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Turbot liver tissue cell in-vitro establishing method and application thereof

A technology of liver tissue and turbot, applied in artificial cell constructs, applications, animal cells, etc., can solve problems such as poor repeatability, slow growth, and cell emigration, and achieve low cost, promote emigration, and rapid cell growth Effect

Inactive Publication Date: 2016-01-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the primary culture of turbot liver tissue cells has been reported before, it has the disadvantages of poor reproducibility, cell migration and slow growth.

Method used

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  • Turbot liver tissue cell in-vitro establishing method and application thereof
  • Turbot liver tissue cell in-vitro establishing method and application thereof
  • Turbot liver tissue cell in-vitro establishing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 A method for primary culture of turbot liver tissue cells.

[0019] Proceed as follows:

[0020] (1) The preparation of culture medium, its step is as follows:

[0021] L-15 liquid medium was purchased from Sigma Company, 100× penicillin and streptomycin solution and 100× sodium pyruvate solution were purchased from Suleibao Company, fetal bovine serum was purchased from Gibco Company, L-alanyl-L-glutamine Amide was purchased from Life Company, and growth factor was purchased from Peprotech Company;

[0022] The preparation method of perch serum: about 2kg of perch blood is taken from the tail vein, and it is made by sterilizing through a 0.22μm filter after bathing in water at 56°C for 30 minutes;

[0023] Preparation of growth factor bFGF and EGF solutions: respectively dissolved in 0.1% bovine serum albumin solution to make 10 mg / ml mother solution, and stored at -20°C after aliquoting.

[0024] Preparation of basal medium: add 1ml of 100× penicillin and...

Embodiment 2

[0046] Example 2: Reporter plasmid transfection of primary cultured cells of turbot liver tissue.

[0047] 72 hours after the start of the primary culture of turbot liver tissue cells, the PIRES2-eGFP reporter plasmid was transfected into the cultured cells in vitro. After 24 hours, the successful expression of green fluorescent protein could be observed, proving that the constructed turbot liver tissue In vitro models of cells are available for molecular cell biology studies in fish.

[0048] Specific steps:

[0049] (1) Primary culture of turbot liver tissue cells, the specific steps are as described above.

[0050] (2) 72 hours after the initiation of the primary culture, the PIRES2-eGFP reporter plasmid was transfected into the cultured cells in vitro.

[0051] Specific transfections include:

[0052] ①The turbot liver tissue cells were mixed with 10 6 The density per well was inoculated into 6-well plates, cultured in a biochemical incubator at 24°C, and the old mediu...

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Abstract

The invention provides a turbot liver tissue cell in-vitro establishing method and application thereof. The method is characterized in that turbot liver tissue serves as the experimental material, the type-II collagenase enzymolysis approach is used for starting the primary culture of fish liver cells, and a novel L-15 compound culture medium where fetal calf serum of certain concentration, various growth factors and other nutritional components are added is used for cultivation in a biochemical incubator at a temperature of 24 DEG C. It is found through lipidosome transfer dyeing that transfer dyeing can be successfully conducted on turbot liver tissue primary culture cells through reporter plasmids such as pIRES2-EGFP. The method is easy and convenient to implement, high in repeatability, low in cost and rapid in cell growth, and an effective in-vitro cell model can be provided for research on the molecular cell biology of fish nutrition metabolism.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to an in vitro construction method of turbot liver tissue cells and its application in molecular cell biology research on nutrition metabolism of fish. Background technique [0002] As a carnivorous fish, turbot needs to add fish oil to its feed during artificial breeding. With the rapid development of the aquatic feed industry, the problem of fish oil shortage has become increasingly prominent. Vegetable oil has become an ideal alternative fat source due to its stable supply, high polyunsaturated fatty acid content and relatively low price. However, if the proportion of vegetable oil replacing fish oil is too high, the health and quality of fish will be affected. In recent years, some progress has been made in the research of replacing fish oil with vegetable oil, but it has not been possible to completely replace fish oil with vegetable oil in the diet of turbot ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/00
Inventor 谭朋郭华荣艾庆辉麦康森
Owner OCEAN UNIV OF CHINA
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