ホ౯఍㆟ሗ࿌᭩
Reactive oxygen species (ROS)࢔ࢵࢭ࢖

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧11 ᭶ 30 ᪥

JaCVAM ホ౯఍㆟

11

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼஭㞝஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥22

Reactive oxygen species (ROS)࢔ࢵࢭ࢖ࡣࠊ໬Ꮫ≀㉁ࡢග཯ᛂᛶࢆ᳨ฟࡍࡿヨ㦂ἲ࡛࠶ࡿࠋග᭚㟢࡟ࡼࡾ⏕ࡌࡿ໬Ꮫ≀㉁ࡢẘᛶࡢከࡃࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫ཯ᛂ࡟క࠺⣽⬊ࡸ⤌⧊ࡢ㓟໬ⓗയᐖࡀⓎ⌧ᶵᗎ࡜ࡉࢀࠊගບ㉳ࡉࢀࡓ໬Ꮫ≀㉁ࡣ୍㔜㡯㓟⣲ࡸࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥ➼ࡢ ROS ࢆ⏘⏕ࡍࡿࠋROS ࢔ࢵࢭ࢖ࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࡓ࡜ࡁࡢ
ROS ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿࡇ࡜࡟ࡼࡾࠊග཯ᛂᛶࢆணࡍࡿࠋ

ROS ࢔ࢵࢭ࢖ࡣࠊJaCVAM ༠ຊࡢୗ࡛ከ᪋タࣂࣜࢹ࣮ࢩࣙࣥࡀᐇ᪋ࡉࢀ 1)ࠊOECD ࡀᐃࡵࡿᅜ㝿ⓗᇶ‽(2005 ᖺ) 2)࡟ᚑࡗ࡚ࠊᑓ㛛ᐙ࠿ࡽ࡞ࡿ⊂❧ࡋࡓ➨୕⪅ホ౯ጤဨ఍࡟ࡼࡾࠊ⛉Ꮫⓗ࡞ホ౯ࡀ⾜ࢃࢀࡓ 3)ࠋJaCVAM ホ౯఍㆟ࡣࠊࡇࢀࢆࡶ࡜࡟ࡋ࡚ JaCVAM ගẘᛶ㈨ᩱ⦅
⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓ ROS ࢔ࢵࢭ࢖ホ౯ሗ࿌᭩㸦2015 ᖺ㸵᭶ 8 ᪥㸧4)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 Reactive oxygen species (ROS)࢔ࢵࢭ࢖
௦ ᭰ ࡍ ࡿ ᑐ ㇟ ẘ ᛶ ヨ 㦂 㸸  In vivo ࡢ ග ẘ ᛶ ヨ 㦂 5) ࠾ ࡼ ࡧ Neutral Red UptakePhototoxicity Test㸦௨ୗࠊ3T3 NRU PT ࡜␎ࡍ )6)ヨ㦂ヨ㦂ἲࡢᴫ␎㸸  ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࠊROS ୰ࡢ୍㔜㡯㓟⣲࡜ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ 2 ✀ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿヨ㦂࡛࠶ࡿࠋ୍㔜㡯㓟⣲ࡣ pnitrosodimethylaniline ࡢኚⰍࢆᣦᶆ࡜ࡋ࡚ࡑࡢ⏘⏕ࢆホ౯ࡍࡿࠋࡍ࡞ࢃࡕ㸪⿕㦂≀㉁ࡀග↷ᑕࢆཷࡅ࡚ບ㉳໬ྜ≀࡜࡞ࡾࠊࡇࢀ࠿ࡽ⏘⏕ࡉࢀࡓ୍㔜㡯㓟⣲ࡀ཯ᛂᾮ୰ࡢ imidazole ࡜཯ᛂࡋ࡚཯ᛂ୰㛫యࢆᙧᡂࡍࡿࠋࡇࢀࡀ p-nitrosodimethylaniline ࢆ㓟໬ࡍࡿࡇ࡜࡛ኚⰍࢆ
ࡶࡓࡽࡋ㸪440 nm ࡢ྾ග್ࡀῶᑡࡍࡿࠋࡇࡢ྾ග್ࡢῶᑡࢆ୍㔜㡯㓟⣲ࡢ⏕ᡂ࡜ࡋ࡚ᐃࡍࡿࠋsinglet oxygen + imidazole Ѝ [peroxide intermediate] Ѝ oxidized imidazole[peroxide intermediate] + p-nitrosodimethylaniline Ѝ oxidized p-nitrosodimethylaniline +productsࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ⏘⏕ࡣࠊnitroblue tetrazolium (NBT) ࡢ㑏ඖࢆᣦᶆ࡜ࡍࡿࠋບ㉳໬ྜ≀࠿ࡽ⏘⏕ࡉࢀࡿ ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡣ୍㟁Ꮚ㌿⛣཯ᛂ࡟ࡼࡾ NBT ࢆ㑏ඖࡋࠊ⏕ᡂࡉࢀࡿ monoformazan ࡣ 560 nm ࡟ UV ྾཰ࢆ᭷ࡍࡿࠋࡇࡢ྾ග್ࡢቑຍࢆࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ⏕ᡂࡢᣦᶆ࡜ࡋ࡚ᐃࡍࡿࠋ

superoxide + NBT ĺ O2 + monoformazan (NBT+)
㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ JaCVAM ༠ຊࡢୗ࡛ᐇ᪋ࡉࢀࡓከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࡀࠊOECD ࡀᐃࡵࡓᅜ㝿ⓗᇶ‽㸦2005 ᖺ)2㸧࡟ᚑࡗ࡚ࠊࣂࣜࢹ࣮ࢩࣙࣥᐇ᪋ᶵ㛵࡜ࡣ⊂❧ࡋࡓᑓ㛛ᐙ࠿ࡽ࡞ࡿ➨33୕⪅ホ౯ጤဨ఍࡟࡚⛉Ꮫⓗ࡞ホ౯ࡸ᳨ドࡀ⾜ࢃࢀࠊࡑࡢ⤖ᯝࢆࡶ࡜࡟ JaCVAM ගẘᛶ㈨ᩱ
⦅⧩ጤဨ఍ࡀሗ࿌᭩࡜ࡋ࡚ࡲ࡜ࡵࡓࡶࡢࢆホ౯㈨ᩱ࡜ࡋࡓࠋ
໬Ꮫ≀㉁࡟ኴ㝧ගࢆ↷ᑕࡋࡓሙྜࠊᚓࡽࢀࡓບ㉳࢚ࢿࣝࢠ࣮ࡣ⏕యෆ≀㉁ࡸ㓟⣲ศᏊ࡟㌿⛣ࡋࡓࡾࠊ࠶ࡿ࠸ࡣ໬ྜ≀ࡑࡢࡶࡢࡀ⏕యෆ≀㉁࡜⤖ྜࡋ࡚ග௜ຍ≀ࢆᙧᡂࡍࡿࠋࡲࡓࠊ
㓟⣲ศᏊࡣບ㉳࢚ࢿࣝࢠ࣮ࡢ࢔ࢡࢭࣉࢱ࣮࡜࡞ࡾࠊࡑࢀ࡟క࠸⏘⏕ࡉࢀࡓ୍㔜㡯㓟⣲ࡸ ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥ➼ࡢ ROS ࡟ࡼࡿ⏕యෆ≀㉁ࡢ㓟໬཯ᛂࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢⓎ⑕ཎᅉ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿ 7)ࠋROS ࡣࠊගẘᛶ≀㉁ࡢࡳ࡛࡞ࡃග࢔ࣞࣝࢠ࣮ࢆ♧ࡍ≀㉁࡛ࡶ
Ⓨ⏕ࡋࠊගࢱࣥࣃࢡ⤖ྜࡲࡓࡣගศゎ࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ 8)ࠋ௨ୖࡢࡼ࠺࡟ࠊᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࠊ⏘⏕ࡍࡿ୍㔜㡯㓟⣲࡜ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ᭷↓ࢆㄪ࡭ࡿග໬Ꮫⓗ཯ᛂᛶヨ㦂࡛࠶ࡿࡇ࡜࠿ࡽࠊගᏳ඲ᛶࡢホ౯ἲ࡜ࡋ࡚ཎ⌮ⓗ࡟ጇᙜᛶࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊື≀࠶ࡿ࠸ࡣື≀⏤᮶ࡢ⏕యヨᩱࢆ౑⏝ࡋ࡞࠸ࡇ࡜࠿ࡽࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ཯ᛂࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࠊከ᳨యฎ⌮ࡀྍ⬟࡛࠶ࡿࠋࡉࡽ࡟ࢥࢫࢺ㠃࡟࠾࠸࡚ࡶඃࢀ࡚࠸ࡿࠋከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜ࠊᮏヨ㦂ἲ࡟ࡼࡿගẘᛶ㝧ᛶ≀㉁ࡢ᳨ฟ⋡ࡣ 100%࡛࠶ࡾࠊഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽࠊගẘᛶࡢࣁࢨ࣮ࢻホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡉࡽ࡟ࠊ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ9)࡟࠾࠸࡚ࠊᮏヨ㦂ἲࡣึᮇホ౯㡯┠ࡢࡦ࡜ࡘ࡜ࡋ࡚グ㍕ࡉࢀࠊᮏヨ㦂ἲ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ㛤Ⓨ୰ࡢ་⸆ရ࡟ࠊࡉࡽ࡞ࡿගẘᛶヨ㦂ࢆ⾜࠺ᚲせࡣ࡞࠸࡜ࡉࢀ࡚࠸ࡿࠋࡑࡢࡓࡵࠊᮏヨ㦂ἲࢆගᏳ඲ᛶホ౯࡟ᑟධࡍࡿࡇ࡜࡟ࡼࡾࠊ௦᭰ࡍࡿගẘᛶヨ㦂ᩘࡢ๐ῶ࡟ࡘ࡞ࡀࡿࡇ࡜
ࡀᮇᚅ࡛ࡁࡿࠋ ḟ࡟ࠊ⣽⬊ࡸື≀ࢆ฼⏝ࡍࡿගᏳ඲ᛶホ౯⣔࡛ࡣࠊࡑࢀ⮬㌟ࡢẘᛶࡀᙉ࠸ UVB ࢆග※࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀ㞴ࡋࡃࠊUVB 㡿ᇦ࡟ࡋ࠿྾཰ࢆ᭷ࡉ࡞࠸໬ྜ≀࡟ᑐࡋ࡚㐺⏝ࡀᅔ㞴࡞ሙྜࡀ࠶ࡿࡀࠊᮏヨ㦂ἲࡣ⿕㦂≀㉁⮬యࡢ≀ᛶࡢホ౯⣔࡛࠶ࡿࡓࡵ࡟ UVB ࡢ↷ᑕࡶྍ⬟࡛࠶ࡿࠋࡲࡓࠊ⏕యヨᩱࢆᚲせ࡜ࡋ࡞࠸ࢩࢫࢸ࣒࡛࠶ࡿࡓࡵࠊࡑࡢ෌⌧ᛶࡶ㧗࠸ࠋ୍᪉ࠊ㐺⏝㝈⏺࡜ࡋ࡚ࠊ཯ᛂᾮ୰࡛ᯒฟࡍࡿ⁐ゎᛶࡢప࠸≀㉁ࡸ⁐ゎ᫬࡟╔Ⰽࡀㄆࡵࡽࢀࡿ໬Ꮫ≀㉁ࡣホ౯࡛ࡁ࡞࠸ࠋࡲࡓ⿕㦂≀㉁ࡣࣔࣝ⃰ᗘ༢఩࡛ヨ㦂ࡍࡿࡼ࠺ࣉࣟࢺࢥࣝࡀタᐃࡉࢀ࡚࠸ࡿࡓࡵࠊᢳฟ≀ࡸΰྜ≀ࡢࡼ࠺࡟ศᏊ㔞ࡀ୙࡛᫂ࣔࣝ⃰ᗘࢆ⟬ฟ࡛ࡁ࡞࠸⿕㦂≀㉁ࡣホ౯࡛ࡁ࡞࠸ࠋࡇࡢࡼ࠺࡞ヨ㦂᪉ἲࡢㄢ㢟࡟ᑐࡋࠊྛࠎࠊ⏺㠃άᛶ๣ࡸ࢔ࣝࣈ࣑ࣥࢆຍ࠼ࡓ᪉ἲ 10-12)ࠊᣦ♧⸆ࢆῧຍࡋ࡞࠸ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢタᐃ 13)ࠊPg/mL ༢఩࡛ヨ㦂ࡍࡿ᪉ἲ14)➼ࡢᑐᛂἲࡀ᳨ウࡉࢀ࡚࠾ࡾࠊᢳฟ≀ࡸΰྜ≀➼ࡢከ࠸໬⢝ရཎᩱࢆࡣࡌࡵࠊ୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆࡟ࡘ࠸࡚ࡢගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚᭷⏝ᛶࡀᮇᚅ࡛ࡁࡿࡀࠊࡲࡔホ౯᪉ἲ࡜ࡋ࡚☜❧ࡣࡉࢀ࡚࠸࡞࠸ࠋࡲࡓࠊගᏳ඲ᛶࡢ㐺⏝⠊ᅖ࡜ࡋ࡚ගẘᛶ࠾ࡼࡧග࢔ࣞࣝࢠ࣮ࡣ࡜ࡶ࡟Ⓨ⌧ᶵᗎࡢୖὶ࡟44ග໬Ꮫ཯ᛂࡀᏑᅾࡍࡿࡇ࡜ࠊࡉࡽ࡟ࠊග࢔ࣞࣝࢠ࣮≀㉁ࡶᮏヨ㦂ἲ᳨࡛ฟ࡛ࡁࡿ࡜ࡢ▱ぢࡀᚓࡽࢀ࡚ࡁࡓࡇ࡜࠿ࡽ 3)ࠊ㐺ษ࡟ᐇ᪋ࡉࢀࡓ ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡛࠶ࢀࡤගẘᛶࠊග࢔ࣞࣝࢠ࣮ࢆ♧ࡉ࡞࠸࡜ุ᩿ࡉࢀࡿࡀࠊ㝧ᛶࡢሙྜࡣࠊู㏵ホ౯ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸 ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜ࡋࡓ⡆౽࠿ࡘື≀࠾ࡼࡧື≀⏤᮶ヨᩱࢆ⏝࠸࡞࠸ヨ㦂ἲ࡛࠶ࡾࠊᨃఝኴ㝧ග↷ᑕ⿦⨨(solar simulator)ࠊUVAᐃ⿦⨨ࠊ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࡀ฼⏝࡛ࡁࡿ᪋タ࡛࠶ࢀࡤᐜ᫆࡟ᐇ᪋࡛ࡁࡿヨ㦂࡛࠶ࡿࠋᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜ࡋࡓගẘᛶ࠾ࡼࡧග࢔ࣞࣝࢠ࣮ࡢⓎ⌧ᶵᗎ࡟࠾ࡅࡿୖὶࡢ㔜せ࡞࢖࣋ࣥࢺࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢගᏳ඲ᛶࢆ⪃࠼ࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ࡞࠾ࠊᮏヨ㦂ἲࡢᐇ᪋࡟ᙜࡓࡗ࡚ࡣࠊ⿕㦂≀㉁ࡢ≉ᛶ࡜ヨ㦂ἲࡢ㐺⏝㝈⏺ࢆぢᴟࡵࠊᚓࡽࢀࡓ⤖ᯝࡢゎ㔘࡟ὀពࢆᡶ࠺ᚲせࡀ࠶ࡿࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸 ᮏヨ㦂ἲࡣࠊ㐺⏝㝈⏺࡟␃ពࡍࢀࡤࠊග໬Ꮫ཯ᛂᛶ࡟㛵ࡋ࡚ഇ㝜ᛶࡢホ౯ࢆࡍࡿࡇ࡜ࡀ࡞࠸ࡇ࡜࠿ࡽࠊࡉࡽ࡞ࡿගᏳ඲ᛶホ౯ࡢせྰࡢุ᩿࡟฼⏝࡛ࡁࡿࠋ2014 ᖺ࡟ᮏヨ㦂ἲࡣ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚᥇ᢥࡉࢀࠊ᪤࡟⾜ᨻⓗ࡞ཷࡅධࢀࡀጞࡲࡗ࡚࠸ࡿࠋᮏヨ㦂ἲࢆගᏳ඲ᛶࡢホ౯ᡓ␎࡟⤌ࡳධࢀࡿࡇ࡜ࡣࠊ3T3 NRU PT ࡸࡑࢀ࡟⥆ࡃື≀ヨ㦂ࡢᐇ᪋ᩘࢆ๐ῶࡍࡿࡇ࡜࡟ࡘ࡞ࡀࡿࠋ໬⢝ရ࣭་⸆㒊እရཎᩱࠊ㎰⸆ࠊࡑࡢ௚୍⯡໬Ꮫ≀㉁࡟
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