CN106701675A - Method for primary culture of lung macrophage of Chinese soft shell turtles - Google Patents
Method for primary culture of lung macrophage of Chinese soft shell turtles Download PDFInfo
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- CN106701675A CN106701675A CN201710054441.3A CN201710054441A CN106701675A CN 106701675 A CN106701675 A CN 106701675A CN 201710054441 A CN201710054441 A CN 201710054441A CN 106701675 A CN106701675 A CN 106701675A
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Abstract
The invention discloses a method for primary culture of lung macrophage of Chinese soft shell turtles. The method comprises the steps of separation of complete lung macrophage of the Chinese soft shell turtles and primary culture of the lung macrophage. The method is simple to operate, convenient to culture and high in culture success rate; particularly in flushing in vitro lung tissues, the method makes it easy and convenient to perform isolated culture of primary cells of the lung tissues, and effective cytokine is provided for research on viruses of the Chinese soft shell turtles.
Description
Technical field
The present invention relates to a kind of cell injuring model method, more particularly to a kind of original cuiture of Shelled Turtle Trionyx Sinensis pulmonary macrophage
Method.
Background technology
Shelled Turtle Trionyx Sinensis, also known as water fish, soft-shelled turtle, the soft-shelled turtle, are common cultivation tortoise kinds.Shelled Turtle Trionyx Sinensis move in rivers, lakes and marhshes, pond
The water flows gently for the pool, reservoir etc., the freshwater of the numerous life of fishes and shrimps, also often haunts in big mountain stream.In quiet, cleaning, sunny
Waterfront side activity relatively frequently, sometimes disembarkation but can not from water source too away from.Can on land creep, climb, also can be in water certainly
By swimming.Happiness is got sun or wind of enjoying the cool.Proverb among the people describe the activity of soft-shelled turtle be " spring shampoo goes on beach, and summer, the scorching shade of willow trees was dwelt,
Autumn is cool to enter the bottom, winter severe cold sludge pool ".Summer has solarization first to be accustomed to, and cold winter meeting hibernation, next year starts revival and seeks
Food.Eating fishes and shrimps, insect etc., the also plant food such as drinking water grass, cereal, and the especially saprophage, resistance to famine such as the smelly fish of addiction, rotten shrimp
Starve, but it is voracious and cruel, and such as bait lacks and can also kill and devour mutually.Property cowardice be afraid of the sound, in daytime latent water or in mud, night
Water outlet is looked for food.
Shelled Turtle Trionyx Sinensis are the domestic main famous-brand and high-quality Special Breed kind of fresh water, the first freshwater fish cell line ZC- in the country
7901 are published on aquatic product journal by Zhang Nianci Yang Guang intelligence (1981).There is the foundation of many freshwater fish cell lines later.And in
Magnificent soft-shelled turtle cell injuring model also has been reported that.Wherein Wu Kang, Xue Ming wait by force (2000) to be published in Chinese aquatic science, Shelled Turtle Trionyx Sinensis embryo
The research of cell injuring model；Li Xiaoli, 5 kinds of tissues of Shelled Turtle Trionyx Sinensis for once making (2010) such as soldiers to be published on Ecology magazine are thin
The cultured in vitro experiment of born of the same parents；Pay Jianping, Nie Pin etc. sets up Shelled Turtle Trionyx Sinensis aortic smooth muscle cell system STA (soft-shelled
Turtle artery), it was published on Fish Pathology in 2013.There is complex operation in the above method, condition is more severe
Carve, the relatively low deficiency of culture success ratio.The cultural method of Shelled Turtle Trionyx Sinensis pulmonary macrophage also has no report.
The content of the invention
It is simple to operate it is an object of the invention to provide a kind of primary culture method of Shelled Turtle Trionyx Sinensis pulmonary macrophage, culture
Convenient, culture success ratio is high, particularly rinses in vitro lung tissue so that lung tissue primary cell is separately cultured easy and convenient, is
Trionyx Sinensis Virus research provides effective cell medium.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of primary culture method of Shelled Turtle Trionyx Sinensis pulmonary macrophage, comprises the following steps：
First, the separation of the complete pulmonary macrophage of Shelled Turtle Trionyx Sinensis：
(1) Shelled Turtle Trionyx Sinensis of 100-500g weights are taken, body surface sterilization is put to death using disconnected cervical approach, dissects and take out complete lung tissue；
(2) lung tissue is placed in precooling plus dual anti-PBS liquid, is removed other unnecessary and is organized, washing 3 times, then
It is put into the HBSS balanced salt solutions of precooling；
(3) use the M199 fluid nutrient mediums containing 3% hyclone to inject lung for cleaning solution, rinse twice, merge
Flushing liquor obtained by twice；
(4) flushing liquor crosses 100 μm of filter screens, is once centrifuged, and removes supernatant, precipitation plus erythrocyte cracked liquid splitting erythrocyte 5-
8min, 100 μm of strainer filterings, secondary centrifuging, secondary centrifuging gained cell precipitation is stand-by；
2nd, the original cuiture of pulmonary macrophage：
Secondary centrifuging gained cell precipitation is resuspended with cell culture fluid, by 1 × 106~5 × 106Individual/ml cells are dispensed to thin
Born of the same parents' blake bottle culture, 28~30 DEG C, 5%CO2Under the conditions of cultivate.
Preferably, in step (1), body surface sterilization soaks 20-30min using 0.1% potassium permanganate.
Preferably, PBS liquid pH in step (2) plus dual anti-is 7.4, dual anti-is 500 μ g/ml penicillin, 500 μ g/ml
Streptomysin.
Preferably, in step (3), flushing operation is that cleaning solution is injected into lung, and cleaning solution is in intrapulmonary and rocks for holding
1-3 minutes, from tracheae pouring out cleaning solution obtained flushing liquor.
Preferably, in step (4), a parameter of noncentricity is that 800r/min is centrifuged 5min, and secondary centrifuging parameter is 800r/
Min is centrifuged 5min.
Preferably, the cell culture fluid is to contain 20-30% hyclones, 100 μ g/mL penicillin and 100 μ g/mL
The M199 fluid nutrient mediums of streptomysin.
Preferably, Tissue Culture Flask is using preceding, first with 1% gelatin bed board, 37 DEG C of 2~4h of incubation are placed in, then remove bright
Glue.So operation is conducive to the adherent growth of pulmonary macrophage.
Preferably, 28~30 DEG C, 5%CO2Under the conditions of cultivate 3~4h after, change cell culture fluid it is not adherent to remove
Red blood cell and dead cell, continue to cultivate 12~16h, change cell culture fluid and further remove residual red blood cells, continue to cultivate.
The beneficial effects of the invention are as follows：The present invention establishes the primitive cell culture method of Shelled Turtle Trionyx Sinensis pulmonary macrophage, its
Feature is simple to operate, and culture is convenient, without specific cell separation liquid.Adherent growth can be effectively obtained with direct douche
Pulmonary macrophage, normal morphology 12-20d is maintained under the condition of culture that can be described in the present invention, can meet follow-up more
Biological experiment, practicality is very high.The original cuiture of other reptiles aquatic animal pulmonary macrophages can be applied to.
Brief description of the drawings
Fig. 1 is the in vitro lung tissue of Shelled Turtle Trionyx Sinensis.
Fig. 2 is the pulmonary macrophage of adherent growth.
Fig. 3 is the ACP dyeing identifications of pulmonary macrophage.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, raw material and equipment for being used etc. is commercially available or commonly used in the art.
Method in following embodiments, unless otherwise instructed, is the conventional method of this area.
Embodiment：
A kind of primary culture method of Shelled Turtle Trionyx Sinensis pulmonary macrophage, comprises the following steps：
First, the separation of the complete pulmonary macrophage of Shelled Turtle Trionyx Sinensis：
(1) Shelled Turtle Trionyx Sinensis of 100-500g weights are taken, body surface sterilization is carried out using 0.1% potassium permanganate immersion 20-30min, adopted
Put to death with disconnected cervical approach, and as far as possible after extrusion blood, dissect and take out complete lung tissue (band part tracheae, see Fig. 1)；
(2) lung tissue be placed in precooling plus dual anti-PBS liquid (pH 7.4, dual anti-is 500 μ g/ml penicillin, 500 μ g/
Ml streptomysins) in, other unnecessary tissues such as removal fat and connective tissue are washed 3 times, are then placed in the HBSS balances of precooling
In salting liquid (commercially available)；
(3) lift the tracheae being connected with lung with aseptic nipper, be by the M199 fluid nutrient mediums containing 3% hyclone
Cleaning solution injects lung, and now tweezers, base of lung portion is lifted with another by lung's expansion, and about 1-3min is gently rocked at two ends, from gas
Cleaning solution is poured out at pipe and completes once flushing, repeat to rinse 1 time, merge the flushing liquor of gained twice；
(4) 100 μm of strainer filterings of flushing liquor, once centrifugation (800r/min is centrifuged 5min), removes supernatant, precipitation plus red blood cell
Lysate (commercially available) splitting erythrocyte 5-8min, 100 μm of strainer filterings, secondary centrifuging (800r/min be centrifuged 5min), it is secondary from
Heart gained cell precipitation is stand-by；
2nd, the original cuiture of pulmonary macrophage：
Secondary centrifuging gained cell precipitation with cell culture fluid (containing 20-30% hyclones, 100 μ g/mL penicillin and
The M199 fluid nutrient mediums of 100 μ g/mL streptomysins) it is resuspended, by 1 × 106~5 × 106Individual/ml cells are dispensed to Tissue Culture Flask
Culture, 28~30 DEG C, 5%CO2Under the conditions of cultivate 3~4h after, cell culture fluid is changed to remove not adherent red blood cell and dead
Cell, continues to cultivate 12~16h, changes cell culture fluid and further removes residual red blood cells, remaining most of for lung macrophage is thin
Born of the same parents, purity continues to cultivate up to 75-90%.Using preceding, first with 1% gelatin bed board, (i.e. gelatin covers cell and trains Tissue Culture Flask
Support bottle), 37 DEG C of 2~4h of incubation are placed in, then remove gelatin.
Cellular morphology is observed and identified：100 × and 400 × phase contrast microscope under observation of cell, wherein macrophage is in
It is easy to assemble agglomerating growth, rough surface mostly；There is a small amount of red blood cell ovalize, surface is relatively smooth (Fig. 2).Macrophage
Because activity of acid phosphatase is higher, after being dyeed through ACP staining kits, cell takes on a red color (Fig. 3).Cell after being separately cultured is big
Red can be dyed by ACP staining reagents more, show the cell mostly pulmonary macrophage being separately cultured.Can be used for subsequent experimental.
Current Shelled Turtle Trionyx Sinensis pulmonary macrophage cell can be cultivated 12-20 days in vitro, and cell still maintains kilter.The present invention
Purity up to 75-90% pulmonary macrophages can be obtained, can be used for the experimental studies such as Trionyx Sinensis Virus infection.
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (8)
1. a kind of primary culture method of Shelled Turtle Trionyx Sinensis pulmonary macrophage, it is characterised in that comprise the following steps：
First, the separation of the complete pulmonary macrophage of Shelled Turtle Trionyx Sinensis：
(1) Shelled Turtle Trionyx Sinensis of 100-500g weights are taken, body surface sterilization is put to death using disconnected cervical approach, dissects and take out complete lung tissue；
(2) lung tissue is placed in precooling plus dual anti-PBS liquid, removes other unnecessary tissues, is washed 3 times, is then placed in
In the HBSS balanced salt solutions of precooling；
(3) use the M199 fluid nutrient mediums containing 3% hyclone to inject lung for cleaning solution, rinse twice, merge twice
The flushing liquor of gained；
(4) flushing liquor crosses 100 μm of filter screens, is once centrifuged, and removes supernatant, precipitation plus erythrocyte cracked liquid splitting erythrocyte 5-8min,
100 μm of strainer filterings, secondary centrifuging, secondary centrifuging gained cell precipitation is stand-by；
2nd, the original cuiture of pulmonary macrophage：
Secondary centrifuging gained cell precipitation is resuspended with cell culture fluid, by 1 × 106~5 × 106Individual/ml cells are dispensed to cell training
Support bottle culture, 28~30 DEG C, 5%CO2Under the conditions of cultivate.
2. primary culture method according to claim 1, it is characterised in that in step (1), body surface sterilization uses 0.1%
Potassium permanganate soaks 20-30min.
3. primary culture method according to claim 1, it is characterised in that PBS liquid pH in step (2) plus dual anti-is
7.4, dual anti-is 500 μ g/ml penicillin, 500 μ g/ml streptomysins.
4. primary culture method according to claim 1 and 2, it is characterised in that in step (3), flushing operation is to wash
Liquid injection lung is washed, cleaning solution is in intrapulmonary and rocks 1-3 minutes for holding, and pouring out cleaning solution from tracheae obtains flushing liquor.
5. primary culture method according to claim 1 and 2, it is characterised in that in step (4), a time parameter of noncentricity is
800r/min is centrifuged 5min, and secondary centrifuging parameter is that 800r/min is centrifuged 5min.
6. primary culture method according to claim 1, it is characterised in that the cell culture fluid is to contain 20-30%
The M199 fluid nutrient mediums of hyclone, 100 μ g/mL penicillin and 100 μ g/mL streptomysins.
7. primary culture method according to claim 1 and 2, it is characterised in that Tissue Culture Flask using preceding, first with 1%
Gelatin bed board, is placed in 37 DEG C of 2~4h of incubation, then remove gelatin.
8. primary culture method according to claim 1 and 2, it is characterised in that 28~30 DEG C, 5%CO2Under the conditions of cultivate 3
After~4h, change cell culture fluid to remove not adherent red blood cell and dead cell, continue to cultivate 12~16h, change cell training
Nutrient solution further removes residual red blood cells, continues to cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710054441.3A CN106701675A (en) | 2017-01-22 | 2017-01-22 | Method for primary culture of lung macrophage of Chinese soft shell turtles |
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| CN201710054441.3A CN106701675A (en) | 2017-01-22 | 2017-01-22 | Method for primary culture of lung macrophage of Chinese soft shell turtles |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8663984B2 (en) * | 2007-01-12 | 2014-03-04 | The Board Of Trustees Of The University Of Illinois | Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus |
| CN105793416A (en) * | 2013-12-12 | 2016-07-20 | 英特维特国际股份有限公司 | Immortalized porcine alveolar macrophage |
-
2017
- 2017-01-22 CN CN201710054441.3A patent/CN106701675A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8663984B2 (en) * | 2007-01-12 | 2014-03-04 | The Board Of Trustees Of The University Of Illinois | Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus |
| CN105793416A (en) * | 2013-12-12 | 2016-07-20 | 英特维特国际股份有限公司 | Immortalized porcine alveolar macrophage |
Non-Patent Citations (1)
| Title |
|---|
| 惠文巧等: "山羊肺泡巨噬细胞分离培养及脂多糖诱导形态学观察", 《中国草食动物科学》 * |
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