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Method for inducing formation of porcine fat cells by cell signal channel inhibitor

A cell signal and inhibitor technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve problems such as biosafety effects, achieve simplicity and safety, prevent gene mutation, and form a mechanism The effect of promotion

Inactive Publication Date: 2012-03-21
JILIN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention provides a method for inducing the formation of pig adipocytes by a cell signaling pathway inhibitor, through which the cell signaling pathway inhibitor acts on the cells, and the obtained pig adipocytes have no insertion of foreign genes, which solves the problem of foreign gene insertion caused by transgenic The question of the impact of mutations on biosafety

Method used

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  • Method for inducing formation of porcine fat cells by cell signal channel inhibitor
  • Method for inducing formation of porcine fat cells by cell signal channel inhibitor
  • Method for inducing formation of porcine fat cells by cell signal channel inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0024] 0.5μM Cell signaling pathway inhibitor SB431542 induces transformation of porcine embryonic fibroblasts into adipocytes

[0025] Pig embryonic fibroblasts were divided into 5 × 10 4 piece / cm 2 The proportion of the cells was evenly inoculated into 6cm cell culture plates and cultured with induction medium. The induction medium formula is: 15% (v / v) Knockout Serum Replacement (Invitrogen) serum, 82.8% (v / v) DMEM / F12 (Invitrogen) medium, 0.1mM non-essential amino acids (Invitrogen), 0.055mM β-sulfhydryl Ethanol (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.5 μM SB431542 (Stemgent). 37°C, 5% CO 2 cultured in an incubator.

[0026] The results showed that after 20 days of induction culture, the formation of bright, lipid-rich cells was observed (see figure 1 , figure 2 ). After oil red O staining, round lipid droplets stained red can be observed, and pig fat-specific genes (PPARγ, C / EBPα, β, δ, LPL, AP2, Adipoq, CFD, SLC2A4) were respectively after PCR Specific...

Embodiment 2

[0030] 0.2μM Cell signaling pathway inhibitor SB431542 induces transformation of porcine embryonic fibroblasts into adipocytes

[0031] Pig embryonic fibroblasts were divided into 5 × 10 4 piece / cm 2 The proportion of the cells was evenly inoculated into 6cm cell culture plates and cultured with induction medium. The induction medium formula is: 15% (v / v) Knockout Serum Replacement (Invitrogen) serum, 82.8% (v / v) DMEM / F12 (Invitrogen) medium, 0.1mM non-essential amino acids (Invitrogen), 0.055mM β-sulfhydryl Ethanol (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.2 μM SB431542 (Stemgent). 37°C, 5% CO 2 cultured in an incubator.

[0032] The results showed that after 20 days of induction culture, the formation of bright cells rich in lipid droplets could be observed. After oil red O staining, round lipid droplets stained red can be observed, which proves that 0.2 μM SB431542 can promote the transformation of porcine embryonic fibroblasts into adipocytes, and the calculate...

Embodiment 3

[0034] 1μM Cell signaling pathway inhibitor SB431542 induces transformation of porcine embryonic fibroblasts into adipocytes

[0035] Pig embryonic fibroblasts were divided into 5 × 10 4 piece / cm 2The proportion of the cells was evenly inoculated into 6cm cell culture plates and cultured with induction medium. The induction medium formula is: 15% (v / v) Knockout Serum Replacement (Invitrogen) serum, 82.8% (v / v) DMEM / F12 (Invitrogen) medium, 0.1mM non-essential amino acids (Invitrogen), 0.055mM β-sulfhydryl Ethanol (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 μM SB431542 (Stemgent). 37°C, 5% CO 2 cultured in an incubator.

[0036] The results showed that after 20 days of induction culture, the formation of bright cells rich in lipid droplets could be observed. After oil red O staining, round lipid droplets stained red can be observed, which proves that 1 μM SB431542 can promote the transformation of porcine embryonic fibroblasts into adipocytes, and the calculated effic...

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Abstract

The invention provides a method for inducing formation of porcine fat cells by a cell signal channel inhibitor. The method comprises the following steps of uniformly inoculating porcine embryo fibroblasts into a Corning cell culture plate according to a ratio of 4-6* 104 cells / cm2; culturing by using an inducing culture medium, and observing the cells, and performing oil red O dyeing to prove that the cells are fat cells after culturing for 20 days in a culture box containing 5% CO2 at 37 DEG C. The cell signal channel inhibitor instead of a transgene is used for inducing cell transformation,so that genic mutation caused by insertion of an exogenous gene into a cell genome can be effectively prevented; by the method, the research on the forming mechanism of the fat cells is facilitated to a certain extent; and the method with convenience and safety can be widely applied and can be used for researching a fat forming mechanism and also can play a roll in disease therapy and other clinical applications.

Description

technical field [0001] The invention discloses a method for inducing the formation of pig adipocytes by a cell signaling pathway inhibitor, which transforms pig embryonic fibroblasts into adipocytes, and belongs to the technical field of cell induction. Background technique [0002] Due to the needs of clinical treatment, people need specially differentiated cells to treat diseases, so stem cell induction and differentiation technology has gradually matured, but it has been found that some cells can directly differentiate into another type of differentiated cells without going through the stem cell state, as reported The methods are all realized by transgenic means, directly transforming terminally differentiated cells into other types of functional cells: such as transforming embryonic fibroblasts, chondrocytes and retinal epithelial cells into muscle cells with contractile properties; Transform beta lymphocytes into macrophages; transform islet exocrine cells into beta isl...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 欧阳红生逄大欣朱建国周杨高飞唐成程全龙泉张明军
Owner JILIN UNIV
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