CN103301162A - Method for establishing fish inflammatory bowel disease model and established model thereof - Google Patents
Method for establishing fish inflammatory bowel disease model and established model thereof Download PDFInfo
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- CN103301162A CN103301162A CN201310134518XA CN201310134518A CN103301162A CN 103301162 A CN103301162 A CN 103301162A CN 201310134518X A CN201310134518X A CN 201310134518XA CN 201310134518 A CN201310134518 A CN 201310134518A CN 103301162 A CN103301162 A CN 103301162A
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Abstract
The invention relates to the technical field of establishment of animal experiment models and in particular relates to a method for establishing a fish inflammatory bowel disease model and the established model thereof. The method comprises the following steps: injecting aeromonas hydrophila with the collection number of CCTCC NO.M2013089 into a soaked anaesthetic healthy fish through anus perfusion, breeding and selecting fish with the symptom of the inflammatory bowel disease as the fish inflammatory bowel disease model. According to the method, the aeromonas hydrophila is directly filled in the intestinal tract of the fish, and the process that the pathogenic bacteria enter blood through intramuscular injection or intraperitoneal injection and act on the intestinal tract through blood circulation is avoided, so that the pathogenic bacteria directly act on the intestinal tract, and the pertinence is improved; furthermore, the established model has high stability and repeatability, and because the aeromonas hydrophila is wide in infection spectrum, the established method can be widely applied to multiple fishes.
Description
Technical field
The present invention relates to animal experimental model constructing technology field, relate in particular to the model of a kind of Fish inflammatory bowel method for establishing model and foundation thereof.
Background technology
The animal protein nutrition that Fish are a large amount of for China consumer has carried, but the occurrence frequency of inflammatory bowel is high in the breeding process, usually diseases such as fish bacterial enteritis and erythroderma, gill rot, intestinal hexamitiasis, balantidiasis, viral hemorrhagic disease, liver and gallbladder syndrome are concurrent, have a strong impact on its survival rate and breeding production performance, hinder the sustainable development of fish culture industry.The mechanism that fish enteritis takes place is still indeterminate at present, so morbidity main cause, disease symptom, the pathomechanism of researching fish inflammatory bowel (IBD) have important significance for theories and huge Practical significance; In the hope of can not taking the effectively preventing measure to safeguard the health of Fish intestinal before the infection inflammation enteropathy Fish at the cause of disease, use medicine less, the maximum growth potential of performance Fish is produced safe fishery-ies product.In recent years, medical circle is very active to the IBD cause of disease and Study on Pathogenesis, and be made as research this sick pathogeny and the drug screening of animal enteritis model provide carrier.
At present, people induce by chemicalses such as acetic acid, trinitro-benzene-sulfonic acid (TNBS), oxazolone, carrageenin, sulfated polysaccharides and set up the IBD model, also induce with gene knockout and transgenic technology and set up the inflammatory bowel disease model.As for the development of aquatic animal Fish inflammatory bowel disease model, have the report of inducing black blue patch Fugu ocellatus enteritis with inflammatory factor IL-16, but the inducer specificity of this model is strong, model versatility on other Fish is not strong.Simultaneously, compare with the medical science inflammatory bowel, the living environment complexity of aquatic animal is subjected to the influence of the factors such as physical and chemical factors such as water body dissolved oxygen, ammonia nitrogen, pathogenic microorganism greater than terrestrial animal, therefore relatively difficult to aquatic animal inflammatory bowel Study of model, now be the starting stage.
As far back as 1958, Wang Deming etc. are separated to a kind of visible peristalsis visible intestinal peristalsis point-like aerogenesis Zymomonas mobilis (Aeromonas punctata f.intestinalis) (Wang Deming from suffer from the sick Ctenopharyngodon idellus of enteritis, 1959), with Aeromonas hydrophila (Aeromonas hydrophila) synonym (Xu Baihai, 1987).This bacterium is a Chang Juzhe in normal fish body intestinal, exists in a large number in natural water body and bed mud, and is a lot of with the touch opportunity of fish.When fish under good water environment condition, do not fall ill though infect.Under the mal-condition of complexity, Aeromonas hydrophila can cause epiphytotics breaking out (as concurrent with the viral hemorrhagic disease), this is because virus impels host's body to change, cause fish body resistance to descend, defense function reduces, thereby antibacterial is invaded in blood and the intestinal easily, and a large amount of breeding causes enteritis or systemic bleeding inflammation.But the mechanism that fish enteritis takes place is still indeterminate.Later discovers, the Aeromonas hydrophila pattern of infection is extensive, and nearly all aquatic animal can both be infected.Xu uncle's last of the twelve Earthly Branches etc. discovers that this bacterium infects by the lumbar injection to healthy grass carp, obtains positive findings, and soaks or the oral healthy grass carp that all can not make infects.From 1964, uncle's Xu last of the twelve Earthly Branches etc. continued and has studied 10 years and but is not separated to mouthful hello a successful malignant bacteria (Xu Baihai, 1987) all the time.
Aeromonas hydrophila can't catch an illness healthy Fish by oral or immersion, and must adopt lumbar injection or intramuscular injection, but, lumbar injection or intramuscular injection meeting cause mechanical damage to the fish body, and adopt the mode of lumbar injection or muscle can make pathogenic bacterium or enter blood, follow blood circulation to cause the morbidity of fish body whole body, the inflammatory bowel disease model that constructs is not pointed, and follow-up study has been caused adverse effect.Have not yet to see and to cause scorching method by the damage of on-mechanicals such as oral, immersion or anus perfusion.
Summary of the invention
Pathogenic factor, pathomechanism, preventing control method for the researching fish inflammatory bowel, the invention provides a kind of Fish inflammatory bowel disease model of simple, good reproducibility, and make its feature that shows as similar Fish inflammatory bowel, for the inflammatory bowel cause of disease, the pathomechanism of Fish, the formulation that prevents and treats method provide the research carrier.
For achieving the above object, the invention provides following technical scheme:
The invention provides a kind of Fish inflammatory bowel method for establishing model, may further comprise the steps:
Pouring into deposit number by anus is that culturing also, option table reveals the fish of inflammatory bowel disease symptoms as Fish inflammatory bowel disease model in the healthy fish body of Aeromonas hydrophila injection through soaking anesthesia of CCTCC NO.M2013089.
The Aeromonas hydrophila that the present invention adopts, strain name is Aeromonas hydrophila SDS-1, hepatopancrease, kidney, intestinal separation acquisition from the Ctenopharyngodon idellus bacterial enteritis belong to Aeromonas Aeromonas hydrophila (Aeromonas hydrophila).
Aeromonas hydrophila SDS-1 bacterial strain of the present invention carries out preservation on March 18th, 2013 at China typical culture collection center (CCTCC), the preservation centre address be Wuhan City, Hubei Province Wuhan University in the school, deposit number is CCTCC NO.M2013089.
As preferably, deposit number is that the concentration of the Aeromonas hydrophila of CCTCC NO.M2013089 is 10
5CFU/mL～10
8CFU/mL.
As preferably, the body weight that is used for making up the fish of Fish inflammatory bowel disease model is 20g～100g.
As preferably, the groundwater increment of anus perfusion is 0.25mL/ tail～0.50mL/ tail.
As preferably, be Ctenopharyngodon idellus or Carassius auratus for the fish that makes up Fish inflammatory bowel disease model
As preferably, in the Fish inflammatory bowel method for establishing model provided by the invention, culture and be specially in the breeding process, 1% feedstuff of the fish body weight of throwing something and feeding, the time of throwing something and feeding is 8:00～9:00 in the morning; Temperature of cultivation is 25 ℃～28 ℃, DO 〉=7.0mg/L, and the batch (-type) oxygenation, the pH value of water is 7.5 ± 0.5, wherein: NH
4 +-N≤0.2mg/L, NO
2-N≤0.05mg/L, H
2S≤0.05mg/L.Change the water frequency: 2d/ time, the water of 1/3 volume of at every turn more exchanging treaties.
As preferably, the inflammatory bowel disease symptoms is: anus and Di redness, intestinal mucosa fester, congested, downright bad, inflammatory cell infiltration, and the enterocyte microvillus comes off.
As preferably, in the Fish inflammatory bowel method for establishing model provided by the invention, the anesthetis that anesthesia is adopted is MS-222.
Preferably, in the Fish inflammatory bowel method for establishing model provided by the invention, narcotic quality-volumetric concentration that anesthesia is adopted is 40mg/L～70mg/L.
A kind of Fish inflammatory bowel disease model that adopts method provided by the invention to set up.
Fish inflammatory bowel disease model provided by the invention shows as acute inflammation in the 3d after foundation, transfer chronic inflammatory disease behind the 7d to.
Pro-inflammatory cytokine mRNA expression raises earlier and afterwards descends in the Fish inflammatory bowel model body provided by the invention.
Activity of myeloperoxidase is significantly higher than healthy fish in the Fish inflammatory bowel model body provided by the invention.
As preferably, spectrophotometer is adopted in the detection of activity of myeloperoxidase.
Because the employing of technique scheme, compared with prior art, the present invention has following advantage:
1. a kind of Fish inflammatory bowel method for establishing model provided by the invention and model thereof, adopting the anus perfusion is in the healthy fish body of Aeromonas hydrophila injection through soaking anesthesia of CCTCC NO.M2013089 with deposit number, avoided the mechanical damage to the fish body, Aeromonas hydrophila directly is fed in the intestinal of fish, Aeromonas hydrophila directly contacts intestinal and infects, avoided intramuscular injection or lumbar injection to make pathogenic bacterium enter blood, make it act on the process of intestinal by blood circulation again, have more specific aim.
2. the survival rate that adopts method provided by the invention to set up the preparation of Fish inflammatory bowel disease model can reach 97%～100%, dissect back observation intestinal 100% and have the enteritis symptom, the 1d～3d shows as acute inflammation, is converted into chronic inflammatory disease behind the 7d, repeatability and stability are all fine, modeling in batches.
3. because the pattern of infection of Aeromonas hydrophila is very extensive, therefore, the method that the present invention sets up can be widely used in multiple Fish.
4. the present invention is for studying the sick pathomechanism of aquatic animal enteritis and researching and developing effective medicine and lay a good foundation.
Biological preservation explanation
Bacterial strain Aeromonas hydrophila SDS-1: classification name: Aeromonas hydrophila SDS-1(Aeromonas hydrophila SDS-1), be deposited in Chinese typical culture collection center (CCTCC) on March 18th, 2013, the preservation centre address be Wuhan City, Hubei Province Wuhan University in the school, deposit number is CCTCC NO.M2013089.
Description of drawings
Fig. 1 show the inverse the 1st, 2 of normal Ctenopharyngodon idellus circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 1 (a) show the inverse the 1st, 2 of normal Ctenopharyngodon idellus circumnutate between middle part intestinal structure,
Show inflammatory cell nuclear, the inflammatory cell rareness; Fig. 1 (b) show the inverse the 1st, 2 of normal Ctenopharyngodon idellus circumnutate between intestinal mucosa surface, middle part microvillus structure, microvillus is arranged closely;
Fig. 2 show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 1d circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 2 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 1d circumnutate between middle part intestinal structure,
Show the surge of inflammatory cell quantity,
Show that microvillus comes off; Fig. 2 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 1d circumnutate between intestinal mucosa surface, middle part microvillus structure,
Show that mucous layer surface, antibacterial intrusion back microvillus comes off, and forms the wound depression;
Fig. 3 show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 3d circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 3 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 3d circumnutate between middle part intestinal structure,
Show that inflammatory cell quantity is slightly less than 1d,
Show the mucous layer cellular edema, goblet cell increases; Fig. 3 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 3d circumnutate between intestinal mucosa surface, middle part microvillus structure,
Show that mucous layer surface, antibacterial intrusion back minority wound depression shoals, wound begins convergence;
Fig. 4 show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 7d circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 4 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 7d circumnutate between middle part intestinal structure
Show that inflammatory cell quantity is still a lot,
Show the mucous layer cellular edema, goblet cell is more; Fig. 4 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 7d circumnutate between intestinal mucosa surface, middle part microvillus structure,
Show that antibacterial intrusion back mucous layer superficial cut depression is more shallow, microvillus begins repairing growth;
Fig. 5 show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 14d circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 5 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 14d circumnutate between middle part intestinal structure,
Show that inflammatory cell quantity is less than 7d,
Show that the mucous layer cellular edema alleviates, goblet cell begins to reduce; Fig. 5 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 14d circumnutate between intestinal mucosa surface, middle part microvillus structure,
Show that antibacterial intrusion back mucous layer superficial cut further recovers;
Fig. 6 show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 21d circumnutate between middle part intestinal structure and mucomembranous surface microvillus structure thereof; Wherein, Fig. 6 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 21d circumnutate between middle part intestinal structure,
Show that inflammatory cell quantity obviously is less than the intestinal of bacterial infection 3-7d,
Show that the mucous layer cellular edema disappears substantially, the goblet cell number recovers to approach normal; Fig. 6 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 21d circumnutate between intestinal mucosa surface, middle part microvillus structure,
Show that antibacterial intrusion back mucous layer superficial cut recovers substantially, only exists slight wound;
Fig. 7 shows the testing result to the intestinal activity of myeloperoxidase variation tendency of the embodiment of the invention 1 each processed group fish, wherein, curve 1 shows behind the perfusion Aeromonas hydrophila positive controls fish intestinal MPO activity change situation in the embodiment of the invention 1, curve 2 shows behind the perfusion Aeromonas hydrophila experimental group fish intestinal MPO activity change situation in the embodiment of the invention 1, curve 3 shows behind the perfusion Aeromonas hydrophila negative control group fish intestinal MPO activity change situation in the embodiment of the invention 1, and curve 4 shows the embodiment of the invention 1 empty matched group fish intestinal MPO activity change situation behind the perfusion Aeromonas hydrophila;
Fig. 8 shows the testing result to the expression of pro-inflammatory cytokine in the embodiment of the invention 1 each processed group, wherein, Fig. 8 (a) shows that the expression of fish intestinal pro-inflammatory cytokine IL-1 β in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes, wherein, post 1 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 2 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 3 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 4 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 5 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 6 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the negative control group fish intestinal, post 7 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 8 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 9 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 10 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 11 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 12 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the experimental group fish intestinal, post 13 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, post 14 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, post 15 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, post 16 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, post 17 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, post 18 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-1 β in the positive controls fish intestinal, * show and negative control group significant difference (p＜0.05) that * * shows and negative control group difference extremely significantly (p＜0.01); Fig. 8 (b) shows that the expression of fish intestinal pro-inflammatory cytokine IL-8 in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes, wherein, post 1 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 2 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 3 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 4 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 5 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 6 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the negative control group fish intestinal, post 7 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 8 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 9 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 10 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 11 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 12 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the experimental group fish intestinal, post 13 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, post 14 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, post 15 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, post 16 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, post 17 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, post 18 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine IL-8 in the positive controls fish intestinal, * show and negative control group significant difference (p＜0.05) that * * shows and negative control group difference extremely significantly (p＜0.01); Fig. 8 (c) shows that the expression of fish intestinal pro-inflammatory cytokine TNF-α in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes, wherein, post 1 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 2 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 3 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 4 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 5 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 6 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the negative control group fish intestinal, post 7 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 8 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 9 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 10 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 11 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 12 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the experimental group fish intestinal, post 13 shows when handling back 0d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, post 14 shows when handling back 1d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, post 15 shows when handling back 3d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, post 16 shows when handling back 7d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, post 17 shows when handling back 14d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, post 18 shows when handling back 21d among the embodiment 1 expression of pro-inflammatory cytokine TNF-α in the positive controls fish intestinal, * show and negative control group significant difference (p＜0.05) that * * shows and negative control group difference extremely significantly (p＜0.01).
The specific embodiment
The invention provides the model of a kind of Fish inflammatory bowel method for establishing model and foundation thereof, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The invention provides a kind of Fish inflammatory bowel method for establishing model, may further comprise the steps:
Pouring into deposit number by anus is that culturing also, option table reveals the fish of inflammatory bowel disease symptoms as Fish inflammatory bowel disease model in the healthy fish body of Aeromonas hydrophila injection through soaking anesthesia of CCTCC NO.M2013089.
Deposit number is that the concentration of the Aeromonas hydrophila of CCTCC NO.M2013089 is 10
5CFU/mL～10
8CFU/mL.
Body weight for the fish of setting up Fish inflammatory bowel disease model is 20g～100g.
In an embodiment of the present invention, the groundwater increment groundwater increment of anus perfusion is 0.25mL/ tail～0.50mL/ tail.
The fish that is used for setting up Fish inflammatory bowel disease model is Ctenopharyngodon idellus or Carassius auratus.
In the Fish inflammatory bowel method for establishing model provided by the invention, culture and to be specially in the breeding process, 1% feedstuff of the fish body weight of throwing something and feeding, the time of throwing something and feeding is 8:00～9:00 in the morning; Temperature of cultivation is 25 ℃～28 ℃, DO 〉=7.0mg/L, and the batch (-type) oxygenation, the pH value of water is 7.5 ± 0.5, wherein: NH
4 +-N≤0.2mg/L, NO
2-N≤0.05mg/L, H
2S≤0.05mg/L.Change the water frequency: 2d/ time, the water of 1/3 volume of at every turn more exchanging treaties.
The inflammatory bowel disease symptoms is: anus and Di redness, intestinal mucosa are festered, hyperemia, downright bad, inflammatory cell infiltration, and the enterocyte microvillus comes off.
In the Fish inflammatory bowel method for establishing model provided by the invention, the anesthetis that anesthesia is adopted is MS-222.
For guaranteeing anaesthetic effect, in the Fish inflammatory bowel method for establishing model provided by the invention, narcotic quality-volumetric concentration that anesthesia is adopted is 40mg/L～70mg/L.
A kind of Fish inflammatory bowel disease model that adopts method provided by the invention to set up.
Fish inflammatory bowel disease model provided by the invention shows as acute inflammation in the 3d after foundation, transfer chronic inflammatory disease behind the 7d to.
Pro-inflammatory cytokine mRNA expression raises earlier and afterwards descends in the Fish inflammatory bowel model body provided by the invention.
Activity of myeloperoxidase is significantly higher than healthy fish in the Fish inflammatory bowel model body provided by the invention.
Spectrophotometer is adopted in detection to activity of myeloperoxidase.
Pouring into deposit number by anus is that culturing also, option table reveals the fish of inflammatory bowel disease symptoms as Fish inflammatory bowel disease model in the healthy fish body of Aeromonas hydrophila injection through soaking anesthesia of CCTCC NO.M2013089.Method provided by the invention has been avoided the mechanical damage to the fish body, Aeromonas hydrophila directly is fed in the intestinal of fish, Aeromonas hydrophila directly contacts intestinal and infects, avoided intramuscular injection or lumbar injection to make pathogenic bacterium enter blood, make it act on the process of intestinal by blood circulation again, have more specific aim.And the model stability that makes up and good repeatability.And, because the pattern of infection of Aeromonas hydrophila is very extensive, all Fish are infected, therefore, the method that the present invention sets up can be widely used in multiple Fish.Experiment shows: the survival rate that adopts method provided by the invention to set up the preparation of Fish inflammatory bowel disease model can reach 97%～100%, dissect back observation intestinal 100% and have the inflammatory bowel disease symptoms, the 1d～3d shows as acute inflammation, is converted into chronic inflammatory disease behind the 7d, and repeatability and stability are all fine.
Examination material of the present invention is all common commercially available product, all can buy in market.Wherein test fish available from Wujiang City, Jiangsu Province Song Ling town Zhu Maogen Aquatic product cooperative society.
Below in conjunction with the drawings and specific embodiments the present invention is further described:
Getting 240 tail specifications is the Ctenopharyngodon idellus of 20g～50g, sets up the inflammatory bowel disease model behind laboratory rearing 20d.To test fish and be divided into 4 groups, be respectively the experimental group of perfusion Aeromonas hydrophila, the normal saline negative control group of perfusion 0.7%, positive controls, the blank group of perfusion TNBS, every group of 60 tails.
The compound method of TNBS solution is: be that 5%TNBS mixes with the dehydrated alcohol equal-volume with volume fraction, namely.
Preparation quality-volumetric concentration is the MS-222 solution of 50mg/L, soaks the anaesthesia experiment fish.
It is the Aeromonas hydrophila solution of CCTCC NO.M2013089 that the experimental group fish is poured into deposit number by anus, and concentration is 10
8The bacterium liquid of CFU/mL.To the physiological saline solution of negative control group by anus perfusion 0.7%, positive controls is poured into TNBS solution by anus.The groundwater increment of anus perfusion is 0.25mL/ tail～0.35mL/ tail, and groundwater increment increases with the increase of fish body weight.Blank is not done any processing.
After the perfusion fish is cultured in aquarium, a small amount of commodity feed of grass carp of throwing something and feeding, feeding volume is 1% of body weight, the time of throwing something and feeding is 8:00～9:00 in the morning; Temperature of cultivation is 27 ± 1 ℃, DO 〉=7.0mg/L, and the batch (-type) oxygenation, pH is 7.5 ± 0.2, NH
4 +-N≤0.2mg/L, NO
2-N≤0.05mg/L, H
2S≤0.05mg/L.Change the water frequency: 2d/ time, 1/3 volume of at every turn more exchanging treaties.
Observe the symptoms and be recorded as motility rate, the survival rate of the prepared Ctenopharyngodon idellus inflammatory bowel of present embodiment disease model is 100%, 100% to have fish inflammatory bowel disease symptoms, is acute inflammation from the 1d～3d, transfers chronic inflammatory disease behind the 7d to.
Getting 240 tail specifications is the Carassius auratus of 20g～50g, sets up the inflammatory bowel disease model behind laboratory rearing 20d.To test fish and be divided into 4 groups, be respectively the experimental group of perfusion Aeromonas hydrophila, the normal saline negative control group of perfusion 0.7%, positive controls, the blank group of perfusion TNBS, every group of 60 tails.
The compound method of TNBS solution is: be that 5%TNBS mixes with the dehydrated alcohol equal-volume with volume fraction, namely.
Preparation quality-volumetric concentration is the MS-222 solution of 70mg/L, soaks the anaesthesia experiment fish.
It is the Aeromonas hydrophila solution of CCTCC NO.M2013089 that the experimental group fish is poured into deposit number by anus, and concentration is 10
7The bacterium liquid of CFU/mL.To the physiological saline solution of negative control group by anus perfusion 0.7%, positive controls is poured into TNBS solution by anus.The groundwater increment of anus perfusion is 0.25mL/ tail～0.35mL/ tail, and groundwater increment increases with the increase of fish body weight.Blank is not done any processing.
After the perfusion fish is cultured in aquarium, a small amount of commercial feed of throwing something and feeding, feeding volume are 1% of body weight, and the time of throwing something and feeding is 8:00～9:00 in the morning; Temperature of cultivation is 27 ± 1 ℃, DO 〉=7.0mg/L, and the batch (-type) oxygenation, pH is 7.5 ± 0.2, NH
4 +-N≤0.2mg/L, NO
2-N≤0.05mg/L, H
2S≤0.05mg/L.Change the water frequency: 2d/ time, 1/3 volume of at every turn more exchanging treaties.
Observe the symptoms and be recorded as motility rate, the survival rate of the prepared Cyprinus carpio inflammatory bowel of present embodiment disease model is 100%, 100% to have fish inflammatory bowel disease symptoms, is acute inflammation from the 1d～3d, transfers chronic inflammatory disease behind the 7d to.
Getting 300 tail specifications is the Ctenopharyngodon idellus of 50g～100g, sets up the inflammatory bowel disease model behind laboratory rearing 20d.To test fish and be divided into 4 groups, be respectively the experimental group of perfusion Aeromonas hydrophila, the normal saline negative control group of perfusion 0.7%, positive controls, the blank group of perfusion TNBS, every group of 100 tails.
The compound method of TNBS solution is: be that 5%TNBS mixes with the dehydrated alcohol equal-volume with volume fraction, namely.
Preparation quality-volumetric concentration is the MS-222 solution of 70mg/L, soaks the anaesthesia experiment fish.
It is the Aeromonas hydrophila solution of CCTCC NO.M2013089 that the experimental group fish is poured into deposit number by anus, and concentration is 10
7The bacterium liquid of CFU/mL.To the physiological saline solution of negative control group by anus perfusion 0.7%, positive controls is poured into TNBS solution by anus.Groundwater increment is 0.4mL～0.5mL, and groundwater increment increases blank with the increase of fish body weight and do not do any processing.
After the perfusion fish is cultured in aquarium, a small amount of commodity feed of grass carp of throwing something and feeding, feeding volume is 1% of body weight, the time of throwing something and feeding is 8:00～9:00 in the morning; Temperature of cultivation is 27 ± 1 ℃, DO 〉=7.0mg/L, and the batch (-type) oxygenation, pH is 7.5 ± 0.2, NH
4 +-N≤0.2mg/L, NO
2-N≤0.05mg/L, H
2S≤0.05mg/L.Change the water frequency: 2d/ time, 1/3 volume of at every turn more exchanging treaties.
Observe the symptoms and be recorded as motility rate, the survival rate of the prepared Ctenopharyngodon idellus inflammatory bowel of present embodiment disease model is 97%, 100% to have fish inflammatory bowel disease symptoms, is acute inflammation from the 1st～3d, transfers chronic inflammatory disease behind the 7d to.
In order further to detect Fish inflammatory bowel Preparation of model effect, except the perusal symptom, each each processed group fish among the embodiment 1～3 is carried out fish body indication observe and record the result.
Different disposal group Ctenopharyngodon idellus health indication situation of change in the embodiment of the invention 1 is seen Table 1.
The health indication of table 1 different disposal group Ctenopharyngodon idellus changes
In 21d, there are not the phenomena of mortality.As shown in Table 1, the blank group without any processing that the embodiment of the invention 1 provides all keeps normal in 21d; Several groups of Ctenopharyngodon idellus through perfusion slight organic damage all occurred at 1d, but negative control group disappears substantially to the 3d symptom, and later sign is all normal; The positive controls of perfusion TNBS, pouring into moved about the same day has slowly slightly, and the red and swollen phenomenon of Di is more obvious, during 3d, fin ray occurs and festers, has a small amount of ascites, and 7d moves about and is tending towards normal, but Di also has redness, and intestinal mucosa festers in reparation; What the sign of pathogen group was different with positive controls is that the red and swollen phenomenon 3d of Di is obvious than 1d, but during to 7d, inflammatory bowel no longer develops, and tends towards stability during 14d, and the 21d reparation is normal substantially, has only slight ascites.
To the observed result of the Fish inflammatory bowel disease model that adopts the preparation of other embodiments of the invention provider method similarly.Show that the present invention is not strong to the specificity of variety classes Different Individual.In building process, show good stable and repeatability.
Each each processed group fish among the embodiment 1～3 is carried out fish body indication carries out pathological section and detect, respectively after perfusion the 0th, 1,3,7,14,21d take a sample on (each every group of 10 tails), all soaks with MS-222 before the sampling and anaesthetizes, MS-222 concentration is 60mg/L.
The grass carp intestinal at middle part was cut into slices and electron microscopic observation between the inverse the 1st of different disposal group Ctenopharyngodon idellus in the embodiment of the invention 1,2 circumnutated.Conventional method is carried out the paraffin wax section, and HE dyeing adopts the binocular inverted microscope of Olympus company to observe the intestinal structure for following 400 times; Also get simultaneously the film-making of same area organizational routine scanning electron microscope flaking method, the S-4700 type awkward silence at a meeting emission scan Electronic Speculum of Hitachi, Ltd is observed intestinal mucosa surface microvillus structure for following 5000 times.The section testing result of experimental group such as Fig. 1 in the embodiment of the invention 1～shown in Figure 6.It is dark that the nucleus of inflammatory cell is.
Wherein, Fig. 1 (a) show the inverse the 1st, 2 of normal Ctenopharyngodon idellus circumnutate between middle part intestinal structure, rare inflammatory cell is only arranged between tela submucosa, lamina propria and transversus and the longitudinal muscle; Fig. 1 (b) show the inverse the 1st, 2 of normal Ctenopharyngodon idellus circumnutate between intestinal mucosa surface, middle part microvillus structure, the microvillus arrangement architecture of mucous layer surface epithelial cell is neat;
Fig. 2 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 1d circumnutate between middle part intestinal structure, have a large amount of inflammatory cells between tela submucosa, lamina propria and transversus and the longitudinal muscle; Fig. 2 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 1d circumnutate between intestinal mucosa surface, middle part microvillus structure, antibacterial is invaded mucous layer surface, back microvillus and comes off, and forms the wound depression;
Fig. 3 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 3d circumnutate between middle part intestinal structure, the inflammatory cell between tela submucosa, lamina propria and transversus and the longitudinal muscle has minimizing slightly than 1d, the mucous layer cellular edema, goblet cell increases; Fig. 3 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 3d circumnutate between intestinal mucosa surface, middle part microvillus structure, mucous layer surface minority wound depression shoals, wound begins convergence;
Fig. 4 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 7d circumnutate between middle part intestinal structure, the inflammatory cell between tela submucosa, lamina propria and transversus and the longitudinal muscle is still more, the mucous layer cellular edema, the goblet cell cell is more than 3d; Fig. 4 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 7d circumnutate between intestinal mucosa surface, middle part microvillus structure, mucous layer superficial cut depression is more shallow, microvillus begins repairing growth;
Fig. 5 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 14d circumnutate between middle part intestinal structure, inflammatory cell between tela submucosa, lamina propria and transversus and the longitudinal muscle slightly reduces than 7d, the mucous layer cellular edema alleviates, and goblet cell begins to reduce; Fig. 5 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 14d circumnutate between intestinal mucosa surface, middle part microvillus structure, the mucous layer superficial cut further recovers;
Fig. 6 (a) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 21d circumnutate between middle part intestinal structure, approach when the inflammatory cell quantity between tela submucosa, lamina propria and transversus and the longitudinal muscle and 14d, the mucous layer edema disappears substantially, and goblet cell quantity is recovered to approach normal; Fig. 6 (b) show the inverse the 1st, 2 of the Ctenopharyngodon idellus behind the perfusion Aeromonas hydrophila 21d circumnutate between intestinal mucosa surface, middle part microvillus structure, the mucous layer superficial cut recovers substantially, only has slight wound.
The result shows: after causing inflammation, the inflammation at experimental group and positive controls rear portion is fairly obvious, and the model construction success is described, difference is that the inflammatory symptom of positive controls is acute inflammation, and 1d is the most serious, and 3d recovers to some extent; And experimental group is the most obvious at 3d, and then recovers, and begins behind the 7d to recover.In the later stage, experimental group is consistent with the symptom of positive controls, is chronic inflammatory disease.
To the section testing result of the Fish inflammatory bowel disease model that adopts the preparation of other embodiments of the invention provider method similarly.Show that the present invention is not strong to the specificity of variety classes Different Individual.In building process, show good stable and repeatability.
Each each processed group fish among the embodiment 1～3 is carried out the active detection of intestinal myeloperoxidase (MPO) (MPO).Respectively after perfusion the 0th, 1,3,7,14,21d takes a sample on (each every group of 10 tails), all soaks anesthesia with MS-222 before the sampling, MS-222 concentration is 60mg/L.The active test kit operation instructions that build up according to Nanjing that detect of intestinal myeloperoxidase (MPO) (MPO) carry out, to the testing result of the intestinal activity of myeloperoxidase variation tendency of the embodiment of the invention 1 each processed group fish as shown in Figure 7, wherein, curve 1 shows behind the perfusion Aeromonas hydrophila positive controls fish intestinal MPO activity change situation in the embodiment of the invention 1, curve 2 shows behind the perfusion Aeromonas hydrophila experimental group fish intestinal MPO activity change situation in the embodiment of the invention 1, curve 3 shows behind the perfusion Aeromonas hydrophila negative control group fish intestinal MPO activity change situation in the embodiment of the invention 1, and curve 4 shows the embodiment of the invention 1 empty matched group fish intestinal MPO activity change situation behind the perfusion Aeromonas hydrophila.The result shows: the variation tendency of the intestinal activity of myeloperoxidase of experimental group fish and positive control basically identical, and with histopathology performance basically identical, and with space management or negative control notable difference is arranged, show: the Ctenopharyngodon idellus inflammatory bowel disease model that the method that the embodiment of the invention 1 experimental group provides makes up is created as merit.
To the active testing result of intestinal myeloperoxidase (MPO) (MPO) of the Fish inflammatory bowel disease model that adopts the preparation of other embodiments of the invention provider method similarly.Show that the present invention is not strong to the specificity of variety classes Different Individual.In building process, show good stable and repeatability.
Adopt the mode of real-time fluorescence quantitative PCR reaction, detect among the embodiment 1～3 each processed group Fish intestinal IL-1 β, the IL-8 in each, the expression of TNF-α inflammatory factor changes.Respectively after perfusion the 0th, 1,3,7,14,21d takes a sample on (each every group of 10 tails), all soaks anesthesia with MS-222 before the sampling, MS-222 concentration is 60mg/L.At first, extract the mRNA in the Fish inflammatory bowel disease model intestinal that adopts method foundation provided by the invention, and be translated into cDNA.
Real-time fluorescence quantitative PCR overall reaction system 20 μ L, wherein: water 4.4 μ L, 10 * master Mix10 μ L, concentration is each 0.3 μ L of upstream and downstream primer of 10 μ mol/L, cDNA solution 5 μ L.
Response procedures: pre-94 ℃ of 2min of degeneration; 94 ℃ of 20s of degeneration, 60 ℃ of 30s of annealing temperature extend 72 ℃ of 30s, 40 circulations.
Experiment is as shown in table 2 with primer.Adopt β-actin as reference.To the testing result of the expression of pro-inflammatory cytokine in the embodiment of the invention 1 each processed group as shown in Figure 8, Fig. 8 (a) shows that the expression of fish intestinal pro-inflammatory cytokine IL-1 β in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes, Fig. 8 (b) shows that the expression of fish intestinal pro-inflammatory cytokine IL-8 in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes, and Fig. 8 (c) shows that the expression of fish intestinal pro-inflammatory cytokine TNF-α in the metainfective embodiment of the invention 1 each processed group of Aeromonas hydrophila changes.As shown in the figure: after being subjected to the virulence factor stimulation, the pro-inflammatory cytokine of experimental group and positive controls all has the process of rise on the mRNA expression, but both distinguish to some extent, experimental group reaches the highest at 3d, and wherein, inflammatory factor IL-1 β relative expression quantity reaches 22.13 times, be significantly higher than 5.94 times (p＜0.05) of negative control group, obviously descend behind the 7d, slight rebound to 11.59 times behind the 14d also is significantly higher than 5.50 times of matched groups (p＜0.05); The summit of positive controls is 3149.19 times (p＜0.01) at 1d, and 3d drops to 189.35 times (p＜0.01), similar with experimental group behind the 14d, slight rebound to 14.79 times (p＜0.05) shows as chronic inflammatory disease, and the symptom of positive controls is more serious than experimental group.Inflammatory factor IL-8 is littler than IL-1 β with the amplitude of variation of TNF-α, but variation tendency is similar to IL-1 β, experimental group 3d peaks, be respectively 30.39 times (p＜0.05) and 15.50 times (p＜0.01), all be significantly higher than 8.78 times and 2.07 times of matched group, 7d descends, during to 14d, slight rebound is arranged again, be respectively 10.46 times and 3.52 times, also be significantly higher than 7.21 times of matched group and 1.96 times (p＜0.05).Still be higher than matched group during to 21d, but difference is not remarkable.
The result shows: it is consistent with positive control to adopt method provided by the invention to cause the variation tendency of expression of scorching back grass carp intestinal IL-1 β, IL-8, TNF-α inflammatory factor, and obviously is different from negative control.The fish gastrointestinal model that shows the method foundation that the embodiment of the invention 1 provides successfully constructs.
To intestinal IL-1 β, the IL-8 of the Fish inflammatory bowel disease model that adopts the preparation of other embodiments of the invention provider method, testing result that TNF-α inflammatory factor expression changes similarly.Show that the present invention is not strong to the specificity of variety classes Different Individual.In building process, show good stable and repeatability.
The primer of table 2 real-time fluorescence quantitative PCR overall reaction system
| The primer title | Sequence numbering |
| IL-1βF | SEQ?ID?NO:1 |
| IL-1βR | SEQ?ID?NO:2 |
| IL-8F | SEQ?ID?NO:3 |
| IL-8R | SEQ?ID?NO:4 |
| TNF-αF | SEQ?ID?NO:5 |
| TNF-αR | SEQ?ID?NO:6 |
| β-actinF | SEQ?ID?NO:7 |
| β-actinR | SEQ?ID?NO:8 |
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a Fish inflammatory bowel method for establishing model is characterized in that, may further comprise the steps:
Pouring into deposit number by anus is that culturing also, option table reveals the fish of inflammatory bowel disease symptoms as Fish inflammatory bowel disease model in the healthy fish body of Aeromonas hydrophila injection through soaking anesthesia of CCTCC NO.M2013089.
2. method according to claim 1 is characterized in that, described deposit number is that the concentration of the Aeromonas hydrophila of CCTCC NO.M2013089 is 10
5CFU/mL～10
8CFU/mL.
3. method according to claim 1 is characterized in that, the body weight of described fish is 20g～100g.
4. method according to claim 1 is characterized in that, the groundwater increment of described anus perfusion is 0.25mL/ tail～0.50mL/ tail.
5. method according to claim 1 is characterized in that, described fish is Ctenopharyngodon idellus or Carassius auratus.
6. method according to claim 1 is characterized in that, described inflammatory bowel disease symptoms is: anus and Di redness, intestinal mucosa are festered, hyperemia, downright bad, and inflammatory cell infiltration or enterocyte microvillus come off.
7. according to the described method of claim 1, it is characterized in that described anesthetis is MS-222.
8. method according to claim 1 is characterized in that, described narcotic concentration is 40mg/L～70mg/L.
9. one kind as the claim 1～8 Fish inflammatory bowel disease model that method is set up as described in each.
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