Supernatant of brown adipocytes, method for preparing same and utilization thereof

A brown fat, cell technology, applied in embryonic cells, animal cells, cell culture medium, etc., can solve problems such as hormone group difficulties

Pending Publication Date: 2021-05-25
NAT CENT FOR GLOBAL HEALTH & MEDICINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is extremely difficult to obtain BAT-producing hormones in a non-damaged state (Non-Patent Document 18)

Method used

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  • Supernatant of brown adipocytes, method for preparing same and utilization thereof
  • Supernatant of brown adipocytes, method for preparing same and utilization thereof
  • Supernatant of brown adipocytes, method for preparing same and utilization thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0360] [Example 1] Hypoglycemic effect of subcutaneous administration of brown adipocyte supernatant derived from human ESCs prepared in a "buffer containing only salts and high-concentration glucose" to mice:

[0361] Using the KhES-3 strain of human ESC (Suemori et al., Biochem Biophys Res Commun 345:926-932, 2006) maintained on mouse fetal fibroblasts (MEF), the method described in [Reference Example 4] was used. Induction of differentiation into brown adipocytes (brownadipocyte: BA). The differentiation medium was removed from the mature BA on the 10th day of differentiation induction (after 8 days of planktonic, and the second day of adherent culture on a 6 cm dish), and Krebs-Ringer-HEPES (KRH) containing 16.8 mM glucose was added. ) buffer (NaCl: 128mM, KCl: 5mM, CaCl 2 2.7mM, MgSO 4 1.2mM,Na 2 HPO 4: 1mM, HEPES (pH7.4): 20mM, glucose 16.8mM) 2ml, in a carbon dioxide gas culture device (37 ℃, 5% CO 2 ) after culturing for 16 hours, the supernatant (BA-SUP) was reco...

Embodiment 2

[0364] [Example 2] Induction of expression of glucose transporter gene Glut4 / GLUT4 when human ESC-derived BA supernatant prepared in "BA differentiation medium" was added to skeletal muscle cells:

[0365] BA was prepared by the method described in [Reference Example 4] using human ESCs (KhES-3 strain) maintained in culture on mouse fetal fibroblasts (MEF). And the differentiation medium was removed from mature BA on day 10 and fresh differentiation medium was added (medium exchange). Next, in a carbon dioxide gas culture device (37°C, 5% CO 2 ) after culturing for 16 hours, the supernatant (BA-SUP) was recovered. For the control group, place fresh differentiation medium, gelatin-coated dishes in a carbon dioxide gas culture device (37°C, 5% CO 2 ), and the supernatant recovered after 16 hours was used as the supernatant of the control group (ControlSUP).

[0366] Using the mouse muscle bud cell line C2C12 obtained from the American Type Culture Collection (ATCC), according...

Embodiment 3

[0370] [Example 3] Insulin secretion-promoting effect of BA supernatant derived from human ESC / iPSC prepared by "buffer containing only salts and high-concentration glucose" on islet β cells:

[0371] In the same manner as in [Example 1], SUP of BA derived from human ESC was prepared. That is, in order to induce BA differentiation from human ESCs maintained and cultured on MEFs (Non-Patent Document 19), the differentiation medium was removed from mature BAs on day 10, and KRH buffer containing 16.8 mM glucose was added, and cultured in carbon dioxide gas. device (37°C, 5% CO 2 ) after culturing for 16 hours and recover the supernatant. On the other hand, MIN6 cells (obtained from Jun Miyazaki, a special professor at Osaka University's Industry-University Cooperation Headquarters (currently)) which are mouse islet β cell lines were treated according to the recommended method (Miyazaki et al., Endocrinology 127:126-132, 1990) in 96-well plates. After it was washed with PBS bu...

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Abstract

Provided is a composition that comprises a supernatant of brown adipocytes or a purified product thereof and has a metabolism improving effect. Also provided is a method for preparing the aforesaid supernatant without using a liquid culture containing glucose at a high concentration. Also provided is a method for producing brown adipocytes using pluripotent stem cells, said method being useful for preparing a supernatant of brown adipocytes. In the present invention, a supernatant having a metabolism improving effect was successfully obtained from brown adipocytes. Also, this supernatant could be obtained without using a liquid culture containing glucose at a high concentration. In the present invention, moreover, brown adipocytes were successfully formed from pluripotent stem cells using a feeder-free culture system without adding a cytokine cocktail, etc. The supernatant of brown adipocytes according to the present invention is expected as applicable to the development of therapeutic drugs for sugar metabolism-related diseases and cosmetics.

Description

technical field [0001] The present invention relates to a preparation method of brown adipocyte supernatant which has the effect of promoting insulin secretion, the effect of increasing the glucose absorption of pancreatic beta cells, skeletal muscle cells and cardiomyocytes, the effect of improving mitochondrial function, and / or the effect of promoting the healing of skin damage, etc. A composition comprising a supernatant of brown fat cells, and correction of metabolic disorders using the composition as an objective medical therapy and the like. Background technique [0002] The population presenting obesity as well as being overweight is increasing worldwide. According to the "Global Burden of Diseases, Injuries, and Risk Factors Study" in 2017, 1 in 3 people in the world is obese or overweight (Non-Patent Document 1). Obesity or overweight will increase the risk of life-threatening diseases such as type 2 diabetes, ischemic heart disease, cerebrovascular disorder, and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/35A61K35/545A61P3/00A61P3/04A61P3/10A61P5/50A61P43/00C12N5/074
CPCA61K35/545A61P3/10A61K35/35A61P3/04C12N5/0653C12N2506/02C12N2500/34C12N2502/1335C12N2502/1305C12N2533/90C12N2513/00C12N2533/54C12N2502/28A61P17/02C12N5/0031
Inventor 佐伯久美子小林德彦冈雅子松村和典西尾美和子朱亚峰森豊隆
Owner NAT CENT FOR GLOBAL HEALTH & MEDICINE
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