CN103937736A - Method for establishing fish gill cell line - Google Patents
Method for establishing fish gill cell line Download PDFInfo
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- CN103937736A CN103937736A CN201410133077.6A CN201410133077A CN103937736A CN 103937736 A CN103937736 A CN 103937736A CN 201410133077 A CN201410133077 A CN 201410133077A CN 103937736 A CN103937736 A CN 103937736A
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Abstract
The invention discloses a method for establishing a fish gill cell line. The method for establishing the fish gill cell line comprises the following steps: treating temporarily cultured healthy fishes with antibiotic at room temperature, anesthetizing fish bodies with ice water, immerging the fish bodies into 5% dichloroether to be disinfected for 5 minutes, then sterilizing the surfaces of the fish bodies by adopting 70% ethanol cotton balls, then chopping gill tissues into slices with the size of 0.8-1.51mm<3> in a super clean bench, flushing with an antibiotic containing Leibovitz's L-15 culture medium, inoculating into a 25cm<2> cell culture bottle containing 5ml Leibovitz's L-15 complete culture medium, culturing in a culture box at the temperature of 28 DEG C, replacing the complete culture medium once per 3-4 days; when 95% of gill tissue cells are fused, according to a standard trypsin digestion method, subculture in bottles is carried out in ratio of 1:2 until 50 generations are formed, so that the fish gill cell line is established. The method for establishing the fish gill cell line has the advantages that fish gill cells difficult to culture are smoothly cultured and can stably grow in vitro, a cell line is successfully established, passage of more than 50 generations can be formed, stable multiplication is realized, and an infinite cell line can be formed.
Description
Technical field
The invention belongs to fish cell culture technique field, particularly a kind of establishment method of fish Gill cell line.
Background technology
Fish Tissue is cultivated and the development of clone has great importance for separation and stechiology, the genetic expression etc. of virus, poisonous substance, carcinogenic substance.Wolf and Quimby etc. (1962) from rainbow trout sexual gland separation and Culture first fish cell system-RTG-2.Up to now, this clone has been widely used in virusology and toxicological studies.Significant contribution has been made in fields such as fish immunology, toxicology, ecotoxicology, incretology, viral, biomedical research, diseases prevention and treatment, biotechnology, aquaculture and radiation biologies by fish cell system.Cell culture system is also for the application of banding technique provides best method of chromosome preparation.That fish cell system has been widely used as is viral, the external model of genetic expression and aquatic products toxicologic study.After this, the work of exploitation fish cell system has made great progress, and clone quantity significantly increases to 283.Most of fish clone derives from fresh water or anadromous fish.Knowledge to fish cell system or cell cultures is very ripe in developed country, but documents and materials demonstration, and China is very limited in the data in this field, particularly more rare aspect the cultivation of fish cell system.
The fish gill is to maintain homeostatic very important organ.Gill epithelial cell has a lot of functions, comprises the excretion of gaseous interchange, acid base equilibrium and ion adjusting, refuse etc.Water, ion and water are passed through by three kinds of complex cells (breathing cell, chloride cell, myxocyte) these functions thereby the epithelium forming directly contacts and is very important with external environment.When contacting with water, gill epithelium must adapt to outside atmosphere, as temperature, and dissolved oxygen, pH, or pollutent, thus maintain or regulate its normal function.Manyly show function and the regulating effect thereof of the fish gill in the time running into ambient interference in body research.For example, use verified some ion transporter of probe (antibody, cDNA) or antagonist to participate in osmoregulation and acid-alkali accommodation.But, due to fish gill epithelial structure complexity, want to understand better the interaction between function, particularly cell type and the cell of the gill, carry out external fish Gill cell line/foundation or the cultivation of the fish gill extremely urgent.
Several fish gill extracorporeal culturing methods used have its relative merits separately at present.The first extracorporeal culturing method is to separate fresh gill epithelial cell to obtain relevant breathing cell and to be rich in the ion transport information of plastosome cell.Another kind of extracorporeal culturing method is that organ culture system (separates the gill filament and in substratum, cultivates 1 ~ 4 d).This technology has likely been measured the impact on several gill parameters of hydrocortisone or copper.The third method is to have set up several fish Gill cell lines.RTgill-W1 clone has been used to toxicology and pathogenic agent experimental study.
Summary of the invention
The object of the present invention is to provide a kind of establishment method of fish Gill cell line, for the researchs such as physiology of fishes, immunology, toxicology, ecotoxicology, incretology, viral, biomedical research provide technical support and guarantee.
This invention completes by following technical proposal.
The establishment method of fish Gill cell line of the present invention, comprises the steps:
(1) with at room temperature process the healthy fish of supporting temporarily containing the microbiotic water of 500 IU/mL penicillin and 500 μ g/mL Streptomycin sulphates;
(2) the fish body of (1) being processed is with after frozen water anesthesia, immerses 5% dichloroethyl ether, 5 min that sterilize, and then with the cotton balls of 70% ethanol, sterilising treatment carried out in fish surface;
(3) the fish body of (2) being processed is placed in super clean bench, gill tissue is cut into 0.8-1.51 mm under aseptic condition
3fritter, then rinse 3 times with containing antibiotic Leibovitz ' s L-15 substratum;
(4) tissue fragment (3) being obtained is inoculated into 25 cm that contain 5 mL Leibovitz ' s L-15 perfect mediums
2in Tissue Culture Flask, put into temperature and be the incubator of 28 ° of C and cultivate, change perfect medium liquid 1 time every 3-4 d;
(5) in the time that the histocyte of (4) reaches 95% fusion, according to the tryptic digestion method of standard, with the cultivation of going down to posterity of the ratio sub-bottle of 1:2, so go down to posterity and be cultured to for 50 generations, just set up fish Gill cell line.
Antibiotics and dosage in described step (3) are respectively: 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates and 2.5 μ g/mL amphotericins.
Leibovitz ' s L-15 substratum used in described step (3) is commercial product.
In described step (4), the component of Leibovitz ' s L-15 perfect medium used is: Leibovitz ' s L-15 substratum, 15%(V/V) foetal calf serum, 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates, 2.5 μ g/mL amphotericins.
Foetal calf serum in described step (4) is commercial inactivated fetal bovine serum.
In described step (5), in tryptic digestion method used, reagent used is trypsinase-EDTA solution phosphoric acid buffer, that is: 0.25% trypsinase and 0.02%ETDA.
The present invention has following advantages and effect: adopt technical scheme of the present invention, fish gill cell has been carried out to the primary and cultivation of going down to posterity, obtain good culture effect, thereby set up fish Gill cell line, the fish gill cell that makes to be difficult to cultivate has obtained smooth cultivation, in vitro stable growth build and be tied to form merit.In the fish gill cell of cultivating, due to the propagation cultivation of going down to posterity, and allow clone spend the crisis of going down to posterity, go down to posterity and exceeded for 50 generations, and can stablize propagation, become infinite cell line.Thereby for the researchs such as physiology of fishes, immunology, toxicology, ecotoxicology, incretology, viral, biomedical research provide technical support and guarantee.
Embodiment
The establishment method of fish Gill cell line of the present invention, comprises the steps:
(1) the healthy carp juvenile fish of Laboratory Acclimation is placed in the antibiotic treatment water containing 500 IU/mL penicillin and 500 μ g/mL Streptomycin sulphates to 22.0-24.0 ° of C of temperature; Then with frozen water by after juvenile fish anesthesia, immerse 5% chlorex, 5 min that sterilize, finally with 70% ethanol cotton balls cotton ball soaked in alcohol, sterilized in fish surface;
(2) juvenile fish after surface sterilization is put into super clean bench dissect get the gill, by gill tissue in aseptic condition incision into about 1 mm
3fritter, and rinse 3 times with commercial Leibovitz ' the s L-15 nutrient solution that contains 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates and 2.5 μ g/mL amphotericins;
(3) prepare Leibovitz ' s L-15 perfect medium, its component comprises commercial Leibovitz ' s L-15 substratum, 15%(V/V) commercial inactivated fetal bovine serum, 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates and 2.5 μ g/mL amphotericins; Then carp gill tissue fragment is inoculated into 25 cm that contain 5 mL Leibovitz ' s L-15 perfect mediums
2in Tissue Culture Flask, put into temperature and be the incubator of 28 ° of C and cultivate, change perfect medium liquid 1 time every 3 d or 4 d; In the time that the cell of carp gill tissue reaches 95% fusion, according to the tryptic digestion method of standard, with the cultivation of going down to posterity of the ratio sub-bottle of 1:2; Wherein in tryptic digestion method, reagent used is trypsinase-EDTA solution phosphoric acid buffer, that is: 0.25% trypsinase and 0.02%ETDA.
(4) so go down to posterity and be cultured to for 50 generations, set up fish Gill cell line.
Claims (6)
1. the establishment method of fish Gill cell line, comprises the steps:
(1) with at room temperature process the healthy fish of supporting temporarily containing the microbiotic water of 500 IU/mL penicillin and 500 μ g/mL Streptomycin sulphates;
(2) the fish body of (1) being processed is with after frozen water anesthesia, immerses 5% dichloroethyl ether, 5 min that sterilize, and then with the cotton balls of 70% ethanol, sterilising treatment carried out in fish surface;
(3) the fish body of (2) being processed is placed in super clean bench, gill tissue is cut into 0.8-1.51 mm under aseptic condition
3fritter, then rinse 3 times with containing antibiotic Leibovitz ' s L-15 substratum;
(4) tissue fragment (3) being obtained is inoculated into 25 cm that contain 5 mL Leibovitz ' s L-15 perfect mediums
2in Tissue Culture Flask, put into temperature and be the incubator of 28 ° of C and cultivate, change perfect medium liquid 1 time every 3-4 d;
(5) in the time that the histocyte of (4) reaches 95% fusion, according to the tryptic digestion method of standard, with the cultivation of going down to posterity of the ratio sub-bottle of 1:2, so go down to posterity and be cultured to for 50 generations, just set up fish Gill cell line.
2. the establishment method of fish Gill cell line according to claim 1, is characterized in that, Antibiotics and dosage in described step (3) are respectively: 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates and 2.5 μ g/mL amphotericins.
3. the establishment method of fish Gill cell line according to claim 1, is characterized in that, Leibovitz ' s L-15 substratum used in described step (3) is commercial product.
4. the establishment method of fish Gill cell line according to claim 1, it is characterized in that, in described step (4), the component of Leibovitz ' s L-15 perfect medium used is: Leibovitz ' s L-15 substratum, 15%(V/V) foetal calf serum, 500 IU/mL penicillin, 500 μ g/mL Streptomycin sulphates and 2.5 μ g/mL amphotericins.
5. the establishment method of fish Gill cell line according to claim 1, is characterized in that, the foetal calf serum in described step (4) is commercial inactivated fetal bovine serum.
6. the establishment method of fish Gill cell line according to claim 1, it is characterized in that, in described step (5), in tryptic digestion method used, reagent used is trypsinase-EDTA solution phosphoric acid buffer, that is: 0.25% trypsinase and 0.02%ETDA.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971700A (en) * | 2019-03-12 | 2019-07-05 | 南京师范大学 | A kind of primary gill cell culture processes of fugu obscurus |
| CN111876369A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Rapid primary culture method and application of haliotis discus gill cells |
| CN114410569A (en) * | 2021-03-11 | 2022-04-29 | 青海大学 | Construction method of gill cell line of Gymnocypris przewalskii |
| CN117070438A (en) * | 2023-06-13 | 2023-11-17 | 中国水产科学研究院长江水产研究所 | Acipenser sinensis gill cell line and construction method and application thereof |
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| CN102154211A (en) * | 2010-11-17 | 2011-08-17 | 中山大学 | Branchiostoma belcheri tsingtaunese cell line and constructing method thereof |
| CN102399743A (en) * | 2011-12-16 | 2012-04-04 | 中国水产科学研究院长江水产研究所 | Cell line of pterygiophore tissue of cryprinus carpiod and construction method |
| CN102492650A (en) * | 2011-11-24 | 2012-06-13 | 大连海洋大学 | Construction method for Hexagrammos otakii cell line |
| CN102757932A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of sinocyclocheilus grahami grahami fin cell line |
| CN103122333A (en) * | 2013-01-31 | 2013-05-29 | 浙江工商大学 | Method for separation, purification, culture and passage of gill epithelial cells of hybridized prussian carp |
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2014
- 2014-04-04 CN CN201410133077.6A patent/CN103937736A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154211A (en) * | 2010-11-17 | 2011-08-17 | 中山大学 | Branchiostoma belcheri tsingtaunese cell line and constructing method thereof |
| CN102492650A (en) * | 2011-11-24 | 2012-06-13 | 大连海洋大学 | Construction method for Hexagrammos otakii cell line |
| CN102399743A (en) * | 2011-12-16 | 2012-04-04 | 中国水产科学研究院长江水产研究所 | Cell line of pterygiophore tissue of cryprinus carpiod and construction method |
| CN102757932A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of sinocyclocheilus grahami grahami fin cell line |
| CN103122333A (en) * | 2013-01-31 | 2013-05-29 | 浙江工商大学 | Method for separation, purification, culture and passage of gill epithelial cells of hybridized prussian carp |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971700A (en) * | 2019-03-12 | 2019-07-05 | 南京师范大学 | A kind of primary gill cell culture processes of fugu obscurus |
| CN109971700B (en) * | 2019-03-12 | 2021-09-28 | 南京师范大学 | Culture method of primary gill cells of takifugu obscurus |
| CN111876369A (en) * | 2020-07-24 | 2020-11-03 | 中国海洋大学 | Rapid primary culture method and application of haliotis discus gill cells |
| CN114410569A (en) * | 2021-03-11 | 2022-04-29 | 青海大学 | Construction method of gill cell line of Gymnocypris przewalskii |
| CN114410569B (en) * | 2021-03-11 | 2023-12-19 | 青海大学 | Construction method of Qinghai lake naked carp gill cell line |
| CN117070438A (en) * | 2023-06-13 | 2023-11-17 | 中国水产科学研究院长江水产研究所 | Acipenser sinensis gill cell line and construction method and application thereof |
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Application publication date: 20140723 |