CN104762252A - Grass goldfish pelvic fin cell line constructing method - Google Patents
Grass goldfish pelvic fin cell line constructing method Download PDFInfo
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- CN104762252A CN104762252A CN201510206830.4A CN201510206830A CN104762252A CN 104762252 A CN104762252 A CN 104762252A CN 201510206830 A CN201510206830 A CN 201510206830A CN 104762252 A CN104762252 A CN 104762252A
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Abstract
The invention relates to a grass goldfish pelvic fin cell line and a constructing method thereof. Tissue blocks are digested through trypsin, EDTA and hyaluronidase according to the features of grass goldfish pelvic fin tissue histocyte, the digested tissue blocks are transplanted to a cultivation bottle, special multiplication cultivation solution is added to the cultivation bottle, primary culture is conducted, and then the grass goldfish pelvic fin cell line is successfully constructed. By means of the method, a large number of cells can be separated three days after primary culture is conducted, and the cells have the continuous passage capability. After passage is stable, the cells are in a typical fiber form, it proves that the Chinese grass goldfish pelvic fin cell line is stable in character and uniform in component and is a good material for studying the aquatic animal virology and the immunology. At present, the grass goldfish pelvic fin cell line has 60 generations, and the cells can stably increase and have the continuous passage capability.
Description
Technical field
The present invention relates to cell biology, be specifically related to a kind of construction process of grass goldfish abdomeinal fin clone.
Background technology
Fish cell system is through development for many years, and various fresh water, seawater fish clone have become the important in vitro study system of multiple ambit, and many important experiments and new technology all need to study further on the basis of fish cell system.The range of application of fish cell system is increasingly extensive, expands to the numerous areas such as environmental pollutant mechanism of toxication and monitoring, fish immunity, Genetic improvement, conservation of resources from initial virus purification, purification, qualification, vitro propagation.Fish cell system not only can be used as the good in vitro study system of carrying out fundamental research at multiple subjects such as cytobiology, fish virology, physiology of fishes, fish immunology, environmental toxicology, genetics of fishes, fish genetics, pharmacology; also can at fishes virus vaccine development; environmental pollutant toxicity detection; fish gene Engineering Breeding; Fish Tissue embryo studies; Species of Rare Fish from Qingdao conservation of resources, applied research is carried out in the fields such as natural active matter exploitation.No matter in theoretical investigation, or practical application aspect, all there is far reaching significance.
The raising of aquarium fish has a long history in China, and wherein grass goldfish is one of cultivation amount and the maximum kind of export volume in China aquarium fish.Grass goldfish (Carassius auratus) is under the jurisdiction of carp type order (Percoiformers), Cyprinidae (Sparidae), crucian carp genus (Carassius).Wolf and Quimby establishes the first strain fish cell system, i.e. rainbow trout gonadal cell system RTG-2 in the world in the sixties in 20th century.The fish cell system reported at present has more than 300 to plant (Lakra W S, Swaminathan T R, Joy K P.Development, characterization, conservation and storage of fish cell lines:a review [J] .Fish Physiology and Biochemistry, 2011,37 (1): 1-20).The cell strain of the goldfish wherein reported mainly contains GFSK-S1 Goldfish kidney cell line (Dicky L Y, Chow B K C, Chan C B, et al.Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus) [J] .Life sciences, 2000, 66 (7): 593-605), GAKS goldfish squamous cell system (Akimoto K, Takaoka T, Sorimachi K.Development of a simple culture methodfor the tissues contaminated with microorganisms and application to establishment ofa fish cell line [J] .Zoological science, 2000, 17 (1): 61-63), GFM goldfish muscle cell system (Roug é e L, Ostrander G K, Richmond R H, et al.Establishment, characterization, and viral susceptibility of two cell lines derived from goldfish Carassius auratusmuscle and swim bladder [J] .Diseases of aquatic organisms, 2007, 77 (2): 127), GH goldfish heart cell system (CGMCC NO.9334), goldfish caudal fin cell system (Hashimoto H, ToyoharaH, Yokoyama Y, et al.Effects of carp serum on the growth of goldfish fin cells inearly passage [J] .Journal of fish biology, 1997, 50 (1): 201-207).
Cell culture technology is life science, the research means that cell engineering, genetically engineered and biomedical engineering are main.The biologic material that the Study on etiology being configured to fishes virus disease of fish cell system and the vaccine research of virus provide the foundation has important researching value.Identifying that with being separated responsive virus strain be the most effectual way that OIE Office International Des Epizooties (OIE) is recommended by cell strain, is that fishes virus screening sensitive cell line provides abundant biologic material by setting up different types of fish cell strain.At present, the goldfish cell system built is described above, and have not been reported about grass goldfish abdomeinal fin clone.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of construction process of grass goldfish abdomeinal fin clone is provided.
Second object of the present invention provides the purposes of described clone
For achieving the above object, the technical scheme that the present invention takes is:
A construction process for grass goldfish abdomeinal fin clone, is characterized in that, comprise the steps:
1) process of tissue block: clip grass goldfish abdomeinal fin tissue, be soak 30m minute in the dual anti-solution of 1000IU/ml Pen .-Strep in concentration, with 75% alcohol disinfecting process, be that the D-PBS of the amphotericin B of 2.5ug/ml rinses 3 times by concentration, under aseptic condition, abdomeinal fin tissue be cut into 1mm
3tissue block, be immersed in organizing fritter digest 10min in 0.25% trypsinase and 0.02%EDTA after, then use 0.5% hyaluronidase digestion 1h;
2) original cuiture: tissue block being transplanted to floorage is 25cm
2culturing bottle in, keep temperature to be 26 DEG C, after tissue block is transplanted, within the 15th hour, add 2ml multiplication culture liquid, within the 24th hour, add 3ml multiplication culture liquid, every day half, amount changed nutrient solution once;
3) Secondary Culture: after primary cultured cell grows up to individual layer, adds trypsinase and leaves standstill digestion, has then hanged cell with multiplication culture liquid, has gone down to posterity, carry out Secondary Culture in 26 DEG C with 1:2.
Described multiplication culture liquid is the M199 nutrient solution containing 18v/v% foetal calf serum, 15ng/ml human alkaline fibroblast somatomedin, 2ng/ml Urogastron, 1ng/ml I Insulin-Like cell growth factor, 200IU/ml penicillin, 200ug/ml Streptomycin sulphate, 0.5ug/ml amphotericin B, 10mM 2 mercapto ethanol.
The trypsinase concentration added in described step 3 is 0.25%, and digestion time is 2min.
After sub-bottle goes down to posterity, the cultivation of the incubator without carbonic acid gas culturing bottle being placed in 26 DEG C can be covered with for 5 days; Stablize after going down to posterity, cell is in typically becoming fibre shape; This clone is the Chinese grass goldfish abdomeinal fin clone of a kind of stability of characteristics, uniform component, is research aquatic animal virusology and immunologic good material.
Further, described goldfish abdomeinal fin clone has following biological characteristics:
A. the cell near primary separation self-organization block, in epithelioid cell, go down to posterity the stable rear Morphological Features in fibroblast-like cell;
B. ability of cell proliferation is strong, and each tissue block after treatment about 3d all can isolate a large amount of cell, all has the ability of continuous passage;
C. this clone culture condition is 26 DEG C of constant temperature cell culture incubators, without the need to carbonic acid gas.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
Grass goldfish abdomeinal fin clone as above is detecting the application in simplexvirus CyHV-2.
The invention has the advantages that:
1, the present invention is directed to the feature of grass goldfish abdomeinal fin cell, optimization is applicable to the substratum of this growth and proliferation of cell and suitable culture condition, successfully constructs grass goldfish abdomeinal fin clone; This cell proliferation ability is strong, cultivates and can cover with culturing bottle in 5 days after sub-bottle goes down to posterity.At present, this clone, more than continuous passage to 60 generation, can provide a large amount of grass goldfish abdomeinal fin derived cells.Method of the present invention not only can be used for building abdomeinal fin tissue lines, and the method is also applicable to the fin tissue lines (as pectoral fin, dorsal fin, tail fin etc.) building other positions.
2, the abdomeinal fin cell constructed by the present invention is molecular cell level launched fishes virus route of infection, infect mechanism and develop virus vaccines etc. to provide the foundation.
Accompanying drawing explanation
Accompanying drawing 1 is the cell (original cuiture the 3rd day) moved out near the Chinese grass goldfish abdomeinal fin tissue block under microscope.
Accompanying drawing 2 is stablize the Chinese grass goldfish abdomeinal fin clone after going down to posterity under microscope.
Accompanying drawing 3 is for containing crucian simplexvirus II infection experiment result.Wherein, Fig. 3 (A): normal grass goldfish abdomeinal fin clone, scale bar.=100 μm; (B, C) infects the grass goldfish abdomeinal fin clone scale bar.=100and 50 μm of crucian simplexvirus II (CyHV-2); (D) there is cytopathic effect (cytopathic effect, CPE), scale bar.=100 in the grass goldfish abdomeinal fin clone infecting crucian simplexvirus II (CyHV-2).
Accompanying drawing 4 is PCR detected result.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
Embodiment 1
(1) process of tissue block: cut grass goldfish abdomeinal fin, putting into concentration is the dual anti-solution of 1000IU/ml Pen .-Strep (distilled water configuration), after placing 30min, sterilizes 2 minutes, sterilize first in 75% alcohol; Then rinse 3 times with the D-PBS of the amphotericin B containing 2.5ug/ml, carry out secondary sterilization; After flushing, with scissors, abdomeinal fin is cut into about 1mm under sterile conditions
3fritter, be immersed in organizing fritter digest 10min in 0.25% trypsinase and 0.02%EDTA after, then use 0.5% hyaluronidase digestion 1h;
(2) original cuiture: collect and postdigestively organize fritter, is transplanted to bottom for 25cm
2tissue Culture Flask in, keep bottom temp be 25 DEG C.After 15 hours, add the special multiplication culture liquid of 2ml grass goldfish abdomeinal fin cell (nutrient solution formula is the M199 nutrient solution containing 18v/v% foetal calf serum, 15ng/ml human alkaline fibroblast somatomedin, 2ng/ml Urogastron, 1ng/mlI Insulin-Like cell growth factor, 200IU/ml penicillin, 200ug/ml Streptomycin sulphate, 0.5ug/ml amphotericin B, 10mM 2 mercapto ethanol); After 24 hours, add the special multiplication culture liquid of 3ml grass goldfish abdomeinal fin cell, change the fresh medium of 50% afterwards every day.
Start the 3rd day of original cuiture, can observe under microscope around tissue block and have cell to move out, the cell quantity of moving out is many, and homogeneous is bright, has and significantly breeds phenomenon, division growth vigorous (Fig. 1).
(3) Secondary Culture: after the grass goldfish abdomeinal fin cell monolayer confluent culture bottle of original cuiture, old nutrient solution removed by suction pipe, with PBS cleaning twice, adds 1 ml concn containing being 0.25% trypsinase, leave standstill digestion 2min, after basis of microscopic observation cell rounding, add the special multiplication culture liquid that 10ml configures, repeatedly blow and beat, make cell suspension, go down to posterity with 1:2, add special multiplication culture liquid 5ml/ bottle, put into 26 DEG C of incubators and go down to posterity.
After sub-bottle goes down to posterity, cultivate in without the incubator of carbonic acid gas and can cover with for 5 days, this clone has stronger fecundity.Basis of microscopic observation (Fig. 2), cell is in typically becoming fibre shape, and this clone is the Chinese grass goldfish abdomeinal fin clone of a kind of stability of characteristics, uniform component, is research aquatic animal virusology and immunologic good material.At present, this grass goldfish abdomeinal fin clone went down to posterity for 60 generations, and cell can stablize propagation, has the ability of continuous passage.
Comparative example 1
One, experimental technique
1, the determination of the most applicable substratum
By L-15, DMEM and M199 substratum of adding in the culturing bottle inoculating abdomeinal fin tissue block containing 18% foetal calf serum (volume ratio) to 5ml/ bottle, Primary culture at being placed in 26 DEG C, the form of observation of cell, adherent property, growth division situation.
2, the impact of different culture temperature cell growth
The M199 substratum containing 18% foetal calf serum (volume ratio) will be added in the culturing bottle inoculating abdomeinal fin tissue block, be placed in 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C incubators respectively and carry out original cuiture, observe the moving out of cell under differing temps, adherent and proliferative conditions.
3, the impact of different concns FBS cell growth
In order to detect the FBS of different concns to the impact of grass goldfish abdomeinal fin Growth of Cells, add respectively in the culturing bottle inoculating abdomeinal fin tissue block concentration be 14%, 16%, 18%, 20%, the M199 substratum of 22%FBS, Primary culture at being placed in 26 DEG C, the form of observation of cell, adherent property, growth division situation.
4, the impact of different somatomedin cell growth
In order to detect the impact of different somatomedin on abdomeinal fin Growth of Cells, on the basis of M199, be configured with following 4 kinds containing different culture media, within 3 days, observe the moving out of cell in different culture media, adherent and proliferative conditions afterwards.
Substratum 1:18%FBS (volume ratio)+15ng/ml bFGF+2ng/ml EGF+1ng/ml IGF;
Substratum 2:18%FBS (volume ratio)+15ng/ml bFGF+1ng/ml EGF+1ng/ml IGF;
Substratum 3:18%FBS (volume ratio)+15ng/ml bFGF+1ng/ml IGF;
Substratum 4:18%FBS (volume ratio)+10ng/ml bFGF+2ng/ml EGF+1ng/ml IGF;
Substratum 5:18%FBS (volume ratio)+15ng/ml bFGF+2ng/ml EGF+10mM 2 mercapto ethanol+1ng/ml IGF.
Two, result
(1) grass goldfish abdomeinal fin cell tissue is starting original cuiture after 3 days respectively in L-15, DMEM and M199 tri-kinds of different culture medias, result display (table 1), cultivate in M199 substratum abdomeinal fin tissue have a large amount of cell to move out, the cell of moving out accounts for more than 12% of bottle floorage, fast growth; And cultivate quantity that abdomeinal fin histocyte in L-15 and DMEM substratum moves out and vitro growth rates lower.Visible M199 substratum is more suitable for the vitro culture of grass goldfish abdomeinal fin clone.
Table 1
Note :+represent that the cell of moving out around tissue block after 6 days accounts for 2% of culturing bottle base area.
(2) grass goldfish abdomeinal fin is organized in differing temps and starts original cuiture after 3 days, result display (table 2), the quantity that when abdomeinal fin cultivated in M-199 substratum is organized in 26 DEG C, cell is moved out is maximum, cell proliferation is the fastest, is obviously better than the abdomeinal fin tissue being incubated at 22 DEG C, 24 DEG C and 28 DEG C.Visible culture temperature is 26 DEG C and is more suitable for the histiocytic vitro culture of grass goldfish abdomeinal fin.
Table 2
Note :+represent that the cell of moving out around tissue block after 6 days accounts for 2% of culturing bottle base area.
(3) grass goldfish abdomeinal fin is organized in the M199 substratum of different FBS concentration and starts original cuiture after 3 days, result display (table 3), be that to cultivate the quantity that abdomeinal fin histocyte moves out in the substratum of 18%FBS maximum in concentration, rate of propagation is the fastest, obviously due to FBS substratum that concentration is 14%, 16%, 20%, 22%.In visible substratum, FBS concentration is 18% be best suited for the histiocytic vitro culture of grass goldfish abdomeinal fin.
Table 3
Note :+represent that the cell of moving out around tissue block after 6 days accounts for 2% of culturing bottle base area.
(4) grass goldfish abdomeinal fin is organized in above-mentioned different culture media and starts original cuiture after 3 days, result display (table 4), the quantity that the abdomeinal fin histocyte cultivated in substratum 1 and substratum 5 is moved out is maximum, cell proliferation is the fastest, obviously be better than other substratum, illustrate that the proliferated culture medium in the present invention can not only meet grass goldfish abdomeinal fin Growth of Cells, and can effectively promote cell fission and fast breeding.
Table 4
Note :+represent that the cell of moving out around tissue block after 3 days accounts for 5% of culturing bottle base area.
Comparative example 2
(1) process of tissue block: method with embodiment 1, after tryptic digestion 10min, without hyaluronidase digestion.
(2) original cuiture: collect and postdigestively organize fritter, is transplanted to bottom for 25cm
2tissue Culture Flask in, keep bottom temp be 25 DEG C.After 15 hours, add the special multiplication culture liquid of 2ml grass goldfish abdomeinal fin cell; After 24 hours, add the special multiplication culture liquid of 3ml grass goldfish abdomeinal fin cell, change the fresh medium of 50% afterwards every day.
Start the 3rd day of original cuiture, only have around little tissue block and have cell to move out, cell quantity of moving out is few, and form is slightly different from the cellular form of moving out in embodiment 1, and does not substantially observe cell proliferation.
This clone of embodiment 2 is applied to the detection of crucian simplexvirus II (CyHV-2)
One, experimental technique
1, the infection of virus
Cultured grass goldfish abdomeinal fin clone is reached in 6 orifice plates, adherent growth overnight.Respectively by positive to be detected (containing crucian simplexvirus II, and negative sample (alkaline phosphate buffer CyHV-2), PBS) 500 μ l cells infecteds are got, hatch supernatant discarded after 1 hour, 3 times are washed with PBS, add perfect medium 500 μ l to be placed in incubator and to cultivate, every day observation of cell growing state.
2, the extraction of viral DNA
(1) add isopyknic PBS damping fluid, mix, with 8000rpm centrifugal 5 minutes, abandon supernatant;
(2) add 20 μ L proteolytic enzyme k. and be incubated 3 hours in 56 DEG C of water-baths;
(3) centrifugal 5 minutes of 8000rpm, to get in 750 μ L supernatant liquors in a new centrifuge tube, adds isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1); 10 minutes are mixed with the speed of 50 revs/min, more centrifugal 5 minutes with 8000 revs/min, get 650 μ L supernatant liquors in a new centrifuge tube; Repeat 1 time;
(4) get 600 μ L supernatant liquors in a new centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24:1).Mix 10 minutes with 50 revs/min of speed, 8000 revs/min centrifugal 5 minutes;
(5) get 480 μ L supernatant liquors in a new centrifuge tube, add the sodium-acetate of 40 μ L 3mol/L and the cooling ethanol of 960 μ L4 DEG C; Jog, visible white floss;
(6) subzero 20 DEG C place 20-30 minute, after centrifugal 10 minutes with 12000 revs/min, abandoning supernatant;
(7) by the washing with alcohol of 200ul 75%, 14000 revs/min centrifugal 5 minutes, abandons supernatant; Repeat to do once;
(8) milli-Q water with 50ul after 10 minutes is dried, in subzero 20 DEG C of preservations.
3, PCR detects
(1) pcr amplification primer: (according to No. GenBank, the CyHV-2 virus series reported being: the primer of JQ067603.1 design);
Upstream primer CyHV-2F (5 '-3 '): CTTTAGCGTCAGGTCCATAGAGG
Downstream primer CyHV-2R (5 '-3 '): CGTCAGTCCCTGGCAGAAATAAG
Reaction system:
(2) PCR reaction conditions: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 62 DEG C of renaturation 30s, 72 DEG C extend 1min, 34 circulations, and last 72 DEG C extend 10min;
(3) agarose gel electrophoresis
Configuration: 1.0% agarose gel (50mL TAE buffer, 0.5g agar Icing Sugar) shakes up, and microwave oven about 1min seethes with excitement, room temperature places about 2min down in race offset plate, and place comb, room temperature is solid to gelling;
Sample preparation: PCR sample 5 μ L; Dyestuff (SYBR GREEN) 1 μ L; Loading buffer (2 ×) 2.5 μ L, about 30s is placed in mixing;
Loading: sample correspondence added in loading hole, record is good suitable.
(4) glue 100V about 30min is run.
(5) Taking Pictures recording result is observed.
4, experimental result
As shown in Figure 3A, negative infection group (uninfecting virus) Growth of Cells is good; Fig. 3 B, C, infect virus about 12 days and shrink formation cavity sample; Fig. 3 D, infect about 15 days cells formed obvious virus disease cash resemble (CPE).
PCR detected result is shown in Fig. 4, and positive all can increase and obtain the PCR band that fragment is about 640bp.
Above detected result shows: this grass goldfish abdomeinal fin clone can be applied to the detection of crucian simplexvirus II (CyHV-2).
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (3)
1. a construction process for grass goldfish abdomeinal fin clone, is characterized in that, comprises the steps:
1) process of tissue block: clip grass goldfish abdomeinal fin tissue, be soak 30m minute in the dual anti-solution of 1000IU/ml Pen .-Strep in concentration, with 75% alcohol disinfecting process, be that the D-PBS of the amphotericin B of 2.5ug/ml rinses 3 times by concentration, under aseptic condition, abdomeinal fin tissue be cut into 1mm
3tissue block, be immersed in organizing fritter digest 10min in 0.25% trypsinase and 0.02%EDTA after, then use 0.5% hyaluronidase digestion 1h;
2) original cuiture: tissue block being transplanted to floorage is 25cm
2culturing bottle in, keep temperature to be 26 DEG C, after tissue block is transplanted, within the 15th hour, add 2ml multiplication culture liquid, within the 24th hour, add 3ml multiplication culture liquid, every day half, amount changed nutrient solution once;
3) Secondary Culture: after primary cultured cell grows up to individual layer, adds trypsinase and leaves standstill digestion, has then hanged cell with multiplication culture liquid, has gone down to posterity, carry out Secondary Culture in 26 DEG C with 1:2;
Described multiplication culture liquid is the M199 nutrient solution containing 18v/v% foetal calf serum, 15ng/ml human alkaline fibroblast somatomedin, 2ng/ml Urogastron, 1ng/ml I Insulin-Like cell growth factor, 200IU/ml penicillin, 200ug/ml Streptomycin sulphate, 0.5ug/ml amphotericin B, 10mM 2 mercapto ethanol.
2. method according to claim 1, is characterized in that, the trypsinase concentration added in step 3 is 0.25%, and digestion time is 2min.
3. the grass goldfish abdomeinal fin clone constructed by the method described in claim 1 or 2 is detecting the application in simplexvirus CyHV-2.
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