CN110527661A - A kind of Xiphophorus helleri prelarva cell line and its construction method and application - Google Patents
A kind of Xiphophorus helleri prelarva cell line and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of Xiphophorus helleri prelarva cell lines, construct to obtain by originally culture and secondary culture, and the present invention also provides Xiphophorus helleri prelarva cell lines in aquatic livestock virus purification and the application in environmental contaminants monitoring.The present invention carries out cell culture to Xiphophorus helleri prelarva, the condition of culture of cell is primarily determined, chromosome analysis is carried out, cell is had detected to the sensibility of a variety of Aquatic animals virus, and inductive effect of the antibiotics environmental contaminants rifampin to cell is studied, toxicology correlative study of the research for being established as later period Aquatic animals virus and cell line of the cell line as in vitro toxicology cell model applied to environmental contaminants is laid a good foundation.
Description
Technical field
The present invention relates to a kind of cell line and its construction method and applications, and in particular to a kind of Xiphophorus helleri prelarva cell line and
Its construction method and application, belong to medical biotechnology field.
Background technique
Xiphophorus helleri (Xiphophorus helleri, swordtail fish) is tropical and subtropical zone small fishes, taxology
Status be Osteichthyes (Osteichthyes), Medaka shape mesh (Cyprinodontiformes), TaiMedaka section (Poeciliidae),
Xiphophorus helleri category (Xiphophorus).Xiphophorus helleri is small, viviparous, the breeding cycle is short, convenient for feeding management in laboratory, and has
There are the features such as more sensitive to poisonous substances such as Multiple Pesticides, heavy metals, is monitoring water environment, malicious combability measurements and Aquatic animals virus inspection
The ideal test aquatic animal of survey.
Cell model of the fish cell as its Fish tissue, is widely used in field of biomedical research and environmental pollution
The detection of object and toxicity research are laid a good foundation to develop related vaccines for fish and the research of environmental pollution analyte detection.Fish are thin
Born of the same parents have the advantages that very important (waiting 2003 in vast) compared with live fish sample as scientific research material: (1) experimental cost is lower, tests fish
Cultivation need to add cultivation equipment and oxygen increasing equipment of large volume etc., culture fish cell only need to add necessary training for cell
It supports base and serum and has saved experimental cost without putting into a large amount of manpower and material resources；(2) favorable repeatability is carried out real using live fish
It tests, different upgrowth situations is all likely to result in experimental result between the fish body of different batches and the fish body of same batch
Difference, and when using fish cell as experimental material, the i.e. repeatable experiment of experiment condition only need to be accurately controlled, the knot of acquisition is made
Otherness is smaller before and after fruit.
Application of the fish cell culture in terms of virology mainly includes the following aspects: (1) viral separation and mirror
It is fixed: pathological material of disease acquisition being carried out to the fish body of illness, using fish cell as culture materials, is inoculated with suspected virus, carries out point of virus
From, and fluorescent antibody technics and neutralization test are generally used when identifying suspected virus.When more than one in pathological material of disease
Virus, or there are the not homophyletics of identical virus, the separation of virus can be realized by the multiple secondary culture of cell, finally
Obtain the virus of single plant.(2) research of viral pathogenesis mechanism: being separated and identified to virus, can be inoculated with to fish cell
Virus, by observing the upgrowth situation of cell, physiological change and cell be cope with this kind it is viral infect made by it is intracellular
A series of biochemical reactions, come study virus pathogenesis.(3) increasing of virus viral proliferation: is carried out using cell
It grows, the rate of positive infection is high；By the proliferation of virus, research and application in terms of molecular biology being carried out to virus, including
Research to the peptide segment structure of virus, it is later subsequent to prepare antibody and relevant vaccine development.(4) virus susceptibility is studied:
Some aquatic viruses can make a variety of fish illness, and some fish can infect several kinds of aquatic viruses simultaneously.Therefore, thin using fish
Born of the same parents carry out the research of virus susceptibility, to screen and establish sensitive cells strain, to Antibody preparation, vaccine development and are directed to fish
Clinical application, the vaccine dispensing of body are of great significance.(5) epidemiological study: some Aquatic animals virus may have more
The strain of kind serotype, the virus of even same serotype is also with the difference of toxicity power.Pass through fish cell culture skill
Art can carry out the parting of strain, the confirmation side of playing of diagnosis and pathogeny to the state of an illness to the virus with different serotypes
The directive function of tropism.
Drug and personal-care supplies (Pharmaceuticals and personal care products, abbreviation
PPCPs problem of environmental pollution), by Daughton (Daughton&Ternes, 1999) in " Environmental Health
Perspectives " in be put forward for the first time after, just cause extensive concern.PPCPs includes various medicinal compounds, such as in
Medicine, analgesic, antibiotic, contraceptive, B2 beta blocker, tranquillizer etc. and daily nursing articles, as individual's skin is protected
Reason and cosmetic product, aromatic, preservative, detergent, opacifier, hair style finalization agent, tooth care products etc., covering scope pole
To be extensive, and a large amount of uses in daily life.Pharmaceuticals are after human body or animal intake, and only small part is metabolized, greatly
Part is entered in sewage with original shape eventually by urine or excrement, and PPCPs has stronger persistence, biology in the ecosystem
The characteristics of activity, bioaccumulation and slow biological degradability, it is exposed to human body and aquatic, terrestrial organism body for a long time, people can be given
Class health and ecological environment bring potential danger.
Cytochrome P 450 enzymes are the important enzyme systems that body participates in organic pollutant metabolism, play important work in body removing toxic substances
With being the excellent measure of pollutant early stage toxicity assessment.Through long-term squamous subculture, cell function tends to be special for fish cell system
Change, may cause the forfeiture of certain major functions.Studies have shown that many fish cell systems do not have expression cell cytochrome p 450
The function of enzyme makes it have a greatly reduced quality in organic pollutant evaluation application.
CYP 3A gene belongs to the Cytochrome P450 family in I phase metabolic enzyme, and Cytochrome P450 family is to many interior
Especially to environment harmful chemical, there are oxidative metabolism effects for source property and exogenous chemical substance.CYP 3A is a kind of heavy
CYP450 enzyme system is wanted, the rich content in liver and enteron aisle, CYP 3A plays an important role in exogenous drug metabolism, faces
There are about 60% drugs in bed is metabolized via CYP 3A.
Since viral disease causes significant damage to freshwater fish culturing, numerous domestic research institution is from cause of disease, Su Zhuhe
Actively research is unfolded to fresh water fish virus in the various aspects of environment, it is intended to seek to control the effective ways of the disease.Up to the present fish
For virosis there are no special effect medicine therapeutic, immunoprophylaxis is the control most effective control method of Disease.It establishes
The sensitive cell line of virus is the first step for researching and developing vaccine, and research virus infection approach, infection mechanism and development virus
The important system of vaccine.Therefore, sensitive cell line is established, is the separation and identification for carrying out fresh water fish virus, studies viral pathogenesis
Mechanism, the important foundation of vaccine preparation and immunoprophylaxis technology.Meanwhile with the continuous development of fish cell Vitro Culture Techniques
Progress, people begin to use detection and the toxicity research of the fish cell system progress environmental contaminants of in vitro culture.Organism exists
Will appear " group effect ", the fish cell system cell of in vitro culture such as self removing toxic substances, toxin expelling, immune when contact stain object has
Collimation is good, and no individual difference, homogeneity is good, direct to the effect of pollutant, targetedly, and is convenient for the features such as observing,
Biological detection can be effectively carried out to environmental pollutants with rapid sensitive.
The establishing techniques of fish primary cell line are currently a kind of more mature technology, but to be obtained quick for virus
Sense simultaneously there is the cell line of the gene expression of response pollutant still to deposit very big contingency and difficulty again, so establishing one
It is with quantity to quality that strain, which needs the effective approach of fish cell system, that is, large batch of primary cell strain is prepared, to filter out
Sensitive cell line.
So far, have the document report of several cell lines such as Xiphophorus helleri brain cell line, Xiphophorus helleri embryo cell line, but
It is the report for having not yet to see Xiphophorus helleri prelarva cell line.
Summary of the invention
In view of this, the present invention provides a kind of Xiphophorus helleri prelarva cell line and its construction method and application, it is specific to use
Following technical solution:
Xiphophorus helleri prelarva cell line SFF was preserved in Wuhan, China, Wuhan University, Chinese Typical Representative training on August 16th, 2019
Object collection is supported, preservation accession designation number is CCTCC NO:C2019161.
Xiphophorus helleri prelarva cell line of the invention has following biological characteristics:
Xiphophorus helleri prelarva cell line has stronger fertility, and typical epithelioid cell's form is presented；Cell has connected
Continuous secondary culture 8 years, still maintain epithelial cell Morphological Features, growth characteristics；Even in 100 generations of continuous culture, still keep stronger
Growth characteristics and multiplication characteristic.
Cell line stability of characteristics of the present invention reached for 100 generations at present, and cell can maintain good growth conditions, can be right
It carries out freezen protective.Cell line of the present invention has sensibility to a variety of fishes virus, cytopathy occurs after connecing poison, can be with
Directly apply to cause of disease research.It can be applied to the research of detection environmental contaminants simultaneously.
The construction method of above-mentioned Xiphophorus helleri prelarva cell line, steps are as follows:
1) it takes the Xiphophorus helleri prelarva being just born to sterilize 5~10s in 75% ethyl alcohol, to be shredded after PBS rinsed clean, is added
The digestive juice A of 3~5 times of volumes, even spread to outer wall is coated in the culture bottle of 0.1% gelatin after mixing, and will training
It supports bottle and is inverted 27 DEG C of constant temperature in incubator, saturated humidity, 5%CO2It is inverted 6~8h of dry doubling, is then added into culture bottle complete
Culture solution, which is just being set, continues 10~15h of culture, and addition complete culture solution continues culture 2~5 days, obtains primary cell；
2) after primary cell confluent monolayers, complete culture solution is removed, with PBS cleaning 2~3 times, it is thin that pancreatin digestion is added
Complete culture solution is added in born of the same parents, and cell has been hanged in piping and druming, and 27 DEG C of constant temperature carry out secondary culture, obtain Xiphophorus helleri prelarva cell line.
The present invention carries out cell culture to Xiphophorus helleri prelarva, has primarily determined the condition of culture of cell, has carried out chromosome
Analysis, has detected cell to the sensibility of a variety of Aquatic animals virus, and to antibiotics environmental contaminants rifampin to cell
Inductive effect studied, the research for being established as later period Aquatic animals virus of the cell line and cell line are as external poison
The toxicology correlative study that cell model of science is applied to environmental contaminants is laid a good foundation.
Further, the digestive juice A in step 1) is Collagenase I (w/v) and 5% fetal calf serum (w/v) containing 0.1%
DMEM/F-12 culture solution.
Further, complete culture solution be fetal calf serum, the filtered fluid after 24~48h of Xiphophorus helleri brain cell culture, alkalinity at
Fiber like growth factor, epidermal growth factor, penicillin, streptomysin, amphotericin B, DMEM/F-12 culture solution mixed liquor；
Preferably, complete culture solution is 20% fetal calf serum, 10%~20% Xiphophorus helleri brain cell growth-promoting media, 20ng/L basic fibroblast
Like growth factor, the epidermal growth factor of 1ng/ml, 100U/ml penicillin, 100 μ g/ml streptomysins, 0.25 μ g/ml both sexes are mould
Plain B, the mixed liquor for the DMEM/F-12 culture solution that pH value is 7.2~7.4.
The preparation method of above-mentioned Xiphophorus helleri brain cell growth-promoting media is: the Xiphophorus helleri brain cell of 24~48h of culture is taken, by cell
Culture solution is placed in 50mL centrifuge tube, and at 4 DEG C, 5000~6000rpm/min of revolving speed is centrifuged 5~10min, takes supernatant filter membrane, and 4
DEG C save.
The present invention also provides application of the above-mentioned Xiphophorus helleri prelarva cell line in aquatic livestock virus purification.
The present invention also provides application of the above-mentioned Xiphophorus helleri prelarva cell line in environmental contaminants monitoring.
Detailed description of the invention
Fig. 1 is 1 Xiphophorus helleri prelarva cellular morphology of embodiment of the present invention observation figure；
Fig. 2 is the optimum condition inquiry experiment result of 2 Xiphophorus helleri prelarva cell line of the embodiment of the present invention；
Fig. 3 is 3 Xiphophorus helleri prelarva cell line chromosome analysis result of the embodiment of the present invention；
Fig. 4 is that 4 Xiphophorus helleri prelarva cell line of the embodiment of the present invention ties the sensitivity experiments CPE of different Aquatic animals virus
Fruit；
Fig. 5 is sensitivity experiments electrophoresis of the 4 Xiphophorus helleri prelarva cell line of the embodiment of the present invention to different Aquatic animals virus
As a result；
Fig. 6 is the exposure Xiphophorus helleri prelarva cell CYP3A gene expression for 24 hours of 5 various concentration rifampin of the embodiment of the present invention
As a result.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The building of Xiphophorus helleri prelarva cell line SFF
1. preparing cell culture fluid:
It takes GIBCO company DMEM/F-12 culture medium, is added the fetal calf serum for accounting for total volume 10~20% used, 10%~
20% Xiphophorus helleri brain cell growth-promoting media, the Basic Fibroblast Growth Factor (bFGF) of 20ng/mL, the epidermal growth factor of 1ng/mL
Sub (EGF), 4 DEG C of storages are spare；
2 originally cultures:
5~6 tail of Xiphophorus helleri prelarva (viviparity) being just born is taken, is placed in 75% ethyl alcohol and sterilizes 5~10 seconds, PBS rinsing is dry
Only it is placed in the sterile penicillin bottle of 5mL, PBS containing 200ul in bottle is shredded with operating scissors；3~5 times of volumes are added
Digestive juice A, be uniformly mixed；Outer wall is coated to preparatory 0.1% gelatin with by the broken tissue block even spread of mixture slaking enzyme
Culture bottle in；Culture bottle is inverted into 27 DEG C of saturated humidity, 5%CO2In incubator, after being inverted 6~8h of dry doubling, slowly
2mL complete medium is added and is just setting and continues 10~15h of culture, adds culture medium to 5~6mL, continues culture 2~5 days, given birth to
The long primary cell converged to 80%~90%；Wherein, the ingredient of digestive juice A is that 0.1% Collagenase I adds 5% tire ox blood
Clearly.
3. secondary culture:
After primary prelarva cell confluent monolayers, old culture solution is removed with suction pipe, is cleaned twice with PBS, 0.25% pancreas is added
Enzyme (containing EDTA), 10mL complete culture solution was added after microscopic observation cell rounding in 1mL vitellophag, and cell has been hanged in piping and druming,
It is passed on 1:2, complete culture solution is added to 5mL/ bottles, be put into 27 DEG C containing 5%CO2Secondary culture is carried out in incubator；With
Passage in about 3~5 days is primary afterwards；When reaching for 10 generation, fetal calf serum content is kept to the 10% of total volume in cell culture fluid, training
No longer added in nutrient solution Xiphophorus helleri brain cell growth-promoting media, penicillin, streptomysin, amphotericin B, Basic Fibroblast Growth Factor and
(A is primary cell to epidermal growth factor in Fig. 1, Fig. 1；B is 15 generation cells；C is 50 generation cells；D is 100 generation cells).
Complete medium formula is 20% fetal calf serum, 10%~20% Xiphophorus helleri brain cell growth-promoting media, 20ng/L alkalinity
At fiber like growth factor (bFGF), the epidermal growth factor (EGF) of 1ng/mL, 100U/mL penicillin, 100 μ g/mL strepto-s
Element, 0.25 μ g/mL amphotericin B, the DMEM/F-12 culture solution that pH value is 7.2~7.4.
The preparation of Xiphophorus helleri brain cell growth-promoting media: the Xiphophorus helleri brain cell of 24~48h of culture is taken, cell culture fluid is placed in
50mL centrifuge tube, at 4 DEG C, 5000~6000rpm/min of revolving speed is centrifuged 5~10min, takes supernatant filter membrane, 4 DEG C save backup.
Embodiment 2
The optimum condition of Xiphophorus helleri prelarva cell line determines
1, the determination of Xiphophorus helleri prelarva cell line optimal medium
Tetra- kinds of DMEM/F-12, M199, DMEM/F-12 and L-15 mixed culture medium (1:1), L-15 cell culture mediums are selected,
And it adds final concentration of 10% FBS and prepares cell culture fluid.Adjusting cell density is 5 × 104mL-1, four kinds of culture mediums respectively press
The amount in the hole 2.5mL/ is inoculated in 6 orifice plates, cultivates in 27 DEG C of incubator.Every 1d takes out 3 hole cells in each experimental group, uses
Trypsin-EDTA digestion method is collected cell and is counted, and co-cultures 7d, continuous counter 7 times, draws its growth curve.Determine it most
Suitable culture medium is DMEM/F-12 and L-15 mixed culture medium (1:1) (Fig. 2A).
2, the determination of the most suitable serum-concentration of Xiphophorus helleri prelarva cell line
The culture solution that FBS concentration is 5%, 10%, 15%, 20% is prepared respectively, and adjustment cell density is 5 × 104mL-1,
Four kinds of serum-concentration culture mediums are respectively inoculated in 6 orifice plates by the amount in the hole 2.5mL/, cultivate in 27 DEG C of incubator.Every 1d is from each
3 hole cells are taken out in experimental group, is collected and cell and is counted with Trypsin-EDTA digestion method, co-culture 7d, continuous counter 7 times,
Its growth curve is drawn, discovery FBS concentration is 10%~20% culture (Fig. 2 B) for being suitable for Xiphophorus helleri prelarva cell line.
As shown in Figure 2, DMEM/F-12 and L-15 mixed culture medium of the Xiphophorus helleri prelarva cell line in fetal calf serum 15%
In (1:1), it is proliferated best.
Embodiment 3
Cell freezes and recovers
1, cell freezes
1 bottle of (25cm is collected with trypsin digestion2) it is in the Xiphophorus helleri prelarva cell of logarithmic growth phase, 1000g centrifugation
It is discarded supernatant after 5min, freezes protection liquid (DMEM/F-12 and L-15 mixing (1:1) containing 20%FBS and 10%DMSO with 1mL
Culture solution) cell resuspension is placed in cryopreservation tube, cryopreservation tube is placed in program temperature reduction box in -80 DEG C overnight, is finally put into
Liquid nitrogen (- 196 DEG C) medium-term and long-term preservation, and make a record.
2, the recovery of cell
When recovery cell, it is removed from liquid nitrogen cryopreservation tube, is thawed rapidly in 37 DEG C of water-baths, 1000g is centrifuged 5min and collects
25cm is inoculated in after cell2Culture bottle in, be placed in incubator 27 DEG C of cultures, supernatant, replacement training abandoned after cell is adherent
Nutrient solution continues to cultivate.
Cell after recovery is subjected to Trypan Blue, with (the figure of survival rate > 90% of cell counter measurement cell
3)。
Embodiment 4
Sensibility of the Xiphophorus helleri prelarva cell to 5 kinds of Aquatic animals virus
The good Xiphophorus helleri prelarva of upgrowth situation is passed on, is dispensed after mixing well into T25 Tissue Culture Flask, when
Cell is paved with bottom of bottle 80-90% when just having covered with culture bottle bottom when, the cell of identical growth conditions is selected, inhales and abandons old culture
Base, HBSS buffer solution for cleaning twice, are respectively connected to 4 kinds of 1mL common freshwater fish viruses: huichun viremia virus
(Spring viraemia of carp virus, SVCV), Basidiobolus spp (Frog virus3, FV3), Genotype I grass carp
Reovirus (Grass Carp Reovirus, GCRV), Tilapia mossambica lake virus (TiLV-2017A), PBS is as blank control
Group sets up blank control according to cultivation temperature difference respectively, after virus incubation 1h, abandons virus liquid, 5mL cell maintenance medium is added
The Tissue Culture Flask of (complete medium containing 5%FBS), access kind SVCV is placed in 22 DEG C of constant incubators, other cell bottles are put
28 DEG C of incubator is set, cell is observed daily and grows CPE situation, monitor whether lesion occur, once there is CPE lesion, thin
Then born of the same parents, which collect cell suspension before not completely falling off and carry out nucleic acid extraction, carries out PCR detection, do not occur lesion connects malicious group
Sample is collected after 3 generation of blind passage and carries out PCR experiment, through detection this analysis of virus transcription virus whether in Xiphophorus helleri prelarva cell
Proliferation.Culture bottle is taken out when collecting sample, is placed in -20 DEG C of refrigerator overnights, it is rear to take out after culture medium thawing, firmly beat
Culture bottle completely falls off cell, after piping and druming mixes freeze thawing, takes out 200 μ L cell suspensions and carries out nucleic acid extraction in centrifuge tube.
After 4 kinds of Aquatic animals virus of Xiphophorus helleri prelarva cell inoculation, as shown in Figure 4, wherein A is SFF blank control, B
It is inoculated with the CPE generated after TiLV for SFF, C is that SFF is inoculated with the CPE generated after SVCV, and D is that SFF is inoculated with the CPE generated after FV3,
E is that SFF is inoculated with the CPE generated after GCRV, it is seen then that occurs apparent cytopathy (CPE) into the cell, collects in inoculation 5d each
200 μ L of viral suspension extracts nucleic acid and carries out PCR detection.Wherein TiLV-2017A purpose band is 700bp, and SVCV's is detected as half
Shell type reaction, after reaction, the product of twice PCR can detect that the segment of 714bp and 606bp, FV3 purpose band are respectively
The purpose band of 531bp, Genotype I GCRV are 661bp.According to PCR and gel electrophoresis experimental result, it is being inoculated with various viruses
Detect corresponding purpose band (Fig. 5) in cell, in Fig. 5, M:1000bp molecular weight standard；2,4,6,8,10: normal
SFF cell；1: infecting the SFF cell 3 of TiLV-2017A: infecting the SFF cell (sleeve type PCR product) of SVCV；5: infection SVCV
SFF cell (regular-PCR product)；7: infecting the SFF cell of FV3；9: the SFF cell of infection Genotype I GCRV illustrates sword tail
Anchovies fry cell system can virus of proliferation TiLV, FV3, GCRV, SVCV, provide strong material for the research of aquatic animal.
Embodiment 5
Influence after rifampin RIF exposure Xiphophorus helleri brain cell to cytochrome P450 gene expression
By Xiphophorus helleri brain cell inoculation of suspension liquid into six well culture plates, until density is 2 × 105A/mL.To contain 10%
(v/v) DMEM/F12 and L-15 (1:1) culture medium of FBS is placed in 5%CO2, in 27 DEG C of cell incubators preculture for 24 hours, to thin
After born of the same parents' adherent growth, suck culture medium, rejoin the RIF containing various concentration or the culture medium of control, continue cultivate 48h into
Row exposure experiment.RIF is dissolved in dimethyl sulfoxide DMSO, and the ultimate density of solvent is no more than 0.1%.DMSO exposure group is set as pair
According to.RIF exposure concentrations gradient is 10,20,50,100nmolL-1.After exposure, culture medium is removed, is buffered with D-hanks
Liquid cleaning, cell is used for CYP3A gene expression detection after processing.
The Xiphophorus helleri prelarva cell sample induced through RIF extracts total serum IgE, expands through Real-time RT-PCR, selects pipe
Family's gene (β-actin) carries out relative expression quantity analysis to CYP3A gene mRNA.Through Paired- in 16.0 statistical software of SPSS
Samples T-Test test samples variance analysis.
Fig. 6 is the influence that various concentration RIF expresses Xiphophorus helleri prelarva cell CYP3A mRNA, the experimental results showed that, it lures
Lead all concentration induction groups of rear 48h compared with the control group, CYP3A mrna expression amount conspicuousness increases (p < 0.01), and 10~
100nmol·L-1There are good dose-effect relationship between CYP3A mrna expression amount, with the increasing of induced concentration,
The expression quantity of gene increases, in concentration group 50nmolL-1Reach maximum gene expression amount.The display of this result of study,
In 10~100nmolL-1Under concentration RIF exposure, CYP3A mRNA level in-site is significantly raised.
To have carried out associated biomarkers thin for rifampin to environmental pollutants by Xiphophorus helleri prelarva cell line for above-described embodiment
As a result the detection of born of the same parents' cytochrome p 450 gene level confirms that the cell line can effectively detect rifampin RIF to biomarker
Inductive effect, can be used as detection environmental contaminants experimental material, for Xiphophorus helleri be used for environmental contaminants detection provide
One ideal in vitro study system.
Claims (10)
1. a kind of Xiphophorus helleri prelarva cell line, which is characterized in that Xiphophorus helleri prelarva cell line SFF, in the preservation on the 16th of August in 2019
In Wuhan, China, Wuhan University, China typical culture collection center, preservation accession designation number is CCTCC NO:C2019161.
2. a kind of construction method of Xiphophorus helleri prelarva cell line described in claim 1, which is characterized in that steps are as follows:
1) it takes the Xiphophorus helleri prelarva being just born to sterilize 5~10s in 75% ethyl alcohol, to shred after PBS rinsed clean, digestion is added
Liquid A, even spread is into culture bottle after mixing, and culture bottle is inverted in incubator and is inverted 6~8h of dry doubling, then to training
It supports that complete culture solution is added in bottle and is just setting and continues 10~15h of culture, addition complete culture solution continues culture 2~5 days, obtains primary
Cell；
2) after primary cell confluent monolayers, complete culture solution is removed, with PBS cleaning 2~3 times, trypsin digestion cell is added, adds
Enter complete culture solution, cell has been hanged in piping and druming, carries out secondary culture, obtains Xiphophorus helleri prelarva cell line.
3. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 2, which is characterized in that institute in step 1)
Stating digestive juice A is containing 0.1% Collagenase I (w/v) and the DMEM/F-12 culture solution of 5% fetal calf serum (w/v).
4. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 2, which is characterized in that the complete training
Nutrient solution is fetal calf serum, the filtered fluid after 24~48h of Xiphophorus helleri brain cell culture, basic fibroblast like growth factor, epidermis are raw
The long factor, penicillin, streptomysin, amphotericin B, DMEM/F-12 culture solution mixed liquor.
5. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 4, which is characterized in that the complete training
Nutrient solution be 20% fetal calf serum, 10%~20% Xiphophorus helleri brain cell growth-promoting media, 20ng/L basic fibroblast like growth factor,
The epidermal growth factor of 1ng/ml, 100U/ml penicillin, 100 μ g/ml streptomysins, 0.25 μ g/ml amphotericin B, pH value are
The mixed liquor of 7.2~7.4 DMEM/F-12 culture solution.
6. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 4 or 5, which is characterized in that Xiphophorus helleri
The preparation method of brain cell growth-promoting media is: taking the Xiphophorus helleri brain cell of 24~48h of culture, cell culture fluid is placed in 50mL centrifugation
It manages, at 4 DEG C, 5000~6000rpm/min of revolving speed is centrifuged 5~10min, takes supernatant filter membrane, 4 DEG C of preservations.
7. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 2, which is characterized in that institute in step 1)
The additional amount for stating digestive juice A is 3~5 times for shredding rear Xiphophorus helleri prelarva volume；
The outer wall of the culture bottle is coated with 0.1% gelatin.
8. a kind of construction method of Xiphophorus helleri prelarva cell line according to claim 2, which is characterized in that institute in step 1)
Stating and being inverted the condition of dry doubling is CO2Concentration 5%, saturated humidity, 27 DEG C of constant temperature；
The temperature of secondary culture described in step 2) is 27 DEG C of constant temperature.
9. a kind of application of Xiphophorus helleri prelarva cell line described in claim 1 in aquatic livestock virus purification.
10. a kind of application of Xiphophorus helleri prelarva cell line described in claim 1 in environmental contaminants monitoring.
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