JP2020072658A - Non-human model animal affected by non-alcoholic fatty liver disease - Google Patents
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Abstract
Description
本発明は、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の非ヒトモデル動物およびその作製方法に関する。 The present invention relates to a non-human model animal for non-alcoholic fatty liver disease (NAFLD) and a method for producing the same.
非アルコール性脂肪性肝疾患（Non-Alcoholic Fatty Liver Disease：ＮＡＦＬＤ）は脂肪肝を基本病変とし、飲酒歴が乏しいにもかかわらず、アルコール性肝障害に類似する肝実質の炎症・壊死、線維化などの組織変化を呈する病態である。ＮＡＦＬＤは基本的に無症候性であり、病態の進行に伴い、脂肪肝から脂肪性肝炎、さらに肝硬変を経て肝癌に移行する。ＮＡＦＬＤにおける脂肪性肝炎を非アルコール性脂肪性肝炎（Non-Alcoholic SteatoHepatitis：ＮＡＳＨ）と称する。特に近年、肥満または糖尿病などを背景とするメタボリック症候群が社会問題となっており、ＮＡＳＨもメタボリック症候群の一つであると考えられている。ＮＡＦＬＤおよびＮＡＳＨには、合併症として、肥満、糖尿病、高脂血症および高血圧などの生活習慣病が認められ、その臨床病態の主な特徴としては、血中のアラニンアミノトランスフェラーゼ（ＡＬＴ）やヒアルロン酸濃度の上昇、それに対して、血中の総コレステロールやアルブミン濃度の低下などが挙げられる。しかしながら、ＮＡＦＬＤおよびＮＡＳＨの発症機序は未だ不明な点も多く、その効果的な治療法および治療薬は確立されていないのが現状である。その原因の一端は、ＮＡＦＬＤおよびＮＡＳＨはヒトの生活習慣病を発症の基盤とする為に、ＮＡＦＬＤおよびＮＡＳＨの研究のための適当な非ヒトモデル動物が未だ確立されていないことにある。 Non-Alcoholic Fatty Liver Disease (NAFLD) is a basic lesion of fatty liver. Inflammation / necrosis and fibrosis of liver parenchyma similar to alcoholic liver injury despite a poor drinking history. It is a pathological condition that exhibits tissue changes such as. NAFLD is basically asymptomatic, and changes from fatty liver to steatohepatitis, and then to liver cancer through cirrhosis with the progress of the disease state. Steatohepatitis in NAFLD is called non-alcoholic steatohepatitis (NASH). Particularly in recent years, metabolic syndrome due to obesity or diabetes has become a social problem, and NASH is also considered to be one of the metabolic syndromes. Lifestyle-related diseases such as obesity, diabetes, hyperlipidemia and hypertension are recognized as complications in NAFLD and NASH, and the main clinical features thereof are blood alanine aminotransferase (ALT) and hyalurone. An increase in acid concentration, on the other hand, a decrease in total cholesterol or albumin concentration in blood can be mentioned. However, the pathogenic mechanisms of NAFLD and NASH are still unclear, and the effective therapeutic method and therapeutic agent for the same have not yet been established. One of the causes is that, since NAFLD and NASH are the basis of human lifestyle-related diseases, the non-human model animal suitable for studying NAFLD and NASH has not been established yet.
肝硬変、肝癌など致死性の疾患に進展する可能性のあるＮＡＦＬＤおよびＮＡＳＨの病態の解明は、効果的な治療法および治療薬の開発に必須であり、そのためには適当なＮＡＦＬＤおよびＮＡＳＨの非ヒトモデル動物が必要である。これまでに、ＮＡＳＨのモデル動物については報告されているが（例えば、特許文献１および２）、ＮＡＦＬＤの非ヒトモデル動物、特に大型動物についてはＮＡＳＨも含め、ほとんど報告されていない。このような状況下、疾患の進展の観点上、より基盤となるＮＡＦＬＤの非ヒトモデル動物が求められている。とりわけ、ヒトにおけるＮＡＦＬＤの治療法および治療薬の確立を目的として、生理学的、解剖学的および遺伝学的にヒトにより近い非ヒト動物のＮＡＦＬＤモデルが求められていると言える。 Elucidation of the pathological conditions of NAFLD and NASH, which may progress to fatal diseases such as cirrhosis and liver cancer, is essential for the development of effective therapeutic methods and therapeutic agents, and for that purpose, suitable NAFLD and NASH non-human A model animal is needed. So far, although the model animals of NASH have been reported (for example, Patent Documents 1 and 2), non-human model animals of NAFLD, especially large animals, including NASH, have hardly been reported. Under such circumstances, a non-human model animal of NAFLD, which becomes a base, is required from the viewpoint of disease progression. In particular, it can be said that a NAFLD model of a non-human animal that is physiologically, anatomically, and genetically closer to that of humans is required for the purpose of establishing a therapeutic method and therapeutic agent for NAFLD in humans.
そこで、本発明は、ＮＡＦＬＤの非ヒトモデル動物およびその作製方法、ならびに、その作製に用いられる飼料を提供することを目的とする。 Therefore, an object of the present invention is to provide a non-human model animal of NAFLD, a method for producing the same, and a feed used for the production.
本発明者は、上記課題を解決すべく鋭意研究を行った。本発明者は、マイクロミニブタを用いて、特定の組成のコリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料を与えることにより、生理学的、解剖学的および遺伝学的にヒトにより近いマイクロミニブタで、ＮＡＦＬＤモデル動物（モデルブタ）の作製に成功し、本発明を完成させた。当該モデルブタにおいては、血中の総コレステロールやアルブミンの濃度の低下、アラニンアミノトランスフェラーゼ（ＡＬＴ）やアスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）の濃度の上昇、および、ヘパプラスチンテスト（ＨＰＴ）の値の低下など、ＮＡＦＬＤにおいて特徴的な血中パラメータの変化が認められた。 The present inventor has conducted diligent research to solve the above problems. The present inventor uses a microminiature pig to provide a choline-free L-amino acid-defining (CDAA) diet having a specific composition, thereby enabling the microminiature pig to be physiologically, anatomically, and genetically closer to humans. The NAFLD model animal (model pig) was successfully produced, and the present invention was completed. In the model pig, the concentration of total cholesterol and albumin in blood was decreased, the concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was increased, and the value of hepaplastin test (HPT) was decreased. As described above, characteristic changes in blood parameters were observed in NAFLD.
すなわち、本発明としては、具体的には以下のものが例示される。
［１］非ヒト動物にコリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料を与えて飼育する工程を含む、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の非ヒトモデル動物を作製する方法であって、
前記ＣＤＡＡ飼料が、タンパク質を実質的に含有しないことを特徴とする、方法。
［２］前記非ヒト動物が、霊長類、げっ歯類、食肉類および偶蹄類からなる群より選択される哺乳類目の動物である、［１］に記載の方法。
［３］前記非ヒト動物がブタである、［１］または［２］に記載の方法。
［４］［１］〜［３］のいずれかに記載の方法により作製される、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の非ヒトモデル動物。
［５］以下の所見（１）〜（６）の少なくとも１つを示す、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の非ヒト動物モデル：
（１）血中総コレステロール濃度の低下
（２）血中アルブミン濃度の低下
（３）ヘパプラスチンテスト（ＨＰＴ）の値の低下
（４）血中ＩＶ型コラーゲン濃度の上昇
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度の上昇
（６）血中ヒアルロン酸濃度の上昇。
［６］非ヒト動物において非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）を惹起するために用いられる、タンパク質を実質的に含有しない、コリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料。
That is, the following are specifically exemplified as the present invention.
[1] A method for producing a non-human model animal for non-alcoholic fatty liver disease (NAFLD), which comprises a step of feeding a non-human animal with choline-free / L-amino acid-defining (CDAA) feed ,
The method, wherein the CDAA feed is substantially free of protein.
[2] The method according to [1], wherein the non-human animal is an animal of the order Mammalia selected from the group consisting of primates, rodents, carnivores and artiodactyla.
[3] The method according to [1] or [2], wherein the non-human animal is a pig.
[4] A non-human model animal of non-alcoholic fatty liver disease (NAFLD) produced by the method according to any one of [1] to [3].
[5] Non-human animal model of non-alcoholic fatty liver disease (NAFLD) showing at least one of the following findings (1) to (6):
(1) Reduction of total blood cholesterol concentration (2) Reduction of blood albumin concentration (3) Reduction of hepaplastin test (HPT) value (4) Increase of blood type IV collagen concentration (5) Blood alanine Increase in aminotransferase (ALT) concentration (6) Increase in blood hyaluronan concentration.
[6] A choline-free L-amino acid defined (CDAA) feed, which is used to induce non-alcoholic fatty liver disease (NAFLD) in a non-human animal and is substantially free of protein.
本発明により、生理学的、解剖学的および遺伝学的にヒトにより近い非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の非ヒトモデル動物およびその作製方法が提供される。また、本発明により、非ヒト動物において非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）を惹起するために用いられる飼料が提供される。 The present invention provides a non-human model animal of non-alcoholic fatty liver disease (NAFLD) which is physiologically, anatomically and genetically closer to that of humans and a method for producing the same. The present invention also provides a feed used for inducing non-alcoholic fatty liver disease (NAFLD) in non-human animals.
本発明は、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）を発症する非ヒトモデル動物（以下、「本発明のモデル動物」ともいう）を提供する。 The present invention provides a non-human model animal that develops non-alcoholic fatty liver disease (NAFLD) (hereinafter, also referred to as “model animal of the present invention”).
本発明のモデル動物は、例えば、後述する特定の組成のコリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料を非ヒト動物に与えて飼育することにより作製することができる。 The model animal of the present invention can be prepared, for example, by feeding a choline-free / L-amino acid-defining (CDAA) feed having a specific composition described below to a non-human animal and raising the animal.
本発明のモデル動物を作製するために使用される非ヒト動物（以下、「本発明で用いられる非ヒト動物」ともいう）は、実験動物として一般に使用される非ヒト動物であれば特に制限されないが、好ましくは非ヒト脊椎動物であり、より好ましくは非ヒト哺乳動物であり、さらに好ましくは偶蹄類動物である。本発明で用いられる非ヒト動物としては、具体的には、ブタ、ミニブタ、マイクロミニブタ等を例示することができる。本発明で用いられる非ヒト動物は、好ましくは、ミニブタまたはマイクロミニブタであり、特に好ましくはマイクロミニブタである。市販されているマイクロミニブタとしては、例えば、富士マイクラ株式会社製のマイクロミニブタ（系統：Fuji Micromini Pig）が挙げられる。 The non-human animal used for producing the model animal of the present invention (hereinafter, also referred to as “non-human animal used in the present invention”) is not particularly limited as long as it is a non-human animal generally used as an experimental animal. Are preferably non-human vertebrates, more preferably non-human mammals, and even more preferably cloven-hoofed animals. Specific examples of the non-human animal used in the present invention include pigs, miniature pigs, and microminiature pigs. The non-human animal used in the present invention is preferably a mini pig or a micro mini pig, and particularly preferably a micro mini pig. Examples of commercially available micro mini pigs include micro mini pigs manufactured by Fuji Micra Co., Ltd. (system: Fuji Micromini Pig).
本発明で用いられる非ヒト動物とは、ヒトと同様に肥満、糖尿病、高脂血症または高血圧などの生活習慣病を有していてもよい。本発明で用いられる非ヒト動物が生活習慣病を有する場合には、該非ヒト動物が有する生活習慣病の種類は、１種であってもよく、２種以上であってもよい。これらの生活習慣病は、遺伝的に先天的に有するものであってもよく、後天的に獲得されたものであってもよい。 The non-human animal used in the present invention may have a lifestyle-related disease such as obesity, diabetes, hyperlipidemia or hypertension like humans. When the non-human animal used in the present invention has a lifestyle-related disease, the non-human animal may have one type of lifestyle-related disease or two or more types. These lifestyle-related diseases may be genetically congenital or acquired.
本発明において用いられるコリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料（以下、「本発明で用いられる飼料」ともいう）は、タンパク質を実質的に含有しないことを特徴とする。「タンパク質を実質的に含有しない」とは、具体的には、飼料全体に対するタンパク質の含有量が、好ましくは１×１０−７重量％未満であり、より好ましくは１×１０−８重量％未満であり、特に好ましくは０重量％であることを意味する。 The choline-free / L-amino acid defined (CDAA) feed used in the present invention (hereinafter, also referred to as “feed used in the present invention”) is characterized by containing substantially no protein. The phrase "substantially free of protein" specifically means that the content of protein in the entire feed is preferably less than 1 x 10 -7 wt%, more preferably less than 1 x 10 -8 wt%. And particularly preferably 0% by weight.
本発明で用いられる飼料の組成は、上述したタンパク質の含有量の範囲を満たし、本発明で用いられる非ヒト動物においてＮＡＦＬＤの症状を誘発できる限り特に制限されず、例えば、本発明で用いられる非ヒト動物の種類に合わせて適宜設定することができる。例えば、従来のコリン不含・Ｌ−アミノ酸規定（ＣＤＡＡ）飼料の組成を基本にして、本発明で用いられる飼料の組成を設定すればよい。本発明の飼料の組成を設定し、該飼料を作製することは、当業者であれば本明細書の記載および公知の情報や技術、例えば、米国RESEARCH DIETS社製のA15022101およびA02082002Bなどを参照して容易に実施することができる。本発明で用いられる飼料は、公知の情報や技術に基づいて作製してもよく、上記市販を応用・改良したものであってもよい。 The composition of the feed used in the present invention is not particularly limited as long as it satisfies the range of the above-mentioned protein content and can induce the symptoms of NAFLD in the non-human animal used in the present invention. It can be appropriately set according to the type of human animal. For example, the composition of the feed used in the present invention may be set based on the composition of the conventional choline-free / L-amino acid defined (CDAA) feed. For setting the composition of the feed of the present invention and producing the feed, those skilled in the art can refer to the description and known information and techniques in the present specification, for example, A15022101 and A02082002B manufactured by US RESEARCH DIETS. Can be implemented easily. The feed used in the present invention may be produced based on publicly known information or technology, or may be the one obtained by applying / improving the commercially available product.
本発明の方法において、本発明で用いられる非ヒト動物に本発明で用いられるＣＤＡＡ飼料を与え始める時期は、ＮＡＦＬＤを誘発できる限り特に制限されず、例えば非ヒト動物の種類や合併症の有無に応じて適宜設定することができる。例えば、本発明で用いられる非ヒト動物が糖尿病等の生活習慣病を有する場合には、生活習慣病を発症した後、摂餌を開始するのが好ましい。また、該飼料の摂取量は、ＮＡＦＬＤを誘発できる限り特に制限されず、例えば本発明で用いられる非ヒト動物の種類、大きさまたは体重等に応じて適宜設定することができる。例えばマイクロミニブタを用いる場合には、１日当たりの該飼料の摂取量は３００ｇ程度である。また、該飼料を餌として用いて飼育する期間は、ＮＡＦＬＤを誘発できる限り特に制限されず、例えば本発明で用いられる非ヒト動物の種類に応じて適宜設定することができる。例えばマイクロミニブタを用いる場合には、通常６週間以上、好ましくは８週間以上、より好ましくは１２週間以上、該飼料を餌として用いて飼育することが好ましい。このようにして、本発明で用いられる飼料を用いて飼育することにより、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）を誘発することができる。また、例えばマイクロミニブタを用いる場合には、該飼料で飼育を続けると、およそ１４週以降で腹水がたまり、死に至ることがある。 In the method of the present invention, the time to start feeding the CDAA feed used in the present invention to the non-human animal used in the present invention is not particularly limited as long as it can induce NAFLD. For example, it depends on the type of non-human animal and the presence or absence of complications. It can be set as appropriate. For example, when the non-human animal used in the present invention has a lifestyle-related disease such as diabetes, it is preferable to start feeding after the development of the lifestyle-related disease. The intake of the feed is not particularly limited as long as it can induce NAFLD, and can be appropriately set depending on, for example, the type, size or weight of the non-human animal used in the present invention. For example, when micro miniature pigs are used, the daily intake of the feed is about 300 g. Further, the period for breeding using the feed as a feed is not particularly limited as long as NAFLD can be induced, and can be appropriately set depending on, for example, the type of non-human animal used in the present invention. For example, in the case of using a micro miniature pig, it is preferable to feed the feed for 6 weeks or longer, preferably 8 weeks or longer, more preferably 12 weeks or longer. In this way, non-alcoholic fatty liver disease (NAFLD) can be induced by breeding with the feed used in the present invention. Further, for example, when using micro miniature pigs, if the animals are kept on the feed, ascites may be accumulated after about 14 weeks, resulting in death.
なお、本発明の方法において、「飼料を与えて飼育する」とは、本発明で用いられるＣＤＡＡ飼料自体を与える場合に限らず、該ＣＤＡＡ飼料の各成分を別個に与える場合も含む。また、ＮＡＦＬＤを誘発できる限りにおいて他の飼料を併用してもよい。 In the method of the present invention, "feeding and raising" is not limited to the case where the CDAA feed itself used in the present invention is given, but also the case where each component of the CDAA feed is given separately. Further, other feeds may be used in combination as long as NAFLD can be induced.
本発明の方法により作製され得るＮＡＦＬＤモデル動物は、ＮＡＦＬＤ、特に、ヒトのＮＡＦＬＤの特徴的な臨床病態をよく反映し、また、該モデル動物においてはヒトのＮＡＦＬＤと同様の緩やかな病態の進行が認められる。よって、本発明の方法により作製され得るＮＡＦＬＤモデル動物は、ヒトへの外挿性が高く有用である。また、飼料を摂餌させるだけで、ＮＡＦＬＤ症状を生じさせることができるため作製が簡便である。 The NAFLD model animal that can be produced by the method of the present invention well reflects the characteristic clinical pathology of NAFLD, particularly human NAFLD, and in the model animal, the gradual progression of pathological conditions similar to human NAFLD is observed. Is recognized. Therefore, the NAFLD model animal that can be produced by the method of the present invention is highly extrapolative to humans and is useful. Further, the production is simple because NAFLD symptoms can be caused only by feeding the feed.
本発明のモデル動物は、以下の（１）〜（６）から選択される少なくとも１つの所見を示す。
（１）血中総コレステロール濃度の低下
（２）血中アルブミン濃度の低下
（３）ヘパプラスチンテスト（ＨＰＴ）の値の低下
（４）血中ＩＶ型コラーゲン濃度の上昇
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度の上昇
（６）血中ヒアルロン酸濃度の上昇。
なお、本明細書においては、上記所見（１）〜（６）を総称して、「ＮＡＦＬＤの所見」と称する場合がある。
The model animal of the present invention exhibits at least one finding selected from the following (1) to (6).
(1) Decrease in total blood cholesterol concentration (2) Decrease in blood albumin concentration (3) Decrease in hepaplastin test (HPT) value (4) Increase in blood type IV collagen concentration (5) Blood alanine Increase in aminotransferase (ALT) concentration (6) Increase in blood hyaluronan concentration.
In the present specification, the above findings (1) to (6) may be collectively referred to as “findings of NAFLD”.
上記所見（１）〜（６）において、「低下」または「上昇」とは、各所見に該当するパラメータが、ＮＡＦＬＤを惹起しない飼料を用いて飼育された正常個体における「標準値」と比較して「低下」または「上昇」していることを意味する。詳細には、各所見について「標準値」と比較した際の統計学的有意差に基づき判断できる。なお、各所見において、統計解析手法としては公知の統計解析手法を適宜選択して使用することができ、統計解析手法としては、例えば、多群間の検定の場合にはＡＮＯＶＡが挙げられるが、本発明の場合、Ｂｏｎｆｅｒｒｏｎｉの多重比較が好ましく用いられる。上記検定法においてＰ値が０．０５未満であった場合に「低下」または「上昇」が認められたと判断する。一方、Ｐ値が０．０５以上であった場合に「低下」または「上昇」が認められなかったと判断することができる。また、ＮＡＦＬＤを惹起しない飼料、すなわち通常飼料としては、例えば固形飼料ＭＰ−Ａ（オリエンタル酵母工業株式会社製）などが挙げられる。なお、通常飼料を用いて飼育した場合には、ＮＡＦＬＤの所見や病態が認められないことが、実験動物販売者および研究者等の当業者間では共有された認識であることから、本発明のモデル動物を作製する上で、上記各所見に該当するパラメータの経時的な変化量の有意性を検討することが非常に好ましいと言える。 In the above findings (1) to (6), “decrease” or “increase” means that the parameter corresponding to each finding is compared with “standard value” in a normal individual bred with a feed that does not induce NAFLD. Means “decreasing” or “increasing”. Specifically, it can be judged based on the statistically significant difference when each finding is compared with the “standard value”. In each of the findings, a known statistical analysis method can be appropriately selected and used as the statistical analysis method. As the statistical analysis method, for example, in the case of a test between multiple groups, ANOVA can be mentioned. In the present invention, Bonferroni's multiple comparison is preferably used. When the P value is less than 0.05 in the above assay method, it is judged that “decrease” or “increase” is recognized. On the other hand, when the P value is 0.05 or more, it can be determined that “decrease” or “increase” was not observed. In addition, examples of the feed that does not induce NAFLD, that is, normal feed include solid feed MP-A (manufactured by Oriental Yeast Co., Ltd.) and the like. It should be noted that it is a common recognition among those skilled in the art such as experimental animal sellers and researchers that no findings or pathological conditions of NAFLD are observed when they are raised using normal feed. In producing a model animal, it can be said that it is very preferable to examine the significance of the amount of change over time in the parameters corresponding to the above findings.
以下、本発明を実施例により詳細に説明するが、本発明は、これら実施例により制限されるものではなく、本発明の範囲内で種々の変更が可能である。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples, and various modifications can be made within the scope of the present invention.
＜実施例１：ＮＡＦＬＤの非ヒトモデル動物の作製＞
マイクロミニブタを用いて、以下の手順でＮＡＦＬＤの非ヒトモデル動物を作製した。
<Example 1: Preparation of non-human model animal of NAFLD>
Using a micro miniature pig, a non-human model animal of NAFLD was prepared by the following procedure.
約７ヶ月齢（体重約１０ｋｇ）の雄マイクロミニブタ（系統：Fuji Micromini Pig）を富士マイクラ株式会社から購入し、６個体について、自然換気、温度：２０±５℃、相対湿度：５５±２５％、照明サイクル：７：００〜１９：００が明、１９：００〜７：００が暗の飼育環境の下、通常飼料である固形飼料ＭＰ−Ａ（オリエンタル酵母株式会社製、含有コリン：０．２９％、含有粗タンパク質：１５．３％）を飼料として、１日３００ｇ給餌して馴化を行った。なお、給餌は各日の８：００頃に行った。 Approximately 7 months old (body weight: about 10 kg) male micro mini pigs (strain: Fuji Micromini Pig) were purchased from Fuji Micra Co., Ltd., and 6 animals were naturally ventilated, temperature: 20 ± 5 ° C., relative humidity: 55 ± 25%. , Lighting cycle: 7:00 to 19:00 is bright, and 19:00 to 7:00 is dark in a breeding environment, solid feed MP-A (manufactured by Oriental Yeast Co., containing choline: 0. (29%, crude protein content: 15.3%) was used as a feed, and 300 g was fed daily for acclimation. The feeding was performed around 8:00 each day.
使用した通常飼料ＭＰ−Ａの主な成分の組成を表１に示す。 Table 1 shows the composition of the main components of the normal feed MP-A used.
一週間程度の馴化期間の終了後、タンパク質を実質的に含有しないＣＤＡＡ飼料（RESERCH DIETS社製、A15022101）を飼料として、１日３００ｇ給餌して、同一の飼育環境の下、１６週間にわたり自由摂餌により飼育した。なお、給餌は各日の８：００頃に行った。 After the acclimation period of about 1 week, 300 g / day of CDAA feed (A15022101, manufactured by RESERCH DIETS) containing substantially no protein was fed, and the animals were allowed to freely feed for 16 weeks under the same breeding environment. It was bred by food. The feeding was performed around 8:00 each day.
使用したＣＤＡＡ飼料の主な成分の組成を表２に示す。 Table 2 shows the composition of the main components of the CDAA feed used.
詳細な結果については後述するが、タンパク質を実質的に含有しないＣＤＡＡ飼料を与えて飼育することにより、ＮＡＦＬＤに外挿可能なＮＡＦＬＤマイクロミニブタを作製することができた。 Although detailed results will be described later, a NAFLD microminiature pig that can be extrapolated to NAFLD could be produced by feeding a CDAA feed that does not substantially contain protein, and feeding the feed.
＜実施例２：ＮＡＦＬＤの非ヒトモデル動物の臨床病態の検討＞
上記のように飼育された各マイクロミニブタ群について、ＮＡＦＬＤの臨床病態の検討を行った。ＮＡＦＬＤの臨床病態としては、ヒトのＮＡＦＬＤを診断する指標を参考にして、以下の項目（１）〜（６）を設定した（ＮＡＦＬＤの所見）。
（１）血中総コレステロール濃度の低下
（２）血中アルブミン濃度の低下
（３）ヘパプラスチンテスト（ＨＰＴ）の値の低下
（４）血中ＩＶ型コラーゲン濃度の上昇
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度の上昇
（６）血中ヒアルロン酸濃度の上昇。
<Example 2: Examination of clinical pathology of non-human model animal of NAFLD>
The clinical pathology of NAFLD was examined for each group of microminiature pigs bred as described above. As the clinical condition of NAFLD, the following items (1) to (6) were set with reference to the index for diagnosing human NAFLD (findings of NAFLD).
(1) Decrease in total blood cholesterol concentration (2) Decrease in blood albumin concentration (3) Decrease in hepaplastin test (HPT) value (4) Increase in blood type IV collagen concentration (5) Blood alanine Increase in aminotransferase (ALT) concentration (6) Increase in blood hyaluronan concentration.
（ａ）測定方法
実施例１において記載された、通常飼料ＭＰ−Ａによる一週間程度の馴化期間は、ＮＡＦＬＤの病態は認められなかった。従って、馴化期間終了後のマイクロミニブタ６個体について、採血を行い血清を得、得られた血清試料を用いて、以下の（１）〜（６）の各項目について測定を行い、その測定値を「標準値」とした。
（１）血中総コレステロール濃度
（２）血中アルブミン濃度
（３）ヘパプラスチンテスト（ＨＰＴ）の値
（４）血中ＩＶ型コラーゲン濃度
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度
（６）血中ヒアルロン酸濃度
(A) Measuring method No pathological condition of NAFLD was observed during the acclimation period of about 1 week with the normal feed MP-A described in Example 1. Therefore, 6 microminiature pigs after the acclimation period were collected to obtain serum, and the obtained serum sample was used to measure the following items (1) to (6), and the measured values were obtained. It was set to "standard value".
(1) Blood total cholesterol concentration (2) Blood albumin concentration (3) Hepaplastin test (HPT) value (4) Blood type IV collagen concentration (5) Blood alanine aminotransferase (ALT) concentration (6) ) Blood hyaluronic acid concentration
上記（１）〜（６）の各項目の測定は、以下の手法およびキット等を用いて行った。
（１）血中総コレステロール濃度については、コレステロール脱水素酵素（ＵＶ）法を用いて測定した。
（２）血中アルブミン濃度については、ネフェロメトリー（ＢＣＰ改良法）を用いて測定した。
（３）ヘパプラスチンテスト（ＨＰＴ）値については、凝固時間測定を用いて測定した。
（４）血中ＩＶ型コラーゲン濃度については、Type IV collagen ELISA kit, ACB（フナコシ株式会社製）を用いて測定した。
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度については、ＪＳＣＣ標準化対応法を用いて測定した。
（６）血中ヒアルロン酸濃度については、ラテックス凝集免疫比濁法を用いて測定した。
The measurement of each item of the above (1) to (6) was performed using the following methods and kits.
(1) Blood total cholesterol concentration was measured using the cholesterol dehydrogenase (UV) method.
(2) The blood albumin concentration was measured using nephelometry (BCP improvement method).
(3) Hepaplastin test (HPT) values were measured using coagulation time measurement.
(4) The blood type IV collagen concentration was measured using Type IV collagen ELISA kit, ACB (manufactured by Funakoshi Co., Ltd.).
(5) Blood alanine aminotransferase (ALT) concentration was measured using the JSCC standardized method.
(6) Blood hyaluronic acid concentration was measured using the latex agglutination immunoturbidimetric method.
続いて、馴化期間終了後のマイクロミニブタ６個体を、タンパク質を実質的に含有しないＣＤＡＡ飼料（RESEARCH DIETS社製、A15022101）を飼料としてさらに飼育し、タンパク質を実質的に含有しないＣＤＡＡ飼料を給餌開始後１６週間にわたり、２週間ごとに採血を行い血清を得た。得られた血清試料を用いて、上記（１）〜（６）の各項目について測定を行った。 Subsequently, 6 microminiature pigs after the acclimation period were further bred by using a CDAA feed (A15022101, manufactured by RESEARCH DIETS) that does not substantially contain protein as a feed and start feeding a CDAA feed that does not substantially contain protein. Blood was collected every two weeks for serum for 16 weeks thereafter. The obtained serum sample was used to measure each of the above items (1) to (6).
（ｂ）測定結果
各個体の標準値と、タンパク質を実質的に含有しないＣＤＡＡ飼料で飼育開始後１６週までの、上記（１）〜（６）の項目の経時的な変化を表３に示す。
(B) Measurement results Table 3 shows the standard values of each individual and the changes with time in the above items (1) to (6) up to 16 weeks after the start of breeding with a CDAA feed containing substantially no protein. .
（ｃ）測定結果の比較
上記（１）〜（６）各項目について、通常飼料でＭＰ−Ａで一週間程度飼育（馴化）した時点での測定値（標準値）と、タンパク質を実質的に含有しないＣＤＡＡ飼料を給餌後１６週間までの、２週間ごとの測定値とを比較した結果を表４に示す。
(C) Comparison of measurement results For each of the above items (1) to (6), the measurement value (standard value) at the time of breeding (acclimatization) with MP-A in normal feed for about one week and the protein are substantially Table 4 shows the results of comparison with the measurement values every 2 weeks up to 16 weeks after feeding the CDAA feed that did not contain it.
表４中、上向き矢印または下向き矢印で示したものは、６個体の標準値の平均値に対して測定値の平均値が上昇または低下したことを意味する。一本の矢印は、標準値の平均値に対して測定値の平均値が１０％以上上昇または低下したことを意味し、二本矢印は、３０％以上上昇または低下したことを意味し、三本矢印は、５０％以上上昇または低下したことを意味する。また、ハイフンは１０％未満の上昇または低下を意味する。 In Table 4, what is indicated by an upward arrow or a downward arrow means that the average value of the measured values increased or decreased with respect to the average value of the standard values of 6 individuals. A single arrow means that the average value of the measured values increased or decreased by 10% or more with respect to the average value of the standard values, and a double arrow means that the average value of the measured values increased or decreased by 30% or more. This arrow means an increase or decrease of 50% or more. A hyphen means an increase or decrease of less than 10%.
表３および４に示した結果から、タンパク質を実質的に含有しないＣＤＡＡ飼料を用いて飼育した６個体のマイクロミニブタのすべてについて、血中のアルブミン濃度、総コレステロール濃度およびヘパプラスチンテスト（ＨＰＴ）値の著しい低下が認められた。一方、血中のＩＶ型コラーゲン濃度、アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度およびヒアルロン酸濃度の著しい上昇が認められた。 From the results shown in Tables 3 and 4, blood albumin concentration, total cholesterol concentration, and hepaplastin test (HPT) were measured for all 6 microminiature pigs raised on a CDAA diet containing substantially no protein. A significant decrease in the value was observed. On the other hand, a remarkable increase in blood type IV collagen concentration, alanine aminotransferase (ALT) concentration and hyaluronic acid concentration was observed.
（ｄ）肝臓組織の生検
６個体のマイクロミニブタのうちの１個体（個体番号：３）について、肝臓組織を取り出して生検写真を得た。生検写真を図１に示す。
(D) Biopsy of liver tissue With respect to one individual (individual number: 3) of the 6 microminiature pigs, the liver tissue was taken out to obtain a biopsy photograph. The biopsy picture is shown in FIG.
図１の生検写真において、灰色で示す部分は肝細胞を示し、白色の小さな粒状部分は脂肪（脂肪滴）を示す。本発明の方法により得られたマイクロミニブタの肝臓組織においては、正常の肝臓組織では認められないような、多量の脂肪（脂肪滴）の肝細胞への沈着が生じており、生検写真からもＮＡＦＬＤの所見が認められた。 In the biopsy photograph of FIG. 1, the gray portion indicates hepatocytes, and the small white granular portion indicates fat (lipid). In the liver tissue of the microminiature pig obtained by the method of the present invention, a large amount of fat (lipid) is deposited on hepatocytes, which is not observed in normal liver tissue. The findings of NAFLD were observed.
以上より、タンパク質を実質的に含有しないＣＤＡＡ飼料を用いて飼育したマイクロミニブタは、当該飼料の摂餌開始後６〜１２週目において、ＮＡＦＬＤの臨床病態を示すことが明らかとなった。特に、摂餌開始後１０〜１２週目においては、ヒトのＮＡＦＬＤに特徴的な病態の多くを反映しており、タンパク質を実質的に含有しないＣＤＡＡ飼料を与えて飼育することにより、ヒトのＮＡＦＬＤに外挿可能なＮＡＦＬＤモデル動物を作製できることが確認された。このようなタンパク質を実質的に含有しないＣＤＡＡ飼料を用いて飼育したマウスにおいては、ヒトのＮＡＦＬＤと同様の緩やかな病態の進行が認められたことから、本発明のＮＡＦＬＤモデル動物はＮＡＦＬＤの病態の解析に好適であると考えられる。また、タンパク質を実質的に含有しないＣＤＡＡ飼料は、非ヒト動物において非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）を惹起するために用いられ得ると考えられる。 From the above, it was clarified that the microminiature pigs bred with the CDAA feed which does not substantially contain protein show the clinical condition of NAFLD 6 to 12 weeks after the start of feeding of the feed. Particularly, 10 to 12 weeks after the start of feeding, many of the pathological conditions characteristic of human NAFLD are reflected, and human NAFLD was fed by feeding a CDAA feed containing substantially no protein. It was confirmed that a NAFLD model animal that can be extrapolated to was prepared. In mice bred with a CDAA feed that does not substantially contain such a protein, a gradual progression of pathological conditions similar to that of human NAFLD was observed, so that the NAFLD model animal of the present invention is It is considered to be suitable for analysis. It is also believed that a CDAA feed that is substantially free of protein may be used to induce non-alcoholic fatty liver disease (NAFLD) in non-human animals.
本発明により、ヒトと同様の臨床病態を示す非アルコール性脂肪性心疾患（ＮＡＦＬＤ）の非ヒトモデル動物を作製することができる。該非ヒトモデル動物を利用することで、ヒトのＮＡＦＬＤの治療方法および治療薬の開発が可能となる。 According to the present invention, a non-human model animal of non-alcoholic fatty heart disease (NAFLD) showing the same clinical condition as human can be prepared. By using the non-human model animal, it becomes possible to develop a method for treating NAFLD in humans and a therapeutic agent.
Claims (6)
前記ＣＤＡＡ飼料が、タンパク質を実質的に含有しないことを特徴とする、方法。 A method for producing a non-human model animal for non-alcoholic fatty liver disease (NAFLD), which comprises a step of feeding a non-human animal with a choline-free L-amino acid-defining (CDAA) feed.
The method, wherein the CDAA feed is substantially free of protein.
（１）血中総コレステロール濃度の低下
（２）血中アルブミン濃度の低下
（３）ヘパプラスチンテスト（ＨＰＴ）の値の低下
（４）血中ＩＶ型コラーゲン濃度の上昇
（５）血中アラニンアミノトランスフェラーゼ（ＡＬＴ）濃度の上昇
（６）血中ヒアルロン酸濃度の上昇。 Non-human animal model of non-alcoholic fatty liver disease (NAFLD) showing at least one of the following findings (1)-(6):
(1) Decrease in total blood cholesterol concentration (2) Decrease in blood albumin concentration (3) Decrease in hepaplastin test (HPT) value (4) Increase in blood type IV collagen concentration (5) Blood alanine Increase in aminotransferase (ALT) concentration (6) Increase in blood hyaluronan concentration.
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