CN109295103A - A kind of slow virus carrier and its application in building immortalized cells - Google Patents
A kind of slow virus carrier and its application in building immortalized cells Download PDFInfo
- Publication number
- CN109295103A CN109295103A CN201811276840.5A CN201811276840A CN109295103A CN 109295103 A CN109295103 A CN 109295103A CN 201811276840 A CN201811276840 A CN 201811276840A CN 109295103 A CN109295103 A CN 109295103A
- Authority
- CN
- China
- Prior art keywords
- gene
- cell
- tert
- present
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/04—Immortalised cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to gene engineering technology field more particularly to a kind of slow virus carrier and its applications in building immortalized cells.Different immortalization key genes are had selection, specific aim to be integrated into aim cell genome by the present invention by slow-virus infection, not only increase immortalization efficiency, and will not make to generate harmful side product in cell.Due to having imported two kinds of immutalizing genes simultaneously, the application range of the plasmid vector is expanded, can be adapted for more cell categories, and the mechanism that two groups of gene inductions immortalize is variant, complementary can improve and immortalize efficiency.It joined inducible promoter, the expression of TERT can be controlled by inducer, the canceration for reducing cell is possible, can more preferably control the immortalization process of cell.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of slow virus carrier and its in building immortalized cells
In application.
Background technique
Cell immortality refers to that the cell of in vitro culture by the influence of external environment or the change of autogene, has
Unlimited multiplication capacity, to escape the process of normal cell aging death mechanism.The probability of the spontaneous immortalization of cell is extremely low, grinding tooth
Class animal is 10-5~10-6Between, human cell then only has 10-12.Immortalized cells are constructed for drug toxicology, bio-artificial
Organ building and the research of organizational project all have very profound significance.
To establish immortalized cell line, make the normal cell of in vitro culture can be with infinite multiplication, the current most common side
Method is by exogenous virus infection, imports telomerase reverse transcriptase gene or regulate and control the expression of proto-oncogene and tumor suppressor gene
To realize.
MK cells virus SV40 transfection is at present using more immortalization method, and application range is wider, but successfully
Rate is lower.Existing research shows to be successfully established vascular smooth cell strain, superficial cell strain, bone using SV40 transfection cell
Marrow stroma cell strain etc..The mechanism of action of SV40 is sufficiently complex, it mainly contains two kinds of antigens of Large T and Small T,
There is the bond area cancer suppressorfactor P53 and PRb on middle LT antigen, two kinds of cancer suppressorfactors can be made to lose to fissional control,
Promote the cell of in vitro culture that can continue to pass on.
Human papilloma virus HPV transfection is that epithelial cell immortalizes most common method, has height to epithelial cell
Specificity.E6 and E7 is the factor to play an important role in HPV inducing cell immortalization process, and wherein the albumen of E6 coding can be with
P53 is combined, and passes through hydrolase P53.HPV viruse is in mouth epithelial cells, pancreatic epithelial cells, retinal epithelium at present
It there has been certain progress in the immortalization research of cell.
Telomere is located at chromosome both ends, has the critical function for maintaining chromosome structure stable, can constantly dividing with cell
It splits and shortens, cell will start gene damage detection when reaching critical length, to stop dividing, into decline program.Therefore
Telomere length is the important indicator for judging cell ageing.Telomerase is that one kind can be catalyzed telomere and be answered using itself as template
System, maintains the reverse transcriptase of telomere length, and activity is lower in normal cell.And TERT can activate Telomerase, promote telomere
Duplication prevents telomere from shortening, and extends cell survival.
Existing cell immortality method, major defect have:
1, keep cell being immortalized efficiency lower with virus infected cell, and key gene is integrated into cellular genome
With randomness.Part primary cell can temporarily get the ability being constantly proliferated under viral gene effect, smoothly spend M1
Phase；But when cell reached for 20~30 generation, but it can not start to become feeble and die by the M2 phase.And it is incoherent with immortalization in virus
Gene is once integrated into cellular genome the eubolism approach that cell is destroyed there may be by-product.
2, existing frequently-used immortalization method nearly all has the cell being respectively applicable in, and can not be suitable for all cells, make
It uses with limitation.
3, either virus infection still imports the methods of proto-oncogene, tumor suppressor gene, all has and forms tumour cell
Risk has some potential safety problems.
4, the mechanism that existing immortalization process is related to is more complex, and not easy to control, does not have also building controllable forever at present
The case of biochemical carrier.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of slow virus carrier and its being immortalized in building
Application in cell is able to extend the cell immortality time with the carrier, and controllable.
The present invention provides the plasmid vectors comprising TERT gene and immutalizing gene；
The promoter of the TERT gene is inducible promoter；
The immutalizing gene is from SV40 virus, HPV viruse or Epstein-Barr virus.
In the present invention, the promoter of the TERT gene is TRE3G.
In the present invention, the immutalizing gene is SV40LT gene, SV40ST gene, HPV16E6 gene or HPV16E7 base
Cause.
In some embodiments, the plasmid vector includes TERT gene and HPV16E6 gene, the starting of the TERT gene
Son is TRE3G, and the promoter of the HPV16E6 gene is CMV；
In other embodiments, the plasmid vector includes TERT gene and HPV16E6 gene, and the TERT gene opens
Mover is TRE3G, and the promoter of the SV40LT gene is CMV.
The skeleton carrier of plasmid vector of the present invention is pLV-CMV-SV40LT.
Application of the plasmid vector of the present invention in building immortalized cells.
The present invention also provides the slow virus packed with plasmid vector of the present invention.
The immortalized cells as made from slow-virus infection of the present invention.
The present invention also provides it is a kind of construct immortalized cells kit comprising plasmid vector of the present invention or
Slow virus.
In some embodiments, in the kit of building immortalized cells provided by the invention, including matter of the present invention
Grain carrier, slow virus packaging plasmid and slow virus.
In other embodiments, in the kit of building immortalized cells provided by the invention, including it is of the present invention
Slow virus.
It further include the inducer of inducible promoter in kit of the present invention.
It is thin with slow-virus infection of the present invention the present invention also provides a kind of method for constructing immortalized cells
Born of the same parents, being immortalized cell.
Method of the present invention, with the expression of inducer induction TERT.
The induction processing of the inducer all may be used before passage, or in succeeding generations.
Different immortalization key genes are had selection, specific aim to be integrated by the present invention by plasmid transfection or slow-virus infection
In aim cell genome, immortalization efficiency is not only increased, and will not make to generate harmful side product in cell.Due to simultaneously
Two kinds of immutalizing genes have been imported, the application range of the plasmid vector is expanded, can be adapted for more cell categories, and
And the mechanism that two groups of gene inductions immortalize is variant, complementary can improve and immortalize efficiency.It joined inducible promoter, it can
To control the expression of TERT by inducer, the canceration for reducing cell is possible, can more preferably control the immortalization process of cell.
Detailed description of the invention
Fig. 1 shows the Vector map that sets out slowly；Wherein, Fig. 1-a shows that the Vector map of pLVX-CMV600, Fig. 1-b show pLV-CMV-
The Vector map of SV40LT；
Fig. 2 shows slow virus carrier pLV-CMV-HPV16E6 map；
Fig. 3 shows slow virus carrier pLV-TRE3G-hTERT-CMV-SV40LT map；
Fig. 4 shows slow virus carrier pLV-TRE3G-hTERT-CMV-HPV16E6 map；
Fig. 5 shows form when 5 two groups of cells of embodiment reached for 8 generation, wherein Fig. 5-a shows cellular control unit form；Fig. 5-
B shows infected group cellular morphology；
Fig. 6 shows flow cytometer detection result when 5 two groups of cells of embodiment reached for 8 generation, wherein Fig. 6-a shows cellular control unit
Testing result；Fig. 6-b shows the testing result of infected group cell；
Fig. 7 shows the expression of hTERT in two groups of cells of Western bolt detection when 5 two groups of cells of embodiment reached for 8 generation
Amount；
Fig. 8 shows that 5 infected group cell of embodiment reaches the cellular morphology in the 35th generation；
Fig. 9 shows form when 6 two groups of cells of embodiment reached for 10 generation, wherein Fig. 9-a shows cellular control unit form；Figure
9-b shows infected group cellular morphology；
Figure 10 shows flow cytometer detection result when 6 two groups of cells of embodiment reached for 10 generation, wherein Figure 10-a shows that control group is thin
The testing result of born of the same parents；Figure 10-b shows the testing result of infected group cell；
Figure 11 shows that 6 two groups of cells of embodiment reached for the 26th generation, and Western bolt detects the expression of hTERT in two groups of cells
Amount；
Figure 12 shows that 6 two groups of cells of embodiment reach the testing result of the 26th generation two groups of cells after osteogenic induction；Wherein, scheme
12-a shows the testing result of cellular control unit；Figure 12-b shows the testing result of infected group cell；
Figure 13 shows that 6 two groups of cells of embodiment reach the testing result of the 26th generation two groups of cells after adipogenic induction；Wherein, scheme
13-a shows the testing result of cellular control unit；Figure 13-b shows the testing result of infected group cell.
Specific embodiment
The present invention provides a kind of slow virus carrier and its application in building immortalized cells, those skilled in the art
Present disclosure can be used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications pair
It is it will be apparent that they are considered as being included in the present invention for those skilled in the art.Method and application of the invention is
Through being described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to this
The methods and applications of text are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention provides the plasmid vectors comprising TERT gene and immutalizing gene；
The promoter of the TERT gene is inducible promoter；
The immutalizing gene is from SV40 virus, HPV viruse or Epstein-Barr virus.
The TERT gene is telomerase reverse transcriptase gene.Reverse transcriptase of telomere can activate Telomerase, make cell
Telomere will not shorten with cell Proliferation, to realize the immortalization of cell.But the function of TERT is more single, is not pair
All cells are all suitable for, and larger limitation is received in terms of the scope of application.
The TERT gene that the present invention uses is the TERT gene of source of people, i.e. hTERT, and genbank accession number is NM_
198253.2, sequence is as shown in SEQ ID NO:1.
" immutalizing gene " of the present invention refers to can enable the cell for promoting in vitro culture continue from virus
The gene of passage.Carrying out infection to cell with SV40 virus, Epstein-Barr virus or HPV viruse is that cell enters the common of immortalization process
Mode.But HPV viruse infection is common in the research of the immortalization of epithelial cell；Epstein-Barr virus is suitable for the immortality of lymphocyte
Change；Though SV40 can be such that most cells immortalize, immortal rate is very low.Also, virus makes it forever in infection cell
In biochemical process, many reactions are triggered at random, for example have activated the expression of proto-oncogene or inhibit the table of tumor suppressor gene
It reaches, has activated Telomerase etc., specific mechanism is not yet completely clear, therefore, can not with the mode that virus infection reaches immortalization
Control.It is building up to the side for transfecting cell on carrier and reaching immortalization purpose simultaneously using the key gene of virus-mediated immortalization process
Method is more stably and controllable.
In the present invention, the immutalizing gene is SV40LT gene, SV40ST gene, HPV16E6 gene or HPV16E7 base
Cause.
SV40 mainly contains two kinds of antigens of Large T (LT) and Small T (ST) to the zone of action of cell immortality,
Wherein there is the bond area cancer suppressorfactor P53 and PRb on LT antigen, two kinds of cancer suppressorfactors can be made to lose to fissional control
System promotes the cell of in vitro culture that can continue to pass on.E6 and E7 is to play important work in HPV viruse inducing cell immortalization process
The factor, wherein the albumen of E6 coding can be in conjunction with P53, and passes through hydrolase P53.
In some specific embodiments, the immutalizing gene is SV40LT gene or HPV16E6 gene.
Wherein, the nucleotide sequence of the HPV16E6 gene is as shown in SEQ ID NO:3.
The nucleotide sequence of the SV40LT gene is as shown in SEQ ID NO:4.
The inducible promoter refers under certain specific physically or chemically stimulations of signal, can significantly mention
The promoter of the transcriptional level of high gene, for the expression of accurate controlling gene, the present invention uses the starting of chemical signal induction
Son.In the present invention, the promoter of the TERT gene is TRE3G.TRE3G promoter is one of inducible promoter, energy
Enough by tetracycline (DOX) come the expression of controlling gene.Studies have shown that the promoter is for the present invention program, the most
It is suitable for.The present invention is using the nucleotide sequence of TER3G promoter as shown in SEQ ID NO:2.
The present invention introduces TERT gene and immutalizing gene in slow virus plasmid vector, not only can break through virus
Transfection mediates the application limitation immortalized, the carrier can be made to apply in more cells, and substantially increase cell immortality
Change successful probability.However, the segment due to insertion is larger, while two biggish segments of expression can make cell transfecting success rate
It reduces, and the growth in succeeding generations to cell causes to bear.Therefore, TERT gene is regulated and controled using inducible promoter
Expression, after cell smoothly spends the M1 phase, if apoptosis phenomenon occurs in cell, can start hTERT's by evoked promoter
Expression, promotes cell to enter and spends the M2 phase, realizes and immortalizes.
In some embodiments, the plasmid vector includes TERT gene and HPV16E6 gene, the starting of the TERT gene
Son is TRE3G, and the promoter of the HPV16E6 gene is CMV；
The present invention experiments have shown that, it is thin that the positive that mesenchymal stem cell obtains is infected using the slow virus of the vector construction
Cell proliferation rate slows down when born of the same parents reached for 26 generation, and in culture medium hereafter for 24 hours with fortimicin induction, cell telomere length is extensive
It is multiple to stablize, immortalised state is maintained, there is good stemness through detection.Continue to reach for 40 generations, has no aging phenomenon.
In other embodiments, the plasmid vector includes TERT gene and HPV16E6 gene, and the TERT gene opens
Mover is TRE3G, and the promoter of the SV40LT gene is CMV.
The present invention experiments have shown that, the positive cell that people's esophageal epithelial cell obtains is infected using the slow virus of the vector construction
For 24 hours with fortimicin induction, it reached for the 35th generation after, has no aging phenomenon.
The plasmid is the double chain DNA molecule of small, annular, external source target gene can be imported host as carrier
Cell.In the present invention, the plasmid vector of external source target gene part, referred to as skeleton carrier will not included.The plasmid vector
Skeleton carrier be pLV-CMV-SV40LT.
The map of the carrier pLV-CMV-SV40LT such as Fig. 1-b is constructed with pLVX-CMV600 (addgene, #
110723) plasmid, in multiple cloning sites, is inserted into SV40LT segment if Fig. 1-a is initial plasmid.
Application of the plasmid vector of the present invention in building immortalized cells.
Plasmid vector of the present invention is suitable for that the cell context of building is more extensive, in the embodiment of the present invention, is used for
Immortal human esophageal epithelial cell or the building for immortalizing mesenchymal stem cell.Further, can also be applied to
The building of liver cell, retinal epithelial cells, Cardiac Fibroblasts, skin fibroblasts or fat stem cell.
The present invention also provides the slow virus packed with plasmid vector of the present invention.
The construction method of slow virus of the present invention, using plasmid vector provided by the invention and the slow disease of packaging plasmid packaging
Poison.The packaging plasmid is pMD2.G, psPAX2.
It can be improved the transfection efficiency of purpose plasmid using slow virus as carrier.
The immortalized cells as made from slow-virus infection of the present invention.
The present invention also provides it is a kind of construct immortalized cells kit comprising plasmid vector of the present invention or
Slow virus.
In some embodiments, in the kit of building immortalized cells provided by the invention, including matter of the present invention
Grain carrier and slow virus packaging plasmid.
In other embodiments, in the kit of building immortalized cells provided by the invention, including it is of the present invention
Slow virus.
It further include the inducer of inducible promoter in kit of the present invention.
It is thin with slow-virus infection of the present invention the present invention also provides a kind of method for constructing immortalized cells
Born of the same parents, being immortalized cell.
Method of the present invention, with the expression of inducer induction TERT.
The induction processing of the inducer all may be used before passage, or in succeeding generations.
The inducer is tetracycline (fortimicin).
The dosage of the inducer is 1 μ g/mL, and the time of the induction is for 24 hours.
The culture medium of the passage is the primary culture medium that cell is applicable in.
In some embodiments, induction processing is carried out before passage.
In other embodiments, induction processing cell in succeeding generations occurs to carry out when aging phenomenon.
The aging phenomenon includes but is not limited to: cell proliferation rate slows down, and cellular morphology is irregular, and cell starts to wither
It dies, G2 phase Leukopenia, S phase cytosis, telomere length shortens.
Different immortalization key genes are had selection, specific aim to be integrated by the present invention by plasmid transfection or slow-virus infection
In aim cell genome, immortalization efficiency is not only increased, and will not make to generate harmful side product in cell.Due to simultaneously
Two kinds of immutalizing genes have been imported, the application range of the plasmid vector is expanded, can be adapted for more cell categories, and
And the mechanism that two groups of gene inductions immortalize is variant, complementary can improve and immortalize efficiency.It joined inducible promoter, it can
To control the expression of TERT by inducer, the canceration for reducing cell is possible, can more preferably control the immortalization process of cell.
The test material that the present invention uses is all common commercially available product, can all be bought in market.
Wherein the construction method of carrier pLV-CMV-SV40LT is synthesis SV40LT sequence, and adds Pme I at both ends
(GTTTAAAC), Sma I (CCCGGG) restriction enzyme site sequence, is building up to pLVX- for SV40LT by way of double digestion
On CMV600.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
1, it is basic expression vector with slow virus carrier pLV-CMV-SV40LT, constructs pLV-CMV-HPV16E6 carrier.
2, HPV16E6 sequence (NC_001526.4, SEQ ID NO:4) is synthesized, and adds Pme I at both ends
(GTTTAAAC), Sma I (CCCGGG) restriction enzyme site sequence
3, the SV40LT sequence in pLV-CMV-SV40LT carrier (Fig. 1) is substituted for by way of double digestion
HPV16E6 sequence obtains pLV-CMV-HPV16E6 carrier (Fig. 2).
4, TRE3G sequence (SEQ ID NO:2) is synthesized, and adds BamH I (GGATCC), EcoR I at both ends
(GAATTC) restriction enzyme site sequence.
5, pLV- is obtained by TRE3G sequence construct to pLV-CMV-HPV16E6 carrier with the mode for crossing double digestion
TRE3G-CMV-HPV16E6 carrier.
6, synthesize hTERT sequence (NM_198253.2, SEQ ID NO:1), and both ends plus EcoR I (GAATTC),
Xba I (TCTAGA) restriction enzyme site sequence.
7, hTERT sequence construct to pLV-TRE3G-CMV-HPV16E6 is obtained into pLV- by way of double digestion
TRE3G-hTERT-CMV-HPV16E6 carrier (Fig. 4).
Embodiment 2
1, it is basic expression vector with slow virus carrier pLV-CMV-SV40LT, constructs pLV-TRE3G-hTERT-CMV-
SV40LT carrier.
2, TRE3G sequence (SEQ ID NO:2) is synthesized, and adds BamH I (GGATCC), EcoR I at both ends
(GAATTC) restriction enzyme site sequence.
3, the mode of double digestion is crossed by TRE3G sequence construct to pLV-CMV-SV40LT carrier, obtains pLV-TRE3G-
CMV-SV40LT carrier.
4, synthesize hTERT sequence (NM_198253.2, SEQ ID NO:1), and both ends plus EcoR I (GAATTC),
Xba I (TCTAGA) restriction enzyme site sequence.
5, hTERT sequence construct to pLV-TRE3G-CMV-SV40LT is obtained into pLV- by way of double digestion
TRE3G-hTERT-CMV-SV40LT carrier (Fig. 3).
Embodiment 3
PMD2.G carrier, psPAX2 carrier will be used, by pLV-TRE3G-hTERT-CMV-HPV16E6 carrier package at slow
Virus, packing method are as follows:
1,293T cell is pressed 7 × 106A/ware inoculation, is inoculated with 3 wares altogether.The DMEM containing 10% fetal calf serum is added to cultivate
Base is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
2, after for 24 hours, by 10 μ g pMD2.G, 12 μ g psPAX2,10 μ g pLV-TRE3G-hTERT-CMV-HPV16E6 matter
Grain is transfected into jointly in 293T cell.
3, the cell conditioned medium for collecting 48h, 72h after transfecting after being filtered to remove impurity, is concentrated into 1ml, packing 5 with super filter tube
Pipe, is stored in -80 DEG C.
Embodiment 4
PMD2.G carrier, psPAX2 carrier will be used, by pLV-TRE3G-hTERT-CMV-SV40LT carrier package at slow
Virus, packing method are as follows:
1,293T cell is pressed 7 × 106A/ware inoculation, is inoculated with 3 wares altogether.The DMEM containing 10% fetal calf serum is added to cultivate
Base is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
2, after for 24 hours, by 10 μ g pMD2.G, 12 μ g psPAX2,10 μ g pLV-TRE3G-hTERT-CMV-SV40LT plasmids
It is transfected into 293T cell jointly.
3, the cell conditioned medium for collecting 48h, 72h after transfecting after being filtered to remove impurity, is concentrated into 1ml, packing 5 with super filter tube
Pipe, is stored in -80 DEG C.
Embodiment 5
With slow-virus infection people esophageal epithelial cell described in embodiment 3, method is by people's esophageal epithelial cell with 3 × 104
A/hole is seeded in 24 orifice plates, according to MOI=10 virus infection, changes liquid afterwards for 24 hours, observation cell can detect glimmering after infection 72h
Light, efficiency of infection about 80%.
Screening positive clone, for 24 hours with culture medium (1640+10%FBS) processing containing 1 μ g/ml DOX (fortimicin)
To induce the expression of hTERT, then persistently secondary culture observes cell quality.
People's esophageal epithelial cell without slow-virus infection is set as control.
Culture cell is detected:
1, by control group and the cell of infected group while secondary culture, when cell reached for 8 generation, cellular control unit has started
There is aging, and infected group still maintains normal morphology.Cellular morphology is as shown in Figure 5.
2, with the period of two groups of cells of flow cytometer detection, as a result as shown below.As can be seen from the results, cellular control unit
Period is checked, G2 phase Leukopenia, S phase cytosis.Infected group is normal.Detection such as Fig. 6.
3, WB detects the expression quantity of hTERT in two groups of cells, as a result such as Fig. 7.The expression of hTERT is obvious in infected group cell
Higher than cellular control unit.
4, when infected group cell reached for 35 generation, cell state is good, still being capable of normal proliferative.Illustrate that the cell has had
The standby ability immortalized, Fig. 8 show P35 for the form of cell.Reached for 60 generations, cell still maintains immortalised state.
Embodiment 6
With slow-virus infection mesenchymal stem cell (BMSC) described in embodiment 4, method is to do human bone marrow mesenchymal
Cell is with 2 × 104A/hole is seeded in 24 orifice plates, according to MOI=15 virus infection, changes liquid afterwards for 24 hours, is observed carefully after infecting 72h
Born of the same parents can detect fluorescence, efficiency of infection about 70%.
Screening positive clone carries out secondary culture.
BMSC without slow-virus infection is set as control.
When control group and the BMSC cell of infected group reached for 10 generation, form is as shown in Figure 9.With infected group cellular morphology phase
Than the existing part of cellular control unit enters differentiation state.
Identify that the results are shown in Figure 10 with period of the flow cytometer to two groups of cells.Control group is thin as the result is shown
Born of the same parents, which receive in the period, to check, and the period of infected group is normal.
When cell reached for 26 generation, the cell proliferation rate of infected group slows down, with contain 1 μ g/ml DOX (fortimicin)
Culture medium (DMEM/F12+10%FBS) handle for 24 hours to induce the expression of hTERT, then persistently secondary culture observe cellularity
Shape.
After cell restoration ecosystem speed, WB detects the expression of hTERT in non-induced group and induction group BMSC cell,
As a result as shown in figure 11.By the induction of DOX, cell is overexpressed hTERT, and cell telomere length restores to stablize, and cell maintains forever
Biochemical profile.Reached for 40 generations, cell still maintains immortalised state.
Skeletonization, adipogenic induction carried out to the BMSC cell of induction group and non-induced group of the 40th generation, and with alizarin red and oil red O
Dyeing identification respectively, as a result as shown in Figure 12~13.The cell of induction group still has a stemness, and non-induced group of cell is not
Has differentiation capability.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Sequence table
<110>Hunan Feng Hui Biotechnology Co., Ltd
<120>a kind of slow virus carrier and its application in building immortalized cells
<130> MP1821745
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4018
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat 60
gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt 120
gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg 180
cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga 240
cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt 300
ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt 360
cgcgctgctg gacggggccc gcgggggccc ccccgaggcc ttcaccacca gcgtgcgcag 420
ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct 480
gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct 540
ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc 600
cactcaggcc cggcccccgc cacacgctag tggaccccga aggcgtctgg gatgcgaacg 660
ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc 720
gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg 780
cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag 840
gacgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga 900
agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg 960
ccagcaccac gcgggccccc catccacatc gcggccacca cgtccctggg acacgccttg 1020
tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct 1080
gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt 1140
ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg 1200
cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc 1260
gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc 1320
agcagccggt gtctgtgccc gggagaagcc ccagggctct gtggcggccc ccgaggagga 1380
ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt 1440
gtacggcttc gtgcgggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag 1500
gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc 1560
caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg 1620
caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct 1680
ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt 1740
ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg 1800
gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct 1860
gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact 1920
ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg 1980
agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact 2040
gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct 2100
gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga 2160
cccgccgcct gagctgtact ttgtcaaggt ggatgtgacg ggcgcgtacg acaccatccc 2220
ccaggacagg ctcacggagg tcatcgccag catcatcaaa ccccagaaca cgtactgcgt 2280
gcgtcggtat gccgtggtcc agaaggccgc ccatgggcac gtccgcaagg ccttcaagag 2340
ccacgtctct accttgacag acctccagcc gtacatgcga cagttcgtgg ctcacctgca 2400
ggagaccagc ccgctgaggg atgccgtcgt catcgagcag agctcctccc tgaatgaggc 2460
cagcagtggc ctcttcgacg tcttcctacg cttcatgtgc caccacgccg tgcgcatcag 2520
gggcaagtcc tacgtccagt gccaggggat cccgcagggc tccatcctct ccacgctgct 2580
ctgcagcctg tgctacggcg acatggagaa caagctgttt gcggggattc ggcgggacgg 2640
gctgctcctg cgtttggtgg atgatttctt gttggtgaca cctcacctca cccacgcgaa 2700
aaccttcctc aggaccctgg tccgaggtgt ccctgagtat ggctgcgtgg tgaacttgcg 2760
gaagacagtg gtgaacttcc ctgtagaaga cgaggccctg ggtggcacgg cttttgttca 2820
gatgccggcc cacggcctat tcccctggtg cggcctgctg ctggataccc ggaccctgga 2880
ggtgcagagc gactactcca gctatgcccg gacctccatc agagccagtc tcaccttcaa 2940
ccgcggcttc aaggctggga ggaacatgcg tcgcaaactc tttggggtct tgcggctgaa 3000
gtgtcacagc ctgtttctgg atttgcaggt gaacagcctc cagacggtgt gcaccaacat 3060
ctacaagatc ctcctgctgc aggcgtacag gtttcacgca tgtgtgctgc agctcccatt 3120
tcatcagcaa gtttggaaga accccacatt tttcctgcgc gtcatctctg acacggcctc 3180
cctctgctac tccatcctga aagccaagaa cgcagggatg tcgctggggg ccaagggcgc 3240
cgccggccct ctgccctccg aggccgtgca gtggctgtgc caccaagcat tcctgctcaa 3300
gctgactcga caccgtgtca cctacgtgcc actcctgggg tcactcagga cagcccagac 3360
gcagctgagt cggaagctcc cggggacgac gctgactgcc ctggaggccg cagccaaccc 3420
ggcactgccc tcagacttca agaccatcct ggactgatgg ccacccgccc acagccaggc 3480
cgagagcaga caccagcagc cctgtcacgc cgggctctac gtcccaggga gggaggggcg 3540
gcccacaccc aggcccgcac cgctgggagt ctgaggcctg agtgagtgtt tggccgaggc 3600
ctgcatgtcc ggctgaaggc tgagtgtccg gctgaggcct gagcgagtgt ccagccaagg 3660
gctgagtgtc cagcacacct gccgtcttca cttccccaca ggctggcgct cggctccacc 3720
ccagggccag cttttcctca ccaggagccc ggcttccact ccccacatag gaatagtcca 3780
tccccagatt cgccattgtt cacccctcgc cctgccctcc tttgccttcc acccccacca 3840
tccaggtgga gaccctgaga aggaccctgg gagctctggg aatttggagt gaccaaaggt 3900
gtgccctgta cacaggcgag gaccctgcac ctggatgggg gtccctgtgg gtcaaattgg 3960
ggggaggtgc tgtgggagta aaatactgaa tatatgagtt tttcagtttt gaaaaaaa 4018
<210> 2
<211> 379
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag 60
agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc 120
tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta 180
cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag 240
agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg 300
aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa ctttccgtac 360
cacttcctac cctcgtaaa 379
<210> 3
<211> 477
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60
cagttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120
aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggattt atgcatagta 180
tatagagatg ggaatccata tgctgtatgt gataaatgtt taaagtttta ttctaaaatt 240
agtgagtata gacattattg ttatagtttg tatggaacaa cattagaaca gcaatacaac 300
aaaccgttgt gtgatttgtt aattaggtgt attaactgtc aaaagccact gtgtcctgaa 360
gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420
ggtcgatgta tgtcttgttg cagatcatca agaacacgta gagaaaccca gctgtaa 477
<210> 4
<211> 2127
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa 60
aggagtgcct gggggaatat tcctctgatg agaaaggcat atttaaaaaa atgcaaggag 120
tttcatcctg ataaaggagg agatgaagaa aaaatgaaga aaatgaatac tctgtacaag 180
aaaatggaag atggagtaaa atatgctcat caacctgact ttggaggctt ctgggatgca 240
actgagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc ctttaatgag 300
gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac tgctgactct 360
caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga ctttccttca 420
gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc ttgctttgct 480
atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct 540
gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt tcttactcca 600
cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac ctttagcttt 660
ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac tagagatcca 720
ttttctgtta ttgaggaaag tttgccaggt gggttaaagg agcatgattt taatccagaa 780
gaagcagagg aaactaaaca agtgtcctgg aagcttgtaa cagagtatgc aatggaaaca 840
aaatgtgatg atgtgttgtt attgcttggg atgtacttgg aatttcagta cagttttgaa 900
atgtgtttaa aatgtattaa aaaagaacag cccagccact ataagtacca tgaaaagcat 960
tatgcaaatg ctgctatatt tgctgacagc aaaaaccaaa aaaccatatg ccaacaggct 1020
gttgatactg ttttagctaa aaagcgggtt gatagcctac aattaactag agaacaaatg 1080
ttaacaaaca gatttaatga tcttttggat aggatggata taatgtttgg ttctacaggc 1140
tctgctgaca tagaagaatg gatggctgga gttgcttggc tacactgttt gttgcccaaa 1200
atggattcag tggtgtatga ctttttaaaa tgcatggtgt acaacattcc taaaaaaaga 1260
tactggctgt ttaaaggacc aattgatagt ggtaaaacta cattagcagc tgctttgctt 1320
gaattatgtg gggggaaagc tttaaatgtt aatttgccct tggacaggct gaactttgag 1380
ctaggagtag ctattgacca gtttttagta gtttttgagg atgtaaaggg cactggaggg 1440
gagtccagag atttgccttc aggtcaggga attaataacc tggacaattt aagggattat 1500
ttggatggca gtgttaaggt aaacttagaa aagaaacacc taaataaaag aactcaaata 1560
tttccccctg gaatagtcac catgaatgag tacagtgtgc ctaaaacact gcaggccaga 1620
tttgtaaaac aaatagattt taggcccaaa gattatttaa agcattgcct ggaacgcagt 1680
gagtttttgt tagaaaagag aataattcaa agtggcattg ctttgcttct tatgttaatt 1740
tggtacagac ctgtggctga gtttgctcaa agtattcaga gcagaattgt ggagtggaaa 1800
gagagattgg acaaagagtt tagtttgtca gtgtatcaaa aaatgaagtt taatgtggct 1860
atgggaattg gagttttaga ttggctaaga aacagtgatg atgatgatga agacagccag 1920
gaaaatgctg ataaaaatga agatggtggg gagaagaaca tggaagactc agggcatgaa 1980
acaggcattg attcacagtc ccaaggctca tttcaggccc ctcagtcctc acagtctgtt 2040
catgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 2100
acacctcccc ctgaacctga aacataa 2127
Claims (10)
1. including the plasmid vector of TERT gene and immutalizing gene；
The promoter of the TERT gene is inducible promoter；
The immutalizing gene is from SV40 virus, HPV viruse or Epstein-Barr virus.
2. plasmid vector according to claim 1, which is characterized in that the promoter of the TERT gene is TRE3G；It is described
Immutalizing gene is SV40LT gene, SV40ST gene, HPV16E6 gene or HPV16E7 gene.
3. plasmid vector according to claim 1, which is characterized in that
Comprising TERT gene and HPV16E6 gene, the promoter of the TERT gene is TRE3G, and the HPV16E6 gene opens
Mover is CMV；
Or comprising TERT gene and HPV16E6 gene, the promoter of the TERT gene is TRE3G, the SV40LT gene
Promoter is CMV.
4. described in any item plasmid vectors according to claim 1~3, which is characterized in that its skeleton carrier is pLV-CMV-
SV40LT。
5. application of the described in any item plasmid vectors of Claims 1 to 4 in building immortalized cells.
6. with the slow virus of the described in any item plasmid vector packagings of Claims 1 to 4.
7. immortalized cells made from the slow-virus infection as described in claim 6.
8. a kind of kit for constructing immortalized cells, which is characterized in that including the described in any item plasmids of Claims 1 to 4
Carrier or slow virus as claimed in claim 6.
9. a kind of method for constructing immortalized cells, which is characterized in that with slow virus infected cell as claimed in claim 6, obtain
Obtain immortalized cells.
10. according to the method described in claim 9, it is characterized in that, further including with the expression of inducer induction TERT.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811276840.5A CN109295103A (en) | 2018-10-30 | 2018-10-30 | A kind of slow virus carrier and its application in building immortalized cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811276840.5A CN109295103A (en) | 2018-10-30 | 2018-10-30 | A kind of slow virus carrier and its application in building immortalized cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109295103A true CN109295103A (en) | 2019-02-01 |
Family
ID=65145434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811276840.5A Pending CN109295103A (en) | 2018-10-30 | 2018-10-30 | A kind of slow virus carrier and its application in building immortalized cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109295103A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106201A (en) * | 2019-04-03 | 2019-08-09 | 广州辉园苑医药科技有限公司 | A kind of retroviral vector controllably immortalized and human umbilical cord mesenchymal stem cells and its construction method |
| CN112941033A (en) * | 2021-03-11 | 2021-06-11 | 深圳市人民医院 | Construction method of immortalized feeder layer cell strain, immortalized feeder layer cell strain and application |
| CN112980798A (en) * | 2021-01-12 | 2021-06-18 | 重庆市药物种植研究所 | Construction method of immortalized muskrat gland fibroblast |
| CN113025661A (en) * | 2021-01-12 | 2021-06-25 | 重庆市药物种植研究所 | Construction method of immortalized musk glandular epithelial cells |
| CN113528454A (en) * | 2021-07-12 | 2021-10-22 | 福州载基生物科技有限公司 | Construction method of rat cartilage immortalized cell line |
| CN114807043A (en) * | 2022-03-24 | 2022-07-29 | 上海交通大学医学院附属第九人民医院 | Human-derived vestibular schlemma immortalized cell line and construction method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140442A (en) * | 2010-02-01 | 2011-08-03 | 北京大学深圳医院 | Recombinant lentivirus as well as preparation method and application thereof |
| CN105331635A (en) * | 2015-11-05 | 2016-02-17 | 深圳大学 | Induced slow virus expression system and construction method and application thereof |
-
2018
- 2018-10-30 CN CN201811276840.5A patent/CN109295103A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140442A (en) * | 2010-02-01 | 2011-08-03 | 北京大学深圳医院 | Recombinant lentivirus as well as preparation method and application thereof |
| CN105331635A (en) * | 2015-11-05 | 2016-02-17 | 深圳大学 | Induced slow virus expression system and construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LUIGI BALDUCCI等: "Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors", 《STEM CELL RESEARCH & THERAPY》 * |
| 罗琼等: "1６型人乳头瘤病毒Ｅ６、Ｅ７蛋白和端粒酶在宫颈上皮内瘤变发展中的表达及意义", 《南昌大学学报（医学版）》 * |
| 贺小英等: "hTERT与SV40 LT协同表达对牛乳腺上皮细胞体外增殖的影响", 《中国细胞生物学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106201A (en) * | 2019-04-03 | 2019-08-09 | 广州辉园苑医药科技有限公司 | A kind of retroviral vector controllably immortalized and human umbilical cord mesenchymal stem cells and its construction method |
| CN112980798A (en) * | 2021-01-12 | 2021-06-18 | 重庆市药物种植研究所 | Construction method of immortalized muskrat gland fibroblast |
| CN113025661A (en) * | 2021-01-12 | 2021-06-25 | 重庆市药物种植研究所 | Construction method of immortalized musk glandular epithelial cells |
| CN112941033A (en) * | 2021-03-11 | 2021-06-11 | 深圳市人民医院 | Construction method of immortalized feeder layer cell strain, immortalized feeder layer cell strain and application |
| CN113528454A (en) * | 2021-07-12 | 2021-10-22 | 福州载基生物科技有限公司 | Construction method of rat cartilage immortalized cell line |
| CN114807043A (en) * | 2022-03-24 | 2022-07-29 | 上海交通大学医学院附属第九人民医院 | Human-derived vestibular schlemma immortalized cell line and construction method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109295103A (en) | A kind of slow virus carrier and its application in building immortalized cells | |
| Zimmet et al. | Emerging role for bone marrow derived mesenchymal stem cells in myocardial regenerative therapy | |
| Badillo et al. | Murine bone marrow stromal progenitor cells elicit an in vivo cellular and humoral alloimmune response | |
| CN101928692A (en) | Differentiated cells suitable for human therapy | |
| Devitt et al. | Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age | |
| Verdanova et al. | Evaluation of sericin as a fetal bovine serum-replacing cryoprotectant during freezing of human mesenchymal stromal cells and human osteoblast-like cells | |
| EP1989294A2 (en) | Compostions and populations of cells obtained from the umbilical cord and methods of producing the same | |
| JP6868565B2 (en) | Immortalized stem cells and how to make them | |
| US20070282456A1 (en) | Compositions and Methods for Myogenesis of Fat-Derived Stem Cells Expressing Telomerase and Myocardin | |
| CN109415740A (en) | Express the mescenchymal stem cell and application thereof of TRAIL and CD | |
| CN109477119A (en) | Express the mescenchymal stem cell and application thereof of hepatocyte growth factor | |
| CN111676187B (en) | Isolated mesenchymal stem cell population and uses thereof | |
| Chen et al. | Efficiency of exosome production correlates inversely with the developmental maturity of MSC donor | |
| CN105462913A (en) | Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells | |
| GB2374076A (en) | Antigen expression in undifferentiated stem cells | |
| CN109674818A (en) | Purposes of the hAMSCs in preparation treatment acute graft versus host disease drug | |
| CN103087992A (en) | Improved adipose-derived stem cells for cartilage injury repair | |
| CN106282103A (en) | Animal mescenchymal stem cell immunosuppressant can strengthen serum-free medium | |
| JP2022551248A (en) | Treatment of chronic granulomatosis | |
| CN109593725A (en) | A kind of recombination mescenchymal stem cell and its application | |
| CN112941106B (en) | Method for delaying mesenchymal stem cell senescence through FOXP1 gene editing and mutation | |
| US20180066253A1 (en) | Methods and compositions for modifying endothelial cells | |
| Shi | Skeletal stem cells in regenerative medicine | |
| Forte et al. | Interfacing Sca-1 p o s Mesenchymal Stem Cells with Biocompatible Scaffolds with Different Chemical Composition and Geometry | |
| Nikitina et al. | Risk of genetic transformation of multipotent mesenchymal stromal cells in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190201 |
|
| RJ01 | Rejection of invention patent application after publication |