CN111602630A - Method for establishing broiler chicken small intestine mechanical injury model caused by vomitoxin - Google Patents
Method for establishing broiler chicken small intestine mechanical injury model caused by vomitoxin Download PDFInfo
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- A—HUMAN NECESSITIES
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention relates to a method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin, belonging to the technical field of small intestine mechanical injury model construction. The establishing method comprises the following steps: feeding complete feed containing 10mg/kg of vomitoxin to 1 day old broiler chickens, and feeding for 7 days to obtain a broiler chicken small intestine mechanical injury model caused by vomitoxin. The method takes vomitoxin as an inducer, successfully establishes a model of the mechanical injury of the small intestine of the broiler chicken, and has the advantages of simple and convenient operation, reliable result and good repeatability.
Description
Technical Field
The invention relates to the technical field of small intestine mechanical injury model construction, in particular to a method for establishing a broiler small intestine mechanical injury model caused by vomitoxin.
Background
The poultry intestinal tract has dual functions, namely being an important organ for digesting and absorbing nutrient substances by a body and being a congenital defense barrier for maintaining the steady state of the environment in the body. The intestinal barrier of poultry mainly comprises a mechanical barrier, a chemical barrier, a microbial barrier and an immune barrier, and the four barriers are mutually related and jointly maintain the intestinal health of the poultry. The intestinal mechanical barrier, also called physical barrier, is the first line of defense of the body against foreign microorganisms, and is mainly composed of intestinal epithelial cells and their tight junctions. Occludin (occludins), claudins (claudins) and zonula occludens (ZO-1, ZO-2 and ZO-3) and the like are important components of tight junctions, and when intestinal epithelial cells are invaded by bacteria, toxins and the like, the tight junctions can be displaced to prevent the harmful substances from entering, and the intestinal cells are prevented from being damaged. When the intestinal mechanical barrier is disrupted, bacteria and toxins can invade intestinal cells causing intestinal injury to occur.
At present, reports about the damage of small intestine epithelial cells by vomitoxin mainly focus on cells, the research on the damage of small intestine of animal body is less, and no efficient method for establishing a small intestine mechanical damage model exists.
Disclosure of Invention
The invention aims to provide a method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin, so that the broiler chicken model with vomitoxin injury is simple and convenient to construct and operate and stable and reliable in effect.
The invention provides a method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin, which comprises the following steps:
feeding complete feed containing 10mg/kg of vomitoxin to 1 day old broiler chickens, and feeding for 7 days to obtain a broiler chicken small intestine mechanical injury model caused by vomitoxin.
Preferably, the preparation method of the complete feed containing 10mg/kg of vomitoxin comprises the following steps:
inoculating the fusarium graminearum culture into rice for culturing to obtain rice polluted by vomitoxin, and preparing the rice polluted by the vomitoxin into complete feed containing 10mg/kg of vomitoxin.
Preferably, the rice comprises scented rice.
Preferably, the temperature of the culture is 27 ℃ and the time of the culture is 28 d.
Preferably, the preparation method of the fusarium graminearum culture comprises the following steps:
inoculating fusarium graminearum into a potato glucose agar culture medium, and culturing for 7d at 27 ℃ to obtain a fusarium graminearum solid culture;
inoculating fusarium graminearum into a potato glucose broth culture medium, and culturing at 27 ℃ and 180rpm for 7d to obtain a fusarium graminearum liquid culture.
Preferably, the inoculation comprises: simultaneously inoculating the fusarium graminearum solid culture and the fusarium graminearum liquid culture into the rice.
Preferably, the inoculation amount is that every 200g of rice is inoculated with 20-28 g of fusarium graminearum solid culture and 95-105 mL of fusarium graminearum liquid culture.
Preferably, after a model of mechanical injury of small intestines of broiler chickens caused by vomitoxin is established, the method further comprises the step of determining the injury of the model, and the determination method comprises the following steps: determining the contents of amylase, trypsin and lipase in the small intestine of the injured broiler chicken, determining the ratio of villus height to crypt depth, the villus height and crypt depth of the small intestine, and determining the mRNA expression and protein expression of key tight junction proteins ZO-1, Ocglaudin and claudin in the epithelial tissue of the small intestine.
The invention also provides application of the model for mechanical damage of the small intestine of the broiler chicken caused by the vomitoxin, which is constructed by the establishing method in the technical scheme, in screening of drugs or feed additives for inhibiting the vomitoxin.
The invention also provides application of the model for mechanical damage of the small intestine of the broiler chicken caused by the vomitoxin, which is constructed by the establishment method of the technical scheme, in preparation of a medicament for preventing or treating intestinal diseases.
The invention provides a method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin. The intestinal tract is an important place for the digestion and absorption of nutrient substances of the organism and also is the largest immune organ of the organism, and plays an important role in resisting the invasion of pathogenic microorganisms of the organism. At present, no clear report is provided for a method for using a broiler small intestine injury model caused by vomitoxin. Therefore, the establishment of the model of the mechanical injury of the broiler small intestine by the vomitoxin provides an important way for the research of the prevention or treatment of the intestinal diseases and the research, development and application of related medicaments.
Drawings
FIG. 1 is a graph showing the effect of emetic toxin provided by the present invention on duodenal villus height (V) and crypt depth (C) and the ratio of villus height to crypt depth;
FIG. 2 is a graph showing the effect of emetic toxin provided by the present invention on jejunal villus height (V) and crypt depth (C) and ratio of villus height to crypt depth (V/C);
FIG. 3 is a graph showing the effect of emetic toxin provided by the present invention on ileal villus height (V) and crypt depth (C) and the ratio of villus height to crypt depth (V/C).
Detailed Description
The invention provides a method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin, which comprises the following steps:
feeding complete feed containing 10mg/kg of vomitoxin to 1 day old broiler chickens, and feeding for 7 days to obtain a broiler chicken small intestine mechanical injury model caused by vomitoxin. Vomitoxin, also known as Deoxynivalenol (DON), is a trichothecene compound with the chemical name of 3a, 7a, 15-trihydroxy fusarium solani-9-en-8-one. In the selection of the injury reagent, DON generated by the mildewed feed is selected as the injury reagent, and an injury model of broiler small intestine caused by vomitoxin is established; in addition, DON is used as a damage reagent, and the method also has the advantages of wide reagent source and being close to the actual production. The feeding conditions are not particularly limited, and the broiler chicken can be managed by adopting conventional broiler chicken feeding conditions well known by technical personnel in the field, such as feeding according to the conventional humidity, temperature and normal drinking water of the complete broiler chicken feeding. After the broiler chicken is fed for 7 days, the broiler chicken small intestine damaged by vomitoxin is obtained.
In the present invention, the preparation method of the complete feed containing 10mg/kg of vomitoxin preferably comprises the following steps:
inoculating the fusarium graminearum culture into rice for culturing to obtain rice polluted by vomitoxin, and preparing the rice polluted by the vomitoxin into complete feed containing 10mg/kg of vomitoxin. The composition proportion of the complete feed is not specially limited, and the formula of the complete broiler feed known by technical personnel in the field can be referred. In the present invention, the rice preferably comprises scented rice, and the content of vomitoxin caused by the contamination of the scented rice with fusarium graminearum is high. In the present invention, the temperature of the cultivation is preferably 27 ℃ and the time of the cultivation is preferably 28 d.
In the invention, the preparation method of the fusarium graminearum culture comprises the following steps:
inoculating fusarium graminearum into a potato glucose agar culture medium, and culturing for 7d at 27 ℃ to obtain a fusarium graminearum solid culture; in the present invention, the preparation method of the potato dextrose agar medium preferably comprises: weighing 23g of Potato Dextrose Agar (PDA) culture medium, adding into 500ml of distilled water, autoclaving at 115 deg.C for 20min, cooling to about 50 deg.C, and pouring, and solidifying the culture medium.
Inoculating fusarium graminearum into a potato glucose broth culture medium, and culturing at 27 ℃ and 180rpm for 7d to obtain a fusarium graminearum liquid culture. In the present invention, the preparation method of the potato dextrose broth culture medium preferably comprises: 17.5g potato dextrose broth is weighed and cultured, then added into 500ml distilled water, sterilized by autoclaving at 115 ℃ for 20min, and cooled to 27 ℃ for subsequent inoculation.
The fusarium graminearum solid culture and the fusarium graminearum liquid culture obtained by the invention contain fusarium graminearum with strong infectivity.
In the present invention, the inoculation preferably comprises: simultaneously inoculating the fusarium graminearum solid culture and the fusarium graminearum liquid culture into the rice.
In the invention, the inoculation amount is preferably 20-28 g of fusarium graminearum solid culture and 95-105 mL of fusarium graminearum liquid culture per 200g of rice, and more preferably 25g of fusarium graminearum solid culture and 100mL of fusarium graminearum liquid culture.
In the invention, after establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin, the method preferably further comprises the step of determining the damage of the model, wherein the determination method comprises the following steps: determining the contents of amylase, trypsin and lipase in the small intestine of the injured broiler chicken, determining the ratio of villus height to crypt depth, the villus height and crypt depth of the small intestine, and determining the mRNA expression and protein expression of key tight junction proteins ZO-1, Ocglaudin and claudin in the epithelial tissue of the small intestine. The method uses 10mg/kg of vomitoxin to act on the broiler chickens, so that the contents of amylase, trypsin and lipase in duodenum, jejunum and ileum of the broiler chickens are reduced, the villus height and crypt depth of the duodenum, jejunum and ileum are reduced, the ratio of the villus height to the crypt depth is also reduced compared with that of a control group, and the relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA of the duodenum, jejunum and ileum is also reduced, which indicates that the model construction success of mechanical injury of the small intestine of the broiler chickens caused by the vomitoxin is realized.
The invention also provides application of the model for mechanical damage of the small intestine of the broiler chicken caused by the vomitoxin, which is constructed by the establishing method in the technical scheme, in screening of drugs or feed additives for inhibiting the vomitoxin.
The invention also provides application of the model for mechanical damage of the small intestine of the broiler chicken caused by the vomitoxin, which is constructed by the establishment method of the technical scheme, in preparation of a medicament for preventing or treating intestinal diseases.
The method for establishing the model of mechanical injury of small intestine of broiler chicken caused by vomitoxin is described in further detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
Preparing a potato glucose broth culture medium and a potato glucose agar culture medium, respectively inoculating fusarium graminearum to the potato glucose broth culture medium and the potato glucose agar culture medium, and culturing for 7d to obtain a large amount of fusarium graminearum:
weighing 23g of Potato Dextrose Agar (PDA) culture medium, adding the PDA culture medium into 500ml of distilled water, sterilizing the mixture for 20min at 115 ℃, then cooling the mixture to about 50 ℃, inverting the mixture, solidifying the culture medium, inoculating fusarium graminearum stored in a refrigerator at-80 ℃ onto a plate of the culture medium, culturing the culture medium in an incubator at 27 ℃ for 7d, and growing the fusarium graminearum on the plate to obtain the fusarium graminearum solid culture with strong activity.
Weighing 17.5g of potato glucose broth for culturing, adding into 500ml of distilled water, sterilizing at 115 ℃ for 20min under high pressure, cooling to 27 ℃, inoculating the stored fusarium graminearum into the broth culture medium, culturing at 27 ℃ for 7d at 180 turns, and growing the pilus on the bottle mouth to obtain the fusarium graminearum liquid culture with strong activity.
Inoculating fusarium graminearum into fragrant rice to pollute the fragrant rice, and obtaining fragrant rice containing vomitoxin: simultaneously inoculating a fusarium graminearum solid culture (25g) cultured by a 9cm culture dish and a fusarium graminearum liquid culture cultured by a 100ml broth culture medium into 200g fragrant rice, culturing at 27 ℃ for 28 days to obtain fragrant rice polluted by high-content vomitoxin, preparing feed polluted by the vomitoxin according to a formula of broiler complete feed, namely preparing the fragrant rice containing the vomitoxin into complete feed containing 10mg/kg of vomitoxin, feeding broiler chickens of 1 day age for 7 days, completing the establishment of a mechanical injury model of the dorking small intestine caused by DON, and obtaining the broiler small intestine injured by the vomitoxin.
According to the standard feeding environment, the feed, drinking water and environment conditions of the broiler chickens are checked twice at fixed points every day, and the broiler chickens are fed completely according to the standard feeding mode.
And (3) determining amylase, trypsin and lipase which damage the small intestine of the broiler chicken, villus height, crypt depth of the small intestine, the ratio of the villus height to the crypt depth, mRNA expression and protein expression of key tight junction proteins ZO-1, Ocglaudin and claudin in the epithelial tissue of the small intestine, and judging the effect of vomitoxin on the small intestine damage of the broiler chicken.
Example 2
Effect of DON on broiler small intestine amylase, trypsin and lipase
Obtaining small intestines of the broiler chicken: selecting a 2-3 cm middle intestine section of a dorking, namely a duodenum, a jejunum and an ileum, dividing the dorking into two sections, washing the contents in the intestines by using pre-prepared PBS (phosphate buffer solution), and then cutting three sections with the length of 1cm at the front end of each section of intestine into Bouin's stationary liquid for preparing later-stage tissue slices; cutting the rest part, placing into a freezing tube, quickly freezing with liquid nitrogen, and storing at-80 deg.C for later analysis of intestinal tract.
The duodenum, jejunum and ileum stored at low temperature are naturally thawed, and diluted by 40 times with distilled water, and amylase, lipase and trypsin to be detected are detected by using Nanjing constructed kit, and the detection results are shown in the following table 1.
In tables 1 to 6, the shoulder marks contain different letters indicating significant difference (P <0.05), the same letters indicating insignificant difference (P >0.05), and capital letters indicating significant difference (P < 0.01).
TABLE 1 Effect of emetic toxin on Dodecamylase, Trypsin and Lipase (U/mgprot)
| Item | Control group | Test group | P value |
| Duodenal amylase | 15.39±0.54a | 14.97±0.39b | ＝0.05 |
| Duodenal trypsin | 64.43±0.51A | 63.57±0.31B | <0.0001 |
| Duodenal lipase | 3.01±0.12ab | 2.96±0.14ab | 0.6470 |
The activity of the duodenal amylase, the trypsin and the lipase can reflect the digestive capacity of duodenum of the broiler chicken. The results of the measurement in table 1 show that after the broiler chickens eat complete feed containing 10mg/kg of vomitoxin for 7 days, the activities of the duodenal amylase, the trypsin and the lipase are all reduced, but the activity difference of the test group and the control group compared with the activity difference of the duodenal amylase is obvious, the difference of the trypsin is extremely obvious, and the lipase has no obvious difference.
TABLE 2 influence of emetic toxin on the enzymes amyloliquefaciens, trypsin and lipase (U/mgprot)
| Item | Control group | Test group | P value |
| Jejunal amylase | 445.43±3.66a | 443.64±2.88a | 0.2147 |
| Jejunal trypsin | 88.31±0.80a | 87.37±0.60b | <0.05 |
| Jejunal lipase | 3.93±0.12a | 3.87±0.09a | 0.2250 |
The activity of the jejunum amylase, the trypsin and the lipase can reflect the absorption capacity of the jejunum of the broiler chicken. The results of the measurement shown in table 2 show that after the broiler chickens eat complete feed containing 10mg/kg of vomitoxin for 7 days, the activities of the jejunal amylase, the trypsin and the lipase are all reduced, but the activities of the jejunal amylase and the lipase in the test group and the control group are not obviously different, and the difference of the trypsin is obvious.
TABLE 3 influence of emetic toxin on Enteroacetase, Trypsin and Lipase (U/mgprot)
| Item | Control group | Test group | P value |
| Enterobacter retrogradation amylase | 198.91±1.31a | 195.58±3.99b | <0.05 |
| Ileal trypsin | 172.61±0.63a | 171.88±0.29b | <0.05 |
| Ileum lipase | 10.72±0.44a | 10.47±0.40a | 0.7145 |
The activity of the ileal amylase, the trypsin and the lipase can reflect the absorption capacity of the ileum of the broiler chicken. The results of the measurement in table 3 show that after the broiler chickens eat complete feed containing 10mg/kg of vomitoxin for 7 days, the activities of the retrolental amylase, the trypsin and the lipase are all reduced, but the difference between the retrolental amylase and the trypsin is significant compared with the test group and the control group, and the activity of the lipase is not significantly different.
Example 3
Influence of DON on villus height, crypt depth and ratio of villus height to crypt depth of duodenum of broiler
(1) The making process of the broiler duodenal slice comprises the following steps: taking out the fixed duodenum, placing the fixed duodenum on a flat plate, trimming the thickness of a tissue block to be about 3-5 mm by using a single-sided blade, and placing the tissue block into an embedding box (which is required to be clear); dehydrating by gradient alcohol: dehydrating with 70% ethanol for 2h, 80% ethanol for 2h, 85% ethanol for 1h, 90% ethanol for 1h, 95% ethanol I (30min), 95% ethanol II (30min), 100% ethanol I (20min), and 100% ethanol II (20 min); and (3) transparency: carrying out transparent treatment on the tissue block after dehydration is finished, and the specific process is as follows: anhydrous ethanol: xylene (1:1)20min (soaking in the liquid for a long time), xylene I30 min, xylene II 45 min; embedding: putting the transparent tissue block into an embedding machine, and soaking in wax for two and half hours, and then embedding; slicing: fixing the fixed wax block on a slicer, firstly adjusting the thickness to 20 mu m for block trimming, and when the complete tissue section is exposed, adjusting the thickness to 5 mu m for continuous slicing; spreading and baking: the cut wax sheet is put into warm water (about 45 ℃) by using a toothpick for flattening, then is fished out by using a glass slide and is marked, and the cut wax sheet is put on a sheet spreading machine for spreading.
(2) Hematoxylin-eosin (HE) staining procedure: the spread tissue slices are respectively processed by xylene I before dyeing and xylene II before dyeing for 15min, and wax covered on the tissues is removed; and (3) refining the dewaxed tissue blocks by gradient wine to dehydrate: 5min of 100% absolute ethyl alcohol before dyeing, 3min of 95% absolute ethyl alcohol before dyeing, 3min of 90% absolute ethyl alcohol before dyeing, 2min of 80% absolute ethyl alcohol before dyeing, 2min of 70% absolute ethyl alcohol before dyeing and 2min of 50% absolute ethyl alcohol before dyeing; staining of the tissue mass: firstly, performing hematoxylin dip-dyeing for 6min, then performing acidification treatment by 1% hydrochloric acid alcohol for 30s, then washing for 10min by tap water (note that tissue blocks are not needed to be washed), performing blue returning, then sequentially performing tissue block dyeing, then performing 70% absolute ethyl alcohol 2min, performing 80% absolute ethyl alcohol 2min, performing 90% absolute ethyl alcohol 2min, and finally performing dip-dyeing by alcohol-soluble eosin for 30 s; the stained section is sequentially processed by: carrying out dehydration treatment on the dyed 95% absolute ethyl alcohol I for 4min, the dyed 95% absolute ethyl alcohol II for 4min, the dyed 100% absolute ethyl alcohol I for 5min and the dyed 100% absolute ethyl alcohol II for 5 min; respectively soaking the dehydrated tissue blocks in dyed dimethylbenzene I and dyed dimethylbenzene II for 10min for carrying out transparent treatment; finally, mounting with neutral gum (air bubbles cannot be used between the glass slide and the cover glass), wiping the section with xylene after drying, and observing under a microscope.
(3) Observing each tissue section under a microscope, picking different visual fields for photographing, analyzing and processing the picture by ImageJ software, measuring the height (V) of intestinal villi and the depth (C) of crypt on the picture, and calculating the ratio of the height (V) to the depth (C) of crypt.
Fig. 1 and table 4 show the effect of vomitoxin on the height (V) and depth (C) of the villus in duodenum and the ratio of the height to the depth of the crypt, and it can be seen from fig. 1 that the height of villus is lower, the depth of crypt is higher, the ratio is smaller, the villus edge of challenge group is damaged, villus is enlarged, and the arrangement of outer layer cells is loose in the duodenum challenge group compared with the control group.
TABLE 4 influence of emetic toxin on duodenal villus height (V) and crypt depth (C) and ratio of villus height to crypt depth (V/C)
| Item | Control group | Test group | P value |
| Fluff height (V) | 787.99±31.57A | 657.39±50.50B | <0.0001 |
| Depth of recess (C) | 81.20±16.64A | 124.02±19.55B | <0.0001 |
| V/C | 10.03±1.65A | 5.43±0.92B | <0.0001 |
Compared with the control group, the duodenum toxin attacking group has lower villus height, higher crypt depth and smaller ratio, and the difference is very obvious.
Fig. 2 and table 5 show the effect of vomitoxin on the villus height (V) and crypt depth (C) of jejunum and the ratio of the villus height to the crypt depth (V/C), and it can be seen from fig. 2 that the jejunum toxin-attacking group had a lower villus height, a higher crypt depth, a lower ratio, and a fallen matter in the lumen, compared with the control group.
TABLE 5 Effect of emetic toxin on jejunal villus height (V) and crypt depth (C) and ratio of villus height to crypt depth (V/C)
| Item | Control group | Test group | P value |
| Fluff height (V) | 540.62±17.40A | 462.19±9.67B | <0.0001 |
| Depth of recess (C) | 70.77±10.42a | 63.12±10.17a | 0.1324 |
| V/C | 7.85±1.45a | 7.49±1.04a | 0.5471 |
Compared with a control group, the jejunum toxin attacking group has the advantages that the height of villus is reduced, the depth of crypts is reduced, the ratio is reduced, the difference of the height of the villus is extremely obvious, and the difference of the depth of the crypts and the ratio of the height of the villus to the depth of the crypts is not obvious.
Fig. 3 and table 6 show the effect of vomitoxin on ileal villus height (V) and crypt depth (C) and ratio of villus height to crypt depth (V/C), and it can be seen from fig. 3 that the ileal challenge group had lower villus height, higher crypt depth, lower ratio and irregular villus in the challenge group compared to the control group.
TABLE 6 influence of emetic toxin on ileal villus height (V) and crypt depth (C) and ratio of villus height to crypt depth (V/C)
| Item | Control group | Test group | P value |
| Fluff height (V) | 441.29±25.55a | 428.62±10.32a | 0.1847 |
| Depth of recess (C) | 55.64±10.99b | 66.47±6.70a | <0.05 |
| V/C | 8.30±1.97a | 6.52±0.72b | <0.05 |
Compared with a control group, the ileum attacking group has the advantages that the villus height is lower, the crypt depth is larger, the ratio is smaller, the difference between the villus height and the crypt height is not obvious, the difference between the crypt depth attacking group and the control group is obvious, and the difference between the crypt depth and the ratio between the villus height and the crypt depth is not obvious.
Example 4
Influence of DON on relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA of broiler chicken small intestine
Design of primers, the primers of the reference gene and the target gene required by the test are designed by Shanghai Biotechnology Co., Ltd, and synthesized by Beijing Optimalaceae New Biotechnology Co., Ltd, and the sequences of the primers are shown in Table 7:
TABLE 7 primer sequences
Extraction of total RNA: taking about 50mg of intestinal tract samples, putting the intestinal tract samples into a mortar for grinding, and grinding the intestinal tract samples into powder; placing into 2mL sterile EP tube, adding 1mL TRNzol, shaking vigorously to crack tissue, and standing at room temperature for 5 min; adding 200 μ l chloroform into EP tube, oscillating on oscillator to mix thoroughly, standing at room temperature for 3 min; centrifuging at 12000g for 15min at 4 deg.C (taking care to precool the centrifuge in advance), then extracting the supernatant into a new 1.5mLEP tube (the extraction must be slow, and the boundary line does not need to be shaken), adding isopropanol with the same volume as the supernatant, then mixing the mixture evenly, and standing for 30 min; centrifuging at 12000g for 10min at 4 deg.C, pouring out the supernatant, washing the precipitate with 1mL 75% glacial ethanol; centrifuging at 4 ℃ for 3min at 12000g, pouring out the supernatant, repeatedly washing and centrifuging once by using 75% of glacial ethanol, and slowly pouring out the supernatant after washing; the precipitate was left to stand under natural conditions for 10min, dissolved in about 20uL of DEPC water (on an ice box), and finally the RNA concentration was measured with a nucleic acid protein analyzer.
Synthesizing cDNA through reverse transcription reaction: the method comprises two steps of gDNA removal reaction and cDNA synthesis reaction, the specific operation steps are carried out according to the instructions of a reverse transcription kit, and the reaction systems are shown in tables 7 and 8.
TABLE 7 removal reaction System for genomic DNA
TABLE 8 reverse transcription reaction System
Real-time fluorescent quantitative PCR (qRT-PCR): the relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA in small intestine is detected by fluorescent quantitative PCR. The specific reaction system is shown in Table 9.
TABLE 9 real-time fluorescent quantitative PCR reaction System
The influence of DON on the relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA of the broiler chickens is shown in Table 10.
TABLE 10 influence of DON on the relative expression amounts of ZO-1, Ocglaudin and cladin-1 protein mRNAs in broiler chicken duodenum
| Amount of expression | Control group | Test group | P value |
| ZO-1 | 1.01±0.00A | 0.53±0.03B | <0.0001 |
| Occlaudin | 1.01±0.00A | 0.63±0.06B | <0.0001 |
| cladin-1 | 1.01±0.00A | 0.86±0.02B | <0.0001 |
The influence of DON on the relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA of broiler chickens is shown in Table 11.
TABLE 11 influence of DON on the relative expression amounts of ZO-1, Ocglaudin and cladin-1 protein mRNA of broiler chickens
| Amount of expression | Control group | Test group | P value |
| ZO-1 | 1.01±0.00A | 0.85±0.02B | <0.0001 |
| Occlaudin | 1.01±0.00A | 0.88±0.04B | <0.0001 |
| cladin-1 | 1.01±0.00a | 0.95±0.03b | <0.05 |
The influence of DON on the relative expression quantity of ZO-1, Ocglaudin and cladin-1 protein mRNA of broiler ileum is shown in Table 12.
TABLE 12 influence of DON on the relative expression of ZO-1, Ocglaudin, cladin-1 protein mRNA in broiler ileum
| Amount of expression | Control group | Test group | P value |
| ZO-1 | 1.01±0.00A | 0.87±0.02B | <0.0001 |
| Occlaudin | 1.01±0.00a | 0.95±0.03b | <0.05 |
| cladin-1 | 1.01±0.00a | 0.96±0.03b | <<0.050.05 |
The mechanical barrier is also called a physical barrier, can prevent harmful substances such as bacteria in the cavity from entering, can ensure the passing of nutrient substances, electrolytes and water, and maintains the physiological function of the organism, and mainly comprises a layer of columnar epithelial cells, tight junctions and the like, the link mainly comprises transmembrane proteins consisting of occludin (occludin), junctionaladhesive molecules (JAM) and claudins (claudins) and Zonula Occludins (ZO), and the like, has a particularly important function for the structure and the function of the intestinal mucosa barrier, and is a mark of the intestinal mechanical barrier.
The results of the measurements shown in tables 10, 11 and 12 show that DON can significantly reduce the relative expression amounts of ZO-1, Ocglaudin and cladin-1 protein mRNAs in duodenum; obviously reducing the relative expression quantity of ZO-1 and Ocglaudin protein mRNA in jejunum and reducing the relative expression quantity of cladin-1 protein mRNA; obviously reduces the relative expression quantity of the mRNA of the ZO-1 protein in the ileum and reduces the relative expression quantity of the mRNA of Ocglaudin and cladin-1 proteins.
In conclusion, in the embodiment of the invention, DON of 10mg/kg acts on the small intestine of broiler chicken, so that the contents of amylase, trypsin and lipase in duodenum, jejunum and ileum of broiler chicken are reduced, the villus height and crypt depth of duodenum, jejunum and ileum are reduced, the ratio of villus height to crypt depth is also reduced compared with that of a control group, the relative expression quantity of mRNA of ZO-1, Ocglaudin and cladin-1 proteins of duodenum, jejunum and ileum is also reduced, a model of mechanical injury of broiler chicken caused by vomitoxin is successfully established, and the model has the advantages of wide sources of injury reagents, low cost, simplicity and convenience in operation and stable and reliable effect, and an important way is provided for researching, treating and developing intestinal diseases and researching related medicines.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. A method for establishing a broiler chicken small intestine mechanical injury model caused by vomitoxin comprises the following steps:
feeding complete feed containing 10mg/kg of vomitoxin to 1 day old broiler chickens, and feeding for 7 days to obtain a broiler chicken small intestine mechanical injury model caused by vomitoxin.
2. The method for establishing a complete feed according to claim 1, wherein the preparation method of the complete feed containing 10mg/kg of vomitoxin comprises the following steps:
inoculating the fusarium graminearum culture into rice for culturing to obtain rice polluted by vomitoxin, and preparing the rice polluted by the vomitoxin into complete feed containing 10mg/kg of vomitoxin.
3. The method of establishing as claimed in claim 2 wherein said rice comprises scented rice.
4. The method according to claim 2, wherein the temperature of the culture is 27 ℃ and the time of the culture is 28 days.
5. The method of establishing according to claim 2, wherein the method of preparing the fusarium graminearum culture comprises the steps of:
inoculating fusarium graminearum into a potato glucose agar culture medium, and culturing for 7d at 27 ℃ to obtain a fusarium graminearum solid culture;
inoculating fusarium graminearum into a potato glucose broth culture medium, and culturing at 27 ℃ and 180rpm for 7d to obtain a fusarium graminearum liquid culture.
6. The method of establishing according to claim 2 or 5, wherein said inoculating comprises: simultaneously inoculating the fusarium graminearum solid culture and the fusarium graminearum liquid culture into the rice.
7. The method of establishing according to claim 6, wherein the amount of inoculation is 20-28 g of fusarium graminearum solid culture and 95-105 mL of fusarium graminearum liquid culture per 200g of rice.
8. The establishing method of claim 1, wherein after establishing the model of mechanical injury of small intestine of broiler chicken caused by vomitoxin, the establishing method further comprises the step of determining the injury of the model, and the determining method comprises the following steps: determining the contents of amylase, trypsin and lipase in the small intestine of the injured broiler chicken, determining the ratio of villus height to crypt depth, the villus height and crypt depth of the small intestine, and determining the mRNA expression and protein expression of key tight junction proteins ZO-1, Ocglaudin and claudin in the epithelial tissue of the small intestine.
9. The application of the model for mechanical damage of small intestine of broiler chicken caused by vomitoxin, which is constructed by the establishing method of any one of claims 1-8, in screening of drugs or feed additives for inhibiting vomitoxin.
10. The application of the model for mechanical damage of small intestines of broiler chickens caused by vomitoxin, which is constructed by the establishing method of any one of claims 1-8, in preparation of medicines for preventing or treating intestinal diseases.
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