Construction method for inducing larimichthys polyactis liver cell apoptosis model through high-temperature stress

A technology of liver cells and small yellow croaker, applied in climate change adaptation, animal husbandry, etc., can solve problems such as difficulty in standardization, large differences in research results, and little mutual reference between research results.

Active Publication Date: 2021-05-14
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no uniform technical specification for the in vivo experiments on the effect of high temperature on the apoptosis of fish liver cells. The research results are very different and difficult to standardize, resulting in little mutual reference between the research results.

Method used

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  • Construction method for inducing larimichthys polyactis liver cell apoptosis model through high-temperature stress
  • Construction method for inducing larimichthys polyactis liver cell apoptosis model through high-temperature stress
  • Construction method for inducing larimichthys polyactis liver cell apoptosis model through high-temperature stress

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Experimental processing and sample collection

[0015] Take the 6-month-old small yellow croaker full-sibling family with healthy body and no injury on the body surface, and divide them into control group C and experimental group T, with 3 parallels in each group, and 30 experimental fishes are stocked in each parallel, and they are cultured at 0.3m 3 During the temporary breeding period, the water temperature is controlled at 20±0.5°C, dissolved oxygen>6.0mg / L, pH=7.7-7.9, and fed twice a day (07:00 and 16:00) Artificial compound feed, the diameter of the feed particles is about 3mm, and the feed is full. Half an hour after feeding, each bucket is cleaned and 70% of the water is changed. At the beginning of the experiment, the feeding was stopped, the breeding conditions of the control group remained unchanged, and the experimental group was heated at a constant rate of 2°C / h by means of a heating rod. When the temperature reached 32°C, the temperature wa...

Embodiment 2

[0016] Example 2: Determination and analysis of oxidative stress indicators

[0017] For the samples stored at low temperature, the activities of SOD, CAT and GSH-PX and the content of MDA were detected respectively according to the kit instructions. The results showed that after treatment at 32°C for 0 h, the four oxidative stress indicators were not significantly different from those in the control group ( P >0.05), this is the early stage of body exposure to high temperature stress, and the stress response has not yet been induced. After treatment at 32°C for 6 hours, the antioxidant enzyme system began to function, and the activities of antioxidant enzymes SOD and GSH-PX were significantly increased ( P figure 1 , showing oxidative stress.

Embodiment 3

[0018] Example 3: Determination and analysis of the expression level of the key endoplasmic reticulum gene GRP94

[0019] (1) RNA extraction and cDNA synthesis

[0020] The total RNA of liver samples was extracted according to the Trizol method, and its integrity was detected by 2.0% agarose gel electrophoresis, and the concentration and quality of the extracted RNA were measured using a NanoDrop One ultra-micro spectrophotometer. The first strand of cDNA was synthesized according to the instructions of the Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit, and the obtained cDNA solution was stored in a -20°C refrigerator for later use.

[0021] (2) Cloning and analysis of gene sequence fragments

[0022] According to the transcriptome data of the small yellow croaker, the CDS reference sequence of the GRP94 gene of the small yellow croaker was obtained through bioinformatics analysis, and the primers were designed according to Table 1.

[0023...

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Abstract

The invention belongs to the technical field of oxidative stress models, and particularly relates to a construction method for inducing a larimichthys polyactis liver cell apoptosis model through high-temperature stress. The construction method specifically comprises the following steps: when the culture water temperature is about 20 DEG C, treating larimichthys polyactis according to the heating rate of 2 DEG C / h, raising the water temperature to 32 + / -0.5 DEG C, and treating the larimichthys polyactis at the temperature for 6-12 h, so that the larimichthys polyactis is subjected to oxidative stress reaction to induce apoptosis. According to the construction method, the basic water temperature, the heating rate, the high temperature needing to be reached and the processing time of the high temperature before high-temperature stress are determined, and evaluation indexes for model establishment are provided. The larimichthys polyactis liver cell apoptosis model established through high-temperature stress can be used for researching pathogenesis of the larimichthys polyactis liver oxidative injury, and has important theoretical and practical significance for revealing the larimichthys polyactis liver oxidative injury mechanism and researching and developing efficient liver protection drugs.

Description

technical field [0001] The invention belongs to the technical field of oxidative stress model construction, and in particular relates to a method for constructing a liver cell apoptosis model induced by high temperature stress. Background technique [0002] Small yellow croaker, as one of the "four seafoods" in my country, has very important economic value. With the maturity of large-scale artificial breeding and breeding technology, the small yellow croaker breeding industry will develop rapidly. In the breeding process, the temperature of the water body is directly related to the survival rate and growth rate of the breeding objects. Studies have shown that the water temperature of 32°C will cause oxidative stress in small yellow croakers, and in severe cases, it will lead to a large number of deaths. Therefore, elucidating the molecular mechanism of small yellow croaker's response to high temperature stress and providing effective technical means to relieve high tempera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02
CPCA01K67/02A01K2207/35A01K2227/40A01K2267/03Y02A40/81
Inventor 刘峰楼宝詹炜
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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