CN105087491B - A kind of construction method of fancy carp brain cell line and its application - Google Patents
A kind of construction method of fancy carp brain cell line and its application Download PDFInfo
- Publication number
- CN105087491B CN105087491B CN201510531300.7A CN201510531300A CN105087491B CN 105087491 B CN105087491 B CN 105087491B CN 201510531300 A CN201510531300 A CN 201510531300A CN 105087491 B CN105087491 B CN 105087491B
- Authority
- CN
- China
- Prior art keywords
- cell
- fancy carp
- culture
- carp brain
- cell line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000252233 Cyprinus carpio Species 0.000 title claims abstract description 104
- 210000004958 Brain cells Anatomy 0.000 title claims abstract description 68
- 238000010276 construction Methods 0.000 title claims abstract description 13
- 210000004027 cells Anatomy 0.000 claims abstract description 91
- 230000001079 digestive Effects 0.000 claims abstract description 31
- 210000001519 tissues Anatomy 0.000 claims abstract description 29
- 230000029087 digestion Effects 0.000 claims abstract description 24
- 210000004556 Brain Anatomy 0.000 claims abstract description 21
- 238000004113 cell culture Methods 0.000 claims description 23
- 239000003102 growth factor Substances 0.000 claims description 10
- 229940088598 Enzyme Drugs 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 210000002966 Serum Anatomy 0.000 claims description 9
- 102000004142 Trypsin Human genes 0.000 claims description 7
- 108090000631 Trypsin Proteins 0.000 claims description 7
- 230000001464 adherent Effects 0.000 claims description 7
- 229960001322 trypsin Drugs 0.000 claims description 7
- 239000012588 trypsin Substances 0.000 claims description 7
- 230000001605 fetal Effects 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 5
- 210000000496 Pancreas Anatomy 0.000 claims description 4
- 229940049954 Penicillin Drugs 0.000 claims description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
- 229960000626 benzylpenicillin Drugs 0.000 claims description 4
- 230000003115 biocidal Effects 0.000 claims description 4
- 210000002950 fibroblast Anatomy 0.000 claims description 4
- 102100010813 EGF Human genes 0.000 claims description 3
- 101700033006 EGF Proteins 0.000 claims description 3
- 210000004748 cultured cells Anatomy 0.000 claims description 3
- 229940116977 Epidermal Growth Factor Drugs 0.000 claims description 2
- 241000175212 Herpesvirales Species 0.000 claims 1
- 241001051708 Cyprinid herpesvirus 3 Species 0.000 abstract description 40
- 230000012010 growth Effects 0.000 abstract description 15
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 abstract description 6
- 229960005486 vaccines Drugs 0.000 abstract description 6
- 210000002845 Virion Anatomy 0.000 abstract description 3
- 230000018109 developmental process Effects 0.000 abstract description 3
- 231100000614 Poison Toxicity 0.000 abstract description 2
- 230000001717 pathogenic Effects 0.000 abstract description 2
- 239000002574 poison Substances 0.000 abstract description 2
- 230000001681 protective Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 46
- 238000002360 preparation method Methods 0.000 description 9
- 201000010099 disease Diseases 0.000 description 8
- 239000001963 growth media Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 230000000120 cytopathologic Effects 0.000 description 7
- 241000251468 Actinopterygii Species 0.000 description 4
- 230000037165 Serum Concentration Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 201000009910 diseases by infectious agent Diseases 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000003612 virological Effects 0.000 description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N BRL-49594 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 3
- 239000012593 Hanks’ Balanced Salt Solution Substances 0.000 description 3
- 229940055695 Pancreatin Drugs 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 229960003942 amphotericin B Drugs 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 208000002021 Enophthalmos Diseases 0.000 description 2
- 210000002816 Gills Anatomy 0.000 description 2
- 210000003491 Skin Anatomy 0.000 description 2
- 208000001756 Virus Disease Diseases 0.000 description 2
- 230000001488 breeding Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 206010059512 Apoptosis Diseases 0.000 description 1
- 206010049848 Balance disease Diseases 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 210000002808 Connective Tissue Anatomy 0.000 description 1
- 241000252210 Cyprinidae Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 210000002615 Epidermis Anatomy 0.000 description 1
- 210000003237 Epithelioid Cells Anatomy 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010018987 Haemorrhage Diseases 0.000 description 1
- -1 M199 Substances 0.000 description 1
- 210000003097 Mucus Anatomy 0.000 description 1
- 210000003934 Vacuoles Anatomy 0.000 description 1
- VEGOPIRPQIZFRD-UVHWXNHCSA-N [(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl] (E)-3-(4-hydroxyphenyl)prop-2-enoate Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC(=O)\C=C\C1=CC=C(O)C=C1 VEGOPIRPQIZFRD-UVHWXNHCSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000000249 desinfective Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 231100000880 dysequilibrium Toxicity 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002919 epithelial cells Anatomy 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 239000002609 media Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention discloses a kind of construction method of fancy carp brain cell line and its applications, fancy carp brain tissue is first put into containing digesting in clostridiopetidase A and tryptic digestive juice A, collect cell, it adds in the digestive juice B containing EDTA-4Na and is digested, collect postdigestive cell progress originally culture and secondary culture can be obtained fancy carp brain cell line；The present invention is directed to the feature of fancy carp brain tissue cell, brain tissue cell is separated using clostridiopetidase A and trypsase simultaneous digestion method, carry out originally culture, then fancy carp brain cell line is successfully constructed, it is more than generation that 100 have continuously been reached, it can provide a large amount of fancy carp brain derived cell, and cell can maintain good growth conditions, freezen protective can be carried out to it.Fancy carp brain cell line of the present invention has sensibility to Koi herpesvirus, cytopathy occurs after connecing poison, and Electronic Speculum observation is it can be found that the virion being proliferated, cell line of the present invention may be directly applied to pathogenic characteristic research and vaccine development.
Description
Technical field
The present invention relates to the construction method of one plant of fancy carp brain cell line and the applications in terms of isolated viral, and it is raw to belong to fresh water
Object cell culture application technology.
Background technique
Fancy carp (Cyprinus carpio) also known as " red carp ", " Japanese kampo " be one of large-scale ornamental fish, it is the change of carp
Kind, belong to Cyprinidae, carp category, according to color, speckle equal distribution situation, fancy carp is divided into red white, big positive three color, three color of Showa etc. 13 again
Strain.Fancy carp is bright-colored because its figure is good, and cultivation scale is increasing.As breeding environment is more and more severe, fancy carp it is each
Kind communicable disease especially Koi herpesvirus disease starts wide-scale distribution.Koi herpesvirus disease the first explosion in 1998 with
Color column, but just confirmed that its real cause of disease was Koi herpesvirus (Koi herpesvirus, KHV) in 1999.Hereafter in English
State, Continental Europe, the U.S., Japan, which also have, to break out.In recent years, the wide-scale distribution of Koi herpesvirus disease, causes fancy carp to cultivate
The huge economic loss of industry has become one of an important factor for restricting koi breeding industry sustainable development.
Epidemiological study shows that KHV is propagated rapidly, can infect fancy carp and the carp at any age, infect the sick fish of KHV
Typical clinical symptom is to be slow in action, loss of appetite, dysequilibrium, the more mucus of body surface, in skin and fin base portion haemorrhage occur
Shape, enophthalmos,enophthalmus, the gill are pale or color is irregular and downright bad to the serious gill with moderate, and 7~10d starts to occur after infection
Death, the death rate is up to 100% after 2~3 weeks.The virus causes the death rate of disease fish high, and epidemic situation is difficult to control.
Currently, the whole genome sequence for the KHV strain isolated from Israel, the U.S. and Japanese 3 places is
It comes forth, the conivium from Israel and the U.S. is referred to as European strain, and the KHV conivium from Japan is named as Asia
Strain.
Virus is the basis for studying the prevention and control of Koi herpesvirus disease in the separation and culture of cellular level, is KHV most accurate
Diagnosis and treatment method therefore, establish thin from fancy carp but research shows that KHV can only be grown in being originated from original host cell
Born of the same parents system, is the separation and identification for carrying out Koi herpesvirus, studies its pathogenesis, the weight of vaccine preparation and immunoprophylaxis technology
Want basis.
In the development of vaccine, Israel has now been developed Koi herpesvirus vaccine, and successfully lists, due to different bases
Because type strain has differences, the prevention and treatment whether vaccine is suitable for Chinese Koi herpesvirus disease is also unknown, therefore, establishes energy
The cell line from fancy carp for enough separating current KHV epidemic strain is extremely urgent.
The originally culture technology of fish cell can be divided into two kinds substantially at present, tissue mass cell culture and digestion cultivation.
Tissue mass cell culture is easy to produce mechanical damage, and the cell that cell is not easy to move out, and moves out has selectivity.Digestion method is normal at present
It is pancreatin digestion, but pancreatin can cause to damage to some epithelial cells, while can be invalid to fiber and connective tissue.So far
Until, the document report of logical enzyme digestion preparation fancy carp cell line has: Dong Chuanfu, Xue Shuqun and Xiao Yi are all made of tissue block separation
Method establish fancy carp fin ray cell line (Virus Research, 2011,161: 140-149；Fisheries Science magazine, 2012,
25(4): 16-23；Chinese cell biology journal 2012,34 (8): 767-774), Wang Yingying establishes one using digestion method
Strain fancy carp kiss teloblast system (Aquaculture, 2015,435:310-317), but have not yet to see the report of fancy carp brain derived cell system
Road.It can be seen that need to study the construction method of effective fancy carp brain cell line a kind of at present, it is thin that fancy carp its hetero-organization is established for the later period
Born of the same parents system and the research for carrying out Koi herpesvirus lay the foundation.
Summary of the invention
The purpose of the present invention is to provide a kind of construction methods of fancy carp brain cell line
The purpose of the present invention is to provide the fancy carp brain cell lines of above method building in Koi herpesvirus culture
Using.
The technical solution used in the present invention is:
A kind of construction method of fancy carp brain cell line, this method are as follows: fancy carp brain tissue is put into digestive juice A and is disappeared
Change, collect cell, add in digestive juice B and digested, collects postdigestive cell and carry out originally culture and secondary culture i.e.
It can get fancy carp brain cell line；
The digestive juice A is the D-Hanks solution containing 0.8~1.2g/L clostridiopetidase A and 0.7~0.9g/L trypsase；
The digestive juice B is the D-Hanks solution of the EDTA-4Na containing 0.15~0.25g/L.
A kind of construction method of fancy carp brain cell line, comprising the following steps:
1) tissue digestion: fancy carp brain tissue being put into digestive juice A and is digested, and is collected cell, is added in digestive juice B
It is digested, collects postdigestive cell；
2) originally culture: being added fancy carp brain cell culture solution for cell that upper step is collected into and cell suspension be made, 26~
28 DEG C, 4.5~5.5% CO2Under the conditions of carry out originally culture；
3) secondary culture: when primary cultured cell covers with culture bottle bottom, culture solution is removed, is digested with trypsin solution, is disappeared
Enzyme solution is removed after change, fancy carp brain cell culture solution is added, cell is resuspended, and cell will be resuspended and carry out secondary culture；
The digestive juice A is the D-Hanks solution containing 0.8~1.2g/L clostridiopetidase A and 0.7~0.9g/L trypsase；
The digestive juice B is the D-Hanks solution of the EDTA-4Na containing 0.15~0.25g/L.
Further, fancy carp brain tissue is the sterile fancy carp brain tissue shredded in step 1).
Further, temperature when digestive juice A digests in step 1) is 35~38 DEG C, and digestion time is 40~50min, often
10~17min piping and druming is primary.
Further, the temperature that digestive juice B digests in step 1) is 25~30 DEG C, and digestion time is 2~5min.
Further, during step 1) originally culture, after cell is adherent for the first time, primary culture was changed every 2~3 days
Liquid.
Further, above-mentioned fancy carp brain cell culture solution is the table containing 14~16v/v% fetal calf serum, 9~11ng/ml
Skin growth factor, the basic fibroblast like growth factor of 1.8~2.2ng/ml, 18~22mmol/LHEPES, appropriate penicillin,
The L-15 culture solution of streptomysin and amphotericin B, pH are 7.0~7.4.
Further, contain 0.2~0.3%w/v pancreas enzyme -EDTA in step 3) in trypsin solution.
Further, in step 3) after secondary culture to 8~14 generations, with growth factor is free of, antibiotic, and tire are free of
The fancy carp brain cell culture solution that cow's serum content is 9~11v/v% carries out subsequent secondary culture.
Application of the fancy carp brain cell line of above method preparation in culture Koi herpesvirus.
The beneficial effects of the present invention are:
1) present invention is directed to the feature of fancy carp brain tissue cell, separates brain using clostridiopetidase A and trypsase simultaneous digestion method
Histocyte carries out originally culture, then successfully constructs fancy carp brain cell line, it is more than generation continuously to have reached 100, it is possible to provide
A large amount of fancy carp brain derived cell, and cell can maintain good growth conditions, can carry out freezen protective to it.
2) the fancy carp brain cell line that the present invention constructs can be with continuous passage, and the fancy carp brain cell line passed on through this method is existing
It reached for 100 generations, there is sensibility to Koi herpesvirus (KHV), occur cytopathy (CPE) after connecing poison, Electronic Speculum observation
It can be found that the virion of proliferation, cell line of the present invention may be directly applied to the research of KHV pathogenic characteristic and its vaccine is ground
System.
Detailed description of the invention
Fig. 1 is the form of the fancy carp brain cell line of secondary culture under phase contrast microscope；A is that fancy carp brain cell is primary；B is 45
For fancy carp brain cell；
Fig. 2 is growth curve of the fancy carp brain cell line under different culture medium；
Fig. 3 is growth curve of the fancy carp brain cell line under different serum-concentrations；
Fig. 4 is the growth curve of fancy carp brain cell line at different temperatures；
Fig. 5 is that fancy carp brain cell line infects Koi herpesvirus KHV picture；A is that the fancy carp brain before infecting KHV virus is thin
Born of the same parents, B be infect KHV virus 72h after fancy carp brain cell CPE, C be infection KHV virus 120h after fancy carp brain cell CPE, D,
E is the cell electron microscopic section figure for infecting KHV virus.
Specific embodiment
A kind of construction method of fancy carp brain cell line, this method are as follows: fancy carp brain tissue is put into digestive juice A and is disappeared
Change, collect cell, add in digestive juice B and digested, collects postdigestive cell and carry out originally culture and secondary culture i.e.
It can get fancy carp brain cell line；
The digestive juice A is the D-Hanks solution containing 0.8~1.2g/L clostridiopetidase A and 0.7~0.9g/L trypsase；
The digestive juice B is the D-Hanks solution of the EDTA-4Na containing 0.15~0.25g/L.
A kind of construction method of fancy carp brain cell line, comprising the following steps:
1) tissue digestion: fancy carp brain tissue being put into digestive juice A and is digested, and is collected cell, is added in digestive juice B
It is digested, collects postdigestive cell；
2) originally culture: being added fancy carp brain cell culture solution for cell that upper step is collected into and cell suspension be made, 26~
28 DEG C, 4.5~5.5% CO2Under the conditions of carry out originally culture；
3) secondary culture: when primary cultured cell covers with culture bottle bottom, culture solution is removed, is digested with trypsin solution, is disappeared
Enzyme solution is removed after change, fancy carp brain cell culture solution is added, cell is resuspended, and cell will be resuspended and carry out secondary culture；
The digestive juice A is the D-Hanks solution containing 0.8~1.2g/L clostridiopetidase A and 0.7~0.9g/L trypsase； ,
The digestive juice B is the D-Hanks solution of the EDTA-4Na containing 0.15~0.25g/L.
Preferably, fancy carp brain tissue is the sterile fancy carp brain tissue shredded in step 1).
Preferably, in step 1) digestive juice A digest when temperature be 35~38 DEG C, digestion time be 40~50min, every 10
~17min piping and druming is primary.
Preferably, the temperature that digestive juice B digests in step 1) is 25~30 DEG C, and digestion time is 2~5min.
Preferably, during step 1) originally culture, after cell is adherent for the first time, a culture solution was changed every 2~3 days.
Preferably, above-mentioned fancy carp brain cell culture solution is the epidermis containing 14~16v/v% fetal calf serum, 9~11ng/ml
Growth factor, the basic fibroblast like growth factor of 1.8~2.2ng/ml, 18~22mmol/LHEPES, appropriate penicillin, chain
The L-15 culture solution of mycin and amphotericin B, pH are 7.0~7.4.
Preferably, contain 0.2~0.3%w/v pancreas enzyme -EDTA in step 3) in trypsin solution.
Preferably, in step 3) after secondary culture to 8~14 generations, with growth factor is free of, antibiotic, and tire ox are free of
The fancy carp brain cell culture solution that serum content is 9~11v/v% carries out subsequent secondary culture.
Application of the fancy carp brain cell line of above method preparation in culture Koi herpesvirus.
The present invention is further illustrated combined with specific embodiments below, and however, it is not limited to this.
The construction method of 1 carp brain cell line of embodiment
(1) preparation of cell culture fluid and tissue digestion liquid
The preparation of 1, fancy carp brain cell culture solution:
15v/v% fetal calf serum (GIBCO) will be added in L-15 culture solution (GIBCO), the epidermal growth factor of 10ng/ml
(EGF) the basic fibroblast like growth factor (bFGF) (Sigma) of (Sigma) and 2ng/ml, while adding 100IU/ml mould
Element, 100 μ g/ml streptomysins, 0.25 μ g/ml amphotericin B, the HEPES of 20mmol/L, pH 7.0-7.4.
After cell reached for 10 generations, the fetal calf serum additive amount in fancy carp brain cell culture solution is reduced to 10%, does not add life
The long factor and antibiotic.
2. the preparation of tissue digestion liquid
Private organizations digestive juice A: 100mg type Ⅳ collagen enzyme powder and 80mg trypsase powder are dissolved in 100ml D-
In Hanks liquid, stirring and dissolving 2h, 4 DEG C overnight, and 0.22um filtration disinfection dispenses -20 DEG C of preservations；
Private organizations digestive juice B:20mg EDTA-4Na is dissolved in 100ml D-Hanks liquid, high pressure steam sterilization, packing
At bottle, 4 DEG C of refrigerators are saved.
(2) tissue digestion
1) by fresh fancy carp brain tissue, salt is balanced with HBSS(Hank's Balanced Salt Solution, Hank's
Solution) rinsing 2 times after, be placed in the sterile penicillin bottle of 5ml (culture solution of L-15 containing 0.5ml), be cut into operating scissors
About 1mm3Fragment is added 5ml HBSS and collects to 15ml centrifuge tube, and 1000rpm is centrifuged 3 minutes, discards after tissue block is collected by centrifugation
Supernatant repeats 1-2 times；
2) tissue block that upper step is collected is put into private organizations digestive juice A, 37 DEG C of digestion (were blown for 45 minutes every 15 minutes
Beat once), L-15 culture solution is added and terminates digestion, cell is collected by centrifugation, private organizations digestive juice B is added, 27 DEG C of digestion 2-5 divide
Zhong Hou, 1000 turns/min are centrifuged 3 minutes, remove B digestive juice, collect cell.
(3) originally culture
Upper step is obtained into cell addition fancy carp brain cell culture solution, cell suspension is made, is uniformly inoculated in 25 cm2Culture
In bottle, at 27 DEG C, 5% CO2It is cultivated under condition of culture, after brain cell is adherent for the first time, changed a culture solution, every 2~3 days to go
Except not adherent tissue and dead cell.
(4) secondary culture:
When the cell in originally culture covers with culture bottle bottom, old culture solution is removed with suction pipe, and HBSS is washed twice, with containing
When the enzyme Huaihe River of 0.25% pancreas enzyme -EDTA is by cell dissociation to cell rounding, inhales and abandon pancreatin, fresh fancy carp brain cell culture solution is added
Digestion is terminated, with suspension cell is collected after suction pipe featheriness, is inoculated in culture bottle with the ratio of 1:2, L-15 culture medium is added extremely
5mL/ bottles, 27 DEG C are put into, 5% CO2Secondary culture is carried out in incubator；When fancy carp brain cell grows up to single layer again, still more than
Same procedure secondary culture is stated, splitting ratio is 1:(2~3)；When cultivating to after 8~14 generations, with being free of growth factor, and tire
The fancy carp brain cell culture solution that cow's serum content is 10v/v% carries out subsequent secondary culture.
Fancy carp brain cell line prepared by the present invention reached for 100 generations, and cell can stablize increment, is named as KB.It will
45th generation cell and primary cell are observed with phase contrast microscope, and the two form is as shown in Figure 1, it can be seen that KB is primary
Cell shape is irregular, mostly fibroblast-like cells, and with the increase of passage number, fibroblast-like cells gradually apoptosis is given birth to
Long to stablize, the epithelioid cell of form rule replaces.
Further identification and effect detection are made to the fancy carp brain cell line prepared in embodiment below.
Two, the identification and methods for using them of fancy carp brain cell line: include: 1, cell freeze and recover, 2, cell growth it is special
Property analysis 3, virus infection experiment
One, cell freezes and recovers
1) cell freezes: collecting 2 bottles of fancy carp brain cells for being in logarithmic growth phase, 1000g centrifugation with trypsin digestion
It is discarded supernatant after 5min, freezes protection liquid (the L-15 culture solution containing 20% FBS and 10% DMSO) for cell weight with 1mL
It is outstanding to be placed in cryopreservation tube, cryopreservation tube is placed in program temperature reduction box in -70 DEG C overnight, is finally put into long in liquid nitrogen (- 196 DEG C)
Phase saves, and makes a record.
2) recovery of cell: when recovery cell, being removed from liquid nitrogen cryopreservation tube, thaws rapidly in 37 DEG C of water-baths,
1000g centrifugation 5min is inoculated in 25cm after collecting cell2Culture bottle in, 27 DEG C of cultures are placed in incubator, to cell
Supernatant is abandoned after adherent, is replaced culture solution, is continued to cultivate.
Two, growth characteristics measure
1) determination of fancy carp brain cell optimal medium
Select tetra- kinds of cell culture mediums of DMEM, M199, MEM, L-15, and add final concentration of 10% FBS preparation it is thin
Born of the same parents' culture solution.Adjusting cell density is 1 × 105mL-1, four kinds of culture mediums are respectively inoculated in 6 orifice plates by the amount in 2.5 holes mL/, in 27
DEG C incubator in cultivate.Every 1 d takes out 3 hole cells in each experimental group, is collected with Trypsin-EDTA digestion method thin
Born of the same parents simultaneously count, and co-culture 7 d, continuous counter 7 times, draw its growth curve.As Fig. 2 is most suitable for KB in selected culture medium
Culture medium be L-15 culture medium.
2) determination of the most suitable serum-concentration of fancy carp brain cell
The culture solution that FBS concentration is 5%, 10%, 15 %, 20% is prepared respectively, and adjustment cell density is 1 × 105mL-1, four
Kind serum-concentration culture medium is respectively inoculated in 6 orifice plates by the amount in 2.5 holes mL/, cultivates in 27 DEG C of incubator.Every 1 d is from each
3 hole cells are taken out in experimental group, with collected by trypsinisation cell and are counted, and are co-cultured 7 d, continuous counter 7 times, are drawn its growth
Curve.Growing state (see figure 3) of the fancy carp brain cell line KB under different serum-concentrations.It can be seen from the figure that originally thin
Born of the same parents' vitro growth rates at 20% FBS are most fast, and the doubling time, followed by 15%FBS, and cell was 5% about for 24 hours
The speed of growth is very slow under FBS.But with the extension of time, the cell Proliferation quantity highest in 10% FBS, growth conditions
It is best.Cell culture is carried out with the culture medium containing 10% FBS at present.
3) determination of the most suitable cultivation temperature of fancy carp brain cell
15oC, 22oC, 27oC, 32oC tetra- different cultivation temperatures are selected, are cultivated using the L-15 of 10% FBS of addition
Base, adjustment cell density are 1 × 105ml-1, cell suspension is inoculated in 6 orifice plates by the amount in 2.5 holes ml/ and is placed in four differences
In cultivation temperature incubator.Every 1 d takes out 3 hole cells in each experimental group, collects cell with Trypsin-EDTA digestion method
And count, 7 d are co-cultured, continuous counter 7 times, draw its growth curve.Cell counts count results are shown: being cultivated in 15 oC
Under the conditions of, cell can be normal adherent, but the speed of growth is slow, and cell number is up to 4.1 × 105It is a；Item is cultivated in 22 oC
Under part, cell can stablize proliferation, form single layer, but speed is slow compared with 27oC.Under 27 oC condition of culture, growth and proliferation of cell
Rapidly, cell monolayer is stablized, and culture to the 5th d cell number is up to 8.0 × 105It is a；Under 32 oC condition of culture, ancestral cell is played
Growing multiplication is rapid, and culture to the 5th its number of d is up to 8.0 × 105It is a, but cell monolayer is unstable, occurs falling off or death is thin
Born of the same parents, cell number decline (Fig. 4).The temperature for being most suitable for the growth of fancy carp brain cell is 27 DEG C.
Three, virus infection is tested
In order to study fancy carp brain cell line of the present invention to the viral susceptibility of Koi herpesvirus (KHV), with the 50th generation brocade
Carp brain cell is object, sensitivity of detection Koi herpesvirus (KHV) to the cell.After cell inoculation 24 hours, by 1mL
KHV viral solution is added in Tissue Culture Flask, after standing adsorption 1h, removes viral solution, replaces the fresh cells training containing 3%FBS
Nutrient solution (L-15 culture medium), continues to cultivate, and observes cytopathic effect (CPE).
Testing result as shown in figure 5, with infection KHV before fancy carp brain cell (Fig. 5 A) compared with, infect KHV after fancy carp
There is apparent cytopathic effect CPE (as shown in Fig. 5 B and C) in brain cell, therefrom this it appears that fancy carp brain cell
There is vacuole sample, cell fragment increases, cell detachment, and single layer is in broken fishing net shaped；Cell after infection KHV is done into electron microscopic section sight
It examines, the virion in sample there are KHV in lattice-like arrangement can be observed (as shown in Fig. 5 D and E).The above results illustrate this
The fancy carp brain cell line of invention preparation can be used for the culture of Koi herpesvirus.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of construction method of fancy carp brain cell line, it is characterised in that；The following steps are included:
1) tissue digestion: fancy carp brain tissue is put into digestive juice A, 35~38 DEG C, digests 40~50min, every 10~17min is blown
It beats and is once digested, collect cell, add in digestive juice B 25~30 DEG C, digest 2~5min, collect postdigestive cell；
2) originally culture: being added fancy carp brain cell culture solution for the cell that upper step is collected into and cell suspension be made, at 26~28 DEG C,
4.5~5.5%CO2Under the conditions of carry out originally culture；
3) secondary culture: when primary cultured cell covers with culture bottle bottom, culture solution is removed, is digested with trypsin solution, after digestion
Enzyme solution is removed, fancy carp brain cell culture solution is added, cell is resuspended, cell will be resuspended and carry out secondary culture；Secondary culture is to 8~14 generations
After, be free of growth factor, be free of antibiotic, and fetal calf serum content be 9~11v/v% fancy carp brain cell culture solution into
The subsequent secondary culture of row；
The digestive juice A is the D-Hanks solution containing 0.8~1.2g/L clostridiopetidase A and 0.7~0.9g/L trypsase；
The digestive juice B is the D-Hanks solution containing 0.15~0.25g/LEDTA-4Na；
The fancy carp brain cell culture solution be containing 14~16v/v% fetal calf serum, 9~11ng/ml epidermal growth factor,
Basic fibroblast like growth factor, 18~22mmol/LHEPES, appropriate penicillin, streptomysin and the both sexes of 1.8~2.2ng/ml
The L-15 culture solution of mycin B, pH are 7.0~7.4.
2. according to the method described in claim 1, it is characterized by: fancy carp brain tissue is the sterile fancy carp shredded in step 1)
Brain tissue.
3. according to the method described in claim 1, it is characterized by: cell is adherent for the first time during step 1) originally culture
Afterwards, a culture solution was changed every 2~3 days.
4. according to the method described in claim 1, it is characterized by: containing 0.2~0.3%w/ in step 3) in trypsin solution
V pancreas enzyme -EDTA.
5. the fancy carp brain cell line that the construction method of any fancy carp brain cell line of claim 1-4 obtains is in culture fancy carp
Application in herpesviral.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510531300.7A CN105087491B (en) | 2015-08-26 | A kind of construction method of fancy carp brain cell line and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510531300.7A CN105087491B (en) | 2015-08-26 | A kind of construction method of fancy carp brain cell line and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105087491A CN105087491A (en) | 2015-11-25 |
| CN105087491B true CN105087491B (en) | 2019-07-16 |
Family
ID=
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275925A (en) * | 2013-05-27 | 2013-09-04 | 中国水产科学研究院珠江水产研究所 | Construction method of mandarin fish brain cell system |
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
| CN103789263A (en) * | 2014-02-28 | 2014-05-14 | 中国科学院海洋研究所 | Construction method of bastard halibut brain cell system |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275925A (en) * | 2013-05-27 | 2013-09-04 | 中国水产科学研究院珠江水产研究所 | Construction method of mandarin fish brain cell system |
| CN103667176A (en) * | 2013-12-09 | 2014-03-26 | 中国水产科学研究院长江水产研究所 | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof |
| CN103789263A (en) * | 2014-02-28 | 2014-05-14 | 中国科学院海洋研究所 | Construction method of bastard halibut brain cell system |
Non-Patent Citations (2)
| Title |
|---|
| Studies on Cultured Rat Schwann cells.I.Establishment of Purified Populations from Cultures of Peripheral nerve;J.P.Brockes et al.;《Brain Research》;19791231;第165卷;摘要，第106页最后1段，第107页最后1段-第108页第1段 |
| 水华束丝藻胞外聚合物生物活性的研究;吕莹;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20080815(第08期);第30-31页3.4.1斑马鱼星形神经胶质细胞的获得和培养 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN105543164A (en) | Primary isolated culture method for dairy cow mammary epithelial cells | |
| CN105925523A (en) | Squaliobarbus curriculus fin cell line as well as establishing method and application thereof | |
| CN105907721A (en) | Pig intestinal endothelial cell line for stably expressing CaS9 protein | |
| CN103173365B (en) | Method for indoor culture breeding of Plasmopara viticola | |
| CN104974977B (en) | A kind of epinephelus lanceolatus fish nephridial tissue cell line and its construction method | |
| CN104152408B (en) | The preparation method of Subaerial blue green algae | |
| CN109294974A (en) | A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it | |
| CN107629996A (en) | The construction method of one plant of grass carp pectoral fin cell line | |
| CN102732486B (en) | Porcine circovirus type2 strain and application thereof | |
| CN103571798A (en) | Duck tembusu virus low virulent strain and application thereof | |
| CN111073862B (en) | Bovine viral diarrhea type2 attenuated strain and application thereof | |
| CN114196616B (en) | Fancy carp fin tissue cell line and application thereof | |
| CN105087491B (en) | A kind of construction method of fancy carp brain cell line and its application | |
| CN102911920A (en) | KMB17 cell-adapted strain for II type dengue virus and preparation method thereof | |
| CN102120983B (en) | Separation and culture method of epidermal stem cells of Beijing ducks | |
| CN115044556A (en) | Carp brain cell line and application thereof | |
| CN104911153A (en) | Method for separating acian metapneumovirus | |
| CN108753703A (en) | A kind of flounder embryo muscle satellite cell system method for building up | |
| CN110295137B (en) | Channa argus kidney cell line and construction method and application thereof | |
| CN110283793B (en) | Method for separating and culturing porcine pseudorabies virus | |
| CN106290861B (en) | A kind of detection method of exogenous avian leukosis virus | |
| CN105087491A (en) | Constructing method and application of koi brain cell line | |
| CN116836912B (en) | Lateolabrax japonicus kistrodon cell line and construction method and application thereof | |
| CN116926022B (en) | PiNew castle disease virus mGZ of pigeon origin gene VI, application, preparation method, culture method and vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |