細胞周期移行時の分子発現異常を起点とした短期発がん予測系の開発に関する研究

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Title

細胞周期移行時の分子発現異常を起点とした短期発がん予

_{測系の開発に関する研究( 本文(Fulltext) )}

Author(s)

木村, 真之

Report No.(Doctoral

Degree)

博士(獣医学) 甲第456号

Issue Date

2016-03-14

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/54529

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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⣽⬊࿘ᮇ⛣⾜᫬ࡢศᏊⓎ⌧␗ᖖࢆ㉳Ⅼ࡜ࡋࡓ

▷ᮇⓎࡀࢇண ⣔ࡢ㛤Ⓨ࡟㛵ࡍࡿ◊✲

2015 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉

㸦ᮾி㎰ᕤ኱Ꮫ㸧

ᮌᮧ ┿அ

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1 ┠ḟ ᗎㄽ 3 ➨1 ❶ ࣛࢵࢺࡢ⫢Ⓨࡀࢇ≀㉁ᢞ୚᪩ᮇ࠿ࡽ⏕ࡌࡿ⣽⬊࿘ᮇไᚚ␗ᖖ࡜ࡑࡢฟ⌧᫬ᮇ ࡢ᳨ウ ⥴ゝ 7 ᮦᩱ࠾ࡼࡧ᪉ἲ 9 ⤖ᯝ 15 ⪃ᐹ 26 ᑠᣓ 34 ➨2 ❶ ࣛࢵࢺࡢ᭱኱ 90 ᪥㛫཯᚟ᢞ୚౛࡛ࡢ⫢Ⓨࡀࢇ≀㉁࣭⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮ࡢ ⣽⬊࿘ᮇไᚚ␗ᖖ࡬ࡢ㛵୚ࡢ᳨ウ ⥴ゝ 36 ᮦᩱ࠾ࡼࡧ᪉ἲ 37 ⤖ᯝ 43 ⪃ᐹ 51 ᑠᣓ 61 ➨3 ❶ ࣛࢵࢺࡢ⭈Ⓨࡀࢇ≀㉁ᢞ୚᪩ᮇ࠿ࡽ⏕ࡌࡿ⣽⬊࿘ᮇไᚚ␗ᖖ࡜ࡑࡢฟ⌧᫬ᮇ ࡢ᳨ウ

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2 ⥴ゝ 64 ᮦᩱ࠾ࡼࡧ᪉ἲ 65 ⤖ᯝ 70 ⪃ᐹ 78 ᑠᣓ 86 ⥲ྜ⪃ᐹ 87 ⤖ㄽ 93 ㅰ㎡ 96 ᘬ⏝ᩥ⊩ 97 せ᪨ 111 Abstract 115 ᅗ⾲ 121

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3 ᗎㄽ ࡆࡗṑ㢮ࢆ⏝࠸ࡓ㛗ᮇ㛫ࡀࢇཎᛶヨ㦂ࡣ, ໬Ꮫ≀㉁ࡢⓎࡀࢇᛶண ࡟ᗈࡃ⏝࠸ࡽࢀ ࡚࠸ࡿࡀ, ᢞ୚ࡀ㛗ᮇ㛫࡟ཬࡪࡇ࡜࠿ࡽ, ࢥࢫࢺࡸホ౯ࡢຠ⋡ᛶ➼࡛ㄢ㢟ࡀ࠶ࡿࠋࡇࢀ ࡽࡢ௦᭰ヨ㦂ἲ࡜ࡋ࡚, ࢺࣛࣥࢫࢪ࢙ࢽࢵࢡື≀ࡸ㑇ఏᏊࣀࢵࢡ࢔࢘ࢺື≀ࢆ⏝࠸ࡓ ▷ᮇヨ㦂⣔ [14] , ⫢୰ᮇⓎࡀࢇᛶヨ㦂ἲࡸከ⮚ჾ୰ᮇⓎࡀࢇᛶヨ㦂ࡢࡼ࠺࡞஧ẁ㝵Ⓨ ࡀࢇࣔࢹࣝ [72] ࡀ㛤Ⓨࡉࢀ࡚࠸ࡿࡀ, ࡇࢀࡽࡶࡲࡓ㧗ࢥࢫࢺ࡛, ከࡃࡢ᫬㛫ࢆᚲせ࡜ ࡋ, ホ౯ྍ⬟࡞ᶆⓗ⮚ჾࡀ㝈ࡽࢀ࡚࠸ࡿࠋ⌧ᅾ, ⫢⮚ࡸ⭈⮚᳨࡛ウࡀ㐍ࡵࡽࢀ࡚࠸ࡿࢺ ࢟ࢩࢥࢤࣀ࣑ࢡࢫⓗゎᯒ࡟ࡼࡿⓎࡀࢇᛶண ᡭἲࡣከᩘࡢⓎࡀࢇᶆⓗ࡟ᑐࡋ࡚Ⓨࡀࢇ ᛶࡢண ࡀᮇᚅ࡛ࡁࡿࡀ [41, 42, 77] , ࡇࡢᡭἲࡶࡲࡓ㧗ࢥࢫࢺ࡛࠶ࡾ, ࡉࡽ࡟␗࡞ࡿ ◊✲ᶵ㛵࡛ࢹ࣮ࢱࢆඹ᭷ࡍࡿࡓࡵ࡟ࡣ⤫ྜⓗ᪉ἲࡀᚲせ࡛࠶ࡿࠋࡋࡓࡀࡗ࡚, ▷ᮇ㛫࡛ ྜ⌮ⓗ࡟ㅖ⮚ჾ඲⯡࡟ࢃࡓࡿⓎࡀࢇᛶࢆண ࡋᚓࡿᣦᶆࡢ☜❧ࡀồࡵࡽࢀ࡚࠸ࡿࠋ ᡤᒓ◊✲ᐊࡢඛ⾜◊✲࡟࠾࠸࡚, 」ᩘ✀㢮ࡢⓎࡀࢇᶆⓗᛶࢆᑐ㇟࡜ࡋࡓゎᯒ࡛, 28 ᪥ 㛫ࡢ཯᚟ᢞ୚࡟ࡼࡾⓎࡀࢇࡢᶆⓗ⣽⬊࡟ቑṪάᛶࡢஹ㐍ࢆ♧ࡍⓎࡀࢇ≀㉁ࡣ, ⣽⬊࿘ ᮇࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢάᛶ໬ࢆ཯ᫎࡍࡿࡼ࠺࡞⣽⬊࿘ᮇ㛵㐃ศᏊࡢⓎ⌧ቑຍ࠾ࡼ ࡧ࢔࣏ࢺ࣮ࢩࢫࢆㄏⓎࡍࡿࡇ࡜ࢆぢฟࡋࡓ [73, 74, 82, 83] ࠋࡉࡽ࡟, ⫢⮚࡜⭈⮚ࡢⓎࡀ ࢇ≀㉁ᢞ୚౛࡛, M ᮇ㐍⾜࡟㛵ࢃࡿ ubiquitin D (UBD) ࡢ G2ᮇ࠿ࡽࡢ␗ᖖⓎ⌧ࢆぢฟࡋ ࡓ [74] ࠋ௨ୖࡢ⤖ᯝࡼࡾ, Ⓨࡀࢇ≀㉁ࡣࡑࡢᶆⓗᛶࢆၥࢃࡎ, ▷ᮇ㛫ࡢᢞ୚࡟ࡼࡾⓎ ࡀࢇᶆⓗ⣽⬊࡟ᑐࡋ࡚ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏ࣥࢺᶵᵓࡢ◚⥢ࢆㄏⓎࡍࡿྍ⬟ᛶࢆ

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4 ぢฟࡋࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ, ⣽⬊ቑṪάᛶࢆஹ㐍ࡍࡿⓎࡀࢇ≀㉁ࡀ M ᮇ㐍⾜࡟㛵ࢃࡿ ศᏊࡢᶵ⬟㞀ᐖࢆྵࡴ⣽⬊࿘ᮇไᚚ␗ᖖࢆㄏⓎࡋ, ࡑࢀࡀⓎࡀࢇ࣓࢝ࢽࢬ࣒ࡢ᪩ᮇ㐣 ⛬࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽ, Ⓨࡀࢇ≀㉁≉␗ⓗ࡞⣽⬊࿘ᮇไ ᚚ◚⥢ࡢ⾲⌧ᆺࡢᡂ❧᫬ᮇ࡟ࡘ࠸࡚ࡣᮍ᳨ウ࡞ࡲࡲ࡛࠶ࡿࠋ ᮏ◊✲࡛ࡣ, ໬Ꮫ≀㉁࡟ࡼࡿⓎࡀࢇࡢ୺せ࡞ᶆⓗ⮚ჾ࡛࠶ࡿ⫢⮚࡜⭈⮚ࢆᑐ㇟࡜ࡋ ࡚, 3 ❶❧࡚ࡢ◊✲ࢆᐇ᪋ࡋࡓࠋ➨ 1 ❶࡛ࡣ, ⫢Ⓨࡀࢇ≀㉁ᢞ୚ึᮇ࡟ㄏⓎࡉࢀࡿ⫢⣽⬊ ቑṪ᫬࡟⏕ࡌࡿ, Ⓨࡀࢇ≀㉁≉␗ⓗ࡞⣽⬊࿘ᮇไᚚ◚⥢ࡢ⾲⌧ᆺࡢᡂ❧᫬ᮇ࡜ࡑࡢ୺ せ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ┠ⓗ࡜ࡋ, Ⓨࡀࢇᶆⓗ࡛࠶ࡿ⫢⣽⬊ࢆᑐ㇟࡜ࡋ࡚, ⣽⬊ቑṪάᛶ, ⣽⬊࿘ᮇ㛵㐃ศᏊⓎ⌧࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢኚືࢆ⤒᫬ⓗ࡟ゎᯒࡋࡓࠋࡍ࡞ࢃࡕ, ࣛࢵ ࢺࢆ⏝࠸࡚, ⫢Ⓨࡀࢇ≀㉁࡞࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࢆ 3, 7 ࡞࠸ࡋ 28 ᪥㛫཯᚟ᢞ୚ ࡍࡿ⩌࡜, ෌⏕ᛶ⫢⣽⬊ቑṪࢆㄏⓎࡍࡿ⫢㒊ศษ㝖ࢆ⾜࠺⩌ࢆタᐃࡋ, ᐇ㦂⤊஢ᚋ࡟, ྲྀ ࡾ ฟ ࡋ ࡓ ⫢ ⮚ ࢆ ⏝ ࠸ ࡚, ච␿⤌⧊໬Ꮫᰁ Ⰽ ࠾ࡼࡧ real-time reverse transcription polymerase chain reaction (RT-PCR) ࡟ࡼࡾゎᯒࡋࡓࠋ➨ 2 ❶࡛ࡣ, ➨ 1 ❶࡛ぢฟࡉࢀࡓ཯ ᛂࡀ, ⣽⬊ቑṪㄏⓎᛶ࡟ஈࡋ࠸⫢Ⓨࡀࢇ≀㉁ࡸ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮࡟ࡼࡗ࡚ࡶ⏕ࡌ ࡿ࠿ྰ࠿ࢆ᳨ウࡍࡿࡓࡵ࡟, ࣛࢵࢺ࡟, ⫢Ⓨࡀࢇ≀㉁, ᙅ࠸⫢Ⓨࡀࢇᛶࡀ♧၀ࡉࢀ࡚࠾ ࡾ⣽⬊ቑṪㄏⓎᛶࡀஈࡋ࠸ࡇ࡜ࡀண᝿ࡉࢀࡿ≀㉁, ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮࡞࠸ࡋ㠀Ⓨ ࡀࢇᛶ⫢ẘᛶ≀㉁ࢆ7, 28 ࡞࠸ࡋ 90 ᪥㛫཯᚟ᢞ୚ࡋࡓࠋᐇ㦂⤊஢ᚋ࡟, ྲྀࡾฟࡋࡓ⫢⮚ ࢆ⏝࠸࡚➨ 1 ❶࡜ྠᵝࡢゎᯒࢆ㐍ࡵ, ⤒᫬ⓗ࡞ほᐹ࡟ࡼࡾ, Ⓨࡀࢇ≀㉁࡟≉␗ⓗ࡞ᣦ

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5 ᶆ࡜ࡑࡢ཯ᛂᛶࡢ᭷↓ࢆ᳨ウࡋࡓࠋ➨3 ❶࡛ࡣ, ⫢Ⓨࡀࢇ≀㉁࡛ぢฟࡉࢀࡓ⤒᫬ⓗ࡞཯ ᛂࡀ⭈Ⓨࡀࢇ≀㉁࡛ࡶ⏕ࡌࡿ࠿ྰ࠿ࢆ᳨ウࡍࡿࡓࡵ࡟, ࣛࢵࢺ࡟, ⭈Ⓨࡀࢇ≀㉁࡞࠸ࡋ 㠀Ⓨࡀࢇᛶ⭈ẘᛶ≀㉁ࢆ3, 7 ࡞࠸ࡋ 28 ᪥㛫཯᚟ᢞ୚ࡋࡓࠋᐇ㦂⤊஢ᚋ࡟, ྲྀࡾฟࡋࡓ ⭈⮚ࢆ⏝࠸࡚➨1 ❶࡜ྠᵝࡢゎᯒࢆ㐍ࡵ, 㧊㉁እᖏእᒙ࡟࠾ࡅࡿᒀ⣽⟶ୖ⓶⣽⬊ࢆᑐ㇟ ࡜ࡋࡓ⤒᫬ⓗ࡞ほᐹ࡟ࡼࡾ, Ⓨࡀࢇ≀㉁࡟≉␗ⓗ࡞ᣦᶆ࡜ࡑࡢ཯ᛂᛶࡢ᭷↓ࢆ᳨ウࡋ ࡓࠋ

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6 ➨1 ❶

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7 ⥴ゝ ࣛࢵࢺ࡬ࡢⓎࡀࢇ≀㉁ࡢ཯᚟ᢞ୚᪩ᮇ࠿ࡽ, 㑇ఏẘᛶㄏⓎᛶࡢ᭷↓ࢆၥࢃࡎᶆⓗ⣽ ⬊࡟ᕧ኱᰾ࡢᙧᡂࡀㄏⓎࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾ [3, 4] , ⫢⮚࠾ࡼࡧ⭈⮚࡟࠾࠸ ࡚ࡋࡤࡋࡤㄆࡵࡽࢀࡿࠋ⭈Ⓨࡀࢇ≀㉁ࡣ, ᒀ⣽⟶ᛶ⭈⑕ࢆㄏⓎࡍࡿࢱ࢖ࣉ࡜ᒀ⣽⟶ୖ⓶ ⣽⬊࡟ᕧ኱᰾ࢆㄏⓎࡍࡿࢱ࢖ࣉࡢ2 ࡘ࡟ศ㢮ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [73] ࠋ⭈Ⓨ ࡀࢇ≀㉁࡛࠶ࡿochratoxin A (OTA) ࡣᕧ኱᰾ࢆㄏⓎࡍࡿࡀ, ࡇࡢኚ໬ࡣ᰾ศ⿣ࡢ␗ᖖ࡜ ࡑࢀ࡟క࠺ᰁⰍయ␗ᩘᛶࢆ཯ᫎࡍࡿࡶࡢ࡛࠶ࡾ, ⣽⬊࿘ᮇ㛵㐃ศᏊ, ≉࡟ G2ᮇ࠾ࡼࡧ M ᮇ㛵㐃ศᏊࡢⓎ⌧␗ᖖ࡟㝶కࡋ࡚⏕ࡌࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿ [3] ࠋࡲࡓ, ࡀࢇཎᛶ ヨ㦂ࡢ᪩ᮇẁ㝵࡟࠾࠸࡚⫢Ⓨࡀࢇ≀㉁ࡣࡋࡤࡋࡤ, ⫢⣽⬊ࡢ ”ᕧ኱⣽⬊/ᕧ኱᰾” ࢆㄏ Ⓨࡍࡿࡇ࡜࠿ࡽ [4] , ⣽⬊࿘ᮇ␗ᖖࡀⓎࡀࢇ㐣⛬ࡢ᪩ᮇ࡟⏕ࡌࡿ⌧㇟࡛࠶ࡿࡇ࡜ࡇ࡜ ࡀ᥎ᐹࡉࢀࡓࠋ⣽⬊ቑṪ㐣⛬࡛, DNA ᦆയࡀ⵳✚ࡋ࡚࠸ࡿ≧ែ࡛ G2/M ࢳ࢙ࢵࢡ࣏࢖ࣥ ࢺᶵᵓ࠿ࡽࡢࡍࡾᢤࡅࡀ⏕ࡌࡿࡇ࡜࡟ࡼࡾ, ᰁⰍయࡢ୙ศ㞳ࡸ⣽⬊㉁ศ⿣ࡢ␗ᖖࡀ⏕ ࡌ, ࡑࢀ࡟ࡼࡾ͆tetraploidy/aneuploidy” ࡜ᘬࡁ⥆ࡃᰁⰍయ୙ᏳᐃᛶࡀㄏⓎࡉࢀࡿࡇ࡜ ࡀ♧၀ࡉࢀ࡚࠸ࡿ [26] ࠋࡇࢀࡽࡢ▱ぢࡼࡾ, Ⓨࡀࢇ≀㉁ࡢᢞ୚᪩ᮇ࠿ࡽ⏕ࡌࡿ “ᕧ኱ ᰾” ࡢㄏⓎ࡟㛵୚ࡍࡿ⣽⬊࿘ᮇไᚚ␗ᖖࡀ, Ⓨࡀࢇ࡟㛵୚ࡋ࡚࠸ࡿ࡜࠸࠺௬ㄝࡀᡂ❧ ࡍࡿࠋ ୖグࡢ௬ㄝࢆᇶ࡟ᡤᒓ◊✲ᐊ࡛ࡣ, ඛ⾜◊✲࡟࠾࠸࡚, 28 ᪥㛫ࡢ཯᚟ᢞ୚࡟ࡼࡾᶆⓗ

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8

⣽⬊࡟ቑṪάᛶࡢஹ㐍ࢆ♧ࡍⓎࡀࢇ≀㉁ࡣ, ᶆⓗ⮚ჾࢆၥࢃࡎ, ⣽⬊࿘ᮇࢳ࢙ࢵࢡ࣏࢖ ࣥࢺࡢάᛶ໬ࢆ཯ᫎࡍࡿ⣽⬊࿘ᮇ㛵㐃ศᏊࡢⓎ⌧␗ᖖ࡜࢔࣏ࢺ࣮ࢩࢫࢆㄏⓎࡍࡿࡇ࡜ ࢆぢฟࡋࡓ [73, 74, 82, 83] ࠋࡉࡽ࡟, ⭈ᒀ⣽⟶ୖ⓶⣽⬊ࢆⓎࡀࢇᶆⓗ࡜ࡍࡿ OTA ࠾ࡼ ࡧ⫢⣽⬊ࢆⓎࡀࢇᶆⓗ࡜ࡍࡿ thioacetamide (TAA) ࡀ, M ᮇ㐍⾜࡟㛵ࢃࡿ ubiquitin D (UBD) ࡢ G2ᮇ࠿ࡽࡢ␗ᖖⓎ⌧࡟㉳ᅉࡍࡿ, M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏ࣥࢺᶵᵓࡢ◚⥢ ࢆㄏⓎࡋ࡚࠸ࡿྍ⬟ᛶࡶぢฟࡋ࡚࠸ࡿ [74] ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ, ⣽⬊ቑṪࢆஹ㐍ࡍࡿ Ⓨࡀࢇ≀㉁ࡣ, Ⓨࡀࢇࡢᶆⓗ⮚ჾࢆၥࢃࡎ M ᮇ㐍⾜࡟㛵ࢃࡿศᏊࡢᶵ⬟㞀ᐖࢆྵࡴ⣽ ⬊࿘ᮇไᚚ␗ᖖࢆㄏⓎࡋ, ࡑࢀࡀⓎࡀࢇᶵᗎࡢ᪩ᮇ㐣⛬࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉ ࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽ, Ⓨࡀࢇ≀㉁≉␗ⓗ࡞⣽⬊࿘ᮇไᚚ␗ᖖࡢ⾲⌧ᆺࡢᡂ❧᫬ᮇ࡟ࡘ࠸ ࡚ࡣ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࠋ ᮏ❶࡟࠾ࡅࡿ◊✲࡛ࡣ, Ⓨࡀࢇ≀㉁ࡢ཯᚟ᢞ୚ࡢ᪩ᮇ࠿ࡽ⏕ࡌࡿⓎࡀࢇ≀㉁≉␗ⓗ ࡞⣽⬊࿘ᮇไᚚ◚⥢ࡢ⾲⌧ᆺࡢᡂ❧᫬ᮇ࡜ࡑࡢ୺せ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ┠ⓗ࡜ࡋࡓࠋࡇ ࡢ┠ⓗࡢࡓࡵ࡟, ࣛࢵࢺ࡬⫢Ⓨࡀࢇ≀㉁ࢆ᭱኱ 28 ᪥㛫཯᚟ᢞ୚ࡋ, ⫢⣽⬊ࡢቑṪάᛶ, G1/S ࠾ࡼࡧ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ㛵㐃ศᏊࡢⓎ⌧࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢ⤒ ᫬ⓗ࡞ኚືࢆ, ච␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧ real-time RT-PCR ࡢゎᯒ࡟ࡼࡗ᳨࡚ウࡋࡓࠋࡲ ࡓ, ẚ㍑࡜ࡋ࡚, ⫢⣽⬊ࡢ෌⏕ᛶቑṪࢆㄏⓎࡍࡿ┠ⓗ࡛, ࣛࢵࢺ࡟ᑐࡋ, 㒊ศ⫢ษ㝖࡞ ࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢ཯᚟ᢞ୚ࢆᐇ᪋ࡋࡓࠋ

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9

ᮦᩱ࠾ࡼࡧ᪉ἲ

໬

໬Ꮫ≀㉁

Methyleugenol (MEG) , D-naphthyl isothiocyanate (ANIT) ࡣᮾி໬ᡂᕤᴗᰴᘧ఍♫ (Tokyo, Japan) ࡼࡾ, acetaminophen (APAP) ࡣࢩࢢ࣐࢔ࣝࢻࣜࢵࢳ (St. Louis, MO, USA) ࡼࡾ, TAA, promethazine (PMZ) ࠾ࡼࡧ methyl cellulose 400 ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ (Osaka, Japan) ࡼࡾ㉎ධࡋࡓࠋ ౪ヨື≀ 5 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆ᪥ᮏ࢚ࢫ࢚ࣝࢩ࣮ࡼࡾ㉎ධࡋ, ᗘ 23s3°C, ‵ᗘ 50s 20%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳, ⢊ᮎ CRF-1 (࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ, ᮾி) ࠾ࡼࡧ㣧ᩱỈࡢ⮬⏤⤥Ỉୗ࡛㣫⫱ࡋ, ୍㐌㛫ࡢ㥆໬ᮇ㛫ࡢᚋ, ␗ᖖࡀㄆࡵࡽࢀ࡞࠿ࡗࡓ ື≀ࢆᐇ㦂࡟౪ࡋࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ᮏᐇ㦂࡜ࡋ࡚2 ࡘࡢື≀ᐇ㦂ࢆᐇ᪋ࡋࡓࠋ୧ᐇ㦂࡟࠾࠸࡚ࡶ, ື≀࡟ᑐࡋ࡚ 2/3 㒊ศ ⫢ษ㝖, ⫢Ⓨࡀࢇ≀㉁ࡢ཯᚟ᢞ୚࡞࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢ཯᚟ᢞ୚ࢆᐇ᪋ࡋࡓࠋ MEG ࠾ࡼࡧ TAA ࡣ⫢Ⓨࡀࢇ≀㉁࡜ࡋ࡚ [6, 58] , APAP, ANIT ࠾ࡼࡧ PMZ ࡣ㠀Ⓨࡀࢇᛶ

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10 ⫢ẘᛶ≀㉁࡜ࡋ࡚ [51, 52, 64] 㑅ᐃࡋᐇ㦂࡟⏝࠸ࡓࠋᐇ㦂 1 ࡜ࡋ࡚, 3 ࡞࠸ࡋ 7 ᪥㛫཯᚟ ᢞ୚ヨ㦂ࢆ⾜࠸, ᐇ㦂 2 ࡜ࡋ࡚, 28 ᪥㛫཯᚟ᢞ୚ヨ㦂ࢆ⾜ࡗࡓࠋ࠸ࡎࢀࡢᐇ㦂࡟࠾࠸࡚ ࡶ, ⫢⣽⬊ࡢቑṪάᛶ, ⣽⬊࿘ᮇ㛵㐃ศᏊࡢⓎ⌧࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢ⤒᫬ⓗ࡞ኚືࢆ ච␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧreal-time RT-PCR ࡟ࡼࡾゎᯒࡋࡓࠋ ᐇ㦂1 6 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆึᮇయ㔜࡟ᇶ࡙࠸࡚, ↓ฎ⨨ᑐ↷⩌ (n = 20) , 2/3 㒊ศ⫢ษ 㝖ࢆ⾜࠺⩌ (PH, n = 22) , TAA (400 ppm, n = 20) , APAP (10,000 ppm, n = 20) ࡞࠸ࡋ ANIT (1,000, n = 20) ࡢΰ㣵ᢞ୚ࢆ⾜࠺⩌, MEG (1,000 mg/kg య㔜, n = 22) ࡞࠸ࡋ PMZ (200 mg/kg య㔜, n = 22) ࡢᙉไ⤒ཱྀᢞ୚ࢆ⾜࠺⩌ࡢ 7 ࡘࡢ⩌࡟ศࡅࡓࠋMEG ࠾ࡼࡧ PMZ ࡢ ᢞ୚ࡢ㝿࡟, ⁐፹࡜ࡋ࡚ 0.5 % methyl cellulose ⁐ᾮࢆ౑⏝ࡋࡓࠋANIT ⩌࡟࠾࠸࡚, ᢞ୚ 㛤ጞᚋ 3 ᪥┠࡛ື≀ࡢ೺ᗣ≧ἣࡢᝏ໬ࡀ⏕ࡌࡓࡓࡵ, ⏝㔞ࢆ 600 ppm ࡟ῶᑡࡉࡏࡓࠋ PMZ ⩌࡟࠾࠸࡚, ᢞ୚㛤ጞᚋ 3 ᪥┠࡛ື≀ࡢ೺ᗣ≧ἣࡢᝏ໬ࡀ⏕ࡌࡓࡓࡵ, ⏝㔞ࢆ 100 mg/kg య㔜࡟ῶᑡࡉࡏࡓࠋ↓ฎ⨨ᑐ↷⩌ࡣᇶ♏㣫ᩱ࡜㣧ᩱỈ࡛⥔ᣢࡋࡓࠋᢞ୚㛤ጞᚋ 3 ᪥┠࠾ࡼࡧ7 ᪥┠࡟, ྛ⩌༙ᩘࡎࡘࡢື≀࡟ࡘ࠸࡚, CO2/O2࡟ࡼࡿ῝㯞㓉ୗ࡛኱ື⬦࠿ ࡽࡢᨺ⾑࡟ࡼࡿᏳᴦẅࢆᐇ᪋ࡋ, ⫢⮚ࢆ᥇ྲྀࡋࡓࠋ ᐇ㦂2

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11

6 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆึᮇయ㔜࡟ᇶ࡙࠸࡚, ↓ฎ⨨ᑐ↷⩌ (n=10) , 2/3 㒊ศ⫢ษ 㝖ࢆ⾜࠺⩌ (PH, n = 12) , TAA (400 ppm, n = 10) , APAP (10,000 ppm, n = 10) ࡞࠸ࡋ ANIT (600, n = 10) ࡢΰ㣵ᢞ୚ࢆ⾜࠺⩌, MEG (1,000 mg/kg య㔜, n = 11) ࡞࠸ࡋ PMZ (100 mg/kg య㔜, n = 11) ࡢᙉไ⤒ཱྀᢞ୚ࢆ⾜࠺⩌ࡢ 7 ࡘࡢ⩌࡟ศࡅࡓࠋMEG ࠾ࡼࡧ PMZ ࡢ ᢞ୚ࡢ㝿࡟, ⁐፹࡜ࡋ࡚ 0.5% methyl cellulose ⁐ᾮࢆ౑⏝ࡋࡓࠋMEG ⩌࡟࠾࠸࡚, ᢞ୚ 㛤ጞᚋ10 ᪥┠࡟ 1 ಶయ࡛೺ᗣ≧ἣࡢᝏ໬ࡀ⏕ࡌࡓࡓࡵᏳᴦẅࢆᐇ᪋ࡋ, ࡑࡢᚋ⏝㔞ࢆ 800 mg/kg య㔜࡟ῶᑡࡉࡏࡓࠋ↓ฎ⨨ᑐ↷⩌ࡣᇶ♏㣫ᩱ࡜㣧ᩱỈ࡛⥔ᣢࡋࡓࠋᢞ୚㛤ጞ ᚋ28 ᪥┠࡟, ඲ື≀࡟ࡘ࠸࡚ CO2/O2࡟ࡼࡿ῝㯞㓉ୗ࡛኱ື⬦࠿ࡽࡢᨺ⾑࡟ࡼࡿᏳᴦẅ ࢆᐇ᪋ࡋ, ⫢⮚ࢆ᥇ྲྀࡋࡓࠋ MEG ࠾ࡼࡧ TAA ࡢᢞ୚⏝㔞ࡣⓎࡀࢇ⏝㔞࡞࠸ࡋ᭱኱⪏㔞ࢆタᐃࡋ [6, 58] , ᢞ୚⏝ 㔞ࡢኚ᭦ᚋ࡟࠾࠸࡚ࡶⓎࡀࢇ⏝㔞ࢆୗᅇࡽ࡞࠸ࡼ࠺࡟タᐃࡋࡓࠋAPAP, ANIT ࠾ࡼࡧ PMZ ࡢᢞ୚⏝㔞ࡣ, 13 ࡞࠸ࡋ 16 㐌㛫཯᚟ᢞ୚ヨ㦂࡟࠾࠸࡚ࣛࢵࢺ࡟⫢ẘᛶࢆㄏⓎࡍ ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ⏝㔞ࢆタᐃࡋࡓ [51, 52, 64] ࠋ ື≀ᐇ㦂ィ⏬ࡣ, ᅜ❧኱Ꮫἲேᮾி㎰ᕤ኱Ꮫࡢື≀ᐇ㦂೔⌮ጤဨ఍࡟ᥦฟࡋ࡚ᢎㄆ ࢆཷࡅ, ື≀ࡢྲྀࡾᢅ࠸࡟ࡘ࠸࡚ࡶ, ྠ኱Ꮫࡢᐇ㦂ື≀ᣦ㔪ࢆ㑂Ᏺࡋࡓࠋ ⑓ ⑓⌮⤌⧊ᏛⓗᰁⰍ࠾ࡼࡧච␿⤌⧊໬ᏛᰁⰍ ࣃࣛࣇ࢕ࣥᅛᐃࡋࡓ⫢⮚ࢆ 3 μm ࡢཌࡉ࡟ⷧษࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥ

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12

(HE) ᰁⰍ࠾ࡼࡧච␿⤌⧊໬ᏛᰁⰍ࡟౪ࡋࡓࠋ⫢⮚ษ∦ࢆ⏝࠸࡚, G1ᮇ࠿ࡽM ᮇ࡟⣽⬊

ࡢ᰾࡟Ⓨ⌧ࡍࡿ⣽⬊ቑṪᣦᶆࡢKi-67 [68] , ࢔࣏ࢺ࣮ࢩࢫ㐣⛬ࡢᚋᮇ࡟Ⓨ⌧ࡍࡿ࢔࣏ࢺ ࣮ࢩࢫᣦᶆࡢ cleaved caspase 3 [15] , G2/M ᮇ࡟ DNA ࢹ࢝ࢸࢿ࣮ࢩࣙࣥ࡟㛵୚ࡍࡿ

topoisomerase IIα (TOP2A) [43] , M ᮇ ࡢ ึ ᮇ ࡟ ࢡ ࣟ ࣐ ࢳ ࣥ ࡢ จ 㞟 ࡟ 㛵 ୚ ࡍ ࡿ phosphorylated histone H3 (p-Histone H3) [23] , M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ⺮ⓑ㉁ ࡢmitotic arrest deficient 2 (MAD2) [34] , DNA ᦆയ࡟ᛂ⟅ࡋ࡚Ⓨ⌧ࡍࡿ phosphorylated H2AX (γH2AX) [9] , ࢧ࢖ࢡࣜࣥ౫Ꮡᛶ࢟ࢼ࣮ࢮ㜼ᐖᅉᏊࡢ 1 ࡘ࡛࠶ࡿ p21 Cip1_{[69] ,}

G2/M ᮇ࡟࠾࠸࡚ MAD2 ࡞࡝ࡢࢳ࢙ࢵࢡ࣏࢖ࣥࢺ⺮ⓑ㉁ࡢ࢟ࢿࢺࢥ࢔ᒁᅾࢆῶᑡࡉࡏࡿ

ࡇ࡜࡛ᰁⰍయ୙ᏳᐃᛶࢆㄏᑟࡍࡿUBD [22, 37] , ࠾ࡼࡧ p53 ࡢୗὶศᏊࡢ୍ࡘ࡛ p53 ࡢ ศゎࢆಁ㐍ࡍࡿphosphorylated MDM2 (p-MDM2) [40, 44] ࡟ᑐࡍࡿ୍ḟᢠయࢆ⏝࠸࡚, ච␿⤌⧊໬ᏛᰁⰍࢆᐇ᪋ࡋࡓࠋ⏝࠸ࡓᢠయ, ㈿ά໬᪉ἲ࠾ࡼࡧᢠయᕼ㔘᮲௳ࡣ Table 1-1 ࡟♧ࡋࡓࠋࢩࢢࢼ᳨ࣝฟࡣ VECTASTAINט _{Elite ABC Kit (Vector Laboratories Inc.,}

Burlingame, CA, USA) ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯ᛂࡣ 3,3’-diaminobenzidine/H2O2ࢆ

⏝࠸࡚ྍど໬ࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࡋࡓࠋ

UBD ࡢ⣽⬊࿘ᮇෆ࡛ࡢฟ⌧᫬ᮇࢆ᳨ウࡍࡿ┠ⓗ࡛,

UBD ࡜ TOP2A ࡞࠸ࡋ

p-Histone H3 ࡜ࡢ஧㔜ᰁⰍࢆ⾜ࡗࡓࠋUBD ࡢࢩࢢࢼ᳨ࣝฟࡣ VECTASTAINט_{Elite ABC}

Kit (Vector Laboratories) ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯ᛂࡣ 3,3’-diaminobenzidine/H2O2

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ࡣVECTASTAINט_{Elite ABC-AP kit (Vector Laboratories) ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯}

ᛂࡣVECTOR Red Alkaline Phosphate Substrate Kit I (Vector Laboratories) ࢆ⏝࠸࡚ྍど໬ ࡋࡓࠋ

ච

ච␿⤌⧊໬ᏛᰁⰍࡢᐃ㔞ゎᯒ

ච␿⤌⧊໬ᏛᰁⰍ࡟ࡼࡾ, Ki-67, TOP2A, p-Histone H3, UBD, MAD2, γH2AX, p21 CIp1_࠾

ࡼࡧ p-MDM2 ࡟ᑐࡋ࡚㝧ᛶࢆ♧ࡍ⫢⣽⬊ᩘࡣ, 200 ಸࡢಸ⋡࡛ 1 ಶయᙜࡓࡾࣛࣥࢲ࣒࡟ 10 ど㔝㑅ᢥࡋ, cleaved caspase 3 ࡟ᑐࡋ࡚㝧ᛶࢆ♧ࡍ⫢⣽⬊ᩘࡣ, 100 ಸࡢಸ⋡࡛ 1 ಶయ ᙜࡓࡾࣛࣥࢲ࣒࡟5 ど㔝㑅ᢥࡋ, ࢝࢘ࣥࢺࡋࡓࠋど㔝ࡣ, ⾑⟶ࡸ⤖ྜ⤌⧊ࡀධࡽ࡞࠸ࡼ ࠺࡟㑅ᢥࡋࡓࠋ㛛⬦࿘㎶࡟㝧ᛶ⣽⬊ࡀከࡃศᕸࡍࡿ࡞࡝, 㝧ᛶ⣽⬊ࡀ⫢⮚⤌⧊඲య࡟ᆒ ୍࡟ศᕸࡋ࡞࠸ሙྜࡣ, ೫ࡗࡓ㡿ᇦࢆ㑅ᢥࡋ࡞࠸ࡼ࠺࡟, 㞄᥋ࡋࡓ 5 ࡞࠸ࡋ 10 ど㔝ࢆ㑅 ᢥࡋࡓࠋ㝧ᛶ⣽⬊ᩘࡣどぬⓗ࡟࢝࢘ࣥࢺࡋ, ⥲⣽⬊ᩘࡣ⏬ീゎᯒࢯࣇࢺ WinROOF (୕㇂ ၟ஦, ᮾி) ࢆ⏝࠸࡚ᐃ㔞໬ࡋ, 㝧ᛶ⣽⬊⋡ࢆ⟬ฟࡋࡓࠋ Real-time RT-PCR ࡟ࡼࡿゎᯒ ⣽⬊࿘ᮇ㛵㐃㑇ఏᏊ࠾ࡼࡧDNA ᦆയ㛵㐃㑇ఏᏊࡢ mRNA Ⓨ⌧㔞ࢆ᳨ウࡍࡿࡓࡵ࡟, SYBR®_{Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA) ࢆ⏝࠸, Step One}

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14

mRNA Ⓨ⌧ࡢᐃ㔞ゎᯒࢆᐇ᪋ࡋࡓࠋࣉࣛ࢖࣐࣮ࡣ Primer Express software (Version 3.0; Life Technologies) ࢆ⏝࠸࡚タィࡋ, Table 1-2 ࡟♧ࡋࡓࠋྛ㑇ఏᏊࡢ mRNA Ⓨ⌧㔞ࡣ, ↓ ฎ ⨨ ᑐ ↷ ⩌ ࡛ ࡢ Ⓨ ⌧ ್ ࡟ ᑐ ࡍ ࡿ ┦ ᑐ ್ ࡜ ࡋ ࡚ ồ ࡵ, ෆᅉᛶࢥ ࣥࢺ࣮ࣟࣝ ࡜ࡋ࡚ hypoxanthine phosphoribosyltransferase 1 (Hprt1) ࡢ᳨㔞⥺ࢆồࡵ, 2–ΔΔC T method [39] ࡟࡚ ⟬ฟࡋࡓࠋ ⤫ ⤫ィゎᯒ ඲࡚ࡢᐃ㔞ࢹ࣮ࢱ࡟ࡘ࠸࡚ᖹᆒ್࠾ࡼࡧᶆ‽೫ᕪࢆồࡵࡓࠋᩘ್ࢹ࣮ࢱࡢከ⩌㛫ẚ㍑ ࡣ, Bartlett ᳨ᐃ࡛➼ศᩓࢆ☜ㄆࡋࡓᚋ, ୍ඖ㓄⨨ศᩓศᯒࢆ⾜ࡗࡓࠋBartlett ᳨ᐃ࡛➼ศ ᩓ࡛࠶ࡗࡓሙྜ࡟ࡣDunnett ᳨ᐃࢆ⾜࠸, ➼ศᩓ࡛࡞࠿ࡗࡓሙྜ࡟ࡣ Steel ᳨ᐃࢆ⾜ࡗࡓࠋ ඲࡚ࡢᩘ್ࢹ࣮ࢱࡣ, ↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋࡓࠋ

(17)

15 ⤖ᯝ

య

య㔜࠾ࡼࡧ⫢⮚㔜㔞

3 ᪥┠࡟࠾࠸࡚, PH ⩌, MEG ⩌, TAA ⩌, ANIT ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Table 1-3) ࠋPH ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋMEG ⩌࠾ࡼࡧ APAP ⩌࡛ࡣ↓ฎ⨨ᑐ ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋMEG ⩌, TAA ⩌, APAP ⩌, ANIT ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽ ࢀࡓࠋ

7 ᪥┠࡟࠾࠸࡚, ඲ฎ⨨⩌࡛, ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆ ࡵࡽࢀࡓ (Table 1-3) ࠋPH ⩌࠾ࡼࡧ ANIT ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞 ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋMEG ⩌, TAA ⩌, APAP ⩌, ANIT ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋࡲࡓ, PH ⩌࡟࠾࠸࡚, 6 ᪥ ┠࡟ື≀1 ಶయࡢṚஸࡀ☜ㄆࡉࢀࡓࠋ

28 ᪥┠࡟࠾࠸࡚, MEG ⩌, TAA ⩌, APAP ⩌, ANIT ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ, ↓ฎ⨨ᑐ↷ ⩌࡟ẚ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Table 1-3) ࠋPH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋPMZ ⩌ ࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋMEG ⩌, TAA ⩌,

(18)

16 APAP ⩌, ANIT ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍ ࡀㄆࡵࡽࢀࡓࠋ ⑓ ⑓⌮⤌⧊Ꮫⓗኚ໬ MEG ⩌࡛ࡣ, 3 ᪥┠࡛ࡣ⤌⧊Ꮫⓗ࡞ኚ໬ࡣㄆࡵࡽࢀࡎ, 7 ᪥┠࡛ࡣࡍࡾ࢞ࣛࢫ≧ኚᛶࢆ క࠺ᑠⴥ୰ᚰᛶࡢ⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀࡓࠋࡲࡓࡑࢀ࡟ຍ࠼, 28 ᪥┠࡛ࡣ௨๓ࡢሗ࿌࡜ ྠᵝ࡟, ᰾ࡢ኱ᑠ୙ྠࢆక࠺ “ᕧ኱⣽⬊” ࡀㄆࡵࡽࢀ, ࡧₔᛶ࡟ศᕸࡀㄆࡵࡽࢀࡓ [58] ࠋTAA ⩌࡛ࡣ, 3 ᪥┠࠾ࡼࡧ 7 ᪥┠࡛᰾ᑠయࡢ⫧኱ࢆక࠺⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀ, ࡑ ࢀ࡟ຍ࠼ 7 ᪥┠࡛ࡣ㛛⬦࿘㎶㡿ᇦ࡛⫢⣽⬊ࡢ✵⬊ኚᛶࡀㄆࡵࡽࢀࡓࠋ28 ᪥┠࡛ࡣ௨๓ ࡢሗ࿌࡜ྠᵝ࡟, ᰾ࡢ኱ᑠ୙ྠ, ᭷⣒ศ⿣␗ᖖ࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫ࡜ඹ࡟, “ᕧ኱⣽⬊” ࡀฟ⌧ࡋ, ࡧₔᛶ࡟ࡑࡢ⣽⬊ࡢศᕸࡀㄆࡵࡽࢀࡓ [11] ࠋ᰾ᑠయࡢ⫧኱ࢆక࠺ “ᕧ኱⣽ ⬊” ࡢฟ⌧ࡣࡇࡢ᫬Ⅼ࡟࠾࠸࡚㢧ⴭ࡟ㄆࡵࡽࢀࡓࠋAPAP ⩌࡛ࡣ, 3 ᪥┠࡛᫂ࡽ࠿࡞⤌⧊ ᡤぢࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ7 ᪥┠࠾ࡼࡧ 28 ᪥┠࡛ࡣ, ௨๓ࡢሗ࿌࡜ྠᵝ࡟, ᑠⴥ୰ᚰᛶ ࡢ⫢⣽⬊⫧኱ࡀ㍍ᗘ࡟ㄆࡵࡽࢀࡓ [51] ࠋANIT ⩌࡛ࡣ, 3 ᪥┠࠿ࡽ㛛⬦࿘㎶㡿ᇦ࡛⫹⟶ ቑ⏕ࡀㄆࡵࡽࢀࡓࠋ7 ᪥┠࠿ࡽ, ௨๓ࡢሗ࿌࡜ྠᵝ࡟ᑠሢ≧ࡢ⫢⣽⬊ቯṚࡀᩓᅾᛶ࡟ㄆ ࡵࡽࢀࡓ [64] ࠋPMZ ⩌࡛ࡣ, ௨๓ࡢሗ࿌࡜ྠᵝ࡟ᑠⴥ୰ᚰᛶ࡟ࡍࡾ࢞ࣛࢫ≧ኚᛶࢆక ࠺⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀࡓ [52] ࠋ

(19)

17 ⣽

⣽⬊ቑṪάᛶ࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫኚື

Ki-67 ࡣ⫢⣽⬊ࡢ᰾࡟ᒁᅾࡋ࡚࠸ࡓ (Fig. 1-1A, C ࠾ࡼࡧ E) ࠋ኱㒊ศࡢ౛࡛ࡣ, Ki-67 㝧ᛶ⣽⬊ࡣ⫢⮚⤌⧊඲య࡟ᆒ୍࡟ศᕸࡋ࡚࠸ࡓࡀ, TAA ⩌ࡢ 3 ࠾ࡼࡧ 7 ᪥┠, PMZ ⩌ࡢ 28 ᪥┠࡛ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ࡟ከࡃศᕸࡋ࡚࠸ࡓࠋCleaved caspase 3 ࡣ⫢⣽⬊ࡢ᰾࡜⣽⬊ ㉁࡟ᒁᅾࡋ࡚࠸ࡓ (Fig. 1-1B, D ࠾ࡼࡧ F) ࠋ඲ᢞ୚⩌ࡢ඲ᢞ୚ᮇ㛫࡟࠾࠸࡚, cleaved caspase 3 㝧ᛶ⣽⬊ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ࡟ከࡃศᕸࡋ࡚࠸ࡓࠋ

3 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ PH ⩌, TAA ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛, ↓ฎ⨨ ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, PMZ ⩌࡛ࡣ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-1A) ࠋCleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍ ࡀㄆࡵࡽ, PMZ ⩌࡛ࡣ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-1B) ࠋ

7 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-1C) ࠋCleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-1D) ࠋ

28 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-1E) ࠋCleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌࠾ࡼࡧTAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-1F) ࠋ

(20)

18 ⣽

⣽⬊࿘ᮇ㛵㐃ศᏊⓎ⌧⣽⬊ࡢศᕸ

TOP2A, p-Histone H3, MAD2, γH2AX, p21Cip1_࠾ࡼࡧ_{p-MDM2 ࡣ᰾࡟ᒁᅾࡋ, UBD ࡣ⣽}

⬊㉁࡞࠸ࡋ᭷⣒ศ⿣⣳㗽య࡟ᒁᅾࡋ࡚࠸ࡓ (Fig. 1-2A–G) ࠋ኱㒊ศࡢ౛࡛ࡣ, TOP2A, p-Histone H3, MAD2, γH2AX ࠾ࡼࡧ UBD 㝧ᛶ⣽⬊ࡣ⫢⮚⤌⧊඲య࡟ᆒ୍࡟ศᕸࡋ࡚࠸ ࡓࡀ, TAA ⩌ࡢ 3 ࠾ࡼࡧ 7 ᪥┠, PMZ ⩌ࡢ 28 ᪥┠࡛ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ࡟ከࡃศᕸࡋ࡚࠸ ࡓࠋ඲ᢞ୚⩌ࡢ඲ᢞ୚ᮇ㛫࡟࠾࠸࡚, p21Cip1_࠾ࡼࡧ_{p-MDM2 㝧ᛶ⣽⬊ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ}

࡟ከࡃศᕸࡋ࡚࠸ࡓࠋ

3 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, MEG ⩌࠾ࡼࡧ PMZ ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-2A) ࠋp-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, MEG ⩌࠾ࡼࡧ PMZ ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-2B) ࠋMAD2 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, MEG ⩌࠾ࡼࡧ PMZ ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-2C) ࠋUBD 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ ព࡞ቑຍࡀㄆࡵࡽࢀ, PMZ ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-2D) ࠋγH2AX 㝧ᛶ⣽⬊ ⋡ࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, PMZ ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-2E) ࠋp21Cip1_{㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌}

(21)

19

p-MDM2 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀ ㄆࡵࡽࢀࡓ (Fig. 1-2G) ࠋ

7 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-3A) ࠋp-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽ ࢀ, PMZ ⩌࡛᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-3B) ࠋMAD2 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-3C) ࠋUBD 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, TAA ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-3D) ࠋγH2AX 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-3E) ࠋp21Cip1_{㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌, APAP ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌}

࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-3F) ࠋp-MDM2 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌, APAP ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-3G) ࠋ

28 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, APAP ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-4A) ࠋp-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4B) ࠋMAD2 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌࠾

(22)

20

ࡼࡧPMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4C) ࠋUBD 㝧 ᛶ⣽⬊⋡ࡣ, PH ⩌, MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑ ຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4D) ࠋγH2AX 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4E) ࠋp21Cip1_{㝧ᛶ⣽⬊⋡ࡣ, MEG}

⩌, TAA ⩌࠾ࡼࡧ APAP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4F) ࠋp-MDM2 㝧ᛶ⣽⬊⋡ࡣ, MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព ࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-4G) ࠋ

28 ᪥┠࡟࠾࠸࡚, p-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࡣ, TAA ⩌ ࠾ࡼࡧMEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-5) ࠋ

UBD ࡜࡜TOP2A ࡞࠸ࡋ p-Histone H3 ࡜ࡢඹⓎ⌧

3 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, TAA ⩌࠾ࡼ ࡧANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-6A) ࠋUBD 㝧ᛶ ⣽⬊ࡢ࠺ࡕ TOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋp-Histone H3 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ ๭ྜࡣ, PH ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 1-6B) ࠋUBD 㝧 ᛶ⣽⬊ࡢ࠺ࡕp-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, PH ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋ

(23)

21

7 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, PH ⩌, TAA ⩌࠾ࡼࡧ ANIT ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-6C) ࠋ UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ TOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ ㍑ࡋ࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋp-Histone H3 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍ ࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 1-6D) ࠋUBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛ ↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

28 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, TAA ⩌࠾ ࡼࡧMEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-6E) ࠋUBD 㝧 ᛶ⣽⬊ࡢ࠺ࡕ TOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ ᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋp-Histone H3 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ ࡢ๭ྜࡣ, PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 1-6F) ࠋ UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, MEG ⩌࠾ࡼࡧ TAA ⩌࡛ ↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋ

mRNA ⓎⓎ⌧

28 ᪥┠࡛ቑṪάᛶࡢஹ㐍ࡀㄆࡵࡽࢀࡓ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡟ࡘ࠸࡚, 3, 7 ࠾ࡼࡧ 28 ᪥┠ࡢ᫬Ⅼ࡟࠾ࡅࡿ, Tabe 1-2 ࡟グ㍕ࡉࢀ࡚࠸ࡿ㑇ఏᏊࡢ mRNA Ⓨ⌧㔞ࢆ

(24)

22

real-time RT-PCR ἲ࡟ࡼࡗ࡚ᐃ㔞ࡋ, ↓ฎ⨨ᑐ↷⩌࡟࠾ࡅࡿ mRNA Ⓨ⌧㔞࡜ࡢ┦ᑐ್ࢆ ⟬ฟࡋࡓ (Table 1-4)ࠋ

3 ᪥┠࡟࠾࠸࡚, G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Cdkn1a ࡣ MEG ⩌࠾ࡼࡧ

TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀ, PMZ ⩌࡟࠾࠸ ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋCdkn2a ࠾ࡼࡧ Rb1 ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓

ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋRbl2 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋMdm2 ࠾ࡼ

ࡧTp53 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋ

୍᪉࡛, Tp53 ࡣ MEG ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ ῶᑡࡀㄆࡵࡽࢀࡓࠋM ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࠾ࡼࡧ M ᮇ㛵㐃㑇ఏᏊࡢ࠺ࡕ,

Aurka, Bub1 ࠾ࡼࡧ Plk1 ࡣ MEG ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ

᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋAurkb ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌

࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋMad1l1 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋMad2l1 ࡣ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋDNA ᦆയ㛵㐃㑇ఏᏊࡢ࠺ࡕ Atm ࠾

ࡼࡧChek1 ࡣ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋ

Brca1 ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧

(25)

23

࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋBrcc3 ࡣ TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋEsco1 ࠾ࡼࡧ Rad17 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋGadd45a ࡣ TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀ

ࡓࠋRad50 ࡣ඲ᢞ୚⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ኚືࡣㄆࡵࡽࢀ

࡞࠿ࡗࡓࠋ

7 ᪥┠࡛ࡣ, G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Cdkn1a ࡣ MEG ⩌࠾ࡼࡧ TAA

⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀ, PMZ ⩌࡛᭷ព࡞Ⓨ ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋCdkn2a ࠾ࡼࡧ Tp53 ࡣ MEG ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜

ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋ୍᪉࡛, Tp53 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷ ⩌࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋRb1 ࠾ࡼࡧ Rbl2 ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋ

Mdm2 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀ

ㄆࡵࡽࢀࡓࠋM ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࠾ࡼࡧ M ᮇ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Aurka ࠾

ࡼࡧMad2l1 ࡣ MEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀ

ࡓࠋAurkb, Bub1 ࠾ࡼࡧ Plk1 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA

ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋMad1l1 ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ

(26)

24

Brca1 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀ

ㄆࡵࡽࢀࡓࠋBrca2, Chek1 ࠾ࡼࡧ Chek2 ࡣ MEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ

᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋBrcc3 ࡣ MEG ⩌, TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌

࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋEsco1 ࠾ࡼࡧ Rad17 ࡣ MEG ⩌࠾ࡼ ࡧPMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋ୍᪉࡛,

Esco1, Gadd45a ࠾ࡼࡧ Rad17 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧

ቑຍࡀㄆࡵࡽࢀࡓࠋGadd45a ࡣ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧

ῶᑡࡀㄆࡵࡽࢀࡓࠋRad50 ࡣ MEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶ

ᑡࡀㄆࡵࡽࢀࡓࠋAtm ࡣ඲ᢞ୚⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ኚື

ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

28 ᪥┠࡟࠾࠸࡚, G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Cdkn1a ࠾ࡼࡧ Mdm2 ࡣ

MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀ ࡓࠋ୍᪉࡛, Cdkn1a ࡣ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆ ࡵࡽࢀࡓࠋCdkn2a ࠾ࡼࡧ Tp53 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ

⌧ቑຍࡀㄆࡵࡽࢀࡓࠋRbl2 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA

ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋRb1 ࡣ TAA ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ

࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋM ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࠾ࡼࡧ M ᮇ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Aurka ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑ

(27)

25

ຍࡀㄆࡵࡽࢀࡓࠋAurkb, Mad2l1 ࠾ࡼࡧ Plk1 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜

ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋMad1l1 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋBub1 ࡣ MEG ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋDNA ᦆയ㛵㐃㑇ఏᏊࡢ࠺ࡕ, Brcc3 ࠾ࡼ

ࡧChek1 ࡣ MEG ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍ

ࡀㄆࡵࡽࢀࡓࠋEsco1, Gadd45a, Rad17 ࠾ࡼࡧ Rad50 ࡣ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ

࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ቑຍࡀㄆࡵࡽࢀࡓࠋAtm ࠾ࡼࡧ Esco1 ࡣ MEG ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡜ẚ㍑ࡋ࡚mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋEsco1 ࠾ࡼࡧ Rad17 ࡣ PMZ ⩌࡛↓ ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ῶᑡࡀㄆࡵࡽࢀࡓࠋBrca1, Brca2 ࠾ࡼࡧ

Chek2 ࡣ඲ᢞ୚⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚ mRNA ࡢ᭷ព࡞Ⓨ⌧ኚືࡣㄆࡵࡽࢀ࡞࠿

(28)

26 ⪃ᐹ ᮏ❶࡛ࡣ, ⫢Ⓨࡀࢇ≀㉁≉␗ⓗ࡟ㄏⓎࡉࢀࡿ⣽⬊࿘ᮇไᚚ◚⥢ࡢฟ⌧᫬ᮇ࡜ࡑࡢ୺ せ࣓࢝ࢽࢬ࣒ࢆ᳨ウࡍࡿ┠ⓗ࡛, ⫢Ⓨࡀࢇ≀㉁ࢆࣛࢵࢺ࡟ 3, 7 ࡞࠸ࡋ 28 ᪥㛫཯᚟ᢞ୚ ࡋ, ⣽⬊࿘ᮇ㛵㐃ศᏊࡢⓎ⌧ኚືࢆච␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧ real-time RT-PCR ἲ࡟ࡼࡾ ⤒᫬ⓗ࡟ゎᯒࡋࡓࠋࡑࡢ⤖ᯝ, 7 ᪥┠࡟࠾࠸࡚, ⫢Ⓨࡀࢇᛶ࡟㛵ಀ࡞ࡃ࡯࡜ࢇ࡝ࡢ໬Ꮫ≀ ㉁࡟ࡼࡗ࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡀኚ໬ࡋ࡞࠸ࡶࡋࡃࡣῶᑡࡋ࡚࠾ࡾ, ࡑࢀ࡜࡜ࡶ࡟ p21Cip1 㝧ᛶ⣽⬊⋡࠾ࡼࡧcleaved caspase 3 㝧ᛶ⣽⬊⋡ࡢቑຍࡀㄏⓎࡉࢀࡓࠋࡑࡢ୍᪉࡛, 28 ᪥ ┠࡛ࡣ, ⫢Ⓨࡀࢇ≀㉁ࡢࡳࡀ, ⣽⬊ቑṪάᛶࡢஹ㐍࡜࡜ࡶ࡟ p21Cip1_{㝧ᛶ⣽⬊⋡࠾ࡼࡧ}

cleaved caspase 3 㝧ᛶ⣽⬊⋡ࡢቑຍࢆㄏⓎࡋࡓࠋp21Cip1_{ࡣࢧ࢖ࢡࣜࣥ౫Ꮡᛶ࢟ࢼ࣮ࢮ㜼}

ᐖᅉᏊࡢ1 ࡘ࡛, DNA ᦆയ, 㓟໬ᛶࢫࢺࣞࢫࡸࢧ࢖ࢺ࢝࢖ࣥ࡞࡝ࡢ่⃭࡟ᛂ⟅ࡋ࡚άᛶ ໬ࡉࢀ, G1ᮇ࡟࠾ࡅࡿ⣽⬊࿘ᮇ೵ṆࢆㄏⓎࡍࡿࡇ࡜࠿ࡽ [1, 19, 66, 69] , ᰾ෆ࡟ p21Cip1 ࢆⓎ⌧ࡍࡿ⣽⬊ࡢቑຍࡣ, G1ᮇ࡟࠾࠸࡚⣽⬊࿘ᮇࡀ೵Ṇࡋ࡚࠸ࡿ⣽⬊ࡢቑຍࢆ♧၀ࡍࡿ ࡶࡢ࡛࠶ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋࡲࡓ, 28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁࡟ࡼࡾ, Brca1 ࢆάᛶ໬ࡍࡿ ࡇ࡜࡛DNA ಟ᚟࡟㛵୚ࡍࡿ Brcc3 [10] ࠾ࡼࡧ G2/M ࢳ࢙ࢵࢡ࣏࢖ࣥࢺศᏊ࡛ DNA ᦆയ ࡟ᛂ⟅ࡍࡿChek1 [62] ࡢ mRNA Ⓨ⌧ࡀቑຍࡋࡓࠋࡑࢀ࡜ࡣᑐ↷ⓗ࡟, 3 ࠾ࡼࡧ 7 ᪥┠࡛ ࡣBrcc3 ࠾ࡼࡧ Chek1 ࡢ mRNA Ⓨ⌧ࡣቑຍࡋ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ, ⫢Ⓨࡀࢇ≀ ㉁ࡢᢞ୚㛤ጞᚋ28 ᪥┠࡟, DNA ᦆയࡢ⵳✚ࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡓࡀ, ࡑࢀࡼ

(29)

27

ࡾࡶ▷ᮇ㛫ࡢᢞ୚࡟ࡼࡗ࡚⏕ࡌࡿࡇࢀࡽࡢศᏊࡢ཯ᛂᛶࡣDNA ᦆയ࡜ࡣ㛵㐃ࡀ࡞࠸ࡇ ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓ, p21Cip1_{ࡣ࢔࣏ࢺ࣮ࢩࢫࡢㄏⓎ࡟ᚲせ࡛࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸}

ࡿࡇ࡜࠿ࡽ [33, 38] , 7 ᪥┠࠿ࡽ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡾ⏕ࡌࡓ࢔࣏ࢺ࣮ࢩࢫࡢஹ㐍ࡣ, p21Cip1_{ࡢάᛶ໬࡟㉳ᅉࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ᥎ᐹࡉࢀࡓࠋ୍᪉࡛, 7 ᪥┠࡟࠾࠸࡚㠀Ⓨ}

ࡀࢇᛶ⫢ẘᛶ≀㉁ࡢAPAP ࠾ࡼࡧ ANIT ࡶ p21Cip1_{㝧ᛶ⣽⬊⋡࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢቑຍ}

ࢆㄏⓎࡋࡓࡇ࡜࠿ࡽ, ࡇࡢ᫬Ⅼ࡛ࡢ p21Cip1 _{ࡢάᛶ໬ࡣⓎࡀࢇ≀㉁≉␗ⓗ࡞཯ᛂ࡛ࡣ࡞}

ࡃ, ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶ࡟ᑐࡍࡿ཯ᛂ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ28 ᪥┠࡟࠾࠸࡚, ⫢ Ⓨࡀࢇ≀㉁࡜ྠᵝ࡟㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢPMZ ࡶ Ki-67 㝧ᛶቑṪ⣽⬊ࢆቑຍࡉࡏࡓ ࡀ, p21Cip1_{㝧ᛶ⣽⬊⋡, ࢔࣏ࢺ࣮ࢩࢫ࠾ࡼࡧ DNA ᦆയ㛵㐃㑇ఏᏊࡢ mRNA Ⓨ⌧ࡢቑຍ}

ࡣㄏⓎࡋ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ, p21Cip1_{㝧ᛶ⣽⬊⋡࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢቑຍࡣ,} ௨๓ࡢሗ࿌࡜ྠᵝ࡟ [82] , ⣽⬊ቑṪάᛶࡢஹ㐍ࢆ♧ࡍ⫢Ⓨࡀࢇ≀㉁࡟≉ᚩⓗ࡞཯ᛂ࡛ ࠶ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡓࠋࡋࡓࡀࡗ࡚, 28 ᪥㛫ࡢᢞ୚ᮇ㛫ࡣ, ⫢Ⓨࡀࢇ≀㉁࡜ࡇࡢ᫬Ⅼ࡛ ቑṪάᛶࢆஹ㐍ࡍࡿ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁࡜ࢆ༊ูࡍࡿࡢ࡟༑ศ࡞ᮇ㛫࡛࠶ࡿࡇ࡜ࡀ ♧၀ࡉࢀࡓࠋ ᮏ◊✲࡟࠾࠸࡚, ⫢Ⓨࡀࢇ≀㉁ࡣ඲ᢞ୚ᮇ㛫࡛, RB ࣇ࢓࣑࣮ࣜ⺮ⓑ㉁ࡢ 1 ࡘ࡛ G1ᮇ ࠿ࡽS ᮇ࡬ࡢ㐍⾜ࢆไᚚࡍࡿ Rbl2 㑇ఏᏊࡢ mRNA Ⓨ⌧ࢆῶᑡࡉࡏࡓ [12, 13] ࠋࡋ࠿ࡋ ࡞ࡀࡽ, 㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢ PMZ ࡶ 7 ᪥┠࡛ Rbl2 ࡢ mRNA Ⓨ⌧ࢆῶᑡࡉࡏ࡚࠾ࡾ, 28 ᪥㛫ࡼࡾࡶ᪩࠸ẁ㝵࡛ࡢ Rbl2 ࡢⓎ⌧ᢚไࡣⓎࡀࢇ≀㉁≉␗ⓗ࡞ࡶࡢ࡛ࡣ࡞࠸ࡇ࡜ࡀ

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28

♧၀ࡉࢀࡓࠋ⯆࿡῝࠸ࡇ࡜࡟, PMZ ࡣ 28 ᪥┠࡟࠾࠸࡚, ቑṪάᛶࡢஹ㐍࡜࡜ࡶ࡟, TOP2A, p-Histone H3, MAD2, UBD ࠾ࡼࡧ γH2AX Ⓨ⌧⣽⬊ࢆቑຍࡉࡏࡓࡀ, ࡇࡢ᫬Ⅼ࡟

࠾ࡅࡿRbl2 ࡢ mRNA Ⓨ⌧ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ, ⫢Ⓨࡀࢇ≀㉁ ≉␗ⓗ࡟ㄏⓎࡉࢀࡿ, ⫢⣽⬊࡛ࡢ G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࡣ G1ᮇ࡟࠾ࡅࡿ⣽ ⬊࿘ᮇࡢ㐍⾜ࢆಁ㐍ࡋ, ࡑࢀࡣᢞ୚㛤ጞᚋ 28 ᪥┠ࡼࡾฟ⌧ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ RBL2 ࡢⓎ⌧ᢚไࡣ, ேࡢங⒴ࡸᏊᐑෆ⭷⒴࡟࠾࠸࡚ࡶሗ࿌ࡉࢀ࡚࠸ࡿ [48] ࠋᮏ◊✲ࡢ ඲ᢞ୚ᮇ㛫࡟࠾࠸࡚, ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡾ, p53 ࡢୗὶᅉᏊࡢ 1 ࡘ࡛ࣘࣅ࢟ࢳࣥ໬ࢆಁ㐍 ࡍࡿࡇ࡜࡛p53 ࡸ RB ⺮ⓑ㉁ࡢศゎࢆಁ㐍ࡍࡿ Mdm2 [8, 24, 76] ࡢ mRNA ࡢⓎ⌧ቑຍ࡞ ࠸ࡋቑຍഴྥࡀㄆࡵࡽࢀࡓࠋࡉࡽ࡟, ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡗ࡚, MDM2 ࡢάᛶ໬ᆺ࡛⣽⬊ ㉁࠿ࡽ᰾ෆ࡟⛣ືࡋ p53 ࢆศゎࡍࡿ, 166 ఩ࡢࢭࣜࣥᇶࡀࣜࣥ㓟໬ࡉࢀࡓ MDM2 (p-MDM2) [40, 44] 㝧ᛶ⣽⬊⋡ࡢቑຍࡀ Mdm2 ࡢ mRNA Ⓨ⌧ࡢቑຍ࡜࡜ࡶ࡟ㄆࡵࡽࢀࡓࠋ ᡤᒓ◊✲ᐊࡢඛ⾜◊✲࡟࠾࠸࡚, ⣽⬊ቑṪάᛶࢆஹ㐍ࡍࡿ⫢Ⓨࡀࢇ≀㉁ࡣ p53 Ⓨ⌧⣽⬊ ࢆቑຍࡉࡏ࡚࠾ࡾ, ࡑࢀ࡟ࡼࡗ࡚ Mdm2 ࡢ㌿෗ಁ㐍ࡀㄏⓎࡉࢀ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓ [82] ࠋ୍᪉࡛, 㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁࡛࠶ࡿ APAP ࠾ࡼࡧ PMZ ࡶ 7 ᪥┠࡛ p-MDM2 㝧 ᛶ⣽⬊⋡ࢆቑຍࡉࡏࡓࡀ, 28 ᪥┠࡛ࡣቑຍࡉࡏ࡞࠿ࡗࡓࠋࡇࢀࡽࡢࡇ࡜࠿ࡽ, ⫢Ⓨࡀࢇ ≀㉁≉␗ⓗ࡞, Mdm2 ࡢ mRNA Ⓨ⌧ࡢቑຍ࠾ࡼࡧ᰾ෆ࡟࠾ࡅࡿ p-MDM2 Ⓨ⌧ࡢቑຍࡣ, ࣉࣟࢸ࢔ࢯ࣮࣒ࢆ௓ࡋࡓp53 ࠾ࡼࡧ RB ⺮ⓑ㉁ࡢศゎಁ㐍ࢆ♧၀ࡍࡿࡶࡢ࡛࠶ࡾ, ࡇࡢ ཯ᛂࡣᢞ୚㛤ጞᚋ 28 ᪥┠࠿ࡽฟ⌧ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋp53 ࡣ㑇ఏẘᛶࢫࢺࣞࢫ࡟

(31)

29

ᛂ⟅ࡋ࡚Ⓨ⌧ቑຍ࠾ࡼࡧάᛶ໬ࡋ, p21Cip1 _{࡞࡝ࡢከࡃࡢ㑇ఏᏊⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜࡛,}

G1ᮇ࡛⣽⬊࿘ᮇࢆ೵Ṇࡉࡏ, DNA ಟ᚟ࢆಁࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ [5, 71] ࠋᮏ◊✲࡛ㄆ

ࡵࡽࢀࡓ, p21Cip1_{㝧ᛶ⣽⬊⋡ࡢቑຍ, Cdkn1a, Brcc3 ࠾ࡼࡧ Chek1 㑇ఏᏊࡢ mRNA ࡢⓎ⌧}

ቑຍࡣ, DNA ᦆയࡢ⵳✚ࢆ཯ᫎࡋ, p53 ࡢศゎ࡟㛵㐃ࡋ࡚ㄏⓎࡉࢀ࡚࠸ࡿྍ⬟ᛶࡀ᥎ᐹ ࡉࢀࡓࠋ ᡤᒓ◊✲ᐊࡢඛ⾜◊✲࡟࠾࠸࡚᪤࡟⪃ᐹࡋ࡚࠸ࡿ㏻ࡾ [74] , UBD ࡢ㐣๫Ⓨ⌧࡟ࡼࡾ, M ᮇ࡟࠾࠸࡚ࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࡜ࡋ࡚ᶵ⬟ࡍࡿ MAD2 ࡢ࢟ࢿࢺࢥ࢔࡟࠾ࡅ ࡿᒁᅾࡀᢚไࡉࢀ [22, 37] , ࡑࢀ࡟ࡼࡗ࡚ᰁⰍయ୙ᏳᐃᛶࡀㄏⓎࡉࢀࡿࡶࡢ࡜⪃࠼ࡽ ࢀࡓࠋᡤᒓ◊✲ᐊࡢඛ⾜◊✲࡟࠾࠸࡚, ቑṪάᛶࡢஹ㐍ࢆ♧ࡍⓎࡀࢇ≀㉁ࡢ 28 ᪥㛫཯ ᚟ᢞ୚࡟ࡼࡗ࡚, UBD ࡢ G2ᮇ࠿ࡽࡢ␗ᖖⓎ⌧ࡀㄏⓎࡉࢀࡿࡇ࡜ࢆぢฟࡋ࡚࠾ࡾ, ࡇࢀ ࡣM ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࢆ♧၀ࡍࡿࡶࡢ࡛࠶ࡿࡇ࡜ࡀ⪃࠼ࡽࢀ ࡓ [74] ࠋᮏ◊✲࡛ࡢච␿஧㔜ᰁⰍ࡟ࡼࡿゎᯒࡢ⤖ᯝ, 28 ᪥┠࡟࠾࠸࡚ MEG ⩌࠾ࡼࡧ TAA ⩌࡛, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡀࢃࡎ࠿࡟ቑຍࡋࡓࠋ TOP2A ࡣ G2ᮇ࠾ࡼࡧM ᮇ࡟Ⓨ⌧ࡋ [2, 36, 81] , p-Histone H3 ࡣ M ᮇ࡟Ⓨ⌧ࡍࡿࡇ࡜ࡀ

▱ࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽ [23] , ௨๓ࡢሗ࿌࡜ྠᵝ࡟ [74] , ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡗ࡚ UBD 㝧ᛶ⣽⬊ࡢ G2ᮇ࡟࠾ࡅࡿ๭ྜࡢࢃࡎ࠿࡞ቑຍࡀㄏⓎࡉࢀ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ୍

᪉࡛, MEG ⩌࠾ࡼࡧ TAA ⩌࡛ࡣ, UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ TOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜ ࡣኚ໬ࡋ࡞࠿ࡗࡓࡀ, UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ኱ࡁ

(32)

30 ࡃῶᑡࡋࡓࠋUBD ࡜ MAD2 ࡢ┦஫స⏝ࡀ, ቑṪ⣽⬊ࡢ M ᮇ࡟Ꮡᅾࡍࡿ๭ྜࡢῶᑡ࡜࡜ ࡶ࡟ᰁⰍయࡢᵓ㐀࠾ࡼࡧᩘࡢ␗ᖖࢆㄏⓎࡍࡿࡇ࡜ࡀぢฟࡉࢀ࡚࠾ࡾ, ࡇࢀࡽࡢ⤖ᯝ࠿ ࡽM ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ [75] ࠋ ࡋࡓࡀࡗ࡚, ௒ᅇᚓࡽࢀࡓ⤖ᯝ࠿ࡽ, ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡾ UBD ࡢ G2ᮇ࠿ࡽࡢ␗ᖖⓎ⌧ ࡀㄏⓎࡉࢀ, ࡑࢀࡀ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺࡢṇᖖ࡞᫬ᮇࡼࡾࡶ᪩࠸᫬ᮇ࡛ ࡢUBD ࡢ㐣๫࡞ᶵ⬟ஹ㐍ࢆㄏⓎࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡇࢀࡽࡢ཯ᛂࡣ⫢Ⓨࡀ ࢇ≀㉁≉␗ⓗ࡛࠶ࡾ, 㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢ PMZ ࡟ࡼࡗ࡚ࡣㄏⓎࡉࢀ࡞࠿ࡗࡓࠋࡉ ࡽ࡟, ⫢Ⓨࡀࢇ≀㉁ࡣ 28 ᪥┠࡛ p-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ ྜࢆῶᑡࡉࡏࡓࡇ࡜࠿ࡽ, ቑṪάᛶࢆஹ㐍ࡍࡿ⫢Ⓨࡀࢇ≀㉁ࡣ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵ ࢡ࣏࢖ࣥࢺᶵᵓࢆ୙᏶඲࡞ࡶࡢ࡜ࡍࡿࡇ࡜࡛, ቑṪ⣽⬊ࡢ M ᮇ࠿ࡽࡢ᪩ᮇࡢ⛣⾜ࢆㄏ Ⓨࡋ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀ, ࡲࡓࡇࡢ཯ᛂࡣࣛࢵࢺ࡬ࡢ཯᚟ᢞ୚㛤ጞᚋ 28 ᪥┠࡛ㄏⓎ ࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ3 ᪥┠࡟࠾࠸࡚, PH ࡜ ANIT ࡣ㧗࠸⣽⬊ቑṪㄏⓎᛶࢆ♧ ࡋ࡚࠸ࡓ࡟ࡶ㛵ࢃࡽࡎ, UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ M ᮇ࡟Ꮡᅾࡍࡿ๭ྜࢆ኱ࡁࡃࡣῶᑡࡉࡏ ࡞࠿ࡗࡓࡇ࡜࠿ࡽ, ቑṪ⣽⬊ࡢ M ᮇ࠿ࡽࡢ᪩ᮇࡢ⛣⾜ࡣ, Ⓨࡀࢇ≀㉁࡟ࡼࡾ≉␗ⓗ࡟ㄏ Ⓨࡉࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࡞࠸ࡋ M ᮇ㛵㐃㑇ఏᏊࡢⓎ⌧࡟㛵ࡋ࡚, MEG, TAA ࠾ࡼࡧPMZ ࡣ 3 ᪥┠࠾ࡼࡧ 7 ᪥┠࡟࠾࠸࡚ Aurka, Aurkb, Bub1, Mad1l1, Mad2l1 ࠾ࡼࡧ

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31

Mad2l1 ࠾ࡼࡧ Plk1 ࡢ mRNA Ⓨ⌧ࢆ, TAA ࡣ Aurka, Aurkb, Mad2l1 ࠾ࡼࡧ Plk1 ࡢ mRNA

Ⓨ⌧ࢆቑຍࡉࡏࡓࠋ୍᪉࡛, PMZ ࡣࡇࢀࡽࡢ㑇ఏᏊࡢ mRNA Ⓨ⌧ࡣቑຍࡉࡏ࡞࠿ࡗࡓࠋ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺࡣ⣳㗽⣒ࡀ᥋⥆ࡉࢀ࡚࠸࡞࠸࢟ࢿࢺࢥ࢔ࡀᏑᅾࡍࡿ 㝿࡟άᛶ໬ࡉࢀ, ྛ࢟ࢿࢺࢥ࢔ࡀ᰾ศ⿣⣳㗽య࡟⤖ྜࡍࡿࡲ࡛᰾ศ⿣ࢆ೵Ṇࡉࡏ, ᰁⰍ య␗ᩘᛶࡀ⏕ࡌࡿࡇ࡜ࢆ㜵Ṇࡋ࡚࠸ࡿ [79] ࠋM ᮇ㛵㐃㑇ఏᏊࡢ㐣๫Ⓨ⌧ࡣ, ங⒴ࡸႃ 㢌⒴࠿ࡽసฟࡉࢀࡓᇵ㣴⣽⬊ࡸ, ႃ㢌⒴, ⫶⒴ࡸ⭤⬔⒴⤌⧊࡟࠾࠸࡚ㄆࡵࡽࢀ, ᰁⰍయ ୙Ᏻᐃᛶࡸ⭘⒆ࡢᝏᛶᗘ࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [25, 84, 85, 86] ࠋࡇࢀ ࡽࡢ⤖ᯝ࠿ࡽ, Ⓨࡀࢇ≀㉁ࡢᢞ୚㛤ጞᚋ 28 ᪥┠࡛ㄏⓎࡉࢀࡿ M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ ࣏࢖ࣥࢺ࡞࠸ࡋM ᮇ㛵㐃㑇ఏᏊࡢ mRNA Ⓨ⌧ࡢቑຍࡣ, ᰁⰍయ␗ᖖࡢⓎ⏕ࢆ㜵Ṇࡍࡿ ࡓࡵ࡟M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡀάᛶ໬ࡋࡓࡇ࡜࡛, M ᮇ࡛⣽⬊࿘ᮇࡀ೵ Ṇࡋ࡚࠸ࡿ⫢⣽⬊ࡀቑຍࡋ࡚࠸ࡿࡇ࡜ࢆ཯ᫎࡋ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋࡲࡓ, ࡇࢀࡽ ࡢ⫢⣽⬊ࡣ, M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡀ◚⥢ࡋ࡚࠸ࡿ⫢⣽⬊࡜ࡣูࡢ⣽⬊ 㞟ᅋ࡛࠶ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ ⫢㒊ศษ㝖ࡣ, ฎ⨨ᚋ 2-4 ᪥┠࡟⫢⣽⬊ࡢቑṪάᛶࢆஹ㐍ࡋ, ࡑࡢቑṪάᛶࡣฎ⨨ᚋ 6 ᪥┠ࡲ࡛࡟ṇᖖࣞ࣋ࣝ࡟ᡠࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [18, 35] ࠋᮏ◊✲࡛ࡣ, ⫢Ⓨࡀࢇ ≀㉁ࡢᢞ୚࡟ࡼࡗ࡚⏕ࡌࡿ⣽⬊ቑṪάᛶࡢஹ㐍᫬࡟ㄏⓎࡉࢀࡿ⣽⬊࿘ᮇ㛵㐃ศᏊࡢⓎ ⌧ኚື࡜, PH ฎ⨨࡞࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࡢᢞ୚࡟ࡼࡗ࡚⏕ࡌࡿ෌⏕ᛶቑṪ᫬࡟ ㄏⓎࡉࢀࡿⓎ⌧ኚືࡢ㐪࠸ࢆ⤒᫬ⓗ࡟᳨ウࡋࡓࠋᮏ◊✲࡟࠾࠸࡚, PH, 㠀Ⓨࡀࢇᛶ⫢ẘ

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32

ᛶ≀㉁ࡢAPAP ࠾ࡼࡧ ANIT ࡣ 3 ᪥┠࡛, PMZ ࡣ 28 ᪥┠࡛⫢⣽⬊ࡢቑṪάᛶࢆஹ㐍ࡋ ࡓࠋ28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁ࡢ MEG ࠾ࡼࡧ TAA ࡣ⣽⬊ቑṪάᛶࡢஹ㐍࡜࡜ࡶ࡟ p21Cip1

ࡢάᛶ໬ࢆㄏⓎࡋࡓࡢ࡟ᑐࡋ, ࡇࢀࡽࡢฎ⨨ࡣ 28 ᪥┠࡛ p21Cip1_{ࡢάᛶ໬ࢆక࠺ቑṪά} ᛶࡢஹ㐍ࡣㄏⓎࡋ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ, ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡗ࡚ㄏⓎࡉࢀࡿ p21Cip1_{ࡢάᛶ໬ࢆక࠺⣽⬊ቑṪάᛶࡢஹ㐍ࡣ, Rbl2 ࡢ mRNA Ⓨ⌧ࡢῶᑡ࡜ࡑࢀ࡟క࠺} RBL2 ⺮ⓑ㉁ࡢⓎ⌧ῶᑡ࡟㉳ᅉࡍࡿⓎࡀࢇ≀㉁≉␗ⓗ࡞ G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ ◚⥢ࡀ⏕ࡌࡿ㝿࡟ㄆࡵࡽࢀࡿ཯ᛂ࡛࠶ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋᑐ↷ⓗ࡟, PH ࡸ㠀Ⓨࡀࢇᛶ ⫢ẘᛶ≀㉁ࡣ28 ᪥┠࡟࠾࠸࡚, p21Cip1_{ࡢάᛶ໬ࢆకࢃࡎ, ࠸ࡃࡘ࠿ࡢࣇ࢕࣮ࢻࣂࢵࢡᶵ} ᵓࡀാࡃࡇ࡜࡛⣽⬊ቑṪάᛶࡀᢚไࡉࢀ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ౛࠼ࡤPH ࡟ࡘ࠸࡚ ࡣ, suppressor of cytokine signaling 3 ࡀࢧ࢖ࢺ࢝࢖ࣥࡢࢩࢢࢼࣝ࢝ࢫࢣ࣮ࢻࢆ㈇࡟ไᚚࡍ ࡿࡇ࡜࡛, ⫢⣽⬊ࡢ෌⏕ᛶቑṪࢆᢚไࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [65] ࠋ ⤖ㄽ࡜ࡋ࡚, ⫢Ⓨࡀࢇ≀㉁࡟ࡼࡗ࡚ㄏⓎࡉࢀࡿⓎࡀࢇ≀㉁≉␗ⓗ࡞཯ᛂࡣ, ᢞ୚㛤ጞ ᚋ28 ᪥┠࠿ࡽฟ⌧ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋRbl2 ࡢ mRNA Ⓨ⌧ࡢῶᑡ, Mdm2 ࡢ mRNA Ⓨ⌧࠾ࡼࡧp-MDM2 Ⓨ⌧⣽⬊ࡢቑຍ࡟ࡼࡿ p53 ࠾ࡼࡧ RB ⺮ⓑ㉁ࡢ୙㊊࡜ࡑࢀ࡟ᘬࡁ ⥆ࡃG1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࢆㄏⓎࡋ, ࡑࢀࡀ, ⫢Ⓨࡀࢇ≀㉁ࡢ཯᚟ᢞ୚㛤ጞ ᚋ28 ᪥┠࡛ㄆࡵࡽࢀࡿ⣽⬊ቑṪάᛶࡢஹ㐍࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ᥎ᐹࡉࢀࡓࠋUBD 㝧ᛶ⣽⬊ࡢM ᮇ࡟࠾ࡅࡿ๭ྜࡢῶᑡࡣ, M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࢆ ♧၀ࡋ࡚࠾ࡾ, ቑṪ⣽⬊ࡢ M ᮇ࡟࠾ࡅࡿ๭ྜࡢῶᑡࡣ, ቑṪ⣽⬊ࡢ M ᮇ࠿ࡽࡢ᪩ᮇࡢ

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33

⛣⾜ࢆ཯ᫎࡍࡿࡶࡢ࡛࠶ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋDNA ᦆയࡢ⵳✚ࡣ, ࠾ࡑࡽࡃ MDM2 ࡢ άᛶ໬࡟ࡼࡿp53 ࡢศゎಁ㐍࡟㛵㐃ࡋ࡚⏕ࡌ࡚࠾ࡾ, ࡑࢀࡀ p21 Cip1_{ࡢ␗ᖖ࡞άᛶ໬࠾ࡼ}

(36)

34 ᑠᣓ

➨1 ❶࡛ࡣ, ⫢Ⓨࡀࢇ≀㉁ᢞ୚ึᮇ࡟ㄏⓎࡉࢀࡿ⣽⬊࿘ᮇไᚚ◚⥢࡜ࡑࡢฟ⌧᫬ᮇࡢ ᳨ウࢆ┠ⓗ࡜ࡋ࡚, ⫢Ⓨࡀࢇ≀㉁ࢆࣛࢵࢺ࡬᭱኱ 28 ᪥㛫཯᚟ᢞ୚ࡋࡓࠋࡑࡢ⤖ᯝ, ᢞ୚ 㛤ጞᚋ 28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁ࡣ, ⣽⬊ቑṪάᛶࡢஹ㐍࡜࡜ࡶ࡟ p21Cip1 _{㝧ᛶ⣽⬊⋡,}

p-MDM2 㝧ᛶ⣽⬊⋡, cleaved caspase 3 㝧ᛶ⣽⬊⋡࠾ࡼࡧ DNA ᦆയ㛵㐃㑇ఏᏊࡢ mRNA Ⓨ⌧ࡢቑຍࢆㄏⓎࡋࡓࡇ࡜࠿ࡽ, Ⓨࡀࢇ≀㉁≉␗ⓗ࡞཯ᛂࡣ 28 ᪥┠࠿ࡽฟ⌧ࡍࡿࡇ࡜ ࡀ♧၀ࡉࢀࡓࠋ⫢Ⓨࡀࢇ≀㉁ࡣࡉࡽ࡟, Rbl2 ࡢ mRNA Ⓨ⌧ࡢῶᑡ, Cdkn1a ࠾ࡼࡧ Mdm2 ࡢmRNA Ⓨ⌧ࡢቑຍ, UBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜ࠾ࡼ ࡧp-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࡢῶᑡࢆㄏⓎࡋࡓࠋࡇࢀࡽ ࠿ࡽ, 28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁ࡣ, M ᮇ࡟࠾ࡅࡿ UBD ࡢⓎ⌧ῶᑡ࡟㉳ᅉࡍࡿ M ᮇࢫࣆࣥ ࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢࡜ࡑࢀ࡟క࠺ቑṪ⣽⬊ࡢ M ᮇ࠿ࡽࡢ᪩ᮇࡢ⛣⾜ࢆㄏ Ⓨࡍࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋࡲࡓ, Rbl2 ࡢ mRNA Ⓨ⌧ࡢῶᑡ, Mdm2 ࡢ mRNA Ⓨ⌧࠾ࡼࡧ p-MDM2 Ⓨ⌧⣽⬊ࡢቑຍ࡟ࡼࡗ࡚, p53 ࠾ࡼࡧ RB ⺮ⓑ㉁ࡢ୙㊊࡜ᘬࡁ⥆ࡃ G1/S ࢳ࢙ࢵ ࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢ࡀㄏⓎࡉࢀ, ࡑࢀࡀ⫢Ⓨࡀࢇ≀㉁≉␗ⓗ࡞ቑṪάᛶஹ㐍࡟ᐤ୚ ࡍࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋDNA ᦆയࡢ⵳✚ࡣ, MDM2 ࡢάᛶ໬࡟㉳ᅉࡍࡿ p53 ࡢศゎಁ 㐍࡟㛵㐃ࡋ࡚࠾ࡾ, ࡑࢀࡀ p21Cip1_{ࡢ␗ᖖ࡞άᛶ໬࡜ࡑࢀ࡟క࠺࢔࣏ࢺ࣮ࢩࢫࢆㄏⓎࡋ} ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ

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35 ➨2 ❶

ࣛࢵࢺࡢ᭱኱90 ᪥㛫཯᚟ᢞ୚౛࡛ࡢ⫢Ⓨࡀࢇ≀㉁࣭⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮ࡢ ⣽⬊࿘ᮇไᚚ␗ᖖ࡬ࡢ㛵୚ࡢ᳨ウ

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36 ⥴ゝ

➨1 ❶ࡢ⤖ᯝࡼࡾ, ཯᚟ᢞ୚㛤ጞᚋ 28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁ࡣ, ⣽⬊ቑṪάᛶࡢஹ㐍࡜

࡜ࡶ࡟Rbl2 ࡢ mRNA Ⓨ⌧㔞ࡢῶᑡ, Mdm2 ࡢ mRNA Ⓨ⌧࠾ࡼࡧ phosphorylated-MDM2

(p-MDM2) 㝧ᛶ⣽⬊⋡ࢆቑຍࡉࡏࡓࡇ࡜࠿ࡽ, G1/S ࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢࡟㉳ᅉ ࡋࡓ⣽⬊࿘ᮇ㐍⾜ࡢಁ㐍ࢆㄏⓎࡋ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋࡉࡽ࡟, ubiquitin D (UBD) ࡢM ᮇ࡛ࡢⓎ⌧ࢆῶᑡࡉࡏࡿࡇ࡜࡛, M ᮇࢫࣆࣥࢻࣝࢳ࢙ࢵࢡ࣏࢖ࣥࢺᶵᵓࡢ◚⥢࡜ࡑ ࢀ࡟క࠺ቑṪ⣽⬊ࡢ M ᮇ࠿ࡽࡢ᪩ᮇࡢ⛣⾜ࢆㄏⓎࡋ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋࡋ࠿ࡋ ࡞ࡀࡽ, ᮏ◊✲࡟ඛ❧ࡘඛ⾜◊✲࡟࠾࠸࡚, piperonyl butoxide ࡞࡝ࡢⓎࡀࢇ≀㉁ࡣࣛࢵ ࢺ࡬ࡢ 28 ᪥㛫ࡢ཯᚟ᢞ୚࡟ࡼࡗ࡚ࡶ, ᶆⓗ⣽⬊ࡢቑṪάᛶࡢஹ㐍ࢆ♧ࡉࡎ, ࡇࢀࡽࡢ ≀㉁࡟ࡼࡗ࡚ࡣ⣽⬊࿘ᮇไᚚ␗ᖖࢆ♧၀ࡍࡿࡼ࠺࡞཯ᛂࡣぢฟࡉࢀ࡞࠿ࡗࡓ [74] ࠋ ᮏ❶࡛ࡣ, ➨ 1 ❶ࡼࡾ, ᢞ୚㛤ጞᚋ 28 ᪥┠࡛⫢Ⓨࡀࢇ≀㉁࡟ࡼࡾㄏⓎࡉࢀࡓ⣽⬊࿘ᮇ ไᚚ␗ᖖࡀ, ⣽⬊ቑṪㄏⓎᛶࡢప࠸⫢Ⓨࡀࢇ≀㉁ࡸ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮࡟ࡼࡗ࡚ࡶ ྠᵝ࡟ㄏⓎࡉࢀࡿ࠿ྰ࠿ࢆ᳨ウࡋࡓࠋࡍ࡞ࢃࡕ, ࣛࢵࢺ࡟⫢Ⓨࡀࢇ≀㉁, ᙅ࠸⫢Ⓨࡀࢇ ᛶࡀ♧၀ࡉࢀ࡚࠾ࡾ⣽⬊ቑṪㄏⓎᛶࡢప࠸ࡇ࡜ࡀ᝿ᐃࡉࢀࡿ≀㉁, ⫢Ⓨࡀࢇࣉ࣮ࣟࣔ ࢱ࣮࡞࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࢆ7, 28 ࡞࠸ࡋ 90 ᪥㛫཯᚟ᢞ୚ࡋ, ➨ 1 ❶࡜ྠᵝ࡟ච ␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧreal-time RT-PCR ゎᯒἲࢆ⏝࠸᳨࡚ウࡋࡓࠋ

(39)

37 ᮦᩱ࠾ࡼࡧ᪉ἲ

໬

໬Ꮫ≀㉁

Oxfendazole (OX) ࡣ ᮾ ி ໬ ᡂ ᕤ ᴗ ᰴ ᘧ ఍ ♫ (Tokyo, Japan) ࡼ ࡾ , acetaminophen (APAP) ࠾ࡼࡧ methapyrilene (MP) ࡣࢩࢢ࣐࢔ࣝࢻࣜࢵࢳ (St. Louis, MO, USA) ࡼࡾ, β-naphthoflavone (BNF) , carbadox (CRB) , leucomalachite green (LMG) , thioacetamide (TAA) , promethazine (PMZ) ࠾ࡼࡧ methyl cellulose 400 ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ (Osaka, Japan) ࡼࡾ㉎ධࡋࡓࠋ ౪ヨື≀ 5 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆ᪥ᮏ࢚ࢫ࢚ࣝࢩ࣮ࡼࡾ㉎ධࡋ, ᗘ 23s3°C, ‵ᗘ 50s 20%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳, ⢊ᮎ CRF-1 (࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ, ᮾி) ࠾ࡼࡧ㣧ᩱỈࡢ⮬⏤⤥Ỉୗ࡛㣫⫱ࡋ, ୍㐌㛫ࡢ㥆໬ᮇ㛫ࡢᚋ, ␗ᖖࡀㄆࡵࡽࢀ࡞࠿ࡗࡓ ື≀ࢆᐇ㦂࡟౪ࡋࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ື≀࡟ᑐࡋ࡚, ⫢Ⓨࡀࢇ≀㉁, ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮࡞࠸ࡋ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁ࢆ 7, 28 ࡞࠸ࡋ 90 ᪥㛫཯᚟ᢞ୚ࡋࡓࠋ7 ࡞࠸ࡋ 28 ᪥㛫཯᚟ᢞ୚ヨ㦂࡛ࡣ, MP, CRB, LMG,

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38 BNF, OX ࠾ࡼࡧ PMZ ࢆ⏝࠸, 90 ᪥㛫཯᚟ヨ㦂࡛ࡣ, MP, TAA, CRB, LMG, BNF, OX, APAP ࠾ࡼࡧ PMZ ࢆ⏝࠸ࡓࠋᢞ୚⤊஢ᚋ, ⫢⮚࡟࠾ࡅࡿ⣽⬊ቑṪάᛶ, ⣽⬊࿘ᮇ㛵㐃ศ ᏊࡢⓎ⌧࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫࡢ⤒᫬ⓗ࡞ኚືࢆච␿⤌⧊໬ᏛᰁⰍ࡞࠸ࡋ real-time RT-PCR ἲ࡟ࡼࡾゎᯒࡋࡓࠋ MP, TAA ࠾ࡼࡧ CRB ࡣ⫢Ⓨࡀࢇ≀㉁࡜ࡋ࡚㑅ᐃࡋࡓ [6, 32, 57] ࠋLMG ࡣ 2 ᖺ㛫ࡢ ࡀࢇཎᛶヨ㦂࡛ࡣࢃࡎ࠿࡟⫢⮚⭘⒆ࡢⓎ⏕⋡ࢆቑຍࡉࡏࡿࡢࡳ࡛࠶ࡿࡇ࡜࠿ࡽ, ᙅ࠸ ⫢Ⓨࡀࢇᛶࡀ♧၀ࡉࢀ࡚࠸ࡿ≀㉁࡜ࡋ࡚㑅ᐃࡋࡓ [59] ࠋBNF ࠾ࡼࡧ OX ࡣ⫢Ⓨࡀࢇࣉ ࣮ࣟࣔࢱ࣮࡜ࡋ࡚㑅ᐃࡋ [49, 70] , PMZ ࠾ࡼࡧ APAP ࡣ㠀Ⓨࡀࢇᛶ⫢ẘᛶ≀㉁࡜ࡋ࡚㑅 ᐃࡋࡓ [51, 52] ࠋOX ࡣ㛗ᮇ㛫ࡢ཯᚟ᢞ୚ヨ㦂࡛⫢⮚⭘⒆ࢆㄏⓎࡏࡎ [80] , BNF ࡣኚ␗ ཎᛶࢆ♧ࡉࡎ, ࡲࡓ, ࣛࢵࢺࡢ⫢⮚࡟ᑐࡋ࡚Ⓨࡀࢇ࢖ࢽࢩ࢚࣮ࢩࣙࣥస⏝ࢆ♧ࡉ࡞࠿ࡗ ࡓࡇ࡜࠿ࡽ༢⊂ᢞ୚࡛ࡣⓎࡀࢇᛶࢆ♧ࡉ࡞࠸Ⓨࡀࢇࣉ࣮ࣟࣔࢱ࣮࡜ࡳ࡞ࡉࢀ࡚࠸ࡿ [21, 45] ࠋ 7 ࠾ࡼࡧ 28 ᪥㛫ᢞ୚ヨ㦂࡛ࡣ, 6 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆึᮇయ㔜࡟ᇶ࡙࠸࡚, ↓ฎ ⨨ᑐ↷⩌ (n = 20) , MP (1,000 ppm; n = 20) , CRB (300 ppm; n = 20) , LMG (1,160 ppm; n = 20) , BNF (10,000 ppm; n = 20) ࡞࠸ࡋ OX (500 ppm; n = 20) ࡢΰ㣵ᢞ୚ࢆ⾜࠺⩌, ࡞࠸ ࡋPMZ (100 mg/kg య㔜, n = 22) ࡢᙉไ⤒ཱྀᢞ୚ࢆ⾜࠺⩌ࡢ 7 ࡘࡢ⩌࡟ศࡅࡓࠋPMZ ࡢ ᢞ୚ࡢ㝿࡟, ⁐፹࡜ࡋ࡚ 0.5% methyl cellulose ⁐ᾮࢆ౑⏝ࡋࡓࠋ↓ฎ⨨ᑐ↷⩌ࡣᇶ♏㣫 ᩱ࡜㣧ᩱỈ࡛⥔ᣢࡋࡓࠋᢞ୚㛤ጞᚋ 7 ᪥┠࠾ࡼࡧ 28 ᪥┠࡟, ྛ⩌༙ᩘࡎࡘࡢື≀࡟ࡘ

(41)

39 ࠸࡚, CO2/O2࡟ࡼࡿ῝㯞㓉ୗ࡛኱ື⬦࠿ࡽࡢᨺ⾑࡟ࡼࡿᏳᴦẅࢆ⾜࠸, ⫢⮚ࢆ᥇ྲྀࡋࡓࠋ 90 ᪥㛫ᢞ୚ヨ㦂࡛ࡣ, 6 㐌㱋ࡢ㞝ᛶ F344 ࣛࢵࢺࢆึᮇయ㔜࡟ᇶ࡙࠸࡚, ↓ฎ⨨ᑐ↷⩌ (n = 10) , TAA (400 ppm; n = 10) , MP (1,000 ppm; n = 10) , CRB (300 ppm; n = 20) , LMG (1,160 ppm; n = 10) , BNF (10,000 ppm; n = 10) , OX (500 ppm; n = 10) ࡞࠸ࡋ APAP (10,000 ppm; n = 10) ࡢΰ㣵ᢞ୚ࢆ⾜࠺⩌, ࡞࠸ࡋ PMZ (100 mg/kg య㔜, n = 11) ࡢᙉ ไ⤒ཱྀᢞ୚ࢆ⾜࠺⩌ࡢ9 ࡘࡢ⩌࡟ศࡅ, ཯᚟ᢞ୚ࢆ⾜ࡗࡓࠋPMZ ࡢᢞ୚ࡢ㝿࡟, ⁐፹࡜ ࡋ࡚0.5% methyl cellulose ⁐ᾮࢆ౑⏝ࡋࡓࠋ↓ฎ⨨ᑐ↷⩌ࡣᇶ♏㣫ᩱ࡜㣧ᩱỈ࡛⥔ᣢࡋ ࡓࠋ↓ฎ⨨ᑐ↷⩌ࡣᇶ♏㣫ᩱ࡜㣧ᩱỈ࡛⥔ᣢࡋࡓࠋᢞ୚㛤ጞᚋ 90 ᪥┠࡟, ඲ື≀࡟ࡘ ࠸࡚, CO2/O2࡟ࡼࡿ῝㯞㓉ୗ࡛኱ື⬦࠿ࡽࡢᨺ⾑࡟ࡼࡿᏳᴦẅࢆ⾜࠸, ⫢⮚ࢆ᥇ྲྀࡋࡓࠋ MP, TAA ࠾ࡼࡧ CRB ࡢᢞ୚⏝㔞ࡣⓎࡀࢇ⏝㔞ࢆタᐃࡋࡓ [6, 32, 57] ࠋBNF ࠾ࡼࡧ OX ࡢᢞ୚⏝㔞ࡣ, ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝࣛࢵࢺࢆ⏝࠸ࡓ 6 㐌㛫ࡢ཯᚟ᢞ୚ᚋ࡟ glutathione S-transferase placental ࡟ࡼࡗ᳨࡚ฟࡉࢀࡿ๓ࡀࢇ⑓ኚࡢᙧᡂࢆಁ㐍ࡍࡿ⏝㔞 ࢆタᐃࡋࡓ [49, 70] ࠋLMG, APAP ࠾ࡼࡧ PMZ ࡢᢞ୚⏝㔞ࡣ, 28 ᪥㛫࡞࠸ࡋ 13 㐌㛫཯ ᚟ᢞ୚ヨ㦂࡟࠾࠸࡚ࣛࢵࢺ࡟⫢ẘᛶࢆㄏⓎࡍࡿ⏝㔞ࢆタᐃࡋࡓ [51, 52, 59] ࠋ ື≀ᐇ㦂ィ⏬ࡣ, ᅜ❧኱Ꮫἲேᮾி㎰ᕤ኱Ꮫࡢື≀ᐇ㦂೔⌮ጤဨ఍࡟ᥦฟࡋ࡚ᢎㄆ ࢆཷࡅ, ື≀ࡢྲྀࡾᢅ࠸࡟ࡘ࠸࡚ࡶ, ྠ኱Ꮫࡢᐇ㦂ື≀ᣦ㔪ࢆ㑂Ᏺࡋࡓࠋ ⑓ ⑓⌮⤌⧊ᏛⓗᰁⰍ࠾ࡼࡧච␿⤌⧊໬ᏛᰁⰍ

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40

ࣃࣛࣇ࢕ࣥᅛᐃࡋࡓ⫢⮚ࢆ 3 μm ࡢཌࡉ࡟ⷧษࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥ (HE) ᰁⰍ࠾ࡼࡧච␿⤌⧊໬ᏛᰁⰍ࡟౪ࡋࡓࠋ⫢⮚ษ∦ࢆ⏝࠸࡚, G1ᮇ࠿ࡽM ᮇ࡟Ꮡᅾ

ࡍࡿ⣽⬊ࡢ᰾࡟Ⓨ⌧ࡍࡿ⣽⬊ቑṪᣦᶆࡢKi-67 [68] , ࢔࣏ࢺ࣮ࢩࢫ㐣⛬ࡢᚋᮇ࡟Ⓨ⌧ࡍ ࡿ࢔࣏ࢺ࣮ࢩࢫᣦᶆࡢcleaved caspase 3 [15] , G2/M ᮇ࡟ DNA ࢹ࢝ࢸࢿ࣮ࢩࣙࣥ࡟㛵ࢃࡿ

topoisomerase IIα (TOP2A) [43] , M ᮇ ࡢ ึ ᮇ ࡟ ࢡ ࣟ ࣐ ࢳ ࣥ ࡢ จ 㞟 ࡟ 㛵 ࢃ ࡿ phosphorylated-histone H3 (p-Histone H3) [23] , ࢧ࢖ࢡࣜࣥ౫Ꮡᛶ࢟ࢼ࣮ࢮ㜼ᐖᅉᏊࡢ 1 ࡘ࡛࠶ࡿp21 Cip1_{[69] , G}

2/M ᮇ࡟࠾࠸࡚ mitotic arrest deficient 2 (MAD2) ࡞࡝ࡢࢳ࢙ࢵࢡ ࣏࢖ࣥࢺ⺮ⓑ㉁ࡢ࢟ࢿࢺࢥ࢔ᒁᅾࢆῶᑡࡉࡏࡿࡇ࡜࡛ᰁⰍయ୙Ᏻᐃᛶࢆㄏᑟࡍࡿ ubiquitin D (UBD) [22, 37] , ࠾ࡼࡧ p53 ࡢୗὶศᏊࡢ୍ࡘ࡛ p53 ࡢศゎࢆಁ㐍ࡍࡿ phosphorylated-MDM2 (p-MDM2) [40, 44] ࡟ᑐࡍࡿ୍ḟᢠయࢆ⏝࠸࡚, ච␿⤌⧊໬Ꮫᰁ Ⰽࢆᐇ᪋ࡋࡓࠋ⏝࠸ࡓᢠయ, ㈿ά໬᪉ἲ࠾ࡼࡧᢠయᕼ㔘᮲௳ࡣ Table 2-1 ࡟♧ࡋࡓࠋࢩࢢ ࢼ᳨ࣝฟࡣVECTASTAINט_{Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA)}

ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯ᛂࡣ 3,3’-diaminobenzidine/H2O2 ࢆ⏝࠸࡚ྍど໬ࡋࡓᚋ,

࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࡋࡓࠋ

UBD ࡢ⣽⬊࿘ᮇෆ࡛ࡢฟ⌧᫬ᮇࢆ᳨ウࡍࡿ┠ⓗ࡛,

UBD ࡜ TOP2A ࡞࠸ࡋ

p-Histone H3 ࡜ࡢ஧㔜ᰁⰍࢆ⾜ࡗࡓࠋUBD ࡢࢩࢢࢼ᳨ࣝฟࡣ VECTASTAINט_{Elite ABC}

Kit㸦Vector Laboratories㸧ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯ᛂࡣ 3,3’-diaminobenzidine/H2O2

(43)

41

ࡣVECTASTAINט_{Elite ABC-AP kit (Vector Laboratories) ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸, ච␿཯}

ᛂࡣVECTOR Red Alkaline Phosphate Substrate Kit I (Vector Laboratories) ࢆ⏝࠸࡚ྍど໬ ࡋࡓࠋ

ච

ච␿⤌⧊໬ᏛᰁⰍࡢᐃ㔞ゎᯒ

ච␿⤌⧊໬ᏛᰁⰍ࡟ࡼࡾ, Ki-67, TOP2A, p-Histone H3, UBD, p21 CIp1_{࠾ࡼࡧ p-MDM2}

࡟ᑐࡋ࡚㝧ᛶࢆ♧ࡍ⫢⣽⬊ᩘࡣ, 200 ಸࡢಸ⋡࡛ 1 ಶయᙜࡓࡾࣛࣥࢲ࣒࡟ 10 ど㔝㑅ᢥࡋ, cleaved caspase 3 ࡟ᑐࡋ࡚㝧ᛶࢆ♧ࡍ⫢⣽⬊ᩘࡣ, 100 ಸࡢಸ⋡࡛ 1 ಶయᙜࡓࡾࣛࣥࢲ࣒ ࡟5 ど㔝㑅ᢥࡋ, ࢝࢘ࣥࢺࡋࡓࠋど㔝ࡢ㑅ᢥࡣ, ⾑⟶ࡸ⤖ྜ⤌⧊ࡀධࡽ࡞࠸ࡼ࠺࡟⾜ࡗ ࡓࠋ㛛⬦࿘㎶࡟㝧ᛶ⣽⬊ࡀከࡃศᕸࡍࡿ࡞࡝, 㝧ᛶ⣽⬊ࡀ⫢⮚⤌⧊඲య࡟ᆒ୍࡟ศᕸࡋ ࡞࠸ሙྜࡣ, ೫ࡗࡓ㡿ᇦࢆ㑅ᢥࡋ࡞࠸ࡼ࠺࡟, 㞄᥋ࡋࡓ 5 ࡞࠸ࡋ 10 ど㔝ࢆ㑅ᢥࡋࡓࠋ㝧 ᛶ⣽⬊ᩘࡣどぬⓗ࡟࢝࢘ࣥࢺࡋ, ⥲⣽⬊ᩘࡣ⏬ീゎᯒࢯࣇࢺ WinROOF (୕㇂ၟ஦, ᮾ ி) ࢆ⏝࠸࡚ᐃ㔞໬ࡋ, 㝧ᛶ⣽⬊⋡ࢆ⟬ฟࡋࡓࠋ Real-time RT-PCR ࡟ࡼࡿゎᯒ

⣽⬊࿘ᮇ㛵㐃㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࢆ᳨ウࡍࡿࡓࡵ࡟, SYBR®_{Green PCR Master Mix}

(Life Technologies, Carlsbad, CA, USA) ࢆ⏝࠸, Step One Plus™ Real-time PCR System (Life Technologies) ࡟࡚, 〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚, mRNA Ⓨ⌧ࡢᐃ㔞ゎᯒࢆᐇ

(44)

42

᪋ࡋࡓࠋࣉࣛ࢖࣐࣮ࡣPrimer Express software (Version 3.0; Life Technologies) ࢆ⏝࠸࡚タィࡋ, Table 2-2 ࡟♧ࡋࡓࠋྛ㑇ఏᏊࡢ mRNA Ⓨ⌧ࡣ, ↓ฎ⨨ᑐ↷⩌࡛ࡢⓎ⌧್࡟ᑐࡍࡿ ┦ᑐ್࡜ࡋ࡚ồࡵ, ෆᅉᛶࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚ hypoxanthine phosphoribosyltransferase 1 (Hprt1) ࡞࠸ࡋ actin, beta (Actb) ࡢ᳨㔞⥺ࢆồࡵ, 2–ΔΔC

T method [39] ࡟࡚⟬ฟࡋࡓࠋ ⤫ ⤫ィゎᯒ ඲࡚ࡢᐃ㔞ࢹ࣮ࢱ࡟ࡘ࠸࡚ᖹᆒ್࠾ࡼࡧᶆ‽೫ᕪࢆồࡵࡓࠋᩘ್ࢹ࣮ࢱࡢከ⩌㛫ẚ㍑ ࡣ, Bartlett ᳨ᐃ࡛➼ศᩓࢆ☜ㄆࡋࡓᚋ, ୍ඖ㓄⨨ศᩓศᯒࢆ⾜ࡗࡓࠋBartlett ᳨ᐃ࡛➼ศ ᩓ࡛࠶ࡗࡓሙྜ࡟ࡣDunnett ᳨ᐃࢆ⾜࠸, ➼ศᩓ࡛࡞࠿ࡗࡓሙྜ࡟ࡣ Steel ᳨ᐃࢆ⾜ࡗࡓࠋ ඲࡚ࡢᩘ್ࢹ࣮ࢱࡣ, ↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋࡓࠋ

(45)

43 ⤖ᯝ య య㔜࠾ࡼࡧ⫢⮚㔜㔞 7 ᪥┠࡟࠾࠸࡚, MP ⩌, CRB ⩌, LMG ⩌, BNF ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Table 2-3) ࠋLMG ⩌, BNF ⩌࠾ࡼࡧ OX ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋ඲ᢞ୚⩌࡛↓ ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋ 28 ᪥┠࡟࠾࠸࡚, ඲ᢞ୚⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆࡵ ࡽࢀࡓ (Table 2-3) ࠋMP ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ῶᑡࡀㄆ ࡵࡽࢀࡓࠋLMG ⩌, BNF ⩌࠾ࡼࡧ OX ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋCRB ⩌, LMG ⩌, BNF ⩌, OX ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷ ⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋ 90 ᪥┠࡟࠾࠸࡚, MP ⩌, TAA ⩌, CRB ⩌, LMG ⩌, BNF ⩌, APAP ⩌࠾ࡼࡧ PMZ ⩌࡛ ࡣ, ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ᭱⤊య㔜ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Table 2-3) ࠋMP ⩌ ࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋLMG ⩌, BNF ⩌, OX ⩌, APAP ⩌࠾ࡼࡧ PMZ ⩌࡛ࡣ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ⤯ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍ ࡀㄆࡵࡽࢀࡓࠋ඲ᢞ୚⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚, ┦ᑐ⫢㔜㔞ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽ ࢀࡓࠋ

(46)

44 ⑓ ⑓⌮⤌⧊Ꮫⓗኚ໬ MP ⩌࡛ࡣ, ඲ᢞ୚ᮇ㛫࡛, ᰾ࡢ኱ᑠ୙ྠ, ᭷⣒ศ⿣␗ᖖ࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫ࡜ඹ࡟, “ᕧ኱⣽⬊” ࡀฟ⌧ࡋ, ࡧₔᛶ࡟ࡑࡢ⣽⬊ࡢศᕸࡀㄆࡵࡽࢀࡓ [57] ࠋ28 ᪥┠࠾ࡼࡧ 90 ᪥┠࡛, ⫹⟶ቑ⏕, 㛫㉁ࡢ⥺⥔ቑ⏕ࢆక࠺ oval cell ࡢቑṪ࠾ࡼࡧⰍ⣲ỿ╔ࡀ, 㛛⬦࿘㎶㡿ᇦ࡟㢧ⴭ࡟ㄆࡵࡽࢀࡓࠋࡉࡽ࡟, 90 ᪥┠࡛⫹⟶⥺⥔⑕࠾ࡼࡧዲ㓟ᛶ⫢⣽⬊ቑṪᕢࡀㄆ ࡵࡽࢀࡓࠋCRB ⩌࡛ࡣ, ඲ᢞ୚ᮇ㛫࡛, ᫂ࡽ࠿࡞⤌⧊Ꮫⓗᡤぢࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ LMG ⩌࡛ࡣ, ඲ᢞ୚ᮇ㛫࡛, ௨๓ࡢሗ࿌࡜ྠᵝ࡟, ᑠⴥ୰ᚰᛶ࡟⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀ ࡓ [59] ࠋࡉࡽ࡟, 90 ᪥┠࡟࠾࠸࡚, ⫢⣽⬊ࡢ⬡⫫ኚᛶࡀᩓᅾᛶ࡟ㄆࡵࡽࢀࡓࠋBNF ⩌ ࡛ࡣ, 7 ᪥┠࡛, ᫂ࡽ࠿࡞⤌⧊Ꮫⓗኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ28 ᪥┠࠾ࡼࡧ 90 ᪥┠࡛ࡣ, ௨๓ࡢሗ࿌࡜ྠᵝ࡟, ᑠⴥ୰ᚰᛶ࡟⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀࡓ [70] ࠋࡉࡽ࡟, 90 ᪥┠࡟ ࠾࠸࡚, ⫢⣽⬊ࡢ⬡⫫ኚᛶࡀᩓᅾᛶ࡟ㄆࡵࡽࢀࡓࠋOX ⩌࡛ࡣ, ඲ᢞ୚ᮇ㛫࡛, ௨๓ࡢሗ ࿌࡜ྠᵝ࡟ᑠⴥ୰ᚰᛶ࡟⫢⣽⬊⫧኱࡜⫢⣽⬊ࡢ⬡⫫ኚᛶࡀㄆࡵࡽࢀࡓ [49] ࠋPMZ ⩌࡛ ࡣ, ඲ᢞ୚ᮇ㛫࡛, ௨๓ࡢሗ࿌࡜ྠᵝ࡟, ᑠⴥ୰ᚰᛶ࡟⫢⣽⬊⫧኱ࡀㄆࡵࡽࢀࡓ [52] ࠋ ࡉࡽ࡟, 90 ᪥┠࡟࠾࠸࡚, ⫢⣽⬊ࡢ⬡⫫ኚᛶࡀᩓᅾᛶ࡟ㄆࡵࡽࢀࡓࠋTAA ⩌࡛ࡣ, 90 ᪥ ┠࡛, ௨๓ࡢሗ࿌࡜ྠᵝ࡟, ᰾ࡢ኱ᑠ୙ྠ, ᭷⣒ศ⿣␗ᖖ࠾ࡼࡧ࢔࣏ࢺ࣮ࢩࢫ࡜ඹ࡟, “ᕧ኱⣽⬊” ࡀㄆࡵࡽࢀ, ࡧₔᛶ࡟ศᕸࡀㄆࡵࡽࢀࡓ [

11

] ࠋࡲࡓ, ⫹⟶ቑ⏕, 㛫㉁ࡢ⥺ ⥔ቑ⏕ࢆక࠺oval cell ࡢቑṪ࠾ࡼࡧⰍ⣲ỿ╔ࡀ, 㛛⬦࿘㎶㡿ᇦ࡟㢧ⴭ࡟ㄆࡵࡽࢀࡓࠋࡉ

(47)

45

ࡽ࡟, ⥺⥔ᛶ⤖ྜ⤌⧊࡟༊⏬ࡉࢀࡓ෌⏕ᛶ⤖⠇ࡢᙧᡂࡀㄆࡵࡽࢀࡓࠋAPAP ⩌࡛ࡣ, ᫂ ࡽ࠿࡞⤌⧊Ꮫⓗᡤぢࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

⣽

⣽⬊࿘ᮇ㛵㐃ศᏊⓎ⌧⣽⬊ࡢศᕸ

Ki-67, p-Histone H3, TOP2A, p21Cip1_࠾ࡼࡧ_{p-MDM2 ࡣ᰾࡟ᒁᅾࡋ, cleaved caspase 3 ࡣ}

᰾࠾ࡼࡧ⣽⬊㉁࡟ᒁᅾࡋ, UBD ࡣ⣽⬊㉁࡞࠸ࡋ᭷⣒ศ⿣⣳㗽య࡟ᒁᅾࡋ࡚࠸ࡓ (Fig. 2-1A–G) ࠋ኱㒊ศࡢ౛࡛ࡣ, Ki-67, p-Histone H3, TOP2A ࠾ࡼࡧ UBD 㝧ᛶ⣽⬊ࡣ⫢⮚⤌ ⧊඲య࡟ᆒ୍࡟ศᕸࡋ࡚࠸ࡓࡀ, TAA ⩌ࡢ 7 ᪥┠, PMZ ⩌ࡢ 28 ᪥┠࡛ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ ࡟ከࡃศᕸࡋ࡚࠸ࡓࠋ඲ᢞ୚⩌ࡢ඲ᢞ୚ᮇ㛫࡟࠾࠸࡚, p21Cip1_{, p-MDM2 ࠾ࡼࡧ cleaved} caspase 3 㝧ᛶ⣽⬊ࡣ㛛⬦࿘ᅖࡢ㡿ᇦ࡟ከࡃศᕸࡋ࡚࠸ࡓࠋ 7 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀ ㄆࡵࡽࢀ, CRB ⩌, LMG ⩌࠾ࡼࡧ OX ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-1A) ࠋ p-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, CRB ⩌࠾ࡼࡧ OX ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡ ࡀㄆࡵࡽࢀࡓ (Fig. 2-1B) ࠋTOP2A 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព ࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌࠾ࡼࡧ OX ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-1C) ࠋUBD 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌, LMG ⩌ ࠾ࡼࡧOX ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-1D) ࠋp21Cip1_{㝧ᛶ⣽⬊⋡ࡣ, MP ⩌, CRB} ⩌࠾ࡼࡧ LMG ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-1E) ࠋ

(48)

46

p-MDM2 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌, LMG ⩌, BNF ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-1F) ࠋcleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-1G) ࠋ

28 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-2A) ࠋp-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ PMZ ⩌ ࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-2B) ࠋTOP2A 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽ ࢀࡓ (Fig. 2-2C) ࠋUBD 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-2D) ࠋp21Cip1_㝧ᛶ⣽ ⬊⋡ࡣ, CRB ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-2E) ࠋ p-MDM2 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ LMG ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆ ࡵࡽࢀࡓ (Fig. 2-2F) ࠋcleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-2G) ࠋ

90 ᪥┠࡟࠾࠸࡚, Ki-67 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-3A) ࠋp-Histone H3 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ TAA ⩌ ࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌࠾ࡼࡧ APAP ⩌࡛᭷ព࡞ῶᑡ ࡀㄆࡵࡽࢀࡓ (Fig. 2-3B) ࠋTOP2A 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ TAA ⩌࡛↓ฎ⨨ᑐ↷⩌ ࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-3C) ࠋUBD 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌࠾ࡼࡧ TAA

(49)

47

⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-3D) ࠋp21Cip1_{㝧ᛶ⣽⬊⋡}

ࡣ, TAA ⩌, CRB ⩌, LMG ⩌, OX ⩌, APAP ⩌࠾ࡼࡧ PMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-3E) ࠋp-MDM2 㝧ᛶ⣽⬊⋡ࡣ, TAA ⩌࠾ࡼࡧ LMG ⩌࡛ ↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, OX ⩌࡛᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-3F) ࠋcleaved caspase 3 㝧ᛶ⣽⬊⋡ࡣ, MP ⩌, TAA ⩌࠾ࡼࡧ OX ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-3G) ࠋ p-Histone H3 㝧㝧ᛶ⣽⬊⋡ࡢKi-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜ ቑṪ⣽⬊ࡢ࠺ࡕ M ᮇ࡟Ꮡᅾࡍࡿ⣽⬊ࡢ๭ྜࢆ᥎ᐃࡍࡿࡓࡵ࡟, ྠ୍ಶయࡢ⫢⮚࠿ࡽ ᚓࡽࢀࡓูࡢ⤌⧊ࢫࣛ࢖ࢻࢆ⏝࠸࡚, p-Histone H3 ࡞࠸ࡋ Ki-67 ࡟ࡘ࠸࡚ච␿⤌⧊໬Ꮫᰁ Ⰽࢆᐇ᪋ࡋ, ྛศᏊ࡟࠾ࡅࡿ㝧ᛶ⣽⬊⋡ࢆࡑࢀࡒࢀồࡵࡓᚋ, ᚓࡽࢀࡓࢹ࣮ࢱࢆ⏝࠸࡚, p-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࢆồࡵࡓࠋ 7 ᪥┠࡟࠾࠸࡚, p-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࡣ, ඲ᢞ୚ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 2-4A) ࠋ28 ᪥┠࡟࠾ ࠸࡚, p-Histone H3 㝧ᛶ⣽⬊⋡ࡢ Ki-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷ ⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-4B) ࠋ90 ᪥┠࡟࠾࠸࡚, p-Histone H3 㝧ᛶ ⣽⬊⋡ࡢKi-67 㝧ᛶ⣽⬊⋡࡟ᑐࡍࡿ๭ྜࡣ, MP ⩌, TAA ⩌࠾ࡼࡧ CRB ⩌࡛↓ฎ⨨ᑐ↷ ⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-4C) ࠋ

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48

UBD ࡜࡜TOP2A ࡞࠸ࡋ p-Histone H3 ࡜ࡢඹⓎ⌧

28 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, MP ⩌࠾ࡼ ࡧPMZ ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2-5A) ࠋUBD 㝧ᛶ ⣽⬊ࡢ࠺ࡕ TOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋp-Histone H3 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ ๭ྜࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡟ẚ㍑ࡋ࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀ, CRB ⩌࡛᭷ព࡞ῶᑡ ࡀㄆࡵࡽࢀࡓ (Fig. 2-5B) ࠋUBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜ ࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋ 90 ᪥┠࡟࠾࠸࡚, TOP2A 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, ඲ฎ⨨⩌ ࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 2-5C) ࠋUBD 㝧ᛶ⣽⬊ ࡢ࠺ࡕTOP2A ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, MP ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ῶᑡ ࡀㄆࡵࡽࢀࡓࠋp-Histone H3 㝧ᛶ⣽⬊ࡢ࠺ࡕ UBD ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, OX ⩌࡛ ↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓ (Fig. 2-5D) ࠋUBD 㝧ᛶ⣽⬊ࡢ࠺ࡕ p-Histone H3 ࢆඹⓎ⌧ࡍࡿ⣽⬊ࡢ๭ྜࡣ, MP ⩌࠾ࡼࡧ CRB ⩌࡛↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ ࡚᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋ mRNA Ⓨ⌧