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Method for culturing primary gill cells of takifugu obscurus

A technology of puffer obscurus and cell culture, which is applied in the field of cell culture, can solve the problems of studying the mechanism of disease damage, etc., and achieve the effects of stable physiological state, breaking through research bottlenecks, and high cell yield

Active Publication Date: 2019-07-05
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Fugu obscurus, there has been no successful report on the study of the mechanism of disease damage by culturing corresponding cells.

Method used

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  • Method for culturing primary gill cells of takifugu obscurus
  • Method for culturing primary gill cells of takifugu obscurus
  • Method for culturing primary gill cells of takifugu obscurus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0027] 1. Cell culture

[0028] a. Pufferfish obscurus gill tissue fragmentation

[0029] Grab puffer obscura (breeding condition is 3-5 days in a water environment with a salinity of 5-10), wipe the fish body with 75% alcohol, take out the gills and put them in a cell culture dish containing soaking liquid, soak for 3-5 days 5min; transfer the fish gills to a petri dish containing cleaning solution, and wash them repeatedly for 5-10 times; figure 1 , It can be seen from the figure that the gill tissue after cleaning with the cleaning solution is clear and tidy, the physiological state is good, and there is no blood stain left.

[0030] The preparation method of the soaking solution is: add NaCl to pure water until the salinity is 5-10, and add 50 μl double antibody solution (Penicillin 100Units / ml, Streptomycin 100μg / ml) into 100ml soaking solution, 0.22μm micropore Membrane filtration sterilization;

[0031] Cleaning solution formula: CaCl 2 (Anhydrous) 0.14g; KCl 0.5g; ...

Embodiment 2

[0051] It is basically the same as Example 1, the difference is only as follows:

[0052] The preparation method of the soaking solution is: add NaCl to the pure water until the salinity is 5-10, and add 40 μl double antibody solution (Penicillin 100Units / ml, Streptomycin 100μg / ml) to 100ml soaking solution, 0.22μm micropore Membrane filtration sterilization;

[0053] Cleaning solution formula: CaCl 2 (Anhydrous) 0.14g; KCl 0.4g; KH 2 PO 4 0.06g; MgCl 2 ·6H 2 O 0.10g; MgSO 4 ·7H 2 O 0.10g; NaCl 10.0g; NaHCO 3 0.35g; Na 2 HPO 4 ·7H 2 O 0.06g; D-glucose 1.0g; phenol red 0.02g; ultrapure water to 1 liter. And add 90μl double antibody solution to 100ml cleaning solution, filter and sterilize with 0.22μm microporous membrane, the penicillin in the double antibody solution is 100Units / ml, streptomycin is 100μg / ml;

[0054] The preparation method of complete culture medium: DMEM basal medium, add fetal bovine serum, the volume ratio of DMEM basal medium and fetal bovine...

Embodiment 3

[0057] It is basically the same as Example 1, the difference is only as follows:

[0058] The preparation method of the soaking solution is: add NaCl to the pure water until the salinity is 5-10, and add 60 μl double antibody solution (Penicillin 100Units / ml, Streptomycin 100μg / ml) into 100ml soaking solution, 0.22μm micropore Membrane filtration sterilization;

[0059] Cleaning solution formula: CaCl 2 (Anhydrous) 0.14g; KCl 0.6g; KH 2 PO 4 0.06g; MgCl 2 ·6H 2 O 0.10g; MgSO 4 ·7H 2 O 0.10g; NaCl 12.0g; NaHCO 3 0.35g; Na 2 HPO 4 ·7H 2 O 0.06g; D-glucose 1.0g; phenol red 0.02g; ultrapure water to 1 liter. And add 110μl of double antibody solution to 100ml of cleaning solution, filter and sterilize with 0.22μm microporous membrane, the penicillin in the double antibody solution is 100Units / ml, streptomycin is 100μg / ml;

[0060] The preparation method of the complete culture medium: DMEM basal medium, add fetal bovine serum, the volume ratio of DMEM basal medium and...

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Abstract

The invention discloses a method for culturing primary gill cells of takifugu obscurus. The method comprises the following steps: (1) taking a gill tissue of the takifugu obscurus, soaking with a soaking solution containing double-resistant penicillin and streptomycin, cleaning with a cleaning solution, and cutting gill filaments; (2) digesting the gill filaments by using trypsin, filtering, centrifuging and taking precipitates; and then removing red blood cells by using a red blood cell lysate, and centrifuging to obtain cell precipitates; and (3) adding a complete culture solution into the cell precipitates, blowing the cells, supplementing the complete culture solution, and culturing. Compared with the prior art, the method provided by the invention is applied to culture of the primarygill cells of the takifugu obscurus, tissues can be effectively separated, the cell yield is high after gill digestion treatment, the cultured cells grow well, the physiological state is stable, the experimental research standard is achieved, the research approach of various disease problems possibly occurring in takifugu obscurus culture production can be fundamentally provided, and the bottleneck of research of the takifugu obscurus is broken through.

Description

technical field [0001] The invention relates to a method for culturing primary gill cells of puffer obscura, belonging to the technical field of cell culture. Background technique [0002] Fish gills are important respiratory organs of bony fishes. The direction of blood flow in gills is opposite to that of water flow, which greatly promotes the gas exchange rate of fish. At the same time, fish gills are also one of the important organs to maintain homeostasis, including acid-base balance, osmotic pressure regulation, and excretion of metabolic waste. In water, gill filaments are fully unfolded, and water temperature, pH value, salinity, metal ions, etc. all directly or indirectly affect the physiological state of gill filaments. By observing the damage of gill filaments and gill arches, the physiological state of fish can be initially reflected. Many studies have confirmed that certain proteins in gill cells are involved in body homeostasis regulation when subjected to ex...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2509/00
Inventor 王涛胡亚东尹绍武魏小珍褚鹏
Owner NANJING NORMAL UNIVERSITY
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