CN103756952B - The structure of a kind of Cynoglossus semilaevis ovary cell line and application process - Google Patents
The structure of a kind of Cynoglossus semilaevis ovary cell line and application process Download PDFInfo
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Abstract
It is an object of the invention to provide the structure of Cynoglossus semilaevis ovary tissue lines, comprise the following steps that 1) aseptically remove Cynoglossus semilaevis ovary tissue outermost tunic, tissue is cut into small pieces by culture fluid, piece of tissue PBS solution is rinsed, digest with trypsin solution again, containing cell in solution after trypsinization, cell mass and do not digest piece of tissue completely；2) solution centrifugal is removed supernatant pancreatin, precipitate by culture fluid suspension cell, cell mass and small tissue blocks；After making suspension, it is inoculated in the culture plate handled well with NTx, adds culture fluid and be placed in 24 DEG C of biochemical cultivation cases cultivation, complete the structure of ovary cell line through Secondary Culture.The construction method of the present invention is to utilize Cynoglossus semilaevis ovary to be organized into merit to establish Cynoglossus semilaevis ovary tissue lines, and the cell line of foundation can be with continuous passage, and the passage cell obtained through this method can pass 60 generations more than, be provided that substantial amounts of gonad cell.
Description
Technical field
The invention belongs to seawater fish technical field of cell culture, be specifically related to a kind of seawater fish Cynoglossus semilaevis
The structure of (Cynoglossus semilaevis) ovarian tissue cells system and application process.
Background technology
Cell type in Fish ovary tissue predominantly sexual cell and be centered around somatic cell about.Reproduction
Interacting between cell and somatic cell plays important work in the Sex determination and the development of ovary of Fish early stage
With.In ovary somatic Main Function be for sexual cell provide nutrition and synthesize substantial amounts of steroid swash
The self renewal and the Development And Differentiation that usually regulate ovum are ripe ovum.
Therefore the foundation of ovary somatic cell system is for studying the self renewal of ovum and being divided into mature egg
Regulatory mechanism has an important theory significance, and cell line can normal expression ovary exceptional function gene,
Thus can be widely applied to the functional verification research of sex related gene, for researching fish sex differentiation and property
Jue Ding not have important practical significance.Additionally, ovary cell line also can be widely applied to virusology, immunity
The research fields such as, genomics, developmental biology and environmental toxicology.
At present, Fish ovary cell line is essentially from freshwater fish in the world, mainly has following: calendar year 2001
India scholar G.Sunil Kumar etc. are by adding fish tissue extracting solution, prawn extracting solution, agglutinin, fat
Polysaccharide, glucose and ovary extracting solution etc. establish Africa Nian ovary cell line, Japanese scholars Shogo in 2013
Higaki etc. by add fish embryo extract, epidermal growth factor, basic fibroblast growth factor and β-
Mercaptoethanols etc. establish Japan's palpus ovary cell line, and B.Crespo in 2013 etc. establish Europe perch ovum
Nest cell injuring model system.But the foundation of seawater fish ovarian tissue cells system is always fish cell training
Difficult point in Yanging, the most only has been reported, up to the present China in atlantic salmon and blueback salmon
Seawater fish ovarian tissue cells system does not still set up.
Cynoglossus semilaevis (Cynoglossus semilaevis) is northern China coastal economy seawater fish, male and female
There is obvious speed of growth difference between individuality, the raun speed of growth is 2～4 times of milter.Additionally, half is sliding
Tongue sole raun has special-shaped W chromosome, belongs to ZW/ZZ type Sex Determination Mechanism, therefore studies half sliding-tongue
Sole Sex Determination Mechanism contributes to disclosing the essence of female milter speed of growth difference, and can be fish sex control
Research processed is provided fundamental basis.Therefore, set up Cynoglossus semilaevis ovary cell line have important theory significance and
Using value.
Summary of the invention
It is an object of the invention to provide the foundation side of a kind of marine fish Cynoglossus semilaevis ovary tissue lines
Method, thus effectively set up Cynoglossus semilaevis ovary tissue lines, for research Cynoglossus semilaevis Sex determination and ovum
Sub-Mature Regulation mechanism provides basis.
The structure of the Cynoglossus semilaevis ovary tissue lines of the present invention, comprises the following steps that
1) the Cynoglossus semilaevis ovary tissue except outermost tunic is aseptically removed, by group in culture fluid
Knit and be cut into small pieces, piece of tissue PBS solution is rinsed, then is 0.25%～0.5% trypsin solution by concentration
Digestion, containing cell in the solution after trypsinization, cell mass and do not digest piece of tissue completely
2) solution centrifugal is removed supernatant pancreatin, with DMEM/F12 special culture solution suspension cell, cell mass
Precipitate with small tissue blocks；After making suspension, it is inoculated in the culture plate handled well with 0.1% NTx, adds
Add culture fluid and be placed in 24 DEG C of biochemical cultivation cases cultivation, complete the structure of ovary cell line through Secondary Culture.
Wherein, culture fluid is containing 20% hyclone (FBS), 100IU/ml penicillin, 100 μ g/ml chains
The people of recombinant human epidermal growth factor (EGF), 10～the 20ng/ml of mycin, 40～60ng/ml recombinates alkali
Property fibroblast growth factor (bFGF), 10～the para-insulin like cell growth factor I of 20ng/ml
(IGF-1) the DMEM/F12 complete culture solution of and 10～20IU/ml human chorionic gonadotropins (HCG).
The trypsin solution digestion of step 1), the suitableeest digestion time is 5～15 minutes；
Step 2) Secondary Culture, the training after the steps include: that gonad cell grows up to monolayer, in sucking-off culture hole
Nutrient solution, adds the trypsin solution of 0.25%, after standing digestion in each culture hole；Suck trypsin
Solution, adds culture fluid in each culture hole, gonad cell suspension made by clock at the bottom of piping and druming culture hole；Take out ovum
Nest cell suspension, joins in new culture hole, and each culture hole is added culture fluid and cultivated.
The construction method of the present invention is to utilize Cynoglossus semilaevis ovary to be organized into merit to establish Cynoglossus semilaevis ovary tissue
Cell line, the cell line of foundation can be with continuous passage, and the passage cell obtained through this method can pass 60
More than Dai, it is provided that substantial amounts of gonad cell.Can be widely used for the transfection of exogenous gene, plasmid, sex phase
Close the checking of functional gene it can also be used to the breeding of Fish common virus and Interaction between virus and cell are machine-processed
Research, it is also possible to carry out environmental pollution analyte detection etc., there is important actual application value.
Accompanying drawing explanation
Fig. 1: Cynoglossus semilaevis ovary cell line cell is primary and passes on light microscopic photo；
Wherein: A, Cynoglossus semilaevis ovary cell primary grows up to monolayer；B, Cynoglossus semilaevis ovary cell the 20th
Generation；C, Cynoglossus semilaevis ovary cell the 60th generation, scale=50 μm；
Fig. 2: Cynoglossus semilaevis ovary cell line cell cryopreservation resuscitation light microscopic photo；
Wherein: A, light microscopic photo before ovary cell line the 8th generation cell cryopreservation；B, ovary cell line the 8th generation cell
Light microscopic photo after recovery, scale=50 μm；
Fig. 3: Cynoglossus semilaevis ovary cell line cell growth curve；
Fig. 4: Cynoglossus semilaevis ovary cell line cell karyotyping；
Fluorescence photo after Fig. 5: Cynoglossus semilaevis ovary cell line cell transfection pEGFP-N3 plasmid；
Fig. 6: the expression analysis of the sex correlation function genes such as Cynoglossus semilaevis ovary cell line cell p450arom.
Detailed description of the invention
The general steps of construction method of the present invention is as follows:
1. healthy Cynoglossus semilaevis is sterilized 1 minute in 70% ethanol, takes off ovary tissue in aseptic super-clean bench,
Peel off ovary tissue outermost tunic, in 5%FBS-DMEM/F12 complete culture solution, tissue is cut into 1mm3
Fritter, PBS rinse 3 times, 0.25%～0.5% trypsinization 5～15 minutes, (if digestion time is less than
5 minutes then Digestion the most weak, the tissue cell that loosens not cannot digest, if digestion time is longer than
Within 15 minutes, then digest and excessively can make loss cell even death).It is centrifuged 10 points at 1000rpm/min after digestion
Clock removes pancreatin, with the 20%FBS-DMEM/F12 special culture solution suspension cell of 1～1.5ml, makes thin
Born of the same parents' suspension (if culture fluid volume is less than 1ml, cell growing nutrient needs after inoculating, can not be met, if
Culture fluid volume is unfavorable for cell, cell mass and piece of tissue quick wall attaching after then inoculating more than 1.5ml), inoculation
In the six porocyte culture plates that 0.1% NTx is handled well, it is placed in biochemical cultivation case cultivation.
Add special culture solution 1ml after 2.24 hours, after 72 hours, change the old culture fluid of half.Cell is long
Becoming after monolayer and use trypsin digestion Secondary Culture, being passaged to 60 generations more than completes Cynoglossus semilaevis ovary tissue carefully
The structure of born of the same parents system.
3. the preparation of ovary cell line special culture solution: take DMEM/F12 culture medium powder tri-distilled water and dissolve
Rear tune pH value is 7.0, degerming standby with the filtering with microporous membrane of 0.22 μm；Take what 80ml prepared
DMEM/F12 fluid medium, add hyclone 20ml, the people of 4～6 μ g recombinate epidermal growth because of
Son (EGF), 1～2 people's recombination basic fibroblast growth factor (bFGF), 1～2 class islets of langerhans of μ g of μ g
Element like cell growth factor I (IGF-1) and 1000～2000IU human chorionic gonadotropins (HCG).It is
The gonad cell special culture solution of the present invention.Three kinds of somatomedin in culture fluid provided by the present invention and
HCG is the key factor promoting gonad cell division growth, if lacking interim a kind of somatomedin, cell
The speed of growth slows down.
4, the successive transfer culture of Cynoglossus semilaevis ovary cell: after gonad cell grows up to monolayer, in sucking-off culture hole
Culture fluid, in each culture hole add 0.5 milliliter of concentration be the trypsin solution of 0.25%, standing disappears
Change 0.5～1.5 minute；Suck trypsin solution, every culture hole add the above-mentioned special culture solution of 2 milliliters,
Within 1～3 minute, gonad cell suspension is made at the bottom of pipettor piping and druming culture hole；1 milliliter of ovum is taken out from culture hole
Nest cell suspension, joins in new culture hole, and each culture hole adds above-mentioned special culture solution 1 milliliter,
It is allowed to final volume to 2 milliliters；After gonad cell grows up to monolayer again, still carry out with above-mentioned same procedure
Secondary Culture.When cell was cultivated to 3 generation, collect in six orifice plate culture hole by the method for above-mentioned trypsinization
Cell, the cell suspension of every 2 culture hole proceeds to 1 25cm2Tissue Culture Flask continues cultivate, this
Rear propagating method is ibid.
Embodiment 1
Healthy Cynoglossus semilaevis is sterilized 1 minute in 70% ethanol, takes off ovary tissue in aseptic super-clean bench, stripping
From ovary tissue outermost tunic, in 5%FBS-DMEM/F12 complete culture solution, tissue is cut into 1mm3
Fritter, PBS rinse 3 times, 0.25% trypsinization 15 minutes, 1000rpm/min is centrifuged 10 minutes and goes
Fall pancreatin, with the 20%FBS-DMEM/F12 special culture solution suspension cell of 1ml, make cell suspension,
It is inoculated in the six porocyte culture plates that 0.1% NTx is handled well, is placed in biochemical cultivation case cultivation.Join
100ml special culture solution processed, takes DMEM/F12l fluid medium 80ml, adds hyclone 20ml,
1～4 μ g recombinant human epidermal growth factor (EGF), 0.5～1.5 people's recombination basic fibroblast cells of μ g
Somatomedin (bFGF), 0.5～1.5 para-insulin like cell growth factor I (IGF-1), 500～2000IU of μ g
Human chorionic gonadotropin (HCG).Add special culture solution 1ml after 24 hours, change half after 72h special
Use culture fluid.After gonad cell grows up to monolayer, the culture fluid in sucking-off culture hole, in each culture hole
Adding 0.5 milliliter of concentration is the trypsin solution of 0.25%, stands digestion 1 minute；Suck trypsin molten
Liquid, added the above-mentioned special culture solution of 2 milliliters in every culture hole, with at the bottom of pipettor piping and druming culture hole 2 minutes
Make gonad cell suspension；From culture hole, take out 1 milliliter of gonad cell suspension, join new culture hole
In, each culture hole adds above-mentioned special culture solution 1 milliliter, is allowed to final volume to 2 milliliters；Treat ovary
After cell grows up to monolayer again, still carry out successive transfer culture with above-mentioned same procedure, when cell reached for 3 generation
Cell is proceeded to 25cm from six well culture plates2In successive transfer culture in the same way.
Embodiment 2
Healthy Cynoglossus semilaevis is sterilized 1 minute in 70% ethanol, takes off ovary tissue in aseptic super-clean bench, stripping
From ovary tissue outermost tunic, in 5%FBS-DMEM/F12 complete culture solution, tissue is cut into 1mm3
Fritter, PBS rinse 3 times, 0.4% trypsinization 10 minutes, 1000rpm/min is centrifuged 10 minutes and removes
Pancreatin, with the 20%FBS-DMEM/F12 special culture solution suspension cell of 1.25ml, makes cell suspension,
It is inoculated in the six porocyte culture plates that 0.1% NTx is handled well, is placed in biochemical cultivation case cultivation；20%
FBS-DMEM/F12 special cultivation compound method is ibid；Special culture solution 1ml, 72h is added after 24 hours
Rear replacing half special culture solution；After gonad cell grows up to monolayer, the culture fluid in sucking-off culture hole, to
Adding 0.5 milliliter of concentration in each culture hole is the trypsin solution of 0.25%, stands digestion 0.5 minute；
Suck trypsin solution, every culture hole adds the above-mentioned special culture solution of 2 milliliters, blows and beats with pipettor
Within at the bottom of culture hole 1 minute, make gonad cell suspension；From culture hole, take out 1 milliliter of gonad cell suspension, add
Entering in new culture hole, each culture hole adds above-mentioned special culture solution 1 milliliter, is allowed to final volume to 2
Milliliter；After gonad cell grows up to monolayer again, still carry out successive transfer culture, cell with above-mentioned same procedure
When reaching for 3 generation, cell is proceeded to 25cm from six well culture plates2In successive transfer culture in the same way.
Embodiment 3
Healthy Cynoglossus semilaevis is sterilized 1 minute in 70% ethanol, takes off ovary tissue in aseptic super-clean bench, stripping
From ovary tissue outermost tunic, in 5%FBS-DMEM/F12 complete culture solution, tissue is cut into 1mm3
Fritter, PBS rinse 3 times, 0.25% trypsinization 20 minutes, 1000rpm/min is centrifuged 10 minutes and goes
Fall pancreatin, with the 20%FBS-DMEM/F12 special culture solution suspension cell of 1.5ml, make cell suspension,
It is inoculated in the six porocyte culture plates handled well, is placed in biochemical cultivation case cultivation；20%
FBS-DMEM/F12 special cultivation compound method ibid adds special culture solution 1ml, after 72h after 24 hours
Change half special culture solution.After gonad cell grows up to monolayer, the culture fluid in sucking-off culture hole, to often
Adding 0.5 milliliter of concentration in individual culture hole is the trypsin solution of 0.5%, stands digestion 1 minute；Suck
Trypsin solution, adds the above-mentioned special culture solution of 2 milliliters in every culture hole, cultivate with pipettor piping and druming
Within at the bottom of hole 3 minutes, make gonad cell suspension；From culture hole, take out 1 milliliter of gonad cell suspension, join
In new culture hole, each culture hole adds above-mentioned special culture solution 1 milliliter, is allowed to final volume to 2 millis
Rise；After gonad cell grows up to monolayer again, still carrying out successive transfer culture with above-mentioned same procedure, cell passes
During to 3 generation, cell is proceeded to 25cm from six well culture plates2In successive transfer culture in the same way..
The Cynoglossus semilaevis ovary cell line that present embodiment 1 is set up reached for 72 generations, growth division vigorous (as
Fig. 1, shown in 3), within every 3～4 days, pass on once, after cryopreservation, activity is basically unchanged (such as Fig. 2 institute
Show)；And still there is normal diploid karyotype (as shown in Figure 4), can normal expression ovary tissue
The functional genes such as P450arom (as shown in Figure 6), can be widely used for the transfection of exogenous gene, plasmid, sex
The checking of correlation function gene is it can also be used to the breeding of Fish common virus and Interaction between virus and cell machine
The research of system, it is also possible to carry out environmental pollution analyte detection etc., there is important actual application value.Utilize
This cell line cell has carried out the transfection of pEGFP-N3 plasmid, and GFP green fluorescent protein has substantial amounts of expression
(as shown in Figure 5), result shows have higher transfection efficiency.Therefore, the Cynoglossus semilaevis that the present invention builds
Ovary cell line has broad application prospects.
Claims (3)
1. a construction method for Cynoglossus semilaevis ovary tissue lines, comprises the following steps that
1) the outermost tunic of Cynoglossus semilaevis ovary tissue is aseptically removed, by tissue shear in culture fluid
Become fritter, piece of tissue PBS solution rinsed, then be 0.25%～0.5% trypsin solution digestion by concentration,
Solution after trypsinization contains cell, cell mass and does not digest piece of tissue completely；
2) solution centrifugal is removed supernatant trypsin, with DMEM/F12 special culture solution suspension cell, carefully
Born of the same parents group and small tissue blocks precipitate；After making suspension, it is inoculated in the culture plate handled well with 0.1% NTx,
Add culture fluid and be placed in 24 DEG C of biochemical cultivation cases cultivation, complete the structure of ovary cell line through Secondary Culture
Build；
Described culture fluid, is to be added with 20% hyclone (FBS), 100IU/ml penicillin, 100 μ g/ml
People's recombinant basic of the recombinant human epidermal growth factor of streptomycin, 40～60ng/ml, 10～20ng/ml becomes
Para-insulin like cell growth factor I (IGF-1) of fibroblast growth factor, 10～20ng/ml and
The DMEM/F12 culture fluid of 10～20IU/ml human chorionic gonadotropins (HCG).
2. construction method as claimed in claim 1, it is characterised in that described step 1) Trypsin
Enzymatic solution digests, and its digestion time is 5～15 minutes.
3. construction method as claimed in claim 1, it is characterised in that described step 2) pass on training
Supporting, after the steps include: that gonad cell grows up to monolayer, the culture fluid in sucking-off culture hole, to each culture hole
The trypsin solution of middle addition 0.25%, after standing digestion；Suck trypsin solution, in each culture hole
Add culture fluid, at the bottom of piping and druming culture hole, make gonad cell suspension；Take out gonad cell suspension, join new
Culture hole in, each culture hole is added culture fluid and is cultivated.
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| CN104152402A (en) * | 2014-08-26 | 2014-11-19 | 中国水产科学研究院黄海水产研究所 | Construction and application method of cynoglossus semilaevis pseudo male gonad cell line |
| CN105861417A (en) * | 2016-06-06 | 2016-08-17 | 中国计量大学 | Establishment method for pomacea canaliculata ovary cell line |
| CN106508733A (en) * | 2016-09-21 | 2017-03-22 | 华南农业大学 | Application of recombining IGF-1 and hCG of epinephlus coioides to jointly promote ovary maturity of epinephlus coioides |
| CN106834207A (en) * | 2016-12-14 | 2017-06-13 | 中国水产科学研究院珠江水产研究所 | A kind of Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method |
| CN110499280B (en) * | 2018-11-26 | 2021-05-11 | 北京市水产科学研究所（国家淡水渔业工程技术研究中心） | In-vitro construction method of Acipenser parvum thecal cell line and reagent used by same |
| CN112877277B (en) * | 2021-03-12 | 2022-09-23 | 集美大学 | Large yellow croaker ovary tissue cell line and application thereof |
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| CN103060259A (en) * | 2012-12-06 | 2013-04-24 | 沙珍霞 | Building method of fish head and kidney tissue derived cell lines |
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