CN109792972A - A kind of cysticercus extracorporeal culturing method - Google Patents
A kind of cysticercus extracorporeal culturing method Download PDFInfo
- Publication number
- CN109792972A CN109792972A CN201910172991.4A CN201910172991A CN109792972A CN 109792972 A CN109792972 A CN 109792972A CN 201910172991 A CN201910172991 A CN 201910172991A CN 109792972 A CN109792972 A CN 109792972A
- Authority
- CN
- China
- Prior art keywords
- culture
- cysticercus
- time
- added
- culture solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of cysticercus extracorporeal culturing method, especially a kind of cysticercus pisiformis in-vitro culture method.Method of the invention is: separating after complete cysticercus cleans up, is transferred in culture vessel in it added with culture solution, culture vessel is placed in carbon dioxide cell incubator, in 37 DEG C of temperature, CO out of host animal body2It is cultivated under the conditions of concentration 5%, relative humidity 70%, polypide starts the 6h after culture and 12h is respectively changed liquid 1 time, removes outmoded culture solution when changing liquid every time as far as possible, fresh complete culture solution 8ml/ bottle/time is added, and later every 48h is changed liquid 1 time.The present invention solves the problems, such as that cysticercus pisiformis is survived for a long time in vitro, can easily obtain sufficient research material, polypide survival time in vitro is long, can meet the screening period of drug screening and Pharmacological Evaluation, while can directly observe polypide-drug effect.Independent of host, Experimental Background is easily controllable for culture of the invention, and more correct assessment can be made to larval stage specific experiment.
Description
Technical field
The present invention relates to a kind of helminth extracorporeal culturing method, helminth cultural method of the present invention refers to cysticercus
Extracorporeal culturing method, especially a kind of cysticercus pisiformis in-vitro culture method.
Background technique
Cysticercosis is a kind of common parasitic disease, and distribution is extensive, seriously threatens animal husbandry development and public
It is safe and healthy.Cysticercus pisiformis disease therein is parasitized in rabbit body by middle silk ribbon phase larva-cysticercus pisiformis of Taenia Pisiformis
Caused by a kind of common parasitic parasitosis.In China, rabbit average rate is up to 40%, the death rate 4.0%~23.69%,
Seriously threaten the development of rabbit keeping.Cysticercus pisiformis main parasitic is in the liver of rabbit, stomach nethike embrane, mesenterium or rectum rear wall；
Disease rabbit liver is caused to damage when a large amount of infection, the diseases such as clinical manifestation digestive disorders, loss of appetite, spiritual depressed, thin, anaemia
Shape, die by visitation of God when serious；Furthermore can secondary pasteurellosis etc., lead to the reduction of feed for rabbit return rate, economic benefit decline.
The Taenia Pisiformis history of life is complicated, and the complete history of life, which needs to convert 2 hosts, to complete.Adult is usually parasitic
In the small enteral of canine, worm's ovum is developed in adult gravid proglottid, and is fallen off and be discharged into external environment with gravid proglottid, contaminated food or water
Source.Worm's ovum reaches liver with blood flow after being eaten by rabbit and migrates to omentum majus, mesenterium etc. development is infectious beans shape capsule tail
The larva of a tapeworm or the cercaria of a schistosome.China still faces very big problem to the prevention and treatment of the disease at present, needs to screen and excavate specific drugs target and vaccine candidate
Molecule provides new tool for the effective prevention and control and radical cure of the disease.As the broad scale research of helminth functional genome develops, base
Because functional verification and newtype drug are parsed into the bottleneck for the research of current helminth.In terms of Taenia Pisiformis research, nothing has
The stable cell lines of effect also not yet establish feasible in-vitro evaluation model for utilizing.
In vitro culture is a valuable technology platform in parasitology research, can under intuitive and controlled condition into
The correlative studys such as row parasite antigen, drug effect, immunological effect and helminth and host's interaction.In recent decades, with
Cultural method continuous improvement and updating, pass through imitate host body certain factors and condition establish be suitable for culture side
Method promotes the development of the artificial culture technique research of helminth.However, parasite species are various, the history of life is complicated, without general training
Nutrient solution or unified cultural method carry out in vitro culture to helminth.
Relative to protozoon, fluke and nematode, the progress of tapeworm Vitro Culture Techniques is more slow.Currently, being used for beans
The work worm sample of shape cysticercus research, is mainly collected in the rabbit of nature or experimental infection, the method has the following shortcomings:
(1) at high cost by dog-rabbit infection cycle guarantor's worm method, heavy workload, bothersome laborious, investment personnel are more；Pacifying
There are problems in terms of full property and economy.In addition, protecting, the worm test period is long, and generally requiring 4 months or so could complete.
(2) it is not easy to carry out drug screening and medicine under intuitive or controlled condition by the worm sample living of cysticercus in rabbit body
The researchs such as effect evaluation.
(3) enough metabolic secretion antigen or excretion body cannot be collected at any time by the worm sample living of cysticercus in rabbit body.
Therefore it provides a kind of feasible and stable cysticercus pisiformis extracorporeal culturing method, be substantially shorter the scientific research time with
Cost helps to carry out the research such as antigen collection, drug screening and effective drug effect analysis in vitro；For the research with tapeworm
And its Study of Prevention Technology has important learning value and application value.
Summary of the invention
The present invention provides a kind of effective cysticercus in-vitro culture method, and present invention particularly provides a kind of achievable beans shape capsules
The cysticercus cultural method that cercaria survives for a long time in vitro.In addition, the present invention also provides one kind to be convenient for cysticercus pisiformis generation
Thank to the cysticercus pisiformis in vitro culture side of the needs such as secretion antigen preparation, the preparation of excretion body, polypide metabolism detection, drug screening
Method can be conducive to carry out metabolic secretion antigen preparation, the preparation of excretion body, polypide metabolism using the polypide of method culture of the invention
Detection, drug screening etc..The alternative model that can also be used as studying other important tapeworm gene functions, such as cysticercosis cellulosae, ox capsule
Cercaria disease etc..But Process of in vitro only keeps cysticercus Larva Morpho. Logy, and not being related to the development of in vitro culture larva is adult.
To achieve the above object, the present invention adopts the following technical scheme:
Cysticercus in-vitro culture method separates complete cysticercus out of host animal body first, aseptically uses
The PBS that at least one antibiotic is wherein added is cleaned up, and is then transferred in the culture vessel in it added with culture solution, culture is held
Device is placed in carbon dioxide cell incubator, in 37 DEG C of temperature, CO2It is cultivated under the conditions of concentration 5%, relative humidity 70%,
Polypide starts the 6h after culture and 12h is respectively changed liquid 1 time, removes outmoded culture solution when changing liquid every time as far as possible, is added fresh
Complete culture solution 8ml/ bottle/time, later every 48 h are changed liquid 1 time.
A kind of cysticercus pisiformis in-vitro culture method of the invention is: the complete beans of fresh separated from the abdominal cavity of infected rabbits
Shape cysticercus aseptically, is cleaned up with the PBS that at least one antibiotic is wherein added, and is then transferred in it added with training
In the culture vessel of nutrient solution, culture vessel is placed in carbon dioxide cell incubator, in 37 DEG C of temperature, CO2It is concentration 5%, opposite
It is cultivated under the conditions of humidity 70%, polypide starts the 6h after culture and 12h is respectively changed liquid 1 time, removes as far as possible when changing liquid every time
Outmoded culture solution is removed, fresh complete culture solution 8ml/ bottle/time is added, later every 48h is changed liquid 1 time.
Preferably, in cysticercus pisiformis in-vitro culture method of the invention, added with the mould of 100U/ml in PBS used
The streptomysin of element and 100 μ g/ml, the culture solution are wherein to be added with fetal calf serum, penicillin, streptomysin, anphotericin
B, the RPMI1640 culture solution of L-Glutamine and sodium bicarbonate, in which: fetal calf serum content is 10% (v/v)；The antibiosis
Element final concentration of penicillin 100U/ml, 100 μ g/ml of streptomysin, 1.25 μ g/mL of amphotericin B；The L-Glutamine is dense eventually
Degree is 2.5mM；It is 7.2~7.4 that sodium bicarbonate adjustment culture solution pH, which is added,.
Preferably, cysticercus pisiformis in-vitro culture method of the invention, the additional amount of initial incubation liquid are as follows: 6 orifice plates add
Enter that amount is the hole 3ml/ or cell bottle additional amount is 8ml/ (25cm2) bottle.
Cysticercus pisiformis in-vitro culture method for collecting cysticercus pisiformis excretion body of the invention is: will be from infected rabbits
Abdominal cavity in the complete cysticercus pisiformis of fresh separated aseptically cleaned with the PBS that at least one antibiotic is wherein added
Completely, it is then transferred in it added with carrying out in the culture vessel for removing the culture solution of excretion body processing, culture vessel is placed in two
In carbonoxide cell incubator, in 37 DEG C of temperature, CO2It is cultivated under the conditions of concentration 5%, relative humidity 70%, polypide starts
6h and 12h after culture are respectively changed liquid 1 time, remove outmoded culture solution when changing liquid every time as far as possible, and fresh complete culture is added
Liquid 8ml/ bottle/time changes liquid 1 time for every 48 hours later.
Preferably, used in the cysticercus pisiformis in-vitro culture method for collecting cysticercus pisiformis excretion body of the invention
PBS in added with the penicillin of 100U/ml and the streptomysin of 100 μ g/ml, the culture solution be wherein added with penicillin,
Streptomysin, amphotericin B, L-Glutamine and sodium bicarbonate and the fetal calf serum handled except excretion body of carrying out over
RPMI1640 culture solution, in which: fetal calf serum content is 10% (v/v)；The final concentration of penicillin 100U/ml of antibiotic,
100 μ g/ml of streptomysin, 1.25 μ g/mL of amphotericin B；The final concentration of 2.5mM of L-Glutamine；Sodium bicarbonate is added
Adjusting culture solution pH is 7.2~7.4.
Preferably, cysticercus pisiformis in-vitro culture method of the invention, the additional amount of initial incubation liquid are as follows: 6 orifice plates add
Enter that amount is the hole 3ml/ or cell bottle additional amount is 8ml/ (25cm2) bottle.
The method of collection cysticercus pisiformis metabolic secretion antigen and excretion body of the invention is to carry out beans by method above-mentioned
The culture of shape cysticercus, after changing liquid for the second time, every 48h collection polypide culture solution is primary, the methods of concentrated or ultracentrifugation
Further obtain metabolic secretion antigen or excretion body.
The present invention has the advantages that
(1) a kind of in-vitro culture method for cysticercus pisiformis is provided, cysticercus pisiformis is efficiently solved and grows in vitro
The problem of time survives；
(2) sufficient research material can be easily obtained, such as cysticercus pisiformis metabolic secretion antigen and excretion body；
(3) polypide survival time in vitro is long, can meet the screening period of drug screening and Pharmacological Evaluation, while can directly see
Examine polypide-drug effect；
(4) in vitro culture realizes independent of host and carries out cysticercus pisiformis generation under controllable, intuitive and known conditions
Thank to the research such as antigen, immunological effect and drug screening；
(5) formula of liquid is easily cultivated, Experimental Background is easily controllable；
(6) Index Establishment is reasonable, and in vitro culture strict regulations only keep cysticercus larvae alive state, and it is raw not to be related to polypide
Long development and reproduction concept, if larva growth in vitro is adult；More correct assessment can be made to larval stage specific experiment.
Detailed description of the invention
Fig. 1 is that the light microscopic representative configuration of cysticercus pisiformis in vitro culture of the present invention observes figure.Wherein × 40 observe o:
Polypide before cultivating；A:RPMI-1640+6% lapin bile culture group；B:PBS culture group；C:RPMI-1640 culture group；D: completely
1 culture group of culture solution.× 100 observation e:a and b group polypide cephalomere forms；F:c and d group cephalomere form.Sc indicates cephalomere；Su table
Show sucker；BW indicates vesica；Ho indicates small hook.
Fig. 2 is cysticercus pisiformis metabolic secretion antigen westernblot qualification result of the present invention.Wherein M: albumen point
Son amount marker；ESP: metabolic secretion antigen；Tpeno: cysticercus pisiformis enolase, native protein molecular weight about 48kDa；
Tp14-3-3: cysticercus pisiformis 14-3-3, native protein molecular weight about 28kDa.
Fig. 3 is cysticercus pisiformis culture supernatant excretion body immuno-electron microscope qualification figure of the present invention.Wherein arrow is shown
CD63 positive mark's excretion body particle.
Specific embodiment
For a better understanding of the present invention, the content that the present invention is furture elucidated in conjunction with the accompanying drawings and embodiments, but the present invention
Content be not limited solely to following embodiment.
Reagent and material as used in the following examples commercially obtain unless otherwise specified.Tire ox blood
Clearly, Gibco company is purchased from without excretion body fetal calf serum, RPMI1640 culture solution and 7.5% sodium bicarbonate solution；L- glutamy
Amine, penicillin, streptomysin and amphotericin B are purchased from Sigma company.
The long-acting culture in vitro of 1 cysticercus pisiformis of embodiment
1. the preparation of complete culture solution
The complete culture solution is made of mother liquor and additive, process for preparation strict aseptic technique.
1.1 complete culture solutions 1
Using RPMI1640 culture solution as mother liquor；The mother liquor is synthetic media, includes glucose, amino acid, balance salt, dimension
He such as orders at the multiple nutritional components.Additive includes fetal calf serum, penicillin, streptomysin, amphotericin B, L-Glutamine and carbon
Sour hydrogen sodium.Content of the fetal calf serum in complete medium is 10% (v/v)；The final concentration of penicillin of antibiotic
100U/ml, 100 μ g/ml of streptomysin, 1.25 μ g/mL of amphotericin B；The final concentration of 2.5mM of L-Glutamine；It is described
7.5% sodium bicarbonate is appropriate, and adjustment complete culture solution pH is 7.2~7.4.
1.2 complete culture solutions 2
Serum additive in complete culture solution 2 is no excretion body fetal calf serum, mother liquor and other additives with training completely
Nutrient solution 1.
2. the acquisition and pretreatment of cysticercus pisiformis
Specific step is as follows for cysticercus pisiformis acquisition and progress aseptic process: parasitism is collected from infected rabbits abdominal cavity
Cysticercus pisiformis is removed simultaneously together with the parasitic sites such as stomach nethike embrane tissue.It is placed in superclean bench, with the PBS that sterilizes (containing double
It is anti-) sufficiently clean for several times, tissue is transferred in another clean plate, carefully separates polypide with eye scissors and tweezers, carefully
Extra host tissue is removed, pays attention to that polypide complete structure cannot be destroyed, that is, ensures the integrality of cephalomere and vesica；With sterilizing
PBS (containing dual anti-) is cleaned 5 times repeatedly, then is washed 3 times with RPMI l640 culture solution, and it is spare to be transferred to new clean plate.
3. the external sterile culture of cysticercus pisiformis
In super-clean bench, complete cysticercus pisiformis is selected, it is careful to move into steril cell bottle (25cm2), it is aforementioned that 8 ml are added
Complete culture solution 1,100/bottle of culture density；Bottle cap is tightened, carbon dioxide cell incubator culture is placed in.
Preferably, sugared part, sterile and anaerobic environment are the prerequisites for guaranteeing polypide Motility.It therefore, is raising beans
Condition of culture is arranged in the culture efficiency of shape cysticercus are as follows: and 37 DEG C of constant temperature, 5%CO2, relative saturation humidity about 70%.After culture
1,6,12,24,36,48h and it is carried out continuously observation daily, cyst wall under swing and microscope is routed up with cysticercus cephalomere and is stretched for worm
Body survival judgment criteria, observes and records cysticercus pisiformis survival time in vitro and morphologic change, measures body size and simultaneously takes pictures.
Preferably, change liquid method are as follows: the 6h and 12h that polypide starts culture are respectively changed liquid 1 time, when changing liquid every time as far as possible with
Aseptic straw removes outmoded culture solution, and fresh complete culture solution 8ml/ bottle/time is added, continues to train with aforementioned preferred condition of culture
It supports, changes every other day later liquid 1 time.Period observes polypide activity condition in time, can also microscopic observation cephalomere mobility and cyst wall it is flexible
State understands polypide survival condition.
Preferably, when collecting metabolic secretion antigen and excretion body, polypide culture 6h is changed into liquid, discards culture solution；It is added new
Fresh culture solution is further cultured for 12h, discards culture solution, the culture solution more renewed, and every 48h is collected 1 time.
Preferably, incubation strict aseptic technique guarantees pollution-free；Antibiotic concentration is added and maintains threshold value, it must not
It is excessively high, avoid toxic reaction from causing polypide structural collapse, death.
Preferably, the cultural method can provide physico chemical factor, nutriment and the germ-free condition that polypide is suitable for existence.Worm
Body survival 3 months or more, meet the needs of the test periods such as drug screening.
Embodiment 2 uses the comparative studies of three kinds of culture solutions and culture solution culture cysticercus pisiformis provided by the invention
In superclean bench, size and more consistent pretreatment polypide 40 of state are selected, are randomly divided into 4 groups, 10
A/group is put into corresponding culture solution culture, including PBS culture group, RPMI-1640 culture group, RPMI-1640+10% lapin bile
1 culture group of culture group and complete culture solution of the present invention.Condition of culture setting are as follows: 37 DEG C, 5%CO2, in each group culture solution
Containing dual anti-(penicillin 100U/mL, 100 μ g/mL of streptomysin) and amphotericin B (1.25 μ g/mL).Repeat culture 3 times；Yu Pei
5min after supporting, 1,2,12,24,36,48h and be carried out continuously observation daily, capsule under swing and microscope is routed up with cysticercus cephalomere
Wall stretches survives judgment criteria for polypide, when observing and recording time that cysticercus pisiformis cephalomere routs up, routing up rate, Motility
Between and morphologic change, measurement body size simultaneously take pictures.
Each culture group cysticercus general form changes as follows: the cysticercus before culture is oval, and vesica is transparent, fullness degree
Good, size is about 0.4cm × 0.7cm, and capsule intracavity liquid is limpid, and the dotted cephalomere of visible milky.The visible head of light microscopic observation
It is good (× 40, Fig. 1, o) to save stretching motion.It is low that PBS culture group polypide cephalomere routs up rate, is only 33% after 12h；It can generally survive
To 7d, polypide wriggle and stop (Fig. 1, a)；The small hook of microscopic observation cephalomere is obvious, marshalling.And lapin bile is to cysticercus pisiformis
Cephalomere is routed up with significant facilitation, and it is fast that polypide cephalomere routs up speed.Cultivate 15min, RPMI-1640+10% lapin bile
The neck section of group cysticercus can be routed up from capsule naturally completely, and it is 100% that cephalomere, which routs up rate,；Necktie knot section is thinner, stretches out about 4
~6mm, voluntarily twisting is obvious；Vesica becomes smaller, and body wall and cephalomere sucker mobility are good (Fig. 1, b)；Microscopic observation neck section surface light
Sliding, gauffer is uniformly fine and closely woven, and clavula is prominent obvious, and quality is thick and solid, is serrated.For 24 hours, this group of polypide mobility weakens, worm for culture
Somatocyst bubble becomes smaller, partial disappearance, and no liquid is full；The visible obvious sucker of microscopic observation cephalomere, rostellum and two punctuate the small hook of shape (figure
1, e), thin cyst, structural fuzzy, degree of shrinkage weaken.With the extension of incubation time, polypide activity stops, biliary culture group
Cysticercus pisiformis longest can survive to 48h.RPMI-1640 (Fig. 1, c) and complete culture solution 1 (Fig. 1, d) culture group polypide cephalomere
Rout up slow, it is low to rout up rate, culture 12h only 20%；But as incubation time extends, cephalomere is routed up rate and be can be improved to 70%.Completely
1 group of polypide energy of culture solution is good；Microscopic observation cephalomere sucker is high-visible, but has no small hook (Fig. 1, f).Temperature and light are equal
Can influence polypide cephalomere routs up state；Body size after culture is different, differs greatly, polypide length between 1.0~
3.8cm, but be mostly 1.5~2cm.
With the extension of incubation time, each culture group polypide most obvious difference is that vesica variation.Vesica and culture
Before compare, morphologic change mainly occurs, shows as length；Wherein 1 group of polypide vesica form of complete culture solution maintains effect
It is good.3 wheat harvesting period of in vitro culture, polypide survival rate is up to 90% or more；By table 1 as it can be seen that the polypide capsule of 1 group of complete culture solution of measurement
Bubble length is 1.83 ± 0.10cm, and width is 0.85 ± 0.05 cm, and vesica fullness degree is good, and cyst fluid is limpid, and cyst wall edge is complete,
It is capable of expansion and contraction；It is significant (P < 0.05) with other culture group polypide vesica difference in size；Remaining culture group polypide vesica obviously becomes
Small, cyst fluid is reduced or is disappeared, and cyst wall is thinning, and loosely, edge is not whole.
Testing result shows that, with the polypide in vitro culture state difference of remaining cultural method culture, the time-to-live is short, and complete
The long-acting cultivating system of culture solution 1 enhances polypide to external environment to maintaining polypide to survive in vitro vigor and extension time-to-live
Adaptability, improving survival rate has good effect.The requirement of different experiments purpose can be met.
Cysticercus pisiformis developmental condition in the different culture solutions of table 1
Note: different capitalizations indicate significant difference (P < 0.05) in same column.
The preparation of 3 cysticercus pisiformis metabolic secretion antigen of embodiment
The culture of 3.1 cysticercus pisiformis
Complete cysticercus pisiformis is selected, it is careful to move into steril cell bottle (25cm2), 8ml complete culture above-mentioned is added
Liquid 1,100/bottle of culture density；Bottle cap is tightened, carbon dioxide cell incubator culture is placed in.It is observed continuously daily, with capsule tail
Larva of a tapeworm or the cercaria of a schistosome cephalomere routs up cyst wall under swing and microscope and stretches for polypide survival judgment criteria.
Preferred condition of culture are as follows: 37 DEG C, 5%CO2, relative saturation humidity about 70%.
Preferably change liquid method are as follows: the 6h and 12h that polypide starts culture are respectively changed liquid 1 time, when changing liquid every time as far as possible with
Aseptic straw removes outmoded culture solution, and fresh complete culture solution 8ml/ bottle/time is added, continues to train with aforementioned preferred condition of culture
It supports, every 48h collects 1 culture supernatant later.
Separation, purifying and the identification of 3.2 cysticercus pisiformis metabolic secretion antigens
By the polypide culture supernatant of collection, 10min is centrifuged in 4 DEG C, 12000g；Supernatant is drawn, through 0.45 μm of filter mistake
Filter；15ml filtrate is gone in the Amicon Ultra-15 super filter tube of PBS Balance Treatment (molecular cut off 10kDa), 25 DEG C,
After 5000g is centrifuged 30min, the liquid in collecting pipe is discarded, PBS is added in inner tube and carries out buffer exchange, tenderness inhales product of drawing a design
For several times, 25 DEG C, 5000g centrifugation 30min after, draw inner tube in concentrating sample.
It is anti-with anti-Tpeno monoclonal using the metabolic secretion antigen in SDS-PAGE separation cysticercus pisiformis culture supernatant
Body 1C4 and anti-Tp14-3-3 polyclonal antibody are probe, using Western blot method to its marker protein enolase and
14-3-3 albumen has carried out immune-blotting method.As a result: the metabolic secretion antigen in cysticercus pisiformis culture supernatant is successfully concentrated,
Westernblot utilizes specific antibody that polypide enolase and 14-3-3 positive band, size point can be detected as the result is shown
Not about 48kDa and 28kDa is consistent with expected size.
The preparation of 4 cysticercus pisiformis excretion body of embodiment
The culture of 4.1 cysticercus pisiformis
Complete cysticercus pisiformis is selected, it is careful to move into steril cell bottle (25cm2), 8ml complete culture above-mentioned is added
Liquid 2,100/bottle of culture density；Bottle cap is tightened, carbon dioxide cell incubator culture is placed in.It is observed continuously daily, with capsule tail
Larva of a tapeworm or the cercaria of a schistosome cephalomere routs up cyst wall under swing and microscope and stretches for polypide survival judgment criteria.
Preferred condition of culture are as follows: 37 DEG C, 5%CO2, relative saturation humidity about 70%.
Preferably change liquid method are as follows: the 6h and 12h that polypide starts culture are respectively changed liquid 1 time, when changing liquid every time as far as possible with
Aseptic straw removes outmoded culture solution, and fresh complete culture solution 8ml/ bottle/time is added, continues to train with aforementioned preferred condition of culture
It supports, every 48h collects 1 culture supernatant later.
4.2 cysticercus pisiformis excretion bodies isolate and purify
Excretion body in cysticercus pisiformis culture supernatant is extracted using supercentrifugation, steps are as follows: collecting polypide culture
Supernatant, 4 DEG C, 300g centrifugation 10min；Supernatant is drawn, is centrifuged 20min in 4 DEG C, 2000g；Take supernatant, then through 4 DEG C, 10000g from
Heart 40min collects supernatant；Supernatant is filtered and transferred to ultracentrifugation pipe through 0.22 μm of filter, 12ml/ pipe, in 4 DEG C,
110000g is centrifuged 120min；Merging precipitating is washed with PBS (0.22 μm of filter filtering), is centrifuged again in 4 DEG C, 110000g
120min；Supernatant is abandoned, precipitating is dissolved in 10 μ l PBS (0.22 μm of filter filtering) and mixes.
The identification of 4.3 cysticercus pisiformis immune colloid gold electronic speculums
It takes 2.5% paraformaldehyde, 10 μ l that the excretion liquid solution prepared in 4.2 is added to be uniformly mixed, the front of Electronic Speculum copper mesh connects
Drop is touched, 30min is incubated at room temperature；Copper mesh is washed with PBS and 50mM glycine respectively；After 5%BSA closes copper mesh 10min,
45min is incubated for 20 μ l CD63 antibody (1:40)；PBS wash 3 times, with 20 μ l colloid gold label sheep anti mouse-IgG secondary antibodies (1:
20) it is incubated for 60min；According to a conventional method, copper mesh successively is handled with 0.5%BSA, PBS, 2.5% paraformaldehyde and deionized water；Add
10 μ l phosphotungstic acid negative staining 1min, suck dye liquor with filter paper, dry copper mesh, utilize transmission electron microscope observing excretion volume morphing.
As a result: it is discoid complete vesica of the diameter in 50~150nm that cysticercus pisiformis excretion body is observed under transmission electron microscope；
Immuno-electron microscope as the result is shown film surface significant transmembrane protein CD63 be the positive.
The above description is merely a specific embodiment, but the protection scope of the application is not limited thereto, any
Change or replacement within the technical scope of the present application should all be covered within the scope of protection of this application.Therefore, this Shen
Protection scope please should be using claims as foundation.
Claims (8)
1. cysticercus extracorporeal culturing method, it is characterised in that complete cysticercus is separated out of host animal body, in aseptic condition
It is lower to be cleaned up with the PBS that at least one antibiotic is wherein added, it is then transferred in the culture vessel in it added with culture solution, training
Feeding container is placed in carbon dioxide cell incubator, in 37 DEG C of temperature, CO2It is trained under the conditions of concentration 5%, relative humidity 70%
It supports, polypide starts the 6h after culture and 12h is respectively changed liquid 1 time, removes outmoded culture solution when changing liquid every time as far as possible, is added fresh
Complete culture solution 8ml/ bottle/time, later every 48h changes liquid 1 time.
2. a kind of cysticercus pisiformis in-vitro culture method, it is characterised in that by the complete beans of fresh separated from the abdominal cavity of infected rabbits
Shape cysticercus is aseptically cleaned up with the PBS that at least one antibiotic is wherein added, and is then transferred in it added with training
In the culture vessel of nutrient solution, culture vessel is placed in carbon dioxide cell incubator, in 37 DEG C of temperature, CO2It is concentration 5%, opposite
It is cultivated under the conditions of humidity 70%, polypide starts the 6h after culture and 12h is respectively changed liquid 1 time, removes as far as possible when changing liquid every time
Outmoded culture solution is removed, fresh complete culture solution 8ml/ bottle/time is added, later every 48h is changed liquid 1 time.
3. cysticercus pisiformis in-vitro culture method according to claim 2, it is characterised in that in PBS used added with
The streptomysin of the penicillin of 100U/ml and 100 μ g/ml, the culture solution are wherein to be added with fetal calf serum, penicillin, chain
Mycin, amphotericin B, L-Glutamine and sodium bicarbonate RPMI1640 culture solution, in which: fetal calf serum content be 10%
(v/v)；The final concentration of penicillin 100U/ml of antibiotic, 100 μ g/ml of streptomysin, 1.25 μ g/mL of amphotericin B；It is described
The final concentration of 2.5mM of L-Glutamine；It is 7.2~7.4 that sodium bicarbonate adjustment culture solution pH, which is added,.
4. cysticercus pisiformis in-vitro culture method according to claim 3, it is characterised in that the additional amount of initial incubation liquid
Are as follows: 6 orifice plates additional amount is the hole 3ml/ or cell bottle additional amount is 8ml/ (25cm2) bottle.
5. a kind of for collecting the cysticercus pisiformis in-vitro culture method of cysticercus pisiformis excretion body, it is characterised in that will be from infection
The complete cysticercus pisiformis of fresh separated is aseptically clear with the PBS that at least one antibiotic is wherein added in the abdominal cavity of rabbit
Then wash clean is transferred in it added with carrying out in the culture vessel for removing the culture solution of excretion body processing, culture vessel is placed in
In carbon dioxide cell incubator, in 37 DEG C of temperature, CO2It is cultivated under the conditions of concentration 5%, relative humidity 70%, polypide opens
6h and 12h after beginning culture are respectively changed liquid 1 time, remove outmoded culture solution when changing liquid every time as far as possible, fresh complete training is added
Nutrient solution 8ml/ bottle/time, later every 48h are changed liquid 1 time.
6. it is according to claim 5 for collecting the cysticercus pisiformis in-vitro culture method of cysticercus pisiformis excretion body,
Added with the penicillin of 100U/ml and the streptomysin of 100 μ g/ml in PBS used in being characterized in that, the culture solution is wherein to add
Added with penicillin, streptomysin, amphotericin B, L-Glutamine and sodium bicarbonate and the tire ox carried out over except the processing of excretion body
The RPMI1640 culture solution of serum, in which: fetal calf serum content is 10% (v/v)；The final concentration of penicillin of antibiotic
100U/ml, 100 μ g/ml of streptomysin, 1.25 μ g/mL of amphotericin B；The final concentration of 2.5mM of L-Glutamine；Carbon is added
Sour hydrogen sodium adjustment culture solution pH is 7.2~7.4.
7. cysticercus pisiformis in-vitro culture method according to claim 6, it is characterised in that the additional amount of initial incubation liquid
Are as follows: 6 orifice plates additional amount is the hole 3ml/ or cell bottle additional amount is 8ml/ (25cm2) bottle.
8. the method for collecting cysticercus pisiformis metabolic secretion antigen and excretion body, it is characterized in that by side described in claim 6 or 7
Method carries out cysticercus pisiformis culture, and after changing liquid for the second time, it is primary that every 48h collects polypide culture solution, it is concentrated or exceed the speed limit from
The methods of heart further obtains metabolic secretion antigen or excretion body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910172991.4A CN109792972B (en) | 2019-03-07 | 2019-03-07 | In-vitro culture method for cysticercus cellulosae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910172991.4A CN109792972B (en) | 2019-03-07 | 2019-03-07 | In-vitro culture method for cysticercus cellulosae |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109792972A true CN109792972A (en) | 2019-05-24 |
| CN109792972B CN109792972B (en) | 2021-04-27 |
Family
ID=66561704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910172991.4A Active CN109792972B (en) | 2019-03-07 | 2019-03-07 | In-vitro culture method for cysticercus cellulosae |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109792972B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795484A (en) * | 2021-02-08 | 2021-05-14 | 自然资源部第四海洋研究所(中国—东盟国家海洋科技联合研发中心) | Method for extracting and separating exosome of cryptocaryon irritans of marine parasitic ciliates and application of exosome |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107439493A (en) * | 2017-09-12 | 2017-12-08 | 新疆农垦科学院 | A kind of training mode of micro- mouthful of Hymenolepis |
-
2019
- 2019-03-07 CN CN201910172991.4A patent/CN109792972B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107439493A (en) * | 2017-09-12 | 2017-12-08 | 新疆农垦科学院 | A kind of training mode of micro- mouthful of Hymenolepis |
Non-Patent Citations (2)
| Title |
|---|
| 孙晓林: "兔豆状囊尾蚴和豆状带绦虫头节的组织结构观察", 《中国兽医科学》 * |
| 张念: "兔豆状囊尾蚴的体外培养研究", 《动物医院进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795484A (en) * | 2021-02-08 | 2021-05-14 | 自然资源部第四海洋研究所(中国—东盟国家海洋科技联合研发中心) | Method for extracting and separating exosome of cryptocaryon irritans of marine parasitic ciliates and application of exosome |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109792972B (en) | 2021-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Diamond | Lumen dwelling protozoa: Entamoeba, trichomonads, and Giardia | |
| Ordal et al. | Pathogenic myxobacteria | |
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN101491228B (en) | Sea no-nucleus pearl incubation method | |
| CN101491229B (en) | Vitro culturing method of pearl shell mantle tissue and culture medium | |
| CN106754668A (en) | A kind of stem cell medium and parenteral solution | |
| CN102732482A (en) | In vitro induction culture method for bone marrow-derived macrophages | |
| CN110283777A (en) | A kind of continuous cultural method of prawn cell | |
| CN102304492A (en) | Primary culture method for liver cells of crucian carp | |
| CN102492623A (en) | Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates | |
| CN108048327A (en) | A kind of new method for efficiently separating diatom | |
| CN108251378A (en) | A kind of interstital stem cell excretion body for being overexpressed PTGDS genes and its preparation method and application | |
| CN102382798A (en) | Separation method and culture in vitro method for mouse female germline stem cells | |
| CN109792972A (en) | A kind of cysticercus extracorporeal culturing method | |
| CN111990330B (en) | Method for constructing mouse staphylococcus aureus type mastitis model | |
| Gilbert | Monoxenic cultivation of the rotifer Brachionus calyciflorus in a defined medium | |
| CN109679912A (en) | A kind of isolated culture method of rat olfactory ensheathing cell | |
| CN101732709B (en) | Method for preparing haemorrhagic fever vaccine by culturing primary cells in multilayer culture flask | |
| CN101451122B (en) | Construction method of Epinephelus fuscoguttatus swim bladder cell line | |
| CN106635990A (en) | Primary culturing method for dorsal root ganglion satellite glial cells | |
| CN106906177A (en) | A kind of naked mole interstitial glands is isolated and purified and cultural method | |
| CN105420198A (en) | Application of chicken liver cancer cell system as duck plague virus host | |
| CN106635865A (en) | Culture medium for separation and purification of mycoplasma bovirhinis, preparation method and application of culture medium | |
| CN106434550A (en) | Improved PPCs cell culture method | |
| Meerovitch | A new host of Entamoeba invadens Rodhain, 1934 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |