMI substratum called after A liquid; 36 μ L Lipofectamine 2000 are diluted in the 1.5mL serum-free Opti-MEMI substratum called after B liquid; With A liquid and B liquid mixing, incubated at room made and forms the DNA-liposome complex in 20 minutes; The DNA-liposome complex is transferred in the culture dish that contains 5mL Opti-MEMI growth medium and serum, adds and contain 6 * 10
6The 293FT cell suspension of individual cell is 37 ℃, CO in temperature
2The gas volume mark is to cultivate under 5% the condition, after 6 hours substratum is replaced by 5mL and contains the DMEM substratum that Sodium.alpha.-ketopropionate that concentration is 1mmol/L and volume fraction are 10% foetal calf serum, visible cell generation pathology after 2 days, show as attached cell and become circle, visible round cell is thyrsiform and gathers together and be distributed on the normal 293FT cell after 3 days, virus plaque appears, collection contains the culture supernatant of virus, 4 ℃ of temperature, centrifugal 15 minutes of 3000r/min, collecting supernatant liquor, is the membrane filtration of 0.45 μ m with the aperture, promptly gets Osxp-Apoptin recombinant slow virus suspension, temperature-80 is ℃ frozen, standby.
In 6 well culture plates, cultivate the MC3T3-E1 cell, when treating that cell reaches 50% fusion, add different extent of dilution (10
-7～10
-2) Osxp-Apoptin recombinant slow virus suspension, and final concentration is the polybrene (polybrene) of 6 μ g/mL and the blasticidin (blasticidin) that final concentration is 4.0 μ g/mL, the screening cells infected, through 10～12 days, gained blasticidin resistance colony was 1% violet staining liquid with PBS rinsing 2 times, adding 1mL massfraction, room temperature was placed 10 minutes, use the PBS rinsing again 2 times, calculate indigo plant and dye the clone, the titre that records the Osxp-Apoptin recombinant slow virus is 2～5 * 10
6TU/mL.
Genomic dna with gained Osxp-Apoptin recombinant slow virus is a template, with F3 and R3 be the upstream and downstream primer [F3:5 '-actccgagtcaagagtaggattgtagg-3 ' (SEQ ID No.7); R3:5 '-agaatgggagaa tgggagagaag-3 ' (SEQ ID No.8)] pcr amplification Osxp fragment, with F4 and R4 be the upstream and downstream primer [F4:5 '-atctgcaactgcggacaa-3 ' (SEQ ID No.9); R4:5 '-gctgggagtagtggtaatcaa-3 ' (SEQ ID No.10)] pcr amplification Apo fragment, the result shows, adopts above-mentioned primer can amplify the Osxp fragment of 565bp and the Apo fragment of 157bp.
Get 1 * 10
6Individual C3H10T1/2 cell after the Osxp-Apoptin recombinant slow virus infects 24 hours, PBS with 4 ℃ of precoolings of temperature washs 1 time, be that 4% Paraformaldehyde 96 room temperature is fixed 40 minutes with the 1ml massfraction again, with PBS washing 1 time, contain the PBS re-suspended cell that volume fraction is 5% human serum with 1ml again, hatched on ice 10 minutes, centrifugal 5 minutes of 800rpm, abandon supernatant, add 200 μ l and contain the PBS of anti-V5 epitope antibodies (Invitrogen company) that massfraction is the FITC mark of 0.5% bovine serum albumin and Sa, lucifuge was placed 30 minutes on ice, the centrifugal supernatant of abandoning, add 200 μ l massfractions again and be 1% Paraformaldehyde 96 and fix, detect fluorescent value with flow cytometer, 10000 cells of every pipe counting.The result shows, when infection multiplicity (MOI)=10, and the C3H10T1/2 cell expressing Apoptin more than 85%; When MOI=40, nearly all C3H10T1/2 cell is all expressed Apoptin.
Two, the detection of Osxp-Apoptin recombinant slow virus carrying out targeted induction on osteogenic differentiated cell apoptosis ability
1, to chondrocyte's influence
The SD rat in newborn 1 week is taken off neck execution, cut bilateral hip and knee cartilage under the aseptic condition, with DMEM substratum rinsing 2 times, be cut into 1mm * 1mm size, adding concentration is the collagenase solution of 0.3U/mL, 37 ℃ of digestion of temperature 4 hours, blow and beat into single cell suspension, 150 order stainless steel filtering nets filter, centrifugal 5 minutes of filtrate 1000r/min, abandoning supernatant with the DMEM substratum re-suspended cell that contains the Streptomycin sulphate that volume fraction is 10% foetal calf serum, concentration is 100U/mL penicillin and concentration is 100U/mL, is 10 with cell density
5Individual/mL is inoculated in the culturing bottle, is 37 ℃, CO in temperature
2The gas volume mark is to cultivate under 5% the condition, backsight cell attachment situation was changed fresh culture in 48 hours, changed 1 fresh culture in per 2～3 days later on, after under inverted microscope, observing the chondrocyte and merging into sheet and cover with most of culturing bottle wall, discard substratum, add massfraction and be 0.25% trypsin solution and massfraction and be 0.02% edta solution and carry out had digestive transfer culture, with 5 * 10
5Individual chondrocyte is gone down to posterity into 24 orifice plates, treat that cytogamy reaches at 50% o'clock, infect the Osxp-Apoptin recombinant slow virus and add the polybrene that final concentration is 8mg/L by MOI=40, infect and be replaced by the DMEM substratum that contains the Streptomycin sulphate that volume fraction is 10% foetal calf serum, concentration is 100U/mL penicillin and concentration is 100U/mL in back 24 hours, infect back 72 hours with cell with 1 * 10
4Individual/hole is inoculated in 96 well culture plates, after next day, abundance reached 90%, press MOI=40 and infect the Osxp-Apoptin recombinant slow virus, infect and be replaced by the DMEM substratum that contains the Streptomycin sulphate that volume fraction is 10% foetal calf serum, concentration is 100U/mL penicillin and concentration is 100U/mL in back 24 hours, infect and carried out cell proliferation experiment (WST-8 staining) in back 72 hours, carry out chondrocyte's the blue dyeing of A Erxin simultaneously and observe the stromatin polysaccharide content.
The result: through the epiphyseal cartilage cell that the Osxp-Apoptin recombinant slow virus is handled, there be (Fig. 3) in the visible a large amount of positive staining cells of the blue dyeing of A Erxin.Compare with the normal cartilage cell with empty virus treated cell through the epiphyseal cartilage cell that the Osxp-Apoptin recombinant slow virus is handled, three's propagation and survival rate no significant difference (P＞0.05) (Fig. 4) show that the Osxp-Apoptin recombinant slow virus has the effect of evading to the chondrocyte.
2, mescenchymal stem cell is become the influence of cartilage differentiation
Get the two lower limb long bones of 1 monthly age mouse, fully wash medullary space, is 5: 3 mixings with washing fluid and lymphocyte separation medium with volume ratio, centrifugal 30 minutes of 1500r/min, draw mononuclear cell layer, with the low sugar DMEM substratum that contains the Streptomycin sulphate that volume fraction is 10% foetal calf serum, concentration is 100U/mL penicillin and concentration is 100U/mL carry out former be commissioned to train foster, changed 1 fresh culture in per 3 days, carrying out the 1st time after 12 days goes down to posterity, went down to posterity 1 time in per afterwards 3～5 days, get third generation MSCs, adjusting cell density is 5.0 * 10
5Individual/cm
2Infect the Osxp-Apoptin recombinant slow virus and add the polybrene that final concentration is 8mg/L by MOI=40, infect and be replaced by that to contain volume fraction be 10% foetal calf serum in back 24 hours, concentration is the penicillin of 100U/mL and the DMEM substratum of the Streptomycin sulphate that concentration is 100U/mL, infect and be carried out to the chondrocyte induction differentiation in back 48 hours: add serum-free chondrocyte induction liquid and (contain the transforminggrowthfactor-that concentration is 10ng/mL, concentration is the dexamethasone of 0.1 μ mol/L, concentration is that the xitix and 1 * ITS+1) of 50 μ g/mL carries out the monolayer cell inducing culture, carried out cell proliferation experiment (WST-8 staining) in back 7 days and 14 days respectively at inducing, the II Collagen Type VI detects (SP method), blue dyeing of A Erxin and alkaline phosphatase staining adopt Flow cytometry MSCs to become the cartilage differentiation efficiency simultaneously.
The result: the MSCs propagation of handling through the Osxp-Apoptin recombinant slow virus is normal, the II Collagen Type VI is expressed normal (Fig. 5), the blue dyeing of A Erxin normal (Fig. 6), do not see that there be (Fig. 7) in the alkaline phosphatase positive cell, show that the Osxp-Apoptin recombinant slow virus becomes the cartilage differentiation to have the effect of evading preferably to MSCs.The MSCs that Flow cytometry Osxp-Apoptin recombinant slow virus is handled becomes the cartilage differentiation efficiency up to 85%.
3, to osteoblastic influence
Get 24 hours inner tube of a tyre mouse skull bones, in PBS, remove blood and reticular tissue on every side, be cut into the mud shape with scissors, the adding massfraction is 0.25% trypsin solution, 37 ℃ of digestion of temperature 30 minutes, trypsin solution is abandoned in suction, with PBS washing 1 time, add the collagenase solution that concentration is 0.3U/mL again, 37 ℃ of vibration digestion of temperature 1 hour are inhaled and are abandoned collagenase solution, add the DMEM substratum again, blow and beat osteocomma repeatedly cell is split away off from osteocomma, the gained cell suspension inoculation is 37 ℃, CO in temperature in culturing bottle
2The gas volume mark is to cultivate under 5% the condition, change fresh culture after 24 hours, changed 1 fresh culture in per afterwards 48 hours, treat that former generation scleroblast fusion reaches at 60% o'clock, infect the Osxp-Apoptin recombinant slow virus and add the polybrene that final concentration is 8mg/L by MOI=40, infect and be replaced by that to contain volume fraction be 10% foetal calf serum in back 24 hours, concentration is the penicillin of 100U/mL and the DMEM substratum of the Streptomycin sulphate that concentration is 100U/mL, respectively at infecting back 24 hours and 48 hours collecting cells, extract total RNA, adopt the RT-PCR method to detect the expression of Apoptin, adopt the laser co-focusing method to detect the Subcellular Localization of Apoptin in scleroblast simultaneously, infect and adopted the trypan blue staining to measure inhibitory rate of cell growth in back 72 hours.
Result: be template with the cell total rna that infects back 24 hours and 48 hours respectively, all can amplify Apoptin cDNA, show that Apoptin expresses in cells infected through RT-PCR; And the dna ladder shape band that typical apoptosis has appears in infected cell, and showing has apoptosis to take place.Laser co-focusing detects visible Apoptin and is positioned nucleus.Infected back 72 hours, nearly all scleroblast death shows that the Osxp-Apoptin recombinant slow virus has stronger scavenging(action) to scleroblast.
In addition, the present invention also utilizes cell strain such as mice embryonic scleroblast strain MC3T3-E1, skull scleroblast strain HCO and people's human osteosarcoma cell strain MG-63 to carry out above-mentioned detection, all obtain similar results, confirm that the Osxp-Apoptin recombinant slow virus has stronger scavenging(action) to scleroblast.
4, to the influence of mescenchymal stem cell Osteoblast Differentiation
Get former generation MSCs and carry out the osteogenic induction differentiation: adjusting cell density is 1.0 * 10
5Individual/cm
2, being inoculated in 6 orifice plates, adding contains the skeletonization complementary element, and (concentration is 10
-8The dexamethasone of mol/L, concentration are 10
-3β-the Phosphoric acid glycerol esters of mol/L and concentration are the xitix of 50mg/L) substratum carry out inducing culture, respectively at inducing back 1,3,7 and 14 day by MOI=40 infection Osxp-Apoptin recombinant slow virus, and in infecting back 24 hours collecting cells, extract total RNA, adopt the RT-PCR method to detect the expression of Osterix and Caspase-3, observe the reactivity of the clone of different Osteoblast Differentiation degree Osterix.
The result: along with the Osteoblast Differentiation that infects back MSCs, the expression of Osterix and Caspase-3 increases gradually, shows that the Osxp-Apoptin recombinant slow virus has the good reactivity at Osterix.
5, observe chondrogenetic influence at body
, press MOI=40 and infect the Osxp-Apoptin recombinant slow virus as seed cell with third generation MSCs, infected back 24 hours, tryptic digestion, centrifugal collecting cell, adjusting cell density is 2 * 10
7Individual/mL, mix with chitosan-Sodium Glycerophosphate (C/GP) gel again, fully piping and druming makes and mixes, carry out the subcutaneous plantation of nude mice with the 1mL syringe, respectively at 4,8 and 12 weeks of plantation back taking out sample, carry out formalin fixed, rare nitric acid decalcification, specimens paraffin embedding slices according to a conventional method, and carry out Yihong-phenodin (HE) dyeing, toluidine blue (TB) dyeing and the dyeing of immunohistochemical methods II Collagen Type VI.
The result: visible class cartilage forms different cartilage matrix that dyes of justacrine toluidine blue and cartilage specificity II Collagen Type VI in the Histological section, do not see that (Fig. 8) appears in significantly ossified kitchen range, show that the MSCs that handles through the Osxp-Apoptin recombinant slow virus becomes to have good one-tenth cartilage ability under the cartilage environment in vivo, can not produce ossified kitchen range.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis and its production and application
<160>10
<210>1
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primers F 1
<400>1
ggggtacccc?cacccaggga?agcaagatga?t 31
<210>2
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer R1
<400>2
cggaattccg?ggtcccaagg?agtcaggcag?at 32
<210>3
<211>2188
<212>DNA
＜213〉mouse (Mus musculus)
<400>3
ccacccaggg?aagcaagatg?atttatgaga?ggagctactt?attgaagaat?ggaaattaaa 60
tccctagcca?agagggcatc?ggggatggca?cctggtgact?cagaaggaat?gatagaggct 120
agagcattcc?ctttgggggc?aattaatccc?aagagcctaa?gtgtgggcat?aaatgagttg 180
ctctgtccct?cagtcctgct?tgccttaaat?catctcaggt?tccaggttct?gtcctgcaaa 240
ccgaggtggg?cctaggtctc?attccctaac?agtgaaatcc?agtatgctct?gcaaaccaca 300
gcaagctttt?cccactctct?accacttgct?ccaggctgcc?tctagattgt?aactcattga 360
aattgaaaca?atgacattgc?agtagaagaa?agcacacttg?gcacatcatc?cccctactcc 420
cacaaaacac?acacacacac?acacatacac?acgcacatac?atacacacaa?atacatgatc 480
atgcatgcac?acacatacac?aggcacaaac?acatatatgc?acacacatat?atacatacat 540
atacacacac?acatgcacac?acacatacct?aacatacata?tatatacaca?tgcatacaca?600
tacacacatc?catacacatc?atatacactc?aggcacacac?attcacatat?ccacacaggc?660
acacacacat?gcacacacat?acatacatat?acacacactt?acacatacat?atacacactc?720
acacatatgc?acacacagat?acatgcatgc?atacacatac?acacaaatgc?acactcacac?780
atacacacat?acatatatac?ataggcacac?acatacacat?atatacacag?gcacacacac?840
atacacgaac?acacatacat?atacatacat?tcacatatat?gcacacacat?acacatatac?900
acgcatacac?gtacacacaa?atgcacactc?acacatgcac?acacatacat?aatatacaca?960
ctctcacaca?tgcacataca?cacatacata?cacatacatg?tgcatgcaca?cacacaaata?1020
cacatgcata?catccacatt?cacacagatg?cagacacaaa?tgcacacaca?cacacacaca?1080
cacacacaca?cacacacaca?cacacgcaca?ctgccaccct?gaactagtgg?tggctaaatg?1140
aacaataagt?ctccatcacc?agcttggggg?gaggtaggtg?gtagtgtagg?tgcccccatt?1200
gtgtgatcat?gttcattgta?tgagtttgtc?tgtgttcatt?catcatagtg?acagtcccca?1260
tgtgggtagc?agagagtacg?tgtgcatgca?tcatctccgt?gtttgctcat?gagtgtgtat?1320
gtcagtgtgt?tccagtcttt?ctgtgtgagt?gtcgtcccca?atcccccatc?ccccccccca?1380
gatctctaat?tagtggtttg?gggtttgttc?cttttccctc?ctgttccttt?cctcagcagc?1440
gcggcagcag?cggcggcagc?ctcggtggta?gcagcagcag?cagcagcagc?agcagcagca?1500
gcagcagcag?cagcagcaac?agaagctgcc?gcgccgctga?gtagcagcaa?ggactccgag?1560
tcaagagtag?gattgtagga?ttggatctga?gtgggaacaa?gagtgagctg?gcctgagaga?1620
ggagcagatc?cctcccagcg?ccctcaggcc?acccattgcc?agtaatcttc?aagccagacc?1680
tcttgagagg?agacgggaca?gccaacccta?gcctacccag?gtacagacac?tgggcagttc?1740
tgggggactg?cccacagatg?cctattggat?tcctggggta?tgtaggactc?ccgggtctac?1800
cagccctttt?cacctttccc?catagcaccc?ccaaggaagc?tctgacaact?tgcccatatt?1860
cctgtttccc?acccgtcccc?tgggcacccc?cttttcttct?ctccctccca?gatcccttct?1920
ttggggagct?cagcaaatgg?agcaggaaat?ttggaccctc?tgcctccctc?tctcgccttc?1980
ctcattggat?ccggagtctt?ctccgctggg?aaagctgtaa?ttagagggtg?gatccctaca?2040
gacagagagc?agccccccca?cccccacccc?ccagtccctt?ctaactttag?atctcttctc?2100
tcccattctc?ccattctccc?tccctctccc?ttctccctct?cccactggct?cctcggttct?2160
ctccatctgc?ctgactcctt?gggacccg 2188
<210>4
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primers F 2
<400>4
cggatatccg?agtaatttca?aatgaacgct 30
<210>5
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer R2
<400>5
gctctagagc?cagtcttata?cgccttcttg?c 31
<210>6
<211>374
<212>DNA
＜213〉chicken (Gallus gallus)
<400>6
agtaatttca?aatgaacgct?ctccaagaag?atactccacc?cggaccatca?acggtgttca 60
ggccaccaac?aagttcacgg?ccgttggaaa?cccctcactg?cagagagatc?cggattggta 120
tcgctggaat?tacaatcact?ctatcgctgt?gtggctgcgc?gaatgctcgc?gctcccacgc 180
taagatctgc?aactgcggac?aattcagaaa?gcactggttt?caagaatgtg?ccggacttga 240
ggaccgatca?acccaagcct?ccctcgaaga?agcgatcctg?cgacccctcc?gagtacaggg 300
taagcgagct?aaaagaaagc?ttgattacca?ctactcccag?ccgaccccga?accgcaagaa 360
ggtgtataag?actg 374
<210>7
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primers F 3
<400>7
actccgagtc?aagagtagga?ttgtagg 27
<210>8
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer R 3
<400>8
agaatgggag?aatgggagag?aag 23
<210>9
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primers F 4
<400>9
atctgcaact?gcggacaa 18
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer R4
<400>10
gctgggagta?gtggtaatca?a 21
Claims (9)
1. the recombinant slow virus of carrying out targeted induction on osteogenic differentiated cell apoptosis, it is characterized in that: comprise an Apoptin expression casette in the described recombinant slow virus genome, described Apoptin expression casette comprises Osterix gene promoter, Apoptin gene and terminator, and described Apoptin expression of gene is regulated and control by the Osterix gene promoter.
2. according to the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 1, it is characterized in that: described Apoptin expression casette also comprises V5 epitope gene sequence, and described V5 epitope gene sequence is between Apoptin gene and terminator.
3. according to the recombinant slow virus of claim 1 or 2 described carrying out targeted induction on osteogenic differentiated cell apoptosis, it is characterized in that: described recombinant slow virus is the Apoptin expression casette is inserted the HIV-1 viral genome and to obtain.
4. the preparation method of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 1 is characterized in that: may further comprise the steps:
A, the segmental clone of Osterix gene promoter: the design of primers according to segmental nucleotide sequence of Osterix gene promoter and lentiviral vectors pLenti6/V5-D-TOPO requires to design and synthesize primer, genomic dna with mice embryonic scleroblast MC3T3-E1 is a template, pcr amplification Osterix gene promoter fragment, be cloned into the pGEM-T carrier, get recombinant vectors pGEM-T/Osxp;
The clone of b, Apoptin gene fragment: the nucleotide sequence according to the Apoptin gene designs and synthesizes primer, genomic dna with extraction in the chicken bursa tissue is a template, pcr amplification Apoptin gene fragment is cloned into the pGEM-T carrier, gets recombinant vectors pGEM-T/Apo; In pGEM-T/Apo, go out the Apoptin gene fragment, under the effect of T4DNA ligase enzyme, be connected, get recombinant vectors pcDNA3.1-Apo with same pcDNA3.1 (+) carrier through EcoRV and XbaI double digestion with EcoRV and XbaI double digestion;
The structure of c, Osxp-Apoptin recombined lentivirus vector: in pGEM-T/Osxp, go out the Osxp fragment with KpnI and EcoRI double digestion, with be connected under the effect of T4DNA ligase enzyme through the pcDNA3.1-Apo of KpnI and EcoRI double digestion equally, recombinant vectors pcDNA3.1-Osxp-Apo; In pcDNA3.1-Osxp-Apo, go out the Osxp-Apoptin fragment again, be connected, get recombinant vectors pLenti6/V5-Osxp-Apo with the pLenti6/V5-D-TOPO carrier with KpnI and XbaI double digestion;
The packing of d, Osxp-Apoptin recombinant slow virus: adopt Lipofectamine 2000 reagent with pLenti6/V5-Osxp-Apo and slow virus packaging plasmid mixture cotransfection 293FT cell; after treating that obvious pathology appears in cell; collection contains the culture supernatant of virus, promptly gets the Osxp-Apoptin recombinant slow virus.
5. according to the preparation method of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 4, it is characterized in that: primer sequence described in the step a is:
Upstream primer: 5 '-ggggtacccccacccagggaagcaagatgat-3 ';
Downstream primer: 5 '-cggaattccgggtcccaaggagtcaggcagat-3 '.
6. according to the preparation method of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 5, it is characterized in that: the condition of pcr amplification described in the step a is: 94 ℃ of pre-sex change 5 minutes, 1 minute, 59 ℃ annealing of 94 ℃ of sex change are 1 minute then, 72 ℃ were extended 2 minutes, totally 35 circulations, last 72 ℃ were extended 10 minutes.
7. according to the preparation method of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 4, it is characterized in that: primer sequence described in the step b is:
Upstream primer: 5 '-cggatatccgagtaatttcaaatgaacgct-3 ';
Downstream primer: 5 '-gctctagagccagtcttatacgccttcttgc-3 '.
8. according to the preparation method of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 7, it is characterized in that: the condition of pcr amplification described in the step b is: 94 ℃ of pre-sex change 5 minutes, 1 minute, 60 ℃ annealing of 94 ℃ of sex change are 1 minute then, 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 5 minutes.
9. the application of the recombinant slow virus of the described carrying out targeted induction on osteogenic differentiated cell apoptosis of claim 1 in the medicine of preparation induced osteogenesis noble cells apoptosis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154369A (en) * | 2011-01-27 | 2011-08-17 | 中国人民解放军第三军医大学 | Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus |
| CN115369079A (en) * | 2022-06-22 | 2022-11-22 | 广东省科学院生物与医学工程研究所 | Composition and application thereof in preparation of cell film |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154369A (en) * | 2011-01-27 | 2011-08-17 | 中国人民解放军第三军医大学 | Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus |
| CN102154369B (en) * | 2011-01-27 | 2012-09-12 | 中国人民解放军第三军医大学 | Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus |
| CN115369079A (en) * | 2022-06-22 | 2022-11-22 | 广东省科学院生物与医学工程研究所 | Composition and application thereof in preparation of cell film |
| CN115369079B (en) * | 2022-06-22 | 2023-10-13 | 广东省科学院生物与医学工程研究所 | Composition and application thereof in preparation of cell thin film |
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