JP5522435B2 - Suppression of obesity by inhibition of MXD3 gene expression - Google Patents
Suppression of obesity by inhibition of MXD3 gene expression Download PDFInfo
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Description
本発明は，肥満抑制剤のスクリーニング方法に関する。 The present invention relates to a screening method for an obesity inhibitor.
肥満は体重や体脂肪が増加した状態であるが，放置すると血管障害，糖尿病，脂質異常症，高血圧症等を発症し重大な合併症により死に至る病態である。食事療法，運動療法，薬物療法，行動療法，外科療法等が試みられているが，治療効果は不十分である。この中でも薬物療法は，現時点で膵臓リパーゼ阻害薬，選択的セロトニン／ノルエピネフリン再取り込み阻害薬，ノルアドレナリン作用薬等，その標的分子が限られており，臨床的効果も限界がある。 Obesity is a condition in which body weight and body fat have increased, but if left untreated, it develops vascular disorders, diabetes, dyslipidemia, hypertension, etc., resulting in death due to serious complications. Dietary therapy, exercise therapy, drug therapy, behavioral therapy, surgical therapy, etc. have been tried, but the therapeutic effect is insufficient. Among these, pharmacotherapy has limited target molecules such as pancreatic lipase inhibitors, selective serotonin / norepinephrine reuptake inhibitors, noradrenaline agonists, and has limited clinical effects.
従来の治療法には，多くの重大な問題点が認められる。食事療法，運動療法，行動療法などは，有効性において多くの問題点を残している。外科療法は，手術によるリスクや術後の合併症や副作用が問題点となる。薬物療法は限られた薬理ターゲット分子による治療であるため，有効性や副作用に深刻な問題点を残している。今後薬物療法の有効性を改善し，深刻な副作用を軽減するためには，新しい治療遺伝子を標的にした治療戦略の開発が不可欠である。 There are many serious problems with conventional treatments. Dietary therapy, exercise therapy, behavioral therapy, etc. have left many problems in effectiveness. Surgical therapy is problematic due to the risk of surgery and postoperative complications and side effects. Because pharmacotherapy is treatment with limited pharmacological target molecules, serious problems remain in effectiveness and side effects. In order to improve the effectiveness of pharmacotherapy and reduce serious side effects in the future, it is essential to develop treatment strategies targeting new therapeutic genes.
その発現を阻害することにより肥満を抑制することができるということが発見されている遺伝子としては，MITF（microphthalmia transcription factor）がある。また，肥満に関連した遺伝子として，遺伝子RPL19，POMC，PC2，Prolactin，Leptin Rec.，HSG P25L2G＿lが報告されている（WO2001/029071）。 A gene that has been found to be able to suppress obesity by inhibiting its expression is MITF (microphthalmia transcription factor). Further, genes RPL19, POMC, PC2, Prolactin, Leptin Rec., HSG P25L2G_l have been reported as genes related to obesity (WO2001 / 029071).
本発明は，肥満抑制剤のスクリーニング方法を提供することを目的とする。 An object of this invention is to provide the screening method of an obesity inhibitor.
本発明者らは，体重増加や脂肪増加とＭＸＤ３遺伝子の発現との間に相関があること，およびその発現機能を阻害することにより，肥満の重要な病態指標である体重，体脂肪，血清中性脂肪の増加を抑制することを見出した。 The present inventors have found that there is a correlation between weight gain or fat increase and MXD3 gene expression, and by inhibiting the expression function, weight, body fat, and serum are important pathological indicators of obesity. It was found that the increase in sex fat was suppressed.
すなわち，本発明は，肥満抑制剤の候補物質を選択する方法であって，
試験物質を細胞と接触させ，
細胞におけるＭＸＤ３遺伝子の発現を測定し，そして，
試験物質と接触させたときに接触させていないときと比較してＭＸＤ３遺伝子の発現が抑制されている場合に，その試験物質を肥満抑制剤の候補物質として選択する，
の各工程を含む方法を提供する。
That is, the present invention is a method for selecting a candidate substance for an obesity inhibitor,
Bringing the test substance into contact with the cell;
Measuring the expression of the MXD3 gene in the cell; and
Selecting the test substance as a candidate substance for an obesity inhibitor when the expression of the MXD3 gene is suppressed compared to when not contacting with the test substance,
The method including each of these steps is provided.
別の観点においては，本発明は，肥満抑制剤の候補物質を選択する方法であって，
ゼブラフィッシュに試験物質を投与し，
ＭＸＤ３遺伝子の発現を測定し，そして，
試験物質を投与したときに投与していないときと比較してＭＸＤ３遺伝子の発現が抑制されている場合に，その試験物質を肥満抑制剤の候補物質として選択する，
の各工程を含む方法を提供する。
In another aspect, the present invention provides a method for selecting a candidate substance for an obesity inhibitor,
Administer test substance to zebrafish,
Measuring the expression of the MXD3 gene; and
Selecting the test substance as a candidate substance for an obesity inhibitor when the expression of the MXD3 gene is suppressed compared to when not administering the test substance,
The method including each of these steps is provided.
さらに別の観点においては，本発明は，ＭＸＤ３遺伝子に対するアンチセンス・オリゴヌクレオチド，ＭＸＤ３遺伝子に対するリボザイム，ＭＸＤ３遺伝子に対するｓｉＲＮＡからなる群より選択される物質を有効成分として含有する肥満抑制剤を提供する。 In still another aspect, the present invention provides an obesity inhibitor containing as an active ingredient a substance selected from the group consisting of antisense oligonucleotides against the MXD3 gene, ribozymes against the MXD3 gene, and siRNAs against the MXD3 gene.
本発明のスクリーニング方法により選択された物質は，肥満の抑制，体重増加の抑制，中性脂肪の低下作用を有する薬剤の候補物質として有用である。 The substance selected by the screening method of the present invention is useful as a candidate substance for a drug having an effect of inhibiting obesity, inhibiting weight gain, and reducing neutral fat.
本発明においては，ゼブラフィッシュに肥満誘導を行うと腹腔内脂肪組織内のＭＸＤ３遺伝子発現量が増加し，食事制限を行うと低下することが見いだされた。また，下記の実施例に示されるように，肥満誘導したゼブラフィッシュにＭＸＤ３遺伝子に対するアンチセンスオリゴヌクレオチドを投与すると，体重増加や腹腔内脂肪組織量の増加が抑制され，脂肪組織関連遺伝子発現量が減少することが見いだされた。これらの結果から，ＭＸＤ３遺伝子は肥満の治療標的となりうること，およびＭＸＤ３遺伝子の発現の抑制を指標として，肥満抑制剤の候補物質を同定しうることが明らかとなった。 In the present invention, it was found that when obesity induction is performed in zebrafish, the expression level of MXD3 gene in the intraperitoneal adipose tissue increases, and decreases when dietary restriction is performed. In addition, as shown in the Examples below, when an antisense oligonucleotide against the MXD3 gene was administered to zebrafish induced to obesity, the increase in body weight and the increase in abdominal fat tissue mass was suppressed, and the expression level of adipose tissue-related gene was It was found to decrease. From these results, it became clear that the MXD3 gene can be a therapeutic target for obesity, and that a candidate substance for an obesity inhibitor can be identified using suppression of the expression of the MXD3 gene as an index.
ＭＸＤ３は，ＭＡＤ３またはＭＧＣ２３８３とも称され，Mycスーパーファミリーに属する転写制御因子である。ヒトのＭＸＤ３の遺伝子および蛋白質の配列はNCBI: NM＿001142935.1に記載されている。この蛋白質は，補因子ＭＡＸとともにヘテロダイマーを形成し，標的遺伝子のプロモータに結合してその転写を調節する。ＭＡＸ−ＭＸＤ３複合体を解離させると細胞増殖の制御が異常となるため，ＭＸＤ３は腫瘍発生に関与することが示唆されている。これまで，ＭＸＤ３遺伝子または蛋白質の発現と肥満や脂肪細胞の増殖との関連性についての示唆はない。 MXD3 is also called MAD3 or MGC2383, and is a transcriptional regulatory factor belonging to the Myc superfamily. The sequence of human MXD3 gene and protein is described in NCBI: NM_001142935.1. This protein forms a heterodimer with the cofactor MAX and binds to the promoter of the target gene to regulate its transcription. Dissociation of the MAX-MXD3 complex results in abnormal cell growth control, suggesting that MXD3 is involved in tumorigenesis. To date, there is no suggestion about the relationship between the expression of the MXD3 gene or protein and the growth of obesity or adipocytes.
スクリーニング方法
１つの観点においては，本発明は，多様な試験物質から，肥満抑制剤の候補物質をスクリーニングする方法を提供する。この方法は，試験物質が試験管内または細胞内でＭＸＤ３遺伝子の発現を阻害する能力を測定することにより評価することができる。試験物質がＭＸＤ３遺伝子の発現を阻害する能力は，試験物質をＭＸＤ３遺伝子を発現する細胞と接触させて，ＭＸＤ３のｍＲＮＡ量または蛋白質量を測定することにより評価することができる。試験物質と接触させたときに，接触させないときと比較してＭＸＤ３遺伝子の発現が抑制されれば，その試験物質は肥満抑制剤の候補物質であると考えられる。
Screening Method In one aspect, the present invention provides a method for screening candidate substances for obesity inhibitors from various test substances. This method can be evaluated by measuring the ability of the test substance to inhibit the expression of the MXD3 gene in vitro or in cells. The ability of the test substance to inhibit the expression of the MXD3 gene can be evaluated by contacting the test substance with a cell expressing the MXD3 gene and measuring the amount of mRNA or protein of MXD3. If the expression of the MXD3 gene is suppressed when contacted with a test substance as compared to when it is not contacted, the test substance is considered to be a candidate substance for an obesity inhibitor.
試験物質は，種々の合成または天然の化合物ライブラリー，コンビナトリアルライブラリー，オリゴヌクレオチドライブラリー，ペプチドライブラリー等のライブラリーから得ることができる。また，細菌，真菌類，藻類，植物，動物等の天然物からの抽出物やその部分精製物を試験物質として用いてもよい。 The test substance can be obtained from libraries such as various synthetic or natural compound libraries, combinatorial libraries, oligonucleotide libraries, peptide libraries and the like. Moreover, you may use the extract from natural products, such as bacteria, fungi, algae, a plant, and an animal, or its partially purified product as a test substance.
ＭＸＤ３遺伝子の発現を測定するためには，RT-PCR法，RNA増幅法，ISH（in situ hybridization）法，ISP（in situ PCR）法，レーザーマイクロダイセクション法，レーザーキャプチャーマイクロダイセクション法（LCM）などの，当該技術分野においてよく知られる遺伝子発現分析法を用いることができる。また，ＭＸＤ３の蛋白質量は，抗ＭＸＤ３抗体を用いるＥＬＩＳＡ法やウエスタンブロット法などにより測定することができる。ヒト，ラット，マウス，ゼブラフィッシュなどのＭＸＤ３蛋白質およびこれをコードする遺伝子の配列は公表されている。 To measure the expression of MXD3 gene, RT-PCR, RNA amplification, ISH (in situ hybridization), ISP (in situ PCR), laser microdissection, laser capture microdissection (LCM) And the like well-known gene expression analysis methods in the art can be used. In addition, the protein mass of MXD3 can be measured by an ELISA method using an anti-MXD3 antibody, a Western blot method, or the like. The sequences of MXD3 protein such as human, rat, mouse, zebrafish and the gene encoding the same have been published.
ＭＸＤ３遺伝子を発現する細胞としては，各種の組織に由来する初代培養組織や培養細胞株などの任意のものを用いることができる。ＭＸＤ３遺伝子を強制発現させるよう遺伝子組換えされた細菌，酵母，昆虫細胞，動物細胞等を用いてもよい。 As the cells expressing the MXD3 gene, any cells such as primary cultured tissues and cultured cell lines derived from various tissues can be used. Bacteria, yeast, insect cells, animal cells, etc. genetically modified to force the MXD3 gene to be expressed may be used.
１つの好ましい態様においては，試験物質をゼブラフィッシュに投与して，ＭＸＤ３遺伝子の発現を調べる。ゼブラフィッシュ（属名Danio，例えばDanio rerio）とはインド原産の小型の熱帯魚である。主要臓器・組織の発生・構造などがヒトと良く似ているうえ，身体が透明であるため各種臓器および血管の形態を肉眼で観察することができるため，モデル動物として新薬候補化合物のスクリーニングに用いるのに適している。 In one preferred embodiment, a test substance is administered to zebrafish to examine MXD3 gene expression. Zebrafish (genus name Danio, for example Danio rerio) is a small tropical fish native to India. The main organs and tissues are similar in appearance and structure to humans, and because the body is transparent, various organs and blood vessel morphology can be observed with the naked eye. Suitable for
本発明の方法には，通常の飼育条件で飼育したゼブラフィッシュを用いてもよく，あるいは，高脂肪餌を与えて肥満誘導したモデルゼブラフィッシュを用いてもよい。試験物質をゼブラフィッシュに投与する方法としては，候補物質が水溶性である場合には飼育水中に溶解すればよく，水不溶性である場合には適当な界面活性剤との複合体またはエマルジョンの形で飼育水中に懸濁すればよい。あるいは，ゼブラフィッシュの餌に混ぜて経口投与してもよく，注射などにより非経口投与してもよい。 In the method of the present invention, a zebrafish raised under normal breeding conditions may be used, or a model zebrafish induced by obesity by feeding a high fat diet may be used. The test substance can be administered to zebrafish by dissolving it in the breeding water if the candidate substance is water-soluble, or in the form of a complex or emulsion with an appropriate surfactant if it is water-insoluble. Can be suspended in the breeding water. Alternatively, it may be administered orally in a zebrafish diet, or parenterally by injection or the like.
本発明のスクリーニング方法にゼブラフィッシュを用いることの利点は，試験物質がＭＸＤ３遺伝子の発現に及ぼす影響を測定できると同時に，毒性試験も行うことができることである。さらに，体重の変化，脂肪組織量の変化，心臓などの各種臓器の形態や機能の変化，各種マーカー遺伝子の発現量も測定することができる。 An advantage of using zebrafish in the screening method of the present invention is that the influence of the test substance on the expression of the MXD3 gene can be measured, and at the same time, a toxicity test can be performed. Furthermore, changes in body weight, changes in adipose tissue mass, changes in morphology and function of various organs such as the heart, and expression levels of various marker genes can also be measured.
このようにして，本発明のスクリーニング方法により選択された物質は，肥満の抑制，体重増加の抑制，中性脂肪の低下作用を有する薬剤の候補物質として有用である。 In this way, the substance selected by the screening method of the present invention is useful as a candidate substance for a drug having an action of inhibiting obesity, inhibiting weight gain, and reducing triglycerides.
肥満抑制剤
別の観点においては，本発明は，ＭＸＤ３遺伝子の発現を阻害する物質を有効成分として含有する肥満抑制剤を提供する。遺伝子の発現を阻害しうる阻害剤としては，例えば，
アンチセンスオリゴヌクレオチド，リボザイム，ＲＮＡ干渉（ＲＮＡｉ）を引き起こす分子（例えば，ｄｓＲＮＡ，ｓｉＲＮＡ，ｓｈＲＮＡ，ｍｉＲＮＡ）等の，ＭＸＤ３遺伝子の発現（転写および／または翻訳）の抑制剤を挙げることができる。このような核酸は，ＭＸＤ３の既知のヌクレオチド配列情報に基づいて容易に設計し製造することができ，ＭＸＤ３遺伝子またはＭＸＤ３をコードするｍＲＮＡに結合しその発現を阻害することができる。アンチセンス，リボザイム技術およびＲＮＡｉ技術を用いて遺伝子発現を制御する一般的方法，またはこのようにして外因性遺伝子を発現させる遺伝子治療方法は当該技術分野においてよく知られている。
In another aspect of the obesity inhibitor , the present invention provides an obesity inhibitor that contains a substance that inhibits the expression of the MXD3 gene as an active ingredient. Examples of inhibitors that can inhibit gene expression include:
Examples include inhibitors of expression (transcription and / or translation) of the MXD3 gene, such as antisense oligonucleotides, ribozymes, and molecules that cause RNA interference (RNAi) (eg, dsRNA, siRNA, shRNA, miRNA). Such a nucleic acid can be easily designed and manufactured based on the known nucleotide sequence information of MXD3, and can bind to and inhibit the expression of MXD3 gene or mRNA encoding MXD3. General methods for controlling gene expression using antisense, ribozyme technology and RNAi technology, or gene therapy methods for expressing exogenous genes in this manner are well known in the art.
アンチセンスオリゴヌクレオチドとは，ＭＸＤ３をコードするｍＲＮＡと相補的な配列を有する核酸分子またはその誘導体を表す。アンチセンスオリゴヌクレオチドは，ｍＲＮＡと特異的に結合し，転写および／または翻訳を阻害することにより蛋白質の発現を阻害する。結合はワトソン・クリックまたはフーグスティーン型の塩基対相補性によるものでもよく，トリプレックス形成によるものでもよい。 An antisense oligonucleotide represents a nucleic acid molecule having a sequence complementary to mRNA encoding MXD3 or a derivative thereof. Antisense oligonucleotides specifically bind to mRNA and inhibit protein expression by inhibiting transcription and / or translation. Binding may be by Watson-Crick or Hoogsteen-type base pair complementarity, or by triplex formation.
リボザイムとは，標的とする核酸配列を切断しうる触媒的特性を有するＲＮＡ分子を表す。リボザイムは，一般に，エンドヌクレアーゼ，リガーゼまたはポリメラーゼ活性を示す。種々の二次構造のリボザイム，例えばハンマーヘッドタイプおよびヘアピンタイプのリボザイムが知られている。ＲＮＡ干渉（ＲＮＡｉ）とは，二本鎖ＲＮＡ分子を用いて標的遺伝子をサイレンシングする手法をいう。 A ribozyme represents an RNA molecule having catalytic properties that can cleave a target nucleic acid sequence. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Various ribozymes of secondary structure, such as hammerhead type and hairpin type ribozymes are known. RNA interference (RNAi) refers to a technique of silencing a target gene using a double-stranded RNA molecule.
本発明の肥満抑制剤は，被験者にそのまま投与することも可能であるが，通常，医薬で用いられる担体を用いて製剤して投与する。製剤に用いる担体としては，製剤分野で常用されるいずれのものをも用いることができ，例えば，滅菌水，生理食塩水，賦形剤，安定剤，酸化防止剤，緩衝剤，界面活性剤，結合剤等が好ましく用いられる。さらに，本発明の肥満抑制剤をマイクロカプセルや高分子ゲル中に封入して，徐放性製剤としてもよい。本発明の肥満抑制剤の投与経路としては，経口投与，静脈内投与，皮内投与，皮下投与，筋肉内投与，体腔内投与等が挙げられる。投与量は，肥満抑制剤の種類や投与経路，疾患の程度等に応じて適宜選択されるが，通常，０．１μg〜１０００mg/Kg，好ましくは，１μg〜１０mg/Kgである。 The obesity-suppressing agent of the present invention can be administered to a subject as it is, but is usually formulated and administered using a carrier used in medicine. As the carrier used in the preparation, any of those commonly used in the pharmaceutical field can be used. For example, sterile water, physiological saline, excipients, stabilizers, antioxidants, buffers, surfactants, A binder or the like is preferably used. Furthermore, the obesity inhibitor of the present invention may be encapsulated in a microcapsule or a polymer gel to form a sustained-release preparation. The administration route of the obesity inhibitor of the present invention includes oral administration, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, intracavitary administration and the like. The dose is appropriately selected according to the type of obesity inhibitor, the administration route, the degree of disease, etc., but is usually 0.1 μg to 1000 mg / Kg, preferably 1 μg to 10 mg / Kg.
以下に実施例により本発明をより詳細に説明するが，本発明はこれらの実施例により限定されるものではない。 EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.
実施例１ 肥満誘導ゼブラフィッシュにおける腹腔内脂肪組織のＭＸＤ３遺伝子発現
ゼブラフィッシュ成魚（ＡＢ系統，受精後４ヶ月齢，オス）に，１匹あたりアルテミア６０ｍｇ／日をエサとして与え（肥満誘導），１週間後と２ヶ月後の腹腔内脂肪を回収した。さらに肥満誘導２ヶ月後，食事制限実験として，アルテミア２．５ｍｇ／日に減らした状態にして２週間飼育し，腹腔内脂肪を回収した。回収した脂肪組織からＲＮｅａｓｙ Ｌｉｐｉｄ Ｔｉｓｓｕｅ Ｍｉｎｉ Ｋｉｔ（キアゲン社）を用いて，ｔｏｔａｌ ＲＮＡを精製，Ｓｕｐｅｒ Ｓｃｒｉｐｔ III（インビトロジェン社）を用いて，ｃＤＮＡを合成し，ＭＸＤ３遺伝子発現量を定量的ＰＣＲ法にて測定した。
Example 1 MXD3 Gene Expression of Intraperitoneal Adipose Tissue in Obesity-Induced Zebrafish Zebrafish adult fish (AB strain, 4 months old after fertilization, male) are fed with 60 mg / day of Artemia per animal (obesity induction), 1 Intraperitoneal fat was collected after a week and two months later. Further, 2 months after the induction of obesity, as a diet restriction experiment, Artemia was reduced to 2.5 mg / day, reared for 2 weeks, and intraperitoneal fat was collected. From the collected adipose tissue, RNeasy Lipid Tissue Mini Kit (Qiagen) was used to purify total RNA, Super Script III (Invitrogen) was used to synthesize cDNA, and the MXD3 gene expression level was determined by quantitative PCR. It was measured.
ゼブラフィッシュＭＸＤ３遺伝子特異的モルフォリノアンチセンスオリゴヌクレオタイドの配列は，Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎに掲載されている，ＬＯＣＵＳ ＮＭ＿２０１０５６ ＭＡＸ ｄｉｍｅｒｉｚａｔｉｏｎ ｐｒｏｔｅｉｎ ３ （ｍｘｄ３）を使用し，プライマーをデザインし，ＰＣＲ実験に使用した。
ｍｘｄ３プライマーの配列情報
フォワードプライマー：ＣＣＡＣＡＧＣＴＡＣＴＣＴＡＣＣＴＣＧＧＡＴ（配列番号１）
リバースプライマー：ＧＴＡＡＧＧＣＴＧＴＴＣＴＧＴＧＡＴＡＡＴＧＡＴＧＧＴ（配列番号２）
The sequence of the zebrafish MXD3 gene-specific morpholino antisense oligonucleotide was designed using the primers of LOCUS NM_201056 MAX dimerization protein 3 (mxd3) published in the National Center for Biotechnology Information. did.
Sequence information of mxd3 primer Forward primer: CCACAGCTACTCTACTCTGGAT (SEQ ID NO: 1)
Reverse primer: GTAAGGCTGTTCTGTGATAATGATGGT (SEQ ID NO: 2)
結果を図１に示す。overfed 1Wは肥満誘導１週間目，overfed 2Wは肥満誘導２ヶ月目，overfed + diet restrictionは肥満誘導を２ヶ月処理したのち，２週間の食事制限を行った実験動物群である。これらのＭＸＤ３遺伝子発現量は，ハウスキーピング遺伝子であるＡＣＴＩＮ，ＢＥＴＡの発現量で正規化した相対値で示される。図１に示すように，ＭＸＤ３遺伝子発現量は，肥満誘導２ヶ月目（overfed 2M）では，肥満誘導１週間に比較し，約４倍の有意な増加を認め，食事制限では減少する傾向を示した。 The results are shown in FIG. Overfed 1W is the first week of obesity induction, overfed 2W is the second month of obesity induction, and overfed + diet restriction is a group of experimental animals that have been subjected to obesity induction for two months, followed by a two-week diet restriction. These MXD3 gene expression levels are expressed as relative values normalized by the expression levels of ACTIN and BETA, which are housekeeping genes. As shown in Fig. 1, the MXD3 gene expression level showed a significant increase of about 4 times in obesity induction 2 months (overfed 2M) compared to 1 week in obesity induction, and showed a tendency to decrease in dietary restriction. It was.
以上の結果から，肥満誘導を行うと腹腔内脂肪組織内のＭＸＤ３遺伝子発現量は増加し，食事制限を行うと低下することが明らかとなった。 From the above results, it was clarified that the MXD3 gene expression level in the intraperitoneal adipose tissue increases when obesity is induced and decreases when dietary restriction is performed.
実施例２ ＭＸＤ３遺伝子発現抑制が腹腔内脂肪組織量および脂肪組織関連遺伝子発現量に及ぼす影響
ゼブラフィッシュ成魚（ＡＢ系統あるいはｒｏｓｅ系統，受精後４ヶ月齢，メス）の腹腔内に，１ｋｇ体重あたり１００ｍｇ量の，ゼブラフィッシュＭＸＤ３遺伝子特異的モルフォリノアンチセンスオリゴヌクレオチドを，リポフェクション法を用いて腹腔内投与した。
Example 2 Effect of suppression of MXD3 gene expression on abdominal adipose tissue and adipose tissue-related gene expression 100 mg / kg body weight in the abdominal cavity of adult zebrafish (AB strain or rose strain, 4 months old after fertilization, female) A quantity of zebrafish MXD3 gene-specific morpholino antisense oligonucleotide was administered intraperitoneally using the lipofection method.
ゼブラフィッシュＭＸＤ３遺伝子特異的モルフォリノアンチセンスオリゴヌクレオチドの配列は，Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎに掲載されている，ＬＯＣＵＳ ＮＭ＿２０１０５６ ＭＡＸ ｄｉｍｅｒｉｚａｔｉｏｎ ｐｒｏｔｅｉｎ ３ （ｍｘｄ３），ｍＲＮＡの転写開始点に結合する相補的配列とした。ＭＸＤ３モルフォリノアンチセンスオリゴヌクレオチドの配列情報は下記のとおりである。
ＧＡＴＧＴＴＧＣＡＣＧＴＡＴＴＴＡＣＣＴＣＣＡＴＴ（配列番号３）
The sequence of the zebrafish MXD3 gene-specific morpholino antisense oligonucleotide binds to the transcription start point of LOCUS NM — 201056 MAX dimerization protein 3 (mxd3), which is published in the National Center for Biotechnology Information. . The sequence information of MXD3 morpholino antisense oligonucleotide is as follows.
GATGTTGCACGTATTTACTCCCATT (SEQ ID NO: 3)
比較対照群として，ゼブラフィッシュには存在しない遺伝子を標的としたコントロールモルフォリノアンチセンスオリゴヌクレオチドを同様の方法を用いて腹腔内投与した。コントロールモルフォリノアンチセンスオリゴヌクレオチドの配列情報は下記のとおりである。
ＣＣＴＣＴＴＡＣＣＴＣＡＧＴＴＡＣＡＡＴＴＴＡＴＡ（配列番号４）
As a control group, a control morpholino antisense oligonucleotide targeting a gene not present in zebrafish was administered intraperitoneally using the same method. The sequence information of the control morpholino antisense oligonucleotide is as follows.
CCTCTTACCTCAGTTACAATTTTATA (SEQ ID NO: 4)
リポフェクション法には，リポフェクタミン２０００（インビトロジェン社）の試薬を用いた。各腹腔内投与は週に1回，day1, 7, 14, 21に行い，その期間，肥満誘導としてゼブラフィッシュのエサであるアルテミアを，１日６０ｍｇ投与した。 Lipofectamine 2000 (Invitrogen) reagent was used for the lipofection method. Each intraperitoneal administration was performed once a week on days 1, 7, 14, and 21. During that period, 60 mg of Artemia, a zebrafish feed, was administered daily for obesity induction.
毎週１回，身長と体重を測定した結果，ＭＸＤ３遺伝子発現抑制群では，肥満誘導時の体重増加が有意に抑制された（図２）。身長については差を認めなかった。 As a result of measuring height and weight once a week, in the MXD3 gene expression suppression group, weight gain during obesity induction was significantly suppressed (FIG. 2). There was no difference in height.
また，３週間後の血漿中の中性脂肪値をＬタイプワコー ＴＧ・Ｈ（和光純薬）を用いて測定した。図３に結果を示す。normal fedは通常飼育動物群，overfedは肥満誘導群である。ＭＸＤ３遺伝子発現抑制により，血漿中の中性脂肪の量が減少した。摂食量については差を認めなかった。 In addition, the neutral fat level in plasma after 3 weeks was measured using L-type Wako TG • H (Wako Pure Chemical Industries). The results are shown in FIG. normal fed is a group of domestic animals, and overfed is an obesity induction group. The amount of neutral fat in plasma decreased due to suppression of MXD3 gene expression. There was no difference in food intake.
なおＭＸＤ３遺伝子発現の抑制は，day 21の段階で，腹腔内臓器を摘出し，蛋白質を抽出して，ウェスタンブロット法にて確認した。ＭＸＤ３ ＭＯ投与群ではＭＸＤ３蛋白質量の減少が認められた。 The suppression of the MXD3 gene expression was confirmed on the day 21 by extracting the intraperitoneal organ, extracting the protein, and western blotting. In the MXD3 MO administration group, a decrease in the amount of MXD3 protein was observed.
蛍光色素の１種であるナイルレッドは，脂肪組織を特異的に染色することが知られている。ｒｏｓｅ系統のゼブラフィッシュを用いた場合，ナイルレッド染色によって，腹腔内の脂肪量を蛍光量にて測定できる。ＭＸＤ３ ＭＯを腹腔内投与し，肥満誘導３週間後のゼブラフィッシュに対して，ナイルレッド染色を行った結果，ＭＸＤ３ ＭＯ群ではコントロールと比較してその蛍光量が１／４に減弱していた（図４）。 Nile red, which is one type of fluorescent dye, is known to specifically stain adipose tissue. When a rose zebrafish is used, the amount of fat in the abdominal cavity can be measured by the amount of fluorescence by Nile red staining. MXD3 MO was intraperitoneally administered, and as a result of performing Nile red staining on zebrafish 3 weeks after induction of obesity, the fluorescence amount in the MXD3 MO group was attenuated to 1/4 compared to the control ( FIG. 4).
さらに，腹腔内脂肪組織を剔出し，その組織に存在しているｍＲＮＡの量を定量的ＰＣＲ法で測定したところ，図５に示すように，脂肪細胞のマーカー遺伝子であるＣａｖｅｏｌｉｎ（ＣＡＶ），ＣＤ３６，Ａｄｉｐｏｎｅｃｔｉｎ ｂ遺伝子の発現量の減少を認めた。 Furthermore, when the abdominal fat tissue was excised and the amount of mRNA present in the tissue was measured by quantitative PCR, as shown in FIG. 5, Caveolin (CAV), CD36, which are adipocyte marker genes, were obtained. , A decrease in the expression level of Adiponectin b gene was observed.
また，脂肪前駆細胞から成熟脂肪細胞への分化を誘導するＰｅｒｏｘｉｓｏｍｅ Ｐｒｏｌｉｆｅｒａｔｏｒ−Ａｃｔｉｖａｔｅｄ Ｒｅｃｅｐｔｏｒ−Ｇａｍｍａ（ＰＰＡＲＧ），ＣＣＡＡＴ／Ｅｎｈａｎｃｅｒ−ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ，ａｌｐｈａ（ＣＥＢＰＡ）遺伝子の発現量も減少していた（図６）。
これらの遺伝子の定量的ＰＣＲに用いたプライマー配列は下記のとおりである。
ＣＡＶ
フォワードプライマー：ＧＡＡＧＧＡＧＡＧＧＡＴＧＧＣＧＡＡＧＡＡＧ（配列番号５）
リバースプライマー：ＧＡＡＧＧＡＧＡＧＧＡＴＧＧＣＧＡＡＧＡＡＧ（配列番号６）
ＣＤ３６
フォワードプライマー：ＣＧＡＴＧＡＧＡＣＧＧＣＣＡＡＡＡＴＧＴＴＣＡ（配列番号７）
リバースプライマー：ＣＣＣＡＧＡＡＡＴＡＴＴＧＣＣＧＡＡＣＣＡＧＴＴ（配列番号８）
Ａｄｉｐｏｎｅｃｔｉｎ ｂ
フォワードプライマー：ＣＴＡＣＴＴＣＴＴＣＡＣＧＴＡＣＣＡＴＣＴＧＡＣＣ（配列番号９）
リバースプライマー：ＣＡＴＧＡＴＧＧＣＣＴＴＧＴＣＧＴＴＣＴＴＧＴ（配列番号１０）
ＰＰＡＲＧ
フォワードプライマー： ＴＣＣＡＣＡＧＣＴＡＣＣＡＧＴＣＣＡＧＡＴＣ（配列番号１１）
リバースプライマー：ＣＡＧＣＧＴＣＡＣＣＴＧＧＴＣＧＴＴＣＡ（配列番号１２）
ＣＥＢＰＡ
フォワードプライマー：ＧＴＡＣＣＴＡＧＴＣＣＧＣＡＣＣＡＣＣＡＧ（配列番号１３）
リバースプライマー：ＣＴＧＴＡＣＴＣＧＧＴＧＣＴＧＴＴＣＴＴＧＴ（配列番号１４）
In addition, the expression levels of Peroxisome Proliferator-Activated Receptor-Gamma (PPARG), CCAAT / Enhancer-binding protein, and Alpha (CEBPA) genes that induce differentiation from preadipocytes to mature adipocytes were also reduced (FIG. 6). .
The primer sequences used for quantitative PCR of these genes are as follows.
CAV
Forward primer: GAAGGAGAGAGATGGCGAGAAAG (SEQ ID NO: 5)
Reverse primer: GAAGGAGAGAGATGGCGAAGAAG (SEQ ID NO: 6)
CD36
Forward primer: CGATGGAGCGGCCAAAATGTTCA (SEQ ID NO: 7)
Reverse primer: CCCAGAAAATTGCCCGAACCAGTT (SEQ ID NO: 8)
Adipontin b
Forward primer: CTACTTCTTCACGTACCATCTGACC (SEQ ID NO: 9)
Reverse primer: CATGATGGCCTTGTCGTTCTTGT (SEQ ID NO: 10)
PPARG
Forward primer: TCCACAGCTACCAGTCCCATC (SEQ ID NO: 11)
Reverse primer: CAGCGTCACCTGGTCGTTCA (SEQ ID NO: 12)
CEBPA
Forward primer: GTACCTAGTCCCGCACCACCAG (SEQ ID NO: 13)
Reverse primer: CGTACTCGGGTGCTGTTCTTGT (SEQ ID NO: 14)
以上の結果から，ＭＸＤ３遺伝子の発現を阻害することにより，肥満誘導における体重増加，血漿中の中性脂肪量の増加が抑制され，腹腔内脂肪組織量および脂肪組織関連遺伝子発現量が減少し，前駆脂肪細胞から脂肪細胞への分化が抑制されたことがわかる。 From the above results, by inhibiting the expression of the MXD3 gene, weight gain in obesity induction and increase in plasma neutral fat mass were suppressed, and intraperitoneal adipose tissue mass and adipose tissue-related gene expression were decreased, It can be seen that differentiation from preadipocytes to adipocytes was suppressed.
本発明は，肥満抑制剤の候補物質のスクリーニングならびに肥満抑制剤の治療に有用である。 The present invention is useful for screening candidate substances for obesity inhibitors and treating obesity inhibitors.
Claims (3)
試験物質を細胞と接触させ，
細胞におけるＭＸＤ３遺伝子の発現を測定し，そして，
試験物質と接触させたときに接触させていないときと比較してＭＸＤ３遺伝子の発現が抑制されている場合に，その試験物質を肥満抑制剤の候補物質として選択する，
の各工程を含む方法。 A method of selecting candidate substances for obesity inhibitors,
Bringing the test substance into contact with the cell;
Measuring the expression of the MXD3 gene in the cell; and
Selecting the test substance as a candidate substance for an obesity inhibitor when the expression of the MXD3 gene is suppressed compared to when not contacting with the test substance,
The method including each process of these.
ゼブラフィッシュに試験物質を投与し，
ＭＸＤ３遺伝子の発現を測定し，そして，
試験物質を投与したときに投与していないときと比較してＭＸＤ３遺伝子の発現が抑制されている場合に，その試験物質を肥満抑制剤の候補物質として選択する，
の各工程を含む方法。 A method of selecting candidate substances for obesity inhibitors,
Administer test substance to zebrafish,
Measuring the expression of the MXD3 gene; and
Selecting the test substance as a candidate substance for an obesity inhibitor when the expression of the MXD3 gene is suppressed compared to when not administering the test substance,
The method including each process of these.
An obesity inhibitor comprising as an active ingredient a substance selected from the group consisting of an antisense oligonucleotide against the MXD3 gene, a ribozyme against the MXD3 gene, and an siRNA against the MXD3 gene.
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