CN101955907A - Pig small intestine epithelial cell line and construction method thereof - Google Patents
Pig small intestine epithelial cell line and construction method thereof Download PDFInfo
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Abstract
The invention discloses a pig small intestine epithelial cell line (ZYM-SIEC02), which was preserved in China Center for Type Culture Collection on January 13th, 2010 with a collection number of CCTCC-C201001. The invention also discloses the construction method of the cell line. The cell line has the advantages of low nutrient condition requirements, quick cell growth, easy cultivation and convenient promotion and application.
Description
Technical field
The invention belongs to the zooblast field of engineering technology, relate to and set up a kind of new clone, particularly chitterlings epithelial cell line (ZYM-SIEC02) and establishment method thereof.
Background technology
(Intestinal epithelial cells IEC) is the major function cell of enteron aisle to the small intestinal mucosa epithelial cell, participates in digestion, absorption, immunization barrier and the stress reaction etc. of enteron aisle, and with the inside and outside secreting function of enteron aisle substantial connection is arranged.Therefore, the separation and Culture of carrying out intestinal epithelial cell is one of main means of research Small Intestine, absorption of nutrient ingredients mechanism and regulation and control thereof
People such as the moving Zhang Yanming tutor of academy of sciences of Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology are successively separating and cultural method pig umbilical vein vascular endothelial cell, use the preliminary study that telomerase reverse transcriptase gene makes pig vascular endothelial cell immortalization, successively disclosing pig umbilical vein vascular endothelial cell in the preliminary study of pig umbilical vein vascular endothelial cell immortalization separates and cultural method, adopt will the encode eukaryon expression plasmid pCI-neo-hTERT of hTERT and neo gene of liposome transfection method to import the former foster porcine vein endothelial cell of being commissioned to train and carry out using the G418 screening after the transfection and obtain anti-G418 positive cell continuation enlarged culturing, carry out the positive cell that cell streaming screening obtains expressing with fluorescence antibody and in the in-vitro cultivation case, cultivate, after the sorting the clone of immortalization.In the method 500 μ g/mL G418 were screened to 14 days, add the trysinization monoclonal cell with filter paper, enlarged culturing in changing 96 holes over to, the mono-clonal positive cell is less like this, the quantity of cell growth is considerably less, poor growth, and to add 20% foetal calf serum in process of production, the production cost height is not easy to apply.In June, 2008, people such as Zhang Yanming tutor begin to attempt to change same plasmid over to former generation chitterlings epithelial cell, but when this transfection, find, 600 μ g/mL G418 screening to 14 days, is added trysinization monoclonal cell, enlarged culturing in changing 96 holes over to filter paper, six of the mono-clonals that obtains, the cell growth is rapid, and only needs to add 5% serum (except during cell recovery, needing 7% serum) in process of production.
Reference
[1] Hong Haixia, Zhang Yanming, grandson Pei etc. separate and cultivation [J] pig umbilical vein vascular endothelial cell. Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal (natural science edition) the 32nd the 5th phase of volume of May in 2004.
[2] Su Zhengyuan, Zhang Yanming, Hong Haixia etc. use the preliminary study [J] that telomerase reverse transcriptase gene makes pig vascular endothelial cell immortalization. journal of animal science and veterinary medicine .2007.38 (4): 407～411.
[3] Ma Quanli, Hong Haixia, Zhang Yanming etc., the preliminary study [J] of pig umbilical vein vascular endothelial cell immortalization. Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal (natural science edition) 2007,35 (7).
[4]Hong?H?X，Zhang?Y?M，Xu?H，and?et?al.(2007)Immortalization?of?swine?umbilical?vein?endothelial?cells?with?human?telomerase?reverse?transcriptase.MolCells，24(3)：358-363
Description of drawings
Fig. 1 is former the 3rd generation of generation chitterlings epithelial cell inverted microscope observation figure (* 200);
Fig. 2 is 60 generation chitterlings epithelial cell inverted microscope observation figure (* 100) after the transfection;
Fig. 3 detects the expression of results agarose gel electrophoresis figure (positive) of exogenous hTERT in the transfectional cell for RT-PCR;
Fig. 4 detects hTERT gene (positive) figure for the immunohistochemical methods method;
Fig. 5 detects hTERT gene (feminine gender) figure for the immunohistochemical methods method;
The protein expression that Fig. 6 detects the hTERT gene for western blot is figure as a result;
Fig. 7 is that flow cytometer (FCM) detects chitterlings clones (ZYM-SIEC02) the 8th generation periodogram;
Fig. 8 is that flow cytometer (FCM) detects chitterlings clones (ZYM-SIEC02) the 58th generation periodogram;
Fig. 9 is the mensuration figure of chitterlings clone (ZYM-SIEC02) growth curve.
Summary of the invention
The object of the present invention is to provide a kind of being easy to cultivate fast growth, chitterlings epithelial cell line and establishment method thereof that working method is easy.
This cell lies in and was deposited in Chinese typical culture collection center on January 13rd, 2010, and it abbreviates CCTCC as, and preservation is registered on the books and is numbered CCTCC-C201001, the depositary institution address: Wuhan City, Hubei Province Wuhan University preservation center.
A further object of the invention provides the establishment method of chitterlings epithelial cell line, specifically comprises the following steps:
(1) asepticly takes to separate newborn piglet small intestine ileum or jejunal segment, with tissue block method's separation and Culture intestinal epithelial cells cell, in 37 ℃, 5% CO
2Cultivated 5～7 days in the incubator;
(2) adopt the liposome transfection method, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported former chitterlings epithelial cell of being commissioned to train foster carry out transfection
(3) treat behind the cell transfecting pCI-neo-hTERT eukaryon expression plasmid that pair cell carries out the processing of going down to posterity at 1: 1 after 24 hours, 48h cell attachment and in good condition, add 600 μ g/mL G418 screening, cell began death in the 3rd day, the back was changed a nutrient solution in per three days and was added 600 μ g/mL G418 and continues screening, a large amount of cell vacuolations after 9 days, the 14th day, untransfected group cell was all dead under the G418 effect, and transfection group still has than many cells, and existing G418 resistant cell begins growth, screened one month, G418 concentration is reduced to 300 μ g/mL and is kept screening, treats visible big positive colony under the mirror, after using the filter paper method with positive clone digestion, use infinite dilution method sample that positive colony is separated into individual cells again, and it is planted in 96 orifice plates, every porocyte quantity≤2, the hole of the cell in the every hole of mark, 37 ℃, 5% CO
2Enlarged culturing in the incubator is observed behind the 72h, will transfer to 48 orifice plates by clone's digestion that a cell proliferation obtains, and by that analogy, obtains anti-G418 positive cell.
(4) with positive cell external continuously in 37 ℃, 5% CO
2Cultivate in the incubator, cell presents the growth of paving stone sample monolayer adherence, and there is contact inhibition in iuntercellular, cultivates to surpass the clone that 50 generations promptly get immortalization.
Described pCI-neo-hTERT imports former chitterlings epithelial cell of being commissioned to train foster and carries out transfection, specifically comprises the following steps:
(1) transfection is preceding 48 hours, and ATV digestion reaches the chitterlings chrotoplast and the counting in the 3rd generation, with 1 * 10
4Individual cells/well is inoculated in 12 orifice plates, makes cell can reach 80%～90% degree of converging the same day in transfection;
(2) transfection is preceding 24 hours, changes the high sugared DMEM nutrient solution of fresh serum-free;
(3) with serum-free D-MEM/F-12 nutrient solution dilute 2 μ L, 4 μ L, 6 μ L respectively, 8 μ LpCI-neo-hTERT plasmid to volumes are 50 μ L, plasmid concentration reaches 0.01 μ g/ μ L～0.04 μ g/ μ L, mixing gently, room temperature effect 5 minutes;
(4) (invitrogen company buys, and Chinese is a liposome 2000, and Cat.no.11668-027) 4 μ L are 50 μ L to volume, mixing gently, room temperature effect 5 minutes with serum-free D-MEM/F-12 nutrient solution dilution Lipofectamine 2000 Reagent;
(5) Lipofectamine 2000 of the plasmid of mixed diluting and dilution, this moment, cumulative volume was 100 μ L, mixing gently, room temperature effect 20 minutes;
(6), add 400 μ L serum-free D-MEM/F-12 nutrient solutions with the old nutrient solution sucking-off in 12 orifice plates;
(7) dropwise step (5) gained mixture is joined in the different holes limit edged wave and culture plate, mixing gently; Set up 2 hole untransfected blanks simultaneously;
(8) in 37 ℃, 5% CO
2Hatched in the incubator 4～6 hours, and discarded nutrient solution, add complete D-MEM/F-12 nutrient solution.
Described complete D-MEM/F-12 nutrient solution: buy from the Dulbecco ' s of the GIBCO of invitrogen company Modified Eagle Medium:Nutrient Mixture F-12 (Ham) (1: 1) substratum, add 5% foetal calf serum.
Chitterlings epithelial cell line of the present invention (ZYM-SIEC02) compared with prior art has the following advantages:
(1) nutritional condition of ZYM-SIEC02 of the present invention requires low, existing cytotrophy conditional request is low, originally needs 5% foetal calf serum, various nutritive substances such as heparin, glutamine, and present cell is only required 5% serum, greatly reduces the cost of cultivation.
(2) ZYM-SIEC02 of the present invention is very good by the growth conditions of the cell of application infinite dilution method acquisition; originally went down to posterity needs 3～5 days at 1: 1; present passage goes down to posterity at 1: 1 needs 16-24h, and the vitality of cell is very strong than before, is convenient to the application of mass-producing.
(3) ZYM-SIEC02 of the present invention still can keep division growth more than 50 generations of external infection, and aging and death do not take place, and is a kind of clone of immortalization, now reaches for 89 generations, for research and prevention swine disease provide basic experiment material.
Embodiment
The chitterlings epithelial cell line that provides below in conjunction with the contriver former be commissioned to train support used organization material source, sampling process, the former foster substratum of being commissioned to train with culture condition, the process that goes down to posterity and number of times, go down to posterity cultivate form and the culture condition that goes down to posterity, new clone separate and the embodiment of identification mark further specifies the present invention.
The establishment method of embodiment 1 chitterlings epithelial cell line (ZYM-SIEC02)
(1) asepticly takes to separate newborn piglet small intestine ileum or jejunal segment, with tissue block method's separation and Culture intestinal epithelial cells cell, in 37 ℃, 5% CO
2Cultivated 7～12 days in the incubator.
(2) adopt the liposome transfection method, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported former chitterlings epithelial cell of being commissioned to train foster carry out transfection.
(3) treat behind the cell transfecting pCI-neo-hTERT eukaryon expression plasmid after 24 hours, use ATV digestion, to be passaged to another culture dish at 1: 1, add 600 μ g/mL G418 screening behind the 48h, cell began death in the 3rd day, the back was changed a nutrient solution in per three days and was added 600 μ g/mL G418 and continues screening, a large amount of cell vacuolations after 9 days, the 14th day, untransfected group cell is all dead under the G418 effect, transfection group still has than many cells, and existing G418 resistant cell begin the growth, screened one month, G418 concentration is reduced to 300 μ g/mL and is kept screening, treat under the mirror visible big positive colony, use the filter paper method with positive clone digestion after, use infinite dilution method sample that positive colony is separated into individual cells again, and it is planted in 96 orifice plates, every porocyte quantity≤2, the hole of the cell in the every hole of mark, 37 ℃, 5% CO
2Enlarged culturing in the incubator is observed behind the 72h, will transfer to 48 orifice plates by clone's digestion that a cell proliferation obtains, and by that analogy, obtains anti-G418 positive cell
(4) with positive cell external continuously in 37 ℃, 5% CO
2Cultivate in the incubator, cell presents the growth of paving stone sample monolayer adherence after the sorting, and there is contact inhibition in iuntercellular, cultivates to surpass the clone that 50 generations promptly get immortalization.
The former chitterlings epithelial cell of being commissioned to train foster of pCI-neo-hTERT importing carries out transfection in the described step (2), specifically comprises the following steps:
(1) transfection is preceding 48 hours, and ATV digestion reaches the chitterlings chrotoplast and the counting in the 3rd generation, with 1 * 10
4Individual cells/well is inoculated in 12 orifice plates, makes cell can reach 80%～90% degree of converging the same day in transfection;
(2) transfection is preceding 24 hours, changes the high sugared DMEM nutrient solution of fresh serum-free;
(3) with serum-free D-MEM/F-12 nutrient solution dilute 2 μ L, 4 μ L, 6 μ L respectively, 8 μ LpCI-neo-hTERT plasmid to volumes are 50 μ L, plasmid concentration reaches 0.01 μ g/ μ L～0.04 μ g/ μ L, mixing gently, room temperature effect 5 minutes;
(4) (invitrogen company buys, and Chinese is a liposome 2000, and Cat.no.11668-027) 4 μ L are 50 μ L to volume, mixing gently, room temperature effect 5 minutes with serum-free D-MEM/F-12 nutrient solution dilution Lipofectamine 2000 Reagent;
(5) Lipofectamine 2000 of the plasmid of mixed diluting and dilution, this moment, cumulative volume was 100 μ L, mixing gently, room temperature effect 20 minutes;
(6), add 400 μ L serum-free D-MEM/F-12 nutrient solutions with the old nutrient solution sucking-off in 12 orifice plates;
(7) dropwise step (5) gained mixture is joined in the different holes limit edged wave and culture plate, mixing gently; Set up 2 hole untransfected blanks simultaneously;
(8) in 37 ℃, 5% CO
2Hatched in the incubator 4～6 hours, and discarded nutrient solution, add complete D-MEM/F-12 nutrient solution.Described complete D-MEM/F-12 nutrient solution: buy from the Dulbecco ' s of the GIBCO of invitrogen company Modified Eagle Medium:Nutrient Mixture F-12 (Ham) (1: 1) substratum, add 5% foetal calf serum.
Cell through after the transfection under phase microscope the observation metamorphosis, and compare with former generation chitterlings epithelial cell (as Fig. 1).
ZYM-SIEC02 (as Fig. 2) and primary cell plesiomorphism, the cell central nucleus are obvious, high-visible 2 or 2 above kernels.The most cells form is polygon or oval, and cell is typical pebbles paving stone shape to be arranged, and there is contact inhibition in iuntercellular.
Embodiment 3.RT-PCR detects the expression of exogenous hTERT in the transfectional cell
(1) design of primers is with synthetic
According to 1 pair of specific detection primer of the human telomerase reverse transcriptase gene reference sequences of having delivered among the GenBank (accession number is NM-198255) design, the upstream and downstream primer is respectively P1:5 '-TATGCCGTGGTCCAGAAG-3 '; P2:5 '-TATGCCGTGGTCCAGAAG-3 ', the amplified fragments size is 416bp, primer is synthetic by the biological limited liability company of Beijing three rich polygala roots.
(2) extraction of cell total rna
1. transfectional cell and primary cell with being inoculated in 35 millimeters culture dish after the ATV digestion, are treated to take out after cell is paved with individual layer respectively, and PBS washes twice with sterilization, discards liquid.
2. add 750 μ L Trizol reagent, the mixing that turns upside down, treat that lysis fully after, cell pyrolysis liquid is drawn onto in the 1.5mL sterilization EP pipe of DEPC water treatment, 4 ℃ left standstill 5 minutes.
3. add 200 μ L chloroforms, fully shake to emulsification evenly, 4 ℃ left standstill 15 minutes.
4. centrifugal 15 minutes at 4 ℃ with 12000 rev/mins.
5. gentle aspiration supernatant 450 μ L are transferred to the 1.5mL sterilization EP pipe of another new DEPC water treatment, add the ice-cold Virahol of 500 μ L (20 ℃ of storages), put upside down mixing gently, put-20 ℃ and spend the night.
6. centrifugal 10 minutes at 4 ℃ with 12000 rev/mins.
7. abandon supernatant, add the ice-cold 750mL/L ethanol of 1mL, put upside down gently several times, centrifugal 10 minutes at 4 ℃ with 12000 rev/mins.
8. abandon supernatant, be inverted the EP pipe, drying at room temperature nucleic acid.
9. add 20 μ L DEPC water dissolution RNA.
(3) first chain cDNA are synthetic
Following solution of adding and reagent carry out reverse transcription in 0.5mL PCR reaction tubes:
The piping and druming mixing, centrifugal slightly, add AMV ThermoScript II 1.0 μ L, in 42 ℃ of water-bath effects 1.5 hours.
(4) the double-stranded cDNA of pcr amplification
In the PCR reaction tubes, be formulated as follows reaction system:
Reverse transcription product (cDNA) 10 μ L
10×PCR?buffer(Mg2+free) 5μL
dNTP 4μL
Upstream primer P1 0.5 μ L
Downstream primer P2 0.5 μ L
MgCl
2 3μL
Sterilization deionized water 26.5 μ L
Taq archaeal dna polymerase 0.5 μ L
Mixing is placed in the pcr amplification instrument, 95 ℃ of pre-sex change 5 minutes; 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 30 seconds, carry out 30 circulations; Last 72 ℃ were extended 10 minutes.After amplification was finished, the PCR product (was seen Fig. 3, M.Maker among the figure with 10g/L agarose gel electrophoresis observations; 1.F10; 2.F65; 3. plasmid; 4. former generation .).
Rabbit anti-people hTERT polyclonal antibody and instant SABC immunohistochemical methods test kit specification sheets with reference to Wuhan doctor's moral company carry out, and concrete grammar is as follows:
(1) place aseptic cover glass 0.5cm * 0.6cm in 24 hole plastic culture plate holes, SIECs and hTERT-SIECs are digested to unicellular with ATV, and cell concn to 2 * 10 are adjusted in the counting back
5Individual/mL, inoculation 0.5mL cell suspension in every hole.
When (2) treating that cell grows to nearly fusion state (low density individual layer), wash 3 times with PBS (0.01mol/L, pH 7.4), add 0.4g/L Paraformaldehyde 96 phosphate buffered saline buffer (pH 7.4) or 95% alcohol, cell faces up, and room temperature is fixed 10 minutes, with PBS flushing 3 times, each 5 minutes.
(3) add 50 μ L peroxidase blocking solutions, with the activity of blocking-up endogenous peroxydase, room temperature effect 20 minutes was with PBS flushing 3 times, each 5 minutes.
(4) remove PBS liquid, add 50 μ L BSA confining liquids, hatch 20min under the room temperature.Get rid of unnecessary liquid, do not wash, the anti-people hTERT of the rabbit polyclonal antibody 50 μ L that add 1: 100 times of dilution put into wet box and spend the night for 4 ℃ on cover glass.An anti-equivalent PBS that drips does negative control simultaneously, with PBS flushing 3 times, and each 5min.
(5) remove PBS liquid and add biotin labeled goat anti-rabbit igg, in 37 ℃ hatch 1 hour after, with PBS washing 3 times, each 5min.
(6) remove PBS liquid, add 50 μ L Streptomycin sulphate antibiotin peroxidase solution, incubated at room 15min, with PBS washing 3 times, each 5min.
(7) remove PBS liquid and add the DAB working fluid that 100 μ L newly join, under inverted microscope, observe immediately then, take a picture.Cell among Fig. 4 is pale brown look, and Fig. 5 negative cells does not change, and cultured cells expression hTERT gene is described.
Embodiment 5Western Blot detects
(1) albumen sample preparation: outwell nutrient solution, and bottle tipped upside down on make thieving paper blot nutrient solution on the thieving paper, every bottle of cell adds the PBS (0.01M, pH7.2～7.3) of 3ml4 ℃ of precooling.Keep flat and shake the 1min washed cell gently, discard washing lotion then.Repeat above operation twice, wash cell three times altogether with the flush away nutrient solution.PBS is abandoned clean back to be placed culturing bottle on ice.RIPA lysate (1mM PMSF) shakes up and places on ice.Every bottle of cell adds 250～500 μ l lysates, in cracking 30min on ice.After cracking is intact, cell is scraped a side in culturing bottle, with rifle cell debris and lysate are moved in the 1.5ml centrifuge tube then with clean scraper.And it is no longer sticky until lysate with rifle piping and druming.
(2) SDS-PAGE electrophoresis: install that the working instructions by the Bio-RadMini-Protean electrophoresis system install sheet glass behind the Bio-Rad minigel electrophoresis mould, be fixed on the glue plate; Detect antigenic molecular weight size according to experiment and select suitable electrophoretic separation gum concentration separation gel solution by required acrylamide concentration configuration certain volume in a small beaker, in the gap of two sheet glass, pour into acrylamide soln rapidly, wait for 30-60min, make gel polymerisation.After gel polymerisation, between separation gel and water layer an interface clearly can appear; The preparation spacer gel, and, in spacer gel solution, insert the comb of the pre-design of clean experiment immediately directly pouring into spacer gel on the polymeric separation gel, vertically be positioned over gel under the room temperature; The spacer gel polymerization is back (30min) fully, shifts out comb, and on electrophoresis apparatus, groove respectively adds Tris glycine electrophoretic buffer up and down gel sets; Sample is mixed with gel loading buffer, heat 5min so that protein denaturation at 100 ℃; Design application of sample order is pressed the predefined procedure application of sample.Electrophoresis apparatus and power supply are connected, regulate voltage to 60V, transfer to 100V after running spacer gel, tetrabromophenol sulfonphthalein to be treated is moved to 0.5cm place, separation gel bottom, powered-down.
(3) change film: change over to after in methyl alcohol, soaking pvdf membrane more than the 20min and shift in the liquid.Filter paper is also immersed in the transfer liquid.Glue is unloaded, and upper left corner cut is soaked in shifting liquid slightly, spreads three filter paper of film and every side in order.Attention is evicted bubble from glass rod, and electric turn trough adds the 1L electricity changes liquid.Glue is tiled on the sponge, drips a little electricity commentaries on classics liquid and drive bubble once more, put into electric turn trough after envelope is tight, notice that film is in an anodal side.Cooling places mixture of ice and water with electrophoresis chamber.Constant current 400mA, 4h.
(4) sealing and hybridization
Film is taken out from electric turn trough, and TTBS is rinsing a little, is immersed in slowly to sway in the confining liquid one hour.The confining liquid that contains the anti-people hTERT of rabbit polyclonal antibody drips on the plastic film of shaking table, the Western film is taken out from confining liquid, and filter paper pastes the angle and blots slightly, and face down is attached to one and resists, note not staying bubble, jog was hatched one hour or 4 ℃ of standing over night under the room temperature.Too much evaporate to prevent liquid at reaction system outer mask one moistening plate.One anti-hatch end after, with embathing again three times behind the TTBS rinsing film, 10min at every turn.Goat anti-rabbit igg (HRP), by 1: 10000 dilution proportion, room temperature jog one hour.Two anti-hatch end after, with embathing again three times behind the TTBS rinsing film, 10min at every turn.
(5) luminous evaluation
Use the HRP-ECL luminescence method.With A, B luminescent solution diluted mixture in proportion.Film pastes the angle and blots with deionized water rinsing a little, filter paper, and anti-subsides method is overlying on A, the B mixing drop, turn off the light and blot, place the preservative film internal fixing, cover film rapidly in film magazine to filter paper subsides angle, visible light green fluorescence band (about 5min) back, close the glue box, expose according to the finding fluorescence intensity.Take out film and immerse 1-2min in the developing solution immediately fully, be placed on after the clear water rinsing once in the stop bath to the complete photographic fixing of egative film, clear water washes down and dries, and demarcates Marker, analyzes and scans.(see figure 6)
(1) ZYM-SIEC02 sample to be measured is made single cell suspension, then 1000 rev/mins centrifugal 8 minutes, abandon supernatant.
(2) fix with the cold ethanol of 700mL/L of 4 ℃ of precoolings, 4 ℃ of preservations are fixed 18 hours at least.
(3) adjusting cell concn is 106/mL, gets the 1mL cell suspension, washes 3 times with PBS, and cell is resuspended in the 1m L PI dye liquor (final concentration is 50 μ g/mL), hatches 30 minutes for 37 ℃, carries out flow cytometry analysis (seeing Fig. 7 to 8).
The mensuration of embodiment 7 growth curves
The 10th, 20,30,50 generation hTERT-SIECs after the transfection are inoculated in 24 orifice plates by 1 * 104/hole, changed liquid 1 time in 3～4 days, use the 2.5g/L trypsin digestion and cell every day, the blood cell counting plate counting.Each time point is established 3 parallel holes, and each hole counting 3 times is got the growth curve that its mean value is drawn cell.Fig. 9 illustrates that cell begins to increase the 3rd day quantity, to the 5th, 6 day the time cell quantity reach maximum, be about 5 * 10
5Individual.
Claims (4)
1. chitterlings epithelial cell line, this cell lies in and was deposited in Chinese typical culture collection center on January 13rd, 2010, and preservation is registered on the books and is numbered CCTCC-C201001.
2. the establishment method of the described chitterlings epithelial cell line of claim 1 is characterized in that, comprises the following steps:
(1) asepticly takes to separate chitterlings ileum or jejunal segment,, in 37 ℃, 5% CO2 incubator, cultivated 7～12 days with tissue mass cell culture separation and Culture intestinal epithelial cell;
(2) adopt the liposome transfection method, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported former chitterlings epithelial cell of being commissioned to train foster carry out transfection;
(3) treat behind the cell transfecting pCI-neo-hTERT eukaryon expression plasmid after 24 hours, use ATV digestion, to reach another culture dish at 1: 1, add 600 μ g/mL G418 screening behind the 48h, cell began death in the 3rd day, the back was changed a nutrient solution in per three days and was added 600 μ g/mL G418 and continues screening, a large amount of cell vacuolations after 9 days, the 14th day, untransfected group cell is all dead under the G418 effect, and transfection group still has than many cells, G418 concentration is reduced to 300 μ g/mL keep screening, observe every day, occurs until positive colony; After using the filter paper method with positive clone digestion, use infinite dilution method sample that positive colony is separated into individual cells again, and it is planted in 96 orifice plates, every porocyte quantity≤2, the hole of the cell in the every hole of mark, 37 ℃, 5% CO
2Enlarged culturing in the incubator is observed behind the 72h, will transfer to 48 orifice plates by clone's digestion that a cell proliferation obtains, and by that analogy, obtains anti-G418 positive cell.
(4) with positive cell external continuously in 37 ℃, 5% CO
2Cultivate in the incubator, cell presents the growth of paving stone sample monolayer adherence after the sorting, and there is contact inhibition in iuntercellular, cultivates to surpass the clone that 50 generations promptly get immortalization.
3. the establishment method of chitterlings epithelial cell line according to claim 2 is characterized in that, described pCI-neo-hTERT imports former chitterlings epithelial cell of being commissioned to train foster and carries out transfection, specifically comprises the following steps:
(1) transfection is preceding 48 hours, and ATV digestion reaches the chitterlings chrotoplast and the counting in the 3rd generation, with 1 * 10
4Individual cells/well is inoculated in 12 orifice plates, makes cell can reach 80%～90% degree of converging the same day in transfection;
(2) transfection is preceding 24 hours, changes the high sugared DMEM nutrient solution of fresh serum-free;
(3) with serum-free D-MEM/F-12 nutrient solution dilute 2 μ L, 4 μ L, 6 μ L respectively, 8 μ LpCI-neo-hTERT plasmid to volumes are 50 μ L, plasmid concentration reaches 0.01 μ g/ μ L～0.04 μ g/ μ L, mixing gently, room temperature effect 5 minutes;
(4) be 50 μ L with serum-free D-MEM/F-12 nutrient solution dilution Lipofectamine 2000Reagent 4 μ L to volume, mixing gently, room temperature effect 5 minutes;
(5) Lipofectamine 2000 of the plasmid of mixed diluting and dilution, this moment, cumulative volume was 100 μ L, mixing gently, room temperature effect 20 minutes;
(6), add 400 μ L serum-free D-MEM/F-12 nutrient solutions with the old nutrient solution sucking-off in 12 orifice plates;
(7) dropwise step (5) gained mixture is joined in the different holes limit edged wave and culture plate, mixing gently; Set up 2 hole untransfected blanks simultaneously;
(8) in 37 ℃, 5% CO
2Hatched in the incubator 4～6 hours, and discarded nutrient solution, add complete D-MEM/F-12 nutrient solution.
4. the establishment method of chitterlings epithelial cell line according to claim 3, it is characterized in that, described complete D-MEM/F-12 nutrient solution: buy from the Dulbecco ' s of the GIBCO of invitrogen company Modified Eagle Medium:Nutrient Mixture F-12 (Ham) (1: 1) substratum, add 5% foetal calf serum.
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| CN104726409A (en) * | 2013-12-19 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Preparation method and applications of immortalized duck embryo hepatic cell line |
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| CN109554394A (en) * | 2018-11-23 | 2019-04-02 | 佛山科学技术学院 | A kind of conditionity inducing expression AsCpf1 slow virus carrier and its construction method and the application in being is built in chitterlings epithelial cell |
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| CN101255407A (en) * | 2008-02-04 | 2008-09-03 | 首都医科大学 | Immortalized rat marrow stroma cell system and preparation method thereof |
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Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726409A (en) * | 2013-12-19 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Preparation method and applications of immortalized duck embryo hepatic cell line |
| CN104726409B (en) * | 2013-12-19 | 2017-12-29 | 普莱柯生物工程股份有限公司 | A kind of preparation method and application of the duck embryos hepatic cell line of immortalization |
| 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 |
| CN104531607B (en) * | 2014-12-29 | 2017-11-07 | 南京农业大学 | The primary bronchiole epithelial cell of dog and its application in immortalized cells is prepared |
| CN105462931A (en) * | 2015-12-16 | 2016-04-06 | 山东省农业科学院畜牧兽医研究所 | Porcine intestinal epithelial cell line and application thereof |
| CN105483089A (en) * | 2016-01-15 | 2016-04-13 | 张彦明 | Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof |
| CN106676059A (en) * | 2016-12-23 | 2017-05-17 | 湖南师范大学 | Piglet small intestine epithelial cell classification and separation method |
| CN106676059B (en) * | 2016-12-23 | 2019-11-15 | 湖南师范大学 | A kind of Small Intestine of Piglets epithelial cell fractionation method |
| 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 |
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| CN107653201A (en) * | 2017-08-31 | 2018-02-02 | 浙江美保龙生物技术有限公司 | A kind of lawsonia intracellularis separation method |
| CN107653201B (en) * | 2017-08-31 | 2021-03-16 | 浙江美保龙生物技术有限公司 | Lawsonia intracellularis separation method |
| CN109554394A (en) * | 2018-11-23 | 2019-04-02 | 佛山科学技术学院 | A kind of conditionity inducing expression AsCpf1 slow virus carrier and its construction method and the application in being is built in chitterlings epithelial cell |
| CN109628404A (en) * | 2018-12-18 | 2019-04-16 | 浙江大学 | The construction method and purposes of Preadipocyte immortalized cell line under pigskin |
| CN109628404B (en) * | 2018-12-18 | 2020-04-28 | 浙江大学 | Construction method and application of porcine subcutaneous adipocyte precursor immortalized cell line |
| CN109679893A (en) * | 2019-01-17 | 2019-04-26 | 浙江工商大学 | Enteric epithelium monolayer cultivation and characterizing method based on mouse intestinal stem cell |
| CN111454876A (en) * | 2019-01-18 | 2020-07-28 | 安徽农业大学 | Method for infecting porcine small intestinal mucosal epithelial cell line by porcine epidemic diarrhea virus |
| CN110004144A (en) * | 2019-03-06 | 2019-07-12 | 中国农业大学 | The PGC promoter and its application of chitterlings epithelial cell expression |
| CN115161285A (en) * | 2022-03-09 | 2022-10-11 | 河南科技大学 | Immortalized Holstein bull calf small intestine epithelial cell line and construction method and application thereof |
| CN115161285B (en) * | 2022-03-09 | 2024-06-04 | 河南科技大学 | Immortalized Holstein male calf small intestine epithelial cell line and construction method and application thereof |
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