CN104388391B - Mice paneth's cell hybridoma cell strain, preparation method and applications - Google Patents
Mice paneth's cell hybridoma cell strain, preparation method and applications Download PDFInfo
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- CN104388391B CN104388391B CN201410578367.1A CN201410578367A CN104388391B CN 104388391 B CN104388391 B CN 104388391B CN 201410578367 A CN201410578367 A CN 201410578367A CN 104388391 B CN104388391 B CN 104388391B
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Abstract
The invention discloses one plant of mice paneth's cell hybridoma GCLP cell strain, the preparation method and application of mice paneth's cell hybridoma GCLP cell strains, the cell strain that the present invention is provided can not only be passed in persistence under cell culture condition, also there is paneth's cell physiological function, mice alexin, lysozyme and digestive tract stem cell somatomedin can be secreted.
Description
Field that the present invention belongs to：
The invention belongs to hybridoma technology field, specifically, the present invention relates to mice paneth's cell hybridoma cell strain,
Prepare the application of the method and mice paneth's cell hybridoma cell strain of mice paneth's cell hybridoma cell strain.
Technical background
Paneth's cell is a kind of cell of many animal alimentary canals, be recent studies indicate that：The cell has secretion dynamic
The ability of thing specificity alexin, lysozyme and digestive tract stem cell factor etc. material, these things secreted by which
Mass-energy kills pathogenic microorganism and regulation and control digestive tract cell differentiation, to maintaining gastral health to play extremely important effect
（Tao Kaizhong, Tang Qingjuan, Zheng Ping. paneth's cell progress, modern biomedical progress, 2009,9（4）：794-800）.
At present, with regard to paneth's cell research more adopt histological method, such as immunohistochemical analysis, separation and Culture Pan Shi
Cell has no report.Paneth's cell system for experimentation mostly is the canceration paneth's cell of human or animal, such as T-84 and Caco-
2 etc.（Kuang Wei, State of Zhao's fine jade. several special Intestine Epithelial Cell Lines. Chinese herding magazine, 2007,43（13）：41-43）.It is this kind of
Cell line is got by long-term in vitro culture domestication, does not often possess the secretory function of normal paneth's cell（Discipline Hua Ying, it is old
Its Kui, Zeng Hui. the primary culture in vitro of small intestinal epithelial cells, Chinese herding magazine, 2010,16（9）：1417-
1419）；And from tissue detached paneth's cell cannot long-term cultivation, this causes the research of paneth's cell and using by very
It is big to limit.
Set up animal immortality cell line passage cell to be usually taken and builds is that the method for being is built with cancerous tumor cell（Xue Qingshan.
The philosophy and technique of In vitro culture. Beijing：Science Press, 2001）.Both approaches need longer time or special cancer
Become tissue.In body, the external indissolubility culture such as some well differentiated terminal cell such as lymphocytes is extremely difficult, and sets up
Cancerization cell and normal cell there is essential distinction, have many limitation under study for action（Horse Yulong, Xu Zirong, Guo Tong,
Deng. the separation of Intestinum Gallus domesticus epithelial cell and primary culture method. Chinese veterinary's journal, 2007,27（1）：74- 80.）.
Inventive technique content：
It is an object of the invention to provide a kind of mice paneth's cell hybridoma cell strain, the cell strain can not only be trained in cell
Under the conditions of supporting, persistence is passed on, and also with paneth's cell physiological function, can secrete mice alexin, lysozyme and digestive tract dry thin
Born of the same parents' somatomedin.
It is also another object of the present invention to provide a kind of method for preparing mice paneth's cell hybridoma GCLP cell strains.
It is also another object of the present invention to provide the application of mice paneth's cell hybridoma GCLP cell strains.
The invention discloses one plant of new hybridoma cell strain, its feature is being：Pan Shi of the cell strain by mouse jejunum
Cell and SP2/0 cell hybridizations are merged and are obtained, and can pass in persistence under cell culture condition, can secrete mice alexin, molten
Bacterium enzyme and digestive tract stem cell somatomedin, the cell are mice paneth's cell hybridoma GCLP cell strains, are preserved in China
Type Tissue Collection, deposit number are CCTCC C2014195, and preservation date is on October 24th, 2014.Chinese Typical Representative
Culture collection abbreviation CCTCC, is preservation mechanism that Patent Office of the People's Republic of China specifies, positioned at Wuhan City, Hubei Province Wuhan University
In the school, postcode 430072, network address：Www.cctcc.org, phone：027-68752319, Email:cctcc@
whu.edu.cn.After the present invention is disclosed, nonprofit researcher can ask for this microorganism to collection in accordance with the law
Strain reproduces the present invention.
The invention discloses the method that mice paneth's cell hybridoma GCLP cell strains are set up, the method includes following step
Suddenly：
（1）The digestion of mouse jejunum digestive tract endo cell is separated：After mouse jejunum is removed, with trypsin, neutral egg
The mixture of white enzyme and collagenase is digested, and is flushed out discrete endo cell is digested with cell culture fluid after fully digesting
Come, be configured to the cell suspension of proper density；
（2）Cell fusion：Cell suspension and murine myeloma cell SP2/0 mixed in equal amounts, make fusion agent with PEG, conventional
Method merges, and is inoculated with fused cell, makees screening culture medium with HAT, obtain in the feeder layer made of Raw264.7 cells
Obtain digestive tract endo cell hybridoma group；
（3）The separation and Culture of hybridoma cell strain and screening：Different fused cell clones are suctioned out with thin suction pipe, is trained respectively
Supporting carries out continuous passage in the culture hole of Raw264.7 feeder layers is covered with, and parallel sample carries out phloxine dyeing, stays
The cell clone of the parallel sample of lower those stained positives continues culture, and taking parallel sample carries out the paneth's cell of CK-18 antibody
Specific antigen Testing and appraisal, the fusion cell line with paneth's cell specific antigen of selecting and remain are thin as paneth's cell hybridoma
Born of the same parents' strain continues to cultivate and preserves, and builds up paneth's cell hybridoma cell strain.
In the present invention, PEG refers to Polyethylene Glycol, the abbreviation of English name polyethylene glycol, HAT be containing H-
Hypoxanthine hypoxanthine, A-Aminopterin methotrexates, T --- Thymidine Thymidines
Selective agar medium.Paneth's cell in the present invention refers to the epithelioid cell in small intestinal.
The invention also discloses the application of new paneth's cell hybridoma cell strain.
Murine myeloma cell SP2/0 in the present invention, Raw264.7 feeder cells can be by being given birth to by commercially available business
Thing company or Spawn preservation organization are obtained, and such as China typical culture collection center preservation has the cell, and researcher can be in this
The heart is asked for.
Advantages of the present invention：Compare in prior art, the present invention has the following advantages：
Mice Pan Shi fusion GCLP cell strains can not only be passed in persistence under cell culture condition, also with paneth's cell
Physiological function, can secrete mice alexin, lysozyme and digestive tract stem cell somatomedin.The cell strain gram that the present invention is provided
The situation that hardly possible under ex vivo situation have studied paneth's cell has been taken, and substantial amounts of sample number has been provided for long-term lasting research.
Mice paneth's cell hybridoma GCLP cell strains are the medicine function and thus of developing paneth's cell secretions
Newtype drug is developed there is provided means of production basis.
Description of the drawings
Fig. 1 is the mouse jejunum endo cell group phloxine dyeing picture figure that enzymic digestion is obtained
Fig. 2 is the fused cell clone figure obtained after blind hybridization
Fig. 3 is the Pan Shi fusion cell lines phloxine dyeing picture figure that Jing screenings are obtained
Fig. 4 is Pan Shi fusion cell line karyotyping figures
Fig. 5 is that Pan Shi fusion cell line paneth's cell specific antigen histochemistry detection figure A represent Pan Shi fused cell B
Represent negative cells control
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.It should be appreciated that these embodiments are merely to illustrate
The present invention, and can not limit the scope of the invention.
The foundation of 1 mice paneth's cell hybridoma cell strain of embodiment
The preparation of 1.1 main solutions and vessel
1.1.1 contain dual anti-PBS solution：Penicillin and streptomycin is added in conventional PBS solution so as to which concentration is respectively reached
200 units/ml and 200mg/ml.
1.1.2 0.5% tryptic digestive juice：Trypsin solution is prepared with the PBS solution added with 0.04EDTA, pancreas is made
Final concentration of the 0.5% of protease.
1.1.3 0.25% tryptic digestive juice：Trypsin solution is prepared with the PBS solution added with 0.02EDTA, is made
Tryptic middle concentration is 0.25%.
1.1.4 Collagenase+neutral enzymatic mixture slaking liquid：Collagen protein is prepared respectively with plasma-free DMEM medium
I concentrated solution of Ⅺ type of enzyme and neutral protease, its concentration are respectively 2000u/ml and 0.1mg/ml, with front by the two mixed in equal amounts
Afterwards, 10 times of dilutions are made.
1.1.5 DMEM complete mediums：Hyclone is added in DMEM basal mediums so as to which final concentration reaches
15%。
1.1.6 other cellar culture solution
1.1.7 flush end injection needle：No. 16 injection needle tips are flat by saw and polish smooth, and needle tubing length is maintained at 2cm
More than.
1.1.8 colligation cotton rope：The thin cotton thread for disinfecting
1.1.9 zoopery operating theater instruments
1.1.10 plate, centrifuge tube, suction pipe etc. other separate cell vessel.
1.1.11 all solution Jing 0.2um filtration sterilizations, the process of all vessel Jing autoclave sterilizations.
1.1.12 aseptic bubbler：Saline bottles of the 100ml with flanging rubber plug is taken, a flush end injection on bottle stopper, is inserted
Syringe needle, loads saline or PBS solution, makes liquid level about 2cm with a distance from bottleneck in bottle, load in the plastic bag of high temperature high voltage resistant, prick
Tight bag mouth, sterilizes in putting autoclave, takes out and tighten plastic bag in bottleneck in passing after cooling, with being front preheated to 37 DEG C.
1.2 mices prepare：It was animal of drawing materials to choose 28 age in days kunming mices, using first hungry 24 hours.
The preparation of 1.3 feeder layer cells：The Raw264.7 cells in exponential phase are chosen, Jing 10ug/ml work is dense
The ametycin of degree is processed 3 hours, and digestion adjusts cell density to 10 with culture medium after dispelling5Individual/ml, is inoculated in 96 orifice plates
In, can be used for cultivating hybridoma after 24 hours.
The digestion of 1.4 mouse jejunum endo cells is separated：
1.4.1 jejunum sampling：The disconnected neck of the kunming mice of 28 ages in days is put to death, and sterilizes about 5 minutes in being soaked in 75% ethanol,
Sterilizing room superclean bench is faced upward and is put in the plate of more than 9cm specifications, breaks stomach wall, is separated jejunum with ophthalmic tweezers, is used operating scissorss
From the jejunum of about 5 centimeter length of middle part clip, it is put in another plate.
1.4.2 rinse：Flush end injection needle is mounted on 50ml syringes, about 50ml is drawn and is contained dual anti-PBS solution, note
One end insertion about 0.5cm depth of the needle ejecting tube from jejunum, clamps insertion site with ophthalmic tweezers, and upper decant(-ation) emitter makes jejunum all outstanding
Sky, pushes hard syringe plunger so as in PBS solution quickly through jejunum enteric cavity, content therein is developed, so
Afterwards jejunum is put in another sterilized petri dishes, injection needle is inserted from the other end of jejunum Jing after cotton ball soaked in alcohol cleaning disinfection, weight
Multiple said process back flush jejunum, is so repeated 3 times the above, after the completion of last time is rinsed,
1.4.3 digestion：37 DEG C of aseptic bubbler will be pre-heated to, remove the plastic bag packaging outside which, remove be inserted with it is flat
The bottle stopper of end injection needle, inserts the needle into jejunum one end, is tightened on syringe needle with cotton rope, takes the syringe of a 5ml, draws about
The Collagenase of 2ml+neutral enzymatic mixture slaking liquid, connecting needle first touch syringe plunger so as to inject about in intestinal tube
The Digestive system of 1ml rinses intestinal tube, with the other end of Thread ligation intestinal tube, is further continued for pushing away syringe plunger, by remaining Digestive system
Push in intestinal tube（Stop injecting when intestinal tube is fully filled, in case pressure is excessive to burst intestinal tube）, bottle stopper is vertically above carried, by intestinal tube
Slowly being put in bottle from bottleneck, stoppering bottle stopper, in 37 DEG C, 5%CO2 incubators being put into together with syringe thereon, digestion is about
40 minutes, bubbler is taken out, takes the syringe of a 10nl, drawn 10mlDMEM complete mediums, change the syringe on bottle stopper,
Bottle stopper is gently extracted, intestinal tube is gently vertically proposed, is moved to above a 10ml centrifuge tube mouths of pipe, intestinal tube is gently cut off with eye scissorss
Ligation end, pushes hard syringe plunger so as to which middle culture medium gets intestinal tube express developed, and flushing liquor is collected in centrifuge tube, and this is
First time peptic cell suspension, then repeats above-mentioned digestion process with 0.5% trypsin solution, and digestion time is 15 minutes,
Postdigestive intestinal tube is rinsed with the cell suspension of first time digestion, secondary digestion cell suspension is obtained, is blown and beaten repeatedly carefully with suction pipe
Born of the same parents' suspension so as in piece of tissue it is fully dispersed for individual cells, with the filter cloth of 200 mesh by the cell suspension after piping and druming filter in
In another 10ml centrifuge tubes, counted under microscope individual cells therein.
1.4.3 cell collect and with oncocyte hybrid fusion：The SP2/0 cells taken in exponential phase are some, gently
Dispel and cell is counted in cell counting count board, and calculate the cell density in suspension, according to jejunum endo cell sum：
SP2/0 total cellular score=1:Both cell suspension are mixed by 1 ratio, and mixed liquor is loaded in a centrifuge tube being of moderate size
It is centrifuged 5 minutes with 1000 revs/min of rotating speed, abandons supernatant, and blot the liquid in centrifuge tube with aseptic cotton carrier as far as possible, leave ttom of pipe
Cell deposition thing.Cell fusion PEG0.4ml, about 1 minute used time, immediately with 1000 are slowly added to in cell deposition thing
Rev/min rotating speed be centrifuged 5 minutes, add 10mlDMEM complete mediums immediately after taking out centrifuge tube, then turned with 1000 revs/min
Speed centrifugation 5 minutes, abandons supernatant, leaves ttom of pipe cell deposition thing.
1.4.4 the culture of hybridoma：Adjusted with the cell deposition thing of the resuspended ttom of pipe of hybridoma selective medium
Ganglion cell's density is to 104Individual/ml.96 well culture plates for being covered with Raw264.7 feeder layer cells are sucked into culture medium, hybridoma is used
Cell suspension carries out bed board again to the culture plate, is put in 37 DEG C, cultivates in 5%CO2 incubators.
1.5 filtering hybridoma
1.5.1 the separation and Culture of hybridoma cell clone：After cell culture after bed board 10 days, it can be seen that many
Merge successful cell to grow in cloning.It is attenuated with tip and the bent pasteur pipet of stretch bending, will be grown under the microscope
All hybridoma cell clones are individually suctioned out, and are cultivated to single being lined with 96 orifice plates of Raw264.7 feeder layer cells and are continued
Culture, each clone are put in same hole, allow its continued growth to expand.Cell is covered with after culture hole by 1:2 ratio is passed on, even
After resuming 3-5 generations, depart from feeder layer cells and continue to pass on, routinely hybridoma passes on operation and continues to pass on later.
1.5.2 the identification of hybridoma with select and remain：When individual cells clone is passaged to 2-4 holes cell, to wherein one
Hole carries out phloxine dyeing, leaves the cell clone of those phloxine stained positives and continues training as the primary dcreening operation clone that selects and remain
Support, and they be respectively classified into into 2 pieces of culture plates, one of cell is used for paneth's cell specific antigen Immunofluorescence test,
The cell clone of the corresponding another piece of culture plate of positive cell is selected and remain and is continued culture, and the culture of 48 hours is further passed on to which
Alexin, lysozyme content in liquid detected, using content highest cell clone as final hybridoma stay after
Continuous culture.
1.5.3 the foundation of hybridoma cell strain cell bank：Continue culture according to conventional hybridization oncocyte culture operation final
Hybridoma under selecting and remain, until cell propagation is to sufficient amount, then according to cell bank sets up program by the cell liquid nitrogen
It is frozen, build up can indissolubility application cell bank.
The Secondary Culture of 2 GCLP cells of embodiment
It is just the same with ordinary hybridomas cell culture operations, continuous expanding propagation will be carried out after the GCLP cell thawings of preservation
Pass on, every 5 generations, the content of detection mice paneth's cell specificity alexin α 5 is sampled to culture supernatant, as long as its content
Then visual cell culture is normal to reach more than 50ng/ml, and hereditary change does not occur in hybridoma, should otherwise check incubation with
Cell chromosome.The speed of growth of cell after 10 generations of continuous culture, cellular morphology are unchanged, cell supernatant mice paneth's cell
The content of specificity alexin α 5 is changed between 62-80ng/ml all the time, it was demonstrated that the cytogenetics stable performance, can conduct
Paneth's cell is used in exchange cell strain.
Application of the 3 GCLP cells of embodiment in prevention newborn piglet diarrhoeal diseasess
3.1 cells prepare：According to the method for conventional hybridization oncocyte culture by GCLP cell culture to required cell concentration,
The content that supernatant sample determines its mouse beta-alexin 1 element is taken, standard is reached and is qualified supernatant, can be collected for subsequently should
With.
The process of 3.2 supernatant：After cell growth 48 hours, supernatant is gently collected（Because hybridoma it is adherent loosely
Gu, vibrate excessive when receiving supernatant, easily make cell subsequent operation be affected in being suspended in supernatant in a large number）, more than 1800 revs/min
Rotating speed is centrifuged 10 minutes, takes supernatant, and 0.22um membrane filtrations, filtrate are used as the enforcement sample of prevention of diarrhea in piglets disease.
The selection of 3.3 test pigs：On piglet diarrhoea prevalence pig farm, 10 nest of piglet being born on the same day is chosen, is divided into experiment
Group and matched group, test group pig the 3rd -the between 7 days after birth, the daily morning give the GCLP that every piglet is fed after processing thin
Born of the same parents supernatant 10ml, matched group row conventinal breeding.The diarrhoea incidence and death condition of observation 2 groups of piglets of statistics.According to upper
Method is stated, is tested on Wuhan pig farm respectively between in December, 2013 and 2 months 2014, is as a result shown：Experimental group piglet diarrhea
Incidence rate and the mortality rate arrived because of diarrhoea are respectively 3% and 2%.And matched group piglet diarrhea incidence rate and mortality rate are then respectively
87% and 76%.During test, the matched group of the piglet diarrhoea sickness rate, mortality rate and this test of other swinerys of pig farm is basic
Quite, this illustrates that the GCLP cell culture supernatants used by this experiment have good prevention and control effect to newborn piglet diarrhoeal diseasess.
Claims (2)
1. a strain of hybridoma strain, its feature is being：Paneth's cell and SP2/0 cell hybridization of the cell strain by mouse jejunum
Merge and obtain, can pass in persistence under cell culture condition, mice alexin, lysozyme and digestive tract stem cell can be secreted and promoted
Somatomedin, the cell are mice paneth's cell hybridoma GCLP cell strains, are preserved in China typical culture collection center, are protected
It is CCTCC C2014195 to hide numbering.
2. application of the mice paneth's cell hybridoma GCLP cell strains described in claim 1 in antibacterials production.
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| CN108998405A (en) * | 2018-06-15 | 2018-12-14 | 翁炳焕 | A kind of preparation of HCV-RNA detection monoclonal Quality Control Reference Strains |
| CN109022416A (en) * | 2018-06-15 | 2018-12-18 | 翁炳焕 | The preparation of HBV-DNA detection monoclonal Quality Control Reference Strains |
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| CN103173413A (en) * | 2012-12-07 | 2013-06-26 | 天津三箭生物技术有限公司 | Mouse anti-human beta-catenin monoclonal antibody and hybridoma cell strain for secreting monoclonal antibody |
| CN103289962A (en) * | 2012-12-07 | 2013-09-11 | 天津三箭生物技术有限公司 | Mouse anti-human CD23 monoclonal antibody and hybridoma cell strain secreting monoclonal antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173413A (en) * | 2012-12-07 | 2013-06-26 | 天津三箭生物技术有限公司 | Mouse anti-human beta-catenin monoclonal antibody and hybridoma cell strain for secreting monoclonal antibody |
| CN103289962A (en) * | 2012-12-07 | 2013-09-11 | 天津三箭生物技术有限公司 | Mouse anti-human CD23 monoclonal antibody and hybridoma cell strain secreting monoclonal antibody |
Non-Patent Citations (1)
| Title |
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| 潘氏细胞研究进展;陶凯忠 等;《现代生物医学进展》;20091231;第9卷(第4期);794-800 * |
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