CN108118024A - A kind of Portunus trituberculatus Miers haemocyte separation method - Google Patents
A kind of Portunus trituberculatus Miers haemocyte separation method Download PDFInfo
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Abstract
The invention discloses a kind of Portunus trituberculatus Miers haemocyte separation method, the isotonic solution obtained using the osmotic pressure of sodium chloride solution adjusting PBS buffer solutions dilutes Iodixanol, the Iodixanol liquid of three kinds of various concentrations of gradient configuration；Discontinuous density gradient liquid is formed using cushion layer technique, then gradient of continuous density liquid is formed as cell separating liquid through standing；Gather Portunus trituberculatus Miers haemocyte, the mixed cell suspension obtained after being diluted using isotonic solution as separate object；Mixed cell suspension is taken to be added slowly in cell separating liquid, Portunus trituberculatus Miers haemocyte is divided into three layers after centrifugation, and top layer is hyaline cell, and interlayer is small granular cell, and lowest level is large granule cells；Advantage be only with a step density gradient centrifugation, it is not only easy to operate, but also be found through experiments that the activity for the haemocyte separated is good, available for cell culture.
Description
Technical field
The present invention relates to a kind of cell separation technology, more particularly, to a kind of Portunus trituberculatus Miers haemocyte separation method.
Background technology
Portunus trituberculatus Miers (Portunus trituberculatus) is commonly called as Blue Swimming Crab, rifle crab, is under the jurisdiction of subphylum crustacea
(Crustacea), hapalonychia guiding principle (Malacostraca), Decapoda (Decapoda), Portumidae (Portunidae), swimming crab
Belong to (Portunus), be distributed widely in the marine sites such as China, Japan, Korea and Malaysian archipelago, be the important marine products in China
Economic crab.Portunus trituberculatus Miers disease takes place frequently in recent years, according to statistics especially with diseases such as bacterium, virus and parasites in all kinds of diseases
Based on originality disease, heavy losses are caused to China's culture fishery and coastal economy.At present, related ocean shell-fish is congenital
Property immune-related basic research gradually increase, with accelerate invertebrate disease immune prevention process.Cell isolation and culture
Technology is of crucial importance in research animal immune, the pathogenesis of disease and prevention and control, however the technology is moved in ocean without vertebra
It receives in the research of object and greatly limits, seriously hinder the research of oceanic invertebrate immune function and related mechanism.
The principal immune tissue or organ of crustacean are its hemolymph and haemocyte, and crustacean cellular immunity is then
Mainly undertaken by haemocyte.A viewpoint being widely recognized as at present is：Shrimp waste crustacean haemocyte have hyaline cell,
Little particle (half particle) cell and 3 major class of large granule cells.Wherein, hyaline cell individual is minimum, is practically free of in cytoplasm
Particle is primarily involved in phagocytosis；Small granular cell contains small acidophilic granule, and participate in early stage identification, solidification with
And partial phagocytosis；Large granule cells has pro-phenoloxidase system activity, is being subject to immunostimulation that can activate phenol oxidase
Original, while there is cytotoxic effect.At present, related shrimps haemocyte isolation technics has had corresponding research.Such as：Duan Hu
Deng can be by red claw crayfish (Cherax quadricarinatus) using two step density-gradient centrifugation methods using Percoll separating liquids
Haemocyte separates.For another example：Dantas-Lima et al. is by the use of Iodixanol as cell separating liquid to the blood of litopenaeus vannamei
Cell is separated, its 3 kinds of haemocytes are kept completely separate the two step gradient centrifugations of also the same needs that come by discovery.At present, state
It is inside and outside still that 3 kinds of haemocytes are not kept completely separate the related technology reports to come using a step density-gradient centrifugation method, both at home and abroad
Also still without carrying out the research in relation to crab class haemocyte isolation technics.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of Portunus trituberculatus Miers haemocyte separation method, only need a step
Three kinds of haemocytes of Portunus trituberculatus Miers can be kept completely separate out by density gradient centrifugation, and the activity for the haemocyte separated
It is good.
Technical solution is used by the present invention solves above-mentioned technical problem：A kind of Portunus trituberculatus Miers haemocyte separation side
Method, it is characterised in that comprise the following steps：
Step 1, the preparation of cell separating liquid：
The PBS buffer solutions that 1_1, configuration molarity are 0.1M；
The osmotic pressure that 1_2, the sodium chloride solution for being 3M using molarity adjust PBS buffer solutions is 900mOs
Mol/kg obtains isotonic solution；
1_3, using isotonic solution as dilution, Iodixanol that dilution concentration expressed in percentage by volume is 60%, gradient configuration volume
Percentage concentration is respectively 10%, 15% and 20% three kinds of Iodixanol liquid；
1_4, using cushion layer technique, be respectively 10%, 15% and 20% three kinds of Iodixanol liquid by concentration expressed in percentage by volume
Equivalent is loaded onto in centrifuge tube successively, makes to form parting surface between any two, forms discontinuous density gradient liquid；
1_5, after discontinuous density gradient liquid is placed in when standing 12~18 is small under 4~6 DEG C of temperature environments, formed continuous close
Gradient liquid is spent, using the gradient of continuous density liquid as cell separating liquid；
Step 2, the acquisition of Portunus trituberculatus Miers haemocyte：
2_1, anti-coagulants rinse asepsis injector is used；
2_2, using the asepsis injector after rinse, extract hemolymph from the step joint of temporarily foster Portunus trituberculatus Miers
The anti-coagulants 1 reserved in asepsis injector after liquid, with rinse:1 mixing, then mixed liquor is placed in centrifuge tube；
2_3, after carrying out 1000~1200g centrifugal treatings, 2~3min to mixed liquor at room temperature, supernatant is removed, obtains cell
Sediment；
2_4, using the isotonic solution that step 1_2 is obtained as cell suspension, by the obtained cell pellets of step 2_3 and cell
Portunus trituberculatus Miers whole blood cell suspension is obtained after the abundant mixing of suspension；
Step 3, the separation of Portunus trituberculatus Miers haemocyte：
3_1, Portunus trituberculatus Miers whole blood cell suspension is taken to be added slowly in cell separating liquid, wherein, Portunus trituberculatus Miers whole blood
The volume ratio of cell suspension and cell separating liquid is 1:5~4；
3_2, after carrying out 2200~2400g centrifugal treatings 14~16min under 4~6 DEG C of temperature environments, Portunus trituberculatus Miers blood
Cell is divided into three layers, and top layer is hyaline cell layer, and interlayer is small granular cell layer, and lowest level is large granule cells layer.
Anti-coagulants in the step 2_1 is ACD anti-coagulants.
After the step 3_2, in the hyaline cell layer isolated hyaline cell solution, in small granular cell layer
Small granular cell solution, the large granule cells solution in large granule cells layer carry out cell separating liquid washing, to keep more preferable
Activity, detailed process is：Hyaline cell solution is drawn out from hyaline cell layer, it is the hyaline cell drawn out to add in volume
3 times of isotonic solution of liquor capacity after carrying out 1100~1300g centrifugal treatings, 2~3min under 4~6 DEG C of temperature environments, is gone
Clearly, sediment is obtained；Then 3 times of isotonic solution of the volume for the volume of sediment is added in sediment, in 4~6 DEG C of temperature
After carrying out 1100~1300g centrifugal treatings, 2~3min under environment, supernatant is removed, obtains the hyaline cell after elution cell separating liquid
Solution；In an identical manner, obtain elution cell separating liquid after small granular cell solution and elution cell separating liquid after it is big
Granular cell solution.
Compared with prior art, the advantage of the invention is that：
1) the method for the present invention by three kinds of different volumes percentage concentrations Iodixanol liquid by cushion layer technique and through stand shape
Into gradient of continuous density liquid as cell separating liquid, using cell separating liquid by three kinds of blood of the Portunus trituberculatus Miers haemocyte of acquisition
Cell separates, not only easy to operate only with a step density gradient centrifugation, but also is found through experiments that is separated
The activity of haemocyte is good, available for cell culture.
2) it is found through experiments that the separative efficiency of the method for the present invention is high, can almost separates three kinds of haemocytes,
The separation rate of three kinds of haemocytes has reached more than 95%.
3) the method for the present invention provides for the development of the multinomial researchs such as crab class cellular immunity, cause of disease pathogenic mechanism and disease prevention and control
Preferable instrument.
Description of the drawings
Fig. 1 be Portunus trituberculatus Miers haemocyte be isolated to hyaline cell, small granular cell, large granule cells is in cell
Distribution in separating liquid；
Fig. 2 a are form of the hyaline cell that is isolated to of Portunus trituberculatus Miers haemocyte after Giemsa is dyed；
Fig. 2 b are form of the small granular cell that is isolated to of Portunus trituberculatus Miers haemocyte after Giemsa is dyed；
Fig. 2 c obtain form of the large granule cells after Giemsa is dyed for what Portunus trituberculatus Miers haemocyte was isolated to；
Fig. 3 a are the cell attachment of hyaline cell and in vitro culture form that Portunus trituberculatus Miers haemocyte is isolated to；
Fig. 3 b are the cell attachment of small granular cell and in vitro culture form that Portunus trituberculatus Miers haemocyte is isolated to；
Fig. 3 c obtain the cell attachment and in vitro culture of large granule cells for what Portunus trituberculatus Miers haemocyte was isolated to
Form.
Specific embodiment
The present invention is described in further detail below in conjunction with attached drawing embodiment.
Embodiment one：
A kind of Portunus trituberculatus Miers haemocyte separation method that the present embodiment proposes, comprises the following steps：
Step 1, the preparation of cell separating liquid：
The PBS buffer solutions that 1_1, configuration molarity are 0.1M.
The osmotic pressure that 1_2, the sodium chloride solution for being 3M using molarity adjust PBS buffer solutions is 900mOs
Mol/kg obtains isotonic solution.Wherein, PBS buffer solution 10ml are can use, take sodium chloride solution 1.2ml.
1_3, using isotonic solution as dilution, Iodixanol (OptiPrep that dilution concentration expressed in percentage by volume is 60%TM), ladder
Degree configuration concentration expressed in percentage by volume is respectively 10%, 15% and 20% three kinds of Iodixanol liquid.
1_4, using existing cushion layer technique, be respectively 10%, 15% and 20% three kinds of iodine gram by concentration expressed in percentage by volume
Equivalent is loaded onto in centrifuge tube husky alcohol liquid successively, makes to form parting surface between any two, forms discontinuous density gradient liquid.
Here, the specific acquisition process of discontinuous density gradient liquid is：The syringe that syringe needle is 10cm is used to be used as sample-adding
Device is first 10% with the concentration expressed in percentage by volume of syringe sucking 3.5ml if three kinds of Iodixanol liquid respectively need to be loaded 2.5ml
Iodixanol liquid, discharge part before sample-adding, until 3ml, it is ensured that syringe needle is hydraulically full, prevents bubble from entering；Then needle tubing is dried,
Along centrifuge tube (15ml) wall, syringe needle is slowly moved to bottom of the tube is centrifuged, pushes piston, by the iodine that concentration expressed in percentage by volume is 10%
Gram husky alcohol liquid adds in centrifugation bottom of the tube, until after 0.5ml graduation marks, slowly withdraws needle tubing along centrifuge tube；Needle tubing is dried, with identical
Method injection volume percentage concentration be 15% Iodixanol liquid 2.5ml, pays attention to sample-adding process will slowly, make volume basis dense
It spends between the Iodixanol liquid for being 15% for 10% Iodixanol liquid and concentration expressed in percentage by volume and forms parting surface；Dry needle tubing,
Injection volume percentage concentration is 20% Iodixanol liquid 2.5ml in the same way, notices that sample-adding process is slow, makes volume
Parting surface is formed between the Iodixanol liquid that the Iodixanol liquid and concentration expressed in percentage by volume that percentage concentration is 15% are 20%.
1_5, discontinuous density gradient liquid is placed in when standing 12~18 is small under 4 DEG C of temperature environments after (or staying overnight), shape
Into gradient of continuous density liquid, using the gradient of continuous density liquid as cell separating liquid.
Step 2, the acquisition of Portunus trituberculatus Miers haemocyte：
2_1, ACD anti-coagulants rinse asepsis injectors are used.
2_2, using the asepsis injector after rinse, extract hemolymph from the step joint of temporarily foster Portunus trituberculatus Miers
The anti-coagulants 1 reserved in asepsis injector after liquid, with rinse:1 mixing, then mixed liquor is placed in the centrifuge tube of 1.5ml.
2_3, after carrying out 1100g centrifugal treatings 2.5min to mixed liquor at room temperature, supernatant is removed, obtains cell pellet；
2_4, using the isotonic solution that step 1_2 is obtained as cell suspension, by the obtained cell pellets of step 2_3 and cell
Portunus trituberculatus Miers whole blood cell suspension is obtained after the abundant mixing of suspension.
Step 3, the separation of Portunus trituberculatus Miers haemocyte：
3_1, Portunus trituberculatus Miers whole blood cell suspension is taken to be added slowly in cell separating liquid, wherein, Portunus trituberculatus Miers whole blood
The volume ratio of cell suspension and cell separating liquid is 1:5.Mixed cell suspension 2ml is taken in experiment.
3_2, after carrying out 2300g centrifugal treatings 15min under 4 DEG C of temperature environments, Portunus trituberculatus Miers haemocyte is divided into three
Layer, top layer are hyaline cell layer, and interlayer is small granular cell layer, and lowest level is large granule cells layer.Fig. 1 gives three warts
Hyaline cell layer that Portunus Trituberculatus cell is isolated to, small granular cell layer, large granule cells layer are in cell separating liquid
Distribution.
To the hyaline cell in the hyaline cell layer of top layer, the small granular cell in the small granular cell layer in interlayer,
Large granule cells in undermost large granule cells layer carries out Giemsa dyeing, observes each confluent monolayer cells type.Take out top layer
Hyaline cell, drip on glass slide, even spread, after air-drying at room temperature, with the paraformaldehyde fixer that concentration is 4%
Then fixed 10min is added dropwise Giemsa working solutions covering smear, room temperature dyeing 30min, then is slowly rinsed with distilled water, aqueous
Mountant mounting, finally observes the coloration result of hyaline cell under the microscope, and takes pictures.In an identical manner, small is obtained
The coloration result of granulocyte is simultaneously taken pictures and is obtained the coloration result of large granule cells and take pictures.Fig. 2 a give Portunus trituberculatus Miers blood
Form of the hyaline cell that cell is isolated to after Giemsa is dyed, Fig. 2 b give Portunus trituberculatus Miers haemocyte through separation
Form of the obtained small granular cell after Giemsa is dyed, what Fig. 2 c gave that Portunus trituberculatus Miers haemocyte is isolated to
To form of the large granule cells after Giemsa is dyed, pass through separated hyaline cell compared with granular cell from coloration result is visible
Small, cytoplasm is few, and nucleocytoplasmic ratio is big；And small granular cell volume is medium, cytoplasm is more and wherein has macroscopic dyed particles,
But particle is smaller；Large granule cells volume is maximum, cytoplasm it is more and in have a macroscopic dyed particles, the larger dyeing of particle compared with
It is deep.
In concrete operations, the temperature and time in step 1_5, rotating speed and time in step 2_3, in step 3_2
Temperature, rotating speed and time can suitably adjust in a small range, and the influence to separating resulting is not very big, the temperature in step 1_5
The scope of degree is 4~6 DEG C, when the scope of time is 12~18 small；The scope of rotating speed in step 2_3 is 1000~1200g, when
Between scope be 2~3min；The scope of temperature in step 3_2 is 4~6 DEG C, and the scope of rotating speed is 2200~2400g, the time
Scope be 14~16min.The scope of mixed cell suspension and the volume ratio of cell separating liquid in step 3_1 is 1:5~4,
It is found through experiments that and the volume ratio of mixed cell suspension and cell separating liquid is set to 1:It is separated when 5 better.
Embodiment two：
The present embodiment is on the basis of embodiment one, has carried out further processing.I.e.：After step 3_2, to separation
The small granular cell solution in hyaline cell solution, small granular cell layer in the hyaline cell layer gone out, in large granule cells layer
Large granule cells solution carry out cell separating liquid washing, to keep preferably activity, detailed process is：From hyaline cell layer
Hyaline cell solution is drawn out, 3 times of the isotonic solution that volume is the hyaline cell liquor capacity drawn out is added in, in 4 DEG C of temperature
After carrying out 1200g centrifugal treatings 2.5min under environment, supernatant is removed, obtains sediment；Then it is heavy volume to be added in sediment
3 times of isotonic solution of the volume of starch after carrying out 1200g centrifugal treatings 2.5min under 4 DEG C of temperature environments, removes supernatant, obtains
Elute the hyaline cell solution after cell separating liquid；In an identical manner, the small granular cell after elution cell separating liquid is obtained
Large granule cells solution after solution and elution cell separating liquid.Hyaline cell solution, elution after cell separating liquid is eluted
Each middle addition is isotonic for the large granule cells solution after small granular cell solution and elution cell separating liquid after cell separating liquid
Liquid is resuspended, and cell is resuspended according to 1:The hyclone mixing that 10 ratio and the L15 culture mediums addition concentration configured are 10%,
Then cell is layered on to be positioned in common biochemical cultivation case in 12 orifice plates and is cultivated in 25 DEG C, to thin after culture 4h and 12h
Born of the same parents are observed and are taken pictures in inverted microscope.Fig. 3 a give the hyaline cell that Portunus trituberculatus Miers haemocyte is isolated to
Cell attachment and in vitro culture form, Fig. 3 b give the thin of the small granular cell that Portunus trituberculatus Miers haemocyte is isolated to
Born of the same parents are adherent and in vitro culture form, and what Fig. 3 c gave that Portunus trituberculatus Miers haemocyte is isolated to obtains the thin of large granule cells
Born of the same parents are adherent and in vitro culture form, can be adherent from the visible three kinds of cells of cell culture form, but it is adherent after cellular morphology
Short shuttle-type is presented in difference after hyaline cell is adherent, and long shuttle-type is presented in small granular cell, and the adherent rear major part of large granule cells is still
Rounded or triangle still has macroscopic particle into the cell.
Claims (3)
1. a kind of Portunus trituberculatus Miers haemocyte separation method, it is characterised in that comprise the following steps：
Step 1, the preparation of cell separating liquid：
The PBS buffer solutions that 1_1, configuration molarity are 0.1M；
The osmotic pressure that 1_2, the sodium chloride solution for being 3M using molarity adjust PBS buffer solutions is 900mOsmol/
Kg obtains isotonic solution；
1_3, using isotonic solution as dilution, Iodixanol that dilution concentration expressed in percentage by volume is 60%, gradient configuration volume basis
Concentration is respectively 10%, 15% and 20% three kinds of Iodixanol liquid；
1_4, using cushion layer technique, by concentration expressed in percentage by volume be respectively 10%, 15% and 20% three kinds of Iodixanol liquid successively
Equivalent is loaded onto in centrifuge tube, makes to form parting surface between any two, forms discontinuous density gradient liquid；
1_5, after discontinuous density gradient liquid is placed in when standing 12~18 is small under 4~6 DEG C of temperature environments, continuous density ladder is formed
Liquid is spent, using the gradient of continuous density liquid as cell separating liquid；
Step 2, the acquisition of Portunus trituberculatus Miers haemocyte：
2_1, anti-coagulants rinse asepsis injector is used；
2_2, using the asepsis injector after rinse, extract liquid of haemolymph from the step joint of temporarily foster Portunus trituberculatus Miers, with
The anti-coagulants 1 reserved in asepsis injector after rinse:1 mixing, then mixed liquor is placed in centrifuge tube；
2_3, after carrying out 1000~1200g centrifugal treatings, 2~3min to mixed liquor at room temperature, supernatant is removed, obtains cell precipitation
Object；
2_4, using the isotonic solution that step 1_2 is obtained as cell suspension, by the obtained cell pellets of step 2_3 and cell suspension
Portunus trituberculatus Miers whole blood cell suspension is obtained after abundant mixing；
Step 3, the separation of Portunus trituberculatus Miers haemocyte：
3_1, Portunus trituberculatus Miers whole blood cell suspension is taken to be added slowly in cell separating liquid, wherein, Portunus trituberculatus Miers whole blood cells
The volume ratio of suspension and cell separating liquid is 1:5~4；
3_2, after carrying out 2200~2400g centrifugal treatings 14~16min under 4~6 DEG C of temperature environments, Portunus trituberculatus Miers haemocyte
It is divided into three layers, top layer is hyaline cell layer, and interlayer is small granular cell layer, and lowest level is large granule cells layer.
A kind of 2. Portunus trituberculatus Miers haemocyte separation method according to claim 1, it is characterised in that the step 2_1
In anti-coagulants be ACD anti-coagulants.
3. a kind of Portunus trituberculatus Miers haemocyte separation method according to claim 1 or 2, it is characterised in that in the step
After rapid 3_2, to the hyaline cell solution in the hyaline cell layer isolated, the small granular cell solution in small granular cell layer,
Large granule cells solution in large granule cells layer carries out cell separating liquid washing, and to keep preferably, activity, detailed process are：
Draw out hyaline cell solution from hyaline cell layer, add in that volume is the hyaline cell liquor capacity drawn out 3 times etc.
Sepage after carrying out 1100~1300g centrifugal treatings, 2~3min under 4~6 DEG C of temperature environments, removes supernatant, obtains sediment；So
Afterwards in sediment add in volume for sediment volume 3 times of isotonic solution, under 4~6 DEG C of temperature environments carry out 1100~
After 2~3min of 1300g centrifugal treatings, supernatant is removed, obtains the hyaline cell solution after elution cell separating liquid；With identical side
Formula obtains the small granular cell solution after elution cell separating liquid and the large granule cells solution after elution cell separating liquid.
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|---|---|---|---|---|
| CN110029143A (en) * | 2019-04-28 | 2019-07-19 | 宁波大学 | A kind of screening technique of resistance Portunus trituberculatus Miers |
| CN110129254A (en) * | 2019-04-23 | 2019-08-16 | 河南师范大学 | A method of Babylonia areolata (Lamarck) granular cell and hyaline cell are separated by medium of Percoll |
| CN110713969A (en) * | 2019-10-15 | 2020-01-21 | 浙江大学 | Blue crab blood cell grouping culture method |
| CN114032280A (en) * | 2021-11-29 | 2022-02-11 | 北部湾大学 | Preparation method of marine invertebrate blood cell suspension |
| CN115074306A (en) * | 2022-07-12 | 2022-09-20 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
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2017
- 2017-11-22 CN CN201711171477.6A patent/CN108118024B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129254A (en) * | 2019-04-23 | 2019-08-16 | 河南师范大学 | A method of Babylonia areolata (Lamarck) granular cell and hyaline cell are separated by medium of Percoll |
| CN110029143A (en) * | 2019-04-28 | 2019-07-19 | 宁波大学 | A kind of screening technique of resistance Portunus trituberculatus Miers |
| CN110713969A (en) * | 2019-10-15 | 2020-01-21 | 浙江大学 | Blue crab blood cell grouping culture method |
| CN114032280A (en) * | 2021-11-29 | 2022-02-11 | 北部湾大学 | Preparation method of marine invertebrate blood cell suspension |
| CN114032280B (en) * | 2021-11-29 | 2023-06-23 | 北部湾大学 | Preparation method of blood cell suspension of marine invertebrate |
| CN115074306A (en) * | 2022-07-12 | 2022-09-20 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
| CN115074306B (en) * | 2022-07-12 | 2023-09-22 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of crab cells |
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| CN108118024B (en) | 2021-03-02 |
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