JP2020145983A - Alopecia areata model animal - Google Patents
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Abstract
Description
本発明は、円形脱毛症モデル動物及び円形脱毛症発症予防又は治療薬のスクリーニング方法に関する。 The present invention relates to alopecia areata model animals and a method for screening a preventive or therapeutic agent for the onset of alopecia areata.
円形脱毛症は、頭皮に円形の脱毛斑を生じる疾患であり、脱毛が頭髪全体に及ぶケースや、頭髪以外の体毛にまで及ぶケースもみられる。このような外観の変化により、患者のＱＯＬは著しく損なわれる。円形脱毛症は自己免疫疾患であると推定されているが、その発症機序はいまだ明らかになっていない。 Alopecia areata is a disease that causes circular bald spots on the scalp, and there are cases where hair loss extends to the entire hair and to body hair other than the hair. Such changes in appearance significantly impair the patient's quality of life. Alopecia areata is presumed to be an autoimmune disease, but its pathogenic mechanism remains unclear.
これまでに、円形脱毛症に関与する遺伝子座として、ヒト白血球型抗原（ＨＬＡ）遺伝子座（非特許文献１）や、自己免疫調節（ａｕｔｏｉｍｍｕｎｅ ｒｅｇｕｌａｔｏｒ：ＡＩＬＥ）遺伝子等の非ＨＬＡ遺伝子座（非特許文献２）が報告されている。また、ＨＬＡ−Ｃ遺伝子座の特定アレルと円形脱毛症とが強く相関することも報告されている（非特許文献３）。 So far, as loci involved in round alopecia, non-HLA loci (non-patent) such as human leukocyte antigen (HLA) locus (Non-Patent Document 1) and autoimmune regulator (AILE) gene. Document 2) has been reported. It has also been reported that a specific allele at the HLA-C locus is strongly correlated with alopecia areata (Non-Patent Document 3).
本発明の課題は、新たな円形脱毛症モデル非ヒト動物、及び該モデル非ヒト動物を用いた円形脱毛症発症予防又は治療薬のスクリーニング方法を提供することにある。 An object of the present invention is to provide a new alopecia areata model non-human animal and a method for screening a preventive or therapeutic agent for the onset of alopecia areata using the model non-human animal.
本発明者らは、これまでに、ｃｏｉｌｅｄ−ｃｏｉｌ α−ｈｅｌｉｃａｌ ｒｏｄ ｐｒｏｔｅｉｎ １（ＣＣＨＣＲ１）タンパク質の特定のアミノ酸を置換した変異型タンパク質を発現する遺伝子改変マウスが、円形脱毛症を発症することを見出した。該マウスは、その約５０％が生後４〜８か月後に背部にパッチ状の脱毛を呈し、円形脱毛症の症状を再現するものであったが、さらに、発症段階を含めたヒトにおける円形脱毛症の病態をよりよく再現したモデル動物の作出が望まれる。
そこで、本発明者らは、ＣＣＨＣＲ１タンパク質に注目し、かかる遺伝子のノックアウトマウスを作製したところ、全く意外にもＣＣＨＣＲ１遺伝子改変マウスとは異なり、ＣＣＨＣＲ１遺伝子ノックアウトマウスは、胎生期及び第一期の毛成長が正常である一方、ストレス負荷により初めて円形脱毛症を発症して円形脱毛症に特徴的な症状を呈し、発症段階を含めたヒトの円形脱毛症の病態をよりよく再現できることを見出し、本発明を完成した。
The present inventors have previously found that genetically modified mice expressing a mutant protein in which a specific amino acid of the coiled-coil α-helical rod protein 1 (CCHCR1) protein is substituted develops alopecia areata. It was. Approximately 50% of the mice exhibited patch-like hair loss on the back 4 to 8 months after birth, reproducing the symptoms of alopecia areata, but further, alopecia areata in humans including the onset stage. It is desired to create a model animal that better reproduces the pathophysiology of the disease.
Therefore, when the present inventors focused on the CCHCR1 protein and produced knockout mice of such a gene, surprisingly, unlike the CCHCR1 gene-modified mouse, the CCHCR1 gene knockout mouse had hair in the embryonic stage and the first stage. While the growth is normal, we found that alopecia areata develops for the first time due to stress load and exhibits the symptoms characteristic of alopecia areata, and that the pathophysiology of alopecia areata in humans including the onset stage can be better reproduced. The invention was completed.
すなわち、本発明は、次の〔１〕〜〔２〕を提供するものである。 That is, the present invention provides the following [1] to [2].
〔１〕ｃｏｉｌｅｄ−ｃｏｉｌ α−ｈｅｌｉｃａｌ ｒｏｄ ｐｒｏｔｅｉｎ １（ＣＣＨＣＲ１）遺伝子がノックアウトされた円形脱毛症モデル非ヒト動物。
〔２〕〔１〕に記載の円形脱毛症モデル非ヒト動物に被験物質を投与することを特徴とする、円形脱毛症発症予防又は治療薬のスクリーニング方法。
[1] Alopecia areata model non-human animal in which the coiled-coil α-helical rod protein 1 (CCHCR1) gene is knocked out.
[2] A method for screening a preventive or therapeutic agent for the onset of alopecia areata, which comprises administering the test substance to the alopecia areata model non-human animal according to [1].
本発明の円形脱毛症モデル非ヒト動物は、ストレス等の発症誘因因子非負荷時には、正常な表現型を示す一方、発症誘因因子負荷により初めて円形脱毛症を発症して円形脱毛症に特徴的な症状を呈し、円形脱毛症の病態をよりよく再現できる。該円形脱毛症モデル非ヒト動物は、新たな円形脱毛症の発症予防又は治療薬の探索に用いることができる。 The alopecia areata model non-human animal of the present invention exhibits a normal phenotype when the onset-inducing factor such as stress is not loaded, but develops alopecia areata for the first time due to the onset-inducing factor loading, which is characteristic of alopecia areata. It presents symptoms and can better reproduce the pathophysiology of alopecia areata. The alopecia areata model non-human animal can be used to prevent the onset of new alopecia areata or to search for a therapeutic agent.
本発明の円形脱毛症モデル非ヒト動物は、全身でｃｏｉｌｅｄ−ｃｏｉｌ α−ｈｅｌｉｃａｌ ｒｏｄ ｐｒｏｔｅｉｎ １（ＣＣＨＣＲ１）遺伝子の機能を欠損したＣＣＨＣＲ１遺伝子ホモノックアウト非ヒト動物である。様々な種のＣＣＨＣＲ１遺伝子及びＣＣＨＣＲ１遺伝子を含むゲノムの塩基配列がデータベースに登録されており、例えばマウスの場合、ＣＣＨＣＲ１遺伝子は配列番号１に示される塩基配列からなる。ここで、「ＣＣＨＣＲ１遺伝子の機能を欠損する」とは、染色体上のＣＣＨＣＲ１遺伝子が例えば破壊又は除去されてノックアウトされ、ＣＣＨＣＲ１タンパク質を産生することができないか或いは遺伝子産物が得られても正常に機能しないことをいう。具体的には、ＣＣＨＣＲ１遺伝子のプロモーター領域又はコード領域の塩基配列の欠失、挿入又は置換等によってその機能が破壊されることをいう。すなわち、「ＣＣＨＣＲ１遺伝子の機能を欠損した非ヒト動物」とは、ＣＣＨＣＲ１遺伝子を遺伝子工学的な手法を用い改変（操作）して作出された、ＣＣＨＣＲ１遺伝子発現不全非ヒト動物を意味する。 The alopecia areata model non-human animal of the present invention is a CCHCR1 gene homo-knockout non-human animal in which the function of the coiled-coil α-helical rod protein 1 (CCHCR1) gene is deficient throughout the body. The nucleotide sequences of the genomes containing the CCHCR1 gene and the CCHCR1 gene of various species are registered in the database. For example, in the case of a mouse, the CCHCR1 gene consists of the nucleotide sequence shown in SEQ ID NO: 1. Here, "deficient in the function of the CCHCR1 gene" means that the CCHCR1 gene on the chromosome is, for example, disrupted or removed and knocked out, so that the CCHCR1 protein cannot be produced or the gene product is obtained, but the function is normal. Say not. Specifically, it means that the function is destroyed by deletion, insertion or substitution of the base sequence of the promoter region or coding region of the CCHCR1 gene. That is, the "non-human animal lacking the function of the CCHCR1 gene" means a non-human animal in which the CCHCR1 gene is deficiently expressed, which is produced by modifying (manipulating) the CCHCR1 gene using a genetic engineering technique.
「非ヒト動物」としては、非ヒト哺乳動物が好ましい。非ヒト哺乳動物としては、例えば、ウシ、ブタ、ヒツジ、ヤギ、ウサギ、イヌ、ネコ、モルモット、ハムスター、マウス、ラットなどが挙げられるが、病態動物モデル系の作製の面から、個体発生及び生物サイクルが比較的短く、また、繁殖が容易な齧歯動物、とりわけマウス又はラットが好ましい。 As the "non-human animal", a non-human mammal is preferable. Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, etc. From the viewpoint of creating a pathological animal model system, individual development and organisms Mammalians, especially mice or rats, which have a relatively short cycle and are easy to breed, are preferred.
非ヒト動物において染色体上のＣＣＨＣＲ１遺伝子をノックアウトするには、通常染色体上の遺伝子をノックアウトするのに用いられている方法を適用することができる。好ましくは、相同組換え領域とリコンビナーゼの標的配列とを有するターゲティングベクターを用いる方法が採用される。
リコンビナーゼと標的配列の組み合わせとしては、ＣｒｅリコンビナーゼとｌｏｘＰ配列（Ｃｒｅ−ｌｏｘＰシステム）又はＦＬＰリコンビナーゼとＦＲＴ配列（ＦＬＰ−ＦＲＴシステム）等が挙げられる。このうち、Ｃｒｅ−ｌｏｘＰシステムは、バクテリオファージＰ１の有する部位特異的組換えシステムを利用したもので、ｌｏｘＰ配列と称されるバクテリオファージＰ１のゲノム由来の３４塩基からなるＤＮＡ配列に対し、ＤＮＡ組換え酵素であるＣｒｅリコンビナーゼが働き、ｌｏｘＰ配列同士の間で部位特異的組換えを生じる。ＦＬＰ−ＦＲＴシステムは、出芽酵母由来のＦＲＴ配列にＦＬＰリコンビナーゼが働き、ＦＲＴ配列同士の間で部位特異的組換えを生じる。
To knock out the CCHCR1 gene on the chromosome in non-human animals, the methods normally used to knock out the gene on the chromosome can be applied. Preferably, a method using a targeting vector having a homologous recombination region and a target sequence of recombinase is adopted.
Examples of the combination of the recombinase and the target sequence include Cre recombinase and loxP sequence (Cre-loxP system), FLP recombinase and FRT sequence (FLP-FRT system), and the like. Of these, the Cre-loxP system utilizes the site-specific recombination system of bacteriophage P1 and is a DNA set for a DNA sequence consisting of 34 bases derived from the genome of bacteriophage P1 called a loxP sequence. Cre recombinase, a recombinase, acts to cause site-specific recombination between loxP sequences. In the FLP-FRT system, FLP recombinase acts on FRT sequences derived from Saccharomyces cerevisiae to cause site-specific recombination between FRT sequences.
本発明の円形脱毛症モデル非ヒト動物は、例えば、ターゲティングベクターによる相同組換えを利用してリコンビナーゼ標的配列に前後を挟まれたＣＣＨＣＲ１遺伝子をヘテロ接合型で有する非ヒト動物を作製し、これと該標的配列を認識するリコンビナーゼを発現する非ヒト動物とを交配して、ＣＣＨＣＲ１遺伝子ヘテロノックアウト非ヒト動物を作製し、さらに該ＣＣＨＣＲ１遺伝子ヘテロノックアウト非ヒト動物同士を交配して得ることができる。以下に、本発明の円形脱毛症モデル非ヒト動物の代表的な作製方法について、マウスを例にとり詳細に説明するが、これに限定されるものではなく、また他の非ヒト動物でも同様にして作製し得る。 For the circular alopecia model non-human animal of the present invention, for example, a non-human animal having the CCHCR1 gene sandwiched between the recombinase target sequence in a heterozygous manner was prepared by using homologous recombination with a targeting vector. It can be obtained by crossing with a non-human animal expressing a recombinase that recognizes the target sequence to prepare a CCHCR1 gene heteroknockout non-human animal, and further crossing the CCHCR1 gene hetero-knockout non-human animal with each other. Hereinafter, a typical method for producing an alopecia areata model non-human animal of the present invention will be described in detail using a mouse as an example, but the present invention is not limited to this, and other non-human animals are similarly described. Can be made.
まず、マウスＣＣＨＣＲ１遺伝子の機能を欠損させるための相同組換え用ターゲティングベクターを構築する。ターゲティングベクターは、データベース等より得た塩基配列情報をもとに、欠失させるＣＣＨＣＲ１遺伝子の領域を決め、その上流及び下流にリコンビナーゼ標的配列を同じ向きで配置し、さらにその上流及び下流に欠失させる領域の両端のゲノム領域（１〜８ｋｂ程度）を相同組換えに必要な相同領域として配置して設計すればよい。欠失させるＣＣＨＣＲ１遺伝子の領域は、特に制限されず、ＣＣＨＣＲ１遺伝子の全部でも一部でもよいが、一部を用いる場合、エクソン等のその領域が失われると機能する遺伝子産物が得られなくなる領域が好ましく、例えば、エクソン４を含む領域が挙げられる。相同領域については、相同組換え効率、相同組換えを起こした細胞の同定方法等を考慮して設計するのが好ましい。
Ｃｒｅ−ｌｏｘＰシステムを利用する場合、ターゲティングベクターは、例えば、同じ向きのｌｏｘＰ配列で前後を挟まれたＣＣＨＣＲ１遺伝子の全部又は一部を含み、その上流及び下流に相同領域を含む。
First, a targeting vector for homologous recombination for deleting the function of the mouse CCHCR1 gene is constructed. In the targeting vector, the region of the CCHCR1 gene to be deleted is determined based on the nucleotide sequence information obtained from a database or the like, the recombination target sequence is arranged upstream and downstream thereof in the same direction, and the region is further deleted upstream and downstream. The genomic regions (about 1 to 8 kb) at both ends of the region to be subjected to the region may be arranged and designed as homologous regions required for homologous recombination. The region of the CCHCR1 gene to be deleted is not particularly limited and may be all or part of the CCHCR1 gene. However, when a part of the CCHCR1 gene is used, there is a region such as an exon in which a functional gene product cannot be obtained if the region is lost. Preferably, for example, a region containing exon 4 can be mentioned. It is preferable to design the homologous region in consideration of the homologous recombination efficiency, the method for identifying the cells that have undergone homologous recombination, and the like.
When utilizing the Cre-loxP system, the targeting vector contains, for example, all or part of the CCHCR1 gene sandwiched between loxP sequences in the same orientation, with homologous regions upstream and downstream thereof.
また、ターゲティングベクターには、ベクターが取り込まれた細胞や目的とする相同組換えの起こっている可能性の高い細胞を選択するためのマーカー遺伝子を含む。マーカー遺伝子としては、例えば、ネオマイシン耐性遺伝子（ｎｅｏ）、ハイグロマイシン耐性遺伝子（ｈｙｇ）、ヘルペスウイルスチミジンキナーゼ遺伝子（ＨＳＶ−ｔｋ）、ジフテリアトキシンＡフラグメント遺伝子（ＤＴ−Ａ）、チミジンキナーゼ遺伝子（ｔｋ）等の薬剤選択に通常用いられる遺伝子が例示される。例えば、ネオマイシン耐性遺伝子は、ネオマイシン類似体であるＧ４１８を用いることにより相同組換えが生じた細胞の選抜を可能にするポジティブ選択用マーカー遺伝子である。また、目的細胞を選抜するためのネガティブ選択に用いるマーカー遺伝子、例えばチミジンキナーゼ遺伝子（選択剤としてガンシクロビル等を用い、それに対する感受性により非相同組換え体を選抜除去する）、ジフテリアトキシンＡフラグメント遺伝子（ＤＴ−Ａにより発現されたジフテリア毒素により、非相同組換え体を選抜除去する）等をポジティブ選択用マーカー遺伝子と共に用いることもできる。ターゲティングベクターにおいて、ポジティブ選択用マーカー遺伝子は、上流と下流の相同領域に挟まれた領域に位置するのが好ましく、リコンビナーゼ存在下でマーカー遺伝子が切り出されることからリコンビナーゼ標的配列に挟まれた領域に位置するのがさらに好ましい。また、ポジティブ選択用マーカー遺伝子を、ＣＣＨＣＲ１遺伝子の切り出しに用いるのとは異なるリコンビナーゼ標的配列で挟んでおけば、後で該リコンビナーゼによりマーカー遺伝子のみを除去することもできる。ネガティブ選択用マーカー遺伝子は、上流又は下流の相同領域の外側の領域に位置するのが好ましい。 In addition, the targeting vector contains a marker gene for selecting a cell into which the vector has been taken up or a cell having a high possibility of homologous recombination of interest. Examples of the marker gene include a neomycin resistance gene (neo), a hyglomycin resistance gene (hyg), a herpesvirus thymidine kinase gene (HSV-tk), a diphtheriatoxin A fragment gene (DT-A), and a thymidine kinase gene (tk). Genes commonly used for drug selection such as are exemplified. For example, the neomycin resistance gene is a positive selection marker gene that enables selection of cells in which homologous recombination has occurred by using the neomycin analog G418. In addition, a marker gene used for negative selection for selecting a target cell, for example, a thymidine kinase gene (using gancyclovir or the like as a selection agent and selecting and removing an illegitimate recombine depending on its sensitivity), a diphtheriatoxin A fragment gene ( Illegitimate recombines are selectively removed by the diphtheria toxin expressed by DT-A) and the like can also be used together with a marker gene for positive selection. In the targeting vector, the marker gene for positive selection is preferably located in the region sandwiched between the upstream and downstream homologous regions, and is located in the region sandwiched between the recombinase target sequences because the marker gene is excised in the presence of recombinase. It is more preferable to do so. Further, if the marker gene for positive selection is sandwiched between a recombinase target sequence different from that used for excising the CCHCR1 gene, only the marker gene can be removed later by the recombinase. The marker gene for negative selection is preferably located in the region outside the homologous region upstream or downstream.
斯かるターゲティングベクターの調製は、通常のＤＮＡ組換え技術により行うことができ、例えば、常法に従ってクローニングしたＣＣＨＣＲ１遺伝子やＢＡＣクローンを利用して、これを適当な制限酵素で切断して得られる断片、又はＰＣＲ法などにより増幅して調製したＤＮＡ断片等を、合成されたリンカーＤＮＡやレポーター遺伝子、薬剤耐性マーカー遺伝子を含む断片等と、前記のような設計に従って適当な順序で結合させればよい。 The preparation of such a targeting vector can be carried out by a conventional DNA recombination technique. For example, a fragment obtained by cleaving the CCHCR1 gene or BAC clone cloned according to a conventional method with an appropriate restriction enzyme. Or, a DNA fragment or the like prepared by amplification by a PCR method or the like may be bound to a synthesized linker DNA, a reporter gene, a fragment containing a drug resistance marker gene, or the like in an appropriate order according to the above design. ..
次に、調製した相同組換え用ターゲティングベクターを、通常キメラマウスの作出に用いられる適当な細胞に導入する。ここで用いる細胞は、例えば卵細胞や胚性幹細胞（ＥＳ細胞）が挙げられるが、生体のあらゆる種類の細胞に分化することができる多分化能を有している点でＥＳ細胞が好ましい。 Next, the prepared targeting vector for homologous recombination is introduced into suitable cells usually used for producing chimeric mice. Examples of the cells used here include egg cells and embryonic stem cells (ES cells), and ES cells are preferable because they have pluripotency capable of differentiating into all types of cells in the living body.
ＥＳ細胞は、胚盤胞の内部細胞塊（ＩＣＭ）から樹立され、未分化状態を保ったまま増殖・培養可能な細胞株であり、ＥＳ細胞を用いる遺伝子導入の方法は、マウスについては確立されている（Mansour,S.L. et al., Nature 336:348(1988)）。ＥＳ細胞としては、既に樹立された細胞株を用いてもよく、例えばマウスの場合、ＴＴ２、Ｃ５７ＢＬ／６等のマウス系統由来のＥＳ細胞が挙げられる。あるいは、新たに樹立したものを用いてもよい。ＥＳ細胞は、常法に従って継代培養すればよい。 ES cells are cell lines established from the inner cell mass (ICM) of blastocysts and capable of proliferating and culturing while maintaining an undifferentiated state, and a method for gene transfer using ES cells has been established for mice. (Mansour, SL et al., Nature 336: 348 (1988)). As the ES cells, an already established cell line may be used. For example, in the case of mice, ES cells derived from mouse strains such as TT2 and C57BL / 6 can be mentioned. Alternatively, a newly established one may be used. ES cells may be subcultured according to a conventional method.
ターゲティングベクターの細胞への導入は、通常の方法、例えば、エレクトロポレーション法、マイクロインジェクション法、リン酸カルシウム法、リポフェクション法、ＤＥＡＥ−デキストラン法等によって行うことができる。このうち、簡便で多数の細胞を処理できることから、エレクトロポレーション法が好適に用いられる。エレクトロポレーション法による遺伝子導入の条件は、通常の動物細胞への遺伝子導入の条件を用いればよい。 The introduction of the targeting vector into cells can be carried out by a usual method, for example, an electroporation method, a microinjection method, a calcium phosphate method, a lipofection method, a DEAE-dextran method or the like. Of these, the electroporation method is preferably used because it can easily treat a large number of cells. As the conditions for gene transfer by the electroporation method, the conditions for gene transfer into normal animal cells may be used.
ターゲティングベクターを導入したＥＳ細胞は、常法に従い培養することで、単一細胞由来のコロニーを得ることができる。このとき、相同組換えが生じたＥＳ細胞では、ベクター中のＣＣＨＣＲ１遺伝子とともにマーカー遺伝子も染色体に組み込まれているため、マーカー遺伝子の発現に基づいて、例えば適当な期間薬剤存在下で培養することにより、目的のＥＳ細胞を選択することができる。また、該コロニーから抽出したゲノムＤＮＡを用いて、ＰＣＲ法、サザンハイブリダイゼーション法等により、所望の相同組換えが生じたＥＳ細胞を選択することもできる。ＰＣＲ法の場合は、例えば、相同領域の外側に設計したプライマーと、マーカー遺伝子内に設計したプライマーを用い、ゲノムＤＮＡを鋳型としてＰＣＲを行い、所望のサイズの増幅産物が得られるかどうかで判断することができる。サザンハイブリダイゼーション法の場合は、例えば、相同組換えによる切断パターンの変化を観察しやすい制限酵素でゲノムＤＮＡを消化したＤＮＡ断片と、相同領域の外側に設計したプローブ及び／又はマーカー遺伝子に設計したプローブを用いてサザンハイブリダイゼーションを行い、目的の位置にバンドが得られるかどうかで判断することができる。 ES cells into which a targeting vector has been introduced can be cultured according to a conventional method to obtain colonies derived from a single cell. At this time, in the ES cells in which homologous recombination has occurred, the marker gene is integrated into the chromosome together with the CCHCR1 gene in the vector. Therefore, based on the expression of the marker gene, for example, by culturing in the presence of a drug for an appropriate period of time. , The desired ES cell can be selected. In addition, using the genomic DNA extracted from the colony, ES cells in which the desired homologous recombination has occurred can be selected by a PCR method, a Southern hybridization method, or the like. In the case of the PCR method, for example, PCR is performed using genomic DNA as a template using a primer designed outside the homology region and a primer designed inside the marker gene, and it is judged whether or not an amplification product of a desired size can be obtained. can do. In the case of the Southern hybridization method, for example, a DNA fragment obtained by digesting genomic DNA with a restriction enzyme that makes it easy to observe changes in the cleavage pattern due to homologous recombination, and a probe and / or a marker gene designed outside the homologous region were designed. Southern hybridization can be performed using a probe, and it can be determined whether or not a band can be obtained at a desired position.
次に、相同組換えを生じたＥＳ細胞を用いて、インジェクション法又はアグリゲーション法等の通常のキメラマウスの作出に用いられる方法に従い、キメラマウスを作製する。具体的には、相同組換えを生じたＥＳ細胞を胚形成の初期の適当な時期、例えば８細胞期の胚又は胚盤胞に注入し、得られた胚を偽妊娠状態の雌の仮親の子宮内に移植することにより、正常なＣＣＨＣＲ１遺伝子座をもつ細胞と相同組換えＥＳ細胞由来のＣＣＨＣＲ１遺伝子座をもつ細胞とから構成されるキメラマウスが得られる。宿主胚をどのような系統の動物から得るかの選択は、常法に従い毛色等の表現型により相同組換えＥＳ細胞由来の細胞と宿主胚由来の細胞とを区別することができるように行えばよい。その後の交配には、ＥＳ細胞寄与率の高いキメラマウスを、毛色等を指標に選抜して用いるのが好ましい。 Next, using the ES cells that have undergone homologous recombination, a chimeric mouse is prepared according to a method used for producing a normal chimeric mouse such as an injection method or an aggregation method. Specifically, ES cells that have undergone homologous recombination are injected into embryos or blastocysts at an appropriate stage in the early stage of embryogenesis, for example, 8-cell stage embryos or blastocysts, and the obtained embryos are injected into pseudopregnant female foster mothers. By transplanting into the embryo, a chimeric mouse composed of cells having a normal CCHCR1 locus and cells having a CCHCR1 locus derived from homologous recombinant ES cells can be obtained. The selection of the strain of the animal from which the host embryo is obtained should be made so that the cells derived from the homologous recombinant ES cells and the cells derived from the host embryo can be distinguished by a phenotype such as hair color according to a conventional method. Good. For subsequent mating, it is preferable to use chimeric mice having a high ES cell contribution rate by selecting them using coat color or the like as an index.
キメラマウスの生殖細胞の一部がＥＳ細胞由来のＣＣＨＣＲ１遺伝子座をもつ場合には、キメラマウスを正常マウスと交配することにより、得られた個体群から、相同組換えＥＳ細胞由来ＣＣＨＣＲ１遺伝子座を一方の相同染色体に有するヘテロマウスを毛色等の判別法により選別することができる。ヘテロマウスの確認は、マウスより抽出したゲノムＤＮＡを鋳型として、適宜設計したプライマーを用いるＰＣＲにて容易に行うことができる。 When some of the germ cells of the chimeric mouse have the CCHCR1 locus derived from ES cells, the CCHCR1 locus derived from the homologous recombinant ES cells can be obtained from the obtained population by mating the chimeric mouse with a normal mouse. Heterozygous mice having one homologous chromosome can be selected by a method for discriminating hair color or the like. The heterozygous mouse can be easily confirmed by PCR using genomic DNA extracted from the mouse as a template and an appropriately designed primer.
次に、得られた相同組換えＥＳ細胞由来のＣＣＨＣＲ１遺伝子座をヘテロ接合型で有するヘテロマウスを、全身でリコンビナーゼを発現するマウスと交配し、リコンビナーゼ認識配列で挟まれた領域が切り出されたＣＣＨＣＲ１遺伝子ヘテロノックアウトマウスを作製する。全身でリコンビナーゼを発現するマウスとしては、全身で機能するプロモーターによりドライブされるリコンビナーゼ遺伝子を有するマウスである限り、特に限定されず、既存のマウスを用いてもよい。例えば、Ｃｒｅ−ｌｏｘＰシステムを利用する場合には、既存のＣＡＧ−Ｃｒｅマウス等を利用でき、具体的には、Ｂ６；ＣＢＡ−Ｔｇ（ＣＡＧ−Ｃｒｅ）４７Ｉｍｅｇマウス（Araki K. et al. Nucleic Acids Res. 2002, 30(19): e103）等を好適に用いることができる。ＣＣＨＣＲ１遺伝子の欠損は、マウスより抽出したゲノムＤＮＡを鋳型として、適宜設計したプライマーを用いるＰＣＲにて容易に確認することができる。かくして得られたＣＣＨＣＲ１遺伝子ヘテロノックアウトマウス同士を交配すれば、ＣＣＨＣＲ１遺伝子の欠損をホモ接合型で有するＣＣＨＣＲ１遺伝子ホモノックアウトマウスを得ることができる。該マウスの遺伝子型については、マウスより抽出したゲノムＤＮＡを鋳型として、適宜設定したプライマーを用いたＰＣＲにて容易に確認することができる。 Next, a heterozygous mouse having the obtained homologous recombination ES cell-derived CCHCR1 locus in a heterozygous manner was crossed with a mouse expressing recombinase systemically, and the region sandwiched by the recombinase recognition sequence was excised. Gene heterozygous knockout mice are generated. The mouse that expresses the recombinase systemically is not particularly limited as long as it has a recombinase gene driven by a promoter that functions systemically, and an existing mouse may be used. For example, when using the Cre-loxP system, existing CAG-Cre mice and the like can be used. Specifically, B6; CBA-Tg (CAG-Cre) 47Imega mice (Araki K. et al. Nucleic Acids) Res. 2002, 30 (19): e103) and the like can be preferably used. Defects in the CCHCR1 gene can be easily confirmed by PCR using genomic DNA extracted from mice as a template and appropriately designed primers. By mating the CCHCR1 gene heterozygous knockout mice thus obtained, a CCHCR1 gene homozygous knockout mouse having a CCHCR1 gene defect can be obtained. The genotype of the mouse can be easily confirmed by PCR using genomic DNA extracted from the mouse as a template and an appropriately set primer.
本発明者らが、このＣＣＨＣＲ１遺伝子ノックアウトマウスを飼育したところ、胎生致死ではなく、また胎生期及び第一期毛成長は正常であった。しかしながら、円形脱毛症の発症誘因因子の負荷により、初めて円形脱毛症を発症し、目視で観察できる脱毛の他、ダーモスコピーで観察できる感嘆符毛、折れた毛髪、走査電子顕微鏡で観察できる感嘆符毛、毛幹形成の異常等を呈した。これらは、円形脱毛症に特徴的な病理像である。該マウスをさらに飼育したところ、脱毛は回復したが、脱毛巣に再生した毛は白く細くなっており、これも円形脱毛症の特徴と合致するものであった。したがって、ＣＣＨＣＲ１遺伝子ノックアウト非ヒト動物は、円形脱毛症の発症及び病理像をよりよく再現するものであり、円形脱毛症モデル非ヒト動物として有用である。ここで、円形脱毛症の発症誘因因子としては、特に限定されないが、主要なメディエーターとして知られるストレスが好ましい。ストレスの負荷方法は、常法に従えばよく、例えば、拘束水浸ストレス（ｗａｔｅｒ ａｖｏｉｄａｎｃｅ ｓｔｒｅｓｓ：ＷＡＳ）試験等が挙げられる。 When the present inventors bred this CCHCR1 gene knockout mouse, it was not lethal to the embryo and the embryonic and first stage hair growth was normal. However, due to the load of alopecia areata inducing factors, alopecia areata develops for the first time, and in addition to visually observable hair loss, exclamation hair that can be observed with dermoscopy, broken hair, and exclamation hair that can be observed with a scanning electron microscope. , Abnormal hair shaft formation, etc. These are pathological features characteristic of alopecia areata. When the mice were further bred, the hair loss recovered, but the hair regenerated in the bald lesions became white and thin, which was also consistent with the characteristics of alopecia areata. Therefore, the CCHCR1 gene knockout non-human animal better reproduces the onset and pathological image of alopecia areata and is useful as a model non-human animal for alopecia areata. Here, the incentive factor for the onset of alopecia areata is not particularly limited, but stress known as a major mediator is preferable. The stress loading method may follow a conventional method, and examples thereof include a water evaidance stress (WAS) test and the like.
ＣＣＨＣＲ１遺伝子がノックアウトされた円形脱毛症モデル非ヒト動物を用いれば、円形脱毛症の発症予防又は治療薬のスクリーニングに応用することができる。例えば、該モデル非ヒト動物に、被験物質を投与してその経過を観察すれば、その被験物質が円形脱毛症の発症予防又は治療薬として有用であるか否かが容易に判定できる。
該モデル非ヒト動物に被験物質を投与した場合に、投与後の脱毛巣面積が投与前の脱毛巣面積に比して小さくなれば、好ましくは投与後の脱毛巣面積が投与前の脱毛巣面積に比して５０％以下になれば、より好ましくは投与後に脱毛がみられなくなれば、その被験物質は、円形脱毛症の治療薬であると判定できる。かかる治療薬は、円形脱毛症に罹患していない対象や罹患する可能性が高い対象などに対する円形脱毛症の発症予防薬としても用いられる。また、かかる研究は、円形脱毛症に罹患していない対象や罹患する可能性が高い対象などに対する円形脱毛症の予防開発法としても用いられる。
Alopecia areata model non-human animals in which the CCHCR1 gene has been knocked out can be used for prevention or screening of therapeutic agents for alopecia areata. For example, by administering a test substance to the model non-human animal and observing the progress thereof, it can be easily determined whether or not the test substance is useful as a preventive or therapeutic agent for the onset of alopecia areata.
When the test substance is administered to the model non-human animal, if the hair loss lesion area after administration is smaller than the hair loss lesion area before administration, the hair loss lesion area after administration is preferably the hair loss lesion area before administration. If the amount is 50% or less, more preferably, if hair loss is not observed after administration, it can be determined that the test substance is a therapeutic agent for alopecia areata. Such a therapeutic agent is also used as a preventive agent for the onset of alopecia areata for subjects who do not have alopecia areata or who are likely to suffer from alopecia areata. Such studies are also used as a preventive development method for alopecia areata for subjects who do not have or are likely to suffer from alopecia areata.
被験物質としては、円形脱毛症に対する発症予防又は治療効果を予測したい薬物であればよく、特に限定されない。被験物質の投与方法としては、特に制限はなく、投与される動物種や被験物質の特性に応じて適宜選択すればよい。例えば、皮膚への塗布、噴霧等による外用の他、経口投与、静脈内投与、筋肉内投与、皮内投与、皮下投与、経皮投与、腹腔内投与、直腸内投与等が挙げられる。被験物質の投与量も、投与される動物種や被験物質の特性に応じて適宜設定すればよい。 The test substance may be any drug for which the onset prevention or therapeutic effect on alopecia areata is to be predicted, and is not particularly limited. The method of administering the test substance is not particularly limited, and may be appropriately selected depending on the animal species to be administered and the characteristics of the test substance. For example, in addition to external application by application to the skin, spraying, etc., oral administration, intravenous administration, intramuscular administration, intradermal administration, subcutaneous administration, transdermal administration, intraperitoneal administration, rectal administration and the like can be mentioned. The dose of the test substance may also be appropriately set according to the animal species to be administered and the characteristics of the test substance.
次に実施例を挙げて本発明を更に詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.
実施例１ ＣＣＨＣＲ１遺伝子ノックアウトマウスの作製
（１）ターゲティングベクターの構築
マウスＣ５７ＢＬ／６Ｎ由来ＲＥＮＫＡ ＥＳ細胞（Mishina M. et al. Neurosci Res. 2007, 58(2): 105-12）のゲノムＤＮＡを鋳型として、マウスＣＣＨＣＲ１遺伝子（配列番号１）のエクソン２及び３を含む２．８ｋｂのＤＮＡ断片を、配列番号２及び３のプライマーセットを用いてＰＣＲにて増幅した。同様に、エクソン４を含む０．９ｋｂのＤＮＡ断片を、配列番号４及び５のプライマーセットを用いて、エクソン５−１０を含む５．０ｋｂのＤＮＡ断片を、配列番号６及び７のプライマーセットを用いてＰＣＲにて増幅した。なお、各プライマーには、クローニングのための制限酵素認識サイトを含む。
XhoI_5_af_5: 5'-ctcgagcacaccacccacattcctgtaag-3'（配列番号２）
HindIII_5_ar_2: 5'-aagcttacctaagggctagtgagatcagg-3'（配列番号３）
EcoRI_F_af_1: 5'-gaattcctgagcccagaaagtggatgac-3'（配列番号４）
EcoRI_F_ar_2: 5'-gaattcgctatgggcgccattcatgag-3'（配列番号５）
XhoI_AscI_3_af_1: 5'-tcgagggcgcgccgcctgcatgggacacacttaatg-3'（配列番号６）
PmeI_3_ar_5: 5'-gtttaaacctgagcctgatccagctccatc-3'（配列番号７）
（各下線部は、制限酵素認識サイトを示す。）
ポジティブ選択用マーカーとしてＰＧＫプロモーターでドライブされるネオマイシン耐性遺伝子の両端にＦＲＴ配列を有する遺伝子カセット（ＰＧＫ＿ｎｅｏカセット）と、ネガティブ選択用マーカーとしてＭＣ１プロモーターでドライブされるＤＴ−Ａ遺伝子を有する遺伝子カセット（ＭＣ１＿ＤＴＡカセット）と、２つのｌｏｘＰ配列とを含むベクターのｌｏｘＰ配列間に、上記で増幅した０．９ｋｂのＤＮＡ断片をクローニングした。次いで、上記で増幅した２．８ｋｂのＤＮＡ断片と５．０ｋｂのＤＮＡ断片も同ベクターにクローニングした。最終的に、ＭＣ１＿ＤＴＡカセット、２．８ｋｂの５’相同領域、第一のｌｏｘＰ配列、０．９ｋｂのｆｌｏｘゲノム領域、ＰＧＫ＿ｎｅｏカセット、第二のｌｏｘＰ配列、及び５．０ｋｂの３’相同領域をこの順で含むターゲティングベクターを得た。
Example 1 Preparation of CCHCR1 gene knockout mouse (1) Construction of targeting vector Genomic DNA of mouse C57BL / 6N-derived RENKA ES cells (Mishina M. et al. Neurosci Res. 2007, 58 (2): 105-12) was used as a template. A 2.8 kb DNA fragment containing exons 2 and 3 of the mouse CCHCR1 gene (SEQ ID NO: 1) was amplified by PCR using the primer sets of SEQ ID NOs: 2 and 3. Similarly, a 0.9 kb DNA fragment containing exons 4 using the primer sets of SEQ ID NOs: 4 and 5, a 5.0 kb DNA fragment containing exons 5-10, and a primer set of SEQ ID NOs: 6 and 7. Amplified by PCR using. In addition, each primer contains a restriction enzyme recognition site for cloning.
XhoI_5_af_5: 5'- ctcgag cacaccacccacattcctgtaag-3'(SEQ ID NO: 2)
HindIII_5_ar_2: 5'- aagctt acctaagggctagtgagatcagg-3'(SEQ ID NO: 3)
EcoRI_F_af_1: 5'- gaattc ctgagcccagaaagtggatgac-3'(SEQ ID NO: 4)
EcoRI_F_ar_2: 5'- gaattc gctatgggcgccattcatgag-3'(SEQ ID NO: 5)
XhoI_AscI_3_af_1: 5'- tcgagggcgcgcc gcctgcatgggacacttaatg-3'(SEQ ID NO: 6)
PmeI_3_ar_5: 5'- gtttaaac ctgagcctgatccagctccatc-3'(SEQ ID NO: 7)
(Each underlined part indicates a restriction enzyme recognition site.)
A gene cassette (PGK_neo cassette) having FRT sequences at both ends of a neomycin resistance gene driven by the PGK promoter as a marker for positive selection, and a gene cassette (MC1_DTA) having a DT-A gene driven by the MC1 promoter as a marker for negative selection. The 0.9 kb DNA fragment amplified above was cloned between the loxP sequence of the vector containing the cassette) and the two loxP sequences. Then, the 2.8 kb DNA fragment and the 5.0 kb DNA fragment amplified above were also cloned into the same vector. Finally, this MC1_DTA cassette, 2.8 kb 5'homologous region, first loxP sequence, 0.9 kb flox genomic region, PGK_neo cassette, second loxP sequence, and 5.0 kb 3'homologous region. A targeting vector containing in order was obtained.
（２）相同組換え
（１）で構築したターゲティングベクターを１本鎖化し、ＲＥＮＫＡ ＥＳ細胞にエレクトロポレーション法にて導入した。ジェネティシン（Ｉｎｖｉｒｏｇｅｎ製）を用いて細胞を選抜し、ジェネティシンに耐性を有するクローンを単離した。単離クローンのＤＮＡと配列番号８及び９のプライマーセットを用いたＰＣＲにて、相同組換えを生じたクローンをスクリーニングした。
sc_5AF2: 5’-caactgaggcccgttacagag-3’（配列番号８）
neo_108r: 5’-cctcagaagaactcgtcaagaag-3’（配列番号９）
ＰＣＲ陽性ＥＳクローンを培養した。該クローンよりＤＮＡを抽出し、配列番号８及び９のプライマーセット（５’領域増幅用）、配列番号１０及び１１のプライマーセット（３’領域増幅用）、並びに配列番号１２及び１３のプライマーセット（第一のｌｏｘＰ領域増幅用）を用いたＰＣＲにてさらに解析した。
sc_3AR4: 5’-cgaggctcttccaggcaacag-3’（配列番号１０）
neo_100: 5’-atcaggacatagcgttggctac-3’（配列番号１１）
lox_5_fw_6: 5’-tagcctctgctatgtactg-3’（配列番号１２）
lox_5_ar_1: 5’-aagtccctactaagcacacgtgg-3（配列番号１３）
これらのクローンで相同組換えが正しく生じたことは、ネオマイシン耐性遺伝子をプローブとしたゲノムサザンハイブリダイゼーションによっても確認した。
(2) Homologous recombination The targeting vector constructed in (1) was positive-strand RNA and introduced into RENKA ES cells by an electroporation method. Cells were selected using Genetisin (manufactured by Invirogen) and clones resistant to Genetisin were isolated. Homologous recombination clones were screened by PCR using the DNA of the isolated clones and the primer sets of SEQ ID NOs: 8 and 9.
sc_5AF2: 5'-caactgaggcccgttacagag-3' (SEQ ID NO: 8)
neo_108r: 5'-cctcagaagaactcgtcaagaag-3' (SEQ ID NO: 9)
PCR-positive ES clones were cultured. DNA was extracted from the clone, and the primer sets of SEQ ID NOs: 8 and 9 (for 5'region amplification), the primer sets of SEQ ID NOs: 10 and 11 (for 3'region amplification), and the primer sets of SEQ ID NOs: 12 and 13 (for amplification of the 3'region). Further analysis was performed by PCR using the first loxP region amplification).
sc_3AR4: 5'-cgaggctcttccaggcaacag-3' (SEQ ID NO: 10)
neo_100: 5'-atcaggacatagcgttggctac-3' (SEQ ID NO: 11)
lox_5_fw_6: 5'-tagcctctgctatgtactg-3' (SEQ ID NO: 12)
lox_5_ar_1: 5'-aagtccctactaagcacacgtgg-3 (SEQ ID NO: 13)
The correct occurrence of homologous recombination in these clones was also confirmed by genomic Southern hybridization using the neomycin resistance gene as a probe.
（３）ＣＣＨＣＲ１遺伝子ノックアウトマウスの作出
アグリゲーション法により、（２）で得られた相同組換えＥＳ細胞クローンとＩＣＲマウス由来８細胞期胚を融合させてキメラマウスを作製した。ＲＥＮＫＡ ＥＳ細胞寄与率の高いキメラマウスをＣ５７ＢＬ／６Ｎマウスと交配し、ｇｅｒｍｌｉｎｅ ｔｒａｎｓｍｉｓｓｉｏｎを生じたＣＣＨＣＲ１遺伝子ヘテロｆｌｏｘマウスを得た。目的アレルの存在は、配列番号８及び９のプライマーセット、並びに配列番号１２及び１３のプライマーセットを用いたＰＣＲにて確認した。
次いで、ＣＣＨＣＲ１遺伝子ヘテロｆｌｏｘマウスを、Ｂ６；ＣＢＡ−Ｔｇ（ＣＡＧ−Ｃｒｅ）４７Ｉｍｅｇマウス（Araki K. et al. Nucleic Acids Res. 2002, 30(19): e103）と交配し、エクソン４及びＰＧＫ＿ｎｅｏカセットを欠失させた。エクソン４を欠失したＣＣＨＣＲ１遺伝子ヘテロノックアウトマウスは、配列番号１４及び１５のプライマーセットを用いたＰＣＲにて同定した。
5A-F2: 5’-ctcacctagaattcagacatcc-3’（配列番号１４）
3A-R1: 5’-agttgcaactggctatagctgc-3’（配列番号１５）
かくして得られたＣＣＨＣＲ１遺伝子ヘテロノックアウトマウス同士を交配し、ＣＣＨＣＲ１遺伝子ホモノックアウトマウスを得た。該マウスの遺伝子型は、配列番号１４及び１５のプライマーセットを用いたＰＣＲにて判定した。ＣＣＨＣＲ１遺伝子ホモノックアウトマウスは、胎生致死ではなく、また胎生期及び第一期毛成長は正常であった。
なお、ＣＣＨＣＲ１遺伝子ホモノックアウトマウスの作出は、株式会社トランジェニックにて実施した。
(3) Creation of CCHCR1 gene knockout mouse A chimeric mouse was prepared by fusing the homologous recombinant ES cell clone obtained in (2) with an 8-cell stage embryo derived from an ICR mouse by an aggregation method. Chimeric mice with a high contribution rate of RENKA ES cells were crossed with C57BL / 6N mice to obtain CCHCR1 gene heteroflux mice that produced germline transmission. The presence of the target allele was confirmed by PCR using the primer sets of SEQ ID NOs: 8 and 9 and the primer sets of SEQ ID NOs: 12 and 13.
CCHCR1 gene heteroflux mice were then crossed with B6; CBA-Tg (CAG-Cre) 47Imega mice (Araki K. et al. Nucleic Acids Res. 2002, 30 (19): e103) for exon 4 and PGK_neo cassettes. Was deleted. Exon 4 deficient CCHCR1 gene heteroknockout mice were identified by PCR using the primer sets of SEQ ID NOs: 14 and 15.
5A-F2: 5'-ctcacctagaattcagacatcc-3'(SEQ ID NO: 14)
3A-R1: 5'-agttgcaactggctatagctgc-3' (SEQ ID NO: 15)
The CCHCR1 gene heterozygous knockout mice thus obtained were crossed with each other to obtain CCHCR1 gene homozygous knockout mice. The genotype of the mouse was determined by PCR using the primer sets of SEQ ID NOs: 14 and 15. CCHCR1 gene homo-knockout mice were not lethal and had normal embryonic and first-stage hair growth.
The CCHCR1 gene homo-knockout mouse was produced by Transgenic Co., Ltd.
実施例２ 拘束水浸ストレス（ｗａｔｅｒ ａｖｏｉｄａｎｃｅ ｓｔｒｅｓｓ：ＷＡＳ）試験
（１）ＷＡＳ試験
本試験には、６−８週齢の実施例１で作出したＣＣＨＣＲ１遺伝子ホモノックアウトマウス（ｎ＝３）と、対照として６−８週齢の野生型マウス（ｎ＝３）を用い、各マウスについてＷＡＳ試験を行った。ＷＡＳ試験は、プラスチックコンテナにプラットフォーム（高さ１１ｃｍ、直径７ｃｍ）を設置し、コンテナ内をプラットフォームの高さの１ｃｍ下まで滅菌水で満たし、該プラットフォームの中央にマウスを２時間置くことで行った。この操作を週に５回、２週間実施した。
試験実施８週間後、マウスの外観を肉眼観察し、さらに毛の形態学的特徴をダーモスコピーを用いて観察した。また、患部の毛をゆっくり引っ張る牽引試験（ｈａｉｒ ｐｕｌｌ ｔｅｓｔ）を行った。さらに、試験実施１８週間後、マウスを再度肉眼観察及びダーモスコピーを用いて観察した。
Example 2 Water avaidance stress (WAS) test (1) WAS test In this test, the CCHCR1 gene homozygous knockout mouse (n = 3) produced in Example 1 at the age of 6-8 weeks was used as a control. A 6-8 week old wild-type mouse (n = 3) was used as a stress test, and each mouse was subjected to a WAS test. The WAS test was performed by placing a platform (height 11 cm, diameter 7 cm) in a plastic container, filling the inside of the container with sterile water to 1 cm below the platform height, and placing the mouse in the center of the platform for 2 hours. .. This operation was performed 5 times a week for 2 weeks.
Eight weeks after the test was conducted, the appearance of the mice was visually observed, and the morphological characteristics of the hair were further observed using a dermoscopy. In addition, a traction test (hair pull test) in which the hair of the affected area was slowly pulled was performed. Furthermore, 18 weeks after the test was conducted, the mice were observed again with the naked eye and using dermoscopy.
（２）免疫組織染色
ＷＡＳ試験実施８週間後のノックアウトマウスの毛包のパラフィン包埋切片を調製し、ＩｍｍＰＲＥＳＳ ＲＥＡＧＥＮＴ（ベクターラボラトリー製）を用い、製品添付のマニュアルに従って、ＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球について免疫組織染色を行った。抗体としては、ｒａｂｂｉｔ ａｎｔｉ−ＣＤ４（ａｂ１８３６８５、ａｂｃａｍ）と、ｂｉｏｔｉｎ ａｎｔｉ−ｍｏｕｓｅ ＣＤ８ａ（１００７０３、ＢｉｏＬｅｇｅｎｄ）を用いた。
(2) Immunohistochemical staining Prepare paraffin-embedded sections of hair follicles of knockout mice 8 weeks after the WAS test, and use ImMPRESS REAGENT (manufactured by Vector Laboratory) according to the manual attached to the product, CD4 + T lymphocytes and CD8. Immunohistochemical staining was performed on + T lymphocytes. As the antibody, rabbit anti-CD4 (ab183685, abcam) and biotin anti-mouse CD8a (100703, BioLegend) were used.
（３）走査電子顕微鏡観察
ＷＡＳ試験実施８週間後のノックアウトマウスの毛幹を走査電子顕微鏡で観察した。
(3) Observation with scanning electron microscope The hair shaft of the knockout mouse 8 weeks after the WAS test was carried out was observed with a scanning electron microscope.
（４）結果
ＷＡＳ試験実施８週間後、野生型マウスに比べ、全てのノックアウトマウスでは、背部に局所的な脱毛が認められた（図１（Ａ））。ダーモスコピー観察では、脱毛部に、折れた毛髪及び感嘆符毛という円形脱毛症に特徴的な表現型がみられた（図１（Ｂ））。牽引試験の結果、異栄養性成長期毛の増加がみられた。
免疫組織染色では、ノックアウトマウスの毛包の球周囲領域には炎症性リンパ球浸潤はほとんど認められなかったが、毛球にはＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球浸潤が認められた（図３）。また、形質細胞の浸潤、毛包数の増加、成長期毛包の小型化がみられた。
走査電子顕微鏡観察では、全てのノックアウトマウスで、感嘆符毛（図４（Ａ））と毛幹の異常（図５（Ｂ））が明らかとなった。
ＷＡＳ試験実施１８週間後、局在脱毛は、自然に回復した（図５（Ａ））。脱毛後、１〜３ヶ月で毛が再生し、その後同じ又は異なる領域に脱毛が生じた。
マウスの円形脱毛症の予後は、様々であり繰り返された。円形脱毛症を長期間維持したマウスでは、脱毛巣内の毛の再生は、円形脱毛症に特徴的な形で生じており、ダーモスコピー観察によると、新たに成長した毛は白く先細りしており、毛包の多くが退行期又は休止期にあった（図５（Ｂ））。
(4) Results Eight weeks after the WAS test was performed, local hair loss was observed on the back of all knockout mice compared to wild-type mice (Fig. 1 (A)). In the dermoscopy observation, a phenotype characteristic of alopecia areata, broken hair and exclamation mark hair, was observed in the hair loss area (Fig. 1 (B)). As a result of the traction test, an increase in allotrophic anagen hair was observed.
Immunohistochemical staining revealed almost no inflammatory lymphocyte infiltration in the perispherical region of the hair follicles of knockout mice, but CD4 + T lymphocytes and CD8 + T lymphocyte infiltration were observed in the hair follicles ( Figure 3). Infiltration of plasma cells, an increase in the number of hair follicles, and miniaturization of growing hair follicles were observed.
Scanning electron microscopy revealed exclamation point hairs (FIG. 4 (A)) and hair shaft abnormalities (FIG. 5 (B)) in all knockout mice.
Eighteen weeks after the WAS test was performed, localized hair loss recovered spontaneously (Fig. 5 (A)). Hair regenerated 1 to 3 months after hair loss, after which hair loss occurred in the same or different areas.
The prognosis for alopecia areata in mice was variable and repetitive. In mice that maintained alopecia areata for a long period of time, hair regrowth in the bald lesions occurred in a manner characteristic of alopecia areata, and dermoscopy observations showed that newly grown hair was white and tapered. Most of the hair follicles were in the degeneration or telogen phase (Fig. 5 (B)).
ストレスは、円形脱毛症発症の古典的な主要メディエーターであることから、ＷＡＳをストレスの誘因として利用した。その結果、上述したように、ＷＡＳは、ＣＣＨＣＲ１遺伝子ノックアウトマウスにおいて、円形脱毛症の病理学的表現型を誘導した。また、免疫組織染色像及び電子顕微鏡観察で認められた感嘆符毛といった特徴は、円形脱毛症のものと合致した。よって、ＣＣＨＣＲ１遺伝子ノックアウトマウスは、円形脱毛症モデルマウスとして有用であることが示された。 Since stress is a classic major mediator of the development of alopecia areata, WAS was used as an incentive for stress. As a result, as described above, WAS induced the pathological phenotype of alopecia areata in CCHCR1 gene knockout mice. In addition, features such as immunohistochemical stained images and exclamation mark hairs observed by electron microscopy were consistent with those of alopecia areata. Therefore, it was shown that the CCHCR1 gene knockout mouse is useful as a model mouse for alopecia areata.
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| CN115918610A (en) * | 2023-02-24 | 2023-04-07 | 中日友好医院(中日友好临床医学研究所) | Preparation method and application of stress liver depression and spleen deficiency type alopecia areata-like mouse model |
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| CN115251009B (en) * | 2022-06-23 | 2023-06-20 | 宁夏医科大学总医院 | Construction method of rat alopecia areata model |
| CN115918610A (en) * | 2023-02-24 | 2023-04-07 | 中日友好医院(中日友好临床医学研究所) | Preparation method and application of stress liver depression and spleen deficiency type alopecia areata-like mouse model |
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