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Method for constructing spleen tissue cell line of acipenser dabryanus

A tissue cell and construction method technology, which is applied in the field of construction of sturgeon spleen tissue cell line, can solve the problems of difficult to obtain surviving individuals, difficult egg or embryo cryopreservation technology to overcome, difficult to mature parents and other problems, and achieves proliferation fast effect

Active Publication Date: 2015-09-09
CHINESE STURGEON RES INST CHINA THREE GOR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is usually difficult to obtain paired mature parents of wild Dabry's sturgeon, and it is even difficult to obtain surviving individuals, and the cryopreservation technology of eggs or embryos is difficult to overcome, and other technologies are urgently needed to make up for the shortcomings of existing technologies

Method used

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  • Method for constructing spleen tissue cell line of acipenser dabryanus
  • Method for constructing spleen tissue cell line of acipenser dabryanus
  • Method for constructing spleen tissue cell line of acipenser dabryanus

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Implementation object:

[0027] The spleen tissue of 1-year-old artificially cultured juvenile Dabry's sturgeon. The total length of the experimental fish is 48cm, the body length is 37.5cm, and the weight is 0.4kg.

[0028] Preparation of special culture medium for spleen cells of Acipenser dabryi:

[0029] Select healthy Dabry's sturgeon, take 2ml of blood from the tail vein, filter the separated serum with a 0.22-micron microporous membrane for future use, take Minimum Essential Medium (MEM) basal culture medium 31.55ml, add to it the 250μl of Sturgeon dabry's serum, 15ml of fetal bovine serum, penicillin, streptomycin, amphotericin B, human epidermal growth factor and human basic fibroblast-like cell growth factor, so that the volume concentration of sturgeon serum accounted for the special 5% of the volume of the culture medium, the volume concentration of fetal bovine serum accounts for 30% of the volume of the special culture medium, the mass concentration of pe...

Embodiment 2

[0037] Implementation object:

[0038] The spleen tissue of 1-year-old artificially cultured juvenile Dabry's sturgeon. The total length of the experimental fish is 43.5 cm, the body length is 34.5 cm, and the weight is 0.4 kg.

[0039] Preparation of special culture medium for spleen cells of Acipenser dabryi:

[0040] Select healthy Acipenser dabryi, extract 1.5ml of tail vein blood, filter and sterilize the separated serum for later use, take Minimum Essential Medium (MEM) basal culture medium 31.4ml, add sterilized Dabry's serum to it 500μl, 15ml of fetal bovine serum, penicillin, streptomycin, amphotericin B, human epidermal growth factor and human basic fibroblast-like cell growth factor, so that the serum volume concentration of Dabry's sturgeon accounts for 10% of the volume of the special culture medium, The volume concentration of fetal bovine serum accounts for 30% of the volume of the special culture medium, the mass concentration of penicillin and streptomycin is...

Embodiment 3

[0048] Implementation object:

[0049] Spleen tissue of 1-year-old artificially cultured juvenile Dabry's sturgeon. The total length of the experimental fish is 40cm, the body length is 31.5cm, and the weight is 0.36kg.

[0050] Preparation of special culture medium for spleen cells of Acipenser dabryi:

[0051] Select healthy Dabry's sturgeon, take 2.5ml of blood from the tail vein, filter and sterilize the separated serum for later use, take 31.45ml of Minimum Essential Medium (MEM) basal culture medium, add the filtered and sterilized Dabry's sturgeon serum to it 400μl, 15ml of fetal bovine serum, penicillin, streptomycin, amphotericin B, human epidermal growth factor and human basic fibroblast-like cell growth factor, so that the serum volume concentration of Dabry's sturgeon accounts for 8% of the volume of the special culture medium, The volume concentration of fetal bovine serum accounts for 30% of the volume of the special culture medium, the mass concentration of pen...

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Abstract

The invention provides a method for constructing a spleen tissue cell line of acipenser dabryanus. The method mainly comprises the following steps: (1), an MEM (minimum essential medium) culture solution containing fetal calf serum, human epidermal growth factors, human fibroblast-like cell growth factors and acipenser dabryanus serum and having the pH of 7.2-7.4 is prepared and stored in a refrigerator at the temperature of 4 DEG C for standby application; (2), spleen tissue is separated from the acipenser dabryanus body and subjected to primary culture with a tissue block culture method; (3), when the cell growth forms a single layer and the bottom coverage rate is higher than 70%, 0.25% trypsin digestion is adopted for subculture; (4), vigorously growing spleen cells with uniform forms are subjected to liquid nitrogen cryopreservation and resuscitaion culture. The spleen tissue cell line constructed with the method is fast to reproduce, has multiple forms, is fibroblast-like, and can realize continuous passage and cryopreservation resuscitation. The construction method is simple in technology, easy to operate, can be popularized to culture of spleen cells of other sturgeons and can be expected to be used for acipenser dabryanus species resource conservation as well as separation and identification of sturgeon diseases especially virus pathogens.

Description

technical field [0001] The invention relates to a method for constructing a sturgeon spleen tissue cell line, belonging to the technical field of cell culture. Background technique [0002] Dabry's sturgeon (Acipenser dabryanus), also known as the Yangtze River sturgeon, is mainly distributed in the upper and middle reaches of the Yangtze River and the lower reaches of the Jinsha River. Due to overfishing, water pollution, construction of water conservancy projects, etc., the resources of Dabry's sturgeon in the Yangtze River Basin are extremely scarce. Preserving its eggs or embryos is undoubtedly of great significance to the protection of the endangered Dabry's sturgeon species. However, it is usually difficult to obtain paired mature parents of wild Dabry's sturgeon, and it is even difficult to obtain surviving individuals, and the cryopreservation technology of eggs or embryos is difficult to overcome, so other technologies are urgently needed to make up for the deficie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 刘娟娟杜合军肖衎刘雪清赵珣
Owner CHINESE STURGEON RES INST CHINA THREE GOR
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