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Construction method and application of immortalized cell lines of porcine subcutaneous adipose precursor cells

A fat precursor cell, immortalized cell line technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as unfavorable passage, no pig fat cells, etc., to reduce the number of separations, slow down The rate at which cells enter clonal senescence, the effect of broad application prospects

Active Publication Date: 2019-04-16
ZHEJIANG UNIV
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  • Application Information

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Problems solved by technology

At present, the culture of pig primary adipocytes is not conducive to subculture, and every time the primary adipocytes are isolated, piglets aged 3-7 days need to be slaughtered, which greatly increases the cost of the experiment and the success rate of the experiment
However, there are no cell lines or immortalized cells of porcine adipocytes

Method used

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  • Construction method and application of immortalized cell lines of porcine subcutaneous adipose precursor cells
  • Construction method and application of immortalized cell lines of porcine subcutaneous adipose precursor cells
  • Construction method and application of immortalized cell lines of porcine subcutaneous adipose precursor cells

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Experimental program
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Embodiment Construction

[0039] (1) Isolation and culture of primary porcine subcutaneous adipocytes

[0040] 1. Separate subcutaneous adipose tissue under sterile conditions, rinse with PBS (containing penicillin and streptomycin) three times, cut into 1-2mm in PBS 3 organization block. The preparation method of PBS (containing penicillin and streptomycin) is: adding penicillin and streptomycin with a final concentration of 100U / ml in every 100ml of PBS buffer solution (pH 7.2-7.4).

[0041] 2. Add about 15ml of digestion solution containing 0.15% type I collagenase to 10g of tissue block and digest in a 37°C water bath for 70min-90min.

[0042] The formula of the digestive solution is: add 0.15 g type I collagenase to 100 ml of the digestive solution to obtain a digestive solution containing 0.15% type I collagenase; the type I collagenase can be purchased from Gibco, for example.

[0043] 3. After the digestion is completed, add an equal volume of DMEM complete medium (DMEM+10% fetal bovine serum...

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Abstract

The invention provides a construction method of immortalized cell lines of porcine subcutaneous adipose precursor cells. The method includes the following steps that primary porcine subcutaneous adipocytes are obtained; cells are cultured, and pBABE-puro-hTERT plasmids are transfected when the cell growth density is 60%-70%; after transfection for 48+ / -2 h, a 30%-serum DMEM medium containing 4 microgram / mL purinomycin used for screening; after screening for 48+ / -2 h, and liquid is changed for cultivation of the DMEM medium; after cells are cloned, the cloned cells are digested and cultured, and identification is performed until the immortalized porcine subcutaneous adipose precursor cell model is obtained. The immortalized cell lines of porcine subcutaneous adipose precursor cells can be used for adipose precursor cell proliferation, or polyester mechanism differentiation and regulation mechanism research.

Description

technical field [0001] The invention constructs pig subcutaneous fat precursor cells overexpressing the telomerase reverse transcription gene by transfecting the telomerase plasmid to obtain an immortalized cell line, which belongs to the application technology in the technical field of biology and modern agriculture. Background technique [0002] Adipose tissue plays an important role in the energy metabolism of the animal body and is an important place for storage and energy supply. Adipose tissue, as an endocrine organ, secretes some adipokines such as adiponectin and leptin, which play an important role in metabolic diseases such as obesity and diabetes. Among them, fat deposition is mainly determined by the proliferation, differentiation and polyester maturation of preadipocytes, which directly affects glucose and lipid metabolism and human health. On the other hand, from the perspective of animal husbandry, subcutaneous fat deposition directly affects the quality of p...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/54C12N15/85C12Q1/6851
CPCC12N5/0653C12N9/1276C12N15/85C12N2510/04C12Q1/6851C12Y207/07049C12Q2531/113C12Q2563/107
Inventor 单体中刘嘉琪有文静徐子叶农秋雲汪以真
Owner ZHEJIANG UNIV
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