JP2013190410A - Method for preparing coexistence system of trichophyton and three-dimensional cultured skin and evaluation of antifungal agent using the same - Google Patents
Method for preparing coexistence system of trichophyton and three-dimensional cultured skin and evaluation of antifungal agent using the same Download PDFInfo
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本発明は３次元培養皮膚と白癬菌を共培養し、培養皮膚の角層下に白癬菌を生きたまま埋め込んだ感染モデル系およびそれを用いた抗菌剤の効果の評価方法に関する。 The present invention relates to an infection model system in which a three-dimensional cultured skin and ringworm bacteria are co-cultured, and the ringworm bacteria are embedded alive under the horny layer of the cultured skin, and a method for evaluating the effect of an antibacterial agent using the infection model system.
白癬はあらゆるヒトの疾患の中でもっとも罹患率が高く、ほぼ世界のすべての地域において人口の１０％以上が常時罹患しているといわれ、日本においても患者数は１千万人を超えるものと推定されている。患者数がもっとも多いのが俗に「水虫」といわれている足白癬で、次いで爪白癬が多い。現在でも患者数が極めて多く、難治性で再発しやすい白癬に対し、新しい治療法および薬剤の開発は重要な社会的課題である。 Ringworm is the most prevalent of all human diseases, and it is said that more than 10% of the population is constantly affected in almost all parts of the world. In Japan, the number of patients exceeds 10 million. It is estimated. The largest number of patients is tinea pedis, commonly referred to as “athlete's foot”, followed by onychomycosis. The development of new therapies and drugs is an important social issue for tinea tinea, which is still very refractory and recurrent.
白癬は、トリコフィトン属等の真菌の感染によって引き起こされるが、起因菌としてはトリコフィトン ルブルム、トリコフィトン メンタグロフィテス、エピデルモフィトン フロックスムの順に多い。白癬菌は角化した皮膚の組織、すなわち角質層や毛髪および爪に侵入・発育し、局所の感染（白癬）を引き起こす。 Ringworm is caused by infection with fungi of the genus Trichophyton, etc., and the most common causes are Trichophyton rubulum, Trichophyton mentagrophytes, and Epidermophyton Phloxum. Ringworm fungus invades and develops keratinized skin tissue, that is, the stratum corneum, hair and nails, and causes local infection (ringworm).
白癬に対する抗菌剤の開発において抗菌力の評価が必要不可欠とされている。従来の抗菌力評価は、ｉｎ ｖｉｔｒｏにおいて直接白癬菌と抗菌剤と作用させる方法がある（特許文献１）。しかし、白癬菌は感染すると角層内に潜り込み、抗菌剤の抗菌力に加え、角層内への浸透力も必要とされているため、ｉｎ ｖｉｔｒｏでの評価はかならずしも十分とはいえない。これに対し、実状況に近い感染動物モデルの使用は必要不可欠とされ、これまで白癬菌感染のモデル実験は専らモルモットやマウス（特許文献２）を用いて行われてきた。 Evaluation of antibacterial activity is indispensable in the development of antibacterial agents against ringworm. Conventional antibacterial activity evaluation includes a method of directly reacting ringworm and antibacterial agent in vitro (Patent Document 1). However, since ringworm bacteria sneak into the stratum corneum when infected, antibacterial activity of the antibacterial agent and penetration into the stratum corneum are also required, so in vitro evaluation is not always sufficient. On the other hand, the use of an infected animal model that is close to the actual situation is indispensable, and until now, model experiments for ringworm infection have been carried out exclusively using guinea pigs and mice (Patent Document 2).
しかしながら、当該動物では感染および病態に関する宿主側の免疫学的解析、遺伝学的解析が充分かつ効率的には行われ難いという問題点があった。また、最近動物愛護の観点より次第に動物試験法の使用が制限されるようになってきている。このため、それらの解析をする必要がない新しい感染モデルの開発が求められていた。 However, the animal has a problem that it is difficult to perform sufficient and efficient host-side immunological analysis and genetic analysis on infection and disease state. In recent years, the use of animal testing methods has been increasingly restricted from the viewpoint of animal welfare. For this reason, development of a new infection model which does not need to analyze them was called for.
一方、３次元培養皮膚は古くから開発され（非特許文献１）、実際の皮膚に近い構造および生理活性を有することからやけどの治癒時の代替皮膚（特許文献３）や、皮膚刺激性試験（特許文献４）や経皮吸収促進試験（特許文献５）などの動物代替試験として用いられているが、白癬菌などの感染モデル系に使用された報告はない。 On the other hand, three-dimensional cultured skin has been developed for a long time (Non-patent Document 1), and has a structure and physiological activity close to that of actual skin. Although it is used as an animal substitute test such as Patent Document 4) and percutaneous absorption promotion test (Patent Document 5), there is no report used for an infection model system such as ringworm.
上述したように、従来の技術では、抗菌剤の実際の病態に対する作用状況を十分に反映しているとは考えにくい抗菌剤と白癬菌を直接作用させる方法、あるいは実験動物を犠牲にした上、免疫学的解析、遺伝学的解析が充分かつ効率的には行われ難い方法が実施されてきた。これらが本発明が解決しようとする課題である。 As mentioned above, the conventional technology sacrifices the method of direct action of antibacterial agents and ringworm fungi, which are unlikely to sufficiently reflect the action status of the antibacterial agents against the actual pathological condition, Methods have been implemented in which immunological analysis and genetic analysis are difficult to perform sufficiently and efficiently. These are the problems to be solved by the present invention.
本発明者らは、動物を用いず、３次元培養皮膚と白癬菌を共培養し、培養皮膚の角層下に白癬菌を生きたまま埋め込んだ感染モデル系を創出し、さらにこのモデルを使えば白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価できることを見出し、本発明を完成するに至った。 The present inventors co-cultured three-dimensional cultured skin and ringworm bacteria without using animals, and created an infection model system in which ringworm bacteria were alive and embedded under the horny layer of the cultured skin. As a result, the inventors have found that the antibacterial effect against ringworm and the toxicity to the three-dimensional cultured skin can be simultaneously evaluated, and the present invention has been completed.
すなわち、動物を用いた白癬菌感染モデルを代替するｉｎ ｖｉｔｒｏモデルとして、生きた白癬菌と３次元培養皮膚を共存したことを特徴とする新規な皮膚白癬菌感染モデルを提供するものである。このモデルに抗真菌製剤等を塗布し、白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価することを特徴とする。 That is, the present invention provides a novel skin ringworm fungus infection model characterized by the coexistence of live ringworm bacteria and three-dimensional cultured skin as an in vitro model that replaces the ringworm fungus infection model using animals. An antifungal preparation or the like is applied to this model, and the antibacterial effect against trichophyton and the toxicity to the three-dimensional cultured skin are simultaneously evaluated.
本発明は、抗菌剤と白癬菌を直接作用させる従来の技術に比べ、実際の感染状況に近いｉｎ ｖｉｔｒｏ実験条件を提供し、さらに実験動物を用いることなく実際の状況に近い薬剤の抗菌作用を評価する方法を提供できる。 The present invention provides in vitro experimental conditions that are close to the actual infection situation, compared to the conventional technique in which an antibacterial agent and ringworm fungi are allowed to act directly, and further provides an antibacterial action of a drug that is close to the actual situation without using experimental animals. A method of evaluation can be provided.
本発明において３次元培養皮膚は、表皮構造又は表皮と真皮構造を有するものである。 In the present invention, the three-dimensional cultured skin has an epidermis structure or an epidermis and dermis structure.
本発明において白癬菌は皮膚や爪に感染すると認められているものであれば、特に限定されるものではなく、例えば、トリコフィトン ルブルム、トリコフィトン メンタグロフィテス、エピデルモフィトン フロックスムなどが挙げられる。 In the present invention, the ringworm is not particularly limited as long as it is recognized that it infects the skin and nails, and examples thereof include Trichophyton rubulum, Trichophyton mentagrophytes, Epidermophyton floxum and the like.
これらの白癬菌の３次元培養皮膚試料への感染（接種）にあたっては、定量的な評価を可能とするため、白癬菌を予め斜面培地で培養し、回収後、ガーゼなどでろ過して菌糸を除去して平板培地播種する。 To infect (inoculate) three-dimensional cultured skin samples of these ringworm bacteria, the ringworm bacteria are cultured in a slant medium in advance and collected, then filtered with gauze, etc. Remove and seed with plate medium.
上記白癬菌を回収し、３次元培養皮膚の表皮側に播く。感染及び角層内の封入は、３７℃、５％ＣＯ２の条件で、７日間程度培養を行えばよい。感染が十分に行われたことは、組織を切片にし、顕微鏡下で染色した真菌の発育と角層の形成を確認することにより判断することができる。The ringworm is collected and spread on the epidermal side of the three-dimensional cultured skin. Infection and encapsulation in the stratum corneum may be carried out for about 7 days under conditions of 37 ° C. and 5% CO 2 . It can be judged that the infection has been sufficiently performed by sectioning the tissue and confirming the growth of the fungus stained with a microscope and the formation of the stratum corneum.
このようにして、白癬菌で感染された３次元培養皮膚に、次いで、抗真菌剤又はこれを含む組成物（以下、「抗真菌製剤」という）を作用させる。この作用させる抗真菌製剤の剤型は、外用剤として通常皮膚に用いられる剤型であれば特に限定されず、例えば、液剤、クリーム剤、軟膏剤として塗布するもの、皮膚を清拭するもの、貼付け剤として貼付けるもの等により作用させることができる。 Thus, the antifungal agent or a composition containing the same (hereinafter referred to as “antifungal preparation”) is allowed to act on the three-dimensional cultured skin infected with ringworm. The dosage form of the antifungal preparation to be acted on is not particularly limited as long as it is a dosage form usually used on the skin as an external preparation, for example, a liquid, cream, ointment applied, skin wiping, It can be made to act by what is stuck as a sticking agent.
３次元培養皮膚に抗真菌製剤を作用させるに当たっては、ろ紙や布に事前にしみこませた抗真菌製剤を用いるのが好ましい。一定時間３次元培養皮膚の表皮側に抗真菌製剤を作用させた後、抗真菌製剤を除去する。３次元培養皮膚をホモジナイズし、白癬菌を抽出した後、同様に平板培地にて培養し、菌数を測定する。また、同時に抗真菌製剤の３次元培養皮膚に対する毒性を細胞染色することにより評価することもできる。 When the antifungal preparation is allowed to act on the three-dimensional cultured skin, it is preferable to use an antifungal preparation previously impregnated in filter paper or cloth. After the antifungal preparation is allowed to act on the epidermis side of the three-dimensional cultured skin for a certain time, the antifungal preparation is removed. After homogenizing the three-dimensional cultured skin and extracting trichophyton, the same is cultured in a plate medium and the number of bacteria is measured. At the same time, the toxicity of the antifungal preparation to the three-dimensional cultured skin can be evaluated by cell staining.
白癬菌の播種菌数としては１０４ｃｆｕ／ｃｍ２〜１０６ｃｆｕ／ｃｍ２が最も望ましいとされている。10 4 cfu / cm 2 to 10 6 cfu / cm 2 is most preferable as the number of inoculated bacteria of ringworm.
以下、実施例に基づいて本発明を詳説する。本発明は特にこれらにより限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples. The present invention is not particularly limited by these.
１．白癬菌の調製
培地は、ポテト・デキストロース寒天培地（ＰＤＡ培地）３．９％となるように蒸留水に添加、攪拌後、オートクレーブで滅菌（１２０℃、１５分）し作製した。
トリコフィトン メンタグロファイテスを斜面培地に播種し、３０℃で１週間培養した。この斜面培地に１０ｍＬの０．０５％ツィーン８０を加えた生理食塩水を添加した。軽くピペッティングして遊離させた後、ガーゼでろ過し菌糸を除去した。３０００ｒｐｍ、５分間遠心で分生子を集め、０．０５％ツィーン８０−生理食塩水で３回洗浄し、４ｍＬの０．０５％ツィーン８０−生理食塩水に懸濁した。この懸濁液を血球計算板にて菌量を測定した。1. Preparation of ringworm bacteria The medium was prepared by adding potato dextrose agar medium (PDA medium) to distilled water to 3.9%, stirring, and then sterilizing (120 ° C., 15 minutes) with an autoclave.
Trichophyton mentagrophytes was inoculated on a slant medium and cultured at 30 ° C. for 1 week. To this slant medium, 10 mL of physiological saline with 0.05% Tween 80 was added. After releasing by light pipetting, the mixture was filtered through gauze to remove the mycelium. Conidia were collected by centrifugation at 3000 rpm for 5 minutes, washed 3 times with 0.05% Tween 80-saline, and suspended in 4 mL of 0.05% Tween 80-saline. The bacterial mass of this suspension was measured with a hemocytometer.
上記１．で調製した接種菌液を平板培地に画線し、３０℃で１週間培養した。同様に１０ｍＬの０．０５％ツィーン８０を加えた生理食塩水を添加した。軽くピペッティングして遊離させた後、ガーゼでろ過し菌糸を除去した。３０００ｒｐｍ、５分間遠心で分生子を集め、０．０５％ツィーン８０−生理食塩水で３回洗浄し、４ｍＬの０．０５％ツィーン８０−生理食塩水に懸濁した。この懸濁液を血球計算板にて菌量を測定した。 Above 1. The inoculum prepared in step 1 was streaked on a plate medium and cultured at 30 ° C. for 1 week. Similarly, 10 mL of physiological saline supplemented with 0.05% Tween 80 was added. After releasing by light pipetting, the mixture was filtered through gauze to remove the mycelium. Conidia were collected by centrifugation at 3000 rpm for 5 minutes, washed 3 times with 0.05% Tween 80-saline, and suspended in 4 mL of 0.05% Tween 80-saline. The bacterial mass of this suspension was measured with a hemocytometer.
２．感染モデルの作製
３次元培養皮膚（クラボウ製）の表皮側にトリコフィトン・メンタグロファイテスを１０５ｃｆｕ／ｃｍ２となるように接種し、３７℃、５％ＣＯ２の条件で、７日間程度培養した。感染モデルを切片にし、角層の観察および真菌染色法による白癬菌の観察を行った。2. Preparation of infection model Trichophyton mentagrophytes is inoculated at 10 5 cfu / cm 2 on the epidermis side of three-dimensional cultured skin (manufactured by Kurabo Industries), and is maintained at 37 ° C. and 5% CO 2 for 7 days. Cultivated to a certain extent. The infection model was sectioned and the stratum corneum was observed and fungus staining was used to observe ringworm.
白癬菌感染３次元培養皮膚の角層中に白癬菌が観察され（図１）、白癬菌未感染の３次元培養皮膚（図２）と同様に角層が観察された。 Ringworms were observed in the stratum corneum of the three-dimensional cultured skin infected with ringworm fungus (FIG. 1), and the stratum corneum was observed in the same manner as the three-dimensional cultured skin not infected with ringworm fungus (FIG. 2).
１．抗真菌製剤の評価
直径８ｍｍのろ紙に抗真菌製剤を５０μＬしみ込ませ、３次元培養皮膚の表皮側にのせ、３７℃、５％ＣＯ２の条件で、２４時間培養した。実施例１は塩化ベンザルコニウム含有抗真菌製剤、実施例２はミコナゾール硝酸塩含有抗真菌製剤、実施例３は０．２％ポリヘキサメチレンビグアニド含有抗真菌製剤である。1. Evaluation of antifungal preparation 50 μL of the antifungal preparation was impregnated into a filter paper having a diameter of 8 mm, placed on the epidermis side of the three-dimensional cultured skin, and cultured at 37 ° C. and 5% CO 2 for 24 hours. Example 1 is an antifungal preparation containing benzalkonium chloride, Example 2 is an antifungal preparation containing miconazole nitrate, and Example 3 is an antifungal preparation containing 0.2% polyhexamethylene biguanide.
２．白癬菌の菌数測定と３次元培養皮膚の細胞毒性
抗真菌製剤を塗布２４時間後、ろ紙を除去し、リン酸緩衝生理食塩水にて洗浄した。３次元培養皮膚をホモジナイズし、白癬菌を抽出した後、平板培地に播き、培養し、３０℃で１週間培養し菌数を測定する。また、別の抗真菌製剤処理後の３次元培養皮膚を１ｍｇ／ｍＬのＭＴＴ［３−（４，５−ジメチルチアゾール−２−イル）２，５−ジフェニルテトラゾリンブロマイド］入り培地に浸し、３時間処理し、イソプロパノールで色素であるホルマザンを抽出し５７０ｎｍの吸光度を測定した。細胞賦活率は式１で求めた。
式１
2. Measurement of the number of tinea fungi and cytotoxicity of 3D cultured skin 24 hours after application of the antifungal preparation, the filter paper was removed and washed with phosphate buffered saline. Three-dimensional cultured skin is homogenized to extract ringworm fungi, then plated on a plate medium, cultured, and cultured at 30 ° C. for 1 week, and the number of bacteria is measured. Further, the three-dimensional cultured skin after treatment with another antifungal preparation was immersed in a medium containing 1 mg / mL of MTT [3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazoline bromide]. After time treatment, formazan, a pigment, was extracted with isopropanol, and the absorbance at 570 nm was measured. The cell activation rate was determined by Equation 1.
Formula 1
抗菌力試験の結果を図３に示す。実施例１〜３のいずれも抗菌力が認められた。さらに時間的に抗菌力が高まることから、角層への浸透に伴い、抗菌力が増加したことがわかった。 The results of the antibacterial activity test are shown in FIG. In any of Examples 1 to 3, antibacterial activity was observed. Furthermore, since the antibacterial activity increased with time, it was found that the antibacterial activity increased with the penetration into the stratum corneum.
抗菌力試験の結果を図４に示す。実施例３のみに毒性が低いことがわかり、選択的に白癬菌を死滅させることがわかった。 The results of the antibacterial activity test are shown in FIG. Only Example 3 was found to be less toxic and was found to selectively kill ringworm.
本発明方法によれば、白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価することができ、皮膚に影響がなく、選択的に菌を死滅させる抗菌製剤の評価に応用しうることがわかった。 According to the method of the present invention, the antibacterial effect against trichomycosis and the toxicity to the three-dimensional cultured skin can be simultaneously evaluated, and the method can be applied to the evaluation of an antibacterial preparation that selectively kills bacteria without affecting the skin. all right.
本発明は３次元培養皮膚と白癬菌を共培養し、培養皮膚の角層下に白癬菌を生きたまま埋め込んだ感染モデル系およびそれを用いた抗菌剤の効果の評価方法に関する。 The present invention relates to an infection model system in which a three-dimensional cultured skin and ringworm bacteria are co-cultured, and the ringworm bacteria are embedded alive under the horny layer of the cultured skin, and a method for evaluating the effect of an antibacterial agent using the infection model system.
白癬はあらゆるヒトの疾患の中でもっとも罹患率が高く、ほぼ世界のすべての地域において人口の１０％以上が常時罹患しているといわれ、日本においても患者数は１千万人を超えるものと推定されている。患者数がもっとも多いのが俗に「水虫」といわれている足白癬で、次いで爪白癬が多い。現在でも患者数が極めて多く、難治性で再発しやすい白癬に対し、新しい治療法および薬剤の開発は重要な社会的課題である。 Ringworm is the most prevalent of all human diseases, and it is said that more than 10% of the population is constantly affected in almost all parts of the world. In Japan, the number of patients exceeds 10 million. It is estimated. The largest number of patients is tinea pedis, commonly referred to as “athlete's foot”, followed by onychomycosis. The development of new therapies and drugs is an important social issue for tinea tinea, which is still very refractory and recurrent.
白癬は、トリコフィトン属等の真菌の感染によって引き起こされるが、起因菌としてはトリコフィトン ルブルム、トリコフィトン メンタグロフィテス、エピデルモフィトン フロックスムの順に多い。白癬菌は角化した皮膚の組織、すなわち角質層や毛髪および爪に侵入・発育し、局所の感染（白癬）を引き起こす。 Ringworm is caused by infection with fungi of the genus Trichophyton, etc., and the most common causes are Trichophyton rubulum, Trichophyton mentagrophytes, and Epidermophyton Phloxum. Ringworm fungus invades and develops keratinized skin tissue, that is, the stratum corneum, hair and nails, and causes local infection (ringworm).
白癬に対する抗菌剤の開発において抗菌力の評価が必要不可欠とされている。従来の抗菌力評価は、ｉｎ ｖｉｔｒｏにおいて直接白癬菌と抗菌剤と作用させる方法がある（特許文献１）。しかし、白癬菌は感染すると角層内に潜り込み、抗菌剤の抗菌力に加え、角層内への浸透力も必要とされているため、ｉｎ
ｖｉｔｒｏでの評価はかならずしも十分とはいえない。これに対し、実状況に近い感染動物モデルの使用は必要不可欠とされ、これまで白癬菌感染のモデル実験は専らモルモットやマウス（特許文献２）を用いて行われてきた。
Evaluation of antibacterial activity is indispensable in the development of antibacterial agents against ringworm. Conventional antibacterial activity evaluation includes a method of directly reacting ringworm and antibacterial agent in vitro (Patent Document 1). However, ringworms can enter the stratum corneum when infected, and in addition to the antibacterial activity of antibacterial agents, penetration into the stratum corneum is also required.
In vitro evaluation is not always sufficient. On the other hand, the use of an infected animal model that is close to the actual situation is indispensable, and until now, model experiments for ringworm infection have been carried out exclusively using guinea pigs and mice (Patent Document 2).
しかしながら、当該動物では感染および病態に関する宿主側の免疫学的解析、遺伝学的解析が充分かつ効率的には行われ難いという問題点があった。また、最近動物愛護の観点より次第に動物試験法の使用が制限されるようになってきている。このため、それらの解析をする必要がない新しい感染モデルの開発が求められていた。 However, the animal has a problem that it is difficult to perform sufficient and efficient host-side immunological analysis and genetic analysis on infection and disease state. In recent years, the use of animal testing methods has been increasingly restricted from the viewpoint of animal welfare. For this reason, development of a new infection model which does not need to analyze them was called for.
一方、３次元培養皮膚は古くから開発され（非特許文献１）、実際の皮膚に近い構造および生理活性を有することからやけどの治癒時の代替皮膚（特許文献３）や、皮膚刺激性試験（特許文献４）や経皮吸収促進試験（特許文献５）などの動物代替試験として用いられているが、白癬菌などの感染モデル系に使用された報告はない。 On the other hand, three-dimensional cultured skin has been developed for a long time (Non-patent Document 1), and has a structure and physiological activity close to that of actual skin. Although it is used as an animal substitute test such as Patent Document 4) and percutaneous absorption promotion test (Patent Document 5), there is no report used for an infection model system such as ringworm.
上述したように、従来の技術では、抗菌剤の実際の病態に対する作用状況を十分に反映しているとは考えにくい抗菌剤と白癬菌を直接作用させる方法、あるいは実験動物を犠牲にした上、免疫学的解析、遺伝学的解析が充分かつ効率的には行われ難い方法が実施されてきた。これらが本発明が解決しようとする課題である。 As mentioned above, the conventional technology sacrifices the method of direct action of antibacterial agents and ringworm fungi, which are unlikely to sufficiently reflect the action status of the antibacterial agents against the actual pathological condition, Methods have been implemented in which immunological analysis and genetic analysis are difficult to perform sufficiently and efficiently. These are the problems to be solved by the present invention.
本発明者らは、動物を用いず、3次元培養皮膚と白癬菌を共培養し、培養皮膚の角層下に白癬菌を生きたまま埋め込んだ感染モデル系を創出し、さらにこのモデルを使えば白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価できることを見出し、本発明を完成するに至った。 The present inventors co-cultured three-dimensional cultured skin and ringworm bacteria without using animals, and created an infection model system in which ringworm bacteria were embedded alive under the horny layer of the cultured skin. As a result, the inventors have found that the antibacterial effect against ringworm and the toxicity to the three-dimensional cultured skin can be simultaneously evaluated, and the present invention has been completed.
すなわち、動物を用いた白癬菌感染モデルを代替するｉｎ ｖｉｔｒｏモデルとして、生きた白癬菌と3次元培養皮膚を共存したことを特徴とする新規な皮膚白癬菌感染モデルを提供するものである。このモデルに抗真菌製剤等を塗布し、白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価することを特徴とする。 That is, the present invention provides a novel skin ringworm fungus infection model characterized by the coexistence of live ringworm bacteria and three-dimensional cultured skin as an in vitro model that substitutes for a ringworm fungus infection model using animals. An antifungal preparation or the like is applied to this model, and the antibacterial effect against trichophyton and the toxicity to the three-dimensional cultured skin are simultaneously evaluated.
本発明は、抗菌剤と白癬菌を直接作用させる従来の技術に比べ、実際の感染状況に近いｉｎ ｖｉｔｒｏ実験条件を提供し、さらに実験動物を用いることなく実際の状況に近い薬剤の抗菌作用を評価する方法を提供できる。 The present invention provides in vitro experimental conditions that are close to the actual infection situation, compared to the conventional technique in which an antibacterial agent and ringworm fungi are allowed to act directly, and further provides an antibacterial action of a drug that is close to the actual situation without using experimental animals. A method of evaluation can be provided.
本発明において３次元培養皮膚は、表皮構造又は表皮と真皮構造を有するものである。 In the present invention, the three-dimensional cultured skin has an epidermis structure or an epidermis and dermis structure.
本発明において白癬菌は皮膚や爪に感染すると認められているものであれば、特に限定されるものではなく、例えば、トリコフィトン ルブルム、トリコフィトン メンタグロフィテス、エピデルモフィトン フロックスムなどが挙げられる。 In the present invention, the ringworm is not particularly limited as long as it is recognized that it infects the skin and nails, and examples thereof include Trichophyton rubulum, Trichophyton mentagrophytes, Epidermophyton floxum and the like.
これらの白癬菌の３次元培養皮膚試料への感染（接種） にあたっては、定量的な評価を可能とするため、白癬菌を予め斜面培地で培養し、回収後、ガーゼなどでろ過して菌糸を除去して平板培地播種する。 To infect (inoculate) three-dimensional cultured skin samples of these ringworm bacteria, the ringworm bacteria are cultured in a slant medium in advance and collected, then filtered with gauze, etc. to obtain the hyphae. Remove and seed with plate medium.
上記白癬菌を回収し、３次元培養皮膚の表皮側に播く。感染及び角層内の封入は、３７ ℃、５％ＣＯ２の条件で、７ 日間程度培養を行えばよい。感染が十分に行われたことは、組織を切片にし、顕微鏡下で染色した真菌の発育と角層の形成を確認することにより判断することができる。 The ringworm is collected and spread on the epidermal side of the three-dimensional cultured skin. Infection and encapsulation in the stratum corneum may be carried out for about 7 days under conditions of 37 ° C. and 5% CO 2 . It can be judged that the infection has been sufficiently performed by sectioning the tissue and confirming the growth of the fungus stained with a microscope and the formation of the stratum corneum.
このようにして、白癬菌で感染された３次元培養皮膚に、次いで、抗真菌剤又はこれを含む組成物（ 以下、「抗真菌製剤」という） を作用させる。この作用させる抗真菌製剤の剤型は、外用剤として通常皮膚に用いられる剤型であれば特に限定されず、例えば、液剤、クリーム剤、軟膏剤として塗布するもの、皮膚を清拭するもの、貼付け剤として貼付けるもの等により作用させることができる。 In this way, the antifungal agent or a composition containing the same (hereinafter referred to as “antifungal preparation”) is allowed to act on the three-dimensional cultured skin infected with ringworm. The dosage form of the antifungal preparation to be acted on is not particularly limited as long as it is a dosage form usually used on the skin as an external preparation, for example, a liquid, cream, ointment applied, skin wiping, It can be made to act by what is stuck as a sticking agent.
３次元培養皮膚に抗真菌製剤を作用させるに当たっては、ろ紙や布に事前にしみこませた抗真菌製剤を用いるのが好ましい。一定時間３次元培養皮膚の表皮側に抗真菌製剤を作用させた後、抗真菌製剤を除去する。３次元培養皮膚をホモジナイズし、白癬菌を抽出した後、同様に平板培地にて培養し、菌数を測定する。また、同時に抗真菌製剤の３次元培養皮膚に対する毒性を細胞染色することにより評価することもできる。 When the antifungal preparation is allowed to act on the three-dimensional cultured skin, it is preferable to use an antifungal preparation previously impregnated in filter paper or cloth. After the antifungal preparation is allowed to act on the epidermis side of the three-dimensional cultured skin for a certain time, the antifungal preparation is removed. After homogenizing the three-dimensional cultured skin and extracting trichophyton, the same is cultured in a plate medium and the number of bacteria is measured. At the same time, the toxicity of the antifungal preparation to the three-dimensional cultured skin can be evaluated by cell staining.
白癬菌の播種菌数としては１０４ｃｆｕ／ｃｍ２〜１０６ｃｆｕ／ｃｍ２が最も望ましいとされている。 10 4 cfu / cm 2 to 10 6 cfu / cm 2 is most preferable as the number of inoculated bacteria of ringworm.
以下、実施例に基づいて本発明を詳説する。本発明は特にこれらにより限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples. The present invention is not particularly limited by these.
１．白癬菌の調製
培地は、ポテト・デキストロース寒天培地（ＰＤＡ培地）３．９ ％となるように蒸留水に添加、攪拌後、オートクレーブで滅菌（ １ ２ ０ ℃ 、１
５ 分） し作製した。トリコフィトン メンタグロファイテスを斜面培地に播種し、３０ ℃ で１ 週間培養した。この斜面培地に１ ０ ｍＬの０ ． ０ ５ ％ ツィーン８
０ を加えた生理食塩水を添加した。軽くピペッティングして遊離させた後、ガーゼでろ過し菌糸を除去した。３ ０ ０ ０ ｒ ｐ ｍ 、５ 分間遠心で分生子を集め、０
． ０ ５ ％ ツィーン８ ０ − 生理食塩水で３ 回洗浄し、４ ｍＬの０ ． ０ ５ ％ ツィーン８ ０ − 生理食塩水に懸濁した。この懸濁液を血球計算板にて菌量を測定した。
1. The tinea fungus preparation medium was added to distilled water so as to be 3.9% potato dextrose agar medium (PDA medium), stirred, and then sterilized by autoclaving (120 ° C., 1
5 minutes). Trichophyton mentagrophytes was inoculated on a slant medium and cultured at 30 ° C. for 1 week. Into this slant medium, 10 mL of 0. 0 5% Tween 8
Saline with 0 added was added. After releasing by light pipetting, the mixture was filtered through gauze to remove the mycelium. 3 0 0 0 r pm, collect conidia by centrifugation for 5 minutes, 0
. 0. 5% Tween 80-Wash 3 times with saline, 4 mL of 0. Suspended in 0.5% Tween 80-saline. The bacterial mass of this suspension was measured with a hemocytometer.
上記１．で調製した接種菌液を平板培地に画線し、３０ ℃ で１ 週間培養した。同様に１ ０ ｍＬの０ ． ０ ５ ％ ツィーン８ ０ を加えた生理食塩水を添加した。軽くピペッティングして遊離させた後、ガーゼでろ過し菌糸を除去した。３
０ ０ ０ ｒ ｐ ｍ 、５ 分間遠心で分生子を集め、０ ． ０ ５ ％ ツィーン８ ０ − 生理食塩水で３ 回洗浄し、４ ｍＬの０ ． ０ ５ ％ ツィーン８
０ − 生理食塩水に懸濁した。この懸濁液を血球計算板にて菌量を測定した。
Above 1. The inoculum prepared in step 1 was streaked on a plate medium and cultured at 30 ° C. for 1 week. Similarly, 10 mL of 0. Saline supplemented with 0 5% Tween 8 0 was added. After releasing by light pipetting, the mixture was filtered through gauze to remove the mycelium. 3
Collect conidia by centrifugation at 0 0 r pm for 5 minutes. 0. 5% Tween 80-Wash 3 times with saline, 4 mL of 0. 0 5% Tween 8
0—Suspended in saline. The bacterial mass of this suspension was measured with a hemocytometer.
２．感染モデルの作製
３次元培養皮膚（クラボウ製）の表皮側にトリコフィトン・メンタグロファイテスを１０５ｃｆｕ／ｃｍ２となるように接種し、３７ ℃、５％ＣＯ２の条件で、７日間程度培養した。感染モデルを切片にし、角層の観察および真菌染色法による白癬菌の観察を行った。
2. Preparation of infection model Trichophyton mentagrophytes is inoculated to 10 5 cfu / cm 2 on the epidermis side of the three-dimensional cultured skin (manufactured by Kurabo Industries), and is maintained at 37 ° C. and 5% CO 2 for 7 days. Cultivated to a certain extent. The infection model was sectioned and the stratum corneum was observed and fungus staining was used to observe ringworm.
白癬菌感染３次元培養皮膚の角層中に白癬菌が観察され（図１）、白癬菌未感染の３次元培養皮膚（図２）と同様に角層が観察された。 Ringworms were observed in the stratum corneum of the three-dimensional cultured skin infected with ringworm fungus (FIG. 1), and the stratum corneum was observed in the same manner as the three-dimensional cultured skin not infected with ringworm fungus (FIG. 2).
１．抗真菌製剤の評価
直径８ｍｍのろ紙に抗真菌製剤を５０μＬしみ込ませ、３次元培養皮膚の表皮側にのせ、３７ ℃、５％ＣＯ２の条件で、２４時間培養した。実施例１は塩化ベンザルコニウム含有抗真菌製剤、実施例２はミコナゾール硝酸塩含有抗真菌製剤、実施例３は０．２％ポリヘキサメチレンビグアニド含有抗真菌製剤である。
1. Evaluation of Antifungal Formulation 50 μL of the antifungal formulation was impregnated into a filter paper having a diameter of 8 mm, placed on the epidermis side of the three-dimensional cultured skin, and cultured at 37 ° C. and 5% CO 2 for 24 hours. Example 1 is an antifungal preparation containing benzalkonium chloride, Example 2 is an antifungal preparation containing miconazole nitrate, and Example 3 is an antifungal preparation containing 0.2% polyhexamethylene biguanide.
２．白癬菌の菌数測定と３次元培養皮膚の細胞毒性
抗真菌製剤を塗布２４時間後、ろ紙を除去し、リン酸緩衝生理食塩水にて洗浄した。３次元培養皮膚をホモジナイズし、白癬菌を抽出した後、平板培地に播き、培養し、３０
℃ で１ 週間培養し菌数を測定する。また、別の抗真菌製剤処理後の３次元培養皮膚を１ｍｇ／ｍＬのＭＴＴ［３−（４，５−ジメチルチアゾール−２−イル）２，５−ジフェニルテトラゾリンブロマイド］入り培地に浸し、３時間処理し、イソプロパノールで色素であるホルマザンを抽出し５７０ｎｍの吸光度を測定した。細胞賦活率は式１で求めた。
2. 24 hours after application of the number of tinea fungus and 3D cultured skin cytotoxic antifungal preparation, the filter paper was removed and washed with phosphate buffered saline. After homogenizing the three-dimensional cultured skin and extracting trichophyton, the seeds are plated and cultured on a plate medium. 30
Incubate at ℃ 1 week and count the number of bacteria. Further, the three-dimensional cultured skin after treatment with another antifungal preparation was immersed in a medium containing 1 mg / mL of MTT [3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazoline bromide]. After time treatment, formazan, a pigment, was extracted with isopropanol, and the absorbance at 570 nm was measured. The cell activation rate was determined by Equation 1.
細胞賦活率（％）＝（試験物質処理後の吸光度）／（試験物質未処理の吸光度）×１００％ Cell activation rate (%) = (absorbance after treatment with test substance) / (absorbance without treatment with test substance) × 100%
抗菌力試験の結果を図３に示す。実施例１〜３のいずれも抗菌力が認められた。さらに時間的に抗菌力が高まることから、角層への浸透に伴い、抗菌力が増加したことがわかった。 The results of the antibacterial activity test are shown in FIG. In any of Examples 1 to 3, antibacterial activity was observed. Furthermore, since the antibacterial activity increased with time, it was found that the antibacterial activity increased with the penetration into the stratum corneum.
抗菌力試験の結果を図４に示す。実施例３のみに毒性が低いことがわかり、選択的に白癬菌を死滅させることがわかった。 The results of the antibacterial activity test are shown in FIG. Only Example 3 was found to be less toxic and was found to selectively kill ringworm.
本発明方法によれば、白癬菌に対する抗菌効果と３次元培養皮膚に対する毒性を同時に評価することができ、皮膚に影響がなく、選択的に菌を死滅させる抗菌製剤の評価に応用しうることがわかった。 According to the method of the present invention, the antibacterial effect against trichomycosis and the toxicity to the three-dimensional cultured skin can be simultaneously evaluated, and the method can be applied to the evaluation of an antibacterial preparation that selectively kills bacteria without affecting the skin. all right.
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| CN112574943B (en) * | 2020-12-14 | 2023-09-01 | 深圳钰捷生物医学科技有限公司 | Model for simulating dermatophyte infection in vitro and establishment method and application thereof |
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