CN105820994A - Primary culture method of ampullaria gigas hemocyte - Google Patents
Primary culture method of ampullaria gigas hemocyte Download PDFInfo
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Abstract
The invention relates to a primary culture method of ampullaria gigas hemocyte and belongs to the field of freshwater organisms' cell culture technology. The method of the invention comprises the following steps: preparing a cell culture solution; selecting 40-50 g of ampullaria gigas and raising in sterile water; sterilizing ampullaria gigas surface; dissecting ampullaria gigas; sucking up ampullaria gigas blood and placing the blood into a cell culture flask containing a HBSS cleaning fluid, and cleaning hemocyte; adding the cleaned hemocyte into a cell culture fluid, placing the culture flask into a 5% CO2 incubator, carrying out primary culture at the constant temperature of 28 DEG C; staying overnight, adding a cell culture fluid, sucking out the cell culture fluid and exchanging the same amount of a cell culture fluid every 5-7 days until the culture flask is filled with expanded and proliferative cells. According to the invention, primary culture of ampullaria gigas hemocyte can be rapidly and repeatedly established within a short time. The required cell culture equipment is simple and has strong maneuverability. The method of the invention is an important supplement for aquatic invertebrate cell culture and provides basic information for the researches on aquatic invertebrate's immunologic mechanism.
Description
Technical field
The invention belongs to limnobios technical field of cell culture, particularly relate to the primary culture method of a kind of Pomacea canaliculata hemocyte.
Background technology
Since the extracorporeal culturing method that Harrison and Carrel at the beginning of last century foundes animal tissue and cell, cell culture technology has obtained significant progress, all serves irreplaceable important function in fundamental research and applied research.But, up to the present, the research for cell culture technology itself focuses primarily upon vertebrates (particularly mammal) and insecticide, and the concern cultivated for aquatic invertebrate cell is the most relatively fewer.In the paper disclosing the relevant cell cultivation problem that publication is delivered, the overwhelming majority is vertebrates and insecticide.
From the sixties in 20th century, researcher begins to focus on the cell of aquatic invertebrate and cultivates problem, and the Various Tissues of aquatic invertebrate and cell are attempted and carry out In vitro culture, including epithelial cell, hemocyte, digestive gland, body and embryonal tissue etc..Just double umbilicus spiral shell embryo (Biomphalariaglabrataembryonie, BGE) cell line (Hansen, 1976) of aquatic invertebrate is established at 20 century 70s.BGE cell line is Hansen to be set up from the double umbilicus spiral shell embryo of fresh water in 1976 to study schistosomicide born of the same parents' col infection cell mechanism, after by the perfect cryopreservation methods such as Bayne and submit to ATCC (CRL-1494).
Although just obtaining such gratifying achievements aquatic invertebrate cell is cultivated early stage, but decades do not have big progress relative to eighties of last century after in the past the seventies, up to the present, except BGE cell line, the most successfully build the report being, most cells cultivate generally the most in vitro can only survival ratio relatively short period of time, the several months although some report cell can be survived but several generations can only be divided and the most no longer divide, whole cultivation is last the most also to come to an end because microorganism is polluted.
Pomacea canaliculata has another name called Ampullaria gigas, Fructus Mali pumilae spiral shell, originates in South America Amazon River basin.Before and after 1980, because of its rich in protein, nutritional labeling is high and fertility is strong, and is introduced into Taiwan, Philippine and Japan as a kind of aquatic economic animal, and rapidly diffuses into East Asia and remaining country (Halwart, 1994) of Southeast Asia.Later due to marketization failure, Pomacea canaliculata is lost one's parents and is spread rapidly result population outbreak, serious harm crop production (Naylor, 1996).2000, World Conservation Union (WorldConservationUnion;InternationalUnionforConservationofNatureandNaturalResources;IUCN) Pomacea canaliculata is classified as one of 100 kinds of pernicious Invasive Alien Species in the world (Loweetal, 2000) by the Invasive Alien Species Committee of Experts, is also wherein unique a kind of freshwater snails.In China mainland, Pomacea canaliculata was incorporated into Guangdong cultivation in 1981, in Guangdong after 1984, Guangxi, the ground such as Fujian starts extensively to cultivate, it is generalized to the ground such as Zhejiang, Jiangxi, Yunnan, Sichuan subsequently, the serious agricultural pests (Yu Xiao equality, 2001) of major part provinces and regions on the south the Changjiang river are become at present.2003, Pomacea canaliculata was listed in " blacklist " of 16 kinds of alien species of first batch of invasion China by State Environmental Protection Administration and the Chinese Academy of Sciences (2003)
During the preventing and treating of Pomacea canaliculata, in addition to cultural control and physical control, screening and the qualification of novel biopesticide are increasingly becoming research emphasis.The activity bioassay of biological pesticide needs the Pomacea canaliculata that a large amount of growth is consistent, strict requirements are had to raising place, workload is big, if and have the Pomacea canaliculata cell of isolated culture, then can study the biological pesticide effect to two Pomacea canaliculatas in vitro, thus contribute to studying the insecticide mechanism of action to Pomacea canaliculata at cellular level, contributing to the improvement of current insecticide and the invention of new pesticides, the cultivation about Pomacea canaliculata hemocyte yet there are no relevant report.Therefore, by the present invention, set up Pomacea canaliculata cell line, quantity and the kind of China's cell line can not only be increased, moreover it is possible to offer reference experience for the foundation of such biological cell system in the future.
Summary of the invention
It is an object of the invention to provide the primary culture method of a kind of Pomacea canaliculata hemocyte, provide technical support and guarantee for Pomacea canaliculata physiological and biochemical research, toxicologic study etc..
The present invention completes by the following technical programs:
(1) preparation cell culture fluid；
(2) spiral shell choosing body weight 40-50 gram is raised 12 hours in sterilized water；
(3) by the mucus on spiral shell surface aseptic cotton carrier scrub, spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 10-20 minute；
(4) spiral shell is cleaned 3-5 time with sterile distilled water；
(5) spiral shell surface moisture is blotted with aseptic filter paper；
(6) spiral shell is placed in the dissecting pan crossed with 75% alcohol disinfecting, dissects Pomacea canaliculata；
(7) at Pomacea canaliculata heart, extract blood with disposable sterilized injector, put into the T-25cm containing 1-2mLHBSS cleanout fluid2Tissue Culture Flask, stands 10-15 minute, after hemocyte is adherent, absorbs HBSS, adds fresh HBSS, repeat 3-5 time；
(8), after removing HBSS last 1 time, add cell culture fluid 1-1.5mL, culture bottle is placed in the CO of 5%2In incubator, 28 DEG C of constant temperature culture；
(9) after overnight, 2-1.5mL cell culture fluid is added, every sucking-off in 5-7 days and the cell culture fluid that changes to 60%--70% amount, until extending and the cell bred is full of culture bottle；
Whole operating process is the most aseptically carried out.
Described cell culture fluid is RPMI1640 culture medium and penicillin, streptomycin, amphotericin B and the mixture of animal serum, and the mixture of saturated phenylthiourea, pH value 7.0-7.4；Described animal serum is hyclone；Described Penicillin Content is 200U/ml, and content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml；Described animal serum content is 10-20%(volume ratio)；Described saturated phenylthiourea solution is that autoclaved phenylthiourea solid excess joins in aseptic RPMI1640 culture medium formulated.
The invention has the beneficial effects as follows:
Use such scheme, Pomacea canaliculata hemocyte has been carried out original cuiture, has achieved preferable culture effect.The present invention is quickly, effective, repeatability is strong, is the important supplement cultivating fresh water invertebral living creature cell.Pomacea canaliculata is important Strategy of Alien Invasive Species, and the present invention contributes to studying Pomacea canaliculata in vitro, provides help for the research of its controlling way and the exploitation of novel biopesticide.
In the present invention, the biological property of Pomacea canaliculata blood cell line is observed and is measured
1., through laboratory observation, this cell line major part adherent growth, the shape major part of cell is circular, (Fig. 1)；
2. with 2x105The concentration of cell/mL is inoculated into T-25cm2In culture bottle, 27 DEG C, without illumination cultivation, take one bottle every day and measure cell concentration, draw growth curve (Fig. 2), and according to formula T=tlg2/ [lg (N/N0)] calculate population doubling time, it is 96 hours through calculating the cell population doublings time of eighth generation.Wherein
T=is in one times of required time of logarithmic (log) phase balanced growth
T=is inoculated into the time measuring cell number
N0Cell number during=inoculation
The total cellular score that N=moment t is measured
3. designing primer with aldolase, cell line that the present invention sets up derives from Pomacea canaliculata hemocyte really to utilize the method for DAF-PCR to identify, rather than the pollution of other cell lines (Fig. 3).Banding pattern after the DNA band provided by cell line expands with Fu Shou blood DNA is identical, and significantly different with compared with control cells system sf9 and s2 banding pattern.
Accompanying drawing explanation
Fig. 1 cultivate 40 days after the cultivation form of hemocyte
The growth curve of Fig. 2 the 8th generation hemocyte
The DAF-PCR of Fig. 3 cell line identifies
Detailed description of the invention
Below in conjunction with example, the present invention is described in further detail:
Embodiment 1
The preparation of cell culture fluid
(preparation 500mL):
Composition quantity
RPMI1640 culture medium 400mL
Hyclone 50.0mL
Phenylthiourea saturated solution 50.0mL
Penicillin 100U
Streptomycin 0.1 μ g
Amphotericin B 0.25 μ g
In aseptic operating platform, being mixed by mentioned component, 4 DEG C save backup.
The original cuiture of embodiment 2 Pomacea canaliculata hemocyte:
Through the following step:
(1) preparation cell culture fluid；
(2) spiral shell choosing body weight 40 grams is raised 12 hours in sterilized water；
(3) by the mucus on spiral shell surface aseptic cotton carrier scrub, spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 10 minutes；
(4) spiral shell is cleaned 3 times with sterile distilled water；
(5) spiral shell surface moisture is blotted with aseptic filter paper；
(6) spiral shell is placed in the dissecting pan crossed with 75% alcohol disinfecting, dissects Pomacea canaliculata；
(7) at Pomacea canaliculata heart, extract blood with disposable sterilized injector, put into the T-25cm containing 1mLHBSS cleanout fluid2Tissue Culture Flask, stands 10 minutes, after hemocyte is adherent, absorbs HBSS, adds fresh HBSS, be repeated 3 times；
(8), after removing HBSS last 1 time, add cell culture fluid 1mL, culture bottle is placed in the CO of 5%2In incubator, 28 DEG C of constant temperature culture；
(9) after overnight, 2mL cell culture fluid is added, every sucking-off in 5 days and the cell culture fluid that changes to 60% amount, until extending and the cell bred is full of culture bottle.
The original cuiture of embodiment 3 Pomacea canaliculata hemocyte:
Through the following step:
(1) preparation cell culture fluid；
(2) spiral shell choosing body weight 50 grams is raised 12 hours in sterilized water；
(3) by the mucus on spiral shell surface aseptic cotton carrier scrub, spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 20 minutes；
(4) spiral shell is cleaned 5 times with sterile distilled water；
(5) spiral shell surface moisture is blotted with aseptic filter paper；
(6) spiral shell is placed in the dissecting pan crossed with 75% alcohol disinfecting, dissects Pomacea canaliculata；
(7) at Pomacea canaliculata heart, extract blood with disposable sterilized injector, put into the T-25cm containing 2mLHBSS cleanout fluid2Tissue Culture Flask, stands 15 minutes, after hemocyte is adherent, absorbs HBSS, adds fresh HBSS, be repeated 5 times；
(8), after removing HBSS last 1 time, add cell culture fluid 1.5mL, culture bottle is placed in the CO of 5%2In incubator, 28 DEG C of constant temperature culture；
(9) after overnight, 1.5mL cell culture fluid is added, every sucking-off in 7 days and the cell culture fluid that changes to 70% amount, until extending and the cell bred is full of culture bottle；
The individual cells in a large number bred the most to the periphery extension be can be observed after 3 days until extending and the cell bred is full of culture bottle after aforesaid operations；After 40th day, proliferative cell is paved with cell culturing surfaces (Fig. 1), the success of Pomacea canaliculata hemocyte original cuiture；The cell containing added value, put in new culture bottle together with whole cell culture fluid sucking-offs, according to 1:5(cell volume: last culture fluid volume) carry out Secondary Culture.Pomacea canaliculata hemocyte original cuiture is tentatively successfully established.Cell colony times total time is 96h(Fig. 2).
The Observation of biological characteristics of embodiment 4 cell line and mensuration；
(1) morphological characteristic: observe through microscope, this cell line adherent growth, major part is circular, and small part fusiformis is less like macrophage-shaped (Fig. 1).Round cell accounts for 95%, average diameter 21 μm;
(2) growth of cell: growth curve is in typical " S ", and population doubling time is 96 hours (Fig. 2)；(3) DAF-PCR identifies: design primer with aldolase, and utilize the method for DAF-PCR to identify cell line that the present invention sets up derives from the hemocyte of Pomacea canaliculata really, rather than the pollution of other cell lines (Fig. 3).Banding pattern after the DNA band provided by cell line expands with Pomacea canaliculata blood DNA is identical, and significantly different with compared with control cells system sf9 and s2 banding pattern.
Claims (6)
1. the primary culture method of a Pomacea canaliculata hemocyte, it is characterised in that the method comprises the following steps:
(1) preparation cell culture fluid；
(2) spiral shell choosing body weight 40-50 gram is raised 12 hours in sterilized water；
(3) by the mucus on spiral shell surface aseptic cotton carrier scrub, spiral shell is immersed in 0.1%KMnO4In solution, carry out surface sterilization 10-20 minute；
(4) spiral shell is cleaned 3-5 time with sterile distilled water；
(5) spiral shell surface moisture is blotted with aseptic filter paper；
(6) spiral shell is placed in the dissecting pan crossed with 75% alcohol disinfecting, dissects Pomacea canaliculata；
(7) at Pomacea canaliculata heart, extract blood with disposable sterilized injector, put into the T-25cm containing 1-2mLHBSS cleanout fluid2Tissue Culture Flask, stands 10-15 minute, after hemocyte is adherent, absorbs HBSS, adds fresh HBSS, repeat 3-5 time；
(8), after removing HBSS last 1 time, add cell culture fluid 1-1.5mL, culture bottle is placed in the CO of 5%2In incubator, 28 DEG C of constant temperature culture；
(9) after overnight, 2-1.5mL cell culture fluid is added, every sucking-off in 5-7 days and the cell culture fluid that changes to 60%--70% amount, until extending and the cell bred is full of culture bottle；
Whole operating process is the most aseptically carried out.
Method the most according to claim 1, it is characterised in that described cell culture fluid is RPMI1640 culture medium and penicillin, streptomycin, amphotericin B and the mixture of animal serum, and the mixture of saturated phenylthiourea, pH value 7.0-7.4.
Method the most according to claim 2, it is characterised in that described animal serum is hyclone.
Method the most according to claim 2, it is characterised in that described Penicillin Content is 200U/ml, content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml.
5. according to the method described in claim 2,3, it is characterised in that described animal serum content is 10-20%(volume ratio).
Method the most according to claim 2, it is characterised in that described saturated phenylthiourea solution is that autoclaved phenylthiourea solid excess joins in aseptic RPMI1640 culture medium formulated.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820380A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis blood lymphocytes |
| CN103820381A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis cells |
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2015
- 2015-01-06 CN CN201510003499.6A patent/CN105820994A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820380A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis blood lymphocytes |
| CN103820381A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis cells |
Non-Patent Citations (3)
| Title |
|---|
| JUAN A. CUETO, ET AL.: "Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| 姜德勋 等: "福寿螺足组织和外套膜组织细胞培养的初步研究", 《淡水渔业》 * |
| 杨淞 等: "不同壳色福寿螺血细胞的比较研究", 《四川动物》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
| CN114134099B (en) * | 2021-11-29 | 2023-06-16 | 北部湾大学 | Balanced salt solution for blood cells of marine invertebrate |
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Application publication date: 20160803 |