CN112997926B - Method for evaluating components for relieving food-borne enteritis based on zebra fish juvenile fish imaging model - Google Patents

Method for evaluating components for relieving food-borne enteritis based on zebra fish juvenile fish imaging model Download PDF

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CN112997926B
CN112997926B CN202011464150.XA CN202011464150A CN112997926B CN 112997926 B CN112997926 B CN 112997926B CN 202011464150 A CN202011464150 A CN 202011464150A CN 112997926 B CN112997926 B CN 112997926B
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fish
enteritis
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food
zebra fish
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CN112997926A (en
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夏晓勤
吴南
黎明
程莹寅
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Institute of Hydrobiology of CAS
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/22—Animal feeding-stuffs from material of animal origin from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention discloses a method for evaluating a component for relieving food-borne enteritis based on a zebra fish juvenile fish imaging model. The method is characterized in that a food-borne enteritis model is constructed on the basis of Tg transgenic fluorescence labeled zebra fish of an innate immune cell double-labeled strain and an acquired immune cell single-labeled strain, and the behaviors of various immune cells participating in the process of food-borne enteritis are effectively presented by adopting a fluorescence imaging visual mode at the time stage of acting of the innate/acquired immune cells in a feed feeding mode, so as to evaluate components for relieving the food-borne enteritis of the fish. The method effectively shortens the evaluation period, completely shows and relieves the influence of the food-borne enteritis component on the innate immunity and the acquired immunity, overcomes the defects of long period and complex operation of the traditional adult fish model test, and also avoids the problem of systemic mucosal tissue immunoreaction caused by soaking juvenile fish in a short period.

Description

Method for evaluating components for relieving food-borne enteritis based on zebra fish juvenile fish imaging model
Technical Field
The invention belongs to the field of fish nutritional immunology, and particularly relates to a method for establishing a food-borne enteritis model based on a zebra fish juvenile fish immune cell imaging mode, and a method for evaluating or screening ingredients capable of relieving fish food-borne enteritis by using the model.
Background
Food-borne enteritis is an important limiting factor causing fish diseases and growth inhibition in aquatic product production and application, attention of academic circles is gradually paid, researches on related fish nutrition, immunology and intestinal microbiology at home and abroad are carried out on model animal zebra fish in large quantities, and 50% of bean pulp feed replacing protein sources is used as a common formula for modeling zebra fish enteritis. As a faster way, modeling of enteritis in zebrafish juvenile fish is also explored by multiple teams. Intestinal flora colonization of zebra fish juvenile fish at the 5-8dpf stage is completed, and the intestinal function of the juvenile fish is gradually perfected. In the intestinal mucosa of the zebra fish juvenile fish, signals can be observed by innate immune cells within one week, signals can be observed by the innate immune cells after the innate immune cells are out of the membrane for 3 weeks, and the composition of the intestinal immune cells of the zebra fish juvenile fish and adult fish is similar to that of the intestinal immune cells of the zebra fish juvenile fish at the age of about one month.
At present, the enteritis of the zebra fish juvenile fish is mainly modeled by 2 modes, namely soaking or feeding the zebra fish juvenile fish with feed, and the strains used are immune cell fluorescence labeling strains, such as a strain for labeling innate immune cells (neutrophils and macrophages) and a strain for labeling acquired immune T lymphocytes. In the zebra fish enteritis model, there are many strains reported for fluorescently labeled neutrophils, such as Tg (BACmpo: GFP, Tg (mpxI: Dendra 2)), and labeled macrophages are fluorescently labeled with the mpeg gene, and there are two fluorescently labeled strains Tg (mpeg1: mCherry/mpx: eGFP) that are seen in cross with the neutrophil strain, and the only strain of labeled T lymphocytes that is observed in the labeled acquired immune cells is Tg (lck: EFP).
T lymphocytes in the intestinal mucosa play a very important role in intestinal health. The soybean meal induced enteritis of zebra fish has been revealed to be an inflammation type dominated by Th17 type cytokines, T cells are increased when inflammation occurs in intestinal tracts and rapid cell migration occurs, Th17 response-related cytokines are up-regulated, and transcription factors and effector factors of regulatory T cells serving as the balance at the other end are down-regulated. These results in turn suggest that the inflammation-inhibiting action of regulatory T cells should be the focus of the development of drugs for enteritis. The research team has established a food-borne enteritis model of adult zebrafish and has applied it to evaluate the enteritis alleviating effect of feed additives, such as galantamine, which has been published, by activating regulatory lymphocytes via cholinergic pathways to resist enteritis. However, in fish intestinal mucosal immunity, besides regulatory T lymphocytes, B lymphocytes also actively participate in infection resistance, and natural immunoglobulin IgM equivalent response factors secreted by the B lymphocytes play an important role in the inflammation generation and recovery of food-borne enteritis. Rag2 is an indispensable important factor for T/B lymphocyte development, and fluorescent labeling of zebra fish strains can indicate intestinal tract acquired immune cells, but is not used for research of enteritis mechanism and related drug development at present. Meanwhile, lck is used as a marker of T lymphocyte maturation, and a fluorescence-labeled strain is only rarely used for researching the intestinal innate immune stage of zebra fish, and the level of cells in the acquired immune stage and the research of evaluating a remission component by applying the cell level to an enteritis model are lacked.
At present, enteritis research is carried out by imaging zebra fish juvenile fish, and the problems are also shown in the following: (1) the systemic mucosal reaction caused by short-term soaking may weaken the response of intestinal immune cells, and the expression of central granulocytes with short half-life is greatly different from fish body to fish body, on the other hand, the phagocytosis (phagocyte) of macrophages is very important for maintaining the intestinal health of fish, while the reaction of macrophages caused by soaking is mainly in the foregut which is acted by swallowing, but not in the middle and rear intestines which are main reaction sites of intestinal mucosa; (2) the selection of the feed feeding time period has an important decisive role in the normal development of the intestinal tract and the presentation of immune response, but the existing reports are that the open feed feeding is carried out from 5dpf to 7dpf, the period can only cause the response of intestinal tract innate immune cells, the research of the period from the development of the intestinal tract to the retention period of acquired immune cells is very limited by 3 weeks to 4 weeks, and the individual report that only T cells participate in soybean meal to cause enteritis is based on 9 dpf.
The invention comprehensively considers the development stages and the cell behaviours of the innate immunity and the acquired immunity of the zebra fish juvenile fish, adopts a feed feeding mode to effectively present the cell behaviors of various immune cells participating in the food-borne enteritis process at the time stage of the action of the innate/acquired immune cells and adopts a fluorescence imaging visualization mode based on a proper transgenic zebra fish strain, and provides a specific mode and a method for application in the effect evaluation of the feed added with fish enteritis drugs or components for relieving the food-borne enteritis.
Disclosure of Invention
The invention aims to provide a method for constructing a food-borne enteritis model by genetically and fluorescently labeling zebra fish with innate immune cell double-labeled strain Tg (lyz: DsRED2/mpeg1: EGFP), acquired immune cell single-labeled strain Tg (rag2: DsRed) and Tg (lck: lck-eGFP) based on the first development stage of innate immunity and acquired immunity of the zebra fish juvenile fish, and a method for evaluating or screening components for relieving the food-borne enteritis of the fish by using the model.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for evaluating a component for relieving food-borne enteritis based on a zebra fish juvenile fish imaging model comprises the following steps:
(1) imaging analysis of zebra fish juvenile fish marked by innate immune cell fluorescence
Hybridizing transgenic zebra fish Tg (lyz: DsRED2) and Tg (mpeg1: EGFP) to obtain transgenic zebra fish Tg (lyz: DsRED2/mpeg1: EGFP) with double markers of neutrophils and macrophages, dividing the transgenic zebra fish fries which are double marked after hatching for 5 days into 3 groups, feeding the groups with experimental feeds, namely a fish meal group, a 50% soybean meal alternative protein source feed group and components to be tested, feeding the groups for 4 days, and performing living imaging on the fries of different treatment groups under a laser confocal microscope on the 9 th day to analyze the quantity and the form change of the neutrophils marked by red fluorescence in intestinal tracts and the macrophages marked by green fluorescence.
In the specific embodiment of the invention, taking sinomenine as an example, 35ppm of sinomenine is added into a 50% bean pulp replacement protein source feed group, compared with the 50% bean pulp replacement protein source feed group, the sinomenine is added, so that the aggregation of neutrophils and macrophages in the middle and rear intestines is remarkably reduced, the macrophage morphology is changed from an oval shape to a slender strip shape, more macrophage differentiation is indicated, and the sinomenine can reduce the acute inflammatory reaction of the middle and rear intestines.
(2) Imaging analysis of zebra fish juvenile fish marked by acquired immune cell fluorescence
And (2) respectively crossing the lymphocyte marker strain Tg (rag2: DsRed) or T lymphocyte marker strain Tg (lck: lck-eGFP) homozygote adult fish with wild zebra fish to obtain heterozygotes, incubating the transgenic zebra fish Tg (rag2: DsRed) and Tg (lck: lck-eGFP) for 5 days, feeding commercial feeds for the young fish until 17 days, dividing the transgenic zebra fish into 3 groups on the 17 th day, feeding experimental feeds, namely a fish meal group, a 50% soybean meal replacement protein source feed group and components to be detected, obtaining a food-borne enteritis zebra fish young fish post-day immunoassay model, carrying out living body imaging on the fries of different treatment groups under a laser confocal microscope on the 27 th day, and analyzing the quantity and morphological change of intestinal red fluorescence labeled lymphocytes and green fluorescence labeled mature T lymphocytes.
In the specific embodiment of the invention, compared with a feed group with 50% of soybean meal replacing protein source, the aggregation of red fluorescence labeled lymphocytes and green fluorescence labeled mature T lymphocytes in the middle and rear intestines can be remarkably relieved after the sinomenine is added, and the two cell forms are more oval undifferentiated types, so that the differentiated type with immunological synapse shape is reduced, which indicates that the sinomenine can relieve the food-borne enteritis to a certain extent through relieving the adaptive immune process.
(3) Imaging analysis of intestinal slices of zebra fish juvenile fish
Establishing a food-borne enteritis zebra fish juvenile fish acquired immune analysis model according to the method in the step (2), and taking the 27 th-day fry to perform HE staining pathological analysis on the middle and rear intestinal tissue slices, wherein the HE staining pathological analysis comprises inherent layer thickness, intestinal villus folding degree and crypt depth.
In the specific embodiment of the invention, compared with a feed group with 50% of soybean meal replacing protein source, the addition of sinomenine can restore the folding degree of intestinal mucosa, restore the natural layer to the normal thickness, show obvious crypt depth and restore the layer number of goblet cells in intestinal epithelium.
Further, the degree of aggregation of immune cells and the histological pathology in the mucosal tissue of the middle and rear intestine were analyzed in the above steps (1), (2) and (3).
Further, compared with a 50% soybean meal replacement protein source feed group, if the number of immune cells in the treatment groups of the steps (1) and (2) is reduced and the aggregation degree is reduced, the lamina propria is restored to the normal thickness and the folding degree of the intestinal mucosa is restored in the step (3), and obvious crypt depth and a certain number of goblet cells are seen, the component to be detected has the effect of relieving the food-borne enteritis.
Compared with the prior art, the advantages are as follows:
(1) techniques using imaging of young fish can reveal neutrophils, macrophages, Rag2 from the immune cell level+DsRed 2-labeled lymphocytes, lck+The dynamic change of cell behavior of eGFP marked mature T lymphocytes in the food-borne enteritis, overcomes the defects of long-period modeling of individual level in the adult fish stage, complicated immunofluorescence technology depending on tissue level and the like, and obviously shortens the evaluation period of ingredients for relieving the food-borne enteritis.
(2) From the perspective of the development stage of the immune system of the intestinal mucosa tissue, the influence of feeding the soybean meal feed on the innate immunity and the acquired immunity is clearly shown in different stages of the completion of the development of the innate immunity and the acquired immunity cells, and the roles of various main immune cells in the process of participating in the food-borne enteritis in the inflammatory reaction and the adaptive immunity process of the innate immunity are effectively shown; and through the feeding mode, the defects of long analysis period and complex analysis steps of the traditional adult fish modeling are overcome, and meanwhile, the immunoreaction of the mucous membrane tissues of the whole body caused by short-term soaking of juvenile fish is avoided, so that a more accurate evaluation scheme for relieving the intestinal mucosa immune effect related to inflammation is provided. Therefore, the model can provide a set of convenient and quick tools for screening the medicines for relieving the food-borne enteritis, and can provide an accurate and reliable evaluation method for evaluating the effect of the components for relieving the food-borne enteritis.
Drawings
Fig. 1 is a model of the enteritis caused by soybean meal of zebra fish juvenile fish. The modeling strategy comprises an innate immunity stage of 0-9dpf and an acquired immunity stage of 0-27dpf, wherein in the innate immunity stage in figure 1A, the Tg (lyz: DsRED2/mpeg1: EGFP) fertilized eggs of double transgenes (which is the starting point of 0 dpf) are used, no feed is fed in 0-5dpf, fish meal Feed (FM), bean pulp feed (50SBM) and bean pulp feed (50SBM +35ppm SN) added with sinomenine are fed in groups every day between 6-9dpf, and fixation, imaging and analysis are carried out after 9dpf is finished; in the acquired immune stage shown in FIG. 1A, two different kinds of fish with Tg (rag2: DsRed) and Tg (lck: lck-eGFP) are fed with no feed within 0-5dpf, basic feed (AP100) is fed at 6-17dpf, FM, 50SBM and 50SBM +35ppm SN are fed in groups of 10dpf every day between 18dpf and 27dpf, and then fixation, imaging and analysis are carried out. FIG. 1B shows the zebrafish lying on its side during imaging, and SP8 laser forward scanning; the section of intestine in the red box of fig. 1B represents the area imaged and analyzed, being the middle and posterior intestine of zebrafish.
FIG. 2 is a typical signal of innate immune cell imaging in a model of enteritis caused by soybean meal from young zebra fish. FIGS. 2A-2D show intestinal conditions (scale bar 100 μm) with 6-9dpd fish meal feed, 2A1-2D1 are partial enlargements of different signals (scale bar 20 μm); FIGS. 2E-2H show intestinal tract conditions (scale bar 100 μm) under the condition of feeding bean pulp feed at 6-9dpd, and 2E1-2H1 is a partial enlarged view (scale bar 20 μm) of different signals after feeding bean pulp; red-labeled centromeres in fig. 2B, 2B1, 2F1, green-labeled macrophages in fig. 2C, 2C1, 2G 1; fig. 2A, 2A1, 2E1 are middle and posterior bowel conditions under bright field conditions, and fig. 2D, 2D1, 2H1 are superimposed views under different channels. FIG. 2I shows the number of neutrophils in different individuals, FIG. 2J shows the number of macrophages in different individuals, and the area counted between them is the area circled by the red frame in FIG. 1B; tn0 represents the 5dpf time point at the previous immunization stage and Tn4 represents the 9dpf time point. 0.05< p means that the difference is not significant, 0.01< p.ltoreq.0.05 means that the difference is significant, and p.ltoreq.0.01 means that the difference is extremely significant.
FIG. 3 is a typical signal of cells in the food-borne enteritis model at the post-natal immune stage of zebra fish larvae. FIGS. 3A-3G show Rag2+Cases where DsRed-labeled lymphocytes were imaged at the post-immune phase, and FIGS. 3H-3N show lck+-situation of hindgut imaging of eGFP-labeled mature T lymphocytes in the acquired immune phase. FIGS. 3A-3C (3A1-3C1) and FIGS. 3D-3F (3D1-3F1) show the feeding of fish meal feed and soybean meal feed, respectively, over a period of 18-27dpf, wherein FIGS. 3A-3C, 3D-3F are the middle and rear intestines in different channels (scale bar 100 μm), FIGS. 3A1-3C1, 3D1-3F1 are the enlarged views of the red inner frame area of the middle and rear intestines (scale bar 20 μm), and FIGS. 3B-3B1, 3E-3E1 are marked with a red signal Rag2+DsRed-labeled lymphocytes, 17dpf at the time point denoted by the abscissa Td0 and 27dpf at the time point denoted by Td10 in FIG. 3G, and Rag2 showing red fluorescence in the hindgut of each fish at the ordinate+DsRed-labeled lymphocyte counts. FIGS. 3H-3J (3H1-3J1) and 3K-3M (3K1-3M1) show fish meal and soybean meal feeds, respectively, being fed at 18-27 dpf; FIGS. 3H, 3K represent imaging in the bright field channel, 3I, 3L represent lck+-eGFP-labeled mature T lymphocytes in the case of green fluorescence channel, fig. 3J, 3M represent overlapping images under both channels; FIGS. 3H1-3M1 (scale bar 20 μ M) are enlarged views of signals within the red circle under the corresponding channel of FIGS. 3H-3M (scale bar 100 μ M); FIG. 3N shows the post-intestinal appearance of lck in each fish in the Fish meal and Soybean meal feed groups+-eGFP-labeled lymphocyte number. 0.05<p represents no significant difference, 0.01<p is less than or equal to 0.05, and p is less than or equal to 0.01, representing that the difference is very significant.
FIG. 4 is an image analysis of the effect of sinomenine on cells in the innate immune phase in a food-borne enteritis model and the morphological imaging of innate immune cells. FIGS. 4A-4D (4A1-4D1) show the effect of tenine concentration at 35ppm on gut innate immunity based on soybean meal feed addition over a 6-9dpf period, FIGS. 4A-4A1 show the morphology of the gut in the brightfield channel, FIGS. 4B-4B1 show the accumulation of neutrophils in the gut, FIGS. 4C-4C1 show the accumulation of green-labeled macrophages in the mid-hind gut, and FIGS. 4D-4D1 show the overlay of the three channels; FIGS. 4A1-4D1 (scale bar 20 μm) are enlarged views of signals within the red circle under the corresponding channel of FIGS. 4A-4D (scale bar 100 μm); FIG. 4E shows the number of neutrophils on the left of the abscissa, the number of macrophages on the right of the abscissa, and the number of cells marked with signals in the hindgut of zebrafish corresponding to the different treatment groups on the ordinate. 0.05< p means that the difference is not significant, 0.01< p.ltoreq.0.05 means that the difference is significant, and p.ltoreq.0.01 means that the difference is extremely significant.
FIG. 5 is the accumulation of sinomenine in the intestinal tract to relieve enteritis-related lymphocytes and the imaging analysis of sinomenine on the corresponding cell morphology. FIGS. 5A-5D and 5E-5H show the effect of a 35ppm tenine concentration on Rag2, respectively, on soybean meal feed+DsRed-labeled lymphocytes, lck+-eGFP labeling of mature T lymphocytes in case of imaging at the post-immune stage. FIGS. 5A-5C show imaging of the mid-hindgut in bright field, red fluorescence and overlapping channels (scale bar 100 μm), with FIGS. 5A1-5C1 showing magnified views within the red circle (scale bar 20 μm) of the corresponding channel; FIG. 5D shows the abscissa representing the different treatment groups in the acquired immunity and the ordinate representing Rag2+The number of cells present in each fish gut for DsRed-labeled lymphocytes. FIGS. 5E-5G show lck under brightfield, green fluorescence and overlapping three channels+Imaging of the middle and rear intestine by eGFP-labeled mature T lymphocytes (scale bar 100 μm), fig. 5E1-5G1 are enlargements of local signals under the corresponding channels (scale bar 20 μm); FIG. 5H shows the different treatment groups in the acquired immunity in abscissa and lck in ordinate+eGFP-labelling the number of mature T-lymphocytes present in the gut of each fish. 0.05<p represents no significant difference, 0.01<p is less than or equal to 0.05, and p is less than or equal to 0.01, representing that the difference is very significant.
FIG. 6 is analysis of HE staining results of intestinal tissues of zebra fish for alleviating food-borne enteritis by sinomenine. FIGS. 6A to 6C show the HE staining results (scale bar 25 μm) of middle and rear intestine sections of three groups of the fish meal, the soybean meal and the soybean meal added with sinomenine at a concentration of 35ppm at the post-immune stage, and FIG. 6D shows that the heights of intestinal villi in the three groups were statistically analyzed by ImageJ software. 0.05< p means that the difference is not significant, 0.01< p.ltoreq.0.05 means that the difference is significant, and p.ltoreq.0.01 means that the difference is extremely significant. The epithelial layer (IEL) is located on the top layer of the Intestinal mucosa, Goblet Cells (GC) are embedded in the epithelial layer, and the sections appear as empty bubbles, as indicated by the arrows in the figure. The Lamina Propria (LP) is the layer that underlies the epithelial layer, with the crypt in the sunken position of the bottom of the intestinal epithelial mucosal fold.
Detailed Description
The invention is further described with reference to the following drawings and specific examples, which should not be construed as limiting the invention. All molecular biological manipulations referred to in the examples are, unless otherwise specified, conventional procedures well known to those skilled in the art.
Test materials, reagents and instrumentation referred to in the following examples:
1. biological material: the AB line wild type Zebrafish (Danio reio) and the transgenic Zebrafish lines used in this experiment, including Tg (lyz: DsRED2), Tg (mpeg1: EGFP), Tg (rag2: DsRed) and Tg (lck: lck-eGFP) were purchased from the national Zebrafish Resource Center (China Zebraphish Resource Center, http:// zfish. cn /), and Tg (lyz: DsRED2/mpeg1: EGFP) dual-fluorescent line were obtained by crossing the two monochromatic fluorescent lines.
2. Reagents and consumables: low melting point agarose (Ultrapure) required for imaging and fixing fryTMLMP agar, invitrogen), paraffin for sectioning (Sigma), Hematoxylin Eosin (HE) staining kit (petunia), larval feed Larva AP100(Zeigler) for zebrafish, 4% paraformaldehyde (barshrap), MS-222(Sigma Aldrich), N-2-hydroxyethylpiperazine-Ni 2-ethanesulfonic acid (HEPES, Coolaber), sinomenine (purity > 98%): xianhuilin Biotechnology Ltd, confocal glass bottom culture dish (20mm diameter, biosharp), 150mm sterile plastic culture dish (150mm diameter, Haimen morning Xiang), and other reagents such as analytically pure alcohol, xylene and other conventional reagents are all national medicine reagents.
3. The instrument equipment comprises: zebrafish independent single-frame circulating feeding system (Tecnplast); the fluorescence imaging analysis equipment is a laser confocal microscopic imaging system (SP8, Leica); the HE stained intestinal tissue sections taken in bright field are as follows: large screen full automatic digital slide scanner (Aperil VERSA 8, Leica) and multispectral imaging microdissection system (olympus fluorescence microscope & PE); a full-automatic dehydrator (Tissue Processor, Citadel 2000, Thermo); full-automatic Embedding machine (Embedding workbench, Histostar, Thermo).
Example 1: application of young fish imaging of innate immunofluorescence labeled zebra fish strain in analysis of effect of sinomenine on relieving soybean meal induced enteritis
(1) Preparation of double transgenic Zebra fish Tg (lyz: DsRED2/mpeg1: EGFP)
1) Transgenic zebra fish which are separately bred by males and females are fed with Tg (lyz: DsRED2) and Tg (mpeg1: EGFP) one week in advance, and are fed with satiation three times a half a day, the breeding water temperature is controlled to be 28 +/-0.5 ℃, the lighting time is 14h, the darkness is 10h, the environment of a zebra fish breeding system is pH: 7.0-8.0 percent, 0.25-0.50 per mill of salinity, 5-8 mg/L of dissolved oxygen, less than 0.02mg/L of total ammonia nitrogen, and other conditions refer to the requirements provided by the national zebra fish culture center (http:// www.zfish.cn);
2) separating a male and a female corresponding to Tg (lyz: DsRED2) and Tg (mpeg1: EGFP) fluorescent strain adult fish with a fish tank for pairing overnight, pulling out a partition plate in the morning the next day, allowing the adult fish to naturally chase and lay eggs for one hour, collecting fish eggs with 0.3 XDanieau's solution (egg water) working solution containing methylene blue, and putting the fish eggs into glass culture dishes of 12cm X5 cm with the concentration of about 100 eggs/dish to obtain embryos containing double fluorescent markers of Tg (lyz: DsRED2/mpeg1: EGFP). The stock formula of 30 × Danieau's solution used for breeding zebrafish fry is as follows:
TABLE 130 XDanieu's solution stock
Figure GDA0003074304330000081
3) Placing the fish fries in an illumination incubator (28 +/-1 ℃, illuminating for 14h and darkness for 10h) for incubation, changing egg water (containing methylene blue) twice in the morning and at night every day, picking out dead white fish eggs, changing the egg water without the methylene blue after 3 days (3dpf), preparing the fish fries for split charging until the 5dpf is cultured, and starting the subsequent modeling experiment;
(2) preparation of feed for modeling zebra fish fry soybean meal induced enteritis
Establishing a zebra fish bean pulp induced enteritis (SBMIE) model according to the following feed formula, and adding sinomenine with the concentration of 35ppm (the experimental concentration is obtained by a pre-experiment, and data is not shown) on the basis of a positive control group to prepare the sinomenine zebra fish juvenile fish feed capable of relieving enteritis, wherein the specific preparation method is as follows:
TABLE 2 Zebra fish SBMIE modeling and Sinomenine addition experiment feed formula
Figure GDA0003074304330000082
Figure GDA0003074304330000091
Note: 1, FM: fish meal group, 50 SBM: soybean meal group, 50SBM +35ppm SN: adding 35ppm sinomenine group;
2. the vitamins in the vitamin supplement in the formula are referred to as NRC, 1993.
1) Sieving fish meal, bean pulp, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix and the like with a 60-mesh sieve, wherein the fish meal and the bean pulp contain larger particles such as fishbone, bean skin and the like, crushing by a crusher, and sieving with the 60-mesh sieve; for less vitamin and mineral content: VD3, VK3, VB12, thiamine, VB6, folic acid, copper sulfate and sodium selenite need to be diluted by 20 times to prepare a premix; said mineral mixture contains (g): magnesium sulfate (MgSO)4·2H2O)60.530g, ferrous sulfate (FeSO)4·H2O)23.110g, copper sulfate (CuSO)4·5H2O)0.010g, zinc sulfate (ZnSO)4·H2O)0.620g, manganese sulfate (MnSO)4·H2O)1.640g, potassium iodide (KI)0.070g, sodium selenite (NaSeO)3)0.005g, adjusted to 1kg with microcrystalline cellulose; the above vitamin premix contains (g) per kg of vitamin premix: 0.05g of vitamin B1(Thiamin), 0.55g of vitamin B2(Riboflavin), 0.59g of vitamin B6(pyridoxine), 0.83g of vitamin B12(cyanocobalamine), 2.89g of pantothenic acid (pantothenic acid), 0.40g of folic acid (folic acid), 19.39g of inositol (inositol), 2.24g of nicotinic acid (niacin), 4.91g of biotin (biotin), 7.16g of vitamin C (ascorbic) and vitamin A (vitamin)A)2.40g, vitamin D (vitamin D)0.40g, vitamin E (vitamin E)12.55g, vitamin K (vitamin K)0.80g, adjusted to 1kg with microcrystalline cellulose;
2) respectively weighing fish meal, bean pulp, corn starch, wheat flour, microcrystalline cellulose, a mineral premix, a vitamin premix and a sinomenine medicament according to the requirements of the weight percentages of the components in the feed formula in proportion;
3) fully stirring and mixing the weighed fish meal, bean pulp and wheat flour uniformly;
4) fully stirring and uniformly mixing the microcrystalline cellulose, the mineral premix and the vitamin premix, adding the corn starch, uniformly mixing, adding the uniformly mixed mixture into the mixture obtained in the step (3), and continuously stirring until the mixture is uniformly mixed;
5) weighing fish oil according to a formula table, adding the fish oil into the mixture obtained in the step (4), and rubbing off and fully stirring the large oil drops until the fish oil is uniformly distributed in the mixture;
6) adding the weighed sinomenine into purified water accounting for 30 percent of the total weight of the mixture, and fully stirring until the sinomenine is completely dissolved;
7) adding the purified water containing sinomenine in the step (6) into the mixture in the step (5), supplementing water to 5-15% of the total weight of the mixture, and stirring to uniformly distribute water;
8) controlling the extrusion pressure and the corresponding rotating speed and temperature by using a double-screw granulator with the diameter of 2mm, slowly adding the mixture obtained in the step (7) into the granulator after preheating, and drying the granulated feed in a dryer prepared in advance, wherein the temperature of the dryer is controlled to be 55-60 ℃, and the drying moisture is about 10%;
9) crushing the dried feed, sieving the crushed feed by a 80-100-mesh sample sieve to obtain corresponding powdered feed, taking out a small part of the powdered feed for measuring conventional nutritional ingredients, and storing the rest of the powdered feed in a refrigerator at the temperature of-20 ℃ in tubes for later use;
(3) zebra fish fry SBMIE modeling feeding scheme
1) After the double transgenic zebrafish Tg (lyz: DsRED2/mpeg1: EGFP) was incubated for 5 days (5pdf), the two transgenic zebrafish were distributed into circular sterile petri dishes with a diameter of 150mm, and the experiment was divided into three groups, each 30 fish/dish: group 1: FM, group 2: 50SBM, group 3: 50SBM +35ppm SN, three biological replicates per group;
2) every group throws the corresponding fodder respectively, and when throwing something and feeding earlier the fodder dissolve in the egg water, throw something and feed thrice every day, throw something and feed the time quantum and do: 8: 30-9:00, 14: 00-14:30 and 18:00-18:30, changing two thirds of egg water culture solution after feeding for half an hour, and making the feed as the step (2);
3) after 9dpf feeding is finished, all the egg water is replaced, and the culture dish is kept clean and tidy and is used for in-vivo imaging analysis of subsequent fries;
(4) living body imaging method for enteritis and remission degree of zebra fish fries
1) 25X 100mL of MS-222 stock solution (Tricaine Powder 400mg, dd H) was prepared in sterile water2O97.9 ml, 1M Tris-HCl PH 9.02.1 ml), membrane-sterilized, stored at 4 deg.C, and working solution thereof with dd H2O diluted to 1 ×; preparing 1% (w/v) of low-melting-point agarose by using a PBS solution, and placing the agarose at room temperature for imaging and fixing the fry;
2) anesthesia: after the egg water of the corresponding group is removed to be dry as much as possible, 1 x anesthetic is added, about 30s of anesthesia is carried out, and a large amount of fish fries are in an anesthetic state;
3) fixing: heating the prepared 1% low-melting-point agarose in the step (1) by using a microwave oven until the temperature is kept at 29 ℃ after the agarose is dissolved, adding the anesthetized fry in the step (2) into a confocal culture dish with the diameter of 20mm, adding a corresponding low-melting-point agarose solution, quickly adjusting the posture of the fry to be the front side by using small tweezers, namely only seeing eyes on one side, enabling the enlarged front intestine part and the middle and rear intestines not to be covered by egg yolk, and keeping the fry parallel to the confocal culture dish on the same horizontal plane as much as possible;
4) imaging: taking pictures of the front side and the side of all the fries fixed in the step (3) in a laser confocal microscope (SP8, Leica) within 2 hours as soon as possible, setting software, and simultaneously finishing imaging of 3 channels of red, green and white light under a 10 multiplied objective lens;
5) statistical analysis: after data collection, processing of the picture data and counting of the number of cells with fluorescence in the mid-hind intestine (cell size about 10 microns) was performed using Leica Application Suite X and ImageJ software, statistical analysis was performed using GraphPad Prism 7.0.
In this example, first by crossing two homozygous transgenic markers, Tg (lyz: DsRED2) and Tg (mpeg1: EGFP), a Tg (lyz: DsRED2/mpeg1: EGFP) line containing both red and green fluorescent markers could be obtained, and two very important immune cells involved in innate immunity could be evaluated in one line at the same time: neutrophils (red), macrophages (green). By making and feeding the powder feed, the influence of a food-borne enteritis model on acute inflammatory reaction can be quickly established at the juvenile fish level, and a corresponding food-borne enteritis component-sinomenine can be evaluated and relieved on the basis of the bean pulp feed.
According to the modeling strategy of the innate immune phase in fig. 1A, at 9dpf, as shown in fig. 2B-2C, 2F-2G, both FM and 50SBM groups showed corresponding fluorescently labeled neutrophils and macrophages in the hindgut region, and the neutrophils were morphologically indistinguishable but diffusely distributed in the hindgut at the soybean meal replacement protein source (fig. 2B, 2B1, 2F 1); the macrophage morphology appeared more similar to the elongated cell morphology after 50% of the soybean meal replaced the protein source and was distributed in a dispersed manner (fig. 2C, 2C1, 2G 1). As shown in fig. 2I-2J, when the FM group and the 50SBM group were subjected to quantitative counting of the fluorescently labeled cells in the posterior intestinal region (red frame region in fig. 1B) at two time periods of 5dpf and 9dpf, more aggregation of the fluorescently labeled neutrophils and macrophages was found in the 50% soybean meal replacement protein source group compared to the FM group, and the difference in the amount was very significant (p values obtained by T-test statistical analysis were 0.005 and 0.0006, respectively), indicating that feeding soybean meal feed caused an acute inflammatory response. As shown in fig. 4A-4D, the addition of 35ppm sinomenine based on soybean meal feed resulted in a certain alleviation of the accumulation of neutrophils and macrophages in the middle and rear intestine, as compared to the 50SBM group; in terms of cell morphology, the addition of drugs did not alter the morphology of neutrophils, but macrophages were mostly elongated in shape (fig. 4a1-4D1), suggesting that more macrophages were differentiated and involved in acute inflammatory reactions. Quantitative statistical analysis of neutrophils and macrophages present in the gut at the 9dpf time point revealed that sinomenine addition very significantly alleviated neutrophil and macrophage accumulation in the gut, with T-test statistical analysis yielding p values of 0.048 and 0.0005, respectively.
In conclusion, in the embodiment, a food-borne enteritis model is established by feeding fish meal and soybean meal feed in the innate immunity stage and using an imaging method, so that acute inflammatory reaction of juvenile fish can be caused, which is expressed by the aggregation of neutrophils and macrophages in the middle and rear intestines on the one hand and the change of the morphology of the macrophages to a certain extent on the other hand, and the functional differentiation of the juvenile fish is suggested; on the basis of the soybean meal feed, a relieving component, namely sinomenine, is added, so that the aggregation of neutrophils and macrophages in the middle and rear intestines can be relieved, the acute inflammatory reaction of the middle and rear intestines can be reduced, the negative effects caused by global immunoreaction and the fact that the evaluation components are dissolved in water in a large amount in the traditional short-term soaking mode are avoided, and the accuracy of the evaluation effect is enhanced.
Example 2: application of young fish imaging of lymphocyte fluorescence labeling zebra fish strain in analysis of sinomenine-alleviated SBMIE effect
(1) Fish mixing and film-forming seedling maintenance
1) Preparing before fish preparation: the Tg (rag2: DsRed) and Tg (lck: lck-eGFP) feeding of the transgenic zebra fish is enhanced one week in advance, the transgenic zebra fish is fed three times after being saturated in half an hour every day, the cultivation water temperature is controlled at 28 +/-0.5 ℃, the illumination time is 14h, the darkness is 10h, and the rest cultivation conditions and experimental steps are the same as those in (1) in the example 1; rag2 label immature (differentiating) lymphocytes, lck being the marker of T lymphocyte maturation, the major lymphocyte population in the intestine;
2) and (3) test crossing to obtain a fluorescence experimental fish: hybridizing Tg (rag2: DsRed) and Tg (lck: lck-eGFP) homozygote adult fish with wild zebra fish, pairing a male and female fish with a fish tank at intervals overnight, pulling out a partition plate in the morning the next day, allowing the adult fish to naturally lay eggs for one hour, collecting fish eggs by using a 0.3 x Danielau's solution (egg water) working solution containing methylene blue, and putting the fish eggs into a 12cm x 5cm glass culture dish with about 100 grains/dish;
3) placing the fish eggs in an illumination incubator (28 +/-1 ℃, 14h of illumination and 10h of darkness), incubating, changing egg water (containing methylene blue) twice in the morning and at night every day, picking out dead fish eggs, and changing the egg water without the methylene blue after 3 days (3dpf) until young fish of 5dpf cultured starts to feed open feed;
(2) preparation of modeling feed
The same feed as used in example 1;
(3) feeding strategy required by modeling
1) After the transgenic zebra fish Tg (rag2: DsRed) and Tg (lck: lck-eGFP) are incubated for 5 days, the transgenic zebra fish are subpackaged into circular sterile culture dishes with the diameter of 150mm, and every 30 fish/dish are divided into three groups: group 1: FM, group 2: 50SBM, group 3: 50SBM +35ppm SN, three biological replicates per group;
2) feeding larval fish feed of Larva AP100 from 5dpf to 16dpf, feeding the larval fish for 10 days from 17dpf to 27dpf, and feeding 18-27 pdf zebra fish with the three kinds of feed respectively;
(4) fry anesthesia, fixation and living body imaging method
1) After the fry is fed to 27pf, the fry is anesthetized, fixed, imaged and subjected to data analysis, which is the same as (3) in the example 1;
2) statistical analysis: after data collection, the image data were processed and the number of fluorescence signals were counted using the Leica Application Suite X and ImageJ software, and the GraphPad Prism 7.0 was statistically analyzed.
In this example, young fish images of zebrafish lines fluorescently labeled with either Tg (rag2: DsRed) or Tg (lck: lck-eGFP) were used to analyze the effect of sinomenine-mitigating SBMIE.
Imaging of juvenile fish in the Tg (Rag2: DsRed) line As shown in FIG. 3(A-F), the distribution of 27dpf, Rag 2-DsRed-labeled lymphocytes in the mid-hind intestine, red-labeled Rag2 lymphocytes (as shown in FIGS. 3B1, 3E1) is shown; at 27dpf, on the one hand, red-labelled lymphocytes corresponding to Rag2 appeared in both FM and 50SBM groups, and the cell morphology was mostly rounded in the fish meal group (fig. 3B1), whereas Rag2 appeared after feeding soybean meal+Many of the lymphocytes exhibited irregular synapse shapes (FIG. 3E1), suggesting Rag2+The lymphocytes are differentiated in the process of food-borne enteritis; on the other hand, quantitative statistical analysis was performed on the number of lymphocytes appearing in the hindgut region of zebra fish, and as shown in fig. 3G, more Rag2 red fluorescence labeled lymphocytes appeared in the soybean meal feed group and were found to be aggregated in the hindgut compared to the FM group, and the difference was very significant (p)<0.001). Based on the food-borne enteritis model, when sinomenine is added at a concentration of 35ppm, on one hand, the number of round cells is increased in the Rag2 red fluorescence-labeled lymphocytes, and the number of synaptic cells is reduced (fig. 5B-5B 1); on the other hand, the accumulation of the Rag2 red fluorescence labeled lymphocytes in the middle and hind intestines caused by the soybean meal can be relieved, and quantitative statistical analysis (as shown in figure 5D) shows that the quantity of the Rag2 red fluorescence labeled lymphocytes is extremely remarkably reduced (p)<0.001)。
Juvenile fish imaging was performed in the Tg (lck: lck-eGFP) line, and FIG. 3(H-M) shows the accumulation of lck-eGFP green fluorescence labeled cells in the mid-hind intestine, and green labeled mature T lymphocytes in the channel (see FIGS. 3I, 3L). At 27dpf, corresponding lck-eGFP green fluorescence labeled lymphocytes appeared in both FM and 50SBM groups, and the cell morphology was irregular and synaptic, with no major difference in cell morphology between the two groups (fig. 3I1, 3L 1); then, quantitative statistical analysis (figure 3N) is carried out on lck green fluorescence labeled lymphocytes appearing in the middle and rear intestines, and the fact that establishment of a food-borne enteritis model can remarkably cause the mature T lymphocytes to gather in the middle and rear intestines (p is less than 0.0001) is found, and the fact that in the zebra fish juvenile food-borne enteritis model, the adaptive immune response is enhanced by increasing the number of the mature T lymphocytes in the acquired immune stage so as to deal with intestinal inflammation caused by bean pulp. On the one hand, oval cell number was recovered in cell morphology and synaptic cell number was decreased in comparison to 50SBM group after feeding bean meal feed containing 35ppm sinomenine based on food-borne enteritis model (fig. 5F, 5F 1); on the other hand, the accumulation of lck-eGFP green fluorescence labeled lymphocytes in the middle and later intestines can be effectively relieved by the bean pulp, and the cell number is extremely remarkably reduced (as shown in figure 5H, p is less than 0.001), which shows that the addition of the sinomenine can relieve the accumulation of mature T lymphocytes in the middle and later intestines caused by the bean pulp and change the morphology of partial lck green fluorescence labeled lymphocytes (suggesting a functional differentiation state) to relieve the adaptive immune response related to inflammation.
Based on Tg (rag2: DsRed) or Tg (lck: lck-eGFP) zebra fish strains, a food-borne enteritis model of the zebra fish juvenile fish induced by soybean meal in the acquired immune stage is established at present, and a alleviating component of the food-borne enteritis is evaluated by a fluorescence imaging technology, so that the state changes of immature lymphocytes and mature T lymphocytes can be tracked at a cell level, and a visual method can be conveniently and quickly used for accurately providing cytological evidence of acquired immune cells of a fish body under inflammation or alleviating conditions in the enteritis process in real time.
Example 3: application of zebra fish juvenile fish intestinal slice imaging in analysis of sinomenine-alleviated SBMIE effect
(1) Sample fixing and dewatering and wax dipping treatment
1) Taking 27pdf treated zebra fish fries of corresponding different groups (see example 2 for the fries of the acquired immune model), gently putting the fries into 4% paraformaldehyde fixing solution by using forceps, taking two fries of each group, and slicing the fries into posterior paraffin;
2) taking out the whole fish, flattening the whole fish, putting the fish into a dewatering box, putting the dewatering box into a hanging basket, and dewatering in a dewatering machine;
3) and (3) dehydrating: 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-absolute ethanol I30 min-absolute ethanol II 30 min;
4) and (3) transparency: adding ethanol after dehydration: toluene (1:1)5-10 min-xylene I5-10 min-xylene II 5-10 min;
5) wax penetration: melting paraffin I1h-65 ℃ at 65 ℃, melting paraffin II1h-65 ℃, melting paraffin III1 h;
(2) paraffin embedding and continuous section
1) Embedding: firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and corresponding labels are attached; cooling in a cooling table at the temperature of minus 20 ℃, taking out the wax block from the embedding frame after the wax is solidified, and finishing the wax block;
2) slicing: placing the trimmed wax block in a paraffin slicer for slicing to a thickness of 4 μm, floating the slices on 40 deg.C warm water of a spreading machine to spread the tissues, taking out the tissues by a glass slide, baking the slices in a 60 deg.C oven, baking with water to remove wax, taking out, and storing at normal temperature;
3) dewaxing: placing the slices in xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-75% alcohol 5min in sequence, and slowly washing with tap water to obtain samples;
(3) hematoxylin eosin staining
1) Hematoxylin staining: staining the slices with hematoxylin staining solution for 3-5min, washing with tap water, and washing the excess staining solution;
2) differentiation, trans-blue: differentiation of differentiation liquid, washing with tap water, returning blue of blue returning liquid, and immersing the sample in running water for washing;
3) eosin staining: the slices are dehydrated for 5min respectively by adding 85 percent and 95 percent gradient alcohol in sequence, and are dyed for 5min in eosin dye solution;
4) and (3) dehydrating: placing the slices in anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, and anhydrous ethanol III for 5 min;
5) and (3) transparency: placing xylene I5 min-xylene II 5min in sequence after dehydration;
6) sealing: sealing with neutral gum, and air drying in a fume hood;
(4) microscopic observation and imaging
Observing the sample by using a multispectral imaging microdissection system (Orlindus fluorescence microscope & PE), imaging under 10 x and 40 x objective lenses by using a bright field, and performing corresponding picture processing and GraphPad Prism 7.0 statistical analysis by using ImageJ at the later stage;
in this example, the HE staining of the hindgut region was performed at 27dpf using FM, 50SBM and 50SBM +35ppm SN, and the morphology of the hindgut sections was observed at a scale bar of 20 μm. As shown in fig. 6A and 6B, the FM group has a complete intestinal tract shape, has a relatively obvious intestinal villus height and intestinal crypt, the Lamina Propria (LP) has a certain thickness, after 50% of soybean meal replaces a protein source, the fold degree of intestinal villus is reduced after 50SBM group, the lamina propria of intestinal mucosa becomes thin, the position of nucleus of intestinal epithelium appears disorder, the number of goblet cells (vacuoles of epithelium layer after HE staining) of intestinal epithelium layer is reduced, and the depth of crypt becomes very shallow, which indicates that zebra fish juvenile fish can cause intestinal tract injury and destroy the intestinal mucosa immune barrier when eating plant feed with high soybean meal content. Figure 6C shows that, after addition of 35ppm sinomenine based on soybean meal feed, sinomenine restored the degree of intestinal mucosal folding, restored the lamina propria to normal thickness, visible significant crypt depth, and restored the number of goblet cells in the intestinal epithelial layer compared to the 50SBM group. Quantitative analysis of the height of intestinal villi in the three groups FM, 50SBM and 50SBM +35ppm SN showed: compared with a fish meal group, the intestinal villus height (p <0.01) can be remarkably reduced after the soybean meal feed is fed, and the intestinal villus height (p <0.01) can be remarkably increased after 35ppm of sinomenine is added on the basis of the soybean meal feed. In a word, intestinal tissue section observation is carried out on three different feed groups by analyzing the fry for the acquired immunity of the zebra fish juvenile enteritis model, so that the intestinal cavities of the middle and rear intestines develop completely at the time point of 27dpf and have an intestinal mucosa tissue structure similar to adult fish, and therefore, the acquired immunity analysis method of the zebra fish juvenile enteritis model can be adopted to evaluate the relieving effect of the medicament on the food-borne enteritis at the acquired immunity level, and the defect that the intestinal mucosa immunity development of early juvenile fish is not completed and the fish evaluation period is too long is overcome.

Claims (3)

1. The method for evaluating the components for relieving the food-borne enteritis based on the zebra fish juvenile fish imaging model is characterized by comprising the following steps:
(1) imaging analysis of zebra fish juvenile fish marked by innate immune cell fluorescence
Hybridizing transgenic zebra fish Tg (lyz: DsRED2) and Tg (mpeg1: EGFP) to obtain transgenic zebra fish Tg (lyz: DsRED2/mpeg1: EGFP) with double markers of neutrophils and macrophages, dividing the transgenic zebra fish fries which are double marked after hatching for 5 days into 3 groups, feeding the groups with experimental feeds, namely a fish meal group, a 50% soybean meal alternative protein source feed group and components to be tested, feeding the groups for 4 days, performing living imaging on the fries of different treatment groups under a laser confocal microscope on the 9 th day, and analyzing the quantity and the form change of the neutrophils marked by red fluorescence in intestinal tracts and the macrophages marked by green fluorescence;
(2) imaging analysis of zebra fish juvenile fish marked by acquired immune cell fluorescence
Carrying out side crossing on lymphocyte marker strain Tg (rag2: DsRed) or T lymphocyte marker strain Tg (lck: lck-eGFP) homozygote adult fishes with wild zebra fishes to obtain heterozygotes, incubating for 5 days, feeding juvenile fishes with commercial feeds until 17 days, dividing the transgenic zebra fishes into 3 groups on the 17 th day, feeding experimental feeds, namely fish meal groups, 50% bean pulp replacement protein source feed groups and components to be tested to obtain a zebra fish juvenile fish post-day immunoassay model for food-borne enteritis, carrying out in-vivo imaging on fries of different treatment groups on the 27 th day under a laser confocal microscope, and analyzing the number and morphological change of intestinal red fluorescence labeled lymphocytes and green fluorescence labeled mature T lymphocytes;
(3) imaging analysis of intestinal slices of zebra fish juvenile fish
And (3) establishing a food-borne enteritis zebra fish juvenile fish acquired immune analysis model according to the method in the step (2), and taking the 27 th-day fry to perform HE staining pathological analysis on a middle and rear intestinal tissue section, wherein the HE staining pathological analysis comprises inherent layer thickness, intestinal villus fold degree and crypt depth.
2. The method according to claim 1, wherein the mucosal tissue of the middle hindgut is analyzed for the degree of immune cell aggregation and histological pathology in steps (1), (2) and (3).
3. The method according to claim 1, wherein when the number of immune cells is reduced and the aggregation degree is reduced in the treatment groups of steps (1) and (2) compared with the group of 50% soybean meal instead of protein source feed, the lamina propria is restored to normal thickness and the folding degree of intestinal mucosa is restored in step (3), and the depth of crypt is obvious, the component to be detected has the effect of relieving the food-borne enteritis.
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