CN102604835A - Method for establishing in-vitro culture model of cryptosporidium baileyi - Google Patents
Method for establishing in-vitro culture model of cryptosporidium baileyi Download PDFInfo
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- CN102604835A CN102604835A CN2012100156256A CN201210015625A CN102604835A CN 102604835 A CN102604835 A CN 102604835A CN 2012100156256 A CN2012100156256 A CN 2012100156256A CN 201210015625 A CN201210015625 A CN 201210015625A CN 102604835 A CN102604835 A CN 102604835A
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Abstract
The invention discloses a method for establishing an in-vitro culture model of cryptosporidium baileyi. The method comprises the following steps of: purifying cryptosporidium baileyi oocysts, and then carrying out excystation on the cryptosporidium baileyi oocysts at 37-40 DEG C; then innocualting mixture of 5*10<4>-5*10<5> C. baileyi oocysts and sporozoites or 5*10<5>-2*10<6> purified sporozoites into embryonated embryo trachea tissues, and culturing at 37-40 DEG C. According to the method provided by the invention, the growth of the chicken-original cryptosporidium baileyi is researched by using an embryonated embryo trachea tissue culture model; the history of the C. baileyi part is completed in the system; and a new in-vitro C. baileyi culture model is established. The model. can be used for carrying out polypide growth analysis, toxicity test, internal and external gene expression, drug screening and the like.
Description
Technical field
The present invention relates to the foundation of a kind of Cryptosporidium tracheal tissue culture model, also relate to simultaneously and utilize this model to carry out the preparation of Cryptosporidium vaccine antibody, toxicity test, polypide metabolism detection, drug screening, the genetic expression of inside and outside source etc.
Background technology
Cryptosporidium (
Cryptosporidium) be a kind of important water disease pathogen; And can cause immunosuppression patient delay property lethality diarrhoea and immune normal population acute diarrhea, have extensive host range, but more than 260 kind of animals such as infected person, domestic animal, poultry, companion animals and wildlife; And can pass through number of ways such as water source, food, air propagates; Thereby cause that children and immunosuppression patient seriously suffer from diarrhoea, and young animal digestive tract diseases and children's fowl upper disease in age, cause its production performance to descend or death; Identified 24 effective worm kinds of Cryptosporidium at present, wherein Cryptosporidium baileyi (
C.baileyi) be the advantage worm kind of infected poultry; Can infect more than 30 kind of bird; Cause serious respiratory symptom, its sickness rate and mortality ratio are higher, and the bird cryptosporidiosis worldwide has wide coverage; 2010, Amer etc. were separated to through morphology with based on SSU rDNA and HSP70 genetic analysis from Chinese kenaf duck excrement appearance first
C. baileyi, prove
C. baileyiThe wild important biomolecule source of pollution in the bird of migrating have been become.According to Surl etc.
C. baileyiStrong to the environment resistibility, drag is very strong, and the anti-drag of drag that is kept in 4 ℃ of 2.5% SRM 935a 18 months is anti-therefore, research
C. baileyiHaving important medical and veterinary science is worth.And it is right
C. baileyiCharacteristic research can be used as the model of other latent spore worms researchs.
Since nineteen eighty-three Woodmansee and Pohlenz reported first Cryptosporidium vitro culture completed successfully vegetative propagation, people began the research of Cryptosporidium vitro culture, and have obtained the achievement that attracts people's attention.Some effectively plants the not only whole developmental history of completion in vitro culture Cryptosporidium; And have at present whole etap that report utilizes two kinds of Cryptosporidiums of acellular culture model completion, for further biological characteristics and the anti-drugs of research polypide lay the foundation.
But up to the present, less about the research of bird Cryptosporidium vitro culture, only be located away from patient's
C. meleagridis(MDBK) carried out culture studies on bovine kidney cells.Complete
C. baileyiEctogenesis is only accomplished in chicken, turkey, duck, francolin, quail embryo.
The cultivation be used for various respiratory road cause of disease is cultivated by tracheal tissue, like the cultivation of IBV etc.According to Sawaguchi etc.: because the tracheae culture model is simple, fast, so although there is the chicken embryo to can be used for passing a vitro culture of virus, more effective with tracheal tissue's culture model when separating and confirm to pass virus than chicken embryo culture model.Owing in experimental infection chick respiratory tract, can see through electron microscopic observation
C. baileyiThe stage is educated in sexual and other interior hair tonics, and
C. baileyiAplasia in some birds and other mammiferous primary cells and some Mammals continuous cell lines, and in the chicken embryo, grow, but chicken embryo culture model is unfavorable for experimental observation, so available tracheal tissue culture model is attempted cultivating
C. baileyiThis respiratory pathogens.
The present invention will
C. baileyiEgg capsule and sporozoite mixture or sporozoite inoculated into chick embryo tracheal ring, through observing the tracheal ring fibre swing, hair tonic is educated in the polypide, observes
C. baileyiDevelopment condition in chicken embryo tracheal ring culture model is research from now on
C. baileyiVirulence, infectivity, metabolism, endogenous and exogenous gene expression, medicine screening etc. lay the first stone.
Summary of the invention
The object of the present invention is to provide a kind of establishment method of Cryptosporidium baileyi in-vitro culture model.
Technical scheme of the present invention is: a kind of establishment method of Cryptosporidium baileyi in-vitro culture model, earlier with behind the Cryptosporidium baileyi egg capsule purifying, excystation under 37 ~ 40 ℃ condition gets 5 * 10 then
4-5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture or 5 * 10
5~ 2 * 10
6The sporozoite inoculated into chick embryo tracheal tissue of individual purifying cultivates under 37 ~ 40 ℃ condition, observes polypide dynamically growth and the strong and weak variation of tracheae ciliary movement.
The excystation liquid of said excystation is at least a in DMEM substratum, 1640 substratum, trypsinase, the taurocholate.Inoculation can obtain through egg capsule and sporozoite mixture behind the aseptic double-deck 5 μ m polycarbonate membrane filtration excystations with Cryptosporidium purifying sporozoite.
The invention has the beneficial effects as follows: the present invention utilizes chicken embryo tracheal tissue culture model research chicken source Cryptosporidium baileyi to grow, and in this system, has accomplished
C. baileyiThe part developmental history has been set up a kind of new
C. baileyiIn-vitro culture model.Can utilize this model to carry out polypide and grow analysis, toxicity test, inside and outside genetic expression, drug screening etc.
Description of drawings
Fig. 1 is the latent spore infected chicken embryo tracheal ring stereoscan photograph of Bei Shi, A: infected group tracheal ring cilium (3000 *) not; B: infected group tracheal ring cilium (4000 *); C: band worm cavity (1000 *) D on the infected group tracheal ring: infected group tracheal ring schizont (7000 *).
Embodiment
A kind of establishment method of Cryptosporidium baileyi in-vitro culture model, earlier with behind the Cryptosporidium baileyi egg capsule purifying, excystation under 37 ~ 40 ℃ condition gets 5 * 10 then
4-5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture or 5 * 10
5-2 * 10
6The sporozoite inoculated into chick embryo tracheal tissue of individual purifying cultivates under 37 ~ 40 ℃ condition, observes polypide dynamically growth and the strong and weak variation of tracheae ciliary movement.
The excystation liquid of said excystation is at least a in DMEM substratum, 1640 substratum, trypsinase, the taurocholate.Inoculation can obtain through egg capsule and sporozoite mixture behind the aseptic double-deck 5 μ m polycarbonate membrane filtration excystations with Cryptosporidium purifying sporozoite.
Embodiment 1
To cultivate behind 24 h ciliary movement activity more than 80% and the inner pure tracheal ring of tracheal ring inoculate about 5 * 10 respectively
4, 5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture (quantity that refers to complete egg capsule before the excystation), 5 * 10
5, 1 * 10
6, 2 * 10
6The sporozoite of individual purifying, 10 tracheal ringes of every kind of inoculation, other gets 80 ℃ of deactivation 10 min egg capsules 5 * 10 of 10 tracheal ring inoculations
4Individually contrast as cleaning process; At above-mentioned polypide inoculation back 24 h; Remove nutrient solution in each culture hole with pipettor; Draw PBS then with moving in addition in 1 new 24 well culture plates (every hole add 1 mL contain the two anti-DMEM of 200 IU/mL keep liquid) after the tracheal ring flushing 3 times, place 37 ℃ of 5%CO then
2Incubator is cultivated, and situation appears in polypide (newborn egg capsule, sporozoite, schizont, merozoite etc.) in every interval 24 h observation nutrient solution.
Embodiment 2
To cultivate behind 24 h ciliary movement activity more than 80% and the inner pure tracheal ring of tracheal ring inoculate about 5 * 10 respectively
4, 5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture (quantity that refers to complete egg capsule before the excystation), 5 * 10
5, 1 * 10
6, 2 * 10
6The sporozoite of individual purifying, 10 tracheal ringes of every kind of inoculation, other gets 80 ℃ of deactivation 10 min egg capsules 5 * 10 of 10 tracheal ring inoculations
4Individually contrast as cleaning process; At above-mentioned polypide inoculation back 24 h; Remove nutrient solution in each culture hole with pipettor; Draw PBS then with moving in addition in 1 new 24 well culture plates (every hole add 1 mL contain the two anti-DMEM of 200 IU/mL keep liquid) after the tracheal ring flushing 3 times, place 38 ℃ of 5%CO then
2Incubator is cultivated, and situation appears in polypide (newborn egg capsule, sporozoite, schizont, merozoite etc.) in every interval 24 h observation nutrient solution.
Embodiment 3
To cultivate behind 24 h ciliary movement activity more than 80% and the inner pure tracheal ring of tracheal ring inoculate about 5 * 10 respectively
4, 5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture (quantity that refers to complete egg capsule before the excystation), 5 * 10
5, 1 * 10
6, 2 * 10
6The sporozoite of individual purifying, 10 tracheal ringes of every kind of inoculation, other gets 80 ℃ of deactivation 10 min egg capsules 5 * 10 of 10 tracheal ring inoculations
4Individually contrast as cleaning process; At above-mentioned polypide inoculation back 24 h; Remove nutrient solution in each culture hole with pipettor; Draw PBS then with moving in addition in 1 new 24 well culture plates (every hole add 1 mL contain the two anti-DMEM of 200 IU/mL keep liquid) after the tracheal ring flushing 3 times, place 39 ℃ of 5%CO then
2Incubator is cultivated, and situation appears in polypide (newborn egg capsule, sporozoite, schizont, merozoite etc.) in every interval 24 h observation nutrient solution.
Embodiment 4
To cultivate behind 24 h ciliary movement activity more than 80% and the inner pure tracheal ring of tracheal ring inoculate about 5 * 10 respectively
4, 5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture (quantity that refers to complete egg capsule before the excystation), 5 * 10
5, 1 * 10
6, 2 * 10
6The sporozoite of individual purifying, 10 tracheal ringes of every kind of inoculation, other gets 80 ℃ of deactivation 10 min egg capsules 5 * 10 of 10 tracheal ring inoculations
4Individually contrast as cleaning process; At above-mentioned polypide inoculation back 24 h; Remove nutrient solution in each culture hole with pipettor; Draw PBS then with moving in addition in 1 new 24 well culture plates (every hole add 1 mL contain the two anti-DMEM of 200 IU/mL keep liquid) after the tracheal ring flushing 3 times, place 40 ℃ of 5%CO then
2Incubator is cultivated, and situation appears in polypide (newborn egg capsule, sporozoite, schizont, merozoite etc.) in every interval 24 h observation nutrient solution.
Concrete operation method:
1, Cryptosporidium baileyi egg capsule excystation method
Described egg capsule excystation method be 37 ℃ ~ 40 ℃ following DMEM or 1640 substratum and 0.25% ~ 0.5% trypsinase with (or) 0.75% ~ 2.25% taurocholate.
This excystation liquid is formed can be the combination of DMEM or 1640 substratum, trypsinase, taurocholate any or two kinds, three kinds.
Preferred 40 ℃ of excystation 90 min～120 min in DMEM or 1640 substratum wherein.
2, sporozoite purification process
Tissue culture adds DMEM with Cryptosporidium purifying sporozoite through above-mentioned excystation method or 1640 substratum pass through aseptic double-deck 5 μ m polycarbonate membranes again behind excystation 90 min～120 min in 39 ~ 40 ℃ of water-baths, waits to count back inoculated into chick embryo tracheal ring.
3, tracheal ring preparation
According to the bibliographical information method, aseptic separation instar chicken embryo tracheae on the 19th uses scalpel at the thick tracheal ring of aseptic plate inscribe written treaty 0.5～1 mm then.Tracheal ring is placed respectively in the 24 orifice plate culture plates, and 1 tracheal ring in every hole adds 1 mL and contains 200 IU/mL two anti-DMEM or 1640 growth medias, 37 ℃ of 0.5% CO
2After cultivating liquid, under 100 times of inverted microscopes, observe.Ciliary movement vivaciously accounts for 80% above person and supplies to inoculate the Cryptosporidium body and function in the tracheal ring.
4, polypide inoculation and cultivation
To cultivate behind 24 h ciliary movement activity more than 80% and the inner pure tracheal ring of tracheal ring inoculate some amount respectively
C. baileyiEgg capsule with (or) sporozoite; At above-mentioned polypide inoculation back 24 h; Remove nutrient solution in each culture hole with pipettor; Draw PBS then with moving in addition in 1 new 24 well culture plates (every hole add 1 mL contain the two anti-DMEM of 200 IU/mL keep liquid) after the tracheal ring flushing 3 times, place 37～40 ℃ of 5%CO then
2Incubator is cultivated, and situation appears in polypide (newborn egg capsule, sporozoite, schizont, merozoite etc.) in every interval 24 h observation nutrient solution.
5, tracheal ring cilium vigor appraisement system and observation index
The tracheal ring of inoculation behind the egg capsule, strong and weak and cilium comes off degree as tracheal ring cilium vigor appraisement system with fibre swing, promptly with the ciliary movement of chicken embryo tracheal ring as indication mechanism.Ciliation swing part percentage (0～100%) in each tracheal ring of record estimation before and after inoculation; (0 for there not being swing by 0～3 differential the score for the cilium vigor; 3 is active for swinging), every interval 24 h observe the relative energy value that infects back different time tracheal ring ciliated epithelial cell.The relative energy value of tracheal ring ciliated epithelial cell calculates by following formula.Per-cent ratio * cilium vigor score value * 100 of tracheal ring ciliation moving part before and after the relative vigor of tracheal ring ciliated epithelial cell=inoculation polypide.The relative energy value of tracheal ring ciliated epithelial cell is observed and recorded under 100 * inverted microscope all, the ciliary movement situation of promptly under inverted microscope, regularly observing the tracheal ring mucomembranous epithelial cell every day.During observation; Earlier wave and culture bottle gently breaks away from the tracheal ring that is attached on bottle wall, to remove free cell in the tube chamber etc.; And then let the smooth bottle of tracheal ring wall observe; The time that record tracheal ring mucous epithelium exists shared per-cent of ciliary movement scope and ciliary movement to stop fully, every group of relative energy value mean number of the ciliated epithelial cell with 4 rings is end-result, and with statistics software test-results is carried out the significant difference analysis.
6, histological examination behind the chicken embryo tracheal ring inoculation egg capsule
Different time takes out 4 tracheal ringes respectively for every group after inoculation, wherein makes paraffin section after 2 processing, makes scanning electron microscopic observation after 2 processing.Simultaneously each point of observation get 2 do not inoculate the tracheal ring of egg capsule, 2 vaccinal fever deactivation egg capsule tracheal ringes are respectively applied for light microscopic and electron microscopic sample contrast.
7, indirect immunofluorescence inspection
To be kept at-80 ℃ and prepare the paraffin organization section behind fixing 12 h according to the method described above with 10% neutral formalin solution through a fairly large number of time point tracheal ring of polypide on paraffin section and the scanning electron microscopic observation tracheal ring, dewaxing back slicing treatment is laggard to connect the immunofluorescence inspection in the ranks.
8, PCR-RFLP analyzes
Take out and be kept at-80 ℃ of a fairly large number of time point tracheal ringes of polypide on paraffin section and scanning electron microscopic observation tracheal ring, do not inoculate 3 of egg capsule and vaccinal fever deactivation egg capsule tracheal ringes respectively; Put into 1.5 mL centrifuge tubes respectively; Homogenate after jolting is washed 5 times about adding pH 7.2 PBS is carried out RFLP according to the report method then and is identified polypide on the tracheal ring.
More than experiment showed, use Cryptosporidium of the present invention tracheal tissue in-vitro culture model, can accomplish the vitro culture of Cryptosporidium baileyi.
Culture model cultivation results of the present invention:
1, polypide developmental history in the chicken embryo tracheal ring culture model
Newborn polypide is monitored with 400 * inverted microscope respectively organizing in the tracheal ring culture hole of Cryptosporidium baileyi infection; The result finds: under 37 ~ 38 ℃ of culture condition; Egg capsule excystation rate has only about 10%; Behind the chicken embryo tracheal ring inoculation polypide, parasitic to finding that not polypide is arranged through electron microscopic observation; But under 39 ~ 40 ℃ of culture condition, the 4th d behind the inoculation polypide has found that in inoculation mean size is sporozoite or the fragmentation increment of 6.5 * 0.9 μ m polypide that swings in the 24 orifice plate culture hole.The 6th d behind the inoculation polypide can be observed the schizont appearance polypide that mean size is about 3.5 * 3.5 μ m, 39 ℃ with 40 ℃ of culture condition under polypide parasitism quantity no difference of science of statistics.
In addition, inoculation 5 * 10
4, 5 * 10
5Individual
C. baileyiThe quantity of the newborn polypide of tracheal ring tissue of egg capsule and sporozoite mixture is obviously more than inoculation 5 * 10
5, 1 * 10
6, 2 * 10
6The newborn polypide quantity of the tracheal ring tissue of purifying sporozoite, and inoculate 5 * 10
4, 5 * 10
5Individual
C. baileyiTwo newborn polypide quantity no difference of science of statistics of group of egg capsule and sporozoite mixture.
2, behind the polypide infected chicken embryo tracheal ring to the active influence of fibre swing
6～9 d behind the Cryptosporidium baileyi of chicken embryo tracheal ring infected chicken source, each infected group tracheal ring cilium comes off obviously many than the blank group, and the fibre swing activity also weakens than blank group.Statistical result showed: each relative vigor of infected group tracheal ring ciliated epithelial cell and blank group comparing difference tool statistical significance under 39 ~ 40 ℃ of culture condition (
P＜0.05).
3, histology and indirect immunofluorescence inspection behind the chicken embryo tracheal ring infection Cryptosporidium baileyi
Through to the om observation of tracheal ring behind the inoculation polypide, the cytology of tracheal ring changes to appear at the earliest with the polypide parasitism inoculates 72 h behind the polypide.Polypide colonizes in tracheal ring ciliated epithelial cell edge, and the polypide parasitic site causes the cilium lodging, arrangement disorder, even come off, some place causes slight nuclear swelling and chromatin displacement.And find: polypide quantity is more than simple inoculation sporozoite group on inoculation egg capsule and the sporozoite mixture group tracheal ring.With on the blank group of quadrat method inspection and the vaccinal fever deactivation egg capsule group tracheal ring discovery doubtful polypide is arranged.
The indirect immunofluorescence that utilizes anti-chicken source Cryptosporidium baileyi IgY antibody to set up shows: 72 h behind the inoculation polypide; Can see the green fluorescence signal of polypide form on the stronger similar paraffin section at tracheal ring ciliated epithelial cell edge, this is illustrated in the tracheal ring polypide of seeing on the paraffin section and really is chicken source Cryptosporidium baileyi.With on the blank group of quadrat method inspection and the vaccinal fever deactivation egg capsule group tracheal ring discovery the hyperfluorescence signal is arranged.
4, electron microscopic examination behind the chicken embryo tracheal ring infection Cryptosporidium baileyi
Observe as shown in Figure 1 under the Electronic Speculum: from infecting back 16 h; The new life who all has big small number not wait on the infected group tracheal ring mucomembranous epithelial cell free surface under 39 ~ 40 ℃ of culture condition is with the worm cavity; Size is about (1-4) * (1-4) μ m; Infect back 96 h and can see that a small amount of diameter is about the II type schizont of 3.5 * 3.5 μ m (containing 4 merozoites); These polypides parasitize in the tracheal ring cilium clump surface epithelial cell and cause that cilium comes off in a large number or lodges, and infected group tracheal ring cilium obviously lacks than infected group not.And polypide is obviously many than other infected group on the tracheal ring behind infection egg capsule and the sporozoite mixture.
Can know by Fig. 1: cilium marshalling (A) and infected group tracheal epithelial cell cilium comes off in a large number or lodge (B, C, D) on the infected group tracheal ring not.Infect 5 * 10
5On tracheal ring, occur a small amount of polypide behind individual sporozoite group 16 h, and can see cilium lodging or disorderly (B).And infect egg capsule and sporozoite mixture group a large amount of polypides (C, D) occur at 78 h after the infection on tracheal ring, and can see a small amount of schizont (among the figure shown in the arrow).
5, PCR-RFLP detected after chicken embryo tracheal ring infected Cryptosporidium baileyi
Each infection activity polypide group tracheal ring all successfully amplifies Cryptosporidium SSU rRNA gene through Nested PCR, and clip size is about 830～840 bp.Confirm all to amplify the Cryptosporidium baileyi fragment on the infected group tracheal ring according to endonuclease bamhi, and vaccinal fever deactivation egg capsule group does not amplify the polypide fragment.
Claims (2)
1. the establishment method of a Cryptosporidium baileyi in-vitro culture model is characterized in that: earlier with behind the Cryptosporidium baileyi egg capsule purifying, excystation under 37 ~ 40 ℃ condition gets 5 * 10 then
4-5 * 10
5Individual
C. baileyiEgg capsule and sporozoite mixture or 5 * 10
5-2 * 10
6The sporozoite inoculated into chick embryo tracheal tissue of individual purifying cultivates under 37 ~ 40 ℃ condition.
2. according to the establishment method of the said Cryptosporidium baileyi in-vitro culture model of neglecting of claim 1, it is characterized in that: the excystation liquid of said excystation is at least a in DMEM substratum, 1640 substratum, trypsinase, the taurocholate.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113552335A (en) * | 2020-05-25 | 2021-10-26 | 华南农业大学 | Preparation method of immune electron microscope sample for positioning specific expression proteins of cryptosporidium in different intracellular parasitic periods |
-
2012
- 2012-01-18 CN CN2012100156256A patent/CN102604835A/en active Pending
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113552335A (en) * | 2020-05-25 | 2021-10-26 | 华南农业大学 | Preparation method of immune electron microscope sample for positioning specific expression proteins of cryptosporidium in different intracellular parasitic periods |
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Application publication date: 20120725 |