JP4303616B2 - Detection method of melanosome transfer - Google Patents
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本発明は、メラノサイトからケラチノサイトへのメラニン顆粒であるメラノソームの転送を簡便かつ正確に検出する方法に関する。 The present invention relates to a method for easily and accurately detecting the transfer of melanosomes, which are melanin granules, from melanocytes to keratinocytes.
紫外線を受けた皮膚が黒化するのは表皮中のメラノサイト中のメラノソームという細胞内小器官（オルガネラ）でチロシナーゼの作用等によりメラニンが生成することにはじまり、メラノソームが表皮の大部分を占めるケラチノサイトに転送され、当該ケラチノサイトに貯蔵されることによる。従来から美白剤としては、メラニンの生成を抑制するためのチロシナーゼ阻害剤が最も多い。 The skin exposed to ultraviolet rays becomes dark, starting with the formation of melanin by the action of tyrosinase, etc., in the melanocytes in the melanocytes in the epidermis, and the melanosomes occupy most of the epidermis. By being transferred and stored in the keratinocytes. Conventionally, as a whitening agent, there are most tyrosinase inhibitors for suppressing the production of melanin.
一方、メラノサイト中のメラニン生成器官であるメラノソームのケラチノサイトへの転送が制御できれば、有効な美白剤になり得ると考えられる。このような、メラノソーム転送阻害剤を探索するには、メラノソーム転送を測定する手段が必要であり、当該手段としては、ＣＦＤＡ等の蛍光物質存在下でメラノサイトを一定時間培養することによってメラノソームを含む全てのオルガネラを蛍光標識した後、ケラチノサイトとの共培養を行い、ケラチノサイト特異抗体とＣＦＤＡの両方の標識が得られた細胞の割合を求める方法（非特許文献１）、メラノサイト及びケラチノサイトを共培養した細胞又は組織切片を電子顕微鏡を用いて観察し、ケラチノサイト内のメラノソームを数える方法（非特許文献１）、ＨａＣａＴ細胞に単離したメラノソームを添加し、取り込まれたメラノソームをフォンタナ・マッソン染色後、画像解析により定量する方法（非特許文献２）が知られている。 On the other hand, if the transfer of melanosomes, which are melanin producing organs in melanocytes, to keratinocytes can be controlled, it is considered that they can be effective whitening agents. In order to search for such a melanosome transfer inhibitor, a means for measuring melanosome transfer is required, and as such means, all melanosomes including melanosomes are cultured by culturing melanocytes for a certain period of time in the presence of a fluorescent substance such as CFDA. A method of obtaining the ratio of cells from which both keratinocyte-specific antibody and CFDA labels were obtained (Non-patent Document 1), cells co-cultured with melanocytes and keratinocytes Alternatively, a tissue section is observed using an electron microscope, and a method of counting melanosomes in keratinocytes (Non-patent Document 1), adding isolated melanosomes to HaCaT cells, and analyzing the captured melanosomes after Fontana-Masson staining. There is known a method of quantifying by the above (Non-Patent Document 2).
しかしながら、非特許文献１の方法では、ＣＦＤＡによる標識はメラノソームに特異的なものではなく、正確な測定は不可能であった。また、非特許文献１の方法では、電子顕微鏡下にメラノソームを数える方法であり、非常に煩雑であり、多数のサンプルの評価は不可能である。また非特許文献２の方法では、単離したメラノソームを直接添加するため、エンドサイト−シス等による転送が検出できず、正確な評価はできない。
本発明の目的は、正確かつ簡便にメラノサイトからケラチノサイトへのメラノソームの転送を検出する方法及びこの方法を用いたメラノソーム転送阻害剤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 method for detecting melanosome transfer from melanocytes to keratinocytes accurately and simply, and a screening method for melanosome transfer inhibitors using this method.
そこで本発明者は、種々検討した結果、メラノサイトとケラチノサイトを共培養し、メラノソーム特異体とケラチノサイト特異抗体を用いて二重染色することにより、メラノサイトからケラチノサイト中に転送されたメラノソームが選択的に検出できること、さらにこの検出系を用いればメラノソーム転送阻害剤のスクリーニングが可能になることを見出した。
すなわち、本発明は、メラノサイト及びケラチノサイトを共培養し、当該培養細胞と、同時に検出可能で、かつ相互に異なる色に染色できる標識でそれぞれ標識されたメラノソーム特異抗体及びケラチノサイト特異抗体との結合体を得、二重に染色された画像を解析することを特徴とするメラノサイトからケラチノサイトへのメラノソーム転送の検出法を提供するものである。
また本発明は、上記メラノソーム転送の検出系を用い、被験物質によるメラノソーム転送量の変化を検出することを特徴とするメラノソーム転送阻害剤のスクリーニング法を提供するものである。
Therefore, as a result of various studies, the inventor selectively detects melanosomes transferred from melanocytes into keratinocytes by co-culturing melanocytes and keratinocytes and double-staining using melanosome-specific and keratinocyte-specific antibodies. It was also found that screening of melanosome transfer inhibitors becomes possible by using this detection system.
That is, the present invention provides a conjugate of a melanocyte-specific antibody and a keratinocyte-specific antibody that are co-cultured with melanocytes and keratinocytes, and labeled with a label that can be simultaneously detected and stained in different colors. The present invention provides a method for detecting melanosome transfer from melanocytes to keratinocytes, characterized by analyzing double-stained images.
The present invention also provides a screening method for a melanosome transfer inhibitor, characterized in that a change in the amount of melanosome transfer caused by a test substance is detected using the melanosome transfer detection system.
本発明の検出法によれば、簡便な操作により、正確にメラノサイトからケラチノサイトへのメラノソーム転送量を測定できる。また、大量かつ正確にメラノソーム転送阻害剤のスクリーニングが可能となる。 According to the detection method of the present invention, the amount of melanosome transfer from melanocytes to keratinocytes can be accurately measured by a simple operation. In addition, melanosome transfer inhibitors can be screened in large quantities and accurately.
本発明のメラノソーム転送検出法においては、まず、メラノサイト及びケラチノサイトを共培養する。メラノサイトとしては、ヒトメラノサイトが好ましく、さらにヒト正常メラノサイトがより好ましい。このようなヒトメラノサイトとしては、例えばクラボウ社、ＣＡＭＢＲＥＸ社から市販されているものを用いることができる。ケラチノサイトとしては、ヒトケラチノサイト、さらにヒト正常ケラチノサイト、特にメラノソームを有さないヒト正常ケラチノサイトが好ましい。このようなケラチノサイトとしては、例えばクラボウ社、ＣＡＭＢＲＥＸ社から市販されているものを用いることができる。 In the melanosome transfer detection method of the present invention, first, melanocytes and keratinocytes are co-cultured. As the melanocytes, human melanocytes are preferable, and human normal melanocytes are more preferable. As such human melanocytes, for example, those commercially available from Kurabo Industries Co., Ltd. and CAMBREX Corporation can be used. As the keratinocytes, human keratinocytes, human normal keratinocytes, particularly human normal keratinocytes having no melanosomes are preferable. As such keratinocytes, for example, those commercially available from Kurabo Industries Co., Ltd. and CAMBREX Corporation can be used.
メラノサイトとケラチノサイトの共培養は、常法に従って行うことができる。例えば、それぞれ継代培養したメラノサイトとケラチノサイトを、両細胞の培養に適した培地中で培養すればよい。ここで、メラノサイトの単独培養に適する培地としては、Ｍｅｄｉｕｍ１５４Ｓ（Ｃａｓｃａｄｅ Ｂｉｏｌｏｇｉｃｓ社）にＨＭＧＳ（Ｃａｓｃａｄｅ Ｂｉｏｌｏｇｉｃｓ社）を加えたもの等が挙げられる。一方、ケラチノサイトの単独培養に適する培地としては、Ｋｅｒａｔｉｎｏｃｙｔｅ−ＳＦＭ（ＧＩＢＣＯ社）にＳｕｐｐｌｅｍｅｎｔｓ Ｆｏｒ Ｋｅｒａｔｉｎｏｃｙｔｅ−ＳＦＭ（ＧＩＢＣＯ社）を加えたもの等が挙げられる。メラノサイトとケラチノサイトの共培養に適する培地としては、Ｋｅｒａｔｉｎｏｃｙｔｅ−ＳＦＭ（ＧＩＢＣＯ社）Ｓｕｐｐｌｅｍｅｎｔなし、とＭｅｄｉｕｍ１５４Ｓ（Ｃａｓｃａｄｅ Ｂｉｏｌｏｇｉｃｓ社）に特注添加剤ＨＭＧＳ（Ｃａｓｃａｄｅ Ｂｉｏｌｏｇｉｃｓ社）からホルボールエステル（ＰＭＡ）を除く全ての添加剤を加えた培地、を２：１の比率で混合したもの等が挙げられる。共培養の条件としては、ケラチノサイトをケラチノサイトの単独培養に適する培地条件下で播種した２４時間後に、ケラチノサイトの半量のメラノサイトを重ねて播種し、メラノサイトとケラチノサイトの共培養に適する培地に置き換えて４〜７日間培養するのが好ましい。 Co-culture of melanocytes and keratinocytes can be performed according to a conventional method. For example, the subcultured melanocytes and keratinocytes may be cultured in a medium suitable for culturing both cells. Here, examples of the medium suitable for culturing melanocytes include those obtained by adding HMGS (Cascade Biology) to Medium154S (Cascade Biology). On the other hand, examples of the medium suitable for cultivating keratinocytes include Keratinocyte-SFM (GIBCO) plus Supplements For Keratinocyte-SFM (GIBCO). As a medium suitable for co-culture of melanocytes and keratinocytes, there is no Keratinocyte-SFM (GIBCO) Supplement, and Medium 154S (Cascade Biologicals) except for phorbol ester (PMA from Cascade Biologics) excluding HMGS (Cascade Biologics). What mixed the culture medium which added the additive by the ratio of 2: 1 etc. is mentioned. As the conditions for co-culture, 24 hours after seeding keratinocytes under a medium condition suitable for culturing keratinocytes alone, half the amount of melanocytes of keratinocytes is seeded in an overlapping manner, and replaced with a medium suitable for co-culture of melanocytes and keratinocytes. It is preferable to culture for 7 days.
次に、共培養された細胞と、同時に検出可能で、かつ相互に異なる色に染色できる標識でそれぞれ標識されたメラノソーム特異抗体及びケラチノサイト特異抗体との結合体を得る。ここで、当該結合体は、培養細胞とメラノソーム特異抗体及びケラチノサイト特異抗体とを反応させた後、それぞれ前記の標識をする方法、又は培養細胞と予めそれぞれ前記の標識をされたメラノソーム特異抗体及びケラチノサイト特異抗体とを反応させる方法により得ることができる。 Next, a conjugate of the co-cultured cells and a melanosome-specific antibody and a keratinocyte-specific antibody, each labeled with a label that can be detected simultaneously and stained in different colors, is obtained. Here, the conjugate is a method of reacting a cultured cell with a melanosome-specific antibody and a keratinocyte-specific antibody, and then performing the labeling, respectively, or a cultured cell and a melanosome-specific antibody and a keratinocyte previously labeled respectively. It can be obtained by a method of reacting with a specific antibody.
メラノソーム特異抗体としては、メラノソームに特異的に反応する抗体であれば特に制限されず、ポリクローナル抗体であってもモノクローナル抗体であってもよい。このうち、ヒトメラノソームに特異的な抗体が好ましい。ただし、ヒトメラノソームに特異的な抗体は、マウスやウサギ等のヒト以外の動物由来であってもよい。このようなメラノソーム特異抗体としては、例えばマウス抗ヒトメラノソーム抗体 抗ｇｐ１００抗体、抗チロシナーゼ抗体、抗ＴＲＰ２抗体、抗ＴＲＰ１抗体等が挙げられる。中でも、マウス抗ヒトメラノソーム抗体が好ましく、抗ｇｐ１００抗体が特に好ましい。 The melanosome-specific antibody is not particularly limited as long as it is an antibody that specifically reacts with melanosome, and may be a polyclonal antibody or a monoclonal antibody. Of these, antibodies specific for human melanosomes are preferred. However, the antibody specific for human melanosomes may be derived from animals other than humans such as mice and rabbits. Examples of such melanosome-specific antibodies include mouse anti-human melanosome antibody, anti-gp100 antibody, anti-tyrosinase antibody, anti-TRP2 antibody, and anti-TRP1 antibody. Among these, mouse anti-human melanosome antibody is preferable, and anti-gp100 antibody is particularly preferable.
ケラチノサイト標識抗体としては、ケラチノサイトを強く認識する抗体であれば特に制限されず、ポリクローナル抗体であっても、モノクローナル抗体であってもよい。またケラチノサイトに特異的な抗体は、ケラチノサイトには多く発現するがメラノサイト及びメラノソームにはわずかしか発現しない、または発現しない物質を認識する抗体であればよい。具体的には抗Ｅ−カドヘリン抗体、サイトケラチン、Ｋｅｒａｔｉｎ−１４等が挙げられる。 The keratinocyte labeled antibody is not particularly limited as long as it is an antibody that strongly recognizes keratinocytes, and may be a polyclonal antibody or a monoclonal antibody. The antibody specific for keratinocytes may be an antibody that recognizes a substance that is highly expressed in keratinocytes but is scarcely expressed in melanocytes and melanosomes. Specific examples include anti-E-cadherin antibody, cytokeratin, keratin-14, and the like.
標識は、同時に検出可能で、かつ相互に異なる色に染色できる２種の標識であればよく、例えば相互に異なる色を生じる蛍光標識、化学発光、酵素反応による発色等が挙げられ、このうち蛍光標識が検出のしやすさ、二重に染色した場合にそれぞれの色と重なった色の分離性、検出精度等の点で好ましい。蛍光標識の例としては、Ｆｌｕｏｒｅｓｃｅｉｎ（ＦＩＴＣ）、ローダミン（ＴＲＩＴＣ）があり、これらを２種組み合せて使用するのが好ましい。 The label only needs to be two types of labels that can be detected at the same time and can be stained in different colors. Examples include fluorescent labels that produce different colors, chemiluminescence, and color development by enzymatic reaction. The label is preferable in terms of ease of detection, separability of colors that overlap each color when double staining, detection accuracy, and the like. Examples of fluorescent labels include Fluorescein (FITC) and Rhodamine (TRITC), and these are preferably used in combination.
このようにして二重染色すると、メラノソームとケラチノサイトがそれぞれ異なる色に染色される。そのうち、ケラチノサイトの染色とメラノソームの染色の重複した部分が、メラノサイトから転送されたメラノソームである。従って、この二重に染色された部分の色の量、例えば蛍光量を測定するか、数、面積等を画像解析すれば、メラノサイトからケラチノサイトへ転送されたメラノソームのみが選択的に検出、定量できる。
蛍光量の測定は常法により行うことができる。例えば、フローサイトメーターを用いて蛍光強度を数値化することにより測定できる。
画像解析も常法により行うことができる。例えば、Image Pro Plusを用い、画像をＲＧＢ色成分に分解した後、メラノソームを標識した蛍光に対応する特定の色成分のみをグレースケールにて表示し、自動抽出によってメラノソーム領域を抽出することにより測定できる。
When double staining is performed in this manner, melanosomes and keratinocytes are stained in different colors. Among them, the overlapping part of keratinocyte staining and melanosome staining is melanosomes transferred from melanocytes. Therefore, by measuring the amount of color of this double-stained portion, for example, the amount of fluorescence, or analyzing the number, area, etc., only melanosomes transferred from melanocytes to keratinocytes can be selectively detected and quantified. .
The amount of fluorescence can be measured by a conventional method. For example, it can be measured by digitizing the fluorescence intensity using a flow cytometer.
Image analysis can also be performed by conventional methods. For example, using Image Pro Plus, after decomposing an image into RGB color components, display only specific color components corresponding to fluorescence labeled with melanosomes in grayscale, and measure by extracting the melanosome region by automatic extraction it can.
このメラノソーム転送検出系を用いれば、メラノソーム転送阻害剤のスクリーニングが可能である。例えばメラノサイトとケラチノサイトの共培養時に被験物質を添加してメラノソーム転送量を測定し、被験物質非添加の場合と対比すれば、当該被験物質がメラノソーム転送を阻害するか否かが判定できる。被験物質の添加は、メラノサイト又はケラチノサイトの単独培養の時点でもよい。 If this melanosome transfer detection system is used, screening for melanosome transfer inhibitors is possible. For example, when a test substance is added at the time of co-culture of melanocytes and keratinocytes and the amount of melanosome transfer is measured and compared with the case where no test substance is added, it can be determined whether or not the test substance inhibits melanosome transfer. The addition of the test substance may be performed at the time of culturing melanocytes or keratinocytes alone.
実施例１
（１）市販のヒト正常ケラチノサイト(NHEK(F)、クラボウ社)を４０００cells/cm2で播種・培養した。セミコンフルエントの状態でダルベッコのリン酸緩衝液(D-PBS, Invitrogen社)で２回洗浄の後、０．２５％トリプシン−EDTA(Invitrogen社)処理によって回収し、７０００cells/cm2の密度で継代する。
（２）継代した細胞をセミコンフルエントの状態でトリプシン処理によって回収し、２００００cells/cm2の密度で播種・培養した。ケラチノサイト単独の培養にはK-SFM(Invitrogen社)にサプリメント(Invitrogen社)を加えた培地を用いた。
（３）ケラチノサイト播種２４時間後、３継代培養したヒト正常メラノサイト(NHEM(F)、クラボウ社)を１００００cells/cm2の密度で添加し、Medium１５４(クラボウ社)にホルボールエステル（PMA）を除くHMGS(クラボウ社)を添加したメラノサイト至適培地と、サプリメントを除いたK-SFMを１：２の比率で混合した培地を用いて４日間培養した。転送阻害のポジティブコントロールとしては、０．１％大豆トリプシンインヒビター(STI)、１mMナイアシンアミドを用い、それぞれ２４時間毎に１回、１２時間毎に１回添加した。
（４）４日間培養した後、リン酸緩衝液（PBS）で２回洗浄を行ない、４％パラホルムアルデヒド（PFA）/PBSを用いて４℃で１０分間処理することによって細胞を固定し、その後PBSで数回洗浄してPFAを完全に除去した。
（５）３％BSA/PBSを添加し１時間室温にて振盪した後、マウス抗ヒトgp100抗体HMB45(DAKO社)、ウサギ抗ヒトＥ−カドヘリン抗体(Santa Cruz Biotechnology社)を添加し、４℃で一昼夜振盪を行なった。ネガティブコントロールとしてはマウスユニバーサルネガティブコントロール(DAKO社)、ウサギIgG(Sigma社)を等濃度で加えたものを用いた。
（６）０．３％BSA/PBSで３回洗浄し、さらに３％BSA/PBSを添加して１時間室温にて振盪した後、ヤギ抗ウマスIgG FITC標識（CHEMICON社）、ブタ抗ウサギIgs TRITC標識（DAKO社）を添加し、室温にて１時間振盪した。
（７）０．３％BSA/PBSで３回洗浄後、核を染色する目的で３００nM４′，６−ジアミジノ−２−フェニルインドール（DAPI）/PBS(Molecular Probes社)を添加して更に１０分間振盪した。PBSで洗浄した後、蛍光顕微鏡にて観察及びデジタル画像の撮影を行なった（図１）。
（８）撮影した画像を用い、E-cadherin抗体によって赤く染色されるケラチノサイト中に認められるHMB45抗体陽性所見（黄色）の数及び面積を、画像解析ソフトであるImage Pro Plus4.5（MEDIA CYBERNETICS社）を用いて解析し、これをメラニン転送量とした。
PBSを添加したサンプルをコントロールとし、そのメラニン転送量を１００としたとき、メラノソーム転送阻害剤剤であるナイアシンアミドを添加すると、ケラチノサイト１細胞あたりにして約４０％、単位面積あたりで２０％の転送阻害効果が認められた。
Example 1
(1) Commercially available human normal keratinocytes (NHEK (F), Kurabo Industries) were seeded and cultured at 4000 cells / cm 2 . After washing twice with Dulbecco's phosphate buffer (D-PBS, Invitrogen) in a semi-confluent state, the cells were recovered by treatment with 0.25% trypsin-EDTA (Invitrogen) and passaged at a density of 7000 cells / cm 2. To pay.
(2) The passaged cells were collected by trypsin treatment in a semi-confluent state, and seeded and cultured at a density of 20000 cells / cm 2 . For the culture of keratinocytes alone, a medium in which supplement (Invitrogen) was added to K-SFM (Invitrogen) was used.
(3) 24 hours after seeding with keratinocytes, human normal melanocytes (NHEM (F), Kurabo) were added for 3 passages at a density of 10000 cells / cm 2 , and phorbol ester (PMA) was added to Medium 154 (Kurabo). Culture was performed for 4 days using a melanocyte optimal medium to which HMGS (Kurabo Co., Ltd.) was added and a medium in which K-SFM excluding supplements was mixed at a ratio of 1: 2. As a positive control for inhibition of transfer, 0.1% soybean trypsin inhibitor (STI) and 1 mM niacinamide were added once every 24 hours and once every 12 hours.
(4) After culturing for 4 days, the cells were fixed by washing twice with phosphate buffer (PBS) and treating with 4% paraformaldehyde (PFA) / PBS at 4 ° C. for 10 minutes. PFA was completely removed by washing several times with PBS.
(5) After 3% BSA / PBS was added and shaken for 1 hour at room temperature, mouse anti-human gp100 antibody HMB45 (DAKO) and rabbit anti-human E-cadherin antibody (Santa Cruz Biotechnology) were added and 4 ° C. And shaken all day and night. As negative controls, mouse universal negative control (DAKO) and rabbit IgG (Sigma) added at equal concentrations were used.
(6) After washing 3 times with 0.3% BSA / PBS, adding 3% BSA / PBS and shaking for 1 hour at room temperature, goat anti-mass IgG FITC label (CHEMICON), pig anti-rabbit Igs TRITC label (DAKO) was added and shaken at room temperature for 1 hour.
(7) After washing three times with 0.3% BSA / PBS, 300 nM 4 ′, 6-diamidino-2-phenylindole (DAPI) / PBS (Molecular Probes) was added for 10 minutes for the purpose of staining the nucleus. Shake. After washing with PBS, observation and digital image photography were performed with a fluorescence microscope (FIG. 1).
(8) The number and area of HMB45 antibody positive findings (yellow) found in keratinocytes stained red with E-cadherin antibody using the captured images, and the image analysis software Image Pro Plus4.5 (MEDIA CYBERNETICS) ) Was used as the melanin transfer amount.
When the control sample is PBS and the amount of melanin transfer is 100, the addition of niacinamide, a melanosome transfer inhibitor, transfers about 40% per keratinocyte cell and 20% per unit area. An inhibitory effect was observed.
比較例１
実施例においてマウス抗ヒトメラノソーム抗体HMB45(DAKO社)を抗チロシナーゼ抗体であるＭａｂ−１に変えた以外は同じ操作をした。ケラチノサイトに転送されたメラノソームは検出されない。
Comparative Example 1
In the Examples, the same operation was performed except that the mouse anti-human melanosome antibody HMB45 (DAKO) was changed to Mab-1, which is an anti-tyrosinase antibody. Melanosomes transferred to keratinocytes are not detected.
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