CN102174573A - Porcine trachea epithelial cell line and building method thereof - Google Patents
Porcine trachea epithelial cell line and building method thereof Download PDFInfo
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Abstract
The invention discloses a building method of a porcine trachea epithelial cell line. The method comprises the following steps of: (1) primary culture: sterilely acquiring newborn piglet tracheas, clearing adhesive tissues, performing digestion and separation by adopting a pronase perfusion cold digestion method to obtain high-purity trachea epithelial cells, suspending the separated primary trachea epithelial cells in a DF12 culture medium, and culturing the cells at the temperature of 37 DEG C under the condition of 5 percent CO2; (2) transfection and screening: extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, and guiding the eukaryotic expression plasmids pCI-neo-hTERT for coding hTERT and neo genes to the porcine trachea epithelial cell primarily cultured in the step (1) by adopting a liposome transfection method to perform transfection; and (3) screening and amplified culture. The method is easy in culture, the growth speed is high, and the operating method is simple and convenient.
Description
Technical field
The present invention relates to biological field of animal cell lines, be specifically related to pig tracheal epithelial cell system and establishment method thereof.
Background technology
Tracheal epithelium is positioned at tracheae tube chamber internal surface, belongs to pseudostratified ciliated columnar epithelium, is the first road barrier that body externally defends objectionable impurities to invade and harass.It is formed, and cell---tracheal epithelial cell is except that having mechanical defense reaction, can also synthesize and secrete multiple mucus composition and cytokine, be used to regulate the growth of airway epithelia cell, keep the tracheal epithelium integrity, moistening and purify the infringement of respiratory tract, the multiple pathogenic bacteria of opposing to tracheal epithelium, in addition, tracheal epithelial cell also has the reaction of the air flue of participation mucosa-immune, vital role such as bronchitis are eliminated in help.
At present, still the research of not having relevant pig tracheal epithelial cell clone establishment method occurs, and the pig vascular endothelial cell of the immortalization that obtains with existing clone establishment method, because it is less to obtain the mono-clonal positive cell in screening process, the amount of cell growth also seldom, and propagation is slowly, nutritional condition is had relatively high expectations, the production cost height is unfavorable for promoting.
Summary of the invention
Technical problem to be solved by this invention provides a kind of being easy to and cultivates, and the speed of growth is very fast, pig tracheal epithelial cell system and establishment method thereof that working method is easy.
The present invention adopts following technical scheme:
The establishment method of one boar tracheal epithelial cell system may further comprise the steps:
(1) former be commissioned to train foster: the aseptic newborn piglet tracheae that obtains, remove adhesion organization, adopt pronase to pour into cold digestion method digestion and separate and obtain highly purified tracheal epithelial cell; Former generation tracheal epithelial cell that separation is obtained is suspended in the DF12 substratum, places 37 ℃, 5%CO
2Cultivate under the condition;
(2) transfection and screening: extract the eukaryon expression plasmid pCI-neo-hTERT that purifying contains the hTERT gene, adopt the liposome transfection method, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported the former pig tracheal epithelial cell of being commissioned to train foster of step (1) carry out transfection;
(3) screening and enlarged culturing.
Described method, described step (2) is with the former pig tracheal epithelial cell of being commissioned to train foster of pCI-neo-hTERT plasmid transfection, and its concrete steps are as follows:
(1) 24h before the transfection is digested to individual cells with STECs with sucrose EDTA and 0.25% pancreatin, step by step with 1 * 10
5Individual cells/well is inoculated in 12 orifice plates, makes cell can reach 80%～90% fusion the same day in transfection;
(2) DMEM/F12 of 4h replacing serum-free before the transfection;
(3) diluting 5 μ LpCI-neo-hTERT plasmid to volumes with the DMEM/F12 of serum-free is 50 μ L, mixing gently, and plasmid concentration reaches 300ng/ μ L～400ng/ μ L room temperature effect 5min;
(4) DMEM/F12 with serum-free dilutes Lipofectamine 20006 μ L, 9 μ L, 12 μ L respectively to 50 μ L, mixing gently, room temperature effect 5min;
(5) Lipofectamine 2000 of the plasmid of mixed diluting and dilution, this moment, cumulative volume was 100 μ L.Mixing gently, room temperature effect 20min;
(6) with the old nutrient solution sucking-off in 12 orifice plates, use D-Hank ' s to clean, add the DMEM/F12 of 900 μ L serum-frees;
(7) dropwise add liposome/DNA mixture in different holes, waggle culture plate, mixing gently before and after the edged of limit; Set up 2 hole untransfecteds as blank simultaneously;
(8) at 37 ℃, volume fraction than being 5%CO
2Hatch 4～6h in the saturated humidity incubator, discard nutrient solution, add DMEM/F12 substratum completely;
(9) 24h～48h observation of cell, and in time change liquid.
Described method, the concrete operations of described step (3) are:
(1) treats behind the transfection 48h that cell converged at 80% o'clock, passage is digested in another 12 orifice plate, adding 800 μ g/mL G418 then screens, in the time of the 7th day, available pancreatin discards Digestive system and adds screening liquid, two weeks of cultured continuously in former ware peptic cell, commutation in per two days is screened liquid 1 time with concentration G418, and every day, observation of cell was grown and death condition;
(2) treat the whole death of cell of control wells after, the concentration of G418 is reduced by half, reduce to 400 μ g/mL;
(3) continue the screening cell about one month, changed 1 not good liquor in per two days, till positive colony is visible;
(4) with positive colony with filter paper method digestion and be transferred to new 12 orifice plates, enlarged culturing.
The present invention also provides the pig tracheal epithelial cell of setting up according to the method described above system.
The present invention adopts the liposome transfection method to change human telomerase reverse transcriptase's gene over to normal pig tracheal epithelial cell, activate its telomerase activation, with self RNA is that the constantly synthetic telomeric dna of template replenishes and prolongs telomere, obtain the ability of unlimited division and propagation, set up the immortalized cell line of pig tracheal epithelial cell.
Description of drawings
Former being commissioned to train of Fig. 1 supported cell (* 100) the Electronic Speculum figure that reached for the 3rd generation.
The 50th generation cell (* 100) Electronic Speculum figure after Fig. 2 transfection.
The 50th generation cell (* 200) Electronic Speculum figure after Fig. 3 transfection.
The 15th generation after preceding the 3rd generation of Fig. 4 transfection and the transfection, 35 generation immortalization STECs hTERTmRNA RT-PCR detected result figure.
The STECs tracheal epithelial cell 8 type Keratin sulfate immunofluorescences of immortalization detect (* 400) Electronic Speculum figure after Fig. 5 transfection.
The STECs growth curve of former generation STECs of Fig. 6 and immortalization relatively.
Fig. 7 STECs cell growth cycle distribution plan of former the 3rd generation of generation.
STECs the 35th generation cell growth cycle distribution plan of Fig. 8 immortalization.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
1, the aseptic newborn piglet tracheae that obtains is removed adhesion organization, adopts pronase to pour into cold digestion method digestion and separates the highly purified tracheal epithelial cell of acquisition, and form is spherical shape, how single existence, the visible fibre swing of microscopically.
2, the former generation tracheal epithelial cell that separation is obtained is suspended in the DF12 substratum, places 37 ℃, 5%CO
2Cultivate under the condition.6～8h, cell attachment; About 24h, cell is the monolayer adherence growth; 2～4d cell is paved with the plate bottom.Identify that through 8 type Keratin sulfate indirect immunofluorescences nucleus does not have specificity fluorescent, cytolemma has fluorescence, observes in conjunction with cellular form, confirms that the cell of institute's separation and Culture is the pig tracheal epithelial cell.
3, extract the eukaryon expression plasmid pCI-neo-hTERT that purifying contains the hTERT gene, adopt the liposome transfection method, utilize will the encode former pig tracheal epithelial cell of being commissioned to train foster of eukaryon expression plasmid pCI-neo-hTERT importing of hTERT and neo gene of Lipofectamine 2000 Regent transfection reagent boxes to carry out transfection.
4, screen by G418 behind the transfection 48h, cell began death in the 2nd day, the 14th day, untransfected group cell is all dead under the G418 effect, and transfection group still has than many cells, and existing G418 resistant cell begins growth, G418 concentration is reduced to half and is kept screening, continue screening about month, wait to find anti-G418 positive cell clone after, in 37 ℃, 5% CO
2Enlarged culturing in the incubator utilizes limiting dilution assay that positive cell is carried out the mono-clonal purifying, obtains monoclonal tracheal epithelial cell, and with its cultivation of going down to posterity.
5, the RT-PCR technology all detects hTERT mRNA and expresses in transfectional cell in the cell of transfection hTERT, carries out Western Blot with anti-hTERT monoclonal antibody and detects, and finds to have specific band to produce, and former generation cultured cells do not detect.Show that exogenous hTERT gene can express in transfection pig tracheal epithelial cell, and activate the telomerase activation of pig tracheal epithelial cell.
6, with the positive monoclonal tracheal epithelial cell external in 37 ℃, 5% CO
2Cultured continuously in the incubator, monolayer cell is the paving stone sample, and the iuntercellular boundary is clearly demarcated, has contact inhibition, has Normocellular proliferating cycle, cultivates to surpass 80 generations, promptly gets the clone of immortalization.
DMEM/F12 substratum of the present invention is available from invitrogen company, and perfect medium is to add 10% foetal calf serum in former substratum.
Serum-free DMEM/F12 nutrient solution of the present invention is the DMEM/F12 nutrient solution that does not add 10% foetal calf serum.
Embodiment 3
The screening process of above-mentioned positive cell and the purge process of transfectional cell, concrete steps are as follows:
1, the acquisition of G418 screening and positive cell
(1) treats behind the transfection 48h that cell converged at 80% o'clock, passage is digested in another 12 orifice plate, add 800 μ g/mL G418 then and screen, in the time of the 7th day, available pancreatin discards Digestive system and adds screening liquid (the DF12 substratum adds G418 fully) in former ware peptic cell.In two weeks of cultured continuously, commutation in per two days is screened liquid 1 time with concentration G418, and every day, observation of cell was grown and death condition.
(2) treat the whole death of cell of control wells after, the concentration of G418 is reduced by half, reduce to 400 μ g/mL.
(3) continue the screening cell about one month, changed 1 not good liquor in per two days, till positive colony is visible.
(4) with positive colony with filter paper method digestion and be transferred to new 12 orifice plates, enlarged culturing.
2, the purifying of transfectional cell
(1) makes cell suspension after the positive cell digestion with enlarged culturing
(2) cell dilution is become the suspension of 100/mL, 50/mL, 25/mL, 10/mL and 1/mL with complete DMEM/F12 substratum.
(3) cell suspension is planted respectively into 96 well culture plates, every hole 0.1mL, cell content are respectively 10/hole, 5/hole, 2.5/hole and 1/hole.
(4) at 37 ℃, volume fraction than being 5%CO
2Cultivate in the saturated humidity incubator.
(5) observe the clonal growth situation with inverted microscope every day, select to have only the hole of a colony growth, carry out mark, the hole that discards more than two and do not have cell to grow.
When (6) treating that cell reaches 70%～80% fusion in the hole, cell transfer is arrived 12 new orifice plates, carry out enlarged culturing with sucrose EDTA and pancreatin substep digestion method.
Embodiment 4
Cell qualification test process:
1, transfectional cell morphological observation
STECs (Fig. 2 and Fig. 3) plesiomorphism of former be commissioned to train foster STECs (Fig. 1) and immortalization is typical epithelium sample.The cell central nucleus are obvious, and the kernel more than 2 is often arranged, and cell can be paved with individual layer, and monolayer cell is typical pebbles paving stone shape to be arranged, and there is contact inhibition in iuntercellular.
2, RT-PCR detects the expression of exogenous hTERT in the immortalized cells
(1) extracting method of cell total rna is as follows:
1. treat former the 3rd generation of generation, immortalization the 15th generation and 35 generation cell reach at 70%～80% o'clock, cell dissociation is got off to transfer in the Eppendorf pipe of 1.5mL.
2. the centrifugal cell culture fluid that removes counts 750 μ LTrizol, and vibration mixes, and room temperature leaves standstill 10min.
3. add 200 μ L trichloromethanes, put upside down mixing, room temperature leaves standstill 3min.
4. under 4 ℃ of conditions, the centrifugal 15min of 12000r/min.
5. draw supernatant liquor 550 μ L, add isopyknic Virahol, put upside down mixing, room temperature leaves standstill 15min.
6. under 4 ℃ of conditions, the centrifugal 15min of 12000r/min.
7. supernatant discarded adds 70% ethanol of precooling DEPC water treatment.
8. under 4 ℃ of conditions, the centrifugal 15min of 12000r/min.
9. supernatant discarded is instantaneous centrifugal, inhales and abandons unnecessary liquid, drying at room temperature 10min.
10. add 24 μ LDEPC water treatments, melt 10min, put-20 ℃ standby.
(2) design of primers is with synthetic
According to the mRNA sequence of hTERT among the GenBank, utilize Primer Premier 5.0, designed the primer of a pair of detection exogenous human telomerase reverse transcriptase gene, the product that finally obtains is 461bp, primer is synthetic by Nanjing Jin Sirui company.
Table 1PCR primer sequence
(3) RT-PCR amplification
1. cDNA is synthesized in reverse transcription
Reaction system is as follows:
Total RNA extracting solution 7 μ L
Olig(dt)15 2μL
dNTP 2μL
RNase-free?H2O 2.5μL
Rapidly at cooled on ice 2min, add following composition behind the brief centrifugal collection reaction solution behind 70 ℃ of heating 5min
5×First-strand?Buffer 4μL
0.1M?DTT 1μL
RNasin 0.5μL
TiANScript-M-MLV 1μL
42 ℃ of temperature of mixing are bathed 50min, termination reaction behind 95 ℃ of heating 5min, and the liquid feeding process is carried out on ice.
2. pcr amplification
Reaction system is as follows:
The PCR reaction conditions is: 94 ℃ of pre-sex change of 4min; 94 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 40s, totally 30 circulations; Last 72 ℃ are extended 6min, 4 ℃ of preservations.
3. RT-PCR product gel electrophoresis is identified and is got above-mentioned PCR product 5 μ L electrophoresis in 10g/L TAE sepharose, contain 0.5 μ g/mL ethidium bromide in the gel, electrophoretic buffer is 1 * TAE, observes, takes pictures in the ultraviolet gel imaging system after 120V, 15min electrophoresis finish.
The result shows, all can amplify specific fragment in the cell of two different generations of the STECs of immortalization, and fragment length is consistent with expection, is about 461bp, and the negative (see figure 4) of STECs amplification in the 3rd generation.
3, the indirect immunofluorescence of Keratin sulfate 8 is identified
With the cell of immortalization with 1 * 10
5/ cm
2Density discards nutrient solution after being inoculated in the interior 24h of the 60mm culture dish of placing 2 cover glasses in advance, collects 2 creep plates, PBS flushing 3 times, acetone fixed 15min, PBS flushing, get 1 creep plate and drip one anti-(mouse anti human 8 type Keratin sulfate monoclonal antibodies), another is done contrast and drips PBS, hatches 24h for 4 ℃, PBS washing 3 times, drip two anti-(goat-anti mouse FITC-IgG) again, 37 ℃ of lucifuge effect 30min, PBS washing 3 times, fluorescence microscope.
Observations shows that Keratin sulfate is positioned on the cytolemma, and cell membrane is painted, shows specific green fluorescence, and the not painted no fluorescence of nucleus shows that control group does not have fluorescence, and the result shows that the STECs of immortalization is an epithelium source property (see figure 5).
4, mtt assay detects the cell viability situation
(1) cell of immortalization is pressed 1 * 10
4/ cm
2Be inoculated in 96 orifice plates, be connected in 8 rounds, 3 repetitions are done in every hole again, and other meets former generation STECs and does contrast.
(2) treat that cytogamy becomes individual layer, add 20 μ LMTT liquid, continue to cultivate 3～4h to first 3 holes of arranging.
(3) discard substratum, add DMSO 150 μ L, uniform mixing changes in the new enzyme plate.
(4) on microplate reader, the 490nm wavelength is surveyed its absorbancy.
(5) Using such method was surveyed 8 days continuously, and image data is drawn the vigor graphic representation.
As we can see from the figure, the vigor curve of the STECs of immortalization and STECs all is inverted " S " type (see figure 6), and two kinds of cells enter plateau then at the logarithmic phase that postvaccinal the 3rd～5d enlivens for propagation.As seen from the figure, the STECs of immortalization is active than STECs propagation.
5, flow cytometer detects the cell cycle
Be cultured to 35 generations immortalization STECs and be cultured to the former generation STECs in the 3rd generation and digest, collect respectively in the aseptic centrifuge tube of 1.5mL, guarantee that at least cell concn reaches 1 * 10
6Individual, PBS liquid cleans, and the centrifugal 5min of 3000r/min discards PBS liquid, and it is resuspended to add 1mLPBS again, adds the abundant mixing of 2mL dehydrated alcohol, dispels, and makes cell become single, under 4 ℃ of states, delivers to flow cytometer and carries out the cell cycle and detect, and the result is analyzed.
From found that, the STECs caryogram of immortalization is normal, still is 2 times of bodies (seeing Fig. 7 and Fig. 8).The 35th generation immortalization the cell of STECs in the G1 phase account for 53.66%, be less than former generation in the 3rd generation STECs 75%, account for 46.34% at S phase cell,, show that the STECs proliferation activity in STECs more former generation of immortalization increases more than 22.76% of the STECs in former generation in the 3rd generation.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (4)
1. the establishment method of boar tracheal epithelial cell system is characterized in that, may further comprise the steps:
(1) former be commissioned to train foster: the aseptic newborn piglet tracheae that obtains, remove adhesion organization, adopt pronase to pour into cold digestion method digestion and separate and obtain highly purified tracheal epithelial cell; Former generation tracheal epithelial cell that separation is obtained is suspended in the DF12 substratum, places 37 ℃, 5%CO
2Cultivate under the condition;
(2) transfection and screening: extract the eukaryon expression plasmid pCI-neo-hTERT that purifying contains the hTERT gene, adopt the liposome transfection method, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported the former pig tracheal epithelial cell of being commissioned to train foster of step (1) carry out transfection;
(3) screening and enlarged culturing.
2. method according to claim 1 is characterized in that, described step (2) is with the former pig tracheal epithelial cell of being commissioned to train foster of pCI-neo-hTERT plasmid transfection, and its concrete steps are as follows:
(1) 24h before the transfection is digested to individual cells with STECs with sucrose EDTA and 0.25% pancreatin, step by step with 1 * 10
5Individual cells/well is inoculated in 12 orifice plates, makes cell can reach 80%～90% fusion the same day in transfection;
(2) DMEM/F12 of 4h replacing serum-free before the transfection;
(3) diluting 5 μ L pCI-neo-hTERT plasmid to volumes with the DMEM/F12 of serum-free is 50 μ L, mixing gently, and plasmid concentration reaches 300ng/ μ L～400ng/ μ L room temperature effect 5min;
(4) DMEM/F12 with serum-free dilutes Lipofectamine 2,000 6 μ L, 9 μ L, 12 μ L respectively to 50 μ L, mixing gently, room temperature effect 5min;
(5) Lipofectamine 2000 of the plasmid of mixed diluting and dilution, this moment, cumulative volume was 100 μ L.Mixing gently, room temperature effect 20min;
(6) with the old nutrient solution sucking-off in 12 orifice plates, use D-Hank ' s to clean, add the DMEM/F12 of 900 μ L serum-frees;
(7) dropwise add liposome/DNA mixture in different holes, waggle culture plate, mixing gently before and after the edged of limit; Set up 2 hole untransfecteds as blank simultaneously;
(8) at 37 ℃, volume fraction than being 5%CO
2Hatch 4～6h in the saturated humidity incubator, discard nutrient solution, add DMEM/F12 substratum completely;
(9) 24h～48h observation of cell, and in time change liquid.
3. method according to claim 1 is characterized in that, the concrete operations of described step (3) are:
(1) treats behind the transfection 48h that cell converged at 80% o'clock, passage is digested in another 12 orifice plate, adding 800 μ g/mL G418 then screens, in the time of the 7th day, available pancreatin discards Digestive system and adds screening liquid, two weeks of cultured continuously in former ware peptic cell, commutation in per two days is screened liquid 1 time with concentration G418, and every day, observation of cell was grown and death condition;
(2) treat the whole death of cell of control wells after, the concentration of G418 is reduced by half, reduce to 400 μ g/mL;
(3) continue the screening cell about one month, changed 1 not good liquor in per two days, till positive colony is visible;
(4) with positive colony with filter paper method digestion and be transferred to new 12 orifice plates, enlarged culturing.
4. the pig tracheal epithelial cell system that method according to claim 1 is set up.
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| CN103849602A (en) * | 2013-07-08 | 2014-06-11 | 山东省滨州畜牧兽医研究院 | Bovine testicle cell line as well as establishment method and application thereof |
| CN104531607A (en) * | 2014-12-29 | 2015-04-22 | 南京农业大学 | Canine primary bronchial epithelial cell and application thereof in preparation of immortalized cells |
| CN105274050A (en) * | 2015-11-12 | 2016-01-27 | 常州大学 | Culture method of human airway epithelial cells for treating bronchial asthma |
| CN105483089A (en) * | 2016-01-15 | 2016-04-13 | 张彦明 | Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof |
| CN107338225A (en) * | 2017-07-10 | 2017-11-10 | 江苏省农业科学院 | Pig bronchial epithelial cell system, preparation method and applications |
| 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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| CN103849602A (en) * | 2013-07-08 | 2014-06-11 | 山东省滨州畜牧兽医研究院 | Bovine testicle cell line as well as establishment method and application thereof |
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| CN105274050A (en) * | 2015-11-12 | 2016-01-27 | 常州大学 | Culture method of human airway epithelial cells for treating bronchial asthma |
| CN105483089A (en) * | 2016-01-15 | 2016-04-13 | 张彦明 | Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof |
| CN107338225A (en) * | 2017-07-10 | 2017-11-10 | 江苏省农业科学院 | Pig bronchial epithelial cell system, preparation method and applications |
| CN107338225B (en) * | 2017-07-10 | 2020-08-28 | 江苏省农业科学院 | Porcine bronchial epithelial cell line, preparation method and application thereof |
| 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: 20110907 |