CN1590539A - Method of making skin as fiber cell immortalization - Google Patents
Method of making skin as fiber cell immortalization Download PDFInfo
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Abstract
A process for preparing the immortal skin fibroblast features that the skin fibroblast is transfected by the antigen LT of simian virus 40 and human telomerase catalysis subunit hTERT.
Description
Technical field
The invention belongs to the cell-fusion techniques field that pair cell is modified in the genetic engineering, relate to a kind of method that makes the skin flbroblast immortalization.
Background technology
Those can flee from from breed old and feeble crisis, thereby the cell with ability of infinite multiplication are referred to as " immortalization " cell through influence spontaneous or that be subjected to extraneous factor among the human and animal.The probability of spontaneous immortalized cells is very little, and rodent is 10
-5～10
-6, people's cell is then more rare, less than 10
12Therefore, scholars change allogenic gene over to the purpose cell by technology such as gene transfections, or induce the senescence-associated gene sudden change, to increase the generation probability of immortalization.Along with the continuous development of biomedical engineering technology, make the immortalization inoblast as the seed source of the standard cell lines or the organizational project of cytobiology, become the task of top priority that presses for solution.At present, also carried out many work in this technical field both at home and abroad, the method that relates to the skin flbroblast immortalization mainly contains following several:
The SV40 method: the SV40 large T antigen is a kind of monkey-kidney cells virus of finding the sixties, and the people is its natural reservoir (of bird flu viruses), and it has structural protein, and (VP1, VP2 VP3) form with two kinds of antigen LT and st.Nineteen eighty-three lubbe L etc. utilize the SV40 large T antigen to transform after selecting three routine Lesch-Nyhan patients' skin flbroblast to cultivate, and successfully it are cultivated more than 200 generations.Cell performance after the conversion reduces the requirement of serum, presents multiple layer growth, contactless inhibition phenomenon, and no anchorage dependence can be grown in soft agar.Have the people successively to be applied to the fibroblastic conversions of patient skin such as Wilms knurl later on, cell survival obviously prolongs.Because the understanding to SV40 is relatively more comprehensive, so be widely used in the research of lifetime, aging and immortalization mechanism.But above-described cell all derives from sick body, and promptly the cell that is obtained is improper.The gene transfection relative difficult of normal somatic cell all is as medium with virus in the cell of minority transfection success.Utilize the cell of virus transfection can only be used for fundamental research,, then have the possibility of induced tumor or other illness if with its seed cell as treatment usefulness cell or tissue engineering.
The telomere enzyme process: Telomerase is the new focus of life science in recent years.The life cycle that the utilization Telomerase prolongs cell also is a frontier of immortalization.Telomere (telomere) is to be positioned at the DNA cap that end of chromosome has specific function, and it is being stablized karyomit(e) and is preventing that aspects such as end of chromosome fusion have important effect.Along with the increase of frequency dividing cell, the shortening of carrying out property of telomere, when shortening to certain limit and promptly can not keep chromosomal stability, cell loses the ability of division growth, and then aging death.Telomerase (telomerase) then is the ribonucleoprotein that the certain sequence of telomere is synthesized and kept in catalysis, is made up of RNA and albumen, has the activity of reversed transcriptive enzyme.Wherein the catalytic subunit of Telomerase (being named as hTERT) plays a crucial role in the activation of Telomerase.1996, Wight etc. found that first the shortening of telomere can cause the propagation crisis of cell.Homayoun in 1998 etc., in the people's who lacks telomerase activation normally diploid fibroblast, change telomerase catalytic subunit over to, the result shows, activate the expression of normal cell Telomerase by the expression of virus-mediated hTERT, and then caused the growth of telomeric dna and the extension of cell life cycle.The normal people inoblast of Telomerase ectopic expression, its vitro culture surpasses 170PD, so far still in cultivation.
Radioactivity factor method: 1996, the survivor after the radiation of high dosage nuclear bomb such as Razmik obtained the skin flbroblast of spontaneous immortalization on one's body.After this produced in succession X ray, ionizing rays,
50Radioactivity factors such as Co are carried out the method for cell immortalityization, how can make cell life cycle obtain extending.Supposition may be because the broken stability of keeping old and feeble mechanism of having encircled of radioelement has promoted to prolong Expression of Related Genes with life.
Oncogene, cancer suppressor gene method: based on the biological characteristics of oncogene, cancer suppressor gene, human ras is arranged, c-myc, N-myc, c-fos, proto-oncogenes such as Ab5 carry out cell transfecting, or cancer suppressor gene is suddenlyd change, carry out finding after the cell transformation that it is very rare to utilize oncogene to transform the situation of former generation inoblast immortalization separately, even promoted the aging of cell.And certain short Reproduction is arranged for passage cell.For example, the expression of c-myc has recovered the activity of Telomerase, and P53 is undergone mutation, and has blocked the NK4A/ARF path, and then the two-phase incident has promoted the activity of Telomerase again conversely, and then promotes immortalization.
Additive method: the somebody utilizes HPV, tetranitro quinoline monoxide, aflatoxin etc. in addition, or above several method is united to use and changed into fibrocyte.
In above several transform modes, though the variation that all experiments still can not clear and definite cell be taken place in the immortalization process, but us have been given new enlightenment, that is exactly on performance LT antigen deactivation P53 and two kinds of proteic bases of PRb, change ectogenic hTERT again over to, the activity of reset terminal granzyme might make cell generation immortalization change or raising immortalization number turnover.
Summary of the invention
According to above-mentioned prior art situation, the objective of the invention is to, the research work that the forefathers that continue have carried out, propose a kind of on the basis of monkey-kidney cells virus (SV40 large T antigen) transfection human skin fibroblast, again it is carried out the transfection of telomerase catalytic subunit (hTERT), has the skin fibroblast line method that stabilate is learned proterties and can be gone down to posterity for a long time in the hope of obtaining, make the acquisition of this cell that standardized cell can be provided the experiment in vitro of skin tissue engineering, help organization engineering skin and make up establishment of standard.And make every effort to make this cell should have following feature:
(1) compare with normal cell, immortalized cell line can go down to posterity for a long time external, at least more than 50 generations.
(2) has metastable, approaching biological character with normal cell.No tumorigenicity can be at external synthesis secretion I type, III Collagen Type VI, and has the ability to repair the sick tissue that decreases.
Requirement according to above-mentioned design and designed immortalization inoblast feature, the present invention makes the method for skin flbroblast immortalization, it is characterized in that: with complementary DNA (cDNA) cDNA and the cDNA that has the human telomerase catalytic subunit hTERT of fluorescent protein expression gene of simian virus 40 large T antigen LT, common transfection skin flbroblast, to set up immortalized cell line, may further comprise the steps:
Step l: cultivate former generation skin flbroblast;
Step 2: use simian virus 40 large T antigen LT, human telomerase catalytic subunit hTERT transfection inoblast respectively, and carry out identifying after the transfection;
Step 3: the structure that has the carrier pIRES2-EGFP-hTERT of fluorescent protein expression gene and telomerase catalytic subunit hTERT cDNA;
Step 4:LT, hTERT cotransfection inoblast: get that the cell of transfection LT carries out the transfection of pIRES2-EGFP-hTERT, utilize fluorescent microscope to separate and obtain positive colony, and carry out enlarged culturing.
The process that described step 1 skin flbroblast of former generation is cultivated is; Use enzyme digestion and obtain the normal human skin inoblast, be typical fibrocyte sample growth after making cell attachment,, determine skin flbroblast with the histochemical stain of anti-waveform silk-protein Vimentin antibody mediated immunity.
Described step 2 with simian virus 40 large T antigen LT, the fibroblastic process of human telomerase catalytic subunit hTERT transfection is:
(1) will reach the skin flbroblast in the 16th generation, inoculation contains the plasmid of LT and hTERT, with enlarged culturing after the aminoglycosides drug screening;
(2) double digestion contains plasmid psv3-neo and the pCl-Neo-hTERT of LT and hTERT cDNA;
(3) enzyme is cut back gained fragment and is carried out digoxigenin labeled, with it as the thymus nucleic acid dna probe;
(4) get the good probe of mark to transfection after the 3rd generation and the 16th generation cell carry out in situ hybridization and detect, dissecting microscope is observed the expression of LT, hTERT messenger RNA(mRNA) mRNA down.
The process of the carrier pIRES2-EGFP-hTERT that fluorescent protein expression gene and telomerase catalytic subunit hTERTcDNA are arranged that described step 3 makes up is:
(1) pIRES2-EGFP plasmid and PCL-Neo-hTERT plasmid carry out double digestion with restriction endonuclease simultaneously;
(2) the product electrophoresis on sepharose after above-mentioned enzyme is cut reclaims test kit with DNA and reclaims;
(3) the dna segment water-bath connects;
(4) get after the competence bacterium melts, add and connect product, transform;
(5) carry out plasmid after clone's enlarged culturing and extract, the plasmid of extraction carries out double digestion and single endonuclease digestion, carries out agarose gel electrophoresis, and the analytical electrophoresis band is determined the carrier that successfully constructs.
Described step 4 gets that the cell of transfection LT carries out the transfection of pIRES2-EGFP-hTERT, realizes the cotransfection of LT and hTERT cDNA, and process is:
(1) with time be transverse axis, cell multiplication number of times (being PD) is drawn cell growth curve for the longitudinal axis;
(2) take limiting dilution assay, obtain the individual cells clone;
(3) karyotyping: get the transformant that is in logarithmic phase after transfection is also screened, carry out karyotyping and detect;
(4) getting cell after the transfection, to be injected into nude mice subcutaneous, carries out tumorigenicity and detect;
(5) utilizing ias to carry out the collagen secretion ability detects.
Description of drawings
Fig. 1: with time is the cell growth curve that transverse axis, cell multiplication number of times (being PD) are drawn for the longitudinal axis.
Fig. 2: the transformant karyotyping legend that is in logarithmic phase after transfection and the screening.
Fig. 3: three kinds of transfectional cell collagen secretion ability comparison diagrams.
Above-mentioned accompanying drawing can be done further understanding to the present invention in conjunction with following specific embodiment.
Concrete real mode
1. in former generation,, the cultivation detailed process of skin flbroblast was:
Use enzyme digestion and obtain the normal human skin inoblast.Be typical fibrocyte sample growth behind the cell attachment.Anti-waveform silk-protein (being Vimentin) antibody mediated immunity histochemical stain, the result shows that cytoplasm is pale brown look, and karyon is not painted, and expression is positive.Non-coloring in the cellular control unit, all negative, determine that this cell is a skin flbroblast.
Respectively with LT, hTERT transfection inoblast and the authentication method after carrying out transfection be:
1) will reach the skin flbroblast in the 16th generation, and be inoculated into six orifice plates, every porocyte number is 2 * 10
5, carry out transfection next day.Before the transfection nutrient solution is changed to the DMEM nutrient solution of serum-free.Get 50 μ l serum-free DMEM nutrient solutions, grouping adds the plasmid pCl-Neo-hTERT that 0.8-1.0 μ g contains the plasmid psv3-neo of LT cDNA and contains hTERT cDNA, draw 50 μ lDMEM nutrient solutions again, add liposome 1-3 μ l, after the incubated at room 5 minutes. both are mixed, continue to hatch after 20 minutes and directly mixed solution is added nutrient solution, shake up gently.Transfectional cell is put into 37 ℃ of CO
2Cultivate in the incubator.Be changed to normal nutrient solution after the transfection in 6 hours, cell goes down to posterity by 1: 10 density when converging, and continues cultivation. and treat density near 50%～70% o'clock, the G418 that adds 0.4g/L screens, and non-transfected cells is in contrast.Resistance clone to be occurred, filter paper method isolated cell clone, renewed vaccination is cultivated.Get respectively after the transfection the 3rd generation and the 16th generation cell detect.
2) EcoRI/PvuII double digestion plasmid psv3-neo, EcoRI/SalI double digestion plasmid pCl-Neo-hTERT.
Endonuclease reaction liquid is:
Thymus nucleic acid DNA 3 μ g
The enzyme cutting buffering liquid 2 μ l of 10 times of concentration
Restriction endonuclease I 1 μ l
Restriction endonuclease II 1 μ l
Make cumulative volume reach 20 μ l with the deionization distilled water
After above-mentioned enzyme cut after product and carry out the 10g/L agarose gel electrophoresis, isolated fragment reclaimed the test kit specification sheets according to DNA and carries out segment and reclaim, and spectrophotometry reclaims the content of back DNA.
3) thermally denature 10 minutes in boiling water of the DNA after sucking-off (1-3 μ g) is reclaimed is put into the quenching of sodium-chlor ice, reclaim the test kit specification sheets according to DNA and carry out pulsating digoxigenin labeled, with it as dna probe.
4) it is centrifugal to take out the good probe of mark, extrapolates behind the mark probe amount on may mark, and with the hybridization solution dilution, making its concentration is 1-2 μ g/ml.Get respectively after the transfection the 3rd generation and the 16th generation cell climbing sheet, drip probe, carry out in situ hybridization and detect, observe the expression (normal cell is as negative control) of LT, hTERT mRNA.
5) get respectively the 3rd generation after the transfection and the 16th generation the cell climbing sheet routine immunization histological chemistry that carries out anti-LT or anti-hTERT antibody detect (painted replace with PBS simultaneously one anti-) as negative control.
3. the building process that has fluorescent protein expression gene and hTERT cDNA carrier pIRES2-EGFP-hTERT is:
1) pIRES2-EGFP plasmid (containing the fluorescent protein expression gene) and PCL-Neo-hTERT plasmid carry out double digestion with EcoRI, SalI restriction endonuclease simultaneously.
Endonuclease reaction liquid is:
DNA 3μg
The enzyme cutting buffering liquid 2 μ l of 10 times of concentration
Restriction endonuclease I 1 μ l
Restriction endonuclease II 1 μ l
Make cumulative volume reach 20 μ l with the deionization distilled water
37 ℃ of enzymes were cut 3 hours.
2) the product electrophoresis on 1% sepharose after above-mentioned enzyme is cut makes carrier fully separate with exogenous genetic fragment; Downcutting target gene fragment and the length that the length that contains hTERT is 3.45kb respectively is to reclaim the carrier segments adhesive tape of 5.3kb test kit (Shanghai Hua Shun) with DNA and reclaim.
3) dna fragmentation connects, and its reaction solution is:
Target gene fragment 0.3 μ g
Carrier DNA 0.1 μ g
The connection damping fluid 2 μ l of 10 times of concentration
T
4Dna ligase 1 μ l
Making cumulative volume with the deionization distilled water is 20 μ l
16 ℃ water-bath 12-16 hour.
4) get after DH5 α competence bacterium melts, add and connect the product mixing, placed on ice 30 minutes, 42 ℃ of shocks 90 seconds; Cooled on ice 1-2 minute; The LB nutrient solution that adds 450 μ l preheatings, 37 ℃ of slow shaking culture 1 hour; Culture gone on the agar plate that contains kantlex smoothen; Treat that liquid is absorbed the back fully and is inverted plate, cultivated 12-16 hour for 37 ℃.
5) from agar plate picking list colony inoculation with contain in the nutrient solution of kantlex, 37 ℃ of shaking culture are spent the night.Carry out plasmid and extract, the plasmid of extraction carries out EcoRI, SalI double digestion and SalI single endonuclease digestion, 1% agarose gel electrophoresis, and the analytical electrophoresis band is determined the carrier that successfully constructs.
4.LT, hTERT cotransfection inoblast detailed process is:
Get that the cell of transfection LT carries out the transfection (method is the same) of pIRES2-EGFP-hTERT, utilize fluorescent microscope to separate and obtain positive colony, and carry out enlarged culturing.
5. the concrete grammar of the comparison of three kinds of transfectional cell biological behaviours is:
1) with time be transverse axis, cell multiplication number of times (being PD) is drawn the cell growth curve (see figure 1) for the longitudinal axis.Normal cell begins to occur propagation slowly near 60PD the time as can be seen, after this in considerable time, is in this slow vegetative state always; The cell of transfection LT increased rapidly in initial 1-2 month, and its rate of propagation is the fastest in all cells, but near 70PD the time, speed slows down rapidly, is in not vegetative state, and cell quantity does not only increase but also reduces gradually, and is to the last dead; Though the cell of transfection hTERT keeps being similar to Normocellular multiplication rate in for some time, compare with normal group, prolong cells in vitro significantly and cultivated the life-span, finally can not escape old and feeble generation; Have only LT-hTERT group cell to be in vigorous vegetative state, total algebraically of cell surpassed for 50 generations so far always.
2) collect cell to be checked, (every part of cell quantity is 5 * 10 to 4 ℃ of fixed test of 75% ethanol
5～1 * 10
6).It is as follows that flow cytometer detects the cell cycle result:
Group G
1 S G
2+M PrI(S+G
2+M)
P3 N 75 10.8 14.2 25.0
P32?N 80.8 11.8 7.4 19.2
P3 LT 69.9 15.8 14.3 30.1
P16?LT 91.8 5.8 2.4 8.2
P3 hTERT 74.3 20.8 4.9 25.7
P30?hTERT 80.2 18.2 1.5 19.7
P30?LT-hTERT 74.8 15.5 9.7 25.2
In the table, P3 N, P32 N be the 3rd, 32 generation normal cell; P3 LT, P16 LT be behind the transfection LT the 3rd, 16 generation cell; P3 hTERT, P30 hTERT be behind the transfection hTERT the 3rd, 30 generation cell; P30 LT-hTER for dye altogether behind LT, the hTERT the 30th generation cell.G in the table
1, S, G
2, M is the different steps of cell growth cycle.
By in the table as seen, the cell proliferation index maximum of P3 LT group, and the proliferation index that P16 LT organizes becomes minimum, it is descending therebetween that be arranged in order is P3 hTERT, P30 LT-hTERT and P30 hTERT.This is consistent with the trend that growth curve is reflected.
3) transformant that is in logarithmic phase after transfection is also screened is got in karyotyping, and in detecting had digestive transfer culture the day before yesterday, next day, cell reached 60-80%, carried out karyotyping, and the result shows that caryogram all belongs to normally.See Fig. 2.
4) tumorigenicity detects: the transformant in the vegetative period of taking the logarithm, DMEM is prepared into single cell suspension with serum-free.Select 86 nude mices about week, an injection point is respectively selected in both sides, with 10
7The cell of/ml density, it is subcutaneous to be expelled to nude mice.Tongue cancer clone T8113 organizes in contrast.The cell kind is gone into nude mice after subcutaneous February, observes, and find that control cells group nude mice back grows 2 * 2cm size tumor, and LT organizes, hTERT organizes, LT-hTERT there is no tumour and occurs.
5) the collagen secretion ability detects: the transformant digestion counting in the vegetative period of taking the logarithm, adjusting cell suspension are done cell creep plate (normal skin fibroblast in contrast) to same density.4% Paraformaldehyde 96 is fixed after 12 hours, the PBS flushing.Cell is through 0.3%Triton X-100/PBS, H
2O
2/ PBS is divided into two groups respectively with LT, hTERT, LT-hTERT and normal cell after handling, and every group contains 10 samples.First group drips the anti-I type collagen antibodies, and second group drips anti-III Collagen Type VI antibody, and 4 ℃ are spent the night; 37 ℃ of rewarmings 1 hour dripped biotinylation two anti-37 ℃ of incubations 1 hour (between above-mentioned each step all through the abundant rinsing of PBS damping fluid); Colour developing liquid (final concentration is 0.5g/L DAB) 0.05mol/L Tris-Hcl damping fluid (PH7.6) and DAB and fresh H
2O
2Preparation develops the color.(painted anti-with PBS replacement one simultaneously) as negative control.Sample applies image analysis system after the dyeing does the albumen gray-scale value and measures.To record numerical value and carry out statistical analysis, there was no significant difference P＞0.05 between four groups of samples.Illustrate that its collagen secretion of cell after the transfection does not have the significance difference with normal cell, sees Fig. 3.
Adopt in this experiment safer liposome as medium in conjunction with the plasmid that contains goal gene, at SV
40T antigen and the independent transfection inoblast of hTERT cDNA poor effect, the very difficult situation that obtains passage cell steady in a long-term adopts SV in this experiment
40The method of T antigen and hTERT cDNA co-transfection.And human SV arranged
40T and hTERT unite and are used for the breast glandular epithelium, and have experiment to prove, and the effect of The combined transfection is far superior to the effect of independent transfection, has confirmed this method and feasibility indirectly.For fear of two kinds have the false positive results that occurs behind the plasmid transfection of identical screening-gene (if a cell transfection SV
40T or only transfection hTERT, all can have resistance and survive), we join the hTERT cDNA of original plasmid on the new carrier that has the fluorescent protein expression gene to, have set up diverse screening system, reach SV
40T and the fibroblastic purpose of hTERT cDNA co-transfection.
The clone that the inventive method is set up can go down to posterity for a long time external, compare with the skin flbroblast of independent transfection LT cDNA or hTERT cDNA, significant prolongation the vitro culture life-span of skin flbroblast, does not have the old and feeble appearance that characterizes so far.The protein excretion function of transformant and normal cell no significant difference, the similar normal cell of its caryogram and secreting function.Compare with other method, the cell that this method obtains has the biological character approaching with normal cell, normal, the no tumorigenicity of caryogram, can be at external normal synthesis secretion type i collagen.The cell that present method obtains not only helps further to study the mechanism of cell immortality generation, and can become the efficient ways that solves seed cell source in the organizational project.
Claims (5)
1, a kind of method that makes the skin flbroblast immortalization, it is characterized in that: with complementary DNA (cDNA) cDNA and the cDNA that has the human telomerase catalytic subunit hTERT of fluorescent protein expression gene of simian virus 40 large T antigen LT, common transfection skin flbroblast, to set up immortalized cell line, may further comprise the steps:
Step 1: cultivate former generation skin flbroblast;
Step 2: use simian virus 40 large T antigen LT, human telomerase catalytic subunit hTERT transfection inoblast respectively, and carry out identifying after the transfection;
Step 3: the structure that has the carrier pIRES2-EGFP-hTERT of fluorescent protein expression gene and telomerase catalytic subunit hTERT cDNA;
Step 4:LT, hTERT cotransfection inoblast: get that the cell of transfection LT carries out the transfection of pIRES2-EGFP-hTERT, utilize fluorescent microscope to separate and obtain positive colony, and carry out enlarged culturing.
2, method according to claim 1, it is characterized in that: the process that described step 1 skin flbroblast of former generation is cultivated is: use enzyme digestion and obtain the normal human skin inoblast, be typical fibrocyte sample growth after making cell attachment, with the histochemical stain of anti-waveform silk-protein Vimentin antibody mediated immunity, determine skin flbroblast.
3, method according to claim 1 is characterized in that: described step 2 with simian virus 40 large T antigen LT, the fibroblastic process of human telomerase catalytic subunit hTERT transfection is:
(1) will reach the skin flbroblast in the 16th generation, inoculation contains the plasmid of LT and hTERT, with enlarged culturing after the aminoglycosides drug screening;
(2) double digestion contains plasmid psv3-neo and the pCl-Neo-hTERT of LT and hTERT cDNA;
(3) enzyme is cut back gained fragment and is carried out digoxigenin labeled, with it as the thymus nucleic acid dna probe;
(4) get the good probe of mark to transfection after the 3rd generation and the 16th generation cell carry out in situ hybridization and detect, dissecting microscope is observed the expression of LT, hTERT messenger RNA(mRNA) mRNA down.
4, method according to claim 1 is characterized in that: the process of the carrier pIRES2-EGFP-hTERT that fluorescent protein expression gene and telomerase catalytic subunit hTERT cDNA are arranged that described step 3 makes up is:
(1) pIRES2-EGFP plasmid and PCL-Neo-hTERT plasmid are carried out double digestion with restriction endonuclease simultaneously;
(2) the product electrophoresis on sepharose after above-mentioned enzyme is cut reclaims test kit with DNA and reclaims;
(3) the dna segment water-bath is connected;
(4) get after the competence bacterium melts, add and connect product.Transform;
(5) carry out plasmid after clone's enlarged culturing and extract, the plasmid of extraction carries out double digestion and single endonuclease digestion, carries out agarose gel electrophoresis, and the analytical electrophoresis band is determined the carrier that successfully constructs.
5, method according to claim 1 is characterized in that: the method for the inoblast of acquired LT of described step 4 and hTERT cDNA cotransfection being carried out biological assay has:
(1) with time be transverse axis, cell multiplication number of times is that the longitudinal axis is drawn cell growth curve;
(2) karyotyping: get the transformant that is in logarithmic phase after transfection is also screened, carry out karyotyping and detect;
(3) getting cell after the transfection, to be injected into nude mice subcutaneous, carries out tumorigenicity and detect;
(4) utilizing ias to carry out the collagen secretion ability detects.
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| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
| CN103282498A (en) * | 2010-11-02 | 2013-09-04 | 亥姆霍兹感染研究中心有限责任公司 | Methods and vectors for cell immortalisation |
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| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
| CN103282498A (en) * | 2010-11-02 | 2013-09-04 | 亥姆霍兹感染研究中心有限责任公司 | Methods and vectors for cell immortalisation |
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