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Method for constructing sinocyclocheilus rhinocerous heart cell line

A technology of rhinoceros horn, golden thread barb, and construction method, which is applied to artificial cell constructs, microorganism-based methods, animal cells, etc., can solve problems such as no similar reports, and achieves repeatability, improved success rate, and large cell volume. Effect

Active Publication Date: 2016-02-24
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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  • Method for constructing sinocyclocheilus rhinocerous heart cell line
  • Method for constructing sinocyclocheilus rhinocerous heart cell line
  • Method for constructing sinocyclocheilus rhinocerous heart cell line

Examples

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Effect test

Embodiment 1

[0026] 1. Preparation of PBS Disinfection Solution and Cell Culture Solution

[0027] PBS disinfectant: Add antibiotics to PBS to make the concentration of penicillin 100IU / ml, streptomycin 100μg / ml, streptomycin sulfate 40μg / ml and amphotericin B 40μg / ml.

[0028] Basic medium: glutamine and fetal bovine serum were added to the M199 medium to make the concentration of glutamine 300 μg / ml, and the fetal bovine serum accounted for 15% of the total volume.

[0029] Primary culture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium to make the concentration of glutamine 600 μg / ml, the fetal bovine serum to account for 25% of the total volume, and the concentration of penicillin to be 100 IU / ml, The concentration of streptomycin was 100 μg / ml, the concentration of streptomycin sulfate was 40 μg / ml, and the concentration of amphotericin B was 40 μg / ml.

[0030] Subculture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium so that...

Embodiment 2

[0046] 1. Preparation of PBS Disinfection Solution and Cell Culture Solution

[0047] PBS disinfectant: Add antibiotics to PBS to make the concentration of penicillin 300IU / ml, streptomycin 300μg / ml, streptomycin sulfate 20μg / ml and amphotericin B 20μg / ml.

[0048] Basic medium: glutamine and fetal bovine serum were added to the M199 medium to make the concentration of glutamine 400 μg / ml, and the fetal bovine serum accounted for 10% of the total volume.

[0049] Primary culture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium, so that the concentration of glutamine is 800 μg / ml, the fetal bovine serum accounts for 15% of the total volume, and the concentration of penicillin is 200 IU / ml, The concentration of streptomycin was 30 μg / ml, the concentration of streptomycin sulfate was 20 μg / ml, and the concentration of amphotericin B was 20 μg / ml.

[0050] Subculture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium so that t...

Embodiment 3

[0061] 1. Preparation of PBS Disinfection Solution and Cell Culture Solution

[0062] PBS disinfectant: Add antibiotics to PBS to make the concentration of penicillin 300IU / ml, streptomycin 300μg / ml, streptomycin sulfate 20μg / ml and amphotericin B 20μg / ml.

[0063] Basic medium: glutamine and fetal bovine serum were added to the M199 medium to make the concentration of glutamine 400 μg / ml, and the fetal bovine serum accounted for 10% of the total volume.

[0064] Primary culture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium, so that the concentration of glutamine is 800 μg / ml, the fetal bovine serum accounts for 15% of the total volume, and the concentration of penicillin is 200 IU / ml, The concentration of streptomycin was 30 μg / ml, the concentration of streptomycin sulfate was 20 μg / ml, and the concentration of amphotericin B was 20 μg / ml.

[0065] Subculture medium: add glutamine, fetal bovine serum and antibiotics to the M199 medium so that t...

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Abstract

The invention relates to a method for constructing a sinocyclocheilus rhinocerous heart cell line. The method comprises the following steps: 1, obtaining of sinocyclocheilus rhinocerous heart tissues, wherein sinocyclocheilus rhinocerous is sterilized through combination of penicillin and ethyl alcohol; 2, primary culture, wherein a culture solution M199 which contains glutamine, penicillin, streptomycin, streptomycin sulphate and amphotericin is adopted for culture, and the osmotic pressure of the culture solution is 280-330 mOsm / l; 3, secondary culture, wherein when the tenth generation is reached, cell culture fluid is replaced with base culture fluid, and the sinocyclocheilus rhinocerous heart cell line is constructed successfully. According to the method for constructing the sinocyclocheilus rhinocerous heart cell line, the obtained sinocyclocheilus rhinocerous heart cell line is in the fibrous form and can conduct continuous passage and be directly applied to research of biological characteristics, requirements of genetic resource preservation and theoretical research conducted on the rare species, namely the sinocyclocheilus rhinocerous, can be satisfied, meanwhile, the heart cell line is the first heart cell line of cave fishes in China, and the foundation is laid for cave adaptability research at the cellular level.

Description

technical field [0001] The invention relates to a method for establishing a cell line by utilizing the heart tissue of rhinoceros horn, and belongs to the technical field of freshwater aquatic organism cell culture and ultra-low temperature cryopreservation. Background technique [0002] Sinocyclocheilusrhinocerous (Regan, 1904) belongs to the genus Sinocyclocheilus (Cypriniformes), Cyprinidae, and is a new cave species published in 1994. At present, the known distribution points of this species It is an underground river surrounding urban and rural areas in Luoping County, Yunnan Province. It belongs to the Nanpanjiang River system. The research on it can only be found in the description of its taxonomic characteristics, the confirmation of its taxonomic status and its phylogenetic relationship. [0003] Due to the small population of rhinoceros horned barb and difficult to obtain, how to protect and preserve the germplasm resources of this rare cave fish has become an urge...

Claims

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Application Information

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IPC IPC(8): C12N5/071A01N1/02C12R1/91
Inventor 王晓爱潘晓赋杨君兴杨坤凤刘倩
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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