JP2019101025A - Fluorescence-labeled polylysine and observation method using the same - Google Patents
Fluorescence-labeled polylysine and observation method using the same Download PDFInfo
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本発明は、蛍光色素化合物で標識した蛍光標識ポリリジン、及びこれを用いる対象の観察方法に関する。 The present invention relates to a fluorescently labeled polylysine labeled with a fluorescent dye compound, and a method of observing an object using the same.
皮膚外用薬や化粧料を効率的よく開発するため、生体表面組織の状態を精度よく観察することが重要である。従来その一般的な方法として、細胞採取用ブラシやテープストリッピング等の公知の手法により採取した生体材料を染色剤で染色したのち、顕微観察することが行われている。
このうち皮膚角層細胞の観察を目的とする場合には、例えば頬部や上腕内側部よりテープストリッピング法にて採取された角層細胞を、ゲンチアナバイオレット−ブリリアントグリーン染色、ローダミンＢ−メチレンブルー染色、ヘマトキシリン−エオジン染色などの染色方法で角層細胞を染色したのち、顕微鏡観察する方法が広く行われている。
しかしながら、これらの染色方法は操作手順が煩雑で、かつ染色に時間がかかるうえ測定者の技量に負うところが大きく、しばしば染色が不十分となり安定した顕微鏡観察が行えない問題があった。
In order to develop skin external preparations and cosmetics efficiently, it is important to observe the condition of the surface tissue of the living body with high accuracy. Conventionally, as a general method, microscopic observation is performed after a biological material collected by a known method such as a cell collection brush or tape stripping is stained with a staining agent.
Among them, for the purpose of observation of stratum corneum cells, for example, stratum corneum cells collected from the cheek or the upper arm by tape stripping method are stained with gentian violet-brilliant green, rhodamine B-methylene blue, After stratum corneum cells are stained by a staining method such as hematoxylin-eosin staining, a method of microscopic observation is widely used.
However, these staining methods require complicated operation procedures, take a long time for staining, are largely dependent on the skill of the measurer, and often suffer from insufficient staining so that stable microscopic observation can not be performed.
この解決策として、例えば特許文献１では、上記した染色法と併せて、タンニン酸、クロム塩類、アルミニウム塩類、グルタールアルデヒド、ホルマリンの群から選ばれる一種または二種以上を媒染剤として使用した角層細胞の染色方法が提案されている。また、特許文献２では、染色剤を水と混和可能な有機溶剤を含有する溶媒に溶解したのち、角層細胞の染色に供する方法が提案されている。 As this solution, for example, in Patent Document 1, a stratum corneum using, as a mordant, one or more selected from the group of tannic acid, chromium salts, aluminum salts, glutaraldehyde and formalin in combination with the above-mentioned staining method Methods for staining cells have been proposed. Further, Patent Document 2 proposes a method in which a staining agent is dissolved in a solvent containing an organic solvent miscible with water and then used for staining of stratum corneum cells.
これらの他に、染色剤を用いずに角層細胞を直接観察する方法も提案されている。例えば、特許文献３では、紫外線下、顕微鏡乃至はビデオマイクロスコープを介して観察する方法が、特許文献４ではテープストリッピングにて採取した角層細胞を共焦点レーザー顕微鏡で観察する方法が提案されている。 Besides these, methods for directly observing stratum corneum cells without using a staining agent have also been proposed. For example, Patent Document 3 proposes a method of observing through a microscope or a video microscope under ultraviolet light, and Patent Document 4 proposes a method of observing a stratum corneum cell collected by tape stripping with a confocal laser microscope. There is.
ところで、ε−ポリ−Ｌ−リジンは、Ｌ−リジンのε位のアミノ基とカルボキシル基とがペプチド結合により直鎖状に連なったポリマーである。ε−ポリ−Ｌ−リジンは、抗菌作用を示すことが知られており、保存料として食品添加物や化粧品原料等に実用化されている（非特許文献１）。また、ε−ポリ−Ｌ−リジンは、正電荷を帯びていることから、負に帯電している物と相互作用する。そして、角層細胞等にも吸着することが示唆されていたが、その吸着挙動については明らかではなかった。 By the way, ε-poly-L-lysine is a polymer in which an amino group at the ε-position of L-lysine and a carboxyl group are linearly linked by a peptide bond. ε-poly-L-lysine is known to exhibit an antibacterial action, and has been put to practical use as a food additive, a cosmetic raw material and the like as a preservative (Non-patent Document 1). Also, since ε-poly-L-lysine is positively charged, it interacts with negatively charged substances. And, it has been suggested that it also adsorbs to stratum corneum cells etc., but its adsorption behavior was not clear.
角層細胞の観察方法に関して、特許文献１及び２の方法では、従来の染色方法を改良しながらも依然として操作手順は煩雑である。また、特許文献３及び４の方法では、高価な光学機器を要するため、大学等の小規模な研究室では利用が困難である。
このような問題に鑑み、本発明の課題は、操作が簡便で、かつ高価な分析機器を使用しなくても、高い精度で角層細胞を始めとする生体組織の観察方法を提供することである。
With regard to the method of observing stratum corneum cells, the methods of Patent Documents 1 and 2 are still complicated in operation procedure while improving the conventional staining method. Further, the methods of Patent Documents 3 and 4 require expensive optical equipment, so that they are difficult to use in a small laboratory such as a university.
In view of such problems, it is an object of the present invention to provide a method for observing living tissue including stratum corneum cells with high accuracy without using an analytical instrument which is easy to operate and which is expensive. is there.
本発明者らは、前記課題を解決するために鋭意研究を重ねた結果、蛍光化合物で標識した蛍光標識ポリリジンを対象に吸着させることにより、蛍光染色された対象を容易に蛍光顕微鏡で観察できることを見出し、本発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that by allowing fluorescently labeled polylysine labeled with a fluorescent compound to adsorb to the object, the fluorescently stained object can be easily observed with a fluorescence microscope. The present invention has been completed.
すなわち、本発明は以下の通りである。
［１］蛍光色素化合物で標識した蛍光標識ポリリジン。
［２］蛍光色素化合物が、３，６−ジアミノ−９−［２，４−ビス（リチオオキシカルボニル）フェニル］−４−（リチオオキシスルホニル）−５−スルホナトキサンチリウム／３，６−ジアミノ−９−［２，５−ビス（リチオオキシカルボニル）フェニル］−４−（リチオオキシスルホニル）−５−スルホナトキサンチリウム又はその誘導体である、［１］に記載の蛍光標識ポリリジン。
［３］蛍光色素化合物が、９−［２−カルボキシ−４（オア５）−［［（２，５−ジオキソ−１−ピロリジニル）オキシ］−カルボニル］フェニル]−３，６−ビス−（ジメチル
アミノ）−キサンチリウム インナーソルト又はその誘導体である、［１］に記載の蛍光
標識ポリリジン。
［４］蛍光色素化合物の導入率が蛍光標識ポリリジン全量の０．１〜１０重量％である、［１］〜［３］のいずれかに記載の蛍光標識ポリリジン。
［５］ポリリジンが、ε−ポリリジン及び／又はその塩である、［１］〜［４］のいずれかに記載の蛍光標識ポリリジン。
［６］［１］〜［５］のいずれかに記載の蛍光標識ポリリジンを含有する、観察用試薬。［７］［１］〜［５］のいずれかに記載の蛍光標識ポリリジンを対象に吸着させる工程を含む、対象の観察方法。
［８］対象が角層細胞、毛髪、微生物、及び繊維製品からなる群から選択される、［７］に記載の観察方法。
［９］吸着工程がｐＨ６〜８の条件下で行われる、［７］又は［８］に記載の観察方法。［１０］対象がダメージ毛髪である、［８］又は［９］に記載の観察方法。
That is, the present invention is as follows.
[1] Fluorescently labeled polylysine labeled with a fluorescent dye compound.
[2] The fluorescent dye compound is 3,6-diamino-9- [2,4-bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium / 3,6-diamino [9] The fluorescently labeled polylysine according to [1], which is -9- [2,5-bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium or a derivative thereof.
[3] The fluorescent dye compound is 9- [2-carboxy-4 (or 5)-[[(2,5-dioxo-1-pyrrolidinyl) oxy] -carbonyl] phenyl] -3,6-bis- (dimethyl) [10] The fluorescently labeled polylysine according to [1], which is an amino) -xanthylium inner salt or a derivative thereof.
[4] The fluorescently labeled polylysine according to any one of [1] to [3], wherein the introduction rate of the fluorescent dye compound is 0.1 to 10% by weight of the total amount of fluorescently labeled polylysine.
[5] The fluorescently labeled polylysine according to any one of [1] to [4], wherein the polylysine is ε-polylysine and / or a salt thereof.
[6] An observation reagent comprising the fluorescently labeled polylysine according to any one of [1] to [5]. [7] A method of observing a subject, comprising the step of adsorbing the fluorescently labeled polylysine according to any one of [1] to [5] to the subject.
[8] The observation method according to [7], wherein the subject is selected from the group consisting of stratum corneum cells, hair, microorganisms, and textiles.
[9] The observation method according to [7] or [8], wherein the adsorption step is performed under conditions of pH 6-8. [10] The observation method according to [8] or [9], wherein the subject is damaged hair.
本発明の蛍光標識ポリリジンを用いれば、煩雑な染色操作や高価な分析機器を用いることなく、簡便な操作で精度よく、角層細胞を始めとする生体細胞や毛髪等の生体組織、微生物、天然繊維や化学繊維及び合成繊維等の繊維素材、天然樹脂や合成樹脂等の工業素材の状態を、観察することができる。 When the fluorescently labeled polylysine of the present invention is used, biological tissues such as keratinocytes and living tissues such as hair, microorganisms, and natural tissues can be accurately and easily by simple operations without using complicated staining procedures and expensive analytical instruments. The state of fiber materials such as fibers, chemical fibers and synthetic fibers, and industrial materials such as natural resins and synthetic resins can be observed.
本発明の一の形態は、蛍光色素化合物で標識した蛍光標識ポリリジンである。すなわちポリリジンに蛍光色素化合物が結合したものであり、かかる結合は共有結合、イオン結合、配位結合、水素結合等特に限定されない。 One aspect of the present invention is fluorescently labeled polylysine labeled with a fluorescent dye compound. That is, the fluorescent dye compound is bonded to polylysine, and such bond is not particularly limited, such as covalent bond, ionic bond, coordinate bond, hydrogen bond and the like.
本発明において、ポリリジンはα−ポリリジン、ε−ポリリジンのいずれでもよく特に限定されるものではないが、毒性の低さ、入手の容易さからε−ポリリジンが好ましい。また、通常はＬ−リジンのポリマーである。
また、ポリリジンは、通常はリジンのホモポリマーであるが、本発明の効果を損なわない限りにおいて、他のアミノ酸をモノマーとして含んでもよい。
また、ポリリジンの大きさは、特に限定されないが、重量平均分子量が好ましくは３０００以上、より好ましくは４０００以上であり、好ましくは１００００以下、より好ましくは８０００以下、さらに好ましくは６０００以下であり、３０００〜６０００の範囲が特に好ましい。なお、ここで重量平均分子量は、ＧＰＣ−ＬＡＬＬＳ法により測定された値である。
In the present invention, polylysine may be either α-polylysine or ε-polylysine and is not particularly limited. However, ε-polylysine is preferable from the viewpoint of low toxicity and easy availability. Also, it is usually a polymer of L-lysine.
Polylysine is usually a homopolymer of lysine, but may contain other amino acids as monomers as long as the effects of the present invention are not impaired.
Further, the size of polylysine is not particularly limited, but the weight average molecular weight is preferably 3000 or more, more preferably 4000 or more, preferably 10000 or less, more preferably 8000 or less, still more preferably 6000 or less, The range of -6000 is particularly preferred. Here, the weight average molecular weight is a value measured by the GPC-LALLS method.
ε−ポリリジンは、例えば特許第１２４５３６１号に記載の方法で製造することができる。具体的には、ストレプトマイセス・アルプラス・サブスピーシーズ・リジノポリメラスを、その組成が、グルコース５重量％、酵母エキス０．５重量％、硫酸アンモニウム１重量％、リン酸水素二カリウム０．０８重量％、リン酸二水素カリウム０．１３６重量％、硫酸マグネシウム・７水和物０．０５重量％、硫酸亜鉛・７水和物０．００４重量％、
及び硫酸鉄・７水和物０．０３重量％であり、ｐＨが６．８に調整された培地にて培養し、得られた培養物からε−ポリリジンを分離・回収する。
他にも、化学的手法によりポリリジンを製造してもよい。
ε-polylysine can be produced, for example, by the method described in Patent No. 1245361. Specifically, Streptomyces alpras subspecies lizinopolymers, the composition of which is 5 wt% glucose, 0.5 wt% yeast extract, 1 wt% ammonium sulfate, 0.08 wt% dipotassium hydrogen phosphate 0.136% by weight of potassium dihydrogen phosphate, 0.05% by weight of magnesium sulfate heptahydrate, 0.004% by weight of zinc sulfate heptahydrate
And, it is cultured in a medium which is 0.03 wt% of iron sulfate heptahydrate and adjusted to pH 6.8, and ε-polylysine is separated and recovered from the obtained culture.
Alternatively, polylysine may be produced by a chemical method.
また、ε−ポリリジンは、遊離の形であってもよいし、塩酸、硫酸、リン酸及び臭化水素酸から選ばれた少なくとも１種の無機酸、または酢酸、プロピオン酸、フマル酸、リンゴ酸及びクエン酸から選ばれた少なくとも１種の有機酸の塩の形であってもよい。
ポリリジン塩は常法により製造される。例えば含水メタノール溶液に前記ε−ポリリジンを溶解させ、これに前記酸を加え、溶液が中和点を過ぎたところで、冷アセトンを加えて沈澱した塩を乾燥させることによって得られる。
Also, ε-polylysine may be in free form, or at least one inorganic acid selected from hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid, or acetic acid, propionic acid, fumaric acid, malic acid And at least one organic acid salt selected from citric acid.
Polylysine salts are prepared by conventional methods. For example, it can be obtained by dissolving the 含水 -polylysine in a water-containing methanol solution, adding the acid thereto, and adding cold acetone when the solution has passed the neutralization point to dry the precipitated salt.
本発明において、蛍光色素化合物は、ポリリジンと結合するものであれば特に限定されず、任意のものを用いればよい。
蛍光色素化合物の例としては、Ａｌｅｘａ類、フルオレセインイソチオシアネート（ＦＩＴＣ）又はその誘導体、ＴＡＭＲＡ、Ｃｙ３、Ｃｙ５、ローダミン６Ｇ（Ｒ６Ｇ）又はその誘導体（例えば、テトラメチルローダミン（ＴＭＲ））、テキサスレッド、ＢＯＤＩＰＹ類、ＲＯＸ、Ｈｅｘ、ＪＯＥ、ＢＨＱ類等が挙げられる。これらのうち、３，６−ジアミノ−９−［２，４−ビス（リチオオキシカルボニル）フェニル］−４−（リチオオキシスルホニル）−５−スルホナトキサンチリウム／３，６−ジアミノ−９−［２，５−ビス（リチオオキシカルボニル）フェニル］−４−（リチオオキシスルホニル）−５−スルホナトキサンチリウム（商品名Alexa Fluor488（Thermo Fisher Scientific社製））またはその誘導体、及び９−［２−カルボキシ−４（オア５）−［［（２，５−ジオキソ−１−ピロリジニル）オキシ］−カルボニル］フェニル]−３，６−ビス−（ジメチルアミノ
）−キサンチリウム インナーソルト（商品名NHS-Rhodamine（Therrmo Fisher Scientific社製））又はその誘導体が、入手のし易さ、取り扱いの簡便さから、好ましい。これら
の蛍光色素化合物は常法によりポリリジンに反応させることにより、共有結合、イオン結合、配位結合、又は水素結合等でポリリジンを蛍光標識する。これらの蛍光色素化合物はポリリジンの有するアミノ基を介して錯体を形成し、蛍光標識ポリリジンを得ることができる（図１及び図２）。
In the present invention, the fluorescent dye compound is not particularly limited as long as it binds to polylysine, and any desired one may be used.
Examples of fluorochrome compounds include Alexa, fluorescein isothiocyanate (FITC) or derivatives thereof, TAMRA, Cy3, Cy5, rhodamine 6G (R6G) or derivatives thereof (eg tetramethylrhodamine (TMR)), Texas red, BODIPY And ROX, Hex, JOE, BHQs and the like. Among these, 3,6-diamino-9- [2,4-bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium / 3,6-diamino-9- [ 2,5-bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium (trade name Alexa Fluor 488 (manufactured by Thermo Fisher Scientific)) or a derivative thereof, and 9- [2- Carboxy-4 (or 5)-[[(2,5-dioxo-1-pyrrolidinyl) oxy] -carbonyl] phenyl] -3,6-bis- (dimethylamino) -xanthylium inner salt (brand name NHS-Rhodamine ( Therrmo Fisher Scientific)) or a derivative thereof is preferred in view of easy availability and ease of handling. These fluorescent dye compounds fluorescently label polylysine by covalent bond, ionic bond, coordinate bond, hydrogen bond or the like by reacting with polylysine by a conventional method. These fluorescent dye compounds can form a complex via the amino group possessed by polylysine to obtain fluorescently labeled polylysine (FIGS. 1 and 2).
本発明において、蛍光色素化合物のポリリジンへの導入率は、蛍光標識ポリリジン全量の好ましくは０．１〜１０重量％であり、より好ましくは０．３〜３重量％である。この範囲より導入率が小さいと蛍光検出をし難い場合があり、またこの範囲より大きいとポリリジンの対象への吸着が困難になる場合がある。 In the present invention, the rate of introduction of the fluorescent dye compound into polylysine is preferably 0.1 to 10% by weight, more preferably 0.3 to 3% by weight of the total amount of fluorescently labeled polylysine. If the introduction rate is smaller than this range, it may be difficult to detect fluorescence, and if it is larger than this range, it may be difficult to adsorb polylysine to a target.
ポリリジンは塩基性アミノ酸Ｌ−リジンのホモポリマーであり、等電点以下の水中では側鎖アミノ基が正電荷を帯びることから、負電荷を帯びた物質に電気的に吸着してイオン複合体を形成する。また、炭化水素リッチな構造から、疎水性相互作用による対象への吸着も生じ得る。
そのため、本発明の蛍光標識ポリリジンは、ポリリジンが吸着し得る対象の観察用試薬として好適に用いることができる。かかる対象としては、特に限定されないが、角層細胞を始めとする生体細胞や毛髪等の生体組織、微生物、天然繊維や化学繊維及び合成繊維等の繊維素材、天然樹脂や合成樹脂等の工業素材などを好適に挙げられる。
Polylysine is a homopolymer of the basic amino acid L-lysine, and in water below the isoelectric point, the side chain amino group bears a positive charge, so it is electrically adsorbed to a negatively charged substance to form an ionic complex. Form. Also, hydrocarbon-rich structures can result in adsorption to objects via hydrophobic interactions.
Therefore, the fluorescently labeled polylysine of the present invention can be suitably used as an observation reagent for an object to which polylysine can be adsorbed. Such objects are not particularly limited, but biological tissues such as horny cells and biological tissues such as hair, microorganisms, fiber materials such as natural fibers, chemical fibers and synthetic fibers, and industrial materials such as natural resins and synthetic resins Etc. are mentioned suitably.
本発明の他の形態は、本発明の蛍光標識ポリリジンを対象に吸着させる工程を含む、対象の観察方法である。前記吸着工程は、通常は水溶液の態様で蛍光標識ポリリジンを対象に接触させることにより行われる。
吸着工程を行う条件は、特に限定されないが、ｐＨ６〜８が好ましい。また、温度は４〜４０℃が好ましい。また、蛍光標識ポリリジンは０．００１〜０．０１重量％の濃度の水溶液で適用することが好ましい。かかる適用時間は、前記濃度にもよるが、５〜６０分
間で十分に吸着が行われる。
Another aspect of the present invention is a method of observing a target, comprising the step of adsorbing the fluorescently labeled polylysine of the present invention to the target. The adsorption step is usually carried out by contacting the fluorescently labeled polylysine with the object in the form of an aqueous solution.
The conditions for performing the adsorption step are not particularly limited, but pH 6 to 8 is preferable. Moreover, as for temperature, 4-40 degreeC is preferable. In addition, it is preferable to apply the fluorescently labeled polylysine in an aqueous solution with a concentration of 0.001 to 0.01% by weight. The application time depends on the concentration, but the adsorption is sufficiently performed in 5 to 60 minutes.
通常は、蛍光標識ポリリジンを対象に接触させて吸着した後、余剰の蛍光標識ポリリジンを洗浄等により除去した後に、蛍光顕微鏡や蛍光観察用デジタルマイクロスコープ等にて蛍光検出することにより、ポリリジンの対象上での分布状況を観察する。
観察対象は、特に限定されないが、角層細胞を始めとする生体細胞や毛髪等の生体組織、微生物、天然繊維や化学繊維及び合成繊維等の繊維素材、天然樹脂や合成樹脂等の工業素材などを好適に挙げられる。
Usually, after the fluorescent labeled polylysine is brought into contact with the target and adsorbed, the excess fluorescent labeled polylysine is removed by washing etc. Then, the target of the polylysine is detected by fluorescence detection with a fluorescent microscope, a digital microscope for fluorescent observation, etc. Observe the distribution above.
The object to be observed is not particularly limited, but biological tissues such as stratum corneum cells and biological tissues such as hair, microorganisms, fiber materials such as natural fibers, chemical fibers and synthetic fibers, and industrial materials such as natural resins and synthetic resins Are preferably mentioned.
次に実施例を挙げて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will next be described in detail by way of examples, which should not be construed as limiting the invention thereto.
＜実施例１＞蛍光標識ポリリジンの調製１
ε−Ｌ−ポリリジン（以降「ＰＬＬ」と記す、Ｍｎ：４０９０、Ｍｗ：４７００、１０質量％水溶液、ＪＮＣ株式会社製）を０．１Ｍ ＮａＨＣＯ３（ｐＨ９．０）溶液で希釈
して１０ｍｇ／ｍＬとした。これに、Alexa Fluor 488 5-TFP ester （Thermo Fisher Scientific）１００μｇ／１００μＬを加えて室温にて１時間反応させた。反応液を遠心限外ろ過（Amicon Ultra 3K）に供し、未反応の蛍光色素を除去し、蛍光標識されたポリリ
ジンAlexa Fluor 488-PLL（ＡＦ−ＰＬＬ）を得た。得られたＡＦ−ＰＬＬは精製水で所
定の濃度に希釈して、以降の吸着実験に供した。
Example 1 Preparation of Fluorescently Labeled Polylysine 1
10 mg / mL of ε-L-polylysine (hereinafter referred to as “PLL”, Mn: 4090, Mw: 4700, 10% by mass aqueous solution, manufactured by JNC Co., Ltd.) diluted with 0.1 M NaHCO 3 (pH 9.0) solution And To this, 100 μg / 100 μL of Alexa Fluor 488 5-TFP ester (Thermo Fisher Scientific) was added and allowed to react at room temperature for 1 hour. The reaction solution was subjected to centrifugal ultrafiltration (Amicon Ultra 3K) to remove unreacted fluorescent dye to obtain fluorescently labeled polylysine Alexa Fluor 488-PLL (AF-PLL). The obtained AF-PLL was diluted to a predetermined concentration with purified water and subjected to the subsequent adsorption experiment.
＜実施例２＞角層細胞への吸着１
角質チェッカー（日本アッシュ）を用いたテープストリッピングにより、健常人上腕内側より最外層角層の角層細胞を非侵襲的に採取した。これにＡＦ−ＰＬＬ水溶液（５０μｇ／ｍＬ）を添加し、室温にて所定の時間インキュベートした。反応後、水洗した後に、角層に吸着したＡＦ−ＰＬＬを蛍光顕微鏡ＥＶＯＳ−ＦＬ（Thermo Fisher Scientific）により観察した。
反応時間ごとの染色結果を図３に示す。ＡＦ−ＰＬＬにより角層細胞が均一に染色された。また、反応時間に依存して蛍光強度が大きくなり吸着量が増加し、約６０分でほぼ飽和した。
Example 2 Adsorption to Corneal Cells 1
The stratum corneum cells of the outermost stratum corneum were noninvasively collected from the inside of the upper arm of a healthy subject by tape stripping using a horny checker (Nippon Ashe). To this was added AF-PLL aqueous solution (50 μg / mL) and incubated at room temperature for a predetermined time. After the reaction, after washing with water, AF-PLL adsorbed to the stratum corneum was observed with a fluorescence microscope EVOS-FL (Thermo Fisher Scientific).
The staining results for each reaction time are shown in FIG. The stratum corneum cells were uniformly stained by AF-PLL. Also, depending on the reaction time, the fluorescence intensity increased and the adsorption amount increased, and was almost saturated in about 60 minutes.
＜実施例３＞濃度依存性１
ＡＦ−ＰＬＬ水溶液の濃度を変えて（０〜１００μｇ／ｍＬ）、実施例２と同様に角層細胞を染色し（室温、２時間）、観察した。
結果を図４に示す。ＡＦ−ＰＬＬの濃度に依存して、蛍光強度の増大が認められた。
Example 3 Concentration Dependence 1
The concentration of the AF-PLL aqueous solution was changed (0 to 100 μg / mL), and the stratum corneum cells were stained as in Example 2 (room temperature, 2 hours) and observed.
The results are shown in FIG. Depending on the concentration of AF-PLL, an increase in fluorescence intensity was observed.
＜実施例４＞非標識ＰＬＬによる競合阻害１
ＡＦ−ＰＬＬと非標識ＰＬＬとを所定の割合で含有する水溶液（ＡＦ−ＰＬＬ濃度：５０μｇ／ｍＬ）を角層細胞に添加し、実施例２と同様に角層細胞を染色（室温、２時間）した後、観察した。
結果を図５に示す。ＡＦ−ＰＬＬの角層細胞への吸着は、非標識ＰＬＬにより競合的に阻害された。この結果から、蛍光検出により観察されるのがＰＬＬの角層細胞への吸着挙動であることが確認された。
Example 4 Competitive Inhibition by Unlabeled PLL 1
An aqueous solution (AF-PLL concentration: 50 μg / mL) containing AF-PLL and unlabeled PLL at a predetermined ratio is added to the stratum corneum cells, and the stratum corneum cells are stained as in Example 2 (room temperature, 2 hours) ) Was observed.
The results are shown in FIG. The adsorption of AF-PLL to stratum corneum cells was competitively inhibited by unlabeled PLL. From this result, it was confirmed that what is observed by fluorescence detection is the adsorption behavior of PLL on stratum corneum cells.
＜実施例５＞Ｌ−リジンモノマーの影響
ＡＦ−ＰＬＬとＬ−リジンとを所定の割合で含有する水溶液（ＡＦ−ＰＬＬ濃度：５０μｇ／ｍＬ）を角層細胞に添加し、実施例２と同様に角層細胞を染色（室温、２時間）した後、観察した。
結果を図６に示す。１００倍濃度のＬ−リジンモノマー共存下でもＡＦ−ＰＬＬの吸着
はほとんど阻害されなかった。
Example 5 Influence of L-Lysine Monomer As in Example 2, an aqueous solution (AF-PLL concentration: 50 μg / mL) containing AF-PLL and L-lysine in a predetermined ratio was added to the stratum corneum cells, as in Example 2. The stratum corneum cells were stained (room temperature, 2 hours) and then observed.
The results are shown in FIG. The adsorption of AF-PLL was hardly inhibited even in the presence of 100-fold concentration of L-lysine monomer.
＜実施例６＞ｐＨ依存性
種々の緩衝剤を用いてＡＦ−ＰＬＬ水溶液（５０μｇ／ｍＬ）のｐＨを２．５〜９．０に調整し、実施例２と同様に角層細胞を染色（室温、２時間）した後、観察した。
結果を図７に示す。酸性領域（ｐＨ２．５〜５．０）では蛍光が検出されず、ＡＦ−ＰＬＬの吸着は阻害されていた。一方、中性領域（ｐＨ６．０〜８．０)ではＡＦ−ＰＬＬ
の吸着は阻害されなかった。アルカリ性領域（ｐＨ９．０）では吸着は阻害される傾向であったが、用いる緩衝液により吸着挙動が異なっていた。
Example 6 pH Dependence The pH of the aqueous AF-PLL solution (50 μg / mL) was adjusted to 2.5 to 9.0 using various buffers, and the stratum corneum cells were stained as in Example 2 ( It observed after room temperature and 2 hours).
The results are shown in FIG. In the acidic region (pH 2.5 to 5.0), no fluorescence was detected, and the adsorption of AF-PLL was inhibited. On the other hand, AF-PLL in the neutral range (pH 6.0 to 8.0)
Adsorption was not inhibited. In the alkaline region (pH 9.0), adsorption tended to be inhibited, but the adsorption behavior was different depending on the buffer used.
＜実施例７＞塩濃度の影響
ＮａＣｌを０〜１．０ｍｏｌ／Ｌで含有するＡＦ−ＰＬＬ水溶液（５０μｇ／ｍＬ）を調製し、実施例２と同様に角層細胞を染色（室温、２時間）した後、観察した。
結果を図８に示す。０．１ｍｏｌ／Ｌ以上ではＮａＣｌ濃度に依存して蛍光強度の減弱が認められ、１ｍｏｌ／Ｌではほぼ完全に吸着が阻害された。
実施例６及び７の結果から、ポリリジンと角層細胞との間の相互作用の少なくとも一部は、ポリリジンのアミノ基とのイオン結合を介したものであると推測できる。
<Example 7> Influence of salt concentration AF-PLL aqueous solution (50 μg / mL) containing NaCl at 0 to 1.0 mol / L is prepared, and the stratum corneum cells are stained as in Example 2 (room temperature, 2 hours) ) Was observed.
The results are shown in FIG. At 0.1 mol / L or more, the fluorescence intensity was reduced depending on the NaCl concentration, and at 1 mol / L, the adsorption was almost completely inhibited.
From the results of Examples 6 and 7, it can be inferred that at least a part of the interaction between polylysine and stratum corneum cells is through an ionic bond with the amino group of polylysine.
＜実施例８＞カチオン化合物の影響
種々のカチオン性界面活性剤を含有するＡＦ−ＰＬＬ水溶液（５０μｇ／ｍＬ）を調製し、実施例２と同様に角層細胞を染色（室温、２時間）した後、観察した。
結果を表１並びに図９及び１０に示す。いずれのカチオン性界面活性剤においても、炭素鎖長が長くなるほど、蛍光強度の減弱が認められ、ポリリジンと角層細胞との相互作用が阻害されることが認められた。
<Example 8> Influence of cationic compound An AF-PLL aqueous solution (50 μg / mL) containing various cationic surfactants was prepared, and the stratum corneum cells were stained (room temperature, 2 hours) in the same manner as Example 2. I observed it later.
The results are shown in Table 1 and Figures 9 and 10. In any of the cationic surfactants, as the carbon chain length increased, the fluorescence intensity decreased, and it was found that the interaction between polylysine and stratum corneum cells was inhibited.
＜実施例９＞毛髪への吸着１
市販のテスト用毛束（ビューラックス）、および健常人より採取した毛髪を角質チェッカー（日本アッシュ）に瞬間接着剤で固定したのち、精製水またはＡＦ−ＰＬＬ水溶液（１００μｇ／ｍＬ）を添加し、室温にて２時間インキュベートした。反応後、水洗した後に、毛髪に吸着したＡＦ−ＰＬＬを蛍光顕微鏡ＥＶＯＳ−ＦＬ（Thermo Fisher Scientific）により観察した。
染色結果を図１１に示す。ＡＦ−ＰＬＬにより毛髪が均一に染色された。なお、毛髪には自家蛍光を示すものもあり、精製水においてもわずかに蛍光が観察された。
Example 9 Adsorption to Hair 1
After fixing a commercially available test hair bundle (Bureuxx) and hair collected from a healthy subject on a horniness checker (Nippon Ash) with a superglue, add purified water or an aqueous AF-PLL solution (100 μg / mL), Incubate for 2 hours at room temperature. After the reaction, after washing with water, AF-PLL adsorbed to the hair was observed with a fluorescence microscope EVOS-FL (Thermo Fisher Scientific).
The staining results are shown in FIG. Hair was uniformly dyed by AF-PLL. Some hairs showed auto-fluorescence, and a slight fluorescence was observed in purified water.
＜実施例１０＞濃度依存性２
ＡＦ−ＰＬＬ水溶液の濃度を変えて（０〜１００μｇ／ｍＬ）、実施例９と同様に毛髪を染色し（室温、２時間）、観察した。
結果を図１２に示す。ＡＦ−ＰＬＬの濃度に依存して、蛍光強度の増大が認められた。
Example 10 Concentration Dependence 2
The concentration of the aqueous AF-PLL solution was changed (0 to 100 μg / mL), and the hair was dyed as in Example 9 (room temperature, 2 hours) and observed.
The results are shown in FIG. Depending on the concentration of AF-PLL, an increase in fluorescence intensity was observed.
＜実施例１１＞非標識ＰＬＬによる競合阻害２
ＡＦ−ＰＬＬと非標識ＰＬＬとを所定の割合で含有する水溶液（ＡＦ−ＰＬＬ濃度：１００μｇ／ｍＬ）を毛髪に添加し、実施例９と同様に毛髪を染色（室温、２時間）した後、観察した。
結果を図１３に示す。ＡＦ−ＰＬＬの毛髪への吸着は、非標識ＰＬＬにより競合的に阻害された。この結果から、蛍光検出により観察されるのがＰＬＬの毛髪への吸着挙動であることが確認された。
Example 11 Competition inhibition by unlabeled PLL 2
An aqueous solution (AF-PLL concentration: 100 μg / mL) containing AF-PLL and unlabeled PLL in a predetermined ratio is added to the hair, and the hair is dyed (room temperature, 2 hours) as in Example 9, I observed it.
The results are shown in FIG. The adsorption of AF-PLL to hair was competitively inhibited by unlabeled PLL. From these results, it was confirmed that what is observed by fluorescence detection is the adsorption behavior of PLL on hair.
＜実施例１２＞ダメージ毛髪への吸着１
ダメージ毛髪は、市販のテスト用毛束をブリーチ処理して作製した。ブリーチ剤処理は、使用直前に１剤（アルカリ剤）と２剤（酸化剤）を混合して、テスト用毛束に塗布した。３０分程放置し、水洗した後、実施例９と同様に毛髪を染色（室温、２時間）した後、観察した。
結果を図１４に示す。ダメージ毛髪ではＡＦ−ＰＬＬにより蛍光輝度の顕著な上昇が認められた。また、ダメージ毛髪では自家蛍光の増加も観察された。
Example 12 Adsorption to Damaged Hair 1
Damaged hair was prepared by bleaching commercially available test hair bundles. The bleaching agent treatment mixed 1 agent (alkaline agent) and 2 agents (oxidizing agent) immediately before use, and applied to the test hair bundle. After leaving it to stand for about 30 minutes and washing with water, the hair was dyed (room temperature, 2 hours) in the same manner as in Example 9 and observed.
The results are shown in FIG. In the damaged hair, a marked increase in fluorescence intensity was observed by AF-PLL. In addition, an increase in autofluorescence was also observed in the damaged hair.
＜実施例１３＞微生物への吸着
サブロー平板培地にて２５℃／３日培養したSaccharomyces cerevisiae（NBRC10217）
を、滅菌綿棒を用いて、リン酸緩衝生理食塩水（ＰＢＳ）６ｍＬに懸濁し、〜１０７ ＣＦＵ／ｍＬの微生物懸濁液を得た。得られた微生物懸濁液を滅菌ピペットで適当量採取して、１．５ｍＬ容マイクロチューブ（Eppendorf製）に分注した後、１０ ｍｇ/ｍＬ Ａ
Ｆ−ＰＬＬを５０ μＬ加え（最終濃度が５００μg／ｍＬ）、トータル１ｍＬの反応液を調製した。比較対照のため、ＡＦ−ＰＬＬ水溶液に代えて同量の精製水を添加した試験区を調製した。これらを室温にて２時間インキュベートした後、遠心分離機（Eppendorf製
）にて遠心分離（５０００ｒｐｍ、１０分）して上清を除去した。沈殿の菌体に１ｍＬのＰＢＳを加えて懸濁し、再度遠心分離し上清液を除去した。これを更に２回繰り返して洗浄した後、微生物に吸着したＡＦ−ＰＬＬを蛍光顕微鏡ＥＶＯＳ−ＦＬ（Thermo Fisher Scientific）により観察した。
染色結果を図１５に示す。ＡＦ−ＰＬＬを作用させることにより、微生物の形に沿うように蛍光が検出され、ＡＦ−ＰＬＬにより微生物が染色されることが観察された。
<Example 13> Adsorption to microorganisms Saccharomyces cerevisiae (NBRC 10217) cultured at 25 ° C./3 days in subrow plate medium
Was suspended in 6 mL of phosphate buffered saline (PBS) using a sterile cotton swab to obtain a microbial suspension of ̃10 7 CFU / mL. Obtain an appropriate volume of the obtained microbial suspension with a sterile pipette, and dispense it into a 1.5 mL microtube (manufactured by Eppendorf), then 10 mg / mL A
50 μL of F-PLL was added (final concentration is 500 μg / mL) to prepare a total of 1 mL of reaction solution. As a control for comparison, a test section was prepared in which the same amount of purified water was added instead of the AF-PLL aqueous solution. After incubating these at room temperature for 2 hours, they were centrifuged (5000 rpm, 10 minutes) in a centrifuge (manufactured by Eppendorf) to remove the supernatant. The precipitated cells were suspended by adding 1 mL of PBS, centrifuged again, and the supernatant was removed. After washing this twice more, AF-PLL adsorbed to the microorganism was observed with a fluorescence microscope EVOS-FL (Thermo Fisher Scientific).
The staining results are shown in FIG. By causing AF-PLL to act, fluorescence was detected along the shape of the microorganism, and it was observed that the microorganism was stained by AF-PLL.
＜実施例１４＞非標識ＰＬＬによる競合阻害３
実施例１３と同様の方法で〜１０７ＣＦＵ/ｍＬに調製した微生物懸濁液を９５０μＬ
取り、１０ ｍｇ/ｍＬのＡＦ−ＰＬＬを１０ μＬ（最終濃度１００μｇ/ｍＬ）と２５０
ｍｇ/ｍＬの非標識ＰＬＬを４０ μＬ（最終濃度１０ ｍｇ/ｍＬ）それぞれ加え（標識
ポリリジン：非標識ポリリジン＝１：１００）、実施例１３と同様に微生物を染色（室温、２時間）した後、観察した。
結果を図１６に示す。ＡＦ−ＰＬＬの微生物への吸着は、非標識ＰＬＬにより競合的に阻害された。この結果から、蛍光検出により観察されるのがＰＬＬの微生物への吸着挙動であることが確認された。
Example 14 Competitive Inhibition by Unlabeled PLL 3
950 μL of the microbial suspension prepared to ̃10 7 CFU / mL in the same manner as in Example 13.
Take 10 μL of 10 mg / mL AF-PLL (final concentration 100 μg / mL) and 250
After adding 40 μL of unlabeled PLL (mg / mL final concentration 10 mg / mL) (labeled polylysine: unlabeled polylysine = 1: 100) and staining the microorganism as in Example 13 (room temperature, 2 hours) , Observed.
The results are shown in FIG. The adsorption of AF-PLL to microorganisms was competitively inhibited by unlabeled PLL. From this result, it was confirmed that what is observed by fluorescence detection is the adsorption behavior of PLL to the microorganism.
＜実施例１５＞蛍光標識ポリリジンの調製２
蛍光色素化合物をNHS-Rhodamineとした以外は実施例１と同様の処理を行い、蛍光標識
されたポリリジンRhodamine-PLL（Ｒｈｏ−ＰＬＬ）を得た。得られたＲｈｏ−ＰＬＬは
精製水で所定の濃度に希釈して、以降の吸着実験に供した。
Example 15 Preparation of Fluorescently Labeled Polylysine 2
The same treatment as in Example 1 was carried out except that the fluorescent dye compound was changed to NHS-Rhodamine, to obtain fluorescently labeled polylysine Rhodamine-PLL (Rho-PLL). The obtained Rho-PLL was diluted to a predetermined concentration with purified water and subjected to the subsequent adsorption experiment.
＜実施例１６＞角層細胞への吸着２
蛍光標識されたポリリジンをＲｈｏ−ＰＬＬ水溶液（濃度０〜２００μｇ／ｍＬ）とした以外は、実施例２と同様に角層細胞を染色（室温、２時間）した。反応後、水洗した後に、角層に吸着したＲｈｏ−ＰＬＬを、蛍光顕微鏡ＥＶＯＳ−ＦＬ（ＲＦＰフィルター装着）により観察した。
結果を図１７に示す。前述したＡＦ−ＰＬＬ同様に、Ｒｈｏ−ＰＬＬの濃度に依存して蛍光強度の増大が認められた。
Example 16 Adsorption to Corneal Cells 2
The stratum corneum cells were stained (room temperature, 2 hours) in the same manner as in Example 2 except that the fluorescently labeled polylysine was changed to an aqueous solution of Rho-PLL (concentration 0 to 200 μg / mL). After the reaction, after washing with water, Rho-PLL adsorbed to the stratum corneum was observed with a fluorescence microscope EVOS-FL (equipped with an RFP filter).
The results are shown in FIG. Similar to the above-mentioned AF-PLL, an increase in fluorescence intensity was observed depending on the concentration of Rho-PLL.
＜実施例１７＞非標識ＰＬＬによる競合阻害４
Ｒｈｏ−ＰＬＬと非標識ＰＬＬとを所定の割合で含有する水溶液（Ｒｈｏ−ＰＬＬ濃度：２０μｇ／ｍＬ、および２００μｇ／ｍＬ）を角層細胞に添加し、実施例２と同様に角層細胞を染色（室温、２時間）した後、蛍光顕微鏡ＥＶＯＳ−ＦＬ（ＲＦＰフィルター装着）により観察した。
結果を図１８に示す。前述したＡＦ−ＰＬＬ同様に、Ｒｈｏ−ＰＬＬの角層細胞への吸着は、非標識ＰＬＬにより競合的に阻害された。この結果から、蛍光検出により観察されるのがＰＬＬの角層細胞への吸着挙動であることが確認された。
Example 17 Competition Inhibition by Unlabeled PLL 4
An aqueous solution (Rho-PLL concentration: 20 μg / mL and 200 μg / mL) containing Rho-PLL and unlabeled PLL in a predetermined ratio is added to the stratum corneum cells, and the stratum corneum cells are stained as in Example 2. After (room temperature, 2 hours), it observed by fluorescence microscope EVOS-FL (RFP filter installation).
The results are shown in FIG. Similar to the above-mentioned AF-PLL, the adsorption of Rho-PLL to stratum corneum cells was competitively inhibited by unlabeled PLL. From this result, it was confirmed that what is observed by fluorescence detection is the adsorption behavior of PLL on stratum corneum cells.
＜実施例１８＞ダメージ毛髪への吸着２
蛍光標識されたポリリジンをＲｈｏ−ＰＬＬ水溶液（Ｒｈｏ−ＰＬＬ濃度；１００μｇ／ｍＬ）とした以外は、実施例１２と同様に毛髪を染色（室温、２時間）した後、蛍光顕微鏡ＥＶＯＳ−ＦＬ（ＲＦＰフィルター装着）により観察した。
結果を図１９に示す。ダメージ毛髪では、前述したＡＦ−ＰＬＬ同様に、Ｒｈｏ−ＰＬＬにより蛍光輝度の顕著な上昇が認められた。しかしながら、Ｒｈｏ−ＰＬＬではＡＦ−ＰＬＬとは異なり、毛髪自身の自家蛍光はほとんど観察されず安定した観察が可能であった。
Example 18 Adsorption to Damaged Hair 2
A hair was stained (room temperature, 2 hours) in the same manner as in Example 12 except that the fluorescently labeled polylysine was changed to an aqueous Rho-PLL solution (Rho-PLL concentration; 100 μg / mL), and then a fluorescence microscope EVOS-FL (RFP It observed by the filter installation.
The results are shown in FIG. In damaged hair, as in the above-described AF-PLL, a marked increase in fluorescence intensity was observed by Rho-PLL. However, unlike Rho-PLL, unlike the AF-PLL, almost no auto-fluorescence of the hair itself was observed and stable observation was possible.
本発明の蛍光標識ポリリジンを用いれば、煩雑な染色操作や高価な分析機器を用いることなく、簡便な操作で精度よく、角層細胞を始めとする生体細胞や毛髪等の生体組織、微生物、天然繊維や化学繊維及び合成繊維等の繊維素材、天然樹脂や合成樹脂等の工業素材の状態を、観察することができるため、非常に有用である。 When the fluorescently labeled polylysine of the present invention is used, biological tissues such as keratinocytes and living tissues such as hair, microorganisms, and natural tissues can be accurately and easily by simple operations without using complicated staining procedures and expensive analytical instruments. It is very useful because it can observe the state of fiber materials such as fibers, chemical fibers and synthetic fibers, and industrial materials such as natural resins and synthetic resins.
Claims (10)
請求項１に記載の蛍光標識ポリリジン。 The fluorescent dye compound is 3,6-diamino-9- [2,4-bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium / 3,6-diamino-9-. [2,5-Bis (lithiooxycarbonyl) phenyl] -4- (lithiooxysulfonyl) -5-sulfonatoxanthylium or a derivative thereof,
The fluorescently labeled polylysine according to claim 1.
ノ）−キサンチリウム インナーソルト又はその誘導体である、請求項１に記載の蛍光標
識ポリリジン。 The fluorescent dye compound is 9- [2-carboxy-4 (or 5)-[[(2,5-dioxo-1-pyrrolidinyl) oxy] -carbonyl] phenyl] -3,6-bis- (dimethylamino)- The fluorescently labeled polylysine according to claim 1, which is a xanthylium inner salt or a derivative thereof.
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