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Method for isolated culture of intestinal epithelial cells of Takifugu obscurus and establishment of stress model

A technology of fugu obscurus and intestinal epithelial cells, applied in artificial cell constructs, animal cells, vertebrate cells, etc., to achieve the effects of removing intestinal pollutants, improving cell survival rate, and inhibiting bacterial contamination

Pending Publication Date: 2022-07-29
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no good stress model at the intestinal cell level in aquatic animals, and the establishment of intestinal epithelial cell stress model can become one of the effective methods to study the mechanism of oxidative stress damage in fish cells

Method used

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  • Method for isolated culture of intestinal epithelial cells of Takifugu obscurus and establishment of stress model
  • Method for isolated culture of intestinal epithelial cells of Takifugu obscurus and establishment of stress model
  • Method for isolated culture of intestinal epithelial cells of Takifugu obscurus and establishment of stress model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Separation and culture of intestinal epithelial cells of pufferfish with dark stripes:

[0036] (1) Select juvenile puffer fish with a body length of 5±0.5cm, and culture the puffer fish in 10ppt saline for more than 7 days before taking the intestinal tissue. During the period, feed containing 10U / kg neomycin and 20U / kg Ampicillin feed, juveniles were fasted for 24 hours before sampling.

[0037] (2) Aseptically dissect the middle intestine after anesthesia, put it in the self-prepared cleaning solution, remove the mesentery, cut the intestine longitudinally, scrape off the contents with elbow tweezers, and wash twice with the self-prepared cleaning solution . Put it into a 1.5ml sterile centrifuge tube and cut it into a volume of 0.5-0.8mm 3 tissue block. The formula of the self-made cleaning solution used is: Na 2 HPO 4 : 1.42g; K 2 HPO 4 : 0.24g; NaCl: 10g; KCl: 0.3g; vitamin C: 1g; D-glucose: 1g;

[0038] (3) Add 1 ml of 0.25% collagenase IV digestion so...

Embodiment 2

[0050] Example 2 is basically the same as Example 1 (Isolation and Culture of Intestinal Epithelial Cells of Oriental Pufferfish), and the difference is only:

[0051] The feed fed contains 20U / kg neomycin and 15U / kg ampicillin;

[0052] In the self-prepared cleaning solution used, the formula is: Na 2 HPO 4 : 1.42g; K 2 HPO 4 : 0.24g; NaCl: 8g; KCl: 0.2g; Vitamin C: 1g; D-glucose: 1g; .

[0053] The formula of the complete medium used is: DMEM + fetal bovine serum + pufferfish serum: 16:2:2, and 400 μl of double antibody solution (100 Units / ml of penicillin, 100 μg / ml of streptomycin) was added to each 100 ml of culture medium.

[0054] The results were observed microscopically, and cell viability was measured by CCK method (such as Image 6 shown), the results are basically the same as in Example 1, which proves that the selection range is reliable.

Embodiment 3

[0056] Embodiment 3 is basically the same as embodiment 1 (the separation and culture of intestinal epithelial cells of pufferfish with dark stripes), and the difference is only the difference of the cleaning solution, as follows:

[0057] After anesthesia, the intestines of the middle section of the pufferfish with dark stripes were aseptically dissected and removed, washed in PBS, the mesentery was removed, the intestines were cut longitudinally, the contents were scraped off with elbow forceps, and washed twice with PBS. Put it into a 1.5ml sterile centrifuge tube and cut it into a volume of 0.5-0.8mm 3 tissue block.

[0058] The results showed that the cell viability was significantly reduced (e.g. Image 6 shown), compared with Example 1, it shows that the self-prepared cleaning solution can effectively improve the cell viability.

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Abstract

The invention discloses a method for isolated culture of intestinal epithelial cells of Takifugu obscurus and establishment of a stress model. According to the technical scheme, firstly, feed treated with antibiotics is fed in the takifugu obscurus breeding process; secondly, selecting the midgut part of the intestinal tract of takifugu obscurus; then, by optimizing a high-efficiency isolated culture technology of the Takifugu obscurus intestinal epithelial cells and cell primary culture conditions, the Takifugu obscurus intestinal epithelial cells which are relatively high in purity and good in growth are obtained; and finally, utilizing cortisol to stimulate intestinal epithelial cells of Takifugu obscurus so as to establish the oxidative stress model. The invention provides a powerful research material for application of fugu obscurus in research of developmental biology, immunology, toxicology, environment detection and the like.

Description

technical field [0001] The invention belongs to the field of aquaculture, and in particular relates to a method for separating and culturing intestinal epithelial cells of pufferfish with dark stripes and establishing a stress model. Background technique [0002] Takifugu fasciatus is an anadromous migratory fish that spends most of its time in the ocean. Its suitable growth temperature range is 23-32°C. It is one of the three fresh foods in the Yangtze River in China, mainly distributed in China Offshore areas and the middle and lower reaches of the Yangtze River. In recent years, the breeding scale of pufferfish in China has gradually expanded. According to the "China Fisheries Statistical Yearbook", the amount of freshwater aquaculture in China in 2020 will exceed 26,000 tons, of which the aquaculture of pufferfish with dark stripes is relatively large. The intestinal tract of fish is an important organ of permeability and acid-base regulation. The digestive ability and...

Claims

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Application Information

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IPC IPC(8): C12N5/071A01K61/10
CPCC12N5/0679A01K61/10C12N2509/10C12N2509/00C12N2501/39
Inventor 王涛马思思尹绍武孙亦如张莹刘雨曦
Owner NANJING NORMAL UNIVERSITY
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