CN108192869A - A kind of channel catfish head-kidney macrophage primary isolated culture method and application - Google Patents
A kind of channel catfish head-kidney macrophage primary isolated culture method and application Download PDFInfo
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Abstract
The invention belongs to biotechnologies, are related to a kind of channel catfish head-kidney macrophage primary isolated culture method and application.This method includes the following steps：A) fish body anesthesia blood sampling；B) head-kidney tissue is taken；C) tissue grinder, filtering；D) cell liquid containing macrophage is centrifuged with 30% 55% percoll separating liquids, separation is twice；E) constant temperature incubation：In 15 culture solutions of L containing 2% 4%FBS, the constant temperature incubation under the conditions of 26 30 DEG C.This method can obtain the channel catfish head-kidney macrophage that purity is high, quantity is big, activity is good, can be applied to the immunity additive functional study of channel catfish well.
Description
Technical field
The invention belongs to biotechnology, more particularly, to a kind of channel catfish head-kidney macrophage primary point
From cultural method and application.
Background technology
Fish head-kidney is equivalent to secondary lymphoid organ in immuning tissue, and key is played in immune response generating process
Effect, wherein macrophage can swallow the cell of natural apoptosis or aging, be played an important role in congenital immunologic process,
The culture that is successfully separated of external head-kidney macrophage is to study the basis of its immune function.Macrophage is in ecotoxicology, disease
Poison, nutrition immune etc. have a wide range of applications.
Channel catfish delicious meat has good reducing blood lipid, brain tonic and intelligence development, tonifying kidney for improving eyesight, weight-reducing anti-aging, enhancing
Immunity function, it is deep to be welcome by breeding production person, processor and consumer.It but can be frequent in the breeding process of channel catfish
There is various viral and bacteriosis generation, promote its immune function by adding immunity additive, be disease prevention
Primary measure.
In vivo study is time-consuming and laborious, and it is then a kind of fast, accurately research method to establish In vitro cell model.
Invention content
The object of the present invention is to provide a kind of channel catfish head-kidney macrophage primary isolated culture method and applications, should
Method can obtain the channel catfish head-kidney macrophage that purity is high, quantity is big, activity is good, can be applied to spot well and pitch
The immunity additive functional study of Wei Channel-catfish.
To achieve these goals, the present invention provides a kind of channel catfish head-kidney macrophage primary side of being separately cultured
Method, this method include the following steps：
A) fish body anesthesia blood sampling；
B) head-kidney tissue is taken；
C) tissue grinder, filtering；
D) cell liquid containing macrophage is centrifuged with the percoll separating liquids of 30%-55%, separation is twice；
E) constant temperature incubation：In the L-15 culture solutions containing 2%-4%FBS, the constant temperature incubation under the conditions of 26-30 DEG C.
In accordance with the present invention it is preferred that this method further includes：
F) macrophage is purified：Step e) constant temperature incubations are after 4-6 hours, after drawing upper strata culture medium, PBS purging cells,
The L-15 culture solutions containing 2%-4%FBS, the constant temperature incubation under the conditions of 26-30 DEG C are added in again.
In accordance with the present invention it is preferred that step a) includes：
The channel catfish of weight 1kg-1.25kg is chosen, MS-222 deep anaesthesias are carried out to channel catfish, take out fish
Body is taken a blood sample in tail portion, after blood sampling, alcohol disinfecting is sprayed on fish body surface, dissection is for use.
In accordance with the present invention it is preferred that the mass ratio of fish body weight and anesthetic is 180-220：1, anesthesia duration is 5-8 points
Clock；Fish body weight and blood sampling volume ratio (g：Ml) it is 110-140：1.
In accordance with the present invention it is preferred that step b) includes：
Head-kidney tissue is taken out, is soaked in physiological saline, is taken out after 3-5min, then be soaked in AIM liquid, soaking time is
20-30min.It takes out head-kidney tissue and this field conventional method realization can be used.
In accordance with the present invention it is preferred that step c) includes：
Head-kidney tissue is placed in sterile steel mesh, is cut somebody's hair using syringe and it is ground, L- is added in process of lapping
15 culture solutions；It grinds end mark and is only left white lipid matter for head-kidney tissue.General weight about 1000g fish bodies take head-kidney
Lapping liquid is more suitable in 15-20ml.
In accordance with the present invention it is preferred that step d) and step e) include：
The obtained cell pulverized liquids of step c) are dispelled, are added in percoll layering liquid, percoll layering liquid upper strata
For the percoll separating liquids of 32%-34%, lower floor is the percoll separating liquids of 50%-52%, after centrifugation, is taken out
Cell at percoll layering interfaces after collection is added in new above-mentioned percoll layering liquid, centrifuges and take out again
Cell at percoll layering interfaces, centrifuges again, abandons supernatant, and cell is purged with 0.65% physiological saline/L-15 culture mediums
Afterwards, the L-15 culture solution cultures containing 2%-4%FBS are added in.The condition of the centrifugation can determine as needed.
Preferably, in above-mentioned steps, the percoll separating liquids of 32%-34%, the percoll separating liquids of 50%-52% with
The volume ratio of cell pulverized liquid is 0.8-1.2：0.8-1.2：1.
According to a kind of specific embodiment of the present invention, the channel catfish head-kidney macrophage primary isolated culture method
Include the following steps：
1st, fish body anesthesia blood sampling.The channel catfish of weight 1kg-1.25kg is chosen, MS-222 is carried out to channel catfish
(fish body weight and anesthetic mass ratio are about 200 to deep anaesthesia：1 effect is preferable), anesthesia duration 5-8 minutes takes out fish
Body is taken a blood sample in Aseptic sterilisation disk, and blood sampling is using 2.5ml graded syringes, and blood sampling position is at the 5cm of tail portion, syringe edge
Fish body side line is inserted into, and blood sampling volume is depending on fish body size, fish body weight and blood sampling volume ratio (g：Ml it is about) 125：1 suitable (later stage
Dissect more clean in fish body), blood sampling finishes, fish body surface sprinkling alcohol disinfecting, and dissection is for use.
2nd, its head-kidney tissue is taken.It is cut off with scissors along fish maw, removes its internal organ, find head-kidney along air bladder front, head-kidney is
It is flat, positioned at air bladder by below frontal lobe, each one of left and right is red.Head-kidney is taken out, is soaked in 0.65% physiological saline, 3-
It takes out and is soaked in AIM liquid (5-8ml) after 5min, impregnate duration 20-30min.
3rd, tissue grinder, steel mesh filtering.Head-kidney tissue is taken out after immersion, is placed in the sterile steel mesh of 100 mesh, utilizes note
Emitter, which is cut somebody's hair, is ground it, and L-15 culture solutions are added in process of lapping.End mark is ground for head-kidney tissue to be only left in vain
Chromolipoid substance.
4th, the cell liquid of macrophage is centrifuged.Cell pulverized liquid is dispelled with liquid-transfering gun, is added to and has adjusted
It is 2-3ml per the most suitable addition of solencyte liquid in the percoll being located in the 10ml centrifuge tubes layering liquid prepared, it is thin after addition
Born of the same parents divide 3 layers, and the bottom is the percoll separating liquids of 3ml density 50%-52%, and middle layer is 3ml density 32%-34%'s
Percoll separating liquids, top layer be the above-mentioned cell liquid of 2ml, adhesive tape to centrifuge tube carry out sealing be placed in refrigerated centrifuge into
Row centrifugation, centrifugal condition are：Centrifugal force 400g, time 20min, 4 DEG C of temperature.After centrifugation, percoll interfaces are taken out
Go out one layer of cells, be collected in dispel in 10ml centrifuge tubes and be added to deployed be located in 10ml centrifuge tubes again
Be 2-3ml per solencyte liquid most suitable addition in percoll layering liquid, 3 layers of cell point after addition, percoll separating liquids, from
Heart condition is as above.It takes out after centrifugation at percoll layering interfaces in cell and 10ml centrifuge tubes, is centrifuged after leveling again,
Centrifugal force 1000g, time 10min, 4 DEG C of temperature.Centrifugation terminates, and abandons supernatant, with volume ratio 3：1 0.65% physiological saline：
L-15 culture mediums dispel each solencyte, centrifugal force 1000g, time 10min, are centrifuged at 4 DEG C of temperature.Centrifugation terminates, and abandons
Supernatant, each pipe add in 5-7ml L-15 culture mediums, and liquid-transfering gun dispels each solencyte, centrifugal force 1000g, time 10min, temperature
It is centrifuged at 4 DEG C of degree.Centrifugation terminates, and abandons supernatant, adds in the L-15 culture solutions containing 2%-4%FBS and dispels cell, per hole 2ml
It is placed in 6 orifice plates in constant incubator under the conditions of 28 DEG C to be incubated and (sterile creep plate can be put in 6 orifice plates in case later stage dyeing is seen
It examines).
5th, macrophage is purified.After being incubated 4-6 hours, liquid-transfering gun draws upper strata culture medium, and PBS is carefully purged 2-3 times, moves
PBS is removed, 2-3ml L-15 culture solutions are added in per hole, with being incubated under the conditions of in constant incubator 28 DEG C.By falling after culture
Put that the adherent effect of fluorescence microscope cell is good, and cell growth state is good, and cell concentration is big, head-kidney macrophage purifying rate reaches
99%.
Above-mentioned AIM liquid ingredient is：45ml L-15 culture medium+2.5ml mycillin mixed liquor+2.5ml amphotericin Bs
(250 μ g/ml)+0.5ml gentamicin sulphates (50 μ g/ml).
The percoll liquid ingredients of above-mentioned different densities are as follows：
32%percoll liquid：Distilled water 14.5ml；HBSS 2.5ml；NaHCO30.125ml；Percoll liquid 8ml.
52%percoll liquid：Distilled water 10ml；HBSS 2.5ml；NaHCO30.125ml；Percoll liquid 13ml.Wherein 32%
Percoll liquid is mixed colours with sterile 50 μ l-100 μ l of 0.1M hydrochloric acid.
The operating method of above-mentioned institute's score layer liquid is：The percoll separation of 3ml 32%-34% is added in 10ml centrifuge tubes
Liquid, the percoll separating liquids that 50%-52% is drawn using 3ml syringes are slowly injected in centrifugation bottom of the tube, can be layered
Apparent percoll separating liquids.
Above-mentioned 2%fetal calf serum+L15 culture solution ingredients are：1ml fetal calf serum+49ml L15
Culture solution, mixing.
Above-mentioned all operations all carry out in sterile super-clean bench, and wherein AIM liquid, PBS are filtered with disposable aspiration needle formula filter.
Dissecting tool, 10ml centrifuge tubes, pipette tips, distilled water, 0.65% physiological saline sterilize with 120 degrees Celsius in high-pressure sterilizing pot
30min is dried for standby for 60 DEG C in baking oven.
The present invention can obtain the channel catfish head-kidney that purity is high, quantity is big, activity is good by above-mentioned steps and condition
Macrophage.
The above method of the present invention can be used for establishing external channel catfish head-kidney Macrophage Model.It can be applied to spot fork
The immunity additive functional study of Wei Channel-catfish.Preferably, with the medium culture institute of the g/ml yeast cell wall polysaccharide of μ containing 400-600
State channel catfish head-kidney macrophage, incubation time 10-15h.Above-mentioned condition of culture can make cell viability more preferably.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is shown according to the method for the present invention, and macrophage adherent growth is observed under 20 × inverted fluorescence microscope
The image of situation.
Fig. 2 shows being centrifuged only with a percoll separating liquid, observed under 20 × inverted fluorescence microscope huge
The image of phagocyte adherent growth situation.
Fig. 3 shows that various concentration yeast cell wall influences Macrophage Cell energy value.
Fig. 4 shows that various concentration yeast cell wall effect different time influences macrophage energy value.It is left in every group
Lateral column represents 12h, and right side column represents for 24 hours.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe the preferred implementations of the present invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without should be limited by embodiments set forth herein.
Embodiment 1
A kind of channel catfish head-kidney macrophage primary isolated culture method, includes the following steps：
A) fish body anesthesia blood sampling.The channel catfish of weight 1kg is chosen, MS-222 depth fiber crops are carried out to channel catfish
Liquor-saturated, anesthesia duration 5 minutes takes out fish body, takes a blood sample in Aseptic sterilisation disk, and blood sampling is using 2.5ml graded syringes, position of taking a blood sample
It is placed at the 5cm of tail portion, syringe is inserted into along fish body side line, and blood sampling volume is about 8ml, and blood sampling finishes, and fish body surface sprinkling alcohol disappears
Poison, dissection are for use.
B) its head-kidney tissue is taken.It is cut off with scissors along fish maw, removes its internal organ, find head-kidney along air bladder front, head-kidney is
It is flat, positioned at air bladder by below frontal lobe, each one of left and right is red.Head-kidney is taken out, is soaked in 0.65% physiological saline, 3min
It takes out and is soaked in 5ml AIM liquid afterwards, impregnate duration 20min.
C) tissue grinder, steel mesh filtering.Head-kidney tissue is taken out after immersion, is placed in the sterile steel mesh of 100 mesh, utilizes note
Emitter, which is cut somebody's hair, is ground it, and 15ml L-15 culture solutions are added in process of lapping.Grinding end mark is only remained for head-kidney tissue
Lower white lipid matter.
D) cell liquid containing macrophage is centrifuged out.Cell pulverized liquid is dispelled with liquid-transfering gun, is added to
In the deployed percoll being located in 10ml centrifuge tubes layering liquid, it is 2ml per the most suitable addition of solencyte liquid, adds in
3 layers of cell point afterwards, the bottom are the percoll separating liquids of 3ml density 52%, and middle layer is divided for the percoll of 3ml density 32%
Chaotropic, top layer are the above-mentioned cell liquid of 2ml, and adhesive tape carries out sealing to be placed in refrigerated centrifuge centrifuging to centrifuge tube, from
Heart condition is：Centrifugal force 400g, time 20min, 4 DEG C of temperature.After centrifugation, take out percoll interfaces and go out one layer carefully
Born of the same parents, are collected in dispel in 10ml centrifuge tubes and are added to the deployed percoll layerings being located in 10ml centrifuge tubes again
It is 2ml per the most suitable addition of solencyte liquid in liquid, 3 layers of cell point after addition, percoll separating liquids, centrifugal condition are as above.From
It takes out at percoll layering interfaces in cell and 10ml centrifuge tubes, is centrifuged after leveling, centrifugal force 1000g again after the heart, when
Between 10min, 4 DEG C of temperature.Centrifugation terminates, and abandons supernatant, with volume ratio 3：1 0.65% physiological saline：L-15 culture mediums are to each
Solencyte is dispelled, centrifugal force 1000g, time 10min, is centrifuged at 4 DEG C of temperature.Centrifugation terminates, and abandons supernatant, and each pipe adds in
5ml L-15 culture mediums, liquid-transfering gun dispel each solencyte, centrifugal force 1000g, time 10min, are centrifuged at 4 DEG C of temperature.Centrifugation
Terminate, abandon supernatant, add in the L-15 culture solutions containing 2%FBS and dispel cell, be placed in 6 orifice plates in constant temperature incubation per hole 2ml
It is incubated under the conditions of 28 DEG C in case and (sterile creep plate can be put in 6 orifice plates in case later stage dyeing observation).
E) macrophage is purified.After being incubated 4 hours, liquid-transfering gun draws upper strata culture medium, and PBS is carefully purged 2 times, removed
PBS adds in 2ml L-15 culture solutions per hole, with being incubated under the conditions of in constant incubator 28 DEG C.It is glimmering by being inverted after culture
The light microscope observation adherent effect of cell is good, and cell growth state is good, and cell concentration is big, and head-kidney macrophage purifying rate reaches 99%
(as shown in Figure 1).
If only carrying out a percoll layering liquid separation, obtained cell is as shown in Figure 2.As can be seen that use percoll
After centrifugation twice the adherent effect of cell is more preferable, heteroproteose cell is few, it is adherent to be conducive to macrophage.
Above-mentioned AIM ingredients are：45ml L-15 culture medium+2.5ml mycillin mixed liquor+2.5ml amphotericin Bs (250 μ
G/ml)+0.5ml gentamicin sulphates (50 μ g/ml).
The percoll liquid ingredients of above-mentioned different densities are as follows：
32%percoll liquid：Distilled water 14.5ml；HBSS2.5ml；NaHCO30.125ml；Percoll liquid 8ml.52%
Percoll liquid：Distilled water 10ml；HBSS2.5ml；NaHCO30.125ml；Percoll liquid 13ml.Wherein 32%percoll liquid
It is mixed colours with sterile 0.1M hydrochloric acid 70ul.
The operating method of above-mentioned institute's score layer liquid is：The percoll separating liquids of 3ml 32%, profit are added in 10ml centrifuge tubes
The percoll separating liquids that 50% is drawn with 3ml syringes are slowly injected from centrifugation bottom of the tube, and it is apparent can to obtain layering
Percoll separating liquids.
Above-mentioned 2%fetal calf serum+L15 culture solution ingredients are：1ml fetal calf serum+49ml L15
Culture solution, mixing.
Above-mentioned all operations all carry out in sterile super-clean bench, and wherein AIM liquid, PBS are filtered with disposable aspiration needle formula filter.
Dissecting tool, 10ml centrifuge tubes, pipette tips, distilled water, 0.65% physiological saline and 120 DEG C of 30min that sterilize in high-pressure sterilizing pot,
It is dried for standby for 60 DEG C in baking oven.
Embodiment 2
Various concentration yeast cell wall polysaccharide promotes the experiment of channel catfish head-kidney macrophage proliferation.
0,20,40,80,100,200,500,1000,2000,4000 μ g/ml yeast cell wall solution effects are set in spot
MTS methods are measured (as shown in Figure 3) its energy value after point Cha Wei Channel-catfish head-kidney macrophages 12h.The result shows that when yeast is thin
The vigor of channel catfish head-kidney macrophage is best during a concentration of 500 μ g/ml of cell wall, which shows at low concentrations simultaneously
Yeast cell wall has facilitation to the vigor of channel catfish head-kidney macrophage, and yeast cell wall is to spot in higher concentrations
The vigor of Cha Wei Channel-catfish head-kidney macrophages has inhibiting effect, this makees Immune enhancement of the yeast cell wall in fish farming industry
With being proved.
Embodiment 3
Various concentration yeast cell wall polysaccharide promotes channel catfish head-kidney macrophage growth function under different time
Experiment.
0,125,250,500 μ g/ml yeast cell walls solution effects are set in channel catfish head-kidney macrophage 12h/
MTS methods are measured its energy value after for 24 hours, and the results are shown in Figure 4.
The result shows that with the extension of yeast cell wall solution effects time, the vigor of channel catfish head-kidney macrophage
It decreases, illustrates that yeast cell wall solution the best use time is preferably 12h, this is for yeast cell wall in fish farming industry
The time effect of middle application also provides reference.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.In the case of without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes will be apparent from for the those of ordinary skill in art field.
Claims (10)
1. a kind of channel catfish head-kidney macrophage primary isolated culture method, which is characterized in that this method includes following step
Suddenly：
A) fish body anesthesia blood sampling；
B) head-kidney tissue is taken；
C) tissue grinder, filtering；
D) cell liquid containing macrophage is centrifuged with the percoll separating liquids of 30%-55%, separation is twice；
E) constant temperature incubation：In the L-15 culture solutions containing 2%-4%FBS, the constant temperature incubation under the conditions of 26-30 DEG C.
2. according to the method described in claim 1, wherein, this method further includes：
F) macrophage is purified：Step e) constant temperature incubations are after 4-6 hours, after drawing upper strata culture medium, PBS purging cells, again
Add in the L-15 culture solutions containing 2%-4%FBS, the constant temperature incubation under the conditions of 26-30 DEG C.
3. according to the method described in claim 1, wherein, step a) includes：
The channel catfish of weight 1kg-1.25kg is chosen, MS-222 deep anaesthesias are carried out to channel catfish, take out fish body, in
Tail portion is taken a blood sample, and after blood sampling, alcohol disinfecting is sprayed on fish body surface, dissection is for use.
4. according to the method described in claim 3, wherein, the mass ratio of fish body weight and anesthetic is 180-220：1, anesthesia duration
It is 5-8 minutes；Fish body weight and blood sampling volume ratio (g：Ml) it is 110-140：1.
5. according to the method described in claim 1, wherein, step b) includes：
Head-kidney tissue is taken out, is soaked in physiological saline, is taken out after 3-5min, then be soaked in AIM liquid, soaking time 20-
30min。
6. according to the method described in claim 1, wherein, step c) includes：
Head-kidney tissue is placed in sterile steel mesh, is cut somebody's hair using syringe and it is ground, L-15 trainings are added in process of lapping
Nutrient solution；It grinds end mark and is only left white lipid matter for head-kidney tissue.
7. according to the method described in claim 1, wherein, step d) and step e) include：
The obtained cell pulverized liquids of step c) are dispelled, are added in percoll layering liquid, percoll layering liquid upper strata is
The percoll separating liquids of 32%-34%, lower floor is the percoll separating liquids of 50%-52%, after centrifugation, is taken out
Cell at percoll layering interfaces after collection is added in new above-mentioned percoll layering liquid, centrifuges and take out again
Cell at percoll layering interfaces, centrifuges again, abandons supernatant, and cell is purged with 0.65% physiological saline/L-15 culture mediums
Afterwards, the L-15 culture solution cultures containing 2%-4%FBS are added in.
8. according to the method described in claim 7, wherein, the percoll separating liquids of 32%-34%, 50%-52%
Percoll separating liquids and the volume ratio of cell pulverized liquid are 0.8-1.2：0.8-1.2：1.
9. the method in claim 1-8 described in any one is in external channel catfish head-kidney Macrophage Model is established
Using.
10. application according to claim 9, wherein, it is trained with the culture medium of the g/ml yeast cell wall polysaccharide of μ containing 400-600
Support the channel catfish head-kidney macrophage, incubation time 10-15h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115044554A (en) * | 2022-05-11 | 2022-09-13 | 华中农业大学 | Separation and purification and primary culture method of macrophages in rana nigromaculata spleen |
| CN115044554B (en) * | 2022-05-11 | 2023-12-08 | 华中农业大学 | Separation and purification and primary culture method for rana nigromaculata spleen macrophages |
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