Method for preparing brown adipocyte

A brown fat and cell technology, applied in the field of brown fat cells and their preparation, can solve problems such as high cost, complicated induction technology, and cancerous cells

Pending Publication Date: 2018-09-04
KYOTO PREFECTURAL PUBLIC UNIV CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] However, it is difficult to deny the risk of canceration of the transplanted cells after the brown fat cells are transplanted in such techniques of introducing genes to induce brown fat cells.
In addition, the induction technology is relatively complicated, and high costs are required to ensure and prove safety

Method used

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  • Method for preparing brown adipocyte
  • Method for preparing brown adipocyte
  • Method for preparing brown adipocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0206] Fibroblasts derived from normal human skin (human dermal fibroblasts; HDF) were suspended in a normal medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Convert it to 1×10 4 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. The next day, the culture supernatant was aspirated, and 500 μL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with a compound or the like was added as described in the figure.

[0207] Adipocyte induction medium is DMEM+MDI medium supplemented with 10% FBS (DMEM supplemented with 10% FBS supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 0.5 μm dexamethasone and 1 μg / mL insulin) .

[0208] The concentrations of the additives are as follows:

[0209] T3: 1nM

[0210] Rosiglitazone: 1 μm

[0211] D4476: 2μm

[0212] Pifithrin alpha [p53 inhibitor]: 5 μm

[0213] SB431542: 2μm

...

Embodiment 2

[0219] Fibroblasts derived from human normal skin (human dermal fibroblasts; HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Convert it to 1×10 4 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. On the next day, the culture supernatant was aspirated, and 500 μL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with various small molecular compounds or the like was added as described in the figure.

[0220] Adipocyte induction medium is DMEM+MDI medium supplemented with 10% FBS (DMEM supplemented with 10% FBS supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 0.5 μm dexamethasone and 1 μg / mL insulin) .

[0221] The concentrations of the additives are as follows:

[0222] T3: 1nM

[0223] Rosiglitazone: 1 μm

[0224] D4476: 2μm

[0225] Pifithrin alpha [p53 inhibitor]: 5 μm ...

Embodiment 3

[0233] Carry out the same experiment with embodiment 2, prepared the cell that cultured with common culture medium, in the adipocyte induction medium that has added T3 and rosiglitazone, cultivated the cell of 14 days, and added T3, Rogner Cells cultured for 14 days in adipocyte induction medium of Litazone and D4476. To these cells, 10 μM of isoproterenol or FSK was added as described in the figure. A group without addition was also produced as a control. After 5 hours, the culture solution was aspirated from each well, and after washing with PBS(-), total RNA was extracted from the cells using ISOGEN II. qRT-PCR was performed in the same manner as in Example 2. Quantification was performed by the ratio of the mRNA level of the UCP1 gene to the mRNA level of the β-actin gene, and the value of fibroblasts cultured in a normal medium was set to 1 for calculation.

[0234] show the result in image 3 . It can be seen that the cells cultured for 14 days in the adipocyte indu...

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Abstract

The present invention addresses the problem of providing: a brown adipocyte; a method for preparing the brown adipocyte; a transplantation material containing a brown adipocyte; a prophylactic or therapeutic agent for various diseases and conditions, which contains a brown adipocyte; and a use. As a means for solving the problem, a method for preparing a brown adipocyte is provided, said method being characterized by comprising culturing a differentiated somatic cell from a mammal in a culture medium in the presence of at least one compound selected from the group consisting of (1) a TGFbeta / SMAD pathway inhibitor, (2) a casein kinase 1 inhibitor, (3) a cAMP inducer and (4) an MEK / ERK pathway inhibitor to convert the somatic cell into a brown adipocyte.

Description

technical field [0001] Cross References to Related Applications [0002] This application claims priority based on Japanese Patent Application No. 2015-157697 for which it applied on August 7, 2015, and takes in the whole indication of this in this specification by reference. [0003] The invention relates to brown adipocytes and a preparation method thereof. In addition, the present invention relates to a prophylactic or therapeutic agent for obesity, diabetes, impaired glucose tolerance, abnormal lipid metabolism, arteriosclerosis, hypertension, hyperuricemia, gout, nonalcoholic fatty liver disease, metabolic syndrome, and its use. Background technique [0004] Obesity and its related metabolic diseases, such as diabetes, metabolic syndrome, etc., have become a huge medical and social problem in industrialized countries. In obesity, white adipocytes not only store the remaining energy from food in the form of fatty acids, but also produce various hormones and cytokines ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/35A61L27/00A61P3/00A61P3/10
CPCA61K35/35C12N2501/01C12N2501/15C12N2501/727C12N5/0653C12N2501/30C12N2501/999C12N2506/1307A61P1/16A61P19/06A61P3/00A61P3/04A61P3/06A61P3/10A61P9/10A61P9/12A61L27/3604A61L27/3804C12N2500/42C12N2501/375C12N2501/385
Inventor 山本健太岸田纲郎山本俊郎松田修
Owner KYOTO PREFECTURAL PUBLIC UNIV CORP
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