CN105176913A - Dairy cattle mammary epithelial cell immortalizing method - Google Patents
Dairy cattle mammary epithelial cell immortalizing method Download PDFInfo
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- CN105176913A CN105176913A CN201510611756.4A CN201510611756A CN105176913A CN 105176913 A CN105176913 A CN 105176913A CN 201510611756 A CN201510611756 A CN 201510611756A CN 105176913 A CN105176913 A CN 105176913A
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Abstract
The invention provides a dairy cattle mammary epithelial cell immortalizing method. The dairy cattle mammary epithelial cell immortalizing method is characterized in that when primary generation dairy cattle mammary epithelial cells are infected through HPV E6 and E7 viruses to be immortalized, pantropic and facultative virus particles are added, and thus the virus infection efficiency is improved. The invention further provides a method for establishing an immortalized dairy cattle mammary epithelial cell line. The method for establishing the immortalized dairy cattle mammary epithelial cell line comprises the following steps that firstly, dairy cattle mammary tissue is subjected to tissue block primary generation culture in a culture bottle; secondly, the primary generation dairy cattle mammary epithelial cells are infected through the virus method, and the primary generation dairy cattle mammary epithelial cells are immortalized; thirdly, positive clone is selected, and the cell line is cultured. By means of the methods, the dairy cattle mammary epithelial cells can be efficiently immortalized, the dairy cattle mammary epithelial cell line high in growth speed and capable of achieving stable continuous passage is established, the dairy cattle mammary epithelial cells with the physiological function are obtained, and experimental cell materials are provided for researching control over lactation nutrition of the dairy cattle mammary epithelial cells and the pathogenesis of diary cattle mastitis.
Description
Technical field
The invention belongs to biological technical field, relate to that cell primary is cultivated, monoclonal cell obtains, westernblot detects E6, E7 virus in the expression of cow mammary gland epithelial cells with set up the method for cow mammary gland epithelial cells system.
Background technology
Cow mammary gland epithelial cells (BMEC, Bovinemammaryepithelialcell) has the function of synthesis and secretion milk-protein, has defense reaction to microorganism invasion mammary tissue.Meanwhile, it is the important reacting cells of mammary tissue.Therefore, the original cuiture and the cellular immortalization that carry out cow mammary gland epithelial cells are regarded as Lactation of Dairy Cow nutrition regulation and mammitis of cow study of incident mechanism important cells model.
Substantially enzyme digestion separation and Culture original cuiture cow mammary gland epithelial cells (WangMZ is adopted both at home and abroad, XuBL, WangHR, etal., " Effectsofarginineconcentrationontheinvitroexpressionofca seinandmTORpathwayrelatedgenesinmammaryepithelialcellsfr omdairycattle ", PLosOne, 2014 (5): e95985), this method can obtain primary cow mammary gland epithelial cells and inoblast is less fast.But the cow mammary gland epithelial cells of acquisition is owing to digesting for a long time through high-concentration enzyme, and the receptor protein of cytolemma is subject to serious destruction, causes a series of signal transduction pathway to be obstructed, and cannot realize the physiological function of cow mammary gland epithelial cells well.
Cultivation correlation test (the Chen Jianhui of the cow mammary gland epithelial cells carried out at home, Tong Huili, Li Qingzhang etc., " foundation of cow mammary gland epithelial cells system " 2009,40 (5): 743-747) in, these cells can only carry out limited Secondary Culture, can not well preserve cow mammary gland epithelial cells system.By comparison, external cow mammary gland epithelial cells system passage number is more.But utilizing cow mammary gland epithelial cells system to carry out in succeeding generations, along with the increase of passage number, cow mammary gland epithelial cells system will lose mammary tissue physiological function, as: chromosomal karyotypic alteration, presents monoploid or polyploid.Numerical chromosome change all will cause the error of testing data, even make some cow mammary gland epithelial cells system can not express milk-protein at all, so just lose and utilize cow mammary gland epithelial cells to study the meaning of lactation nutrition regulation and lactation mechanism thereof.Therefore, functional immortalization cow mammary gland epithelial cells system is set up extremely urgent.
Virus method is one of method making cellular immortalization, can be effectively incorporated in the genome of host cell by target gene.Compare with electroporation transgenic method with traditional transfection, this method is not poisoned cell, efficiency of infection is high and can stably express.Make the virogene of cellular immortalization more at present, e.g., the virogenes such as hTERT, SV40, HPV16E6E7.Wherein, HPVE6 and E7 virogene is the product coming from the expression of women's cervical cancer cell, human epithelial cell's immortalization (TsaoSA can be made, WangXH, LiuY, " Establishmentoftwoimmortalizednasopharyngealepithelialce lllinesusingSV40largeTandHPV16E6/E7viraloncogenes " .BiochimicaetBiophysicaActa, 2002 (1590): 150-158).HPVE6 and E7 virogene makes two signal of interest path pRb-p16INK4a and the p53 inactivation of human epithelial cell immortalization respectively, thus makes cell walk around M1 and the M2 phase respectively, finally makes cell have unlimited value-added ability.But, there is no the report that HPVE6 and E7 can make cow mammary gland epithelial cells immortalization at present.Thus, above-mentioned HE immortalization method is applied to cow mammary gland epithelial cells there is some difficult point following and doubtful point: 1) when utilizing viral method goal gene transduction to be entered in cow mammary gland epithelial cells, whether the envelope glycoprotein Vsvg on viral backbone can in conjunction with cow mammary gland epithelial cells lipid, although at present in research, Vsvg albumen can in conjunction with the lipid on most people or mouse epithelial cells film, can in conjunction with the report of the lipid on cow mammary gland epithelial cells film but there is not yet it; 2) titre due to virus is low, adds the feature of primary cow mammary gland epithelial cells " cannot sharp separation cause cell proliferation slow ", cells infected efficiency can be caused low.
Summary of the invention
For above-mentioned difficult point of the prior art and doubtful point, the invention provides a kind of HPVE6 and E7 virogene that utilizes expeditiously and the method for immortalization is carried out to cow mammary gland epithelial cells, and a kind of method setting up the cow mammary gland epithelial cells system that fast growth and steady and continuous go down to posterity, thus obtain the cow mammary gland epithelial cells with physiological function, for the lactation nutrition regulation of cow mammary gland epithelial cells and the study of incident mechanism of mammitis of cow provide experimental cell material.
To achieve these goals, technical scheme of the present invention is as follows:
The invention provides a kind of cow mammary gland epithelial cells immortalization method, it is characterized in that, when adopting the primary cow mammary gland epithelial cells of HPVE6 and E7 virus infection to make its immortalization, adding the virion of pantropic and amphophilic.Compared with increasing the virion of single preferendum, like this can transmembrane molecule more with cow mammary gland epithelial cells surface bonding, thus increase virus infection efficiency.
Wherein, adopt the following primary cow mammary gland epithelial cells of viral method immortalization, comprise the following steps:
1) cow mammary gland epithelial cells is inoculated;
2) when Growth of Cells is approximately 50%-70% to density, abandon supernatant liquor, clean cell with PBS;
3) HPV16E6 and the E7 virus liquid of pantropic and amphophilic is added step 2) in cow mammary gland epithelial cells in;
4) by step 3) in obtain mixed solution centrifugal, leave and take supernatant liquor;
5) by step 4) in obtain supernatant liquor place 37 DEG C, 5%CO
224h is hatched in incubator;
6) step 5 is abandoned in suction) the middle supernatant liquor obtaining mixed solution, add DMEM/F12 substratum and continue to cultivate;
7) when cell density converges to 70%-80%, antibiotic-screening positive colony is used.
Alternatively, can in step 3) in pantropic and amphophilic HPV16E6 and E7 virus liquid in add polybrene produce electrical charge rejection between virus and cell after eliminating mixing, make virus can exposing cell film surface better, increase efficiency of infection.
Alternatively, can in step 4) in add the stimulating factors such as Urogastron, Insulin-Transferrin-selenium unit, prolactin and hydrocortisone in the supernatant liquor that obtains, promote that primary cow mammary gland epithelial cells can enter the division stage of cell fast.Owing to being in the cell of division stage more easily by virus infection, thus the final object realizing raising viral transgene efficiency.
Present invention also offers a kind of method setting up immortalization cow mammary gland epithelial cells system, it is characterized in that, comprise the following steps:
(1), after the cow mammary gland collected is organized shredding in the medium, its supernatant liquor is seeded in culturing bottle and carries out tissue block original cuiture.Compared with enzyme digestion conventional at present, such cultured cells has stronger cell viability, and rate of propagation is fast, is the Primary breast cellular processes that a kind of acquisition is similar in Contents in Cows;
(2) adopt the viral method infector described above the present invention for cow mammary gland epithelial cells, to make cow mammary gland epithelial cells immortalization;
(3) screening positive clone, makes cell be consistent on form, heredity, chromosome stability and biological function, carries out the cultivation of clone.
Preferably, in step (2), just carry out virus infection when primary cow mammary gland epithelial cells density reaches 50%.Compared with reaching 70% situation of just carrying out infecting with the mammary epithelial cell of people, carry out virus infection when cell density reaches 50%, more positive colony can be produced, create conditions for next step successfully carries out monoclonal cell.
Technical scheme of the present invention achieves following beneficial effect:
(1) in virus infection cow mammary gland epithelial cells process, because primary cell increment is slow, and can not divide fast, cell is in quiescent stage, and this makes virus be difficult to cells infected.In cow mammary gland epithelial cells immortalization method provided by the invention, use pantropic and amphophilic virion to infect cow mammary gland epithelial cells simultaneously.Because this two-strain particle can the different transmembrane molecule in cells infected film surface, and then the envelope glycoprotein increasing pantropic and amphophilic virion is in conjunction with the lipid acceptor of surface of cell membrane and GALV and RAM1 acceptor, thus improve virus infection efficiency.Meanwhile, in the inventive solutions, also can add Urogastron, Insulin-Transferrin-selenium unit, prolactin and hydrocortisone in the supernatant liquor of virus, thus enable primary cow mammary gland epithelial cells enter the division stage of cell fast.Owing to being in the cell of division stage more easily by virus infection, thus reach the object increasing transgene efficiency.
(2) method setting up immortalization cow mammary gland epithelial cells system provided by the invention adopts the mode of tissue block original cuiture, and make the cell cultivating out have stronger cell viability, rate of propagation is fast.In addition, due in the process of cultivating at this without the need to adding cell growth factor, cost savings cost.
(6) adopt the immortalization cow mammary gland epithelial cells system of method establishment of the present invention permanently to preserve, avoid the trouble that multi collect mammary tissue is brought, can be used as commercial clone.
Accompanying drawing explanation
Fig. 1 is the cellular form electron microscope picture (amplifying 100 times) of cow mammary gland epithelial cells.Wherein, (a) is the cow mammary gland epithelial cells by HPV16E6 and E7 immortalization; B () is the cow mammary gland epithelial cells in the 15th generation not proceeding to HPV16E6 and E7.
Fig. 2 is cow mammary gland epithelial cells Keratin 18 immunofluorescence microscopy photo (amplifying 200 times).
Fig. 3 is cow mammary gland epithelial cells chromosome karyotype analysis.
Fig. 4 is the Westernblot result detecting the expression of HPV16E6 and HPV16E7 viral protein in cow mammary gland epithelial cells.Wherein, BMEC: cow mammary gland epithelial cells; Caski: women's cervical cancer cell; BMEC-E6E7: the cow mammary gland epithelial cells being transfected into HPV16E6 and E7; β-actin: beta-actin.
Embodiment
In order to illustrate technical scheme of the present invention and technical purpose, below in conjunction with embodiment, the present invention is described further.
Adopt viral method infector for cow mammary gland epithelial cells, HPV16E6 and E7 virogene is imported in cow mammary gland epithelial cells: 1) inoculate 1 × 10
5individual cow mammary gland epithelial cells in 6 orifice plates, 2) when Growth of Cells is approximately 50% to density, abandon supernatant liquor, clean cell 3 times with the PBS of 2ml; 3) the pantropic virus liquid of 1ml and 1ml amphophilic virus liquid are added containing in cow mammary gland epithelial cells 6 orifice plate; 4) add 6 μ g/ml polybrenes, to eliminate the charge differences between virus and cell, enable virus exposing cell film surface better, increase efficiency of infection.Although polybrene is poisonous to some cell, also little to cow mammary gland epithelial cells toxicity, swing 6 orifice plates, mixes; 5) six orifice plates are put into whizzer, 36 DEG C of centrifugal 90min; 6) adding final concentration in vial supernatant is after centrifugation the raw sub-factor, 1 μ g/mL hydrocortisone and the 4ug/ml prolactin (Sigma) of 20ng/ml epidermis and 1 times of Insulin-Transferrin-selenium (Invitrigen); 7) 6 orifice plates are placed 37 DEG C, 5%Co
224h is hatched in incubator; 8) after infection is spent the night, supernatant liquor is inhaled and abandons, add DMEM/F12 substratum and continue to cultivate; 9) when cell density converges to 70%, with the G418 antibiotic-screening positive colony of 300 ~ 500 μ g/ml, step sizing 15 days.Carry out changing liquid according to the colour-change of substratum, generally changed liquid every 5 days.Within 1 ~ 3 day, cow mammary gland epithelial cells is dead not obvious under the effect of G418 microbiotic, after the 4th day, starts slowly dead at G418 microbiotic effect cow mammary gland epithelial cells.10th day, the cow mammary gland epithelial cells not importing virogene was all dead.But, also have some cow mammary gland epithelial cells to start propagation and speed in culturing bottle, occur cell colony.
When setting up the cow mammary gland epithelial cells system of immortalization, specifically comprise the steps:
(1) cow mammary gland tissue is put into the DMEM substratum containing about 4 ~ 5 times of penicillin and Streptomycin sulphate.
(2) mammary tissue is shredded, repeatedly clean, until supernatant liquor is limpid, is evenly seeded in culturing bottle, puts into 5%CO
2, cultivate 10 days in 37 DEG C of incubators.Struck off by cell, differ digestion, difference adherent method carrys out purifying cow mammary gland epithelial cells, in proliferation process, some cells senesce.
(3) adopt the viral method described in above-described embodiment 1, use HPV16 type E6 and the primary cow mammary gland epithelial cells of E7 virus infection, make this cellular immortalization.
(4) treat step 3) in cell density after immortalization converge to 80%, with the pancreatin separating digesting cow mammary gland epithelial cells group of 1ml, 2ml substratum stops digestion, piping and druming suspension cell, cell counting.The ultimate density of cell is made to be 5 ~ 10/ml by serial dilution.Draw 200 μ l cell suspension inoculations in 96 orifice plates, make every hole only have 1 cell, altogether inoculate 5 96 orifice plates.After cultivating one week, can there is clone in each 96 orifice plates.Change liquid, continue cultivation one week.When hole inner cell density converges to 50%, trysinization separation of milk bovine mammary epithelial cell, transfers to 25cm
2enlarged culturing also further Secondary Culture in culturing bottle.
Growth rhythm research and morphocytology qualification to the immortalization cow mammary gland epithelial cells cultivated:
In the research carrying out Growth of Cells rule, with 1 × 10
4individual immortalized cells/cm
2inoculate cow mammary gland epithelial cells, after 3 days, the density of cow mammary gland epithelial cells is 60 ~ 80%.Be adapted at 6 orifice plates and 25cm
2, 75cm
2cultivate Deng in culturing bottle, after cultivating 3 days, cell density remains 70%, and this result describes cell in process of growth, meets general cell growth curve rule.
When carrying out cell morphology characteristic and observing, the cow mammary gland epithelial cells form through viral infectious process immortalization of the present invention presents island shape colony growth, and some presents paving stone growth.The cow mammary gland epithelial cells form of immortalization (Fig. 1 is a) similar with the cow mammary gland epithelial cells form of the non-immortalization in front 10 generations, but some difference of cow mammary gland epithelial cells form of non-immortalization after itself and 15 generations.The cell of the non-immortalization after 15 generations senesces, and the tenuigenin (cell volume) increased therefrom can obviously recognize in Figure 1b.
The qualification of cow mammary gland epithelial cells Keratin 18:
(1) by 1 × 10
4individual/cm
2cell density is seeded in 4 hole ChamberSlide;
(2) treat that cell density is that 60 ~ 80%, PBS washes 3 times.With the fixing about 30min of 4%PFA, then PBS washes 3 times;
(3) 3% serum room temperatures close 20 ~ 30min, then remove serum;
(4) add CK18 primary antibodie (1:200 dilution), 4 DEG C of overnight incubation, PBS washes 3 times;
(5) add the anti-room temperature lucifuge of fluorescein two and hatch 1h, PBS washes 3 times;
(6) core carries out DAPI dyeing, room temperature 7min;
(7) PBS washes 3 times, then distills washing 1 time;
(8) mounting medium mounting, lucifuge 24 ~ 48h;
(9) expression of fluorescence microscope CK18 in cow mammary gland epithelial cells film (Fig. 2): in figure, cellular form presents paving stone and squamous etc., and it is epitheliated type that this result describes monoclonal cell.
Cow mammary gland epithelial cells karyotyping:
(1) add colchicine: the cow mammary gland epithelial cells in vegetative period of taking the logarithm, add colchicine in nutrient solution, final concentration is 0.05 ~ 0.1 μ g/ml.Cultivation 4 ~ 5h is continued in 37 DEG C of incubators;
(2) enzymic digestion isolated cell, proceeds to 15ml centrifuge tube, room temperature 1000rpm/min, centrifugal 10min, collecting cell;
(3) suck supernatant liquor, add 37 DEG C of preheatings, 0.075mol/LKCl solution 7ml, piping and druming evenly gently, incubation 18min in 38.5 DEG C of water-baths;
(4) add Fresh fixative (methyl alcohol: Glacial acetic acid=3:1) in suspension, beat (piping and druming prevents cell rupture gently); Room temperature 1000rpm/min immediately, centrifugal 10min, abandons supernatant;
(5) add Fresh fixative, blow and beat mixing gently, fixing 30min;
(6) room temperature 1000rpm/min, centrifugal 10min, abandons supernatant;
(7) (5) and (6) are repeated;
(8) add stationary liquid depending on cell concentration in centrifuge tube, piping and druming evenly;
(9) immediately take out lens wiping paper wipe clean after in the slide glass of precooling on ice.Above slide glass, about 1m eminence suction pipe drips rapidly 1 ~ 3 cell suspension, dispels gently, room temperature airing with mouth;
(10) Giemsa working fluid dyeing 20min, tap water room temperature is dried.Slowly rinse from slide one end (noting first not removing dye liquor or directly rinsing blood film) with tap water, microscopy after airing
(11) first find good split coil method with low power lens, re-use high power oil sem observation and take pictures (Fig. 3):
In figure 3, can observe cow mammary gland epithelial cells and still remain diploid karyotype, this result describes the cow mammary gland epithelial cells after adopting the inventive method immortalization and still has biological function and genetic stability.
Westernblot detects the expression of HPV16E6E7 viral protein in cow mammary gland epithelial cells:
(1) total protein of cell sample preparation: treat positive cow mammary gland epithelial cells raised growth, extracts cow mammary gland epithelial cells and extracts total protein of cell.Meanwhile, extract the total protein (positive control) of primary cow mammary gland epithelial cells total protein (negative control) and Caski cell, carry out Westernblot and verify whether E6 and E7 virogene expresses in cow mammary gland epithelial cells.Suction is abandoned substratum PBS and is cleaned 3 times, trysinization separation of milk bovine mammary epithelial cell, and 4 DEG C of centrifugal 5min of 1000rpm, make cell precipitation, abandon supernatant.Add RIPA lysate (RIPA: proteinase inhibitor=50:1) according to cell quantity, generally add the RIPA lysate of 200 μ l, put cracking 30min on ice, every 10min whirlpool 30s in vortex oscillator, 4 DEG C of centrifugal 10min of 12000g.Careful absorption supernatant, i.e. total protein of cell.Measure the protein concentration of each sample, add RIPA lysate and make the protein concentration between each group consistent with albumen sample-loading buffer.Boiling water bath sex change 10min, makes the abundant sex change of albumen.
(2) SDS-PAGE electrophoresis: install Bio-rad gum-making rack.According to the molecular size range of target protein, the required resolving gel concentration of configuration.By the separation gel of the proportional arrangement 12% of each component, mixing 10 times of turning upside down, makes it abundant reaction.Liquid glue is added between sheet glass, and with alcohol block, separation gel polymerization 40min under room temperature.Configure 5% concentrated glue again, the residual alcohol filter paper above separation gel is exhausted, add 5% concentrated glue and insert comb, forbid that between swimming lane, bubble produces, and is polymerized 40min under room temperature.Glue is put into electrophoresis chamber, carefully extracts comb.Sample and standard substance are added in sample well respectively.The leakage of electricity of 100V constant voltage is swum, until tetrabromophenol sulfonphthalein goes to forward position.
(3) transferring film: NC film and filter paper are cut into bigger fritter the same as glue size.NC film and filter paper are put into precooling transferring film damping fluid and are soaked 20min.Transferring film order is negative electrode carbon plate, sponge, filter paper, glue, film, filter paper, sponge, carbon anode plate.100V, 80min carry out transferring film in ice bath.
(4) close and hybridize: according to target protein and β-actin molecular size range, cut required NC film band with scissors, the confining liquid putting into 5% skim-milk closes 90min.Add β-actin, E6 and E7 primary antibodie (1:250 dilution), 4 DEG C are shaken overnight incubation slowly.TBST scavenging solution cleans 6 times, each 10min.Add two anti-(1:3000 dilution) room temperatures to shake slowly and hatch 90min.TBST scavenging solution cleans 6 times, each 10min.
(5) detection of specific band: the general horseradish peroxidase HRP-ECL luminescence method that uses carrys out testing goal albumen.A liquid and B liquid are pressed 1:1 mixing, the liquid on film is blotted with filter paper, add luminescent solution, be placed in gel imaging system exposure (Fig. 4): this result describes HPV16E6 and E7 gene and to be integrated on genome and stably express E6E7 albumen, and this has vital role to the immortalization of cow mammary gland epithelial cells.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and application claims protection domain is defined by appending claims, specification sheets and equivalent thereof.
Claims (7)
1. a cow mammary gland epithelial cells immortalization method, is characterized in that, comprises the following steps:
1) cow mammary gland epithelial cells is inoculated;
2) when Growth of Cells is approximately 50%-70% to density, abandon supernatant liquor, clean cell with PBS;
3) HPV16E6 and the E7 virus liquid of pantropic and amphophilic is added step 2) in cow mammary gland epithelial cells in;
4) by step 3) in obtain mixed solution centrifugal, leave and take supernatant liquor;
5) by step 4) in obtain supernatant liquor place 37 DEG C, 5%CO
224h is hatched in incubator;
6) step 5 is abandoned in suction) the middle supernatant liquor obtaining mixed solution, add DMEM/F12 substratum and continue to cultivate;
7) when cell density converges to 70%-80%, antibiotic-screening positive colony is used.
2. a kind of cow mammary gland epithelial cells immortalization method as claimed in claim 1, is characterized in that, in step 3) in pantropic and amphophilic HPV16E6 and E7 virus liquid in add polybrene.
3. a kind of cow mammary gland epithelial cells immortalization method as claimed in claim 2, is characterized in that, the concentration of described polybrene is 6 μ g/ml.
4. a kind of cow mammary gland epithelial cells immortalization method as described in claim 1 or 3, is characterized in that, in step 4) in obtain supernatant liquor in add stimulating factor.
5. a kind of cow mammary gland epithelial cells immortalization method as claimed in claim 4, is characterized in that, described stimulating factor is Urogastron, hydrocortisone, prolactin and Insulin-Transferrin-selenium.
6. a kind of cow mammary gland epithelial cells immortalization method as claimed in claim 5, it is characterized in that, the add-on of described stimulating factor is respectively: final concentration is the raw sub-factor, 1 μ g/mL hydrocortisone and the 4ug/ml prolactin of 20ng/ml epidermis and 1 times of Insulin-Transferrin-selenium.
7. set up a method for immortalization cow mammary gland epithelial cells system, it is characterized in that, comprise the following steps:
A), after the cow mammary gland collected is organized shredding in the medium, its supernatant liquor is seeded in culturing bottle and carries out tissue block original cuiture;
B) method immortalization method cow mammary gland epithelial cells according to claim 1 is adopted;
C) screening positive clone, carries out the cultivation of clone.
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| CN115011549A (en) * | 2022-07-12 | 2022-09-06 | 上海交通大学医学院附属仁济医院 | Gallbladder epithelial cell line establishing method, gallbladder epithelial cell line and application thereof |
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| CN115011549A (en) * | 2022-07-12 | 2022-09-06 | 上海交通大学医学院附属仁济医院 | Gallbladder epithelial cell line establishing method, gallbladder epithelial cell line and application thereof |
| CN115011549B (en) * | 2022-07-12 | 2023-08-22 | 上海交通大学医学院附属仁济医院 | Method for establishing gall bladder epithelial cells, gall bladder epithelial cell line and application thereof |
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Application publication date: 20151223 |