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Method for evaluating food-borne enteritis relieving ingredients based on zebrafish larvae imaging model

An imaging model, zebrafish technology, applied in fish farming, analytical materials, food processing, etc., can solve the problems of large differences and weaken the response of intestinal immune cells, so as to shorten the evaluation period, reliable evaluation methods, and overcome analysis The long-term effect

Active Publication Date: 2021-06-22
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the use of zebrafish larvae imaging for intestinal inflammation research still has the following problems: (1) the systemic mucosal reaction caused by short-term immersion may weaken the response of intestinal immune cells and act as a short-lived neutrophils. There are large differences in different fish bodies. On the other hand, the phagocytosis of macrophages is very important for the maintenance of fish intestinal health, and the response of macrophages caused by immersion is mainly in the parts that play a role in swallowing. The foregut, rather than the main reaction site of the intestinal mucosa—the mid-hind gut; (2) The choice of feed feeding time period plays an important decisive role in the normal development of the intestinal tract and the presentation of immune responses, but most of the existing reports are from Open feed feeding at 5dpf to 7dpf can only induce the response of intestinal innate immune cells at this stage, and the research on the intestinal development to acquired immune cell residence stage at 3 to 4 weeks is still very limited, only T cells Participation in individual reports of soybean meal-induced enteritis is based on before 9dpf

Method used

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  • Method for evaluating food-borne enteritis relieving ingredients based on zebrafish larvae imaging model
  • Method for evaluating food-borne enteritis relieving ingredients based on zebrafish larvae imaging model
  • Method for evaluating food-borne enteritis relieving ingredients based on zebrafish larvae imaging model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Application of Imaging of Innate Immunofluorescence-labeled Juvenile Zebrafish Strains in Analyzing the Effect of Sinomenine on Alleviating Soybean Meal-Induced Enteritis

[0036] (1) Preparation of double transgenic zebrafish Tg(lyz:DsRED2 / mpeg1:EGFP)

[0037]1) Feed the transgenic zebrafish Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) that have been reared separately one week in advance, and feed them three times a day for half an hour. The temperature of the breeding water is controlled at 28±0.5°C, and the light time is 14h , dark for 10 hours, the environment of the zebrafish breeding system is pH: 7.0-8.0, salinity 0.25-0.50‰, dissolved oxygen 5-8mg / L, total ammonia nitrogen less than 0.02mg / L, other conditions refer to the National Zebrafish Breeding Center Request (http: / / www.zfish.cn);

[0038] 2) One female and one male corresponding to the fluorescent strains of Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) were paired overnight in a matching fish tank, and the separato...

Embodiment 2

[0071] Example 2: Application of imaging of juvenile zebrafish strains with fluorescently labeled lymphocytes in analyzing the effect of sinomenine on alleviating SBMIE

[0072] (1) Maintenance of fish mating and film-forming seedlings

[0073] 1) Preparation before fish mating: feed the transgenic zebrafish Tg(rag2:DsRed) and Tg(lck:lck-eGFP) one week in advance, and feed them three times a day for half an hour. The time is 14h, dark 10h, all the other cultivation conditions and experimental steps are the same as (1) in the embodiment 1; rag2 marks the immature (divide) lymphocyte, lck is the mark of T lymphocyte maturation, and the main lymphocyte in the intestine taxa;

[0074] 2) Test cross to obtain fluorescent experimental fish: cross Tg(rag2:DsRed) and Tg(lck:lck-eGFP) homozygous adult fish with wild-type zebrafish, one female and one male were paired overnight in a mating tank, and the second Unplug the separator every morning, let it spawn naturally for one hour, us...

Embodiment 3

[0088] Example 3: Application of imaging of larval zebrafish intestinal slices in analyzing the effect of sinomenine on alleviating SBMIE

[0089] (1) Sampling and fixing, dehydration and wax immersion treatment

[0090] 1) Get the zebrafish fry after 27pdf of corresponding different groups of treatments (the fry of the acquired immune model is shown in Example 2), gently put the fry into 4% paraformaldehyde fixative solution with tweezers, and take two for each group. Paraffin section of the hindgut was carried out;

[0091] 2) Take out the whole fish, flatten it and put it into the dehydration box, put the dehydration box into the hanging basket and dehydrate it in the dehydrator;

[0092] 3) Dehydration: 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h-absolute ethanol I 30min-absolute ethanol II 30min;

[0093] 4) Transparent: after dehydration, add ethanol: toluene (1:1) 5-10min-xylene I 5-10min-xylene II 5-10min;

[0094] 5) Through the wax: 65 ℃ melting pa...

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Abstract

The invention discloses a method for evaluating food-borne enteritis relieving ingredients based on a zebrafish larvae imaging model. According to the method, a food-borne enteritis model is constructed on the basis of innate immune cell double-labeling strain and acquired immune cell single-labeling strain Tg transgenic fluorescence-labeled zebrafish; and by feedstuff feeding, in time periods when innate / acquired immune cells act, a fluorescence imaging visualization mode is adopted to effectively present behaviors of various immune cells participating in the food-borne enteritis process, which are used for evaluating the fish food-borne enteritis relieving ingredients. According to the method, the evaluation period is effectively shortened, the influence of the food-borne enteritis relieving ingredients on innate immunity and acquired immunity is more completely shown, meanwhile the defects that a traditional adult fish model is long in test period and complex in operation are overcome, and the problem that whole-body mucosal tissue immunoreaction is caused by short-term soaking of juvenile fishes is also avoided.

Description

technical field [0001] The invention belongs to the field of fish nutritional immunology, and specifically relates to a method for establishing a food-borne enteritis model based on zebrafish juvenile immune cell imaging, and using the model to evaluate or screen components that can alleviate fish food-borne enteritis method. Background technique [0002] Foodborne enteritis is an important limiting factor that causes fish diseases and growth inhibition in aquatic production applications, and has increasingly attracted the attention of the academic community. The researches on fish nutrition, immunology, and intestinal microbiology at home and abroad have all been in the model. Animals have been extensively performed on zebrafish, and diets with 50% soybean meal replacing protein sources have been used as a common formula for modeling enteritis in zebrafish. As a faster way, the intestinal inflammation modeling of juvenile zebrafish is also being explored by multiple teams....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K61/10A23K50/80A23K10/30A23K10/22A23K10/37G01N21/64G01N21/84
CPCA01K61/10A23K50/80A23K10/30A23K10/22A23K10/37G01N21/6428G01N21/84G01N2021/6439Y02P60/87Y02A40/81Y02A40/818
Inventor 夏晓勤吴南黎明程莹寅
Owner INST OF AQUATIC LIFE ACAD SINICA
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