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᪥᫬㸸ᖹᡂ ᖺ ᭶ ᪥㸦ᅵ㸧 ᫬ ศ㛤఍
఍ሙ㸸ி㒔኱Ꮫᴦ཭఍㤋 㝵఍㆟࣭ㅮ₇ᐊ㸦ி㒔ᕷᕥி༊ྜྷ⏣஧ᮏᯇ⏫7(/㸧 >ி㒔㥐ࡼࡾᕷࣂࢫ $ ஌ሙࠊ ⣔⤫ࠕᮾᒣ㏻໭኱㊰ࣂࢫࢱ࣮࣑ࢼࣝࡺࡁࠖ஌㌴ࠊࠕ㏆⾨㏻㸦ࡇࡢ࠼࡝࠾ࡾ㸧ࠖୗ㌴ᚐṌࡍࡄ㸹
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‫ۑ‬ᐑୖἋ㈗ ࠊᮡᾆ༓᫂ ࠊୡཱྀྂṌ⳹ ࠊ๓⏣⣧ኵ ዉⰋዪ኱㝔ே㛫ᩥ໬࣭㣗≀ᰤ㣴ࠊ ዉⰋዪ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴
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‫ۑ‬ୡཱྀྂṌ⳹ ࠊᐑୖἋ㈗ ࠊᰘ⏣᭷ຍ ࠊ๓⏣⣧ኵ 㸦 ዉⰋዪ኱㝔ே㛫ᩥ໬࣭㣗≀ᰤ㣴ࠊ ዉⰋዪ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧
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ࠐ୸㔝᫭Ⴙ ࠊᆏᮏᬛᘺ ࠊᚋ⸨๛ ࠊ㧗ᶫಙஅ ࠊἙ⏣↷㞝 㸦 ி኱㝔㎰࣭㣗ရ⏕≀ࠊ ி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧
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ᅾࡣ㔝⏕ᆺࡢ 㻳㻸㻱㻝 ࡜ྠᵝ࡛࠶ࡿࡇ࡜ࢆ♧၀ࡍࡿ⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿࠋ
mRNA ᰾እ㍺㏦ᅉᏊ UAP56 䛺䜙䜃䛻 URH49 䛾」ྜ
యᙧᡂ⬟䜢ᨭ㓄䛩䜛㡿ᇦ䛾ྠᐃ䛸ᶵ⬟ゎᯒ
㸰
‫⏣⸨ۑ‬㈼୍ ࠊᖹᒣ⍞Ꮨ ࠊᐑ๓཭⟇ ࠊ⚄ᡞ኱᭸ ࠊỌᑿ㞞ဢ ࠊ
ቑ⏣ㄔྖ 㸦 ி኱㝔⏕࿨࣭⤫ྜ⏕࿨⛉Ꮫ㸧㻌
䛆┠ⓗ䛇┿᰾⏕≀䛻䛚䛔䛶᰾ෆ䛷㌿෗䛥䜜䛯๓㥑య 㼙㻾㻺㻭 䛿䚸ᵝ䚻䛺 㼙㻾㻺㻭 䝥䝻䝉䝅
䞁䜾䜢ཷ䛡䛶ᡂ⇍ 㼙㻾㻺㻭 䛸䛺䜚䚸⣽⬊㉁䛻㍺㏦䛥䜜䛶⺮ⓑ㉁䜈䛸⩻ヂ䛥䜜䜛䚹ᡂ⇍
㼙㻾㻺㻭 䛾᰾ෆ䛛䜙⣽⬊㉁䜈䛾㍺㏦䛻䛿 㼀㻾㻱㼄 」ྜయ䛜ᶵ⬟䛩䜛䚹㼁㻭㻼㻡㻢 䛿 㼀㻾㻱㼄 」
ྜయ䛾ᶵ⬟୰ᚰ䛸䛺䛳䛶ാ䛟ᅉᏊ䛷䛒䜚䚸㓝ẕ䛛䜙䝠䝖䜎䛷㐍໬ⓗ䛻ಖᏑ䛥䜜䛶䛔䜛䚹
୍᪉䚸㓝ẕ䜔䝝䜶䛺䛹䛷䚸㼁㻭㻼㻡㻢 䛿༢୍䛷Ꮡᅾ䛩䜛䛾䛻ᑐ䛧䛶䚸့ங㢮䛷䛿 㻥㻜㻑௨ୖ
䜒䛾┦ྠᛶ䜢ᣢ䛴 㼁㻾㻴㻠㻥 䛜Ꮡᅾ䛩䜛䚹ᙜ◊✲ᐊ䛷䛾ඛ⾜◊✲䛛䜙䚸䝠䝖䛻䛚䛔䛶
㼁㻾㻴㻠㻥 䛿䚸㼁㻭㻼㻡㻢 䛾ᙧᡂ䛩䜛 㼀㻾㻱㼄 」ྜయ䛸䛿඲䛟␗䛺䜛᪂つ䛾 㻭㻾㻱㼄 」ྜయ䜢ᙧ
ᡂ䛩䜛䛣䛸䛜᫂䜙䛛䛻䛥䜜䛯䚹䜎䛯୧⪅䛜㍺㏦䛩䜛 㼙㻾㻺㻭 䛻䛿┦㐪䛜䜏䜙䜜䚸㑅ᢥⓗ
㼙㻾㻺㻭 ㍺㏦䛻ᶵ⬟䛩䜛䛣䛸䜒᫂䜙䛛䛻䛥䜜䛯䚹㼙㻾㻺㻭 䛾㑅ᢥⓗ䛺㍺㏦䛻䛿䚸」ྜయᙧ
ᡂ䛾㐪䛔䛜 㼙㻾㻺㻭 䛾㑅ᢥᛶ䜢Ỵᐃ䛧䛶䛔䜛䛸ண᝿䛥䜜䜛䛜䚸㠀ᖖ䛻┦ྠᛶ䛾㧗䛔୧
⪅䛜䚸␗䛺䜛」ྜయ䜢ᙧᡂ䛩䜛ศᏊᶵᵓ䛿୙᫂䛷䛒䜛䚹䛭䛣䛷」ྜయᙧᡂ䛾㐪䛔䜢
ไᚚ䛩䜛䚸㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䛾⺮ⓑ㉁㡿ᇦ䛾ྠᐃ䜢ヨ䜏䛯䚹㻌
㻌
䛆᪉ἲ䞉⤖ᯝ䛇ゎᯒ᪉ἲ䛸䛧䛶䚸୧⪅䛷ẚ㍑ⓗ┦ྠᛶ䛾ప䛔୧ᮎ➃㡿ᇦ䜢䛿䛨䜑䚸
㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䛷␗䛺䜛ᵝ䚻䛺㡿ᇦ䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䜻䝯䝷ኚ␗య䜢
సᡂ䛧䚸」ྜయᙧᡂ⬟䚸䛺䜙䜃䛻⣽⬊ෆ 㼙㻾㻺㻭 ㍺㏦⬟䜢ゎᯒ䛧䛯䚹」ྜయᙧᡂ⬟䛿
ච␿ỿ㝆ἲ䛻䜘䜚䚸ኚ␗య䛜 㼀㻾㻱㼄 」ྜయ䜢ᙧᡂ䛩䜛䛛䚸㻭㻾㻱㼄 」ྜయ䜢ᙧᡂ䛩䜛䛛
ゎᯒ䛧䛯䚹㼙㻾㻺㻭 ㍺㏦⬟䛿 㻾㻺㻭㻙㻲㻵㻿㻴 ἲ䛻䜘䜚䚸㼁㻭㻼㻡㻢 䜎䛯䛿 㼁㻾㻴㻠㻥 䛾 㻷㻰 䛻䜘䜚⏕
䛨䜛᰾ෆ䛾 㻼㼛㼘㼥㻌 㻔㻭㻕㻗㻌 㻾㻺㻭 䛾⵳✚䛜䚸ኚ␗యⓎ⌧᫬䛻ᅇ᚟䛩䜛䛛ゎᯒ䛧䛯䚹౛䛘䜀
㼁㻭㻼㻡㻢 䛾䜻䝯䝷ኚ␗య䛜䚸ᮏ᮶ 㼁㻾㻴㻠㻥 䛾ᙧᡂ䛩䜛 㻭㻾㻱㼄 」ྜయ䜢ᙧᡂ䛧䚸㼁㻾㻴㻠㻥 䛸
ྠ䛨⣽⬊ෆ 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴ሙྜ䚸ኚ␗䜢ධ䜜䛯㡿ᇦ䛜」ྜయᙧᡂ䜢ไᚚ䛩䜛㡿
ᇦ䛸ุ᩿䛷䛝䜛䚹䛣䜜䜙 㻞 ✀㢮䛾ゎᯒἲ䛻䜘䜚ヲ⣽䛺」ྜయᙧᡂ䛾ศᏊᇶ┙ゎ᫂䜢⾜
䛳䛯䚹㻌
䜎䛪୧ᮎ➃㓄ิ䜢Ḟᦆ䛥䛫䛯Ḟᦆኚ␗య䛸䚸ᮎ➃㓄ิ䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥
䛾䜻䝯䝷ኚ␗య䜢ゎᯒ䛧䛯䚹Ḟᦆኚ␗య䛾ゎᯒ䛛䜙䚸୧ᮎ➃㓄ิ䛿」ྜయᙧᡂ䛻ᚲ
せ䛷䛒䜛䛣䛸䛜ุ᫂䛧䛯䚹䛧䛛䛧୧ᮎ➃䛾䜻䝯䝷ኚ␗య䛾ゎᯒ䛛䜙୧ᮎ➃䛿」ྜయᙧ
ᡂ䛾㐪䛔䜢ไᚚ䛧䛺䛔䛣䛸䛜ุ᫂䛧䛯䚹⥆䛔䛶୧ᮎ➃௨እ䛾䝁䜰㡿ᇦ䛷␗䛺䜛䜰䝭䝜㓟
䜢 㻝 ṧᇶ䛪䛴ධ䜜᭰䛘䛯䜻䝯䝷ኚ␗య䜢ゎᯒ䛧䛯䚹䛭䛾⤖ᯝ䛒䜛 㻝 䜰䝭䝜㓟䜢ධ䜜᭰䛘
䛯 㼁㻾㻴㻠㻥 ኚ␗య䛜 㼁㻭㻼㻡㻢 䛸ྠᵝ䛾」ྜయᙧᡂ⬟䛸 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴䛣䛸䛜ุ᫂
䛧䛯䚹⌧ᅾ䚸䛣䛾䠍䜰䝭䝜㓟䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 ኚ␗య䛜 㼁㻾㻴㻠㻥 䛸ྠᵝ䛾」ྜయᙧᡂ
⬟䛸 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴䛾䛛䚸䛥䜙䛻䛺䛬䛣䛾䜰䝭䝜㓟䛜」ྜయᙧᡂ䜢ไᚚ䛩䜛䛾䛛䚸
ゎᯒ䜢㐍䜑䛶䛔䜛䚹
పள㖄ẕங䜢䜒䛯䜙䛩ள㖄䝖䝷䞁䝇䝫䞊䝍䞊ZnT2
䛾」ྜ䝦䝔䝻᥋ྜయኚ␗
㸱
‫ۑ‬㐓ᮧ┤ஓ ࠊ✄ẟᗣྖ ࠊᑎすᩥᜨ ࠊᒸᓮᩥᏊ ࠊᡂ⏣ᏹྐ ࠊඣᓥᾈᏊ ࠊ
⚄ᡞ኱᭸ 㸦 ி኱㝔⏕࿨࣭⤫ྜ⏕࿨ࠊ ᪥኱࣭⦎㤿ගࡀୣ⑓㝔࣭ᑠඣࠊ ிዪ
኱࣭㣗≀ᰤ㣴ࠊ ᖇிᖹᡂ኱࣭೺ᗣ࣓ࢹ࢕࣭࢝ࣝ೺ᗣᰤ㣴㸧㻌
࠙┠ⓗࠚ
ள㖄ࡣࠊࣄࢺ࡟࡜ࡗ࡚Ḟ࠿ࡏ࡞࠸ᚲ㡲ᚤ㔞ඖ⣲࡛࠶ࡿࠋ࡜ࡃ࡟ࠊయ㔜ᙜࡓࡾ
࡛ᡂேࡢ⣙ 3 ಸࡶࡢள㖄ࢆᚲせ࡜ࡍࡿஙඣࡣࠊள㖄Ḟஈ࡟࡞ࡿ࡜㔜⠜࡞⓶⭵⅖
ࡸᡂ㛗ࡢ㐜ᘏ࡜࠸ࡗࡓ⑕≧ࢆ♧ࡍࠋஙඣࡢள㖄Ḟஈࡢせᅉ࡜ࡋ࡚ࠊẕங୰ࡢள
㖄ࡢῶᑡࡀ࠶ࡆࡽࢀࡿࠋᡃࠎࡣࠊள㖄Ḟஈ࡟ࡼࡾ㔜⠜࡞⓶⭵⅖ࢆⓎ⑕ࡋࡓஙඣ
࡜ࠊࡑࡢẕぶ࡛ࠊẕங୰ள㖄㔞ࡀ 90%௨ୖῶᑡࡋࡓపள㖄ẕஙᝈ⪅ࢆぢฟࡋࡓࠋ
పள㖄ẕஙࡢཎᅉ࡜ࡋ࡚ࠊẕங୰࡬ࡢள㖄㍺㏦ࢆᢸ࠺ள㖄ࢺࣛࣥࢫ࣏࣮ࢱ࣮
ZnT2 ࡢ㑇ఏᏊኚ␗ࡀ▱ࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊᡃࠎࡣẕぶࡢ ZnT2 㑇ఏᏊ࡟╔┠
ࡋ࡚ゎᯒࢆ⾜ࡗࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚ
1) ẕぶࡢࢤࣀ࣒ DNA ࠿ࡽࠊZnT2 㑇ఏᏊ࡟ 2 ࡘࡢ᪂つ࣑ࢫࢭࣥࢫኚ␗
(W152R࣭S296L)ࢆࡑࢀࡒࢀูࡢ࢔ࣜࣝ࡟ぢฟࡋࡓࠋࡲࡓࠊZnT2 㑇ఏᏊࡢࣉࣟ
࣮ࣔࢱ࣮㡿ᇦ࡟ኚ␗ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ
2) ྛ ZnT2 ࡢள㖄㍺㏦άᛶࢆࠊள㖄ឤཷᛶࡢ⣽⬊ᰴࢆ⏝࠸࡚ホ౯ࡋࡓ⤖ᯝࠊ
ZnT2 S296L ࡣ ZnT2 㔝⏕ᆺ࡜࡯ࡰྠ➼ࡢள㖄㍺㏦άᛶࢆࡶࡘࡀࠊZnT2 W152R
ࡣள㖄㍺㏦άᛶࢆࡶࡓ࡞࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋ
3) ZnT2 ࡣ஧㔞యࢆᙧᡂࡋ࡚ࡣࡌࡵ࡚ள㖄㍺㏦άᛶࢆⓎ᥹ࡍࡿࠋZnT2 ྛኚ␗ᆺ
ࡢ஧㔞యᙧᡂ⬟ࢆホ౯ࡍࡿࡓࡵࠊ㔝⏕ᆺࡸኚ␗ᆺࡢ ZnT2 ࢆ஧㔜Ⓨ⌧ࡉࡏࡓ⣽
⬊ᰴ࡟ࡼࡾඹච␿ỿ㝆ᐇ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊZnT2 S296L ࡣ㔝⏕ᆺ࡜஧㔞
యࢆᙧᡂࡍࡿࡀࠊZnT2 W152R ࡣ㔝⏕ᆺ࡜஧㔞యࢆᙧᡂࡋ࡞࠸ࡇ࡜ࠊࡲࡓࠊZnT2
W152R ࡜ ZnT2 S296L ࡢ⤌ࡳྜࢃࡏࡶࠊ஧㔞యࢆᙧᡂࡋ࡞࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋ
4) ྛ ZnT2 ࡢࢱࣥࣃࢡ㉁ᏳᐃᛶࢆゎᯒࡍࡿࡓࡵࠊZnT2 Ⓨ⌧⣽⬊ᰴࢆࢱࣥࣃࢡ
㉁ྜᡂ㜼ᐖ⸆࡛ฎ⌮ࡋࠊࢱࣥࣃࢡ㉁㔞ࡢ⥅᫬ⓗኚ໬ࢆồࡵࡓࠋ⤖ᯝࠊZnT2 S296L
ࡢࢱࣥࣃࢡ㉁ࡣࠊ㔝⏕ᆺ࡟ẚ࡭࡚ⴭࡋࡃ୙Ᏻᐃ࡟࡞ࡗ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ
࠙⪃ᐹࠚ
ZnT2 W152R ࡣள㖄㍺㏦άᛶࢆࡶࡓࡎࠊࡲࡓ」ྜయᙧᡂ⬟ࡀ࡞࠸ࡇ࡜࠿ࡽࠊ
ඃᛶ㜼ᐖάᛶࡣ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ୍᪉ࠊZnT2 S296L ࡣࠊள㖄㍺㏦άᛶ
࡜」ྜయᙧᡂ⬟ࢆࡶࡘࡀࠊࢱࣥࣃࢡ㉁ࡀ㠀ᖖ࡟୙Ᏻᐃ࡛࠶ࡿࡇ࡜ࡀุ᫂ࡋࡓࠋ
௨ୖ࠿ࡽࠊẕぶࡢ ZnT2 㑇ఏᏊࡀࠊW152R ࡜ S296L ࡢ」ྜ࣊ࢸࣟ᥋ྜయ࡛࠶
ࡿࡇ࡜ࡀࠊẕぶࡢపள㖄ẕஙࡢཎᅉ࡜⪃࠼ࡽࢀࡓࠋ
㸲
஝⇱⫙䝰䝕䝹䝬䜴䝇ヨ㦂䛻䛚䛡䜛䝟䜲䝘䝑䝥䝹ᯝᐇ⏤
᮶䜾䝹䝁䝅䝹䝉䝷䝭䝗⤒ཱྀᦤྲྀ䛾⓶⭵ᶵ⬟ᨵၿຠᯝཬ
䜃 in vitro ⭠⟶䝰䝕䝹䜈䛾ᙳ㡪㻌
㻌
‫‮ۑ‬ὸᘯᶞ ࠊす㇂ὒ㍜ ࠊⓑ஭ᗣோ ࠊ㤿ሙ೺ྐ ࠊ㔝ᔱ ₶ ࠊ኱ᡞಙ᫂ ࠊ
᱔ཎᾈㄔ ࠊỈ㔝㞞ྐ 㸦 ⚄኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ ⚄኱࣭⮬↛⛉Ꮫࠊ 㜰኱㝔
ᕤ࣭⏕࿨ඛ➃ࠊ ୸ၿ〇⸆㸧
࠙┠ⓗࠚࢫࣇ࢕ࣥࢦ⬡㉁ࡢ୍✀࡛࠶ࡿࢭ࣑ࣛࢻࡣゅ㉁ᒙࡢ⣽⬊㛫㝽ࢆᇙࡵ࡚࠸ࡿ
୺せᡂศ࡛࠶ࡿࠋ୺࡟ಖ‵࡞࡝⓶⭵ࡢࣂࣜ࢔ᶵ⬟࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾ໬
⢝ရ࡞࡝࡟ࡶከࡃྵࡲࢀࡿࠋᮏ◊✲࡛ࡣࣃ࢖ࢼࢵࣉࣝᯝᐇࡼࡾᚓࡽࢀࡓࢢࣝࢥࢩ
ࣝࢭ࣑ࣛࢻྵ᭷⏬ศࢆ஝⇱⫙ࣔࢹ࣐ࣝ࢘ࢫ࡟⤒ཱྀᢞ୚ࡋࠊ⓶⭵ᶵ⬟࡟୚࠼ࡿᙳ㡪
ཬࡧ LQYLWUR ⭠⟶ࣔࢹࣝࢆ⏝࠸࡚ࡑࡢస⏝ᶵᵓࢆ᳨ウࡋࡓࠋ
࠙᪉ἲࠚ+RV+5 ࡬࢔ࣞࢫ࣐࢘ࢫ㸦㞝ᛶ 㐌㱋㸧ࢆ⏝࠸ࡓ஝⇱⫙ࣔࢹ࣐ࣝ࢘ࢫ
ヨ㦂࡛ࡣࠊ≉Ṧ㣫ᩱ㸦+5$'㸧࡛ㄏᑟࡋࡓ஝⇱⫙࡟ᑐࡍࡿ⢒ࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤
᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ 㓄ྜ㣫ᩱ㸦3*㸧ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡉࡽ࡟⤒ཱྀᦤྲྀ
࡟ࡼࡿ⭠⟶ච␿࡬ࡢᙳ㡪ࢆ᳨ウࡍࡿࡓࡵࠊࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭࣛ
࣑ࢻ PJPO ࢆࣄࢺᑠ⭠ୖ⓶ᵝ⣽⬊ &DFR ⣽⬊࡜࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊
5$: ⣽⬊ࡢඹᇵ㣴⣔࡟౪ࡋࡓࠋ⟶⭍ഃ࠿ࡽࢧࣥࣉࣝฎ⌮ ᫬㛫ᚋ࡟ /36 ࡛
5$: ⣽⬊ࢆ่⃭ࡋࠊࡉࡽ࡟ ᫬㛫ᇵ㣴ᚋ 5$: ⣽⬊ࡢ඲ 51$ ࢆᢳฟࡋࠊ
ᐃ㔞 573&5 ἲ࡛ ,/P51$ Ⓨ⌧㔞ࢆ ᐃࡋࡓࠋࡲࡓࠊ⭠⟶ୖ⓶ࢆ௓ࡋࡓ௦ㅰ≀
ࡢᙳ㡪ࢆ᳨ウࡍࡿࡓࡵࠊࢺࣛࣥࢬ࢙࢘ࣝ⭷ୖ࡟ศ໬ࡉࡏࡓ &DFR ⣽⬊࡟ᑐࡋ࡚ࠊ
⟶⭍ഃ࠿ࡽࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻࢆῧຍࡋࠊ ᫬㛫ᚋ࡟ᇶᗏ
⭷ഃ࡟㏱㐣ࡋࡓᇵᆅࢆ 5$: ⣽⬊࡬࡜ῧຍࡋࡓࠋ ᫬㛫ᚋࠊ/36 ่⃭ࢆ⾜࠸ࠊ
ࡉࡽ࡟ ᫬㛫ᇵ㣴ࡋࡓᚋ࡟ ,/P51$ Ⓨ⌧㔞ࢆ ᐃࡋࡓࠋ
࠙⤖ᯝࠚ㏻ᖖ㣫ᩱ⩌࡟ẚ࡭ࠊ≉Ṧ㣫ᩱ⩌࡛ 7(:/ ್ࡢୖ᪼ࡀㄆࡵࡽࢀࡓࠋ୍᪉ࠊ
3* ⩌࡛ࡣ 7(:/ ್ࡢୖ᪼ࢆ᭷ព࡟ᢚไࡋࡓࠋࡲࡓ 㐌㛫㣫⫱ᚋࠊ࣐࢘ࢫࡢ⫼㒊⾲
⓶ࢆほᐹࡋࡓ࡜ࡇࢁࠊ≉Ṧ㣫ᩱ⩌࡛ࡣ㢧ⴭ࡞ࢩ࣡ࡢᙧᡂཬࡧ⓶⭵ࡢ⫧ཌࡀ☜ㄆ࡛
ࡁࡓࡢ࡟ᑐࡋࠊ3* ⩌࡛ࡣ㏻ᖖ㣫ᩱ⩌࡜ྠ➼ࡢእほࢆ♧ࡋࡓࠋࡇࡢࡇ࡜࠿ࡽࣃ࢖
ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ࡟ࡣ⓶⭵ᶵ⬟ᨵၿຠᯝࡀ࠶ࡿࡇ࡜ࡀ♧၀
ࡉࢀࡓࠋࡇࢀࡲ࡛ࡢ◊✲࠿ࡽ⢭〇ࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ
㸦3XUH3*㸧ࢆ LQYLWUR ⭠⟶ࣔࢹࣝ࡟౪ࡍࡿࡇ࡜࡛ 5$: ⣽⬊୰ࡢ ,/P51$
Ⓨ⌧㔞ࡀቑຍࡍࡿࡇ࡜ࡀศ࠿ࡗ࡚࠸ࡿࠋࢺࣛࣥࢬ࢙࢘ࣝ⭷ୖࡢ &DFR ⣽⬊༢ᒙ
⭷࡟ 3XUH3* ࢆῧຍࡋᇶᗏ⭷ഃᇵᆅࢆ 5$: ⣽⬊࡟┤᥋ῧຍࡋࡓ࡜ࡇࢁࠊඹ
ᇵ㣴᫬࡜ྠᵝ࡟ 5$: ⣽⬊୰ࡢ ,/P51$ Ⓨ⌧㔞ࢆஹ㐍ࡋࡓࠋࡇࢀࡲ࡛ࡢᐇ
㦂࠿ࡽࠊ3XUH3* ࢆῧຍࡋࠊ ᫬㛫ᇵ㣴ᚋࡢṧᏑ㔞ࡣ⣙ ࡛࠶ࡗࡓࡇ࡜ࢆ⪃៖
ࡍࡿ࡜ࠊࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻࡣ⭠⟶ୖ⓶ࢆ௓ࡋࡓ௦ㅰࢆཷ
ࡅ࡚࠾ࡾࠊࡑࡢ௦ㅰ≀ࡀ࣐ࢡࣟࣇ࢓࣮ࢪ࡬࡜ᙳ㡪ࢆཬࡰࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉ
ࢀࡓࠋ
ᾘ໬⟶䝩䝹䝰䞁䛻䜘䜛⫢⬡㉁௦ㅰไᚚ䛾ᶵᵓゎᯒ
㸳
㻌
‫ۑ‬㣤ሯࡳ࠶ࡁ ࠊ㧗ᶫಙஅ ࠊ᪂㇂⣪⧊ ࠊ᐀ീᶞᏊ ࠊᚋ⸨๛ ࠊ Ἑ⏣↷㞝 㸦 ி኱㝔࣭㎰࣭㣗ရ⏕≀ࠊ ி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧㻌
࠙┠ⓗࠚ⏕యෆࡢ⬡㉁௦ㅰࡣ⚄⤒ࡸᾮᛶᅉᏊࢆ௓ࡋ࡚ከ⮚ჾ㛫࡛ไᚚࡉࢀࡿࡇ࡜
ࡀ㏆ᖺ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋᙜ◊✲ᐊࡢඛ⾜◊✲࡟ࡼࡾࠊᑠ⭠⏤᮶ࡢᾘ໬⟶࣍ࣝ
࡛ࣔࣥ࠶ࡿ cholecystokinin (CCK)ࡣ⫢⣽⬊࡛ࡢ⬡⫫⵳✚ࢆᢚไࡍࡿࡇ࡜ࡀ♧
ࡉࢀࠊCCK ࡀᑠ⭠ࠊ⫢⮚㛫ࡢ⬡㉁௦ㅰ᝟ሗࢆఏ㐩ࡍࡿ㔜せ࡞ᅉᏊ࡛࠶ࡿྍ⬟ᛶ
ࡀ⪃࠼ࡽࢀࡓࠋ࡜ࡇࢁࡀ᭱㏆ࠊCCK Ḟᦆ࣐࢘ࢫࡣ⬡⫫⫢ࢆⓎ⑕ࡋ࡞࠸ࡇ࡜ࡀሗ
࿌ࡉࢀࠊඛ⾜◊✲࠿ࡽࡢண ࡜▩┪ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡑࡇ࡛ᮏ◊✲࡛
ࡣ CCK ࡜ྠ୍ࡢཷᐜయ࡛࠶ࡿ CCKB Receptor (CCKBR) ࢆㄆ㆑ࡍࡿᾘ໬⟶࣍
ࣝࣔࣥࠊgastrin ࡀ CCK ࡟ࡼࡿస⏝ࢆ௦ൾࡋ࡚࠸ࡿࡶࡢ࡜௬ᐃࡋࠊgastrin ࡀ⫢
⬡㉁௦ㅰ࡟ཬࡰࡍᙳ㡪ࠊ୪ࡧ࡟ CCKBR ࢆ௓ࡋࡓ⫢⬡⫫⵳✚ᢚไᶵᵓࡢヲ⣽ࢆ
ゎ᫂ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚࣄࢺ⫢⣽⬊ࣔࢹ࡛ࣝ࠶ࡿ HepG2 ⣽⬊࡟ gastrin ࢆฎ⌮ࡋࡓ⤖ᯝࠊ
⬡⫫⵳✚ࡣ᭷ព࡟ᢚไࡉࢀࠊࡑࡢస⏝ࡣ CCKBR ࡟ᑐࡍࡿ㜼ᐖ๣࡟ࡼࡾ᭷ព࡟
ᾘኻࡋࡓࠋඛ⾜◊✲ࡼࡾࠊCCK ࡣ⫢⣽⬊࡟࠾࠸࡚⬡⫫㓟 ǃ 㓟໬ࢆஹ㐍ࡉࡏࡿࡇ
࡜࡛⬡⫫⵳✚ࢆᢚไࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊCCKBR ࠿ࡽ⬡⫫㓟 ǃ
㓟໬ࡢஹ㐍࡟⮳ࡿࢩࢢࢼࣝఏ㐩⤒㊰࡟㛵ࡍࡿ᳨ウࢆ⾜ࡗࡓࠋCCKBR ࡣ G
protein coupled receptor ࡛࠶ࡾࠊࡑࡢୗὶࡣ Ca2+ ࡀ఩⨨ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚
࠸ࡿࠋࡑࡇ࡛ࠊHepG2 ⣽⬊ࢆ⏝࠸࡚[Ca2+]i ࡟㛵ࡍࡿ᳨ウࢆ⾜ࡗࡓ⤖ᯝࠊgastrin
ࡣ Ca2+ ࡢ⤒㊰ࢆ௓ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋḟ࡟ࠊCa2+ ࡢୗὶࡢࢩࢢࢼࣝఏ㐩⤒
㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊྛ✀㜼ᐖ๣ࢆ⏝࠸ࡓ᳨ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊgastrin
࡟ࡼࡿ⬡⫫⵳✚ᢚไస⏝ࡣ CaMKK ࠾ࡼࡧ AMPK ࡟ᑐࡍࡿ㜼ᐖ๣࡟ࡼࡾ᭷ព࡟
ᾘኻࡋࠊCCK ࢆฎ⌮ࡋࡓሙྜ࡜ྠᵝࡢ⤖ᯝ࡜࡞ࡗࡓࠋࡉࡽ࡟ࠊCCK ࠾ࡼࡧ
gastrin ࡟ࡼࡿస⏝ࢆ⏕యෆ᳨࡛ウࡍࡿࡓࡵࠊ⫢⮚≉␗ⓗ࡞㑇ఏᏊᑟධヨ⸆ࢆ⏝
࠸࡚ CCKBR ࡟ᑐࡍࡿ siRNA ࢆ࣐࢘ࢫ࡟ᑟධࡋࡓࠋࡑࡢ⤖ᯝࠊCCKBR-siRNA
ࢆᑟධࡋࡓ࣐࢘ࢫࡢ⫢⬡⫫⵳✚ࡀ᭷ព࡟ቑຍࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊgastrin ࡣ
CCK ࡜ྠᵝ࡟⫢⣽⬊࡟࠾࠸࡚ CCKBRń[Ca2+]ińCaMKKńAMPK ࢆ௓ࡋ࡚⬡
⫫⵳✚ᢚไస⏝ࢆ♧ࡍࡇ࡜ࠊ࠾ࡼࡧ⫢⬡⫫⵳✚࡟ᑐࡍࡿ CCKBR ࡢ⏕యෆ࡛ࡢ
㛵୚ࡀ♧၀ࡉࢀࡓࠋ
ABCG1 䛻䜘䜛 HMG-CoA 㑏ඖ㓝⣲䛾᪂つάᛶไᚚ
ᶵᵓ䛾ゎᯒ
㸴
㻌
‫ۑ‬Ώ㑔ኴ㑻{ࠊᖹ஭⤢Ꮚ{ࠊ᳜⏣࿴ග{tࠊᯇᑿ㐨᠇{㸦 ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ
ி኱࣭L&H06㸧
࠙┠ⓗࠚࢥࣞࢫࢸ࣮ࣟࣝࡣ⏕య⭷ࡢ㔜せ࡞ᵓᡂᡂศࡔࡀࠊ㐣๫࡞⵳✚ࡣ⣽⬊ẘᛶ
ࢆ♧ࡍࠋࡑࡢࡓࡵࠊ⣽⬊ෆࡢࢥࣞࢫࢸ࣮ࣟࣝ⃰ᗘࡣཝᐦ࡞ㄪ⠇ࢆཷࡅࡿࠋ
ATP-binding cassette G1㸦ABCG1㸧ࡣࠊ⣽⬊ෆࡢవ๫ࢥࣞࢫࢸ࣮ࣟࣝࢆ ATP
ຍỈศゎ౫Ꮡⓗ࡟⣽⬊እ࡬࡜᤼ฟࡍࡿࡇ࡜࡛ࠊࢥࣞࢫࢸ࣮ࣟࣝᜏᖖᛶ࡟㔜せ࡞ᙺ
๭ࢆᯝࡓࡍࠋ᰾ෆ㌿෗ᅉᏊ Liver X receptor㸦LXR㸧ࡣ࢜࢟ࢩࢫࢸ࣮ࣟࣝࢆࣜ࢞
ࣥࢻ࡜ࡍࡿࡇ࡜࠿ࡽࠊLXR άᛶࡣ⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞࡜ṇࡢ┦㛵㛵ಀࡀ࠶
ࡿࠋࡑࡢࡓࡵࠊLXR άᛶࢆᣦᶆ࡜ࡍࡿࡇ࡜࡛⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞ࢆ㛫᥋ⓗ
࡟ホ౯࡛ࡁࡿࠋABCG1 ࢆ HEK293 ⣽⬊࡟Ⓨ⌧ࡉࡏࡿ࡜⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ
ࡀ᤼ฟࡉࢀࡿࡓࡵࠊLXR άᛶࡣῶᑡࡋࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊATP ຍỈศゎάᛶ
ࢆᣢࡓࡎࢥࣞࢫࢸ࣮ࣟࣝࢆ᤼ฟ࡛ࡁ࡞࠸ ABCG1 ኚ␗యࢆⓎ⌧ࡉࡏࡓሙྜࡶࠊྠ
ᵝ࡟ LXR άᛶࡀ኱ࡁࡃῶᑡࡋࡓࠋࡇࢀࡼࡾࠊABCG1 ࡀ⬡㉁᤼ฟ࡟౫Ꮡࡏࡎࠊ
ࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂࢆᢚไࡍࡿࡇ࡜࡛⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞ࢆㄪ⠇ࡋ࡚࠸
ࡿ࡜௬ㄝࢆ❧࡚ࠊࡇࡢ௬ㄝࢆ᳨ウࡍࡿࡇ࡜ࢆᮏ◊✲ࡢ┠ⓗ࡜ࡋࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚື≀⣽⬊࡟࠾ࡅࡿࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂࡣࠊHMG-CoA 㑏ඖ㓝⣲
㸦HMGCR㸧࡟ࡼࡾ HMG-CoA ࡀ࣓ࣂࣟࣥ㓟࡬ኚ᥮ࡉࢀࡿ཯ᛂࡀᚊ㏿ẁ㝵࡜࡞
ࡿࠋࡑࡇ࡛ࠊABCG1 ࡀࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂ࡟୚࠼ࡿᙳ㡪ࢆࠊHMGCR Ⓨ⌧㔞
࡜ࢥࣞࢫࢸ࣮ࣟࣝྜᡂ㔞ࢆ ᐃࡋ HMGCR άᛶࢆ⟬ฟࡍࡿࡇ࡜࡛ホ౯ࡋࡓࠋࡑ
ࡢ⤖ᯝࠊHEK293 ⣽⬊ࡢ ABCG1 ཬࡧ ABCG1 ኚ␗యᏳᐃⓎ⌧ᰴ࡟࠾࠸࡚ࠊ
HMGCR ࡢάᛶࡀపୗࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ㏫࡟ࠊTHP-1 ࣐ࢡࣟࣇ
࢓࣮ࢪ⣽⬊࡟࠾࠸࡚ ABCG1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ࡜ࠊHMGCR άᛶࡀୖ᪼ࡋࡓࠋ
ࡇࢀࡽࡢ⤖ᯝࡣࠊABCG1 ࡀ⬡㉁᤼ฟ࡟౫Ꮡࡏࡎ࡟ HMGCR ࡢάᛶࢆไᚚࡍࡿ
ࡇ࡜ࢆ♧၀ࡋࡓࠋࡋ࠿ࡋࠊHMGCR ࡢάᛶไᚚᶵᵓ࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ AMPK
࡟ࡼࡿ HMGCR ࣜࣥ㓟໬ࣞ࣋ࣝࡣࠊABCG1 Ⓨ⌧࡟ࡼࡾኚ໬ࡋ࡞࠿ࡗࡓࠋḟ࡟ࠊ
ABCG1 ࡀ HMGCR ࡜┤᥋┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾ HMGCR άᛶࢆไᚚࡍࡿ࡜
࠸࠺ྍ⬟ᛶࢆ᳨ウࡍࡿࡓࡵࠊච␿ඹỿ㝆ᐇ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊABCG1 ࡜
HMGCR ࡀ┤᥋┦஫స⏝ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ௨ୖࡼࡾࠊABCG1 ࡀ HMGCR
࡜┦஫స⏝ࡍࡿࡇ࡜࡛ HMGCR ࡢάᛶࢆᢚไࡍࡿ࡜࠸࠺ࠊ᪂つࡢ HMGCR άᛶ
ไᚚᶵᵓࡢᏑᅾࡀ♧၀ࡉࢀࡓࠋ
Procyanidin-B1-3,3"-di-O-gallate 䛿 EGCG 䛸␗䛺䜛
స⏝䛷 HeLa S3 ⣽⬊䛾ቑṪ䜢㜼ᐖ䛩䜛䠛
㸵
㻌
‫▼ۑ‬ཎἋஓຍ ࠊ⥤㔝⩏༤ ࠊᒸᮏಟᖹ ࠊᅵ஭⩧㤿 ࠊᒸᮏὈ㍜ ࠊ㡲⸨㱟ᙪ ࠊ
㛗⏣⿱அ ࠊ୰ᓥ⠊⾜ ࠊ㰺⸨Ᏻ㈗Ꮚ ኱㜰㟁㏻኱㝔࣭ᕤࠊ ኱㜰㟁㏻኱࣭
ᕤࠊ ⌮◊ᇶᖿ◊࣭ࢣ࣑࢝ࣝࣂ࢖࢜ࣟࢪ࣮ࠊ ᐩᒣ┴኱࣭ᕤ
OH
࠙┠ⓗࠚProanthocyanidin(PAs)ࡣࠊከࡃࡢ᳜≀࡟ྵ
ࡲࢀࡿ㧗ᶵ⬟ᛶ࣏ࣜࣇ࢙ࣀ࣮ࣝ໬ྜ≀࡛࠶ࡾࠊ࠾Ⲕࡸ
࢝࢝࢜ࡢႴዲရࠊࣈࢻ࢘➼ࡢᯝ≀㢮ࠊࢫࢠ➼ࡢᶞ⓶࡞
OH
HO
ࡣࠊ⣽⬊⭷ୖࡢཷᐜయ࡜ࡋ࡚ྠᐃࡉࢀࡓ 67 kDa ࣑ࣛ
ࢽࣥࣞࢭࣉࢱ࣮ (67LR)ࢆ௓ࡋ࡚ᵝࠎ࡞⏕⌮άᛶࢆ♧
ࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ 1ࠋࡉࡽ࡟ࠊEGCG ࡣ 67LR
ࢆ௓ࡍࡿ᝟ሗఏ㐩ࡢ㐣⛬࡛ p38MAPK ࢆྵࡴ MAPK
ࡢࣜࣥ㓟໬ࢆ㜼ᐖࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ 2ࠋᡃࠎࡣࠊ
PAs ࡢᢠ⅖⑕స⏝࡞࡝ࡢάᛶⓎ⌧ࡀ EGCG ࡜ྠᵝࡢ
⤒㊰࡟ࡼࡿࡶࡢ࠿ࠊ໬Ꮫⓗ࣭⏕≀Ꮫⓗ࡟ド᫂ࡍࡿࡓࡵ
◊✲ࢆ㐍ࡵ࡚࠸ࡿࠋ
OH
O
࡝࡟ྵࡲࢀ࡚࠸ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡣᴟࡵ
࡚ᙉ࠸ᢠ㓟໬άᛶ࣭ᢠ⅖⑕స⏝➼ࠊᵝࠎ࡞⏕≀άᛶࢆ
♧ࡍࡀࠊኳ↛࠿ࡽ㧗⣧ᗘࡢࢧࣥࣉࣝࢆᚓࡿࡇ࡜ࡣ㠀ᖖ
࡟ᅔ㞴࡛࠶ࡾࠊ⣔⤫ⓗ࡞ᵓ㐀ïάᛶ┦㛵ࡣ࡯࡜ࢇ࡝ゎ
᫂ࡉࢀ࡚࠸࡞࠸ࠋࡑࡢ୍᪉࡛ࠊ࠾Ⲕ࢝ࢸ࢟ࣥ࡜ࡋ࡚▱
ࡽ ࢀ ࡿ (-)-Epigallocatechin-3-O-gallate (EGCG) 1
O
OH
OH
O
OH
OH
1
OH
O
HO
OH
HO
OH
R2
R1
OH
O
OH
O
OH
OH
O
OH
2: R1 = H, R2 = G
3: R1 = G, R2 = H
OH
O
G=
O
OH
OH
OH
࠙᪉ἲ࡜⤖ᯝࠚ ❧య㑅ᢥⓗྜᡂ࡟ࡼࡗ࡚⣧⢋࡟ᚓࡽ
ࢀࡓ✀ࠎࡢ PAs ࢆ⏝࠸࡚ HeLa S3 ⣽⬊࡟ᑐࡍࡿቑṪᢚไάᛶヨ㦂ࢆ⾜ࡗࡓࠋ
ࡑࡢ⤖ᯝࠊB1-3,3”-di-O-gallate (2
2)࡜ B4-3,3”-di-O-gallate (3
3)ࡀࠊEGCG ࡼࡾࡶ
㧗࠸ቑṪᢚไάᛶࢆ♧ࡋࡓࠋࡉࡽ࡟ࠊࡇࢀࡽࡢ໬ྜ≀ࡀ⣽⬊ෆ p38MAPK 㔞࡟
ᙳ㡪ࢆ୚࠼ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓࠋEGCG ࡛ࡣྠᵝ࡞⌧㇟ࡀぢࡽࢀ࡞࠸஦࠿ࡽࠊ
EGCG ࡜ࡣ␗࡞ࡿᶵᵓ࡛άᛶࢆⓎ᥹ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋຍ࠼࡚ࠊ௚
ࡢᐁ⬟ᇶ࡛ಟ㣭ࡋࡓ໬ྜ≀࡟ࡘ࠸࡚ࡶ᳨ウࢆ⾜ࡗ࡚࠾ࡾࠊࡑࢀࡽࢆࡲ࡜ࡵ࡚ሗ࿌
ࡍࡿࠋ
ཧ⪃ᩥ⊩) 1. Tachibana H, et al., Nat Struct Mol Biol. 2004, 11, 380-381
2. Eui-Baek Byun, et al., Biochem Biophys Res Commun. 2012,
426, 480-485
䜲䝛䛻䛚䛡䜛䜶䝸䝅䝍䞊ㄏᑟᛶ௦ㅰ≀䛾ရ✀㛫ẚ㍑
㸶
‫⥙ۑ‬ᖸ㈗Ꮚ ࠊᯇᮏࡩ࠺㤶 ࠊᑎ▼ᨻ⩏ ࠊዟᮏ⿱ ࠊす⏣ᚊኵ ࠊ᳃┤ᶞ 㸦 ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊி኱㝔㎰࣭㎰Ꮫ㸧
࠙┠ⓗࠚ
⑓ཎ⳦ࡢឤᰁࡸ᳜㣗ᛶ᪻⹸ࡢ㣗ᐖ࠿ࡽ㌟ࢆᏲࡿࡓࡵ࡟ࠊ᳜≀ࡣᢠ⳦άᛶࡸẘ
ᛶࠊᚷ㑊άᛶࡢ࠶ࡿ໬ྜ≀࡞࡝ࢆ⏕ྜᡂࡋࠊ⵳✚ࡋ࡚࠸ࡿࠋ㏆ᖺࠊ᳜㣗ᛶ᪻⹸ࡢ
㣗ᐖࡸ⏘༸࡟ࡼࡾㄏᑟࡉࢀࡿ᳜≀ࡢ௦ㅰ≀ࡀ᢬ᢠᛶࢆ♧ࡍ⌧㇟ࡀሗ࿌ࡉࢀ࡚࠸
ࡿࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊᩘ✀ࡢ᪻⹸ࡢྤࡁฟࡋᾮ㸦⭠⟶ෆᐜ≀㸧୰࠿ࡽࠊࢺ࢘ࣔࣟ
ࢥࢩࠊࢱࣂࢥࡸࢲ࢖ࢬ࡟᥹Ⓨᡂศࢆᨺฟࡉࡏࡿ࢚ࣜࢩࢱ࣮ࡀྠᐃࡉࢀ࡚࠸ࡿࠋᮏ
◊✲࡛ࡣࠊ㫣⩟┠ᗂ⹸ࡢ⭠⟶ෆᐜ≀ࡸയᐖᛂ⟅࡟㛵ࢃࡿࢪࣕࢫࣔࣥ㓟ฎ⌮࡟ࡼࡾ
ㄏᑟࡉࢀࡿ࢖ࢿ୙᥹Ⓨᛶ௦ㅰ≀࡟ὀ┠ࡋࡓࠋࡲࡓࠊࡑࡢရ✀㛫࡟࠾ࡅࡿᛂ⟅ࡢẚ
㍑࠿ࡽࠊᐖ⹸ࡢ㣗ᐖࢆ㜵ࡄㄏᑟᛶ௦ㅰ≀ࡢ⏕ྜᡂ⤒㊰ࡢゎ᫂ࡸࡑࡢ⏕ྜᡂ࡟㛵ࢃ
ࡿ㑇ఏᏊࡢྠᐃࢆ┠ᣦࡋ࡚࠸ࡿࠋ
࠙᪉ἲ࣭⤖ᯝࠚ
᳜≀࠿ࡽࡣࢱࣥࣃࢡ㉁ࢆᵓᡂࡍࡿ 20 ✀ࡢ࢔࣑ࣀ㓟ࡢ௚ࠊከࡃࡢ㠀ࢱࣥࣃࢡᛶ
࢔࣑ࣀ㓟ࡀྠᐃࡉࢀ࡚࠾ࡾࠊ୍㒊ࡣ᳜㣗⪅࡟ᑐࡋ࡚ẘᛶࡸᡂ㛗㜼ᐖάᛶࡀሗ࿌ࡉ
ࢀ࡚࠸ࡿࠋࡑࡇ࡛ࡲࡎࠊ࢚ࣜࢩࢱ࣮ฎ⌮᫬ࡢ࢖ࢿᗂⱑ࡟࠾ࡅࡿ࢔࣑ࣀ㓟ࡢኚືࢆ
ㄪ࡭ࡓࠋ
࢖ࢿᗂⱑ (4 ⴥ᏶඲ᒎ㛤ᮇ) ࡢⴥ࡟✰ࢆ㛤ࡅࠊ࢔࣡ࣚࢺ࢘ᗂ⹸⭠⟶ෆᐜ≀ࡢ⦆
⾪ ᾮ ᢳ ฟ ᾮ ࢆ ሬ ᕸ ࡋ ࠊ 2 ࠊ 4 ࠊ 24 ࠊ 48 ᫬ 㛫 ᚋ ࡟ ࢔ ࣑ ࣀ 㓟 ࢆ ᢳ ฟ ࡋ ࠊ
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate ࡛ㄏᑟయᚋࠊLC/MS ࡟౪ヨࡋ
ࡓࠋ࢖ࢿࡢရ✀ࡣ᪥ᮏᬕࠊ㖟ᆓ୺ࠊkasalath ࢆ⏝࠸ࡓࠋ᪥ᮏᬕ࡛ࡣ⭠⟶ෆᐜ≀ᢳ
ฟᾮሬᕸ 2 ᪥ᚋࠊJ-aminobutylic acid (GABA) ࡀቑຍࡋࡓࡀࠊ㖟ᆓ୺ࠊkasalath ࡛
ࡣቑຍࡏࡎࠊရ✀㛫࡟ࡼࡿᛂ⟅࡟ᕪࡀ࠶ࡗࡓࠋ᪥ᮏᬕ࡛ࡣ GABA ࡢ๓㥑య࡛࠶
ࡿ ࢢ ࣝ ࢱ ࣑ ࣥ 㓟 ࡀ ῶ ᑡ ࡋ ࡚ ࠾ ࡾ ࠊ ⭠ ⟶ ෆ ᐜ ≀ ᢳ ฟ ᾮ ሬ ᕸ ࡟ ࡼ ࡾ glutamine
decarboxylase ࡢάᛶ໬ࡀண᝿ࡉࢀࡓࠋ
୍᪉ࠊᗂⱑ࡟ࢪࣕࢫࣔࣥ㓟ࢆሬᕸࡍࡿ࡜ࠊkasalath ࡟࠾࠸࡚ GABA ࡢቑຍࡀ
☜ㄆࡉࢀࠊฎ⌮᪉ἲ࡟ࡼࡗ࡚ࡶရ✀㛫ࡢᛂ⟅࡟ᕪࡀ⏕ࡌࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋ
㓝ẕ Saccharomyces cerevisiae 䛻䛚䛡䜛䝨䝹䜸䜻䝅
䝋䞊䝮ෆ䝺䝗䝑䜽䝇ไᚚᅉᏊ䛾ゎᯒ㻌
㸷
㻌
‫ۑ‬ᓥ୪♸Ꮚ ࠊዟබ⚽ ࠊᑉ㛵῟ ࠊ㜰஭ᗣ⬟ 㸦 ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ ி
኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧
࠙┠ⓗࠚ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ࡣ࢜ࣞ࢖ࣥ㓟࡞࡝ࡢ⬡⫫㓟ࡢș㓟໬ࢆ⾜࠺⣽⬊ᑠჾᐁ
࡛࠶ࡿࠋ࢜ࣞ࢖ࣥ㓟ࢆ༢୍ࡢⅣ⣲※࡜ࡋࡓᇵᆅ࡛㓝ẕࡢᇵ㣴ࢆ⾜࠺࡜ࠊ࣌ࣝ࢜࢟
ࢩࢯ࣮࣒ෆ࡛⬡⫫㓟ࡢ௦ㅰࡀ⾜ࢃࢀ๪⏘≀࡜ࡋ࡚άᛶ㓟⣲ࡢ୍✀࡛࠶ࡿ H2O2 ࡀ
Ⓨ⏕ࡍࡿࠋάᛶ㓟⣲ࡣ㠀ᖖ࡟཯ᛂᛶࡀ㧗ࡃࠊࢱࣥࣃࢡ㉁ࡢኚᛶࡸ DNA ࡢᦆയࢆ
ᘬࡁ㉳ࡇࡍ࡞࡝⣽⬊࡟ᐖࢆ୚࠼ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࡢࡓࡵ⏕≀࡟ࡣάᛶ㓟
⣲ࢆ↓ẘ໬ࡍࡿᢠ㓟໬㓝⣲ࢆ⏘⏕ࡋࠊ⣽⬊ෆࡢࣞࢻࢵࢡࢫ≧ែࢆ୍ᐃ࡟ಖࡘᶵ⬟
ࡀഛࢃࡗ࡚࠸ࡿࠋࡋ࠿ࡋࠊ⏕໬Ꮫⓗゎᯒࡢ㞴ࡋࡉ࠿ࡽ⣽⬊ᑠჾᐁෆ࡟࠾ࡅࡿࣞࢻ
ࢵࢡࢫไᚚࢩࢫࢸ࣒࡟ࡘ࠸࡚ヲࡋ࠸ࡇ࡜ࡣᮍࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ
ࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ⏕ࡁࡓ⣽⬊ෆࡢ㓟໬㑏ඖ≧ែࢆྍど໬࡛ࡁࡿ⺯ගࢱࣥࣃࢡ㉁
ࣉ࣮ࣟࣈ࡛࠶ࡿ Redoxfluor ࢆ㓝ẕ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡟Ⓨ⌧ࡉࡏࠊ࣌ࣝ࢜࢟ࢩ
ࢯ࣮࣒ෆࡢࣞࢻࢵࢡࢫไᚚ࡟㛵ࢃࡿᅉᏊࡢゎᯒࢆ⾜ࡗ࡚࠸ࡿࠋ
࠙᪉ἲ࣭⤖ᯝࠚ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡛ാࡃ࢝ࢱ࣮ࣛࢮ࡛࠶ࡿ CTA1 㑇ఏᏊ◚ቯ
ᰴࠊࣞࢻࢵࢡࢫไᚚ࡟㔜せ࡞ാࡁࢆ⾜ࡗ࡚࠸ࡿศᏊࡢ 1 ࡘ࡛࠶ࡿ TRX2 㑇ఏᏊ
◚ቯᰴࠊCTA1TRX2 ࡢ஧㔜◚ቯᰴࢆసᡂࡋࡓࠋࡇࢀࡽࡢ㑇ఏᏊ◚ቯᰴ࡟ࡘ࠸࡚
࢜ࣞ࢖ࣥ㓟ࢆ༢୍ࡢⅣ⣲※࡜ࡍࡿᇵᆅ࡛⏕⫱ࢆ⾜ࡗࡓ࡜ࡇࢁࠊCTA1 㑇ఏᏊ◚ቯ
ᰴ࡛ࡣ⏕⫱ᗘࡢపୗࡀぢࡽࢀ CTA1TRX2 ஧㔜◚ቯᰴ࡛ࡣࡉࡽ࡟⏕⫱ᗘࡀపୗࡍ
ࡿ࡜࠸࠺⤖ᯝࡀᚓࡽࢀࡓࠋࡇࢀࡽࡢᰴࡢ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆࣞࢻࢵࢡࢫ≧ែࢆㄪ
࡭ࡿࡓࡵࠊ࣌ࣝ࢜࢟ࢩࢯ࣮࣒࡬ࡢ⛣⾜ࢩࢢࢼࣝ㓄ิࢆ௜ࡅࡓ Redoxfluor ࢆࡑࢀ
ࡒࢀࡢᰴ࡟Ⓨ⌧ࡉࡏ⺯ග㢧ᚤ㙾ほᐹࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ CTA1 ◚ቯᰴ࡟࠾࠸࡚
⺯ගᙉᗘࡢపୗࡀぢࡽࢀࠊCTA1TRX2 ஧㔜◚ቯᰴ࡛ࡣ᭦࡟ࡑࡢపୗࡀ㢧ⴭ࡟ぢ
ࡽࢀࡓࠋࡇࡢ᫬ࡢ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡟࠾ࡅࡿ Redoxfluor ࡢⓎ⌧㔞ࢆㄪ࡭ࡿࡓ
ࡵ⣽⬊ᢳฟᾮࢆ㐲ᚰศ㞳࡟ࡼࡗ࡚⣽⬊㉁⏬ศ࡜࢜ࣝ࢞ࢿࣛ⏬ศ࡟ศ⏬ࡋࠊ࢜ࣝ࢞
ࢿࣛ⏬ศ࡟ྵࡲࢀࡿ Redoxfluor 㔞ࡢẚ㍑ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊCTA1TRX2 ஧㔜
◚ቯᰴ࡟㛵ࡋ࡚ࡣ㔝⏕ᰴ࡜ẚ㍑ࡋ࡚࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆࡢ Redoxfluor 㔞ࡢῶᑡ
ࡀ࠶ࡲࡾぢࡽࢀ࡞࠿ࡗࡓࠋࡇࡢࡇ࡜࠿ࡽ CTA1TRX2 㑇ఏᏊࡢ◚ቯࡀ࣌ࣝ࢜࢟ࢩ
ࢯ࣮࣒ෆ Redoxfluor ࡢ⺯ගࢱࣥࣃࢡ㉁ࡢᶵ⬟పୗࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀศ࠿ࡗ
ࡓࠋ
㸯㸮㻌
Improvement in mitochondrial function by
antioxidants protects cellular oxidation caused by
proteasome inhibition
Sunita Maharjan1, Jun Hoseki1,2, Masahide Oku1, Yasuyoshi Sakai1,2
(1Division of Applied Life Sciences, Graduate School of Agriculture and
2
Research Unit for Physiological Chemistry, the Center for the Promotion of
Interdisciplinary Education and Research, Kyoto University)
࠙ Objectiveࠚ Ubiquitin proteasome system is essential for multiple physiological
processes via selective degradation of target proteins. Proteasome impairment is linked
to aging and many pathological conditions from cancer to neurodegenerative disorders
like Alzheimer’s and Parkinson’s disease. We found that proteasome inhibition resulted
in cellular oxidation. In this study, we aimed to investigate molecular mechanisms
underlying protective effects of several antioxidant compounds from food against
cellular oxidation caused by proteasome inhibition.
࠙ Methods and Results ࠚ Intracellular redox state was observed with Redoxfluor
following treatment with a proteasome inhibitor in mammalian cells, stably transfected
with the redox probe. Redoxfluor detects intracellular redox state by its fluorescence
resonance energy transfer (FRET) efficiency with a concomitant redox-dependent
structural change of the protein. The proteasome inhibition induced cellular oxidation.
Simultaneous treatment with antioxidants in food prevented the cellular oxidation
caused by proteasome inhibition and ultimately improved survival rate of cells under the
proteasomal inhibition. Furthermore, treatment with the antioxidants maintained
mitochondrial membrane potential and significantly reduced radioactive oxygen species
(ROS) in mitochondria by using mitochondrial targeted molecular probes. Moreover,
pretreatment with the antioxidants was found to significantly increase protein level of a
mitochondrial antioxidative enzyme suggesting that the pretreatment of the antioxidants
might stimulate a relevant signaling pathway(s) which subsequently increases
antioxidant capacity in cells. The results suggest that improvement in mitochondrial
function by the antioxidants prevents cellular oxidation caused by proteasome inhibition.
㓝ẕ Saccharomyces cerevisiae 䛻䛚䛡䜛⬡⫫⁲ศゎ
䛾䛯䜑䛾䜸䞊䝖䝣䜯䝆䞊⤒㊰䛾ᶵ⬟ゎᯒ㻌
㸯㸯
㻌
‫ۑ‬ᒣ⏣㯞⾰ࠊዟබ⚽ࠊ㜰஭ᗣ⬟㸦ி኱㝔㎰࣭ᛂ⏝⏕࿨㸧
࠙┠ⓗࠚ⬡⫫⁲ࡣ┿᰾⣽⬊࡟ᗈࡃಖᏑࡉࢀࡓ࢚ࢿࣝࢠ࣮㈓ⶶჾᐁ࡛࠶ࡾࠊࢺࣜ࢔
ࢩࣝࢢࣜࢭ࣮ࣟࣝࡸࢫࢸ࣮࢚ࣟࣝࢫࢸࣝ࡞࡝ࡢ୰ᛶ⬡㉁ࡀࣜࣥ⬡㉁୍㔜⭷࡟そ
ࢃࢀࡓᵓ㐀ࢆࡶࡘࠋ⌧ᅾࠊ⬡⫫⁲ࡢศゎᶵᵓࡢ୍ࡘ࡜ࡋ࡚ࠊ࣮࢜ࢺࣇ࢓ࢪ࣮ࡢᏑ
ᅾࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ࣮࢜ࢺࣇ࢓ࢪ࣮࡜ࡣࠊ⣽⬊㉁ᡂศࢆศゎࢥࣥࣃ࣮ࢺ࣓ࣥࢺ
㸦ᾮ⬊㸭ࣜࢯࢯ࣮࣒㸧࡬㍺㏦ࡍࡿศゎ⣔࡛࠶ࡾࠊ࣐ࢡ࣮ࣟ࢜ࢺࣇ࢓ࢪ࣮࡜࣑ࢡࣟ
࣮࢜ࢺࣇ࢓ࢪ࣮ࡢ㸰✀㢮ࡢᶵᵓࡀᏑᅾࡍࡿࠋ๓⪅࡛ࡣࠊศゎᑐ㇟ࢆ᪂⏕⭷ࡀໟࡳ
㎸ࡳࠊᙧᡂࡉࢀࡓ࣮࢜ࢺࣇ࢓ࢦࢯ࣮࣒࡜࿧ࡤࢀࡿᵓ㐀యࡀࠊᾮ⬊⭷࡜⼥ྜࡍࡿࡇ
࡜࡛ᾮ⬊ෆ࡬ᨺฟࡍࡿࠋ୍᪉ࠊᚋ⪅࡛ࡣᾮ⬊⭷ࡢ㝗ධ࡟ࡼࡾᑐ㇟ࢆ┤᥋ໟࡳ㎸ࡴࠋ
࣮࢜ࢺࣇ࢓ࢪ࣮ࡣ୍⯡ⓗ࡟㠀㑅ᢥⓗ࡞ࢱࣥࣃࢡ㉁ศゎ⤒㊰࡜ࡋ࡚▱ࡽࢀࡿࡀࠊ࣌
ࣝ࢜࢟ࢩࢯ࣮࣒➼ࡢ࢜ࣝ࢞ࢿࣛ≉␗ⓗ࡞ࡶࡢࡶ࠶ࡾࠊ့ங㢮࡟࠾࠸࡚⬡⫫⁲㑅ᢥ
ⓗ࡞ࠕ࣏ࣜࣇ࢓ࢪ࣮ࠖࡀᥦၐࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋ㓝ẕ࡟࠾࠸࡚ࡣࡑࡢᏑᅾࡸ࢔ࢵ
ࢭ࢖⣔ࡣ☜❧ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊฟⱆ㓝ẕࢆ⏝࠸࡚࣏ࣜࣇ࢓ࢪ࣮
ࡢᐃ㔞ⓗ࡞࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋࠊ࣏ࣜࣇ࢓ࢪ࣮࡟ᶵ⬟ࡍࡿ㑇ఏᏊࡢ᥈⣴࠾ࡼࡧゎ
ᯒࢆ⾜࠺ࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚ⬡⫫⁲ࡢᾮ⬊ෆ㍺㏦࠾ࡼࡧศゎࢆ᳨ฟࡍࡿࡓࡵࠊ⥳Ⰽ⺯ගࢱࣥࣃ
ࢡ㉁ GFP ࡢᾮ⬊ෆ࡛ࡢᏳᐃᛶࢆ฼⏝ࡋࡓࣉࣟࢭࢵࢩࣥࢢ࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋ
ࡓࠋࡲࡎࠊ⬡⫫⁲ᒁᅾࢱࣥࣃࢡ㉁࡜ GFP ࡢ⼥ྜࢱࣥࣃࢡ㉁ࢆⓎ⌧ࡍࡿ㓝ẕᰴࢆ
స〇ࡋࡓࠋ⼥ྜࢱࣥࣃࢡ㉁Ⓨ⌧ᰴ࠿ࡽᢳฟࡋࡓࢱࣥࣃࢡ㉁ࢆศ㞳ࡋ GFP ᢠయࢆ
⏝࠸ࡓ࢙࢘ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢࢆ⾜ࡗࡓ࡜ࡇࢁࠊ㔝⏕ᰴ࡛ࡣ⼥ྜࢱࣥࣃࢡ㉁ࡢ
ศゎ⏘≀࡛࠶ࡿ㐟㞳ᆺ GFP ࡀ᳨ฟࡉࢀࡓࠋ୍᪉ࠊᾮ⬊ෆศゎࡢḞᦆࢆ♧ࡍ㑇ఏ
Ꮚ◚ቯᰴ࡛ࡣ㐟㞳ᆺ GFP ࡣ᳨ฟࡉࢀ࡞࠿ࡗࡓࠋࡲࡓ⺯ග㢧ᚤ㙾࡟ࡼࡾࠊᾮ⬊ෆ
ศゎḞᦆᰴ࡟࠾࠸࡚ GFP ࡢᾮ⬊ෆᣑᩓࡀほᐹࡉࢀࡓࡇ࡜࠿ࡽࠊ⬡⫫⁲ࡀ࣮࢜ࢺ
ࣇ࢓ࢪ࣮࡟ࡼࡾᾮ⬊ෆ࡟㍺㏦ࡉࢀࡓᚋࠊศゎࢆཷࡅࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ
ࡉࡽ࡟ࠊࣉࣟࢭࢵࢩࣥࢢ࢔ࢵࢭ࢖࡟ࡼࡾࠊࣜࣥ⬡㉁ PI3P㸦phosphatidylinositol
3-phosphate㸧ࢆ⏘⏕ࡍࡿࢱࣥࣃࢡ㉁ࡸ PI3P ࡟⤖ྜࡍࡿࢱࣥࣃࢡ㉁ࡀ࣏ࣜࣇ࢓
ࢪ࣮࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ
ᅛ┦㻙Ẽ┦䝞䜲䜸䝣䜱䝹䝮ᇵ㣴䛜኱⭠⳦䛾 persister
cell ᙧᡂ䛻ཬ䜌䛩ᙳ㡪
㸯㸰
㻌
‫ۑ‬ᐑୖἋ㈗ ࠊᮡᾆ༓᫂ ࠊୡཱྀྂṌ⳹ ࠊ๓⏣⣧ኵ 㸦 ዉⰋዪᏊ኱Ꮫ኱Ꮫ
㝔 㣗ᰤᑓᨷࠊዉⰋዪᏊ኱Ꮫ 㣗≀ᰤ㣴Ꮫ⛉㸧
࠙┠ⓗࠚ㏆ᖺࠊ⣽⳦࡟࠾࠸࡚ persister cell ࡜࿧ࡤࢀࡿᢠ⏕≀㉁⪏ᛶ⣽⬊ࡢᏑᅾ
ࡀὀ┠ࡉࢀ࡚࠸ࡿࠋpersister cell ࡜ࡣ⣽⳦㞟ᅋ୰ࡢ୍㒊ࡢ⣽⬊ࡀ୍᫬ⓗ࡟ఇ╀
≧ែ࡜࡞ࡾᢠ⏕≀㉁⪏ᛶࢆ♧ࡍࡼ࠺࡟࡞ࡗࡓࡶࡢ࡛࠶ࡿࠋpersister cell ࡣኚ␗
ࡸ㑇ఏᏊࡢ⋓ᚓ࡞࡝ࡢ㑇ఏⓗኚ໬ࢆకࢃ࡞࠸➨୕ࡢᢠ⏕≀㉁⪏ᛶ⣽⬊࡜ࡋ࡚ࠊ⣽
⳦ ⏕ ⌮ ࠾ ࡼ ࡧ ⾨ ⏕ ⟶ ⌮ ࡢ ほ Ⅼ ࠿ ࡽ ࡶ ࡑ ࡢ 㔜 せ ᛶ ࡀ ㄆ ㆑ ࡉ ࢀ ࡘ ࡘ ࠶ ࡿ >@ ࠋ
persister cell ࡣẕ㞟ᅋ࡟ᑐࡍࡿᏑᅾ๭ྜࡢᑠࡉࡉ࠿ࡽࠊࡇࢀࡲ࡛༑ศ◊✲ࡀ㐍
ࢇ࡛࠸࡞࠿ࡗࡓࠋࡋ࠿ࡋ㏆ᖺࠊpersister cell ᙧᡂࡀ㑇ఏⓗ࡟ࣉࣟࢢ࣒ࣛࡉࢀࡓ
⌧㇟࡛࠶ࡿࡇ࡜ࢆ♧၀ࡍࡿሗ࿌ࡀ┦ḟ࠸࡛࡞ࡉࢀࠊࡑࡢ⌧㇟ࡢヲ⣽࠾ࡼࡧᶵᵓࡢ
ゎᯒࡀ◊✲ࡢ↔Ⅼ࡜࡞ࡗ࡚ࡁ࡚࠸ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ᪂ࡓ࡞ほⅬ࠿ࡽ
persister cell ᙧᡂࢆゎᯒࡍࡿࡓࡵࠊ୍⯡ⓗ࡞◊✲࡛⏝࠸ࡽࢀ࡚࠸ࡿᾮయᇵ㣴࡛
ࡣ࡞ࡃࠊᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴࡟╔┠ࡋࠊࡑࡢ᮲௳ୗ࡛ࡢ኱⭠⳦ࡢ
persister cell ᙧᡂࡢヲ⣽ゎ᫂ࢆヨࡳࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚ኱⭠⳦ࢆᢠ⏕≀㉁↓ῧຍࡢ LB ᇵᆅ࡛ Υࠊ ᫬㛫ࡢᾮయᇵ㣴
࠶ࡿ࠸ࡣᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴ᚋࠊࡑࢀࡒࢀࡢ⣽⬊ࢆᢠ⏕≀㉁ῧຍ LB
ᇵᆅ࡟ᠱ⃮ࡋࠊΥࠊѸ ᫬㛫ࡢฎ⌮ࢆ⾜ࡗࡓࠋฎ⌮ᚋࡢ⣽⬊ࢆᢠ⏕≀㉁↓ῧ
ຍࡢ LB ᐮኳୖ࡟ࣉ࣮ࣞࢸ࢕ࣥࢢࡋࠊ⏕ṧ⣽⬊㸦 persister cell㸧ᩘࢆ ᐃࡋࡓࠋ
⏕ṧ⣽⬊࠿ࡽ⏕ࡌࡓࢥࣟࢽ࣮ࡣᢠ⏕≀㉁ࣉ࣮ࣞࢺ࡟෌ࢫࢺ࣮ࣜࢡࡋࠊᜏஂⓗ࡞⪏
ᛶ⋓ᚓኚ␗ࡀ㉳ࡇࡗ࡚࠸࡞࠸ࡇ࡜ࢆ☜ㄆࡋࡓࠋྛ✀᮲௳᳨ウࡢ⤖ᯝࠊᅛ┦Ẽ┦
ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴ᚋࡢ⣽⬊࡛ࡣࠊᢠ⏕≀㉁ฎ⌮ࡢ≉ᐃ᮲௳ୗ᳨࡛ฟࡉࢀࡿ
persister cell ࡀࠊᾮయᇵ㣴ᚋࡢ⣽⬊࡜ẚ࡭᭱኱ ಸࡶࡢ㧗࠸㢖ᗘ࡛ฟ⌧ࡍࡿࡇ
࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡇࡢഴྥࡣస⏝ᵝᘧࡢ␗࡞ࡿ࡯࡜ࢇ࡝ࡢᢠ⏕≀㉁࡟࠾࠸࡚ࡶ
ྠᵝ࡛࠶ࡾࠊᢠ⏕≀㉁ࡢ✀㢮࡟≉␗ⓗ࡞⪏ᛶᶵᵓࡢⓎ⌧࡛ࡣ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀ
ࡓࠋࡲࡓୖグ࡜ྠᵝࡢ≉ᐃ᮲௳ୗ࡛ࡢᢠ⏕≀㉁ฎ⌮᫬㛫ࡢ᳨ウࢆ⾜ࡗࡓ࡜ࡇࢁࠊ
ᾮయᇵ㣴ᚋࡢ⣽⬊࡛ࡣ ᫬㛫௨ෆ࡟⏕⣽⬊ࡀᾘኻࡍࡿࡢ࡟ᑐࡋࠊᅛ┦Ẽ┦ࣂ
࢖࢜ࣇ࢕࣒ࣝᚋࡢ⣽⬊࡛ࡣ ᫬㛫ᚋࡶ㧗࠸๭ྜ࡛ persister cell ࡀᏑᅾࡋ⥆ࡅ
ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽࠊ ᫬㛫ࡢᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ
㣴࡟ࡼࡗ࡚ࠊࡑࡢᚋࠊ⣽⬊ࡀᾮయᾋ㐟≧ែ࡟࡞ࡗ࡚ࡶ༑᪥㛫௨ୖ࡟ࢃࡓࡗ࡚ಖᣢ
ࡉࢀࡿ㠀㑇ఏⓗ࡞⏕⌮ኚ໬ࡀ⏕ࡎࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
[1] Keren, I. et al. FEMS Microbiol lett. 230, 13-18. 2004
[2] Kim, L. Annual Review of Microbiology. 64, 357-372. 2010
኱⭠⳦ᶆ‽㔝⏕ᰴ䛻䛚䛡䜛䝣䜯䞊䝆⏘⏕ᰴ䛾⥙⨶ⓗ
᥈⣴
㸯㸱
㻌
‫ۑ‬ᰘ⏣᭷ຍ ࠊ㧘ᶫᮥዉ ࠊ㬼ஂ᳃༓㔛 ࠊୡཱྀྂṌ⳹ ࠊ๓⏣⣧ኵ 㸦 ዉⰋ
ዪᏊ኱㝔ே㛫ᩥ໬࣭㣗≀ᰤ㣴ࠊዉⰋዪᏊ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧㻌
࠙┠ⓗࠚ㏆ᖺࠊୡ⏺ྛᅜ࡛༢㞳ࡉࢀ࡚࠸ࡿ✀ࠎࡢ⑓ཎᛶ኱⭠⳦ᰴ࡛ࡣࠊ㑇ఏᏊ⩌
ࡢከ㔜⋓ᚓ࡟ࡼࡿ㧗⑓ཎᛶ໬ࡸከ๣⪏ᛶ໬ࡀၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋࡇ࠺ࡋࡓ㑇ఏᏊ
⩌ࡢ⋓ᚓࡣࠊ⳦࠿ࡽ⳦࡬ࡢ㑇ఏᏊࡢỈᖹఏ᧛࡟ࡼࡗ࡚⾜ࢃࢀ࡚࠾ࡾࠊࡑࡢ୺せᶵ
ᵓࡢ୍ࡘ࡟ࠊࣇ࢓࣮ࢪ࡟ࡼࡿᙧ㉁ᑟධࡀ࠶ࡿࠋࡋ࠿ࡋࣇ࢓࣮ࢪ࡟ࡼࡿ㑇ఏᏊỈᖹ
ఏ᧛ࡀࠊᐇ㝿ࡢ⎔ቃ୰࡛࡝ࡢࡼ࠺࡟㉳ࡇࡿࡢ࠿࡜࠸࠺Ⅼ࡟ࡘ࠸࡚ࡣࠊ༑ศ࡞◊✲
ࡀ࡞ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᡃࠎࡣ⎔ቃ୰࡛ࡢ࢖࣋ࣥࢺࢆࡼࡾᛅᐇ࡟཯࡛ᫎࡁࡿࣔ
ࢹࣝᐇ㦂⣔ࢆᵓ⠏ࡍࡿࡓࡵ࡟ࠊ኱⭠⳦ࡢᶆ‽㔝⏕ᰴࢥࣞࢡࢩ࡛ࣙࣥ࠶ࡿ ECOR
(E. coli Collection of Reference) 㸵㸰ᰴ 1) ࡢ฼⏝ࢆ⪃࠼ࡓࠋECOR ᰴࡢࣇ࢓࣮
ࢪಖ᭷࣭⏘⏕࡟㛵ࡋ࡚ࡣࠊ㐣ཤ஧ࠊ୕ࡢሗ࿌౛
ࡣ࠶ࡿࡶࡢࡢᮍࡔ༑ศ࡞ゎ
᫂ࡣ࡞ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣࠊECOR ᰴࢆ⏝࠸ࡓᙧ㉁ᑟධࣔࢹࣝᐇ㦂⣔ࢆ
ᵓ⠏ࡍࡿࡓࡵࡢ➨୍ẁ㝵࡜ࡋ࡚ࠊECOR ᰴ୰ࡢࣇ࢓࣮ࢪಖ᭷࣭⏘⏕ᰴࡢ⥙⨶ⓗ
᥈⣴ࢆヨࡳࡓࠋ
࠙᪉ἲࠚྛ ECOR ᰴࢆ Mitomycin C ῧຍ࡟ࡼࡿ SOS ᛂ⟅ㄏᑟ࠾ࡼࡧ↓ฎ⌮ࡢ
㠀ㄏᑟࡢ 2 ࡘࡢ᮲௳ୗ࡛ᇵ㣴ᚋࠊ㐲ᚰศ㞳࡜ࣇ࢕ࣝࢱ࣮⁛⳦࠶ࡿ࠸ࡣࢡࣟࣟ࣍ࣝ
࣒ฎ⌮࡟ࡼࡾୖΎࢆㄪ〇ࡋࡓࠋࡇࢀࡽࡢୖΎࢆูࡢᐇ㦂ᐊᰴ࡟ῧຍࡋࠊࢫ࣏ࢵࢺ
࢔ࢵࢭ࢖࠾ࡼࡧࣉ࣮ࣛࢡ࢔ࢵࢭ࢖࡟ࡼࡾᨺฟࣇ࢓࣮ࢪࡢ᳨ฟࢆ⾜ࡗࡓࠋ
࠙⤖ᯝࠚୖグ᥈⣴ࡢ⤖ᯝ࠿ࡽࠊECOR㸵㸰ᰴ࠿ࡽィ㸱㸴ᰴࡢࣇ࢓࣮ࢪ⏘⏕ᰴࢆ≉
ᐃࡋࡓࠋࡇࡢෆࠊ㸯㸲ᰴࡣࡇࢀࡲ࡛࡟ࣇ࢓࣮ࢪಖ᭷࣭⏘⏕ࡢሗ࿌ࡢ࡞࠸ᰴ࡛࠶ࡗ
ࡓࠋ⌧ᅾࠊࡇࢀࡽࣇ࢓࣮ࢪࡢ༢㞳࣭ྠᐃࢆ㐍ࡵ࡚࠸ࡿࠋ
2), 3)
1) Ochman, H., and Selander, R. K. 1984. J. Bacteriol. 157: 690-693.
2) Riley, M. A., and Gordon D. M. 1992. J. Gen. Microbiol. 138: 1345-1352
3) Nilsson, A. S., Karlsson, J. L., and Haggard-Ljungquist, E.
2004. Mol. Biol. Evol. 21 : 1-13
㔝⏕኱⭠⳦ᰴ䜢⏝䛔䛯㑇ఏᏊỈᖹఏ᧛䛾ゎᯒ
䇲䝰䝕䝹⣔䛾☜❧䛸㣗ရᡂศ䛾ᙳ㡪ホ౯
㸯㸲
㻌
‫ۑ‬ୡཱྀྂṌ⳹ ࠊᐑୖἋ㈗ ࠊᰘ⏣᭷ຍ ࠊ๓⏣⣧ኵ 㸦 ዉⰋዪ኱㝔ே㛫ᩥ
໬࣭㣗≀ᰤ㣴ࠊዉⰋዪ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧㻌
࠙┠ⓗࠚ㏆ᖺࠊከᩘࡢ⑓ཎᛶࡸᢠ⏕≀㉁⪏ᛶࢆ⋓ᚓࡋࡓ᪂ࡓ࡞⑓ཎᛶ኱⭠⳦ࡢฟ
⌧ࡀୡ⏺ⓗ࡞ၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋ኱⭠⳦࡛ࡣ୍⯡࡟⮬↛⎔ቃୗ࡛ࡢᙧ㉁㌿᥮ࡣ࡯
࡜ࢇ࡝㉳ࡇࡽ࡞࠸࡜ࡉࢀ࡚ࡁࡓࡀࠊ㏆ᖺᡃࠎࡢ◊✲ᐊ࡛ࡣࠊΰྜᇵ㣴ࡢ኱⭠⳦ᰴ
㛫࡛ᙧ㉁㌿᥮࡟ࡼࡾ㠀᥋ྜᛶࣉࣛࢫ࣑ࢻࡀỈᖹఏ᧛ࡍࡿ⌧㇟ࢆⓎぢࡋࡓࠋࡇࡢⓎ
ぢࡣࠊ⎔ቃ୰࡛ࡢ኱⭠⳦ࡢ㑇ఏᏊỈᖹఏ᧛࡟㛵ࡍࡿᐃㄝࡢ෌᳨ウࢆಁࡍࡶࡢ࡛࠶
ࡿࠋ⎔ቃ୰ࡢ⌧㇟ࢆṇ☜࡟෌⌧ࡍࡿࡓࡵ࡟ࡣࠊỗ⏝ࡢᐇ㦂ᐊᰴ࡛ࡣ࡞ࡃ㔝⏕⳦ᰴ
ࢆ⏝࠸ࡓᐇ㦂⣔ࡀᚲ㡲࡜࡞ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ㔝⏕኱⭠⳦ᰴࡢ௦⾲࡜ࡋ࡚ࠊ
ECOR㸦E. coli Collection of Reference㸧ࡢ฼⏝ࢆ⪃࠼ࡓࠋECOR ࡜ࡣ 1984 ᖺ
࡟ Ochman ࡽࡀ㑅ᢤࡋࡓࠊ㔝⏕኱⭠⳦ᰴࡢᩘ༓✀㢮ࢆ 72 ᰴ࡛௦⾲ࡍࡿᶆ‽ᰴࢥ
ࣞࢡࢩ࡛ࣙࣥ࠶ࡿࠋᮏ◊✲࡛ࡣࠊࡲࡎ ECOR ᰴࢆ⏝࠸ࡓ㑇ఏᏊỈᖹఏ᧛ࣔࢹࣝ
ᐇ㦂⣔ࡢ☜❧࡜ఏ᧛ᵝᘧࡢุูࢆ⾜࠸ࠊḟ࠸࡛ྠᐇ㦂⣔ࢆ⏝࠸ࠊ㣗ရࡀᰤ㣴※࡜
࡞ࡿ᮲௳ୗ࡛ࡢ㑇ఏᏊỈᖹఏ᧛ࡢⓎ⏕᳨ド࡜ࠊ࣮ࣚࢢࣝࢺ࡟ྵࡲࢀࡿங㓟ࡸ㓑㓟
࡟╔┠ࡋࡓᙳ㡪ホ౯ࢆヨࡳࡓࠋ
࠙᪉ἲࠚ⳦ᰴࡣࠊኳ↛ࣉࣛࢫ࣑ࢻಖ᭷㸭㠀ಖ᭷ࡢ ECOR ᰴࠊேᕤࣉࣛࢫ࣑ࢻᑟ
ධ㸭ᮍᑟධࡢ ECOR ᰴࠊ࠾ࡼࡧᐇ㦂ᐊᰴࢆ⏝࠸ࡓࠋ㑇ఏᏊỈᖹఏ᧛ࡣࠊ2 ✀ࡢ
⳦ᰴࢆඹᇵ㣴ᚋࠊ᪂ࡓ࡞ᢠ⏕≀㉁⪏ᛶࢆ⋓ᚓࡋࡓࢥࣟࢽ࣮ࡢⓎ⏕ᩘ࠿ࡽᐃ㔞໬ࡋ
ࡓࠋ㑇ఏᏊỈᖹఏ᧛ࡢᶵᵓࡣࠊDNase ࠾ࡼࡧ࣓ࣥࣈࣞࣥࢆ⏝࠸ࡓᐇ㦂࡟ࡼࡾุ
ูࡋࡓࠋ㣗ရᢳฟᾮࡣ㐲ᚰศ㞳ᚋࡢୖΎࢆࣇ࢕ࣝࢱ࣮⁛⳦ࡋ࡚⏝࠸ࡓࠋᅛᙧ㸭༙
ᅛᙧ㣗ရ࡛ࡣ㣗ရୖ࡟⨨࠸ࡓ࣓ࣥࣈࣞࣥୖ࡛⳦ࢆᇵ㣴ࡋࡓࠋ
࠙⤖ᯝࠚୖグ⳦ᰴ࠿ࡽࠊᢠ⏕≀㉁⪏ᛶࡢỈᖹఏ᧛ࡀ㉳ࡇࡿ⤌ࡳྜࢃࡏࢆ㑅ᢥᚋࠊ
ᶵᵓุูᐇ㦂࠿ࡽᙧ㉁㌿᥮࠶ࡿ࠸ࡣ᥋ྜ㸦ྍື໬㸧ࡀ㉳ࡇࡿ௦⾲ⓗ⤌ࡳྜࢃࡏࢆ
ࡑࢀࡒࢀ㸰✀㢮ࡎࡘ㸦ィ㸲✀㢮㸧㑅ᐃࡋࡓࠋḟ࠸࡛ࡇࢀࡽ⳦ᰴࢆ⏝࠸࡚ྛ✀㣗ရ
ᡂศ࡛ࡢᇵ㣴ࢆ⾜࠸ࠊ㑇ఏᏊỈᖹఏ᧛ࡢⓎ⏕ࢆ᳨ドࡋࡓࠋࡑࡢ⤖ᯝࠊྛ✀ᢳฟᾮ
୰ࡸᅛᙧ㣗ရୖ࡛ࡶ㏻ᖖࡢᐇ㦂ᇵᆅ࡜࡯ࡰྠ➼ࣞ࣋ࣝࡢ㑇ఏᏊఏ᧛ࡀ㉳ࡇࡿࡇ
࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡲࡓࡑࡢ୰࡛࣮ࣚࢢࣝࢺࡢᾮయᡂศࡀ㑇ఏᏊỈᖹఏ᧛ࡢ㢧ⴭ
࡞ᢚไຠᯝࢆ♧ࡋࡓࡓࡵࠊࡑࡢ୺࡞ᡂศ࡛࠶ࡿங㓟ࡸ㓑㓟ࡢస⏝࡟㛵ࡋࠊࡉࡽ࡟
ヲ⣽࡞ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊங㓟ࡸ㓑㓟ࡣ㑇ఏᏊỈᖹఏ᧛࡟ᑐࡋࠊሷ㓟࡛ࡣ
ぢࡽࢀ࡞࠸⊂⮬ࡢᢚไస⏝ࢆⓎ᥹ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
࠙ㅰ㎡ࠚᮏ◊✲ࡣࠊ㈈ᅋἲே᪥ᮏ⎔ቃ㈈ᅋࡢ◊✲ຓᡂ㔠࠾ࡼࡧ H23 ዉⰋዪᏊ኱
Ꮫ◊✲᥎㐍ࣉࣟࢪ࢙ࢡࢺ⤒㈝ࡢຓᡂࢆཷࡅ࡚ᐇ᪋ࡋࡓࠋ
䜾䜰䝜䝅䞁䛾⏕యෆ㓟໬ᅉᏊ䛿⬡㉁䝠䝗䝻䝨䝹䜸䜻䝅
䝗䛷䛒䜛
㸯㸳
㻌
‫ۑ‬ᆏᮏᮍ᮶ࠊ⸛ཎ⚈Ꮚࠊᶫᮏᇽྐࠊ㔠ἑ࿴ᶞ㸦⚄ᡞ኱㝔㎰࣭⏕࿨ᶵ⬟㸧
࠙┠ⓗࠚ㑇ఏᏊኚ␗ࡢཎᅉࡢ୍ࡘ࡟ࠊ2’-ࢹ࢜࢟ࢩࢢ࢔ࣀࢩࣥ㸦dG㸧ࡢ 8-ࣄࢻࣟ
࢟ࢩ-2’-ࢹ࢜࢟ࢩࢢ࢔ࣀࢩࣥ㸦8-OHdG㸧࡬ࡢ㓟໬ࡀ࠶ࡿࠋ⏕యෆ㓟໬ᅉᏊࡣࠊ୍
⯡ⓗ࡟ࠊ㐣㓟໬Ỉ⣲࠿ࡽ⏕ࡌࡿࣄࢻࣟ࢟ࢩࣛࢪ࢝ࣝ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ
ࣄࢻࣟ࢟ࢩࣛࢪ࢝ࣝࡢ༙ῶᮇࡣ 1 ࢼࣀ⛊࡜▷ࡃࠊࡲࡓࠊࡑࡢ⏕ᡂ࡟ࡣ㑄⛣㔠ᒓࢆ
ᚲせ࡜ࡍࡿࠋࡋࡓࡀࡗ࡚ࠊ㐣㓟໬Ỉ⣲ࡣ⏕యෆ㓟໬ᅉᏊ࡜⪃࠼࡟ࡃ࠸ࠋ୍᪉ࠊ⬡
㉁ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻࡢศゎ࡛⏕ࡌࡿ࣌ࣝ࢜࢟ࢩࣛࢪ࢝ࣝࡢ༙ῶᮇࡣ 7 ⛊࡛࠶
ࡿࠋᮏ◊✲࡛ࡣࠊ㠀⣽⬊⣔࡜⣽⬊⣔࡛ࠊ㐣㓟໬Ỉ⣲࡜⬡⫫㓟ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻ
ࡢ 8-OHdG ࡢ⏕ᡂ㔞ࢆẚ㍑ࡋࡓࠋࡲࡓࠊdG ࡢ㓟໬ࢆᢚไࡍࡿ⏕యෆ࡛᭷ຠ࡜ᛮ
࠼ࡿᢠ㓟໬ᡂศࢆ᳨⣴ࡋࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚࣜࣀ࣮ࣝ㓟ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻ㸦LAHPO㸧ࡣࠊࣜࣀ࣮ࣝ㓟ࢆ⮬
ື㓟໬ࡋ࡚ᚓࡓ㐣㓟໬≀࠿ࡽࠊHPLC ࡛ 98%௨ୖࡢ⣧ᗘ࡟⢭〇ࡋ࡚⏝࠸ࡓࠋdG
250 μMࠊdG 250 μM ࢆྵࡴ࣏ࣜࢯ࣮࣒ࠊ࠶ࡿ࠸ࡣ 10 μg / ml ࡢ௘࢘ࢩ⬚⭢ DNA
࡟ࠊ50 μM ࡢ LAHPO ࠶ࡿ࠸ࡣ㐣㓟໬Ỉ⣲ࢆῧຍࡋࠊ⏕ᡂࡋࡓ 8-OHdG ࢆ㟁Ẽ໬
Ꮫ᳨ฟჾ࡜ UV ᳨ฟჾࢆഛ࠼ࡓ HPLC ࡛ᐃ㔞ࡋࡓࠋLAHPO ࢆ dG ࡟ῧຍࡍࡿ࡜
105 dG ࠶ࡓࡾ 3.35 ࡢ 8-OHdG ࡀࠊ࣏ࣜࢯ࣮࣒࡛ࡣ 6.04 / 105 dG ࡀࠊ⬚⭢ DNA
࡛ࡣ 27.16 / 105 dG ࡢ⏕ᡂࡀㄆࡵࡽࢀࠊ㐣㓟໬Ỉ⣲ῧຍ࡛ࡣࡑࢀࡒࢀ 1.91 / 105
dGࠊ2.17 / 105 dGࠊ9.28 / 105 dG ࡛࠶ࡗࡓࠋLAHPO ࡣ㐣㓟໬Ỉ⣲ࡼࡾࡶ᭷ព
࡟ 8-OHdG ࢆከࡃ⏕ᡂࡋࡓࠋࡲࡓࠊࡇࢀࡽࡢ⣔࡟ 10 μM ࡢ FeSO4 ࢆຍ࠼ࡿ࡜ࠊ
LAHPO ࡜㐣㓟໬Ỉ⣲ࡢ㛫࡟ 8-OHdG ࡢ⏕ᡂ㔞ࡢᕪࡣ࡞ࡃ࡞ࡗࡓࠋࡑࡇ࡛ࠊࣛࢵ
ࢺ⫢࣑ࢺࢥࣥࢻࣜ࢔࡟ 100 μM ࡢ LAHPO ࢆῧຍࡍࡿ࡜ 34.43 / 105 dG ࡢ 8-OHdG
ࡀࠊ㐣㓟໬Ỉ⣲ࡢῧຍ࡛ࡣ 23.96 / 105 dG ࡀ⏕ᡂࡋࡓࠋࡑࡋ࡚ࠊ࣓ࣝ࢝ࣉࢺࢥࣁ
ࢡ㓟࡛ 24 ᫬㛫ฎ⌮ࡋࡓ HepG2 ⣽⬊࡟ 200 μM ࡢ LAHPO ࢆ୚࠼ࡿ࡜ 0.61 / 105 dG
ࡀࠊ㐣㓟໬Ỉ⣲࡛ࡣ 0.34 / 105 dG ࡢ 8-OHdG ࡀ⏕ᡂࡋࡓࠋࡇࡢࡼ࠺࡟ LAHPO ࡀ
᭷ព࡟ 8-OHdG ࢆ⏕ᡂࡋࡓࠋࡑࡇ࡛ࠊ10 μM ࡢ Į-ࢺࢥࣇ࢙࣮ࣟࣝࠊࣝࢸ࢜ࣜࣥࡲ
ࡓࡣࢣࣝࢭࢳࣥࢆ HepG2 ⣽⬊࡟ 1 ᫬㛫୚࠼ࡓᚋࠊ
ࡑࡢ᰾ࢆ༢㞳ࡋ࡚ࠊ
᰾࡟ 100 μM
ࡢ LAHPO ࢆῧຍࡋ࡚ 8-OHdG ⏕ᡂࢆᐃ㔞ࡍࡿࡇ࡜࡛ᢠ㓟໬ຠᯝࢆ ᐃࡋࡓࠋ࠸
ࡎࢀࡢᡂศࡶ 8-OHdG ⏕ᡂࢆ⣙ 60㸣ᢚ࠼ࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ㑄⛣㔠ᒓࡀ㧗⃰
ᗘ࡛Ꮡᅾࡋ࡞࠸⏕యෆ᮲௳࡛ࡣࠊdG ࡢ㓟໬ᅉᏊࡣ㐣㓟໬Ỉ⣲࡛ࡣ࡞ࡃ⬡㉁ࣄࢻ
ࣟ࣌ࣝ࢜࢟ࢩࢻ࡛࠶ࡾࠊࡲࡓࠊࡑࡢ㓟໬ࢆ Į-ࢺࢥࣇ࢙࣮ࣟࣝࡸ࢝ࢸࢥ࣮ࣝᵓ㐀
ࡢࣇࣛ࣎ࣀ࢖ࢻࡀᢚ࠼ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡓࠋ
ᢪྜᛶ䝣䝷䝪䝜䜲䝗䛾ᣕᢠⓗ⭠⟶྾཰䛻䛴䛔䛶
㻌
㸯㸴
‫ۑ‬ᅵ஭ᙬ཭⨾ ࠊ⸛ཎ⚈Ꮚ ࠊᶫᮏᇽྐ ࠊ㔠ἑ࿴ᶞ 㸦 ⚄ᡞ኱㝔㎰࣭⏕࿨ᶵ
⬟㸧㻌
㻌
࠙┠ⓗࠚ᳜≀ᛶ㣗ရ࡟ྵࡲࢀ࡚࠸ࡿከᵝ࡞ࣇࣛ࣎ࣀ࢖ࢻࡣࠊᢠ㓟໬⬟ࡸࢱࣥࣃࢡ
㉁ᶵ⬟ㄪ⠇స⏝ࢆ௓ࡋࡓ⏕ά⩦័⑓ண㜵ຠᯝࡀᮇᚅࡉࢀ࡚࠸ࡿࠋண㜵ຠᯝࢆᮇᚅ
ࡍࡿ࡞ࡽࡤࠊࡑࡢࣇࣛ࣎ࣀ࢖ࢻࡢయෆ྾཰⃰ᗘࡀ㧗࠸᪉ࡀᮃࡲࡋ࠸ࠋࡲࡓࠊࣇࣛ
࣎ࣀ࢖ࢻࡣయෆ྾཰᫬࡟ࢢࣝࢡࣟࣥ㓟࠶ࡿ࠸ࡣ◲㓟ᢪྜࢆཷࡅࡿࡀࠊᢪྜయࡼࡾ
ࡶ࢔ࢢࣜࢥࣥࡢ᪉ࡀ⏕⌮άᛶࡣ㧗࠸ࡢ࡛ࠊయෆ࢔ࢢࣜࢥࣥ⃰ᗘࡀ㧗࠸ࡇ࡜ࡀዲࡲ
ࡋ࠸ࠋᮏ◊✲࡛ࡣࠊࣇࣛ࣎ࣀ࢖ࢻࡢ୰࡛ࡶᢪྜࢆཷࡅࡸࡍ࠸࡜ሗ࿌ࡉࢀ࡚࠸ࡿࢣ
ࣝࢭࢳࣥ࡟╔┠ࡋࠊࢣࣝࢭࢳࣥࠊࡑࡢㄏᑟయ࠾ࡼࡧ㢮⦕໬ྜ≀ࢆ⤌ࡳྜࢃࡏ࡚ࣛ
ࢵࢺ࡟ᢞ୚ࡍࡿࡇ࡜࡛ࠊࢣࣝࢭࢳࣥࡢయෆᚠ⎔⃰ᗘࢆୖࡆࡿ⤌ࡳྜࢃࡏࢆ᥈ࡋฟ
ࡍࡇ࡜ࢆヨࡳࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚ୍ᬌ⤯㣗ࡉࡏࡓ Wistar ST ࣛࢵࢺ㸦㞝ࠊ11 㐌㱋㸧࡟ࠊ50 mg/kg
య㔜ࡎࡘࡢࢣࣝࢭࢳࣥ࡜࢖ࢯࢣࣝࢭࢳࣥࠊࢣࣝࢭࢳࣥ࡜ࣉࣟࢺ࢝ࢸࢡ㓟ࠊࢣࣝࢭ
ࢳࣥ࡜࢖ࢯࢣࣝࢭࢳࣥ࠾ࡼࡧࣉࣟࢺ࢝ࢸࢡ㓟ࢆ⤌ࡳྜࢃࡏࡓヨᩱࢆࣉࣟࣆࣞࣥ
ࢢࣜࢥ࣮ࣝ࡟⁐ゎࡋ࡚⤒ཱྀᢞ୚ࡋࠊࢣࣝࢭࢳࣥࢆ༢⊂࡛୚࠼ࡓሙྜ࡜ẚ㍑ࡋࡓࠋ
ᢞ୚ᚋ 0.5ࠊ1ࠊ2ࠊ4ࠊ6 ᫬㛫࡟ᑿ㟼⬦᥇⾑ࡋࠊ⾑₢୰ࡢࢣࣝࢭࢳࣥ࢔ࢢࣜࢥࣥ
࡜ࢣࣝࢭࢳࣥᢪྜయ㔞ࢆࠊࢡ࣮ࣟ࢔ࣞ࢖᳨ฟჾࢆഛ࠼ࡓ HPLC ࡛ᐃ㔞ࡋࡓࠋࢣ
ࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ࢖ࢯࢣࣝࢭࢳࣥࢆྠ᫬ᢞ୚ࡍࡿ࡜ࠊ⾑₢୰ࡢࢣࣝ
ࢭࢳࣥᢪྜయ㔞ࡣ᭷ព࡟ቑຍࡋࡓࠋࡲࡓࠊྠ᫬ᢞ୚ࡍࡿ࢖ࢯࢣࣝࢭࢳࣥ㔞ࢆࢣࣝ
ࢭࢳࣥ࡟ᑐࡋ࡚ 1/1ࠊ1/2ࠊ1/4ࠊ1/10 ࡜ࡋ࡚ࠊ0.5ࠊ1ࠊ2ࠊ5 ᫬㛫ᚋࡢࣛࢵࢺࡢ⾑
₢୰ࡢࢣࣝࢭࢳࣥࡢ࢔ࢢࣜࢥࣥ࡜ᢪྜయ㔞ࢆẚ㍑ࡋࡓࠋ࠸ࡎࢀࡢΰྜẚ⋡࡛ࡶࢣ
ࣝࢭࢳࣥᢪྜయ㔞ࡶ࢔ࢢࣜࢥࣥࡢ㔞ࡶኚࢃࡽ࡞࠿ࡗࡓࠋࡋ࠿ࡋࠊࢣࣝࢭࢳࣥ࡜࢖
ࢯࢣࣝࢭࢳࣥࢆ➼㔞ᢞ୚ࡋࡓ⩌ࡢࡳࠊࢣࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ⾑₢୰ࡢ
ࢣࣝࢭࢳࣥᢪྜయ㔞ࡀ᭷ព࡟ቑຍࡋࡓࠋࡑࡇ࡛ࠊࢣࣝࢭࢳࣥ࡟࢔ࣆࢤࢽࣥࠊ࢔ࣆ
ࢤࢽࣥ O-㓄⢾యࡢ࢔ࣆࢤࢺࣜࣥࠊ࢔ࣆࢤࢽࣥ C-㓄⢾యࡢࣅࢸ࢟ࢩࣥࢆΰྜࡋ࡚
ᢞ୚ࡋࡓࠋࢣࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ࢔ࣆࢤࢺࣜࣥࡢྠ᫬ᢞ୚⩌࡛ࢣࣝࢭ
ࢳࣥᢪྜయ㔞ࡢ᭷ព࡞ቑຍࡀほᐹࡉࢀࡓࠋࡑࡋ࡚ࠊࣅࢸ࢟ࢩࣥྠ᫬ᢞ୚࡛ࡣ᭷ព
࡞ᙳ㡪ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ௨ୖࡢࡼ࠺࡟ࠊࣇࣛ࣎ࣀ࢖ࢻ O-㓄⢾యࡢྠ᫬ᢞ୚
ࡣࢣࣝࢭࢳࣥࡢయෆ⃰ᗘࢆቑຍࡉࡏࠊࡑࡢᢪྜయ㔞ࢆቑຍࡉࡏࡿࡀࠊ࢔ࢢࣜࢥࣥ
㔞࡟ࡣᙳ㡪ࢆ୚࠼࡞࠿ࡗࡓࠋ
Interaction of wheat E-amylase with maltose and
glucose as examined by fluorescence
㸯㸵
‫ۑ‬Tadessa Daba, Kenji Kojima, and Kuniyo Inouye 䠄Division of Food
Science and Biotechnology, Graduate School of Agriculture, Kyoto
University䠅
࠙ObjectiveࠚE-Amylases [EC 3.2.1.2] are exo-acting enzymes, with successive removal
of E-anomeric maltose from the non-reducing ends of polysaccharides. The fluorescence
quenching effects of maltose and glucose on wheat E-amylase (WBA) were studied by
fluorescence titration. The temperature and pH-dependences of the dissociation
constants (Kd) of WBA-maltose and WBA-glucose complexes were evaluated.
࠙ Methods and Results ࠚ The source of WBA, Himaltosin which is an
industrially-applicable commercial preparation from wheat bran, was purchased from
HBI Enzymes, Osaka1). The fluorescence emission of WBA was observed by excitation
at 280 nm at 25oC, pH 5.4. The fluorescence intensity (FI) of WBA was partly quenched
by the addition of maltose and glucose. The Kd values of the enzyme-inhibitor
complexes (EI) dissociations determined by titration of FI were 0.20s0.12 M for
maltose and 0.36s0.11 M for glucose. The Kd values slightly increased while the Gibbs
energy changes (¨Go) decreased with increasing temperature. The respective Kd values
were 0.22s0.09, 0.20s0.12, and 0.17s0.04 M for maltose and 0.29s0.04, 0.36s
0.11, 0.26s0.07 M for glucose at pH 3.0, 5.4, and 9.0, respectively at 25oC. The
inhibitor constants (Ki) of our previous inhibition kinetics data2) are in reasonable
agreement with the Kd values obtained in this study. The ¨Ho and ¨Go values indicate
that the EI dissociations are endothermic and enthalpy-driven. The temperature and
pH-dependences of the Kd values suggest that the changes in conformation and the
environment of tryptophan and/or tyrosine residues around the binding site of WBA are
governed by pH and temperature. It is therefore, essential to consider the temperature
and pH of starch hydrolysis to reduce the end-product inhibition.
1. Daba T, Kojima K, Inouye K, Enzyme. Microb. Technol. 51, 245-251 (2012)
2. Daba T, Kojima K, Inouye K, submitted
⫧‶䛻క䛖⅖⑕≧ែ䛜⬡⫫⤌⧊䛻䛚䛡䜛 UCP1 Ⓨ⌧
ㄏᑟ䛻୚䛘䜛ᙳ㡪㻌
㸯㸶
㻌
‫୸ۑ‬㔝᫭Ⴙ ࠊᆏᮏᬛᘺ ࠊᚋ⸨๛ ࠊ㧗ᶫಙஅ ࠊἙ⏣↷㞝 㸦ி኱
㝔࣭㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧
࠙┠ⓗࠚ့ங㢮࡟Ꮡᅾࡍࡿ〓Ⰽ⬡⫫⣽⬊ࡣࠊࡑࡢ࣑ࢺࢥࣥࢻࣜ࢔ෆ⭷࡟Ꮡᅾࡍࡿ
uncoupling protein-1 (UCP1) ࡢാࡁ࡟ࡼࡾ࢚ࢿࣝࢠ࣮ࢆᾘ㈝ࡋࠊ⇕ࢆ⏘⏕ࡍ
ࡿࠋUCP1 ࢆⓎ⌧ࡍࡿ⬡⫫⣽⬊ࡣࠊ〓Ⰽ⬡⫫⤌⧊ (BAT) ࡜ࡋ࡚⫪⏥㦵࿘㎶࡞࡝
࡟Ꮡᅾࡍࡿࠋࡉࡽ࡟ᐮ෭࡞࡝ࡢ่⃭࡟ࡼࡾࠊ㰡㋣㒊ⓑⰍ⬡⫫⤌⧊ (I-WAT) ࡞࡝
୍㒊ࡢⓑⰍ⬡⫫⤌⧊࡟࠾࠸࡚ࡶࠊUCP1 ࡢⓎ⌧㔞ࡀቑຍࡋࠊ࢚ࢿࣝࢠ࣮ᾘ㈝㔞ࡀ
ቑ኱ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ୍᪉࡛ࠊ⫧‶࡜ UCP1 ࡢⓎ⌧㔞ࡢ㛫࡟ࡣ㧗࠸
┦㛵ࡀࡳࡽࢀࠊ⫧‶≧ែࡢ⬡⫫⤌⧊࡛ࡣ UCP1 ࡢⓎ⌧ࡀపୗࡋ࡚࠸ࡿࠋᮏ◊✲
࡛ࡣࠊࡑࡢ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚ࠊ⫧‶≧ែࡢ⬡⫫⤌⧊࡛
⏕ࡌࡿ⅖⑕≧ែ࡟╔┠ࡋࠊ⅖⑕≧ែࡀ BAT ࠾ࡼࡧ I-WAT ࡢ୧⬡⫫⤌⧊࡟࠾ࡅࡿ
UCP1 ࡢⓎ⌧ㄏᑟ࡟୚࠼ࡿᙳ㡪ࢆື≀ಶయ᳨࡛ࣞ࣋ࣝウࡋࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚ⫧‶≧ែࡢ BAT ࠾ࡼࡧ I-WAT ࡢ୧⬡⫫⤌⧊࡟࠾ࡅࡿ UCP1 Ⓨ
⌧ㄏᑟ࡟ࡘ࠸࡚ㄪ࡭ࡿࡓࡵࠊ㧗⬡⫫㣗 (HFD) ࢆ 16 㐌㛫ᦤ㣗ࡉࡏࡓ C57BL/6J
࣐࢘ࢫࢆ⏝࠸ࡓ᳨ウࢆ⾜ࡗࡓࠋ࣐࢘ࢫ࡟ᐮ෭่⃭ (4Υࠊ24 ᫬㛫) ࢆ୚࠼ࡿ࡜ࠊ
HFD ᦤ㣗⩌ࡢ BAT ࠾ࡼࡧ I-WAT ࡛ㄏᑟࡉࢀࡿ UCP1 ࡢⓎ⌧㔞ࡀࠊᬑ㏻㣗 (ND)
ᦤ㣗⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ῶᑡࡋࡓࠋࡲࡓࠊHFD ᦤ㣗⩌ࡢ BAT ࠾ࡼࡧ I-WAT ࡛
ࡣࠊ F4/80 (࣐ࢡࣟࣇ࢓࣮ࢪࡢ࣐࣮࣮࢝㑇ఏᏊ) ࡜⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥ tumor
necrosis factor D (TNFD) mRNA ࡢⓎ⌧㔞ࡀ ND ᦤ㣗⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍ
ࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊ࣐ࢡࣟࣇ࢓࣮ࢪࡀᾐ₶ࡋ⅖⑕≧ែࡀច㉳ࡉࢀࡓ BAT ࠾
ࡼࡧ I-WAT ࡛ࡣࠊᐮ෭่⃭࡟ࡼࡾ⏕ࡌࡿ UCP1 ࡢⓎ⌧ㄏᑟࡀᢚไࡉࢀࡿྍ⬟ᛶ
ࡀ⪃࠼ࡽࢀࡓࠋ
ḟ࡟࣐ࢡࣟࣇ࢓࣮ࢪࡀᾐ₶ࡋ⅖⑕≧ែࡀច㉳ࡉࢀࡓ⬡⫫⤌⧊࡛Ⓨ⌧ࡀቑຍࡍ
ࡿ TNFDࡀࠊBAT ࠾ࡼࡧ I-WAT ࡟࠾ࡅࡿ UCP㸯Ⓨ⌧ㄏᑟ࡟୚࠼ࡿᙳ㡪ࢆㄪ࡭
ࡓࠋ㠀⫧‶≧ែࡢ 5 㐌㱋 C57BL/6J ࣐࢘ࢫ࡟ TNFDࢱࣥࣃࢡ㉁mg/kg body
weightࢆ⭡⭍ෆ࡟ᢞ୚ࡋࡓࠋࡑࡢ⤖ᯝࠊBAT ࠾ࡼࡧ I-WAT ࡟࠾࠸࡚ᐮ෭่⃭
࡟ࡼࡾㄏᑟࡉࢀࡿ UCP1 ࡢⓎ⌧㔞ࡀࠊ⏕⌮㣗ሷỈࢆᢞ୚ࡋࡓ࣐࢘ࢫ࡜ẚ㍑ࡋ࡚
᭷ព࡟ῶᑡࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊ⫧‶࡟ࡼࡗ࡚⏕ࡌࡿ⅖⑕≧ែࡀ BAT ࠾ࡼࡧ
I-WAT ࡢ୧⬡⫫⤌⧊࡟࠾࠸࡚ࠊUCP1 Ⓨ⌧ㄏᑟࢆᢚไࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ
䝯䝍䝪䝻䞊䝮ゎᯒ䜢⏝䛔䛯 PPAR άᛶ໬䛻䜘䜚ኚື䛩
䜛⏕యෆ௦ㅰ≀䛾᥈⣴㻌
㸯㸷
㻌
‫ۑ‬ᒣ㷂㝧ኴࠊ㧗ᶫ᫓ᘺࠊᚋ⸨๛ࠊ㧗ᶫಙஅࠊᰘ⏣኱㍜ࠊἙ⏣↷㞝
㸦 ி኱㝔㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫࠊ ࠿ࡎࡉ '1$ ◊㸧㻌
࠙┠ⓗࠚPeroxisome proliferator-activated receptor㸦PPAR㸧ࡣࠊ᰾ෆཷᐜయ
ᆺ㌿෗ㄪ⠇ᅉᏊ࡛࠶ࡾࠊ⢾࣭⬡㉁௦ㅰࡢ୺せ࡞ไᚚᅉᏊ࡛࠶ࡿࠋPPAR ࡟ࡣࠊĮࠊ
Ȗࠊį ࡢ 3 ࡘࡢࢧࣈࢱ࢖ࣉࡀᏑᅾࡍࡿࠋࡑࡢ୰࡛ࠊPPARȖ ࡣ୺࡟⬡⫫⤌⧊࡟Ⓨ⌧
ࡋࠊ⬡⫫⣽⬊ศ໬ࡸ࢖ࣥࢫࣜࣥឤཷᛶࡢㄪ⠇࡟㛵୚ࡋ࡚࠸ࡿࠋࡑࡢࡓࡵࠊPPARȖ
ࡢ⏕⌮ᶵ⬟ࢆ⌮ゎࡍࡿࡇ࡜ࡣࠊ⢾ᒀ⑓ࡸ⬡㉁␗ᖖ⑕࡜࠸ࡗࡓ⏕ά⩦័⑓ࡢண㜵࣭
ᨵၿ࡟ࡘ࡞ࡀࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿࠋPPARȖ άᛶ໬ࡣࠊ୍⯡ⓗ࡟ࠊࡑࡢᶆⓗ㑇ఏ
ᏊࡢⓎ⌧ኚື࡛ホ౯ࡉࢀࡿࠋࡋ࠿ࡋࠊPPARȖ ࡢ⏕⌮ᶵ⬟ࢆໟᣓⓗ࡟⌮ゎࡍࡿࡓ
ࡵ࡟ࡣࠊ⏕࿨ࢩࢫࢸ࣒ࡢ᭱⤊⏘≀࡛࠶ࡿ௦ㅰ≀ࡢ࡛ࣞ࣋ࣝࠊPPARȖ άᛶ໬ࢆホ
౯ࡍࡿࡇ࡜ࡶ㔜せ࡛࠶ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ௦ㅰ඲యࢆಠ▔ⓗ࡟ᢕᥱࡍࡿࡇ࡜
ࡀ࡛ࡁࡿ࣓ࢱ࣮࣒࣎ࣟゎᯒࢆ⏝࠸࡚ࠊPPARȖ άᛶ໬࡟ࡼࡾኚືࡍࡿ⏕యෆ௦ㅰ
⏘≀ࡢ᥈⣴ࢆ⾜ࡗࡓࠋ
࠙᪉ἲ࣭⤖ᯝࠚPPARȖ ࢔ࢦࢽࢫࢺ࡛࠶ࡿ pioglitazone㸦௨ୗ pio㸧ࢆ⫧‶ࣔࢹࣝ
࣐࢘ࢫ࡟ᢞ୚ࡋࠊ4 㐌㛫㣫⫱ࡋࡓࠋࡑࡢᚋࠊゎ๗ࢆ⾜࠸ࠊ⬡⫫⤌⧊ᢳฟ≀ࡢศᯒ
࡟ࡼࡾࠊ⸆๣ࡀ⬡⫫⤌⧊࡬฿㐩ࡋ࡚࠸ࡿࡇ࡜ࢆ☜ㄆࡋࡓࠋࡉࡽ࡟ࠊ⬡⫫⤌⧊୰ࡢ
ᶆⓗ㑇ఏᏊⓎ⌧ࡀ᭷ព࡟ቑຍࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊPPARȖ ࡀάᛶ໬ࡉࢀ࡚࠸ࡿࡇ
࡜ࢆ☜ㄆࡋࡓࠋࡑࡢᚋࠊࡇࡢ࣐࢘ࢫࡢ⾑₢࣭⬡⫫⤌⧊ࢆ 80% methanol ࡛ᢳฟ
ࡋࠊLC-MS ࢆ⏝࠸࡚௦ㅰ≀ࡢ⥙⨶ⓗ࡞ゎᯒ㸦࣓ࢱ࣮࣒࣎ࣟゎᯒ㸧ࢆ⾜ࡗࡓࠋゎ
ᯒࡢ⤖ᯝࠊpio ࢆᢞ୚ࡋࡓ࣐࢘ࢫࡢ⾑୰࣭⬡⫫⤌⧊୰࡟࠾࠸࡚ࠊ࢔࣑ࣀ㓟ࡀ᭷ព
࡟ቑຍࡍࡿࡇ࡜ࢆぢฟࡋࡓࠋḟ࡟ࠊቑຍࡋࡓ࢔࣑ࣀ㓟ࡀ⬡⫫⣽⬊⏤᮶࡛࠶ࡿ࠿ࢆ
࣐࢘ࢫ⏤᮶๓㥑⬡⫫⣽⬊ 3T3-L1 ࢆ⏝࠸᳨࡚ウࡋࡓࠋศ໬ㄏᑟᚋࡢ 3T3-L1 ⬡⫫
⣽⬊࡟ pio ࢆ୍㐌㛫ῧຍࡋࡓᚋࠊ⣽⬊ෆࡢ௦ㅰ≀ࢆ 80% methanol ࡛ᢳฟࡋࠊ
LC-MS ࢆ⏝࠸࡚ゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊpio ῧຍ࡟ࡼࡾࠊື≀ᐇ㦂࡛ቑຍࡀぢࡽ
ࢀࡓ࢔࣑ࣀ㓟ࡀ᭷ព࡟ቑຍࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊࡇࢀࡽࡢ࢔࣑ࣀ㓟
ࡢኚືࡣࠊpio ᢞ୚࣐࢘ࢫࡢ⫢⮚୰࡟࠾࠸࡚ㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡉࡽ࡟ࠊPPARĮ
࢔ࢦࢽࢫࢺᢞ୚࣐࢘ࢫࡢ⾑୰ࠊ⫢⮚୰ࠊ⬡⫫⤌⧊୰ࡢ࣓ࢱ࣮࣒࣎ࣟゎᯒࢆ⾜ࡗࡓ
⤖ᯝࠊPPARĮ ࢔ࢦࢽࢫࢺࡢᢞ୚࡛ࡣࠊpio ᢞ୚࡟ࡼࡾቑຍࡋࡓ࢔࣑ࣀ㓟ࡢኚື
ࡀㄆࡵࡽࢀ࡞࠸ࠊࡲࡓࡣኚື⋡ࡀᑠࡉ࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ
PPARȖ ࡢ᪂ࡓ࡞ᶵ⬟࡜ࡋ࡚㸪⬡⫫⣽⬊࡟࠾ࡅࡿ࢔࣑ࣀ㓟௦ㅰ࡬ࡢ㛵୚ࡀ♧၀ࡉ
ࢀࡓࠋ