CN107686830A - A kind of grass carp hypothalamus cells extracorporeal culturing method - Google Patents
A kind of grass carp hypothalamus cells extracorporeal culturing method Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, specifically disclose a kind of grass carp hypothalamus cells extracorporeal culturing method, the purifying of grass carp primary hypothalamic cell of the present invention and cultural method, the primary hypothalamic cell that quantity is more, purity is high can be obtained, and the NM nutrient solutions prepared by our applicants can significantly improve obtained grass carp hypothalamus cells vigor condition, the primitive cell culture method can be that further investigation fish neuroendocrine system is taken a firm foundation.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of grass carp hypothalamus cells extracorporeal culturing method.
Background technology
The grass carp fresh-water fishes maximum as China's cultured output (2015 annual productions are 5,680,000 tons), it is in breeding process
It is that germ plasm resource is seriously degenerated to face a subject matter, therefore the seed selection of grass carp new varieties is particularly urgent.Yet with grass carp
The sexal maturity time is longer, and raun, which generally requires 4-5, can reach sexal maturity, seriously constrains the process of its fine-variety breeding.
Therefore disclose the molecular mechanism that grass carp sexal maturity starts regulation and control, find out adjust its gonad development and sexal maturity starts it is crucial because
Son, editor's key factor is oriented by technique for gene engineering on this basis and shortens the grass carp sexal maturity time, is had to breeding grass carp
It is significant.
It is affected by environment that the sexal maturity of fish, which starts,.Sense organ the stimulation of external environment (such as temperature, illumination and
Stimulation by running water etc.) brain is delivered to, hypothalamus is secreted gonadotropin-releasing hormone (GRH) (GnRH), excite pituitary secretion rush property
Glandular hormone (LH＆FSH) acts on sexual gland and promotes sexual gland secretory steroid hormone, to promote gonadal maturation and ovulation to arrange
Essence.The GnRH of hypothalamus secretion plays an important role in fish sexal maturity start-up course among these, but external factor
Be how to regulate and control GnRH in hypothalamus express it is still unclear at present.Fish hypothalamic structures are complicated, studied in body by cell
Between interaction and other neuroendocrine transmitters influence, Isolation and culture grass carp hypothalamus cells turn into research inferior colliculus
The important channel of the expression regulation pattern of brain function and GnRH in hypothalamus.By being separately cultured grass carp hypothalamus cells, build
The in-vitro separation of vertical grass carp hypothalamus cells, cultural method, understand Morphological Features of the hypothalamus cells in vitro under condition of culture
And vigor feature, laid the foundation further to disclose grass carp sexal maturity Initiated Mechanism by vitro study.
Fish cell culture is the same with mammaliancellculture, including two stages of original cuiture and Secondary Culture.It is former
It is commissioned to train to support and refers to directly obtain the culture first that cell done from the histoorgan of animal；Passage cell culture is then to external raw
Long cell is enlarged and subculture.In general cultured cell line increasing with passage number, can gradually lose primary
The biochemical functions characteristic of cell is cultivated, and primary cultured cell still member-retaining portion is even all the same with body cell
Physiological Properties, cell remain in that differentiation and the feature of height, and therefore, primary cultured cell is believed to take
Carry out part life science for living animal.For the application present situation of current cell culture, primitive cell culture is answered
Research field is more broad, is ground in some research fields such as ecotoxicology research, physiology of fishes research and science of heredity
The effect of study carefully etc. is even more that the cell of Secondary Culture can not replace.
Because the physiological structure of fish species, age, different tissues organ is different, the interior environment of different cell lifes also thousand
Poor ten thousand are not, therefore for different fish and method of drawing material, training method and the condition of culture of fish Different Organs material of the same race
Also it is different because of cell.In fish cell original cuiture, the nerve cell of brain tissue is the species of the difficult culture of comparison.In recent years,
Seawater fish brain cell culture achieves obvious progress, establishes cobio brain cell line, grouper brain cell line, lefteye flounder in succession
Brain cell line, turbot brain cell line etc..And the foundation in freshwater fish midbrain cell original cuiture and cell line is more rare；
Foundation for grass carp hypothalamus cells original cuiture and cell line not yet has been reported that at present.The present invention establishes grass carp hypothalamus
The method of cell separation, original cuiture and brain cell line Secondary Culture.
The content of the invention
Object of the present invention is to provide a kind of grass carp hypothalamus cells extracorporeal culturing method, method is simple, easy, institute
Primary cell stable performance is obtained, hypothalamus cells maintain good vigor, and cell growth is than more uniform.
In order to achieve the above object, the present invention takes following technical measures：
A kind of grass carp hypothalamus cells extracorporeal culturing method, comprises the steps：
(1) the healthy fresh grass carp hypothalamus cleaned up is cut into a diameter of 0.5-1.0mm fragment；
(2) by the hypothalamus garbage collection cut into centrifuge tube, the cleaning of WM culture mediums is added；
(3) cleaned hypothalamus fragment is transferred in a new centrifuge tube, adds WM culture mediums and (contain 3-8mg/
Ml trypsin), cleared up in 20-37 DEG C of water-bath；
(4) the WM culture mediums containing Trypsin are sucked, then adds and contain 2-5mg/ml Trypsin inhibitor
WM culture mediums；
(5) the WM culture mediums containing trypsin inhibitor are sucked, addition contains 0.1-0.5mg/ml DNase
II WM culture mediums, it is stored at room temperature to clear up intercellular DNA connections；
(6) the WM culture mediums containing DNase II are sucked, add the BM culture mediums containing 1.5~3.0mM EDTA rock or
It is to siphon away culture medium after standing；
(7) hypothalamus fragment is transferred in new centrifuge tube, BM culture mediums is added into pipe, are gently blown and beaten using suction pipe
Hypothalamus fragment, cell are dispelled, and now will be collected into centrifugation after cell sieve filtering of the upper cell using 40-100 μm of aperture
Guan Zhong；
(8) cell that will filter out is transferred in new centrifuge tube, and 1000-3000rpm centrifugations 10-30min collects cell；
(9) upper strata culture medium is removed, adds PM culture mediums and cell is resuspended；
(10) cell suspending liquid is taken, after adding the blue dyeing of platform phenol, is counted under microscope；
(11) PM culture mediums are added to dilute hypothalamus cells, seeded cells into Tissue Culture Plate, is inoculated with per hole
0.5-3.0×106Cells, add the PM culture mediums containing FBS so that FBS whole content is 5-10% in per hole；
(12) hypothalamus cells cultivated in the PM culture mediums containing 5-10%FBS it is adherent after, culture medium is sucked, added
Enter：
A. the NM culture mediums containing 5-15ng/ml Basic Fibroblast Growth Factors carry out Secondary Culture；Or B. adds NM trainings
Follow-up dosing experiment is carried out after supporting base culture.
In approach described above, it is preferred that the specific method of step (1) includes：Health fresh grass carp hypothalamus be by
The fresh brain tissue of healthy grass carp is placed in WM culture mediums；Hypothalamus is divided from brain tissue in the culture dish containing WM culture mediums
From getting off, the coating on hypothalamus surface is peeled off using tweezers；The hypothalamus of separator well is put into centrifuge tube and adds WM
Culture medium cleans to it；The healthy fresh grass carp hypothalamus cleaned up is put on tissue shredding machine, is cut into
A diameter of 0.5-1.0mm fragment；
In approach described above, it is preferred that in step (12), what it is when addition is given birth to containing 5-15ng/ml basic fibroblasts
When the NM culture mediums of the long factor carry out Secondary Culture, a subculture is changed within every three days；
In the process described above, it is preferred that in step (12), when addition be NM medium cultures after to continue dosing real
When testing, after medicine is dissolved in TM culture mediums, then it is incubated together with cell.
The formula of described WM culture mediums includes：
MEM:10.0-12.0mg/ml,HEPES:6.0-8.0mg/ml,NaHCO3:2.0-3.0mg/ml,BSA:3.0-
5.0mg/ml, penicillin:50-100U/ml, streptomysin：50-100μg/ml；
The formula of described BM culture mediums includes：
S-MEM:10.0-12.0mg/ml,HEPES:6.0-8.0mg/ml,NaHCO3:2.0-3.0mg/ml, BSA:3.0-
5.0mg/ml, penicillin:50-100U/ml, streptomysin：50-100 μ g/ml, described S-MEM are Ca2+freeMEM；
The formula of described PM culture mediums includes：
MEM:10.0-12.0mg/ml,NaHCO3:2.0-3.0mg/ml,HEPES:6.0-8.0mg/ml, penicillin:100-
200 U/ml, streptomysin：100-200μg/ml；
The formula of described TM culture mediums includes：
MEM:10.0-12.0mg/ml,NaHCO3:2.0-3.0mg/ml,HEPES:6.0-8.0mg/ml,BSA:1.0-
3.0mg/ml, penicillin:100-200U/ml, streptomysin：100-200μg/ml；
The formula of described NM culture mediums includes：
MEM:10.0-12.0mg/ml,NaHCO3:2.0-3.0mg/ml,HEPES:6.0-8.0mg/ml, penicillin:100-
200U/ml, streptomysin：100-200 μ g/ml, GlutaMax Supplement:25-50 μM, B27Supplement (50 ×):
2-5%, FBS:5-10%.
Above-described culture medium, preferably：
The formula of described WM culture mediums includes:
MEM:9.6mg/ml,HEPES:6.0mg/ml,NaHCO3:2.2mg/ml,BSA:3.0mg/ml, penicillin:50U/
Ml, streptomysin：50μg/ml；PH=7.7.
The formula of described BM culture mediums includes:
S-MEM:10.4mg/ml,HEPES:6.0mg/ml,NaHCO3:2.2mg/ml,BSA:3.0mg/ml, penicillin:
50U/ml, streptomysin：50μg/ml；PH=7.7.
The formula of described PM culture mediums includes:
MEM:9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml, penicillin:100U/ml, streptomysin：
100μg/ml；PH=7.7.
The formula of described TM culture mediums includes:
MEM:9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml,BSA:1.0mg/ml, penicillin:100U/
Ml, streptomysin：100μg/ml；PH=7.7.
The formula of described NM culture mediums includes
MEM:9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml, penicillin:100U/ml, streptomysin：100
μ g/ml, GlutaMax Supplement:25 μM, 2%B27Supplement (50 ×), 5%FBS；PH=7.7.
Compared with prior art, the present invention has advantages below：
(1) present invention establishes the separation of grass carp hypothalamus cells, original cuiture and Secondary Culture method first, passes through we
Method can quickly obtain a large amount of grass carp hypothalamus primary cultured cells with vigor of stablizing, external further to carry out hypothalamus
Experiment is laid a good foundation；
(2) isolation medium (WM culture mediums, BM culture mediums, PM culture mediums) and in-vitro culture medium used in the present invention
(NM culture mediums and TM culture mediums) is configured according to the characteristic of grass carp nerve cell, extraordinary can meet grass carp hypothalamus
The needs of cell, and cost is cheaply many relative to ready-made culture medium is bought.
Brief description of the drawings
Fig. 1 is different cultivation period grass carp hypothalamus cells schematic diagrames.
Fig. 2 is estradiol to β-actin, TAC3a and TACR3mRNA expression quantity shadows in grass carp hypothalamus primary cultured cell
Ring schematic diagram.
Fig. 3 is the Secondary Culture result cell schematic diagram of grass carp hypothalamus cells.
Embodiment
Technical scheme of the present invention, it is the conventional scheme of this area if not otherwise specified, the reagent or material,
If not otherwise specified, commercial channel is derived from.
Embodiment 1：
A kind of grass carp hypothalamus cells extracorporeal culturing method, comprises the steps：
(1) fresh brain tissue of healthy grass carp is placed in WM culture mediums；
(2) hypothalamus is separated from brain tissue in the culture dish containing WM culture mediums, using tweezers by inferior colliculus
Coating on brain surface peels off；
(3) hypothalamus of separator well is put into addition WM culture mediums in 50ml centrifuge tube to clean three times it；
(4) hypothalamus cleaned up is put on tissue shredding machine, is cut into a diameter of 0.8mm fragment；
(5) by the hypothalamus garbage collection cut into 15ml centrifuge tubes, the cleaning of WM culture mediums is added three times；
(6) cleaned hypothalamus fragment is transferred in new 50ml centrifuge tube, adds 10ml WM culture mediums
(containing 65mg trypsin), clear up 30min in 28 DEG C of water-baths；
(7) the WM culture mediums containing Trypsin are removed using disposable pipette, then adds 10ml and contain 2.5mg/
In ml Trypsin inhibitor WM culture mediums and unreacted trypsin；
(8) the WM culture mediums containing trypsin inhibitor are sucked, added containing 0.1mg/ml DNase II's
WM culture mediums, it is stored at room temperature to clear up intercellular DNA connections；
(9) the WM culture mediums containing DNase II are sucked, adding after the BM culture mediums containing 2.0mM EDTA rock will training
Foster base siphons away；
(10) hypothalamus fragment is transferred in new centrifuge tube, BM culture mediums is added into pipe, are gently blown using suction pipe
Thalamus fragment is laid, cell is dispelled, and now will be collected into centrifuge tube after cell sieve filtering of the upper cell using 45 μm of apertures
In；
(11) cell that will filter out is transferred in new centrifuge tube, and 1000rpm centrifugations 10min collects cell；
(12) upper strata culture medium is removed, adds PM culture mediums and cell is resuspended；
(13) cell suspending liquid is taken, after adding the blue dyeing of platform phenol, is counted under microscope；
(14) PM culture mediums are added to dilute hypothalamus cells, seeded cells into Tissue Culture Plate, the inoculation 3.0 per hole
×106Cells, add the PM culture mediums containing FBS so that FBS whole content is 5% in per hole；Condition of culture：28 DEG C,
5% CO2；
(15) after hypothalamus cells are cultivated 24 hours in the PM culture mediums containing 5%FBS, culture medium is sucked, added
NM culture mediums continue to cultivate 48h.
The hypothalamus cells that as described above the step of is separately cultured；As shown in figure 1, the hypothalamus of separation in first day is thin
Born of the same parents' form is more various, but does not grow nerve synapse；Cell peripheral is gradually grown after adding NM culture medium hot housing within second day
Nerve synapse；By the culture of three days, the hypothalamus cells of the 4th day were paved with whole culture plate, and each iuntercellular passes through god
It is connected with each other through cynapse, now the function of nerve cell is recovered, can carry out next step drug-treated experiment.
The formula of described WM culture mediums includes:
MEM(Gibico):9.6mg/ml,HEPES:6.0mg/ml,NaHCO3:2.2mg/ml,BSA:3.0mg/ml, mould
Element:50U/ml, streptomysin：50μg/ml；PH=7.7.
The formula of described BM culture mediums includes:
S-MEM(Sigma):10.4mg/ml,HEPES:6.0mg/ml,NaHCO3:2.2mg/ml,BSA:3.0mg/ml, it is blue or green
Mycin:50U/ml, streptomysin：50 μ g/ml, described S-MEM are Ca2+FreeMEM, pH=7.7.
The formula of described PM culture mediums includes:
MEM(Gibico):9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml, penicillin:100U/ml, chain
Mycin： 100μg/ml；PH=7.7.
The formula of described TM culture mediums includes:
MEM(Gibico):9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml,BSA:1.0mg/ml, mould
Element:100U/ml, streptomysin：100μg/ml；PH=7.7.
The formula of described NM culture mediums includes
MEM(Gibico):9.6mg/ml,NaHCO3:2.2mg/ml,HEPES:6.0mg/ml, penicillin:100U/ml, chain
Mycin：100 μ g/ml, GlutaMax Supplement:25 μM, 2%B27Supplement (50 ×), 5%FBS；PH=
7.7。
Embodiment 2：
Cultivate the viability examination of cell：
Estradiol (E2) is dissolved in TM culture mediums, is prepared into the drug solution of various concentrations, estradiol concentration is respectively
0.1nM, 1nM, 10nM, 100nM, 1000nM, the hypothalamus cells of culture to the 4th day in embodiment 1 are added, be incubated 24 hours
Afterwards, culture medium is siphoned away, each hole, which adds 0.5ml Trizol, collects cell and simultaneously extract RNA, and reverse transcription is into after cDNA, real-time quantitative
PCR verifies regulating and controlling effect of the estradiol to related gene expression in hypothalamus.Pass through reference gene between detection and analysis discovery each group
β-actin expression quantity does not have significant difference (A in Fig. 2), shows that cell culture system is more stable, can meet medicine
The experiment of processing.In addition, estradiol being capable of TAC3a (B in Fig. 2) and TACR3 (C in Fig. 2) mRNA tables in inducing hypothalamic effects cell
Up to amount, and the standard deviation between every group of four repetitions is controlled within 10%, and the above results show that the hypothalamus of culture is thin
Born of the same parents maintain good vigor, and the cell in each hole compares that growth fraction is more uniform in addition, are suitable as studying the cell of hypothalamus
Model.
Embodiment 3：
The Secondary Culture of grass carp hypothalamus cells：
The step of embodiment 1 (15), after hypothalamus cells are cultivated 24 hours in the PM culture mediums containing 5%FBS, it will train
Foster base is sucked, and adds the NM culture mediums containing 5ng/ml Basic Fibroblast Growth Factors and continues to cultivate, and replacing in every 3 days is once cultivated
Base.
After grass carp hypothalamus cells cultivate two weeks in the NM culture mediums containing 5ng/ml Basic Fibroblast Growth Factors, make
10 minutes collection cells are centrifuged with 0.25%Trypsin vitellophags and with 1000rpm rotating speeds；After cell is resuspended in NM culture mediums,
Take 1/3rd cell to be transferred in new Tissue Culture Flask, add the NM containing 5ng/ml Basic Fibroblast Growth Factors
Culture medium continues to cultivate, and after cell covers with blake bottle, carries out second pass generation；In the manner described above, cell reached for the 9th generation
When, it grows and vigor and primary no significant difference (Fig. 3).
Claims (5)
1. a kind of grass carp hypothalamus cells extracorporeal culturing method, comprises the steps：
（1）The healthy fresh grass carp hypothalamus cleaned up is cut into a diameter of 0.5-1.0 mm fragment；
（2）By the hypothalamus garbage collection cut into centrifuge tube, the cleaning of WM culture mediums is added；
（3）Cleaned hypothalamus fragment is transferred in a new centrifuge tube, added containing 3-8mg/ml trypsin's
WM culture mediums, cleared up in 20-37 °C of water-bath；
（4）WM culture mediums containing Trypsin are sucked, then added containing 2-5 mg/ml Trypsin inhibitor's
WM culture mediums；
（5）WM culture mediums containing trypsin inhibitor are sucked, added containing 0.1-0.5mg/ml DNase II's
WM culture mediums, it is stored at room temperature to clear up intercellular DNA connections；
（6）Suck the WM culture mediums containing DNase II, add the BM culture mediums containing 1.5 ~ 3.0mM EDTA and rock or quiet
Postpone and siphon away culture medium；
（7）Hypothalamus fragment is transferred in new centrifuge tube, BM culture mediums are added into pipe, inferior colliculus is gently blown and beaten using suction pipe
Brain fragment, cell are dispelled, and now will be collected into centrifuge tube after cell sieve filtering of the upper cell using 40-100 μm of aperture
In；
（8）The cell that will filter out is transferred in new centrifuge tube, and 1000-3000 rpm centrifugation 10-30 min collect cell；
（9）Upper strata culture medium is removed, PM culture mediums is added and cell is resuspended；
（10）Cell suspending liquid is taken, after adding the blue dyeing of platform phenol, is counted under microscope；
（11）Add PM culture mediums to dilute hypothalamus cells, seed cells into Tissue Culture Plate, the inoculation 0.5-3.0 per hole
×106Cells, add the PM culture mediums containing FBS so that FBS whole content is 5-10% in per hole；；
（12）Hypothalamus cells cultivated in the PM culture mediums containing 5-10 %FBS it is adherent after, culture medium is sucked, add：
NM culture mediums containing 5-15 ng/ml Basic Fibroblast Growth Factors carry out Secondary Culture；Or add NM medium cultures
After carry out follow-up dosing experiment；
The formula of described WM culture mediums includes:
MEM: 10.0-12.0 mg/ml, HEPES: 6.0-8.0 mg/ml, NaHCO3: 2.0-3.0 mg/ml, BSA:
3.0-5.0 mg/ml, penicillin:50-100 U/ml, streptomysin：50-100 μg/ml;
The formula of described BM culture mediums includes:
S-MEM: 10.0-12.0 mg/ml, HEPES: 6.0-8.0 mg/ml, NaHCO3:2.0-3.0 mg/ml, BSA:
3.0-5.0 mg/ml, penicillin:50-100U/ml, streptomysin：50-100 μ g/ml, described S-MEM are Ca2+freeMEM；
The formula of described PM culture mediums includes:
MEM: 10.0-12.0 mg/ml, NaHCO3: 2.0-3.0 mg/ml, HEPES:6.0-8.0 mg/ml, penicillin:
100-200 U/ml, streptomysin：100-200μg/ml;
The formula of described NM culture mediums includes
MEM:10.0-12.0 mg/ml, NaHCO3:2.0-3.0 mg/ml, HEPES:6.0-8.0mg/ml, penicillin:
100-200 U/ml, streptomysin：100-200 μ g/ml, GlutaMax Supplement:25-50 μM, B27 Supplement
(50×):2-5%, FBS: 5-10%.
2. according to the method for claim 1, step（1）Specific method include：The fresh brain tissue of healthy grass carp is put
In WM culture mediums；Hypothalamus is separated from brain tissue in the culture dish containing WM culture mediums, using tweezers by inferior colliculus
Coating on brain surface peels off；The hypothalamus of separator well is put into addition WM culture mediums in centrifuge tube to clean it；Will
The healthy fresh grass carp hypothalamus cleaned up is put on tissue shredding machine, is cut into the broken of a diameter of 0.5-1.0 mm
Piece.
3. according to the method for claim 1, step（11）In, what it is when addition is given birth to containing 5-15 ng/ml basic fibroblasts
When the NM culture mediums of the long factor carry out Secondary Culture, a subculture is changed within every three days.
4. according to the method for claim 1, step（11）In, when addition be NM medium cultures after continue dosing experiment
When, after medicine is dissolved in TM culture mediums, then it is incubated together with cell；
The formula of described TM culture mediums includes:
MEM: 10.0-12.0 mg/ml, NaHCO3: 2.0-3.0 mg/ml, HEPES: 6.0-8.0 mg/ml, BSA:
1.0-3.0 mg/ml, penicillin:100-200 U/ml, streptomysin：100-200 μg/ml.
5. according to the method for claim 1,
The formula of described WM culture mediums includes:
MEM: 9.6mg/ml, HEPES: 6.0mg/ml, NaHCO3: 2.2mg/ml, BSA:3.0mg/ml, penicillin:
50U/ml, streptomysin：50μg/ml; PH=7.7；
The formula of described BM culture mediums includes:
S-MEM: 10.4mg/ml, HEPES: 6.0mg/ml, NaHCO3: 2.2mg/ml, BSA:3.0mg/ml, mould
Element:50U/ml, streptomysin：50μg/ml; pH=7.7；
The formula of described PM culture mediums includes:
MEM: 9.6mg/ml, NaHCO3: 2.2mg/ml, HEPES:6.0mg/ml, penicillin:100U/ml, streptomysin：
100μg/ml; pH =7.7；
The formula of described TM culture mediums includes:
MEM: 9.6mg/ml, NaHCO3: 2.2mg/ml, HEPES: 6.0mg/ml, BSA:1.0mg/ml, penicillin:
100U/ml, streptomysin：100μg/ml;pH =7.7；
The formula of described NM culture mediums includes
MEM: 9.6mg/ml, NaHCO3: 2.2mg/ml, HEPES:6.0mg/ml, penicillin:100U/ml, streptomysin：
100 μ g/ml, GlutaMax Supplement:25 μM, 2% B27 Supplement (50 ×), 5% FBS；PH=7.7.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109182256A (en) * | 2018-08-31 | 2019-01-11 | 浙江省海洋水产研究所 | A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach |
| CN109182256B (en) * | 2018-08-31 | 2020-07-17 | 浙江省海洋水产研究所 | Separation culture and induction method of precursor adipocytes of nibea albiflora |
| CN113234660A (en) * | 2021-04-02 | 2021-08-10 | 肇庆大华农生物药品有限公司 | Grass carp ureter tissue cell line and application thereof |
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