JP5822189B2 - Differentiation induction promoter from stem cells to brown adipocytes - Google Patents
Differentiation induction promoter from stem cells to brown adipocytes Download PDFInfo
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- JP5822189B2 JP5822189B2 JP2010290024A JP2010290024A JP5822189B2 JP 5822189 B2 JP5822189 B2 JP 5822189B2 JP 2010290024 A JP2010290024 A JP 2010290024A JP 2010290024 A JP2010290024 A JP 2010290024A JP 5822189 B2 JP5822189 B2 JP 5822189B2
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Description
本発明は、幹細胞から褐色脂肪細胞への分化誘導促進剤、幹細胞から褐色脂肪細胞の分化誘導方法、当該方法により生成された細胞、前記分化誘導促進剤および／または前記細胞を含む医薬組成物、並びに、糖尿病等の予防剤及び／又は改善剤のスクリーニング方法に関する。 The present invention relates to an agent for inducing differentiation from stem cells to brown adipocytes, a method for inducing differentiation from stem cells to brown adipocytes, a cell generated by the method, the differentiation induction promoter and / or a pharmaceutical composition comprising the cells, The present invention also relates to a screening method for a preventive agent and / or an improving agent for diabetes and the like.
脂肪組織は、生体に存在する重要な臓器の一つであり、その主な役割はエネルギーを脂肪として蓄えること、外界からの物理的衝撃を吸収して器官を保護すること、外界の温度を断熱して体温を保つことなどである。脂肪組織は、白色脂肪組織と褐色脂肪組織の２つのタイプに大別される。 Adipose tissue is one of the important organs in the body, and its main role is to store energy as fat, to protect the organs by absorbing physical shock from the outside world, and to insulate the temperature of the outside world And keep the body temperature. Adipose tissue is roughly divided into two types: white adipose tissue and brown adipose tissue.
白色脂肪組織は、エネルギーを中性脂質として細胞内に貯蓄する白色脂肪細胞から構成されており、白色脂肪細胞は生体内の余剰エネルギーを蓄える働きがある。白色脂肪細胞は生体がエネルギーを必要とする際に、中性脂質を遊離脂肪酸へと分解し、ミトコンドリアに存在する電子伝達系を介してATP（アデノシン三リン酸）を合成してエネルギーを生体へ補給する役割を持つ。 White adipose tissue is composed of white adipocytes that store energy in cells as neutral lipids, and the white adipocytes have a function of storing surplus energy in the living body. White adipocytes break down neutral lipids into free fatty acids when living organisms need energy, synthesize ATP (adenosine triphosphate) via the electron transport system present in mitochondria, and transfer the energy to living organisms. Have a role to supply.
褐色脂肪組織は褐色脂肪細胞から構成されている。褐色脂肪細胞は、白色脂肪細胞と同様に細胞内に中性脂質を貯蓄するが、ミトコンドリア脱共役タンパク質１（uncoupling protein-1：UCP1）の働きにより、電子伝達系におけるATP合成を経ずに、中性脂質を熱へと変換することができる。生体内においては、白色脂肪細胞によるエネルギーの貯蔵およびATP合成系を介したエネルギーの補給と、褐色脂肪細胞による熱産生によるエネルギーの消費により、全身のエネルギー消費や体脂肪量の調節が行われていると考えられている。 Brown adipose tissue is composed of brown adipocytes. Brown adipocytes store neutral lipids in the same way as white adipocytes, but by the action of mitochondrial uncoupling protein-1 (UCP1), without ATP synthesis in the electron transport system, Neutral lipids can be converted to heat. In the living body, energy storage and body fat volume are regulated by the storage of energy by white fat cells and the supply of energy through the ATP synthesis system and the consumption of energy by heat production by brown fat cells. It is believed that
近年、メタボリックシンドロームが著しい社会問題となっており、そのリスクファクターとして肥満の病態解析と対策が求められている。肥満やメタボリックシンドロームは、エネルギー摂取の増大（例えば多食など）とともに、エネルギー消費の減少が大きな原因であると考えられている。肥満等の対策として生体内のエネルギー消費増大が重要であることが認識されつつあり、そのターゲットとして、熱産生によりエネルギーを消費することのできる褐色脂肪細胞が着目されている。 In recent years, metabolic syndrome has become a significant social problem, and as a risk factor there is a need for pathological analysis and countermeasures for obesity. Obesity and metabolic syndrome are considered to be caused mainly by a decrease in energy consumption as well as an increase in energy intake (for example, polyphagia). Increasing energy consumption in vivo is important as a countermeasure against obesity and the like, and brown adipocytes capable of consuming energy by heat production have attracted attention as targets.
生体内では褐色脂肪細胞は、肩甲骨周辺と首の後ろ、わきの下など限られた場所にしかなく、少量しか存在しない。さらに、褐色脂肪細胞は加齢と共に年々少なくなっていくことが知られている。褐色脂肪細胞を生体内において増加させることができれば、エネルギー消費を増大させることが可能となる。 In vivo, brown adipocytes are present only in limited places such as around the scapula, behind the neck, and under the arm, and are present in small amounts. Furthermore, brown adipocytes are known to decrease year by year with aging. If brown adipocytes can be increased in vivo, energy consumption can be increased.
褐色脂肪細胞は、生体内に存在する幹細胞と呼ばれる未分化な細胞から分化すると考えられている（非特許文献１）。幹細胞から褐色脂肪細胞への分化誘導因子として、BMP7等のいくつかの因子が報告されている（非特許文献２）。また、培養レベルにおいて、幹細胞から褐色脂肪細胞以外の細胞を分化誘導せずに、褐色脂肪細胞を選択的に分化させるような因子として、ペルオキシソーム増殖剤応答性受容体（PPARα）などのリガンドが報告されている（特許文献１）。しかしながら、in vitroおよびin vivoのいずれにおいても、褐色脂肪細胞の分化誘導のメカニズムの詳細は未だ不明のままである。幹細胞から褐色脂肪細胞の選択的分化誘導に関する研究は、肥満やメタボリックシンドロームの予防・改善のための新しい手法の開発に結びつくことが期待され、注目されている。 Brown adipocytes are thought to differentiate from undifferentiated cells called stem cells that exist in the living body (Non-patent Document 1). Several factors such as BMP7 have been reported as differentiation-inducing factors from stem cells to brown adipocytes (Non-patent Document 2). In addition, ligands such as peroxisome proliferator-activated receptor (PPARα) have been reported as factors that selectively differentiate brown adipocytes without inducing differentiation of stem cells other than brown adipocytes at the culture level. (Patent Document 1). However, the details of the mechanism for inducing differentiation of brown adipocytes remain unclear both in vitro and in vivo. Research on selective differentiation of brown adipocytes from stem cells is expected to lead to the development of new methods for the prevention and improvement of obesity and metabolic syndrome.
本発明は、幹細胞から褐色脂肪細胞を効率よく選択的に分化誘導するための、分化誘導促進剤および分化誘導方法を提供することを目的とする。 An object of the present invention is to provide a differentiation induction promoter and a differentiation induction method for efficiently and selectively inducing brown adipocytes from stem cells.
本発明者らは、Gdfファミリーに属するGdf5（Growth/differentiation factor-5）が、幹細胞から褐色脂肪前駆細胞の分化誘導を、および褐色脂肪前駆細胞から褐色脂肪細胞への分化誘導を促進することを確認し、Gdf5を幹細胞から褐色脂肪細胞への分化誘導促進剤として使用し得ることを見出し、本発明を達成した。 The present inventors have shown that Gdf5 (Growth / differentiation factor-5) belonging to the Gdf family promotes differentiation induction of brown adipose precursor cells from stem cells and differentiation induction from brown adipose precursor cells to brown adipocytes. As a result, it was found that Gdf5 can be used as a differentiation induction promoter from stem cells to brown adipocytes, and the present invention has been achieved.
すなわち、本発明は以下の通りである。
１．Gdfを有効成分とする、幹細胞から褐色脂肪細胞への分化誘導促進剤。
２．Gdfが、以下のタンパク質から選択される、前項１に記載の幹細胞から褐色脂肪細胞への分化誘導促進剤：
(a) 配列番号１で表されるアミノ酸配列からなるタンパク質；
(b) 配列番号１で表されるアミノ酸配列において、１個以上のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質；
(c) 配列番号１で表されるアミノ酸配列と７０％以上の同一性を有するアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質。
３．間葉系幹細胞から褐色前駆脂肪細胞への分化能を増強する、前項１または２に記載の分化誘導促進剤。
４．褐色前駆脂肪細胞から褐色脂肪細胞への分化能を増強する、前項１〜３のいずれか１に記載の分化誘導促進剤。
５．幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するGdfの存在下で、細胞を培養することを含む、褐色脂肪細胞の分化誘導方法。
６．前記細胞が、間葉系幹細胞および／または褐色前駆脂肪細胞である、前項５に記載の褐色脂肪細胞の分化誘導方法。
７．前項１〜４のいずれか１に記載の分化誘導促進剤と、薬学的に許容される担体とを含む、糖尿病、メタボリックシンドローム、及び／又は肥満の予防及び／又は改善のための医薬組成物。
８．前項５もしくは６に記載の分化誘導方法を用いる、糖尿病、メタボリックシンドローム、及び／又は肥満の予防剤及び／又は改善剤のスクリーニング方法。
That is, the present invention is as follows.
1. An agent for promoting differentiation from stem cells to brown adipocytes, containing Gdf as an active ingredient.
2. The agent for promoting differentiation induction from stem cells to brown adipocytes according to item 1, wherein Gdf is selected from the following proteins:
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1;
(b) The amino acid sequence represented by SEQ ID NO: 1 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and promotes differentiation induction from stem cells to brown adipocytes Functional protein;
(c) A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a function of promoting differentiation induction from stem cells to brown adipocytes.
3. 3. The differentiation induction promoter according to item 1 or 2, which enhances the differentiation ability from mesenchymal stem cells to brown preadipocytes.
4). 4. The differentiation induction promoter according to any one of items 1 to 3, which enhances differentiation ability from brown preadipocytes to brown adipocytes.
5. A method for inducing differentiation of brown adipocytes, comprising culturing cells in the presence of Gdf having a function of promoting differentiation induction from stem cells to brown adipocytes.
6). 6. The method for inducing differentiation of brown adipocytes according to item 5 above, wherein the cells are mesenchymal stem cells and / or brown preadipocytes.
7). 5. A pharmaceutical composition for preventing and / or improving diabetes, metabolic syndrome, and / or obesity, comprising the differentiation induction promoter according to any one of items 1 to 4 and a pharmaceutically acceptable carrier.
8). 7. A screening method for a prophylactic and / or ameliorating agent for diabetes, metabolic syndrome, and / or obesity using the differentiation induction method according to 5 or 6 above.
本発明の分化誘導促進剤および分化誘導方法によれば、高効率で、間葉系幹細胞から褐色前駆脂肪細胞への分化誘導、および、褐色前駆脂肪細胞から褐色脂肪細胞への分化誘導を促進することができる。 According to the differentiation induction promoter and differentiation induction method of the present invention, the differentiation induction from mesenchymal stem cells to brown preadipocytes and the differentiation induction from brown preadipocytes to brown adipocytes are promoted with high efficiency. be able to.
本発明の分化誘導促進剤の有効成分である「Gdf」とは、Gdf（Growth/differentiation factor）ファミリーに含まれるタンパク質の少なくとも１種もしくは２種以上の混合物を意味する。Gdfファミリーのタンパク質は、骨、軟骨、腱、靭帯、神経および皮膚の成長、分化、再生のような形態形成を誘導することが知られており、Gdf1、Gdf2、Gdf3、Gdf5、Gdf6、Gdf7、Gdf8、Gdf9、Gdf10、Gdf11、Gdf15、Gdf16等の複数のアイソフォームが存在する。本発明におけるGdfは、幹細胞から褐色脂肪細胞への分化誘導を促進しうるものであれば、いずれのアイソフォームのGdfであってもよいが、好ましくは本発明のGdfは、Gdf5である。 “Gdf”, which is an active ingredient of the differentiation induction promoter of the present invention, means at least one protein or a mixture of two or more proteins included in the Gdf (Growth / differentiation factor) family. Gdf family of proteins are known to induce morphogenesis like bone, cartilage, tendon, ligament, nerve and skin growth, differentiation, regeneration, Gdf1, Gdf2, Gdf3, Gdf5, Gdf6, Gdf7, There are multiple isoforms such as Gdf8, Gdf9, Gdf10, Gdf11, Gdf15, Gdf16. Gdf in the present invention may be Gdf of any isoform as long as it can promote differentiation induction from stem cells to brown adipocytes, but preferably Gdf of the present invention is Gdf5.
本発明におけるGdfは、哺乳動物由来、例えば、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等由来のものが例示されるが、好ましくはヒトまたはマウス由来である。マウス由来のものとしては、GenBank Accession No. AAH79555（Gdf1）、GenBank Accession No. AAD40308（Gdf2）、GenBank Accession No. AAI01964（Gdf3）、GenBank Accession No. AAA18778（Gdf5）、GenBank Accession No. AAI41340（Gdf6）、GenBank Accession No. AAK30843（Gdf7）、GenBank Accession No. EDK99985（Gdf8）、GenBank Accession No. AAH52667（Gdf9）、GenBank Accession No. AAH58358（Gdf10）、GenBank Accession No. NP_034402（Gdf11）、GenBank Accession No. AAH67248（Gdf15）、GenBank Accession No. CAD60935（Gdf16）に記載のものが例示される。 Gdf in the present invention is exemplified by mammals, for example, humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc., preferably humans Or from a mouse. Examples of mouse-derived products include GenBank Accession No. AAH79555 (Gdf1), GenBank Accession No. AAD40308 (Gdf2), GenBank Accession No. AAI01964 (Gdf3), GenBank Accession No. AAA18778 (Gdf5), GenBank Accession No. AAI41340 (Gdf6 ), GenBank Accession No. AAK30843 (Gdf7), GenBank Accession No. EDK99985 (Gdf8), GenBank Accession No. AAH52667 (Gdf9), GenBank Accession No. AAH58358 (Gdf10), GenBank Accession No. NP_034402 (Gdf11), GenBank Accession No Examples include those described in AAH67248 (Gdf15) and GenBank Accession No. CAD60935 (Gdf16).
本発明のGdfは、以下の(a)〜(c)より選択されるいずれか１のタンパク質であることが好適である。
(a) 配列番号１（Gdf5：GenBank Accession No. AAA18778）で表されるアミノ酸配列からなるタンパク質。
(b) 配列番号１で表されるアミノ酸配列において、１個以上（好ましくは１〜４個）のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質。
(c) 配列番号１で表されるアミノ酸配列と７０％以上（好ましくは８０％以上、より好ましくは９０％以上、さらに好ましくは９５％以上）の同一性を有するアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質。
The Gdf of the present invention is preferably any one protein selected from the following (a) to (c).
(a) A protein comprising an amino acid sequence represented by SEQ ID NO: 1 (Gdf5: GenBank Accession No. AAA18778).
(b) The amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or more (preferably 1 to 4) amino acids are deleted, substituted, inserted and / or added, and from stem cells to brown fat A protein having a function of promoting differentiation into cells.
(c) consisting of an amino acid sequence having 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 95% or more) identity with the amino acid sequence represented by SEQ ID NO: 1, and from stem cells A protein having a function of promoting differentiation into brown adipocytes.
Gdfは、幹細胞から褐色脂肪細胞への分化誘導過程において、褐色脂肪細胞への分化誘導を促進する機能を発揮する。幹細胞から褐色脂肪細胞への分化誘導過程の模式図を図１に示す。分化誘導過程には、「コミットメント」という分化の方向性が決定される段階と、最終的に褐色脂肪組織としての機能を発揮する褐色脂肪細胞に成熟する「分化」という段階に大きく分けられる。コミットメントの段階では、幹細胞（例えば胚性幹細胞）は、間葉系幹細胞を介して、BMP-7等の作用により、褐色脂肪前駆細胞へと誘導される。その後分化の段階において、褐色前駆脂肪細胞がBMP-7等の作用により、褐色脂肪細胞へと成熟する。本発明の分化誘導促進剤は、幹細胞から褐色脂肪細胞の分化誘導過程のどの時点において作用するものであってもよいが、好ましくは間葉系幹細胞から褐色前駆脂肪細胞への分化能、および、褐色前駆脂肪細胞から褐色脂肪細胞への分化能を増強する機能を有するものである。 Gdf exerts the function of promoting differentiation induction into brown adipocytes in the process of inducing differentiation from stem cells into brown adipocytes. A schematic diagram of the differentiation induction process from stem cells to brown adipocytes is shown in FIG. The differentiation induction process can be broadly divided into a stage in which the direction of differentiation called “commitment” is determined and a stage of “differentiation” that matures into brown adipocytes that finally function as brown adipose tissue. At the commitment stage, stem cells (eg, embryonic stem cells) are induced into brown adipose precursor cells by the action of BMP-7 or the like via mesenchymal stem cells. Thereafter, at the stage of differentiation, the brown preadipocytes mature into brown adipocytes by the action of BMP-7 and the like. The differentiation induction promoter of the present invention may act at any point in the differentiation induction process from stem cells to brown adipocytes, preferably the differentiation ability from mesenchymal stem cells to brown preadipocytes, and It has a function of enhancing the differentiation ability from brown preadipocytes to brown adipocytes.
本発明における幹細胞は、褐色脂肪細胞の分化誘導に用いることのできるものであればいかなるものであってもよい。例えば本発明における幹細胞は、胚性幹細胞（ES細胞）、もしくは、骨髄、血液、皮膚、脂肪、脳、肝臓、膵臓、腎臓、筋肉やその他の組織に存在する、未分化な状態の細胞（総称して、体性幹細胞と記す）（なお、体性幹細胞には間葉系幹細胞が含まれる）、さらには遺伝子導入などにより人工的に作製された幹細胞が例示され、これら幹細胞は、初代培養細胞、継代培養細胞、凍結細胞いずれであってもよい。本発明における幹細胞は、好ましくは、間葉系細胞である。間葉系幹細胞とは、間葉系に属する細胞（骨細胞、心筋細胞、軟骨細胞、腱細胞、脂肪細胞など）へ分化しうる能力を有し、かつ自己複製能力を有する細胞として定義される。 The stem cells in the present invention may be any as long as they can be used for induction of brown adipocyte differentiation. For example, the stem cells in the present invention are embryonic stem cells (ES cells) or cells in an undifferentiated state (generic name) present in bone marrow, blood, skin, fat, brain, liver, pancreas, kidney, muscle, and other tissues. (Somatic stem cells include mesenchymal stem cells), and stem cells artificially prepared by gene transfer or the like. These stem cells are primary cultured cells. Any of subcultured cells and frozen cells may be used. The stem cell in the present invention is preferably a mesenchymal cell. Mesenchymal stem cells are defined as cells that have the ability to differentiate into cells belonging to the mesenchymal system (bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and have the ability to self-replicate. .
また、分化の方向性の決定および分化誘導の過程等について図１に示すものと同等の特性を持つ哺乳動物であれば、全ての哺乳動物の幹細胞を、本発明における分化誘導の対象とすることができる。かかる幹細胞として、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物から得られた幹細胞が例示される。 In addition, as long as the mammal has the same characteristics as those shown in FIG. 1 with respect to the determination of the direction of differentiation and the process of differentiation induction, all mammalian stem cells are targeted for differentiation induction in the present invention. Can do. Examples of such stem cells include stem cells obtained from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs.
本発明において、褐色脂肪前駆細胞とは、褐色脂肪細胞に分化しうる能力を有し、かつ自己複製能力を有する細胞として定義される。褐色脂肪前駆細胞は、胚性幹細胞のようにすべての細胞に分化する多分化能は有していないが、生体内において褐色脂肪細胞を供給する機能を有している。 In the present invention, brown adipose precursor cells are defined as cells that have the ability to differentiate into brown adipocytes and have the ability to self-renew. Brown adipose precursor cells do not have the multipotency to differentiate into all cells like embryonic stem cells, but have the function of supplying brown adipocytes in vivo.
本発明における褐色脂肪細胞とは、電子伝達系と酸化的リン酸化によるATP合成を経ずに、中性脂質を熱へと変換する機能を有する細胞である。褐色脂肪細胞は、脂肪滴が複数あり、この脂肪滴に隣接するようにミトコンドリアを多数持つため、褐色を呈している。電子伝達系と酸化的リン酸化によるATP合成は、電子伝達系によりミトコンドリアの内膜と外膜の間に蓄積された水素イオンが、ATP合成酵素内にある穴を突き抜けてエネルギーを発生させ、このエネルギーによってADPにPO4が結合して高エネルギー化合物であるATPを生成する過程である。一方、褐色脂肪細胞における中性脂質の熱変換は、ミトコンドリアの内膜へ結合したUCP1により、内膜と外膜の間に蓄積した水素イオンがUCP1内にある穴を通じてミトコンドリア内へ流入して熱を産生する過程である。 The brown adipocytes in the present invention are cells having a function of converting neutral lipids to heat without undergoing ATP synthesis by an electron transport system and oxidative phosphorylation. The brown adipocytes have a plurality of fat droplets and have a large number of mitochondria so as to be adjacent to the fat droplets, and thus have a brown color. ATP synthesis by electron transport system and oxidative phosphorylation involves the hydrogen ions accumulated between the inner and outer mitochondrial membranes through the electron transport system, generating energy through the holes in the ATP synthase. This is a process in which PO 4 is bound to ADP by energy to produce ATP, which is a high energy compound. On the other hand, the heat conversion of neutral lipids in brown adipocytes is caused by UCP1 bound to the inner mitochondrial membrane, and hydrogen ions accumulated between the inner and outer membranes flow into the mitochondrion through a hole in UCP1. Is the process of producing.
褐色脂肪前駆細胞及び／又は褐色脂肪細胞は、細胞の形態学的観察やオイルレッドO染色により確認することができる。その他例えば、褐色脂肪前駆細胞で発現が確認されているUCP1やPGC1aや、褐色脂肪細胞のマーカーと考えられているUCP1を指標として検出することもできる。これらのマーカーの検出は例えば抗体を用いて、自体公知の手法により行うことができる。 Brown adipose precursor cells and / or brown adipocytes can be confirmed by morphological observation of cells or oil red O staining. In addition, for example, UCP1 and PGC1a whose expression is confirmed in brown adipose precursor cells, and UCP1 considered to be a marker for brown adipocytes can also be detected as an index. Detection of these markers can be performed by a method known per se using, for example, an antibody.
本発明は、Gdf存在下で細胞を培養することを含む、幹細胞から褐色脂肪細胞を分化誘導する方法にも及ぶ。本発明の分化誘導する方法は、必ずしも褐色脂肪細胞が得られることを必須とするものではなく、褐色脂肪細胞に分化の方向性が決定された細胞、例えば褐色前駆脂肪細胞等が得られればよい。また本発明の分化誘導する方法により得られた細胞は、褐色脂肪前駆細胞と褐色脂肪細胞とが混合しているものであってもよい。すなわち、本発明の褐色脂肪細胞を分化誘導する方法は、褐色前駆脂肪細胞を分化誘導する方法を含むものであり、好ましくは、幹細胞から褐色脂肪前駆細胞及び／又は褐色脂肪細胞を分化誘導する方法である。 The present invention also extends to a method for inducing differentiation of brown adipocytes from stem cells, comprising culturing cells in the presence of Gdf. The method for inducing differentiation of the present invention does not necessarily require that brown adipocytes are obtained, and it is only necessary to obtain cells in which the direction of differentiation is determined, for example, brown preadipocytes. . Moreover, the cell obtained by the method for inducing differentiation of the present invention may be a mixture of brown fat precursor cells and brown fat cells. That is, the method of inducing differentiation of brown adipocytes of the present invention includes a method of inducing differentiation of brown preadipocytes, and preferably a method of inducing differentiation of brown adipose precursor cells and / or brown adipocytes from stem cells. It is.
本発明の分化誘導方法において培養される細胞は、褐色脂肪細胞に分化しうる能力を有する細胞であれば、いかなるものであってもよい。褐色脂肪細胞に分化し得る能力を有する細胞としては、幹細胞もしくは褐色脂肪前駆細胞が例示される。幹細胞は好ましくは間葉系幹細胞である。 The cells cultured in the differentiation induction method of the present invention may be any cells as long as they have the ability to differentiate into brown adipocytes. Examples of cells having the ability to differentiate into brown adipocytes include stem cells or brown adipose precursor cells. The stem cell is preferably a mesenchymal stem cell.
本発明の褐色脂肪細胞を分化誘導する方法は、Gdfの存在下で細胞を培養することを含むものであるが、細胞の培養は本研究分野において実施されている自体公知の方法、あるいは今後開発されるあらゆる方法により行うことができる。 The method for inducing differentiation of brown adipocytes according to the present invention includes culturing cells in the presence of Gdf. Cell culturing is a method known per se that has been carried out in this research field, or will be developed in the future. This can be done by any method.
例えば、本発明の方法に用いることができる培地（培養液）としては、細胞の生存増殖に必要な成分（無機塩、炭水化物、ホルモン、必須アミノ酸、ビタミン）を含む基本培地（例えば、イーグル基礎培地 （BME）、Iscove（IMDM）、RPMI、DMEM、Fischer培地、α培地、Leibovitz培地、L-15培地、NCTC培地、F-12培地、MEM、MoCy培地）に、脂肪細胞分化誘導因子として、デキサメタゾン（DEX）、イソブチルメチルキサンチン（IBMX）、インドメタシン（IDM）、インスリン（Ins）、ビオチンの１種以上を添加し、さらに、Gdfを少なくとも１種添加したものを用いることができる。また、増殖速度を増大させるために、プロゲステロン、プラレッシン、トランスフェリン、セレナイトの添加物、あるいは褐色脂肪細胞培養用添加物と、必要に応じて、ヘパリンやヘパラン硫酸又はこれらの脱硫酸化グリコサミノグリカンを含有していてもよい。また、必要に応じて、抗生物質を含有してもよい。 上記分化誘導用培地には、血清が含まれていてもよい。 For example, as a medium (culture solution) that can be used in the method of the present invention, a basic medium (for example, an eagle basal medium) containing components (inorganic salts, carbohydrates, hormones, essential amino acids, vitamins) necessary for viable growth of cells. (BME), Iscove (IMDM), RPMI, DMEM, Fischer medium, α medium, Leibovitz medium, L-15 medium, NCTC medium, F-12 medium, MEM, MoCy medium) and dexamethasone as an adipocyte differentiation inducing factor One or more of (DEX), isobutylmethylxanthine (IBMX), indomethacin (IDM), insulin (Ins), biotin, and at least one Gdf can be used. In order to increase the growth rate, progesterone, plarescine, transferrin, selenite additive, brown adipocyte culture additive, and if necessary heparin, heparan sulfate or their desulfated glycosaminoglycan You may contain. Moreover, you may contain antibiotics as needed. The differentiation induction medium may contain serum.
細胞の培養期間は、幹細胞から褐色脂肪前駆細胞および／または褐色脂肪細胞を分化誘導し得る期間であればよい。細胞の培養期間は特に限定しないが、少なくとも１日以上、好ましくは３日以上、より好ましくは５日以上、さらに好ましくは１〜２週間程度である。培地にGdfを存在させる時期については特に限定されるものではなく、培養期間の全期間にわたってGdfを存在させてもよいが、培養期間の特定の期間のみGdfを存在させてもよい。例えば間葉系幹細胞を培養する場合には、培養初期、好ましくは培養１日目からGdfを含む培地で培養し、数日後例えば３日後などに、培地を、Gdfを含まない培地に交換してもよい。例えば褐色脂肪前駆細胞を培養する場合には、培養初期例えば培養１〜３日目までは、Gdfを含まない培地で培養し、その後培地を、Gdfを含む培地に交換してもよい。 The cell culture period may be a period in which brown adipose precursor cells and / or brown adipocytes can be induced to differentiate from stem cells. The culture period of the cells is not particularly limited, but is at least 1 day, preferably 3 days, more preferably 5 days, and further preferably about 1 to 2 weeks. The time when Gdf is present in the medium is not particularly limited, and Gdf may be present throughout the entire culture period, but Gdf may be present only during a specific period of the culture period. For example, when culturing mesenchymal stem cells, the cells are cultured in a medium containing Gdf at the beginning of culture, preferably from the first day of culture, and after several days, for example, after 3 days, the medium is replaced with a medium containing no Gdf. Also good. For example, in the case of culturing brown fat precursor cells, culture may be performed in a medium not containing Gdf in the initial stage of culture, for example, until the first to third days of culture, and then the medium may be replaced with a medium containing Gdf.
培地中におけるGdfの濃度は、幹細胞から褐色脂肪前駆細胞および／または褐色脂肪細胞を分化誘導し得る量であればよい。例えば培地中におけるGdfの濃度は、1〜1000ng/mlであり、好ましくは10〜1000ng/ml、より好ましくは100〜1000ng/mlである。1ng/ml以下であれば、褐色脂肪細胞への分化誘導作用が見られない可能性が高く、1000ng/ml以上であれば、細胞の生存増殖が阻害される可能性がある。 The concentration of Gdf in the medium may be an amount that can induce differentiation of brown adipose precursor cells and / or brown adipocytes from stem cells. For example, the concentration of Gdf in the medium is 1-1000 ng / ml, preferably 10-1000 ng / ml, more preferably 100-1000 ng / ml. If it is 1 ng / ml or less, there is a high possibility that differentiation inducing action into brown adipocytes is not observed, and if it is 1000 ng / ml or more, cell viability and proliferation may be inhibited.
本発明は、上記分化誘導方法により生成された、褐色脂肪前駆細胞もしくは褐色脂肪細胞にも及ぶ。 The present invention also extends to brown adipose precursor cells or brown adipocytes produced by the above differentiation induction method.
さらに本発明は、本発明の分化誘導促進剤もしくは本発明の分化誘導方法により生成された上述の細胞と、薬学的に許容される担体とを含む医薬組成物にも及ぶ。本明細書における薬学的に許容される担体としては、通常必要により用いられる補助剤、芳香剤、等張化剤、pH調節剤、安定化剤、噴射剤、粘着剤や防腐剤等を適宜選択して含むことができる。 Furthermore, the present invention extends to a pharmaceutical composition comprising the above-described cell produced by the differentiation induction promoter of the present invention or the differentiation induction method of the present invention and a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier in the present specification, auxiliary agents, fragrances, tonicity agents, pH regulators, stabilizers, propellants, pressure-sensitive adhesives, preservatives and the like that are usually used as appropriate are appropriately selected. Can be included.
本発明の医薬組成物は、多様な形態を取ることが出来、腸溶性コーティングされた組成物、徐放性組成物又は持効性組成物、溶液である組成物、局所用組成物、吸入剤である組成物などが挙げられるが、特に限定されるものではない。医薬組成物の投与方法としては、経口投与、非経口投与、粘膜投与、局所投与などがあり、非経口である場合、皮下、静脈内、皮内、腹腔内へ投与できる。
特に褐色脂肪前駆細胞および／または褐色脂肪細胞を含む医薬組成物は、生体への移植材料として用いることができる。前記細胞を生理食塩水や培養液に、例えば1×103〜1×107個/mlとなるように調製し、腹腔内や皮下などへ注射やカテーテルなどにより局所注入することができる。
The pharmaceutical composition of the present invention can take various forms, such as enteric-coated composition, sustained-release composition or sustained-release composition, composition that is a solution, topical composition, inhalant There are no particular restrictions on the composition. The administration method of the pharmaceutical composition includes oral administration, parenteral administration, mucosal administration, topical administration, and the like, and when parenterally, it can be administered subcutaneously, intravenously, intradermally or intraperitoneally.
In particular, a pharmaceutical composition containing brown adipose precursor cells and / or brown adipocytes can be used as a material for transplantation into a living body. The cells can be prepared in physiological saline or a culture solution so as to have a concentration of, for example, 1 × 10 3 to 1 × 10 7 cells / ml, and can be locally injected into the abdominal cavity or subcutaneous region by injection or catheter.
本発明の医薬組成物は、糖尿病、メタボリックシンドローム、及び／又は肥満の予防剤及び／又は改善剤として用いることが可能である。糖尿病は、急性および慢性の種々の合併症を引き起こすことが知られている。また、メタボリックシンドロームや肥満は、例えば心筋梗塞、狭心症、脳卒中や、肝炎等の原因と考えられている。本発明の医薬組成物は、これらの糖尿病、メタボリックシンドローム、及び／又は肥満に起因する疾患の予防剤および／または改善剤として用いることができる。さらに本発明の医薬組成物は、上記以外の褐色脂肪細胞の分化異常に起因する疾患の予防剤および／または改善剤としても用いることが可能であると考えられる。 The pharmaceutical composition of the present invention can be used as an agent for preventing and / or improving diabetes, metabolic syndrome, and / or obesity. Diabetes is known to cause a variety of acute and chronic complications. Metabolic syndrome and obesity are considered to be causes of myocardial infarction, angina pectoris, stroke, hepatitis, and the like. The pharmaceutical composition of the present invention can be used as a prophylactic and / or ameliorating agent for diseases caused by diabetes, metabolic syndrome, and / or obesity. Furthermore, it is considered that the pharmaceutical composition of the present invention can be used as a preventive agent and / or an ameliorating agent for diseases caused by abnormal differentiation of brown adipocytes other than those described above.
本発明はさらに、前記分化誘導方法または、前記分化誘導方法により生成された細胞を用いる、糖尿病、メタボリックシンドローム、及び／又は肥満の予防剤及び／又は改善剤のスクリーニング方法にも及ぶ。後述する実験例では、肥満モデルマウスにおいて褐色脂肪細胞特異的にGdf発現量が上昇しているという結果が得られている。これらの結果は、生体内においてGdfが、褐色脂肪細胞への分化誘導を促進する役割を担っていることを示唆するものである。よって、Gdfによる褐色脂肪細胞の分化誘導促進経路を刺激しうる物質を選択することにより、新たなメカニズムに基づく糖尿病、メタボリックシンドローム、及び／又は肥満を予防及び／又は改善する薬剤を提供することが可能になると期待される。 The present invention further extends to a method for screening for a prophylactic and / or ameliorating agent for diabetes, metabolic syndrome, and / or obesity using the differentiation induction method or cells generated by the differentiation induction method. In an experimental example to be described later, a result that the Gdf expression level is increased specifically in brown adipocytes in an obese model mouse is obtained. These results suggest that Gdf plays a role in promoting differentiation induction into brown adipocytes in vivo. Therefore, by selecting a substance capable of stimulating the differentiation induction promoting pathway of brown adipocytes by Gdf, a drug for preventing and / or improving diabetes, metabolic syndrome, and / or obesity based on a new mechanism is provided. Expected to be possible.
本発明における糖尿病、メタボリックシンドローム、及び／又は肥満の予防剤及び／又は改善剤をスクリーニングする方法は、例えば、本発明の分化誘導方法を用いて細胞を培養する際に、候補物質を存在させて、褐色脂肪細胞への分化誘導への影響を確認する、あるいは、候補物質を生体（マウス等のヒトを除く哺乳動物）に投与等して、候補物質の、Gdfによる褐色脂肪細胞への分化誘導促進経路に対するin vivoでの影響を確認することにより、行なうことができる。 The method for screening for a prophylactic and / or ameliorating agent for diabetes, metabolic syndrome, and / or obesity according to the present invention includes, for example, the presence of a candidate substance when cells are cultured using the differentiation induction method of the present invention. Confirm the effect on the induction of differentiation into brown adipocytes, or administer the candidate substance to the living body (mammals other than humans such as mice) and induce the differentiation of the candidate substance into brown adipocytes by Gdf This can be done by confirming in vivo effects on the facilitating pathway.
以下、本発明の理解を深めるために実施例および実験例により発明内容を具体的に説明するが、これらは本発明の範囲を限定するものではないことはいうまでもない。 Hereinafter, in order to deepen the understanding of the present invention, the contents of the invention will be specifically described by way of examples and experimental examples, but it goes without saying that these do not limit the scope of the present invention.
（実施例１）間葉系幹細胞から褐色脂肪前駆細胞への分化誘導
間葉系幹細胞を培養して褐色脂肪前駆細胞を分化誘導させた。細胞の形態を確認することにより、褐色脂肪前駆細胞を計数した。
１）培養液IMの調製（細胞増殖用培地）
細胞増殖用培養液として、BME（Basal Medium, Eagle）に、ウシ胎児血清（FBS、10%）、20nMのインシュリン（Ins）、1nMのtriiodothyronin（T3）、0.5μMのデキサメタゾン（DEX）、0.5μMのイソブチルメチルキサンチン（IBMX）、0.125mMのインドメタシンを添加した培養液を作製した（以後、培養液IMと記す）。各添加物の濃度は、培養液中の最終濃度である。
(Example 1) Differentiation induction from mesenchymal stem cells to brown adipose precursor cells Mesenchymal stem cells were cultured to induce differentiation of brown adipose precursor cells. Brown fat precursor cells were counted by confirming cell morphology.
1) Preparation of culture solution IM (medium for cell growth)
BME (Basal Medium, Eagle), fetal bovine serum (FBS, 10%), 20 nM insulin (Ins), 1 nM triiodothyronin (T3), 0.5 μM dexamethasone (DEX), 0.5 μM A culture broth supplemented with isobutylmethylxanthine (IBMX) and 0.125 mM indomethacin was prepared (hereinafter referred to as culture broth IM). The concentration of each additive is the final concentration in the culture medium.
２）培養液DMの調製
BME（Basal Medium, Eagle）に、ウシ胎児血清（FBS、10%）、20nMのインシュリン（Ins）、1nMのtriiodothyronin（T3）を添加した培養液を作製した（以後、培養液DMと記す）。各添加物の濃度は、培養液中の最終濃度である。
2) Preparation of culture fluid DM
A culture medium was prepared by adding fetal bovine serum (FBS, 10%), 20 nM insulin (Ins), and 1 nM triiodothyronin (T3) to BME (Basal Medium, Eagle) (hereinafter referred to as culture medium DM). The concentration of each additive is the final concentration in the culture medium.
３）細胞の培養
5×104個/mlの間葉系幹細胞（C3H10T 1/2細胞）を組織培養用24穴プレートに播種し、各種濃度のGdf5（0.1、1、10、または100ng/mL）（Recombinant mouse GDF-5（R&D systems cat#853-G5-050）を添加したDMEM培養液に懸濁して培養を開始した。培養を開始した日を０日目として３日目まで培養し、Gdf5を含まない培養液IMに培地を交換した。その後、２日間おきに新しい培養液DMに交換し、１０日目まで培養を行った（図２（ａ））。褐色脂肪前駆細胞を、オイルレッドO染色により染色して、計数した。
3) Cell culture
5 × 10 4 cells / ml of mesenchymal stem cells (C3H10T 1/2 cells) are seeded in 24-well plates for tissue culture, and various concentrations of Gdf5 (0.1, 1, 10, or 100 ng / mL) (Recombinant mouse GDF -5 (R & D systems cat # 853-G5-050) was added to the suspension in DMEM culture medium, and the culture was started until the third day, starting on day 0. The medium was replaced with the liquid IM, and then the medium was replaced with a new culture medium DM every two days, and the culture was continued until day 10. (FIG. 2 (a)) Brown fat precursor cells were stained with oil red O staining. And counted.
結果を図２（ｂ）と（ｃ）に示す。Gdf5を10または100ng/mLで添加した場合には、分化誘導された褐色脂肪前駆細胞の数が有意に増加しており、分化誘導が促進されることがわかった。 The results are shown in FIGS. 2 (b) and (c). It was found that when Gdf5 was added at 10 or 100 ng / mL, the number of brown adipose precursor cells induced to differentiate significantly increased, and differentiation induction was promoted.
（実施例２）褐色脂肪前駆細胞から褐色脂肪細胞への分化誘導
褐色脂肪前駆細胞を培養して褐色脂肪細胞を分化誘導させた。培養液DMは、実施例１と同様にして作製した。
(Example 2) Differentiation induction from brown adipose precursor cells to brown adipocytes Brown adipose precursor cells were cultured to induce differentiation of brown adipocytes. The culture solution DM was prepared in the same manner as in Example 1.
2×104個/mlの褐色脂肪前駆細胞（BFP細胞）を組織培養用24穴プレートに播種し、培養液DMに懸濁して培養を開始した。培養を開始した日を０日目として３日目まで培養し、各種濃度のGdf5（0.1、1、10、または100ng/mL）を添加した培養液DMに培地を交換した。その後、２日間おきに新しい培養液DM（各種濃度のGdf5を含む）に交換し、１１日目まで培養を行った（図３（ａ））。褐色脂肪細胞を、オイルレッドO染色により染色して、計数した。 2 × 10 4 / ml brown adipose precursor cells (BFP cells) were seeded in a 24-well plate for tissue culture, suspended in culture medium DM, and culture was started. The day on which the culture was started was defined as day 0 until the third day, and the medium was replaced with a culture medium DM to which various concentrations of Gdf5 (0.1, 1, 10, or 100 ng / mL) were added. Thereafter, the culture medium was replaced with a new culture solution DM (containing various concentrations of Gdf5) every two days, and the culture was continued until the 11th day (FIG. 3A). Brown adipocytes were stained by oil red O staining and counted.
結果を図３（ｂ）と（ｃ）に示す。Gdf5を10または100ng/mLで添加した場合には、分化誘導された褐色脂肪細胞の数が有意に増加しており、分化誘導が促進されることがわかった。 The results are shown in FIGS. 3 (b) and (c). It was found that when Gdf5 was added at 10 or 100 ng / mL, the number of differentiation-induced brown adipocytes was significantly increased, and differentiation induction was promoted.
（実験例１）肥満モデルマウスにおけるGdf5の発現の確認
１）肥満モデルマウスの作製
野生型マウスであるC57BL/6Jマウスにおいて、レプチンを欠損させたマウスを作製し、肥満モデルマウス（ob/obマウス）とした。レプチン欠損マウスは、２型糖尿病のモデルマウスとして知られている。ob/obマウスの作製は、Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 1995 Jul 28;269(5223):540-3.の記載に準じて行った。
(Experimental Example 1) Confirmation of Gdf5 Expression in Obese Model Mice 1) Production of Obese Model Mice In the C57BL / 6J mouse, which is a wild type mouse, a mouse lacking leptin was produced and obese model mouse (ob / ob mouse) ). Leptin-deficient mice are known as model mice for type 2 diabetes. The ob / ob mice were produced according to the description of Effects of the obese gene product on body weight regulation in ob / ob mice. Science. 1995 Jul 28; 269 (5223): 540-3.
肥満モデルマウス（ob/obマウス）は、体重と血中グルコース濃度が、C57BL/6Jマウスと比較して上昇しており、体温はC57BL/6Jマウスと比較して低下しており、典型的な２型糖尿病の症状を示していた（図４）。 Obese model mice (ob / ob mice) have increased body weight and blood glucose levels compared to C57BL / 6J mice, and body temperature decreased compared to C57BL / 6J mice. It showed symptoms of type 2 diabetes (FIG. 4).
２）Gdf5の発現の確認
肥満モデルマウスの、骨（Bone）、筋肉（Muscle）、肝臓（Liver）、褐色脂肪組織（BAT）、白色脂肪組織（WAT）、膵臓（Pancreas）の各組織からISOGEN（ニッポンジーン：cat#317-02503）を用いてmRNAを精製した。
得られたmRNAについて、Mx3000P QPCR system （agilent technology）を用いて、Gdf5遺伝子のmRNA発現量の解析を行った。mRNA発現量の評価をするための内部標準として、364b遺伝子の発現量を確認した。解析には、以下のプライマーを用いた。同様にして、野生型マウス（WT）としてC57BL/6JマウスについてもmRNAの発現量の解析を行った。
Gdf5の発現解析に用いたプライマー：
gdf5-F;tgcccctgacttaggacagag（配列番号２）
mgdf5-R;acaccatagatatgaccccctg（配列番号３）
2) Confirmation of Gdf5 expression ISOGEN from bone, bone, muscle, liver, liver, brown adipose tissue (BAT), white adipose tissue (WAT), pancreas (Pancreas) in obese model mice MRNA was purified using (Nippon Gene: cat # 317-02503).
About the obtained mRNA, the mRNA expression level of Gdf5 gene was analyzed using Mx3000P QPCR system (agilent technology). As an internal standard for evaluating the mRNA expression level, the expression level of the 364b gene was confirmed. The following primers were used for the analysis. Similarly, the expression level of mRNA was also analyzed for C57BL / 6J mice as wild-type mice (WT).
Primers used for Gdf5 expression analysis:
gdf5-F; tgcccctgacttaggacagag (SEQ ID NO: 2)
mgdf5-R; acaccatagatatgaccccctg (SEQ ID NO: 3)
また肥満モデルマウスの褐色脂肪組織（BAT）、白色脂肪組織（WAT）からタンパク質画分を分離し、ウェスタンブロッティングを行い、Gdf5のタンパク質の発現量を確認した。タンパク質の発現量の評価をするための内部標準として、Gapdhおよびβチューブリンの発現量を確認した。ウェスタンブロッティングには、抗GDF-5 antibody（R&D systems; cat#AF853）を用いた。同様にして、野生型マウス（WT）としてC57BL/6Jマウスについてもタンパク質の発現量の解析を行った。 In addition, protein fractions were separated from brown adipose tissue (BAT) and white adipose tissue (WAT) of obese model mice, and Western blotting was performed to confirm the expression level of Gdf5 protein. As internal standards for evaluating the protein expression level, the expression levels of Gapdh and β-tubulin were confirmed. For Western blotting, anti-GDF-5 antibody (R & D systems; cat # AF853) was used. Similarly, protein expression levels were analyzed for C57BL / 6J mice as wild-type mice (WT).
結果を図５（ａ）、（ｂ）に示す。
野生型マウスにおいて、組織によるmRNA発現量の違いは見られなかったが、ob/obマウスにおいては、他の組織に比べて褐色脂肪組織において高いmRNA発現量が認められた。また、ob/obマウスの褐色脂肪細胞においては、白色脂肪細胞に比べて、高いGdf5のタンパク質量が認められた。
The results are shown in FIGS. 5 (a) and 5 (b).
In wild-type mice, no difference in mRNA expression level was found between tissues, but in ob / ob mice, a higher mRNA expression level was observed in brown adipose tissue than in other tissues. In ob / ob mice, brown adipocytes showed higher Gdf5 protein content than white adipocytes.
本発明の分化誘導促進剤または分化誘導方法により、遺伝子導入等の複雑な技術を必要とせずに、幹細胞から褐色脂肪細胞へ分化誘導を促進することができ、効率的に褐色脂肪前駆細胞や褐色脂肪細胞を得ることが可能となる。褐色脂肪細胞は中性脂質を直接熱に変換して消費することができる。本発明の分化誘導促進剤および、本発明により得られた褐色脂肪前駆細胞や褐色脂肪細胞は、メタボリックシンドローム及び肥満の予防及び／又は改善に応用することができると考えられ、有用である。さらに、Gdf5による褐色脂肪細胞の分化誘導メカニズムは本発明において新規に見出されたものである。本発明の分化誘導方法や、本発明により得られた褐色脂肪前駆細胞や褐色脂肪細胞を用いることにより、新たな作用メカニズムにより、メタボリックシンドローム及び肥満を予防及び／又は改善し得ることが期待される。 With the differentiation induction promoter or differentiation induction method of the present invention, differentiation induction from stem cells to brown adipocytes can be promoted without the need for complicated techniques such as gene transfer, and brown adipose precursor cells and brown It becomes possible to obtain fat cells. Brown adipocytes can consume neutral lipids directly converted to heat. The differentiation induction promoter of the present invention and the brown adipose precursor cells and brown adipocytes obtained by the present invention are useful because they can be applied to the prevention and / or improvement of metabolic syndrome and obesity. Furthermore, the mechanism for inducing differentiation of brown adipocytes by Gdf5 was newly found in the present invention. It is expected that metabolic syndrome and obesity can be prevented and / or improved by a new action mechanism by using the differentiation induction method of the present invention and the brown adipose precursor cells and brown adipocytes obtained by the present invention. .
Claims (7)
(a) 配列番号１で表されるアミノ酸配列からなるタンパク質；
(b) 配列番号１で表されるアミノ酸配列において、１個以上のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質；
(c) 配列番号１で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質。 An agent for promoting differentiation induction from stem cells to brown adipocytes, comprising as an active ingredient Gdf 5 which is a protein selected from any of the following (a) to (c) :
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1;
(b) The amino acid sequence represented by SEQ ID NO: 1 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and promotes differentiation induction from stem cells to brown adipocytes Functional protein;
(c) A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a function of promoting differentiation induction from stem cells to brown adipocytes.
(a) 配列番号１で表されるアミノ酸配列からなるタンパク質；
(b) 配列番号１で表されるアミノ酸配列において、１個以上のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質；
(c) 配列番号１で表されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ幹細胞から褐色脂肪細胞への分化誘導を促進する機能を有するタンパク質。 Culturing the cells in the presence of Gdf 5 which is a protein selected from any of the following (a) to (c) having a function of promoting differentiation induction from stem cells to brown adipocytes, Brown adipocyte differentiation induction method :
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1;
(b) The amino acid sequence represented by SEQ ID NO: 1 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and promotes differentiation induction from stem cells to brown adipocytes Functional protein;
(c) A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a function of promoting differentiation induction from stem cells to brown adipocytes.
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