SOD2欠損による酸化ストレスが与えるゲノム安定性
、細胞周期進行への影響
著者
?田 隼也
学位授与機関
Tohoku University
ಞ⒬ㄵᩝ
SOD2 Ḗ࡞ࡻࡾ㓗ࢪࢹࣝࢪ࠻ࡾ
ࢣࢿ࣑Ꮽᏽᛮࠉ⣵⬂࿔㐅⾔ࡡᙫ㡢
᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁ ༡⒬ㄚ⛤๑
⏍ⷾᏕᑍᨯ 㐿ఎᏄⷾᏕฦ㔕
A6YM1073 㧏⏛ 㞑ஒ
┘ḗ
➠ 1 ❮ こ᪠ 2 ➠偐❮ ⥬ゕ 5 ➠1⟿ ࡢࡋࡴ࡞ 5 ➠2⟿ ROS (Ὡᛮ㓗⣪⛸)ࡡⓆ⏍࡛㝎ཡ 6 ➠3⟿ ROS ࡛⣵⬂⑵る 7 ➠4⟿ ROS ࡛ࢣࢿ࣑Ꮽᏽᛮ 10 ➠5⟿ SOD2 10 ➠6⟿ ࢼ࣠ࢹࣛDT40 ⣵⬂ 12 ➠7⟿ ᮇ◂✪ࡡ┘Ⓩ 12 ➠ 3 ❮ ࢼ࣠ࢹࣛ DT40 ⣵⬂ࢅ⏕࠷ࡒ SOD2 ᮪௲◒ቪᰬࡡషᠺ 13 ➠ 4 ❮ ࢼ࣠ࢹࣛ DT40 ⣵⬂ࢅ⏕࠷ࡒSOD2 ᮪௲◒ቪᰬࡡ⾪⌟ᆵゆᯊ 21 ➠㸪❮ ⩻ᐳ 38 ➠㸫❮ ᐁ㥺ᮞᩩ࠽ࡻࡦ᪁Ἢ 43 ㅨ㎙ 71 ᘤ⏕ᩝ⊡ 72
➠偏❮
こ᪠
࠘⫴ᬊ࡛┘Ⓩ࠙ࢪ࣭ࣂ࣭࢛࢞ࢨࢺࢩࢪ࣑ࢰ࣭ࢭ (superoxide dismutase : SOD ) ࡢࢪ࣭ࣂ࣭࢛࢞ࢨ
ࢺࢅ㐛㓗ラ⣪ (H2O2) ࡞ንᥦࡌࡾ㓕⣪࡚࠵ࡽࠉࡆࡡ SOD ࡞ࡻࡖ࡙ࠉ⏍మහࡢࢪ࣭ࣂ࣭࢛࢞ࢨ ࢺࡡࣝ࣊ࣜࡂಕࡒࡿ࡙࠷ࡾࠊSOD ࡢ㧏➴┷ᰶ⏍∸࡚ࡢࠉ⣵⬂㈹ᑻᅹᆵࡡ SOD1ࠉ࣐ࢹࢤࣤ ࢺࣛᑻᅹᆵࡡSOD2ࠉฦἢᆵࡡSOD3 ࡡ3 ⛸㢦ࡡᏋᅹ▩ࡼࡿ࡙࠷ࡾࠊ ୌ᪁ࠉฝⰾ㓕ザ࡚ࡢࡆࡡSOD ࡛ࢣࢿ࣑Ꮽᏽᛮ࡞㛭ࡌࡾ㐿ఎᏄ⩄࡛ࡡ㔔◒ቪᰬྙᠺ⮬ほ ࡞ࡾࠉSOD ࡡࢣࢿ࣑Ꮽᏽᛮࡡ㛭♟ြࡈࡿ࡙࠷ࡾࠊࡱࡒࠉ࣐ࢹࢤࣤࢺࣛᑻᅹᆵࡡ SOD ࡚࠵ࡾSOD2 ࡢ⣵⬂࿔౪ᏋⓏ࡞Ⓠ⌟㔖⑵΅ࡌࡾࡆ࡛▩ࡼࡿ࡙࠽ࡽࠉ⣵⬂࿔ࡡ㐅⾔ ࡞రࡼ࠾ࡡᙲࢅᯕࡒࡊ࡙࠷ࡾྊ⬗ᛮ⩻࠻ࡼࡿࡾࠊ ࡊ࠾ࡊࡼࠉࡆࡿࡼᢘ㓗㓕⣪ࡡࢿࢴࢠࢹ࣏ࢪࡢషᠺࡈࡿ࡙࠷ࡾࡵࡡࡡࠉࡐࡿ⏜ᮮࡡ ⣵⬂ࡢࡌ࡚࡞㐛ᗐ㓗ࢪࢹࣝࢪ࡞㛏㛣࡞ࢂࡒࡽུࡄ࡙࠷ࡾࡒࡴ࡞ࠉと☔࡞ᢘ㓗㓕⣪ࡡࢣࢿ ࣑Ꮽᏽᛮࠉ࠽ࡻࡦ⣵⬂࿔ࡡ㐅⾔ࡡᐞࢅゆᯊ࡚ࡀ࡙࠷ࡾሒ࿈ࡢ࠷ࠊࡐࡆ࡚ࠉᮇ◂✪࡚ࡢࠉ ᐖ᪾࡞㐿ఎᏄ◒ቪ⾔࠻ࡾࢼ࣠ࢹࣛDT40 ⣵⬂ࢅ⏕࠷࡙ࠉᢘ㓗㓕⣪ࡡ᮪௲◒ቪᰬࢅషᠺࡊࠉ㐿 ఎᏕⓏゆᯊࢅ⾔࠹ࡆ࡛࡞ࡊࡒࠊ≁࡞ࠉᮇ◂✪࡚ࡢSOD2 ࡞╌┘ࡊ࡙ゆᯊࢅ⾔ࡖࡒࠊ ࠘⤎ᯕ࡛⩻ᐳ࠙
1)
SOD2 ࡡⓆ⌟ೳでᚃࠉ㐛ᗐࡡ㓗ࢪࢹࣝࢪ࡞ࡻࡖ࡙ᚆࠍ࡞⾪⌟ᆵᝇࡌࡾࠊ DT40 ⣵⬂࡞ࠉTet-off system ࢅᑙථࡊࡒ SOD2 ᮪௲◒ቪᰬࢅష⿿ࡊࠉࢷࢹࣚࢦࢠࣛࣤㄇᑙమ࡚࠵ࡾࢺ࢞ࢨࢦࢠࣛࣤࢅ῟ຊࡊࡒ࡛ࡆࢀࠉTet-off system ࡚Ⓠ⌟ࡈࡎࡒhuman SOD2 ࡢ⣑2 ࡚᳠ฝ㝀⏲௧ୖࡱ࡚Ⓠ⌟㔖ୖࡊࡒࠊࡱࡒࠉࡆࡡ࡛ࡀࡡ⣵⬂⑵る⬗࠽ࡻࡦࠉ࣒ࢲࣜࢬࣜ ࣭ࣞࢪᇰᆀ࡚ࡡࢤࣞࢼ࣭ᙟᠺ⋙ࢅῼᏽࡊࡒ⤎ᯕࠉSOD2 ࡡⓆ⌟ೳでࡌࡾࢺ࢞ࢨࢦࢠࣛࣤ
῟ຊᚃ2 ┘ࡡ⣵⬂࡚ࡢࠉ⣵⬂⑵る⬗→ࡦࢤࣞࢼ࣭ᙟᠺ⋙࡞ࡐࡿ㢟ⴥୖࡢびᐳࡈࡿ࠾
ࢤ࣭ࢹ࡞ᑊࡌࡾវུᛮࢅࠉPI Ⰵ࡞ࡻࡾࣆ࣭ࣞࢦࢹ࣒ࢹࣛѸ࡞ࡻࡖ࡙びᐳࡊࡒ࡛ࡆࢀࠉࡷࡢࡽ
ࢺ࢞ࢨࢦࢠࣛࣤ῟ຊ࡞ࡻࡖ࡙⤊Ⓩ࡞វུᛮ୕᪴ࡊ࡙࠷ࡂࡆ࡛ࢂ࠾ࡖࡒࠊ௧୕ࡡࡆ࡛࠾ࡼࠉ
SOD2 ࡡⓆ⌟ೳでᚃࠉහᅹᛮࡡࢪ࣭ࣂ࣭࢛࢞ࢨࢺࡡ⑵ຊ࡞ࡻࡖ࡙㐛ᗐࡡ㓗ࢪࢹࣝࢪⓆ⏍ࡊࠉ SOD2 ᯜῤ⣵⬂ࡡ⑵る⬗ୖࡊ࡙࠷ࡂࡆ࡛♟ြࡈࡿࡒࠊ
2) SOD2 ᯜῤ࡞ࡻࡽࢣࢿ࣑࡞ DNA ഭᐐࢅ㉫ࡆࡈ࠷ࠊ
SOD2 ᯜῤ᮪௲ୖ࡚ࠉmetyl methanesulphonate ࡷ4-nitroquinolin-1-oxide ࡡDNA ഭᐐ࡞ᑊ ࡌࡾវུᛮࢅῼᏽࡊࡒ࡛ࡆࢀࠉ㢟ⴥវུᛮࢅびᐳࡌࡾࡆ࡛ࡢ࡚ࡀ࠾ࡖࡒࠊࡈࡼ࡞ࠉDNA ഭ ᐐࡡᏋᅹࡡᣞᵾ࡛ࡊ࡙ࠉ╀┶Ⰵฦమஹᥦ(SCE)ࢅῼᏽࡊࡒ࡛ࡆࢀࠉ㔕⏍ᰬ࡛ࡡᕣࡢヾࡴࡼࡿ ࠾ࡖࡒࠊSCE୕᪴ࡢ᭩ࡵ㗞ᨼࢣࢿ࣑ഭᐐࡡᣞᵾ࡚࠵ࡾࡆ࡛࠾ࡼࠉSOD2 ᯜῤࡊ࡙ࡵࠉ├ࢣ ࢿ࣑࡞DNA ഭᐐ㉫ࡆࡾࢂࡄ࡚ࡢ࠷ࡆ࡛♟ြࡈࡿࡒࠊ
ࡱࡒࠉDNA 々⿿㜴ᐐ࡚࠵ࡾhydroxy urea ࡷaphidicoline ࡞ᑊࡌࡾវུᛮࢅびᐳࡊࡒ࡛ࡆࢀࠉ ࡆࡿࡼࡡⷾ࡞ᑊࡊ࡙⪇ᛮࢅ♟ࡌࡆ࡛᪺ࡼ࠾࡛ࡖࡒࠊ 3) SOD2 ࡡⓆ⌟ೳでࡢࠉG1࠾ࡼ S ࡡ⛛⾔࠽ࡻࡦ S ࡡ㐅⾔ࢅ㐔ᘇࡈࡎࡾࠊ SOD2 ᯜῤ࡞ࡻࡾ⣵⬂⑵る⬗ࡡୖࡡⅴᅄࢅ᥀ࡾࡒࡴࠉ⣵⬂࿔ฦᕱࡡንࢅびᐳࡊࡒ࡛ࡆ ࢀࠉG1 ࡡ⣵⬂⩄ࡡྙⱕᖱ⑵ຊࡊࠉS ࡡ⣵⬂⩄ࡡྙୖࡊ࡙࠷ࡒࠊ᭞࡞ G1 ࠽ ࡻࡦG2/M࡞⣵⬂ࢅ⇆ㄢࡊࠉḗ࡞ࠉ⇆ㄢࢅゆ㝎ࡊ࡙⣵⬂࿔ࢅ㐅⾔ࡈࡎࡒ࡛ࡆࢀࠉG1 ࠾ ࡼS ࡡ⛛⾔ࠉS ࡡ㐅⾔࡞㐔ᘇびᐳࡈࡿࡒࠊ ࠘⩻ᐳ࠙ SOD2 ࡡⓆ⌟ࢅೳでࡈࡎ࡙ࡵࠉDNA ഭᐐ࡞ᑊࡌࡾវུᛮ㔕⏍ᰬ࡛⇆⛤ᗐࡓࡖࡒࡆ࡛࠾ࡼࠉ ᢘ㓗㓕⣪ࢣࢿ࣑Ꮽᏽᛮ࡞├㛭ࡌࡾࡆ࡛ࢅ♟ြࡌࡾチᣈࡢᚋࡼࡿ࠾ࡖࡒࠊࡊ࠾ࡊࡼࠉ SOD2 ᯜῤ࡞ࡻࡖ࡙ࠉ⣵⬂⑵る⬗ୖࡊࠉࡐࡡⅴᅄࡢࠉ࡞G1࠾ࡼS ࡡ⛛⾔࠽ࡻࡦS ࡡ㐅⾔࡞࠵ࡾࡆ࡛᪺ࡼ࠾࡛ࡖࡒࠊSOD2 ࢅ㐛Ⓠ⌟ࡈࡎࡒ⣵⬂ࡢG࠾ࡼS ࡡ⛛⾔⬗
㧏ࡱࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠽ࡽࠉᮇ◂✪ࡡ⤎ᯕࡢࡆࡡሒ࿈࡛□┢ࡊ࠷ࠊ᭞࡞ࠉSOD2ᯜῤ⣵⬂ࡢ DNA 々⿿㜴ᐐ࡚࠵ࡾHU ࡷaphidicoline ࡞ᑊࡊ࡙⪇ᛮࢅ♟ࡊࡒࡆ࡛ࠉDNA 々⿿㏷ᗐࡡ㐔ᘇࡢ ࡛ࢆびᐳࡈࡿ࠾ࡖࡒࡆ࡛ࠉDNA 々⿿check point ᶭᵋ࡞㛭ࡌࡾCHK-1 ࡡὩᛮびᐳ ࡈࡿ࠾ࡖࡒࡆ࡛࠾ࡼࠉSOD2 Ḗ࡞ࡻࡖ࡙⏍ࡋࡾ㓗ࢪࢹࣝࢪDNA 々⿿ࡡ㛜╂ࢅ㐔ᘇࡌࡾ ྊ⬗ᛮ♟ြࡈࡿࡒࠊ
➠ 2 ❮ ⥬ゕ
➠ 1 ⟿ ࡢࡋࡴ࡞ Ὡᛮ㓗⣪⛸ (ROS)ࡢ㓗⣪⏍మහࡡ࢙ࢾ࣭ࣜ࢟⏐⏍➴ࡡ㐛⛤࡚⏐⏍ࡈࡿࠉ㓗⣪࡞セࠉࡻࡽ ཬᚺᛮࡡ㧏࠷ฦᏄ⩄࡚࠵ࡾࠊ࡞ROS ࡢ࣐ࢹࢤࣤࢺࣛࡡ㞹Ꮔఎ㐡⣌ㅨࡡ㐛⛤࡞࠽࠷ ࡙Ⓠ⏍ࡌࡾࠉᢘ㓗㓕⣪ࡷᢘ㓗∸㈹࡞ࡻࡖ࡙㝎ཡࡈࡿࠉ⏍మහ࡞࠽ࡄࡾᏋᅹ㔖ࡢᖏ࡞โᚒ ࡈࡿ࡙࠷ࡾࠊࡊ࠾ࡊࡼࠉᨲᑏ⥲ࡷ⣰አ⥲ࡡᬸ㟚ࠉຊ㱃࡞ࡻࡾ㐛ROS ࡡⓆ⏍ࡷࠉ 㝎ཡ⬗ࡡୖࡢ⏍మහ࡞࠽࠷࡙㓗ࢪࢹࣝࢪࢅㄇⓆࡌࡾࠊ ୌ᪁ࠉᢘ㓗㓕⣪ࡡን␏ࡢᵕࠍ⑄ᝀࢅᘤࡀ㉫ࡆࡌࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊࢪ࣭ࣂ࣭࢛࢞ࢨ ࢺࢩࢪ࣑ࢰ࣭ࢭࡡ1 ⛸࡚࠵ࡾSOD1ࡡⅤን␏➵ⴆ⦨ᛮഁ⣬◫ (ALS) ࡡᝀ⩽ࡡᩐ偃࡞び ᐳࡈࡿࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Rosen et al., 1993)ࠊࡱࡒࠉ➠21 ␊Ⰵమࡡࢹࣛࢮ࣐࣭࡞ࡻࡖ࡙ ㉫ࡆࡾࢱࣤು⩄࡚ࡢSOD1 ࡡὩᛮ⑵ຊࡌࡾࡆ࡛▩ࡼࡿ࡙࠽ࡽࠉ♼⤊⑄ᝀࡡୌ㒂ࡡⓆ࡞ᢘ㓗㓕⣪㛭ࡊ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡾ(Avraham et al. 1988)ࠊࡐࡡ࡞ࠉSOD2ࡡmitochondria
targeting sequence ࡞࠵ࡒࡾࠉ㸮␊┘ࡡࣚࢼࣤࡡࣁࣛࣤࡡ⨠ᥦን␏࡞ࡻࡖ࡙ࠉ㐘ິ♼⤊⑄ᝀ ࡷ≁Ⓠᛮᚨ➵ࡡⓆࣛࢪࢠ㧏ࡱࡾࡆ࡛(Hiroi et al., 1999)ࠉࣆࣤࣚࣤࢺெ࡚⒬Ⓠ⏍⋙ 1.5 ಶ⛤ᗐࡱ࡚㧏ࡱࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾ(Mitrune et al., 2001)ࠊࡱࡒࠉ⇆ን␏᪡⩹ᝀ⩽ࡡୌ 㒂࡞ヾࡴࡼࡿ࡙࠽ࡽࠉᙔ◂✪ᐄ࡚◂✪㐅ࡴࡼࡿ࡙࠷ࡾ࢘ࣜࢻѸು⩄ࡷࠉᰶ⭯ࡡᡬࡔࢰ ࣤࣂࢠ㈹࡚࠵ࡾ࣐ࣚࣤ A ࡡን␏ⅴᅄ࡚࠵ࡾࣈࣞࢩ࢘ࣛࠉⰅ⣪ᛮஜ⓮ࡡG ⩄ࡷࢤࢢࣤ ು⩄࡛࠷ࡖࡒ᪡⩹ᝀ⩽࡞ࡵࡆࡡን␏ୌ㒂ヾࡴࡼࡿࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Rosenblum et al., 1996)ࠊ ࡈࡼ࡞ࠉࡆࡿࡼࡡ⮣ᗃ୕ࡡሒ࿈࡞ຊ࠻㎾ᖳࠉฝⰾ㓕ザ࡞࠽࠷࡙ᢘ㓗㓕⣪࡛ࢣࢿ࣑Ꮽᏽᛮ⥌ ᣚࡷ⣵⬂࿔ࡡ㐅⾔࡞㛭ࡌࡾ㐿ఎᏄ࡛ࡡ㔔◒ቪᰬྙᠺ⮬ほ࡞ࡾࡆ࡛ฦ࠾ࡖ࡙ࡀ࡙ ࠷ࡾ(Tong et al., 2001ࠉHuang et al., 2003ࠉPan et al., 2006)ࠊ௧୕ࡡࡆ࡛࠾ࡼࠉᢘ㓗㓕⣪ࡢࢣࢿ
࣑Ꮽᏽᛮ⥌ᣚࡷ⣵⬂࿔ࡡ㐅⾔࡞రࡼ࠾ࡡᙲࢅᢰࡖ࡙࠷ࡾྊ⬗ᛮ㧏࠷ࠊࡱࡒࠉࡆࡿࡼᢘ㓗 㓕⣪ࡡࢿࢴࢠࢹ࣏ࢪࡢషᠺࡈࡿ࡙࠷ࡾࡵࡡࡡࠉࡐࡿ⏜ᮮࡡ⣵⬂ࡢࡌ࡚࡞㐛ᗐ㓗ࢪ ࢹࣝࢪ࡞㛏㛣࡞ࢂࡒࡽུࡄ࡙࠷ࡾࡒࡴ࡞ࠉᢘ㓗㓕⣪ࡡࢣࢿ࣑Ꮽᏽᛮࠉ࠽ࡻࡦ⣵⬂࿔ࡡ㐅 ⾔ࡡᐞࢅと☔࡞ゆᯊ࡚ࡀ࡙࠷࠷≟Ἓ࡞࠵ࡾࠊࡐࡆ࡚ᮇ◂✪࡚ࡢࠉ㐿ఎᏄ◒ቪྊ⬗࡚ࡊ ࠾ࡵࠉ᮪௲◒ቪᰬష⿿ྊ⬗ࢼ࣠ࢹࣛB ⣵⬂⏜ᮮࡡDT40 ⣵⬂ࡡᢘ㓗㓕⣪᮪௲◒ቪᰬࢅష ⿿ࡊࠉゆᯊࢅ⾔࠹ࡆ࡛࡞ࡊࡒࠊ ➠ 2 ⟿ ROS (Ὡᛮ㓗⣪⛸)ࡡⓆ⏍࡛㝎ཡ Ὡᛮ㓗⣪⛸ (ROS) ࡛ࡢࠉࢪ࣭ࣂ࣭࢛࢞ࢨࢺ(O2 -) ࠉ㐛㓗ラ⣪(H2O2) ࠉୌ㔔㡧㓗⣪(O 1 ) ࠉ㓗⣪⏍మහࡡ࢙ࢾ࣭ࣜ࢟⏐⏍➴ࡡ㐛⛤࡚ࠉࡻࡽཬᚺᛮࡡ㧏࠷ฦᏄ⛸࡞ንࡊࡒࡵࡡ࡚ ࠵ࡾࠊⓆ⏍″࡛ࡊ࡙ࠉ࣐ࢹࢤࣤࢺࣛࡡ㞹Ꮔఎ㐡⣌ࡡࠉⅎࡷࠉᨲᑏ⥲ࠉ⣰አ⥲࡞ᬸ 㟚ࡊࡒ⟘ᡜ࠵ࡾࠊࡐࡡࠉ┖୯⌣ࡷ࣏ࢠࣞࣆ࣭ࢩ࡛࠷ࡖࡒ㣏⣵⬂࡞ࡢࠉNADPH ࢛࢞ࢨ ࢱ࣭ࢭ࡞ࡻࡖ࡙⮤ⓆⓏ࡞ROS ࢅⓆ⏍ࡈࡎ࡙ウⳞࡌࡾᶭᵋᏋᅹࡌࡾࠊ ROS ࡢཬᚺᛮ㧏ࡂࠉ㐛㔖Ꮛᅹࡌࡿࡣ⏍మහ࡚DNA ࡷࢰࣤࣂࢠ㈹ࠉ⬙㈹ࢅഭᐐࡌࡾ ࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊࡐࡡ⤎ᯕࠉ⣵⬂࡚ࣝ࣊ࣜࡢ⣵⬂⩹ࡷࠉ⒬ࠉ࣎ࢹ࣭ࢨࢪ࡞ࡻࡾ⣵⬂ ほࢅᘤࡀ㉫ࡆࡊࠉమ࡚ࣝ࣊ࣜࡢ⢶ᑺࡷິ⬞◫ࠉᾐჹ⑄ᝀࡡⅴᅄ࡞ࡾࡆ࡛ሒ࿈ ࡈࡿ࡙࠷ࡾࠊࡐࡡࡒࡴࠉ㐛 ROS ࢅ㝎ཡࡊୌᏽࡡࣝ࣊ࣜ࡞ಕࡗᶭᵋᚪこ࡚࠵ࡾࠊࡐࡡࡥ ࡛ࡗࡢࠉᢘ㓗㓕⣪࡞ࡻࡾࡵࡡ࡚ࠉࢪ࣭ࣂ࣭࢛࢞ࢨࢺࢅ㐛㓗ラ⣪࡞ንᥦࡌࡾࢪ࣭ࣂ࣭࢛࢞ࢨ ࢺࢩࢪ࣑ࢰ࣭ࢭ(SOD) ࡷࠉ㐛㓗ラ⣪ࢅラ࡞ንᥦࡌࡾ࢜ࢰ࣭ࣚࢭࠉࢡࣜࢰࢲ࢛࢛ࣤ࣋ࣜ࢞ࢨࢱ ࣭ࢭࡷࠉ࢛࣋ࣜ࢞ࢨࣝࢺ࢞ࢨࣤࡡᏋᅹ▩ࡼࡿ࡙࠷ࡾࠊࡵ࠹ࡥ࡛ࡗࡢࠉᢘ㓗∸㈹࡞ࡻࡾ ࡵࡡ࡚ࠉࣄࢰ࣐ࣤAࠉEࠉࢡࣜࢰࢲ࢛ࣤࠉࢲ࢛ࣝࢺ࢞ࢨࣤ㛭ࡊ࡙࠷ࡾࠊ
➠ 3 ⟿ ROS ࡛ᢘ㓗㓕⣪࡛ࢣࢿ࣑Ꮽᏽᛮ ➠1 ⟿࡚㏑ࡒࡻ࠹࡞ࠉROS ࡢDNA ࡡഭᐐ࡞㛭ࡊ࡙࠷ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾࠊࡊ࠾ࡊ ࡼࠉࢪ࣭ࣂ࣭࢛࢞ࢨࢺࡷ㐛㓗ラ⣪ࡡࡻ࠹⾪ⓏROS ࡢDNA ࢅ༟≺࡚ഭᐐࡌࡾࡻ࠹ഭ ᐐᛮࢅ᭯ࡊ࡙ࡢ࠽ࡼࡍࠉࡻࡽཬᚺᛮࡡ㧏࠷ࠉࣃࢺࣞ࢞ࢨࣚࢩ࢜ࣜ(HO࣬ ) ࡷ࢛࣋ࣜ࢞ࢨࢻࢹࣚ ࢹ(ONOO -) DNA ഭᐐࡡⅴᅄ࡞ࡖ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡾࠊࡆࡿࡼࡢࠉཬᚺᛮࡡセ㍉Ⓩ ࠷ROS ⣵⬂හࡡฦᏄ࡛ࡡཬᚺ࡚⏍ᠺࡌࡾࠊࡐࡿࡑࡿࠉࣃࢺࣞ࢞ࢨࣚࢩ࢜ࣜࡢ㐛㓗ラ⣪ 㕪ࡷ㖙࢛ࣤࠉࢠ࣑ࣞࡡ㔘ᒌ࢛࡛ࣤཬᚺࡌࡾࡆ࡛࡚Ⓠ⏍ࡊࠉ࢛࣋ࣜ࢞ࢨࢻࢹࣚࢹࡢࢪ ࣭ࣂ࣭࢛࢞ࢨࢺ࡛ୌ㓗✽⣪ཬᚺࡌࡾࡆ࡛࡚Ⓠ⏍ࡌࡾࠊࡆࡿࡼࡡDNA ഭᐐష⏕ࡢᙁງ࡚ࠉ8-ࣃࢺࣞ࢞ࢨࢡࢼࣤࡡ⏍ᠺࠉ⛸ሲᇱࡡഭᐐࡷࠉDNA ᮇ㙈ว᩷ࠉDNA ୌᮇ㙈ว᩷ ኣᵕDNA ഭᐐࢅㄇⓆࡌࡾࠊ Fig.2-1 ROS ࡛ᢘ㓗㓕⣪ࡡ㛭౿ᅒ O2 O2 H2O2 - H2O HO࣬
SOD
Catalase
Peroxiredoxin
Glutathione peroxidase Fe2+ ,Cu2+ (Fenton reaction) NO ONOO- DNA Lipid Proteinୌ᪁࡚㎾ᖳ࡞ࡽࠉฝⰾ㓕ザࢅ⏕࠷ࡒኬぜᶅࢪࢠ࣭ࣛࢼࣤࢡ⾔ࢂࡿࠉ⣵⬂㈹ᑻᅹᆵࡡSOD ࢅࢤ࣭ࢺࡌࡾSOD1 ࠵ࡾ࠷ࡢ TSA1(࢛࣋ࣜ࢞ࢨࣝࢺ࢞ࢨࣤ2ࠉ4 ࡡฝⰾ㓕ザ࣓࣌ࣞࢡ)࡛ࢣࢿ࣑ Ꮽᏽᛮ⥌ᣚ࡞㛭ࡊ࡙࠷ࡾ㐿ఎᏄ⩄࡛ࡡ㔔◒ቪᰬྙᠺ⮬ほ࡞ࡾࡆ࡛♟ࡈࡿࡒࠊTable 2-1 ࡞ྙᠺ⮬ほ࡛ࡖࡒ㐿ఎᏄྞࢅ♟ࡌࠊ
SOD1 SOD2 TSA1
DNA replication POL30 , POL32
SGS1, RAD27
RRM3 POL32, SGS1, TOP3, RMI1 HUR1, RAD27
DNA repair RAD51, RAD52,
RAD54, MRE11, RAD5, RAD18, RTT107, MMS1 OGG1, RAD51, RAD52,MSH2 , RAD18, MUS81, RTT107, MMS4
Checkpoint RAD24, RAD17,
RAD9 , MEC3, TOF1, DDC1
RAD24, RAD17, CHK1, DUN1, MEC3, DCC1
Sister chromatid segrigation SGO1, KAR3,
Chromatin remodeling factor Histone chaperon etc…
HDA1, HIR3, SWR1, ARP8
ASF1, RTT109
Table. 2-1 ฝⰾ㓕ザ࡞࠽࠷࡙ᢘ㓗㓕⣪࡛ྙᠺ⮬ほ࡛ࡾ㐿ఎᏄ⩄
ࡱࡒࠉsod1 ◒ቪᰬࡢMethyl methanesulphonate (MMS) ࡷ4-Nitroquinoline 1-oxide (4NQO) ࡛࠷ࡖ ࡒDNA ഭᐐࡷDNA ྙᠺ㜴ᐐ࡚࠵ࡾࠉHydroxyurea (HU) ࡞ᑊࡊ࡙㟸ᖏ࡞ᙁ࠷វུᛮࢅ♟ ࡊࠉࡐࡡⅴᅄࡢࠉDNA ྙᠺࡡᚂ㏷㓕⣪࡚࠵ࡾRibonucleotide reductase ࡡ㑇ඔ࡞ᚪこNADPH ᯜῤࡊ࡙ࡊࡱࡖࡒࡆ࡛ࠉࡱࡒࠉDNA ഭᐐࢲ࢘ࢴࢠ࣎ࣤࢹࡡὩᛮ΅ᘽࡊ࡙ࡊࡱ࠹ࡒ ࡴ࡚࠵ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Carter et al., 2005)ࠊࡱࡒࠉtsa1 ◒ቪᰬࡢDNA ഭᐐ࡞ᑊࡌࡾវ ུᛮ㔕⏍ᰬ࡛⇆⛤ᗐ࡚࠵ࡾࡵࡡࡡࠉ㟸ᖏ࡞㧏㢎ᗐ࡚DNA ࡞ን␏⏍ࡋࡾࡆ࡛ሒ࿈ࡈࡿ࡙ ࠷ࡾ(Huang et al,. 2005ࠉRagu et al., 2007)ࠊᢘ㓗㓕⣪◒ቪᰬ࡚⏍ࡋࡒ⾪⌟ᆵ࠾ࡼࠉᢘ㓗㓕⣪ࡢ ROS ࡡ㝎ཡࡡࡲࡼࡍࠉㅨࡷࢣࢿ࣑Ꮽᏽᛮ⥌ᣚᶭᵋ⮤మ࡞㛭ࡊ࡙ࠉࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ࡞ 㛭ࡊ࡙࠷ࡾྊ⬗ᛮࡢ㧏࠷ࠊୌ᪁ࠉࡆࡿࡼࡡሒ࿈࡛ࡢื࡞ࠉDNA ഭᐐࢲ࢘ࢴࢠ࣎ࣤࢹ࡞࠽ ࠷࡙ᶭ⬗ࡊダ⣵⾉⟮ᣉᘿᛮㄢ (ataxia telangiectasia ; AT) ࡡⅴᅄ㐿ఎᏄ⏐∸࡚࠵ࡾATMࡷ DNA ᮇ㙈ว᩷ࡡಞᚗᶭᵋࡡࡥ࡛ࡗ࡚࠵ࡾࠉNon-homologous End Joining (NHEJ) ࡞㛭ࡌࡾ KU86 ࡡࢿࢴࢠࢹ࣏ࢪ࡞ SOD1 ࢅ㐛Ⓠ⌟ࡌࡾ࡛⮬ほ࡛ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Peter et al,. 2001ࠉKaranjawala et al., 2002ࠉKaranjawala et al., 2003)ࠊSOD ࡢࢪ࣭ࣂ࣭࢛࢞ࢨࢺࢅ㝎 ཡࡌࡾ࡛⇆࡞ࠉ㐛㓗ラ⣪ࢅ⏍ᠺࡊ࡙࠷ࡾࡒࡴ࡞ࠉ㐛࡞Ꮛᅹࡊ࡙ࡵࢣࢿ࣑Ꮽᏽᛮࢅୖࡈ ࡎࡾࡆ࡛ࡆࡡ⤎ᯕࡻࡽ♟ࡈࡿࠉᢘ㓗㓕⣪Ὡᛮࡡㄢ⟿ࡡ㔔こᛮ♟ြࡈࡿ࡙࠷ࡾࠊ
➠ 4 ⟿ ROS ࡛⣵⬂⑵る ROS ࡢDNA ࡷࢰࣤࣂࢠ㈹ࠉ⬙㈹࡞ᑊࡊ࡙㧏࠷ഭᐐᛮࢅ᭯ࡌࡾࡒࡴ࡞ࠉ᭯ᐐ∸㈹࡛ࡊ࡙ ᤂ࠻ࡼࡿࡾୌ᪁࡚ࠉࢨࢡࢻࣜఎ㐡∸㈹࡛ࡊ࡙ᶭ⬗ࡊࠉࢰࣤࣂࢠ㈹୯ࡡࢲ࢛࣭ࣜᇱࢅ㓗ࠉ❟మ ᵋ㏸ࢅንࡈࡎࡾࡆ࡛࡚ࠉࢰࣤࣂࢠ㈹ࡡ㓕⣪Ὡᛮࠉ⣵⬂හᑻᅹࠉDNA ⤎ྙᛮ➴ࢅንࡈࡎࡾ ࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊ ROS ࡢࢨࢡࢻࣜఎ㐡∸㈹࡛ࡊ࡙ࠉ⣵⬂⑵るࡷ⣵⬂࿔ࡡ㐅⾔࡞ࡵ㛭ࡊ࡙࠷ࡾྊ⬗ᛮ♟ ြࡈࡿ࡙࠽ࡽࠉ⣵⬂⑵る࡞㛭ࡌࡾ⤊㊨ࡷS(DNA 々⿿) ࡷM(ฦ) ࡡ㛜╂ࢅโᚒࡊ ࡙࠷ࡾᅄᏄࡡὩᛮࠉὩࡡ㛭ࢅሒ࿈ࡊ࡙࠷ࡾㄵᩝࡢኣᩐᏋᅹࡊ࡙࠷ࡾࠊ⣵⬂⑵るᅄᏄ ࡛ࡊ࡙ࡢࠉepidermal growth factor (EGF) ࡷplatelet-derived growth factor (PDGF) ࠉprotein Kinase C (PKC) ࡷprotein kinase B (AKT) ࡡ⤊㊨ROS ࢅࡊ࡙ὩᛮࠉὩࡈࡿࡾࡆ࡛ሒ࿈ࡈࡿ ࡙࠷ࡾ(Bae et al., 1997ࠉBrar et al., 1999ࠉJunn et al., 2000ࠉSundaresan et al., 1995)ࠊࡱࡒࠉ⣵⬂࿔ ࡡ㐅⾔࡞㛭ࡊ࡙࠷ࡾᅄᏄ࡛ࡊ࡙ࡢࠉcyclin D1 ࡷp16ࠉp21ࠉCdc25c ሒ࿈ࡈࡿ࡙࠷ࡾࠊ
➠ 5 ⟿ SOD2
SOD ࡢࢪ࣭ࣂ࣭࢛࢞ࢨࢺࢅ㐛㓗ラ⣪࡞ንᥦࡌࡾᢘ㓗㓕⣪ࡡ1 ࡗ࡚࠵ࡽࠉⅴᰶ⏍∸࡚ࡢ㕪
࢛ࣤ㒼నᆵࡡFe-SOD ࡷࢼࢴࢢ࢛ࣜࣤ㒼నᆵࡡNi-SOD ࠉฝⰾ㓕ザୖ➴┷ᰶ⏍∸࡚ ࡢ㖙࢛ࣤ࠽ࡻࡦல㕼࢛ࣤ㒼నᆵࡡCu, Zn-SOD (SOD1)ࠉ࣏࢛ࣤ࢝ࣤࣤ㒼నᆵࡡMn-SOD (SOD2) ࡡ偐⛸㢦Ꮛᅹࡊࠉ᭞࡞㧏➴┷ᰶ⏍∸࡚ࡢฦἢᆵࡡEC-SOD (SOD3) Ꮛᅹࡊ࡙࠷ࡾ ࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊࡆࡡ࠹ࡔࠉSOD2 ࡢFe-SOD ࡛ࡡ㛣࡚┞⇆ᛮࢅ᭯ࡊ࡙࠷ࡾࠊSOD2 ࡢ224 ࣐ࢿ㓗࠾ࡼࡾࢰࣤࣂࢠ㈹࡚ࠉN ᮆ❻ഁ࡞࣐ࢹࢤࣤࢺࣛ⛛⾔ࢨࢡࢻࣜᏋᅹࡊࠉ4 㔖మ࡛ ࡊ࡙࣐ࢹࢤࣤࢺࣛ࡞Ꮛᅹࡊ࡙࠷ࡾࠊFig.2-2 ࡞♟ࡌࡻ࠹ὩᛮࢅᣚࡔࠉὩᛮࡡⓆ⌟࡞ࡢὩᛮ୯ ᚨ࡚࠵ࡾTyr 34 ࡡࠉ࣏࢛ࣤ࢝ࣤࣤᚪこ࡚࠵ࡾࡒࡴࠉ࣏࢛ࣤ࢝ࣤࣤࡡ⤎ྙ㒂న࡚࠵ࡾࠉ
His 26ࠉHis 81ࠉAsp 167ࠉAsp 171 㔔こ࣐ࢿ㓗よᇱ࡛ࡊ࡙ಕᏋࡈࡿ࡙࠷ࡾࠊ Fig. 2-2 SOD ࡡ㓕⣪Ὡᛮ Sod2 ࡡࢿࢴࢠࢹ࣏ࢪࡢࡌ࡚࡞ࡨࡒࡗࡡ⣌⤣࡞࠽࠷࡙ష⿿ࡈࡿ࡙࠽ࡽࠉࡔࡼࡡ⣌ ⤣࡞࠽࠷࡙ࡵᖲᆍⓏᑋࡢ2 㐄㛣⛤ᗐ࡚ࠉᚨ➵➴ഭᐐࡈࡿ⮬ほ࡛ࡾࡆ࡛ሒ࿈ࡈࡿ ࡙࠷ࡾࠊࡈࡼ࡞ࠉࡆࡡࢿࢴࢠࢹ࣏ࢪ⏜ᮮ⣵⬂ࡢⰅమ␏ᖏ㉫ࡀ࡙࠷ࡾࡆ࡛ሒ࿈ ࡈࡿ࡙࠽ࡽࠉSod2 ࢣࢿ࣑Ꮽᏽᛮ࡞ᐞࡊ࡙࠷ࡾྊ⬗ᛮ♟ြࡈࡿ࡙࠷ࡾࠊ ฝⰾ㓕ザ࡚ࡢࠉ5̓Ѝ3̓᪁ྡྷࡡDNA helicase Ὡᛮࢅᣚࡗࢰࣤࣂࢠ㈹࡚࠵ࡽࠉ┷ᰶ⏍∸࡞ ࠽࠷࡙㧏ࡂಕᏋࡈࡿ࡙࠷ࡾPif1 ࡡsubfamily ࡚࠵ࡾRRM3(rDNA recombination mutation) ࡛ SOD2 ࡡ㔔◒ቪᰬྙᠺ⮬ほ࡞ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾࠊࡐࡡࠉSod2 ࡛∸⌦Ⓩ࡞┞ பష⏕ࡌࡾࢰࣤࣂࢠ㈹࡛ࡊ࡙ࠉNon-homologous End Joining (NHEJ) ࡞㛭ࡌࡾYku80ࡷࠉ single-strsanded DNA endonuclease ࡚࠵ࡾRad10 ࣈࣞࢷ࢛࣐ࢠࢪⓏᡥἪ࡞ࡻࡽ⇆ᏽࡈࡿ࡙ ࠷ࡾ(Ho et al. 2002)ࠊ
ࡱࡒࠉSOD2ࡢࠉ⣵⬂࿔౪ᏋⓏ࡞Ὡᛮንິࡌࡾࡆ࡛▩ࡼࡿ࡙࠽ࡽࠉG0/G1࡞࠽࠷
࡙Ὡᛮ᭩ࡵ㧏ࡂࠉS ࡞ථࡖࡒ࡛⇆࡞᛬⃥࡞ୖࡊࠉ⇆࡞ROS ࡵ⑵ຊࡌࡾࡆ࡛ሒ ࿈ࡈࡿ࡙࠷ࡾ(Oberley et al. 1995)ࠊࡈࡼ࡞ࠉSOD2 ࢅHeLa ⣵⬂࡚㐛Ⓠ⌟ࡈࡎࡾ࡛ࢤࣤࣆࣜ ࢙ࣤࢹ࡞ࡽࠉ㏳ᖏࡼ G1 ࡞ೳでࡌࡾ᮪௲࡞ࡖ࡙ࡵࠉ᭞࡞⣵⬂⑵る⬗ࢅಕࡗࡆ࡛ ࡚ࡀࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Sarsour et al., 2005)ࠊ SOD-M2+ +O2 -SOD-M+ +O2 SOD-M++O 2 -+2H+ SOD-M2++H 2O2 O2 -+O2 - 2H+ H2O2+ O2
➠ 6 ⟿ ࢼ࣠ࢹࣛ DT40 ⣵⬂
DT40 ⣵⬂ᰬࡢࠉࢼ࣠ࢹࣛB ࣛࣤࣂ⌣⏜ᮮࡡ⣵⬂࡚࠵ࡽࠉࢼ࣠ࢹࣛࡡඞᢰᙔჹᏻ࡚࠵ࡾࣇ ࣜࢧහࡡ⣵⬂ࢅࢹࣛⓉ⾉ࣜࢪ࡚ほࡈࡎࠉࢼ࣠ࢹࣛ B ࣛࣤࣂ⌣๑㥉⣵⬂ࡡฦࡡ㝷 ࡞⾔ࢂࡿࡾV-J᩷∞࡛അV᩷∞㛣ࡡ┞⇆⤄ᥦ࠻ࡡ⤎ᯕ⏍ࡋࡾlg gene conversionࡡ⬗ງࡡಕᣚࢅ ᣞᵾ࡛ࡊ࡙༟㞫ࡈࡿࡒ⣵⬂࡚࠵ࡾࠊࡆࡡ⣵⬂࡚ࡢࠉᵾⓏ⤄ᥦ࠻ࣚࣤࢱ࣑ࣤࢷࢡ࣭ࣝࢨࣘࣤ ࡛⇆ࡋຝ⋙࡚㉫ࡆࡾࡆ࡛ሒ࿈ࡈࡿࡒ (Buerstedde and Takeda., 1991) ࠊࡐࡡࡒࡴࠉDT40 ⣵⬂ᰬ ࡢ≁ᏽ㐿ఎᏄࡡ◒ቪᰬᐖ᪾࡞ష⿿࡚ࡀࠉ㎷㏷㐿ఎᏕⓏゆᯊྊ⬗࡛ࡾࠊࡈࡼࡾฺⅤ࡛ ࡊ࡙ࡢࠉ(1) 偕⛸㢦ࡡⷾ⪇ᛮ࣏࣭࣭࢜࠻ࡾࡡ࡚᭩ኬ㸨⛸㢦ࡡ㐿ఎᏄࡡ㔔々ࢿࢴࢠࢹ ࡚ࡀࡾࡆ࡛ࠉ (2) ᰶᆵ࡛⣵⬂ࡡ⾪⌟ᆵ㟸ᖏ࡞Ꮽᏽ࡚࠵ࡽࠉࡱࡒࠉ⣵⬂ࡡ⑵る㏷࠷ࡆ࡛ࠉ (3) ⮬ほⓏን␏ࡡᑙථࡡ㝷࡞ࡢࢷࢹࣚࢦࢠࣛࣤࢅ⏕࠷ࡒ on-off ࡡ⣌ࢅᑙථࡌࡾ Tet-on/Tet-off gene expression systemࡷCre-LoxP system ࡡࡻ࠹᮪௲⮬ほᰬࢅష⿿ࡌࡾࡆ࡛࡚ࡀࡾࡆ࡛ᣪ ࡅࡼࡿࡾࠊ
➠ 7 ⟿ ᮇ◂✪ࡡ┘Ⓩ
Sod2 ࡡࢿࢴࢠࢹ࣏ࢪࡢᑋ᭩㛏2ࠉ3 㐄㛣࡛ኬን▯࡚࠵ࡽࠉ࠾ࡗゆᯊ࡞ࡢࡌ࡚࡞ 㛏㛣࡞ῳࡖ࡙㓗ࢪࢹࣝࢪࢅུࡄ⤾ࡄ࡙࠷ࡾ≟ឺ࡞࠵ࡾࡒࡴࠉSOD2 ᮇᮮࡡᶭ⬗ࢅビ౮ࡌࡾࡡ ࡢᅏ㞬࡚࠵ࡾࠊࡐࡆ࡚ᮇ◂✪࡚ࡢࠉࢼ࣠ࢹࣛDT40 ⣵⬂ࡡ㛏ᡜࢅὩ࠾ࡊࠉࢷࢹࣚࢦࢠࣛࣤࢅ⏕ ࠷ࡒon-off ࡡ⣌ࢅᑙථࡌࡾTet-on/Tet-off gene expression system ࢅ⏕࠷ࡾࡆ࡛࡚ࠉSOD2 Ⓠ⌟ೳで ├ᚃࡡ⾪⌟ᆵࢅゆᯊࡌࡾࡆ࡛ࢅྊ⬗࡛ࡌࡾ⣌ࢅ☔❟ࡊࠉSOD2 ᯜῤ├ᚃࡡ㓗ࢪࢹࣝࢪࢣࢿ࣑ Ꮽᏽᛮࡷ⣵⬂࿔㐅⾔࡞࠻ࡾᙫ㡢ࢅゆᯊࡌࡾࡆ࡛ࢅ┘Ⓩ࡛ࡊࡒࠊ
➠3❮
ࢼ࣠ࢹࣛ DT40 ⣵⬂ࢅ⏕࠷ࡒ SOD2 ᮪௲◒ቪᰬࡡష⿿
➠ 1 ⟿ ࡢࡋࡴ࡞ ๑❮࡚㏑ࡒࡻ࠹࡞ࠉ࣐ࢹࢤࣤࢺࣛᑻᅹᆵSOD ࡚࠵ࡾSOD2 ࡡࢣࢿ࣑Ꮽᏽᛮࡡ⥌ᣚࡷ⣵⬂ ࿔ࡡ㐅⾔ࡡ㛭ࢅゆᯊࡌࡾࡒࡴ࡞ࠉDT40 SOD2 ◒ቪᰬࢅష⿿ࡌࡾ࡞࠵ࡒࡖ࡙ၡ㢗࡛ࡾ࡛ ⩻࠻ࡼࡿࡒࡡࠉ⛸ࡡSOD2 ◒ቪᰬ࡚びᐳࡈࡿࡾ⮬ほᛮ࡚࠵ࡾࠊSOD2 ࡡࢿࢴࢠࢹ࣏ࢪ ࡢ⫶⏍⮬ほ࡞ࡼ࠷ࡱ࡚ࡵࠉᚨ➵➴ࡡഭᐐ࠾ࡼᑋࡢ᭩㛏࡚ࡵ2 㐄㛣⛤ᗐ࡚࠵ࡾࡆ࡛ࠉࡱࡒࢿ ࢴࢠࢹ࣏ࢪ⏜ᮮ⣵⬂ࡵ㟸ᖏ࡞⾪⌟ᆵᙁࡂࠉ♼⤊⏜ᮮ⣵⬂ࡢ㏳ᖏᇰ㣬᮪௲ୖ࡚ࡢ⮬ほ࡚࠵ࡾ(Li et al., 1995ࠉLiu et al., 2006)ࠊࡆࡡࡒࡴࠉ㏳ᖏࡡ᪁Ἢ࡚SOD2 㐿ఎᏄࢅ◒ቪࡊ࡙ࡵࠉSOD2 ◒ ቪᰬᚋࡼࡿ࠷ྊ⬗ᛮ㧏࠾ࡖࡒࠊࡱࡒࠉ௫࡞ᶖ❟ྊ⬗ࡓࡖࡒ࡛ࡊ࡙ࡵࠉ㐛ᗐ㓗ࢪࢹࣝࢪ ✒ࡊ࡙࠷ࡀࠉᚆࠍ࡞ࡊ࡙࠷ࡂྊ⬗ᛮ⩻࠻ࡼࡿࡾࡒࡴࠉSOD2 ࡡࢣࢿ࣑Ꮽᏽᛮ⥌ᣚࡷ⣵ ⬂࿔ࡡ㐅⾔ࡡ㛭ࢅと☔࡞㏛ヴࡌࡾࡡࡢᅏ㞬࡚࠵ࡾ࡛⩻࠻ࡼࡿࡒࠊࡐࡆ࡚ࠉࢷࢹࣚࢦࢠࣛ
ࣤㄇᑙమ࡚࠵ࡾࠉࢺ࢞ࢨࢦࢠࣛࣤࡡ῟ຊ࡞ࡻࡖ࡙┘Ⓩࡡࢰࣤࣂࢠ㈹ࡡⓆ⌟ࢅೳでࡈࡎࡾࡆ࡛
࡚ࡀࡾTet-off system ࢅᑙථࡊࠉSOD2᮪௲◒ቪᰬࢅష⿿ࡌࡾࡆ࡛࡞ࡊࡒࠊ ࡐࡆ࡚ࠉSOD2 ᮪௲◒ቪᰬᶖ❟ࡡᡋ␆ࢅ௧ୖࡡࡻ࠹࡞❟࡙ࡒࠊ
ձ SOD2 ◒ቪ⏕࣊ࢠࢰ࣭ࢅష⿿ࡌࡾ
ղ SOD2-/+ ᰬࢅష⿿ࡌࡾࠊ
ճ Tet-off system ࡚ࣃࢹSOD2 ࢅⓆ⌟ࡈࡎࡾࡒࡴࡡ࣊ࢠࢰ࣭(hSOD2/1-118)ࢅష⿿ࡊࠉSOD2-/+ ᰬ
࡞ᑙථࡌࡾࠊ
➠ 2 ⟿
SOD2 ◒ቪ⏕࣊ࢠࢰ࣭ࡡష⿿
3.2.1 ࢼ࣠ࢹࣛSOD2 ࡡࢣࢿ࣑DNA ࡡࢠ࣭ࣞࢼࣤࢡ ࡱࡍࠉࢼ࣠ࢹࣛSOD2◒ቪ⏕࣊ࢠࢰѸࢅష⿿ࡌࡾࡒࡴ࡞ࠉDT40⣵⬂࠾ࡼࢣࢿ࣑DNAࢅฝࡊࠉ ࡆࡿࢅ㗢ᆵ࡛ࡊ࡙SOD2 ࡡࢣࢿ࣑DNA ධ㛏ࢅPCR ࡞ࡻࡖ࡙⑵ᖕࡊࡻ࠹࡛ムࡲࡒࠊࡊ࠾ࡊࠉGC rich 㒼า➠ୌ࢙࢞ࢮࣤ࠾ࡼࣤࢹࣞࣤ࡞࠾ࡄ࡙ᩐⓊሲᇱᏋᅹࡌࡾࡒࡴ࠾ࠉPCR ⏐∸ࢅ࢝ࣞ ࣭ࢪ㞹ポὃິ࡞ࡻࡖ࡙ᒈ㛜ࡊ࡙ࡵࠉᩐᅂ࡞偏ᅂ⛤ᗐࡊ࠾┘Ⓩࡡኬࡀࡈࡡࣁࣤࢺ (⣑7 Kbp) ࢅ☔ ヾࡌࡾࡆ࡛ࡢ࡚ࡀࡍࠉࡊ࠾ࡵ☔ヾ࡚ࡀࡒ࡛ࡊ࡙ࡵࠉ⑵ᖕຝ⋙ᴗࡴ࡙ᝇ࠾ࡖࡒࠊࡐࡆ࡚ࠉࡆࡡ PCR ⏐∸ࢅ㗢ᆵ࡛ࡊ࡙GC rich 㒼าࢅ㝎࠷ࡒࠉ➠࢙࢞ࢮࣤ࠾ࡼ➠࢙࢞ࢮࣤࢅnested PCR ࡞ ࡻࡖ࡙⑵ᖕࡊࡻ࠹࡛ムࡲࡒࠊࡐࡡ⤎ᯕࠉ┘Ⓩࡡ⣑6 Kbp ࡡPCR ⏐∸ࢅᚋࡾࡆ࡛࡚ࡀࡒࠊࡆࡡ PCR ⏐∸ࢅpGEM-T easy vector ࡞⤄ࡲ㎰ࡲࠉࢠ࣭ࣞࢼࣤࢡࡊࡒᚃࠉDNA ሲᇱ㒼าࢅ☔ヾࡊࡒ⤎ ᯕࠉSOD2 ࡡ➠2 ࢙࢞ࢮࣤ࠾ࡼ➠5 ࢙࢞ࢮࣤࢅྱࢆ࡚࠷ࡾࡆ࡛ࢅ☔ヾࡊࡒࠊ3.2.2 SOD2 ◒ቪ⏕࣊ࢠࢰѸࡡష⿿
ᚋࡼࡿࡒSOD2 ࡡࢣࢿ࣑DNA ࡡ࠹ࡔࠉࢰ࣭ࢣࢴࢹ࡛ࡾ㒼าࢅ᳠⣬ࠉSOD2 ࡡὩᛮ୯ᚨ࡚࠵ ࡾࠉMn ࢛ࣤࡡ⤎ྙࢦࢹࢅࢩ࣭ࣤࢰ࣭ࢣࢴࢷࣤࢡࡡᵾⓏ࡞ࡊࡒࠊMn ࢛ࣤࡡ⤎ྙࢦࢹ ࡢࠉSOD2 ࡡ➠2ࠉ3ࠉ4ࠉ5 ࢙࢞ࢮࣤࡐࡿࡑࡿ࡞1 ࡗ࠾ࡼ2 ࡗᏋᅹࡊ࡙࠷ࡾࠊᕞྎ୦❻࡞ᩐ༐ሲ ᇱᑊࡡ┞⇆ᛮࡡ࠵ࡾ㒼า࠵ࡾ࡛ࠉࢩ࣭ࣤࢰ࣭ࢣࢴࢷࣤࢡࡡຝ⋙Ⰳ࠷ࡆ࡛▩ࡼࡿ࡙࠷ࡾࡡ ࡚ࠉࢰ࣭ࢣࢴࢷࣤࢡ࣊ࢠࢰ࣭ࡡᕞྎ࡞༎ฦ㛏ࡈࡡDNA ࢅຊࡌࡾࡒࡴ࡞ࠉ୯ኳࡡ➠3 ࢙࢞ ࢮࣤࡡMn ࢛ࣤࡡ⤎ྙࢦࢹ(His 81)࡞࣏࣭ⷾ࢜Ѹࢅථࡌࡾࡆ࡛࡞ࡊࡒࠊࡐࡆ࡚ࠉFig. 3-1 ࡡ ࡻ࠹ᡥ㡨࡚➠3 ࢙࢞ࢮࣤࡡ୦❻࡞BamHϨࢦࢹࢅථࡊࠉ➠3 ࢙࢞ࢮࣤධ㛏ࢅ㝎࠷ࡒ࣊ࢠࢰ ࣭ࢅష⿿ࡊࡒࠊ᭩ᚃ࡞ⷾ⪇ᛮ࣏࣭࢜Ѹ ࢅථࡊ࡙ࠉSOD2 ࡡ◒ቪ⏕࣊ࢠࢰѸࢅష⿿ࡊࡒࠊ
➠ 3 ⟿ ࣃࢹ SOD2-HA / Tet-off ࣊ࢠࢰѸࡡష⿿
SOD2 ࢿࢴࢠࢹ࣏ࢪࡢࠉమ࡚ࣝ࣊ࣜࡢ⮬ほ࡚࠵ࡽࠉ⣵⬂࡚ࣝ࣊ࣜࡵ㟸ᖏ࡞ࢨࢴࢠ࡚࠵ ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾࠊᚉࡖ࡙ࠉDT40 ࡡSOD2 ◒ቪᰬࡵష⿿๑ࡢ⮬ほ࡚࠵ࡾྊ⬗ᛮ⩻࠻ࡼ ࡿࡒࠊࡱࡒࠉࡵࡊష⿿࡚ࡀࡒ࡛ࡊ࡙ࡵࠉ◒ቪᙔิ࠾ࡼ㓗ࢪࢹࣝࢪࢅུࡄ⤾ࡄࡾࡒࡴࠉと☔⾪ ⌟ᆵࢅびᐳ࡚ࡀ࠷ྊ⬗ᛮ࠵ࡖࡒࠊࡐࡆ࡚ࠉ Tet-off system ࡚Ⓠ⌟ࢅೳで࡚ࡀࡾࣃࢹSOD2 Ⓠ ⌟⣌ࢅᑙථᚃࠉࢹࣛSOD2 ࢅ◒ቪࡌࡾࡆ࡛࡞ࡻࡽࠉSOD2 ᮪௲◒ቪᰬࢅష⿿ࡌࡾࡆ࡛࡛ࡊࡒࠊ 3.3.1 ࣃࢹSOD偐 cDNA ࡡࢠ࣭ࣞࢼࣤࢡ
ࣃࢹᏄᐋ㡹⒬⣵⬂࡚࠵ࡾHeLa ⣵⬂࠾ࡼmRNA ࢅฝࡊࠉcDNA ࢅྙᠺࡊࡒࠊࡆࡿࢅ㗢ᆵ࡛ ࡊ࡙PCR ࢅ⾔࠷ࠉࣃࢹ SOD2 cDNA ࢅ⑵ᖕࡊࡒࠊࡆࡡPCR ⏐∸ࢅpGEM-T easy vector ࡞⤄ࡲ㎰
ࡲࠉࢠࣞѸࢼࣤࢡࡊࡒࠊDNA ሲᇱ㒼าࢅ☔ヾࡊࡒ⤎ᯕࠉࣃࢹSOD2 cDNA ࡚࠵ࡾࡆ࡛ࢅ☔ヾࡊࡒࠊ
3.3.2 ࣃࢹSOD2-HA / Tet-off ࣊ࢠࢰѸࡡష⿿
ᚋࡼࡿࡒࣃࢹSOD2 cDNA ࢅTet-off system ᑙථ⏕࣊ࢠࢰ࣭࡚࠵ࡾ1-118 ࣊ࢠࢰ࣭࡞⤄ࡲ㎰ࡳ ࡒࡴ࡞ࠉFig. 3-2 ࡡࡻ࠹࡞ࣃࢹSOD2 cDNA ࡞โ㝀㓕⣪ࢦࢹࢅᑙථࡊࠉࡐࡡࢦࢹࢅฺ⏕ࡊ࡙ 1-118 ࣊ࢠࢰ࣭࡞⤄ࡲ㎰ࢆࡓࠊ
➠ 4 ⟿ SOD2-᮪௲◒ቪᰬࡡష⿿ 3.4.1 SOD2-/+ᰬࡡష⿿ ࡱࡍࠉDT40 㔕⏍ᰬ࡞ᑊࡊ࡙ࠉFig. 3-1 ࡡࡻ࠹࡞ࡊ࡙ᚋࡼࡿࡒSOD2 ◒ቪ⏕࣊ࢠࢰѸࢅࢹࣚࣤࢪ ࣆ࢘ࢠࢨࣘࣤࡊࠉSOD2-/+ᰬࡡᶖ❟ࢅムࡲࡒࠊࡊ࠾ࡊࡼࠉࢹࣚࣤࢪࣆ࢘ࢠࢨࣘࣤᚃࠉgeneticin ࡞ᑊࡊ࡙⪇ᛮࢅ⋋ᚋࡊࡒࢤࣞࢼ࣭ࡢ⏍࠻࡙ࡂࡾࡵࡡࡡࠉࢣࢿ࣑DNA ࢅฝࡊࢣࢿ࣑PCR ࡞ࡻ ࡖ࡙ SOD2-/+ᰬࡡᶖ❟ࢅ☔ヾࡊࡻ࠹࡛ࡊࡒ࡛ࡆࢀࠉ1 ࢠ࣭ࣞࣤࡵ◒ቪࢅ☔ヾࡌࡾࡆ࡛࡚ࡀ࠾ ࡖࡒࠊࡐࡆ࡚ࠉࣈ࣏ࣚѸࢅ々ᩐムࡊࡒࡵࡡࡡࠉࡐࡿ࡚ࡵ☔ヾࡢྊ⬗ࡓࡖࡒࠊࡆࡆ්࡚ᗐࠉ ࢸ࣭ࢰ࣭࣊ࢪ࡞ࡻࡖ࡙SOD2 ࡡࢣࢿ࣑DNA 㒼าࢅ☔ヾࡊࡒ࡛ࡆࢀࠉ௧๑࡞ࠉ◒ቪ⏕ࢤࣤࢪࢹࣚ ࢠࢹࢅష⿿ࡊࡒ࡞☔ヾࡊࡒ㒼า࡛ࠉࣈ࣏ࣚѸࢅシ゛ࡊ࡙࠷ࡒ➠ୌ࢙࢞ࢮࣤࡡ㒼าኣᑛ␏ ࡖ࡙࠷ࡾࡆ࡛ฦ࠾ࡖࡒࠊࡐࡆ࡚ࠉࡆࡡ㒼าࢅࡵ࡛࡞᩺ࡒ࡞ࣈ࣏ࣚѸࢅシ゛ࡊࠉ◒ቪࢅ☔ヾࡊ ࡒ࡛ࡆࢀࠉSOD2-/+ᰬࡡᶖ❟ࢅ☔ヾࡌࡾࡆ࡛࡚ࡀࡒࠊ(24 ࢠ࣭ࣞࣤ୯2 ࢠ࣭ࣞࣤ: 8.3 %) 3.4.2 Tet-off system ࡡᑙථ
ḗ࡞ࠉSOD2-/+ᰬ࡞Tet-off system ࡚hSOD2 ࢅⓆ⌟ࡈࡎࡾࡒࡴ࡞ࠉష⿿ࡊࡒhSOD2-HA/1-118 ࣊
ࢠࢰѸ࠽ࡻࡦ1-128࣊ࢠࢰѸࢅࢹࣚࣤࢪࣆ࢘ࢠࢨࣘࣤࡊࡒࠊHygromycin B ࡞ᑊࡊ࡙⪇ᛮࢅ⋋ᚋࡊࡒ ࢤࣞࢼѸ࠾ࡼ cDNA ࢅฝࡊࠉhSOD2-HA ࡡⓆ⌟ࢅ RT-PCR ࡞ࡻࡖ࡙☔ヾࡊࡒ⤎ᯕࠉⓆ⌟ࢅび ᐳࡌࡾࡆ࡛࡚ࡀࡒࠊ(4 ࢠ࣭ࣞࣤ୯1 ࢠ࣭ࣞࣤ: 25 %) 3.4.3 SOD2-/+ /hSOD2-HA ᰬࡡష⿿ ᭩ᚃ࡞ࠉよࡈࡿࡒࡵ࠹ 1 ࡗࡡ SOD2 㐿ఎᏄࢅ◒ቪࡌࡾࡒࡴࠉࡵ࠹ୌᗐ SOD2 ◒ቪ⏕࣊ࢠࢰѸࢅࢹࣚࣤࢪࣆ࢘ࢠࢨࣘࣤࡊࠉᚋࡼࡿࡒ࣏ࢤࣆ࢘ࢿ࣭ࣜ㓗⪇ᛮࢤ ࣞࢼ࣭ࡡࢣࢿ࣑ DNA ࢅฝࡊࠉࢣࢿ࣑ PCR ࡞ࡻࡽࢩ࣭ࣤࢰ࣭ࢣࢴࢷࣤࢡࡡ ᭯↋ࢅ☔ヾࡊࡒ⤎ᯕࠉ々ᩐࡡࢠ࣭࡚ࣞࣤ◒ቪ☔ヾࡈࡿࡒࠊ(96 ࢠ࣭ࣞࣤ୯ 3 ࢠ ࣭ࣞࣤ: 3.1 %)᭞࡞ࠉࢣࢿ࣑ PCR ࡡ⤎ᯕ㝟ᛮࡓࡖࡒࢠ࣭ࣞࣤࢅ RT-PCR ࡛ࢦࢧ
ࣤࣇࣞࢴࢷࣤࢡ࡞ࡻࡖ࡙ SOD2-/+/hSOD2-HA ᰬࡡᶖ❟ࢅ☔ヾࡊࡒࠊ
(A)
Fig. 3-3 SOD2 -/-/hSOD2-HA ᰬࡡష⿿ (A) RT-PCR ⣵⬂ ᰬ ࠾ࡼ total RNA ࢅ ฝࡊࠉࣈ ࣚ ࣏ ࣭ࢅ ⏕࠷ ࡙ RT-PCR ࢅ⾔ ࡖࡒࠊ2 ࢠࣞ ࣭ࣤ ࡞ ࠽ ࠷࡙ SOD2 ◒ቪࡈࡿ࡙࠷ࡾࡆ࡛ࢅ☔ヾࡊࡒࠊRAD52 ࡢ positive control ࡚࠵ࡾࠊSOD2 RAD52 Wild-Type SOD2-/+/hSOD2-HA SOD2-/-/hSOD2-HA #1 SOD2-/-/hSOD2-HA #2 +/+ -/+ #1 #2 +/+: -/+: #1: #2:
-/-
(B)
(C) Fig. 3-3 ⤾ࡀ (B) ࢦࢧࣤࣇࣞࢴࢷࣤࢡࡡࢪࢹࣚࢷࢩ࣭ (C) ࢦࢧࣤࣇࣞࢴࢷࣤࢡ ⣵⬂࠾ࡼࢣࢿ࣑ DNA ࢅᅂࡊࠉ(B)ࡡࡻ࠹࡞โ㝀㓕⣪ฌ⌦ࡊࠉࢦࢧࣤࣇࣞࢴࢷࣤࢡࢅ⾔ࡖࡒࠊࡐ ࡡ⤎ᯕࠉSOD2 ࡡ◒ቪ☔ヾࡈࡿࡒࠊ Wild-Type SOD2-/+/hSOD2-HA SOD2-/-/hSOD2-HA #1 SOD2-/-/hSOD2-HA #2 +/+: -/+: #1: #2: +/+ -/+ -/- #1 #2 3.8Kbp SOD2 locus Marker SOD2 SOD2 locus Targeted locus NdeϨ NdeϨ BamHϨ BamHϨ BamHϨ BamHϨ Probe Probe 14 Kb 3.8 Kb 14 Kbp➠ 4 ❮
SOD2 ᮪௲◒ቪᰬࡡ⾪⌟ᆵࡡゆᯊ
➠ 1 ⟿ SOD2 ᯜῤ࡞ࡻࡾ⤊Ⓩን 4.1.1 ࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃࡡhSOD2 ࡡᾐ ᶖ❟ࡊࡒSOD2᮪௲◒ቪᰬ࡞ᑙථࡊࡒtet-off systemとᖏ࡞ᶭ⬗ࡊ࡙࠷ࡾࡡ࠾ࢅ☔ヾࡌࡾࡒࡴ ࡞ࠉࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃࡡhSOD2 Ꮛᅹ㔖ࡡንࢅ㛣ࢅ࠽ࡖ࡙ῼᏽࡊࡒࠊࡐࡡ⤎ᯕࠉᚋࡼ ࡿࡒ2ࡗࡡࢠ࣭ࣞࣤ(#1࡛#2)ࡔࡼࡵࠉࢺ࢞ࢨࢦࢠࣛࣤ῟ຊ࡞ࡻࡖ࡙hSOD2ᾐࡊ࡙࠷ࡀࠉ 2 ┘࡞ࡢ࢙ࢪࢰࣤࣇࣞࢴࢹἪࡡ᳠ฝ㝀⏲௧ୖࡱ࡚Ⓠ⌟㔖ୖࡌࡾࡆ࡛ࢂ࠾ࡖࡒࠊ (Fig.4-1) Fig. 4-1 ࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃࡡhSOD2 ࡡᾐ ⣵⬂ࢅࢺ࢞ࢨࢦࢠࣛࣤ(20 ng/mL)Ꮛᅹୖ࡚ᅒ࡞♟ࡊࡒ㛣ᇰ㣬ࡊࡒᚃࠉ⣵⬂ࢅᅂࡊࠉSDS-PAGE ࡚ࢰࣤࣂࢠ㈹ࢅᒈ㛜 ࡊ࡙ᢘమ࡚᳠ฝࡊࡒࠊ 4.2.2 ⣵⬂⑵る᭜⥲ Sod2 ࢿࢴࢠࢹ࣏ࢪ⏜ᮮ⦼⥌ⰾ⣵⬂ࡢࠉ㔕⏍ᰬ࡛セ㍉ࡊ⣵⬂⑵る⬗㟸ᖏ࡞࠷ࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Samper, E., et al. 2003)ࠊࡱࡒࠉSod2-/-࣏ࢪ⏜ᮮࡡ♼⤊⣵⬂ࡢ㏳ᖏࡡ㓗⣪⃨ᗐୖ࡚ࡢ
⮬ほ࡚࠵ࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾ(Liu, J., et al. 2006)ࠊࡐࡆ࡚ࠉSOD2 ࡡᾐDT40 ࡡ⣵⬂⑵る⬗࡞ hSod2-HA Ș-tubulin 0 1 2 3 4 5 SOD2 -/-/hSOD2-HA #1 DOX 0 1 2 3 4 5 #2 (Days)
ᙫ㡢ࢅ࠻ࡾࡡ࠾᳠ゞࡌࡾࡒࡴ࡞ࠉ1106⣵⬂ࢅࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᇰᆀ࡞᧓ࡀࠉ24 㛣ࡇ ࡛࡞ࢹࣛࣂࣤࣇ࣭࡚ࣜⰅࡈࡿ࠾ࡖࡒ⣵⬂ᩐࢅ゛ῼࡊࠉhSOD2 ࡡᾐ⣵⬂⑵る࡞→ࡌᙫ 㡢ࢅㄢࡒࠊࡐࡡ⤎ᯕࠉhSOD2 ᾐࡌࡾࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃ2 ┘ࡱ࡚ࡢࠉ῟ຊࡊ࠷ ሔྙ࡛セ࡛ࢆ⣵⬂⑵る⬗࡞ንࡢびᐳࡈࡿ࠾ࡖࡒࠉ4 ࠾ࡼ5 ┘࡞ࡾ࡛ᚆࠍ࡞⣵ ⬂⑵る⬗ୖࡊ࡙࠷ࡖࡒࠊ(Fig. 4-2) Fig. 4-2 ⣵⬂⑵る᭜⥲ ⣵⬂ᰬࢅࠉ39Υ࡚ࢺ࢞ࢨࢦࢠࣛࣤ῟ຊࠉ࠵ࡾ࠷ࡢᮅ῟ຊࡡᇰᆀ࡚ᇰ㣬ࡊࠉ24 㛣ࡇ࡛࡞ࢹࣛࣂࣤࣇ࣭ࣜ࡞ࡱࡼ ࠷⏍⣵⬂ᩐࢅ゛ῼࡊࠉ᧓࠷ࡒࡡ⣵⬂ᩐ࡛セ㍉ࡊࡒࠊ 4.1.3 ࢤࣞࢼѸᙟᠺ⋙ ⣵⬂⑵る᭜⥲ࡡ⤎ᯕ࠾ࡼࠉSOD2 ࡡᾐࡢ├ࡔ࡞⣵⬂⑵る࡞ᙫ㡢ࢅ࠻ࡾズ࡚ࡢࡂࠉ⤊Ⓩ ࡞⾪⌟ᆵᙁࡂࡖ࡙࠷ࡂࡆ࡛ࡈࡿࡒࠊࡐࡆ࡚ࠉ⣵⬂ࡡ⏍Ꮛ⋙ࢅῼᏽࡌࡾ┘Ⓩ࡚ࠉࢺ࢞ࢨ ࢦࢠࣛࣤᏋᅹୖ࡚1࠾ࡼ5㛣ᇰ㣬ࡊࡒSOD2᮪௲◒ቪᰬࢅ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ࡞᧓ࡀࠉ (Days) WT 1 1 0 100 100 0 10000 10000 0 1000000 1000000 0 day
デ
ー
タ
#
1
sod2#52 + sod2#52 -sod SOD2-/-㸘1 DOX (-) SOD2 -/-㸘1 DOX (+) SOD2 -/-㸘2 DOX (-) SOD2 -/-㸘2 DOX (+) Wild-type 2 4 6 8 0 10 102 103 104 105 106 107 139 Υ࡚ᇰ㣬ࡊࡒ࡛ࡀࡡࢤࣞࢼ࣭ᙟᠺ⋙ࢅῼᏽࡊࡒࠊ偆㔕⏍ᰬ࡚⏍࠻࡙ࡀࡒࢤࣞࢼ࣭ᩐࢅ100%࡛ ࡊ࡙࠷ࡾࠊ假 ࡐࡡ⤎ᯕࠉࢺ࢞ࢨࢦࢠࣛࣤ῟ຊ๑ࡢ㔕⏍ᰬ࡛⇆⛤ᗐࡓࡖࡒࢤࣞࢼ࣭ᙟᠺ⋙ࠉ⤊Ⓩ࡞ ୖࡊ࡙࠷ࡀࠉ4 ࠾ࡼ5 ┘࡞ࡢ25%⛤ᗐࡱ࡚ୖࡊࡒࠊࡊ࠾ࡊࠉ⣵⬂⑵る⬗࡛⇆ᵕ࡞⾪ ⌟ᆵࡡᝇࡢ4 ࠾ࡼ5 ┘࡚⤂ࡊࠉࡐࡿ௧୕ᝇࡌࡾࡆ࡛ࡢ࠾ࡖࡒࠊࡐࡆ࡚ࡆࡿ௧㜾 SOD2 ࡡ⾪⌟ᆵࢅびᐳࡌࡾ㝷࡞ࡢࠉᇱᮇⓏ࡞ࡢࢺ࢞ࢨࢦࢠࣛࣤ῟ຊ5 ┘ࡡ⣵⬂ࢅ⏕ࡌࡾࡆ ࡛࡞ࡊࡒࠊ(Fig.4-3) Fig. 4-3 ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ୯࡚ࡡࢤࣞࢼ࣭ᙟᠺ⋙ ⣵⬂ࢅࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᇰᆀ࡚ᅒ࡞♟ࡊࡒ㛣ᇰ㣬ᚃᅂࡊ࡙ࠉ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ࡞᧓ࡀࠉ7 㛣ᇰ㣬ᚃࠉ⏍࠻ ࡙ࡀࡒࢤࣞࢼ࣭ᩐࢅ゛ῼࡊࡒࠊ㔕⏍ᰬࡡࢤࣞࢼ࣭ᩐࢅ100 %࡛ࡊࡒࠊᅒ୯ࡡࣁ࣭ࡢᵾ‵೩ᕣࢅ♟ࡌࠊ
➠ 2 ⟿ ⛸ ROS ࡞ᑊࡌࡾវུᛮ 4.2.1 ࣂࣚࢤ࣭ࢹវུᛮ
ࢿࢴࢠࢹ࣏ࢪ⏜ᮮࡡSod2 -/-⣵⬂ࡢࠉࢪ࣭ࣂ࣭࢛࢞ࢨࢺࢅ⣵⬂හ࡚㐛࡞Ⓠ⏍ࡈࡎࡾⷾ
ࡡ1 ⛸࡚࠵ࡾࣂࣚࢤ࣭ࢹ࡞ᙁ࠷វུᛮࢅ♟ࡌࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Huang et al., 1997)ࠊࡐࡡ࡞ ࡵࠉRNAi Ἢ࡞ࡻࡖ࡙ࢨࣘࢩࣘࣁ࢙࠾ࡼSOD2 ࢅࢿࢴࢠࢱࣤࡌࡾࡆ࡛࡞ࡻࡖ࡙ࡵࠉ⇆ᵕࡡ ⤎ᯕᚋࡼࡿ࡙࠷ࡾ(Paul et al., 2007)ࠊࡐࡆ࡚ࠉDT40 SOD2 ᮪௲◒ቪᰬ࡚ࡵ⇆ᵕ࡞ᙁ࠷វུᛮࢅ ♟ࡌ࠾᳠ゞࡌࡾࡒࡴ࡞ࠉࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃ 5 ┘ࡱ࡚ࡡ SOD2 ᮪௲◒ቪᰬࢅࣂࣚࢤ࣭ࢹ (200ȣM) ࡚ 24 㛣ฌ⌦ࡊࡒ࡛ࡀࡡࣂࣚࢤ࣭ࢹវུᛮࢅࣆ࣭ࣞࢦࢹ࣒ࢹࣛѸࢅ⏕࠷࡙ PI ༟ Ⰵ࡞ࡻࡖ࡙びᐳࡊࡒࠊPI ࡢ DNA ࡛々ྙమࢅᙟᠺࡌࡾࡆ࡛࡚⺧කࢅⓆࡌࡾࠉほ⣵⬂࡞ࡡࡲ PI ථࡽ㎰ࡳࡆ࡛ྊ⬗ࡒࡴࠉPI 㝟ᛮࡡ⣵⬂ࢅ᳠ฝࡌࡾࡆ࡛࡚ࠉほ⣵⬂ࡡྙࢅῼᏽ࡚ࡀࡾࠊ ࡐࡡ⤎ᯕࠉ㔕⏍ᰬ࡚ࡢ࡛ࢆほ⣵⬂⑵ຊࡊ࠷᮪௲ୖ࡚ࡵࠉSOD2 Ⓠ⌟ೳでᚃࠉSOD2᮪௲ ◒ቪᰬ࡚ࡢࣂࣚࢤ࣭ࢹฌ⌦࡞ࡻࡖ࡙⤊Ⓩ࡞ほ⣵⬂ࡡྙ㢟ⴥ࡞⑵ຊࡊ࡙࠷ࡂᵕᏄびᐳࡈ ࡿࡒ (Fig. 4-4)ࠊ Fig. 4-4 ドキシサイクリン添加後 のパラコート感受性 細胞をドキシサイクリン(20 ng/mL)存 在下で図に示す時間培養後、パラコ−ト (200 μM) で24 時間処理した。細胞を回 収後、PIで染色し、フローサイトメトリ ーによって PI ෩性の細胞の割合を測定 した。
4.2.2 ࡐࡡROS ࡞ᑊࡌࡾវུᛮ
⛸ࡡSOD2 -/-⣵⬂ࡡ㐛㓗ラ⣪ࠉSOD2 ࡐࡡ㝎ཡ࡞㛭ࡊ࠷ROS ࡞ᑊࡌࡾࡡវུᛮ
ࡢሒ࿈࡞ࡻࡖ࡙␏ࡾࠊࡐࡆ࡚ࠉDT40 ⣵⬂࡚ࡢវུᛮࢅ♟ࡌ࠾᳠ゞࡌࡾࡒࡴ࡞ࠉSOD2 ├ 㝎ཡ࡞㛭ࡊ࠷ROS ࢅⓆ⏍ࡈࡎࡾࠉ㐛㓗ラ⣪ࠉࢭ࢛ࢨࣤࠉ࢙ࢹ࣎ࢨࢺ࡚ฌ⌦ࡊࡒ㝷ࡡほ⣵ ⬂ࡡ⑵ຊࢅ4.2.1 ࡛⇆ᵕ࡞PI ༟Ⰵ࡞ࡻࡾࣆ࣭ࣞࢦࢹ࣒ࢹࣛѸ࡚びᐳࡊࡒࠊ ࡐࡡ⤎ᯕࠉࣂࣚࢤ࣭ࢹ࡛ࡢ␏ࡽࠉ㐛㓗ラ⣪ࠉࣃࢺࣞ࢞ࢨࣚࢩ࢜ࣜࢅⓆ⏍ࡈࡎࡾࢭ࢛ࢨࣤࡷࠉ ࢹ࣎ࢮ࣒࣭ࣚࢭ ϩ㜴ᐐ࡚࠵ࡾ࡛භ࡞ ROS ࢅⓆ⏍ࡈࡎࡾ࢙ࢹ࣎ࢨࢺ࡞ᑊࡌࡾវུᛮࡢࢺ࢞ ࢨࢦࢠࣛࣤࢅຊ࠻࠷ሔྙ࡛⇆⛤ᗐ࡚࠵ࡖࡒࠊ(Fig. 4-5)
Fig. 4-5 SOD2 ᯜῤࡡROS Ⓠ⏍ムⷾវུᛮ
SOD2 ᮪௲◒ቪᰬࢅࢺ࢞ࢨࢦࢠࣛࣤ(20 ng/mL)Ꮛᅹୖ࡚5 㛣ᇰ㣬ࡊࡒᚃࠉࣂࣚࢤѸࢹ(200 ȣM) ࡚24 㛣ࠉ㐛㓗ラ⣪
(500 ȣM)࡚6 㛣ࠉzeocin(500 ȣg/mL) ࡚24 㛣ࠉetoposide(10 ȣM) ࡚6 㛣ฌ⌦ࡊࡒࠊ⣵⬂ࢅᅂᚃࠉPI ࡚Ⰵࡊࠉࣆࣞ
➠ 3 ⟿ ࣂࣚࢤѸࢹវུᛮ⑵ኬࡡⅴᅄ 4.3.1 ࣎ࢹ࣭ࢨࢪ࡛࢛࣭ࢹࣆࢩ࣭ࡡ᳠ฝ
SOD2 ᮪௲◒ቪᰬࣂࣚࢤ࣭ࢹ࡞ᑊࡊ࡙㧏࠷វུᛮࢅ♟ࡌⅴᅄࢅゆᯊࡌࡾࡒࡴ࡞ࠉ4.2.1 ࡛⇆ᵕ ࡡ᮪௲࡚⣵⬂ほࡡ1 ⛸࡚࠵ࡾ࣎ࢹ࣭ࢨࢪࡷ࢛࣭ࢹࣆࢩ࣭ࡡ᳠ฝࢅ⾔ࡖࡒࠊ࣎ࢹѸࢨࢪࡡᣞ ᵾ࡛ࡊ࡙ࡢࠉᰶ⭯ᡬࡔࢰࣤࣂࢠ㈹ࡡLaminB1 ࡡฦゆࠉࢽࢠ࢛ࣝࢮѸ࣑༟న࡚ࡡDNA ࡡ᩷∞ (DNA Ladder) ࠉิ࣎ࢹ࣭ࢨࢪࡡᣞᵾ࡚࠵ࡾAnnexin Ϭ࡛PI ࡞ࡻࡾ2 ḗඔࣆ࣭ࣞࢦࢹ࣒ࢹ ࣭ࣛࢅ⏕࠷ࠉ࢛࣭ࢹࣆࢩ࣭ࡡᣞᵾ࡛ࡊ࡙ࡢLC-3 ࡡ⑵ຊࢅ⏕࠷ࡒࠊ
ࡐࡡ⤎ᯕࠉࢺ࢞ࢨࢦࢠࣛࣤ῟ຊࡊ࡙ 5 㛣ᇰ㣬ࡊࡒ SOD2 ᮪௲◒ቪᰬ࡚ࡢࠉࣂࣚࢤ࣭ࢹ࡞ ࡻࡖ࡙ࠉLaminB1 ࡡฦゆࡷࢽࢠ࢛ࣝࢮѸ࣑༟న࡚ࡡDNA ࡡ᩷∞びᐳࡈࡿࠉ࣎ࢹ࣭ࢨࢪ
Fig. 4-6 ࣎ࢹ࣭ࢨࢪ࡛࢛࣭ࢹࣆࢩ࣭ࡡ᳠ฝ (1)
(A) 㔕⏍ᰬ࠽ࡻࡦSOD2 ᮪௲◒ቪᰬࢅࢺ࢞ࢨࢦࢠࣛࣤ(20 ng/mL)Ꮛᅹୖ࡚5 㛣ᇰ㣬ࡊࡒᚃࠉࣂࣚࢤѸࢹ(200 ȣM) ࡚24
㛣ฌ⌦ࡊࡒࠊ⣵⬂ࢅᅂᚃࠉSDS-PAGE ࡞ࡻࡽᒈ㛜ࡊࠉᢘమ࡚᳠ฝࡊࡒࠊ(B) (A)࡛⇆ᵕࡡ᮪௲࡚ࣂࣚࢤѸࢹࢅฌ⌦ࡊࠉ
⣵⬂ࢅᅂᚃ࡞ࢣࢿ࣑DNA ࢅฝࡊࠉ࣭࢝ࣞࢪ㞹ポὃິ࡚ᒈ㛜ࡊࡒࠊ
Fig. 4-6 ࣎ࢹ࣭ࢨࢪ࡛࢛࣭ࢹࣆࢩ࣭ࡡ᳠ฝ (2)
(A) 㔕⏍ᰬ࠽ࡻࡦSOD2 ᮪௲◒ቪᰬࢅࢺ࢞ࢨࢦࢠࣛࣤ(20 ng/mL)Ꮛᅹୖ࡚5 㛣ᇰ㣬ࡊࡒᚃࠉࣂࣚࢤѸࢹ(200 ȣM) ࡚ 24 㛣ฌ⌦ࡊࡒࠊ⣵⬂ࢅᅂᚃࠉࣆ࣭ࣞࢦࢹ࣒ࢹ࣭ࣛ࡞ࡻࡖ࡙late apoptosis⣵⬂ࢅ᳠ฝࡊࡒࠊ
(B) (A)࡛⇆ᵕࡡ᪁Ἢ࡚ealry apoptosis⣵⬂ࢅ᳠ฝࡊࡒࠊ
A B
4.3.2 ࣂࣚࢤ࣭ࢹฌ⌦ࡡ⣵⬂࿔ࡡᙫ㡢
ḗ࡞ࠉࣂࣚࢤ࣭ࢹ࡞ࡻࡖ࡙ㄇⓆࡈࡿࡾ࣎ࢹ࣭ࢨࢪ⣵⬂࿔≁␏Ⓩ࡞Ⓠ⏍ࡌࡾࡡ࠾᳠ゞࡌࡾ
ࡒࡴ࡞ࠉࣂࣚࢤ࣭ࢹࢅฌ⌦ࡊࡒ㝷ࡡ⣵⬂࿔ࡡንࢅCycle TESTTM
PLUS DNA Reagent Kit ࢅ⏕ ࠷࡙᳠ゞࡊࡒࠊ ࡐࡡ⤎ᯕࠉDOX ฌ⌦⣵⬂ࡢࣂࣚࢤ࣭ࢹฌ⌦ࡌࡾࡆ࡛࡚ࠉS ࡛ G2/M ࡡ⣵⬂⩄㝎ࠍ࡞΅ ᑛࡊ╂ࡴࠉ24 hr ᚃ࡞ࡢS ࡛G2/M࡞࠷ࡾ⣵⬂⩄ࡢࡂࡖࡒࠊࢂࡽ࡞ࠉほ⣵⬂ࢅ♟ ࡌsub G1 ࡡ⣵⬂⩄⑵ຊࡊࠉS ࡛G2/M ࡡ⣵⬂≁␏Ⓩ࡞ࣂࣚࢤ࣭ࢹฌ⌦࡞ࡻࡖ࡙ ࣎ࢹ࣭ࢨࢪ㉫ࡆࡖ࡙࠷ࡾࡆ࡛ฦ࠾ࡖࡒ(Fig. 4-7)ࠊ Fig. 4-7 ࣂࣚࢤ࣭ࢹฌ⌦࡞ࡻࡾ⣵⬂࿔ࡡᙫ㡢
SOD2 Ⓠ⌟ୖ࡛ᯜῤୖ࡚ࠉSOD2 ᮪௲◒ቪᰬ࡞ࣂࣚࢤѸࢹ(200 ȣM) ࢅᅒ࡞♟ࡊࡒ㛣ฌ⌦ࡊࡒࠊ⣵⬂ࢅᅂࡊࠉCycle TESTTMPLUS DNA Reagent Kit ࢅ⏕࠷࡙ࠉ⣵⬂࿔ฦᕱࢅࣆ࣭ࣞࢦࢹ࣒ࢹ࣭࡚ࣛゆᯊࡊࡒࠊ
➠ 4 ⟿ DNA ഭᐐ࡞ᑊࡌࡾវུᛮ 4.4.1 DNA ഭᐐ࡞ᑊࡌࡾវུᛮ
ḗ࡞ࠉSOD2 ࡡᾐ࡞ࡻࡖ࡙㉫ࡆࡾ࡛ῼࡈࡿࡾ㐛ᗐࡡ㓗ࢪࢹࣝࢪࢣࢿ࣑Ꮽᏽᛮ࡞ᙫ㡢ࢅ ࠻ࡾ࠾᳠ゞࡌࡾࡒࡴ࡞ࠉDNA ഭᐐ࠽ࡻࡦࠉ⣰አ⥲偆UV假࡞ᑊࡌࡾវུᛮࢅびᐳࡊࡒࠊ ⏕ࡊࡒⷾࡢࠉDNA ࡚ࣜ࢞ࣜࠉDSBsࢅㄇⓆࡌࡾ࡛ࡈࡿ࡙࠷ࡾMMSࠉUV ࡞㎾࠷ഭᐐࢅ DNA ࡞࠻ࡾ࡛࠷ࢂࡿ࡙࠷ࡾ4-NQO ࡚ࠉDNA ഭᐐ࡞ࡗ࠷࡙ࡢⷾྱ࣒᭯ࢲࣜࢬ࣭ࣜࣞࢪᇰ ᆀ࡚ᇰ㣬ᚃࠉ⏍࠻࡙ࡀࡒࢤࣞࢼ࣭ᩐࢅῼᏽࡊࡒࠊUV ࡡሔྙࡢᇰᆀ࡞ⵏࡂ๑࡞↯ᑏࡊࡒࠊࡐࡡ⤎ ᯕࠉSOD2 ᾐࡊ࡙ࡵࠉDNA ഭᐐ࠽ࡻࡦUV ࡞ᑊࡌࡾវུᛮࡢ㔕⏍ᰬ࡛⇆⛤ᗐ࡚ࠉ㢟 ⴥንࡢびᐳࡈࡿ࠾ࡖࡒ(Fig. 4-8)ࠊ ! !" !"" # $ " % &' ' æ &' !" ! % &' () "æ!%!*+()+% ,-.%/!æ0123 ,-.%/!æ #$0123 #$ ! !" !"" # $ " ' !" !' %" 4 4 5 "æ!!6"+445+7 ,-.%/!æ+01 ,-.%/!æ+ #$01 #$ ! !" !"" # $ " % &' ' æ &' !" ! % &' () "æ!%!*+()+% ,-.%/!æ0123 ,-.%/!æ #$0123 #$ S u rvi va l ( % ) 100 10 1 0 2.5 5 7.5 10 12.5 UV (J/m2) 100 10 1 0 5 10 15 20 MMS (v/v !10-6) S u rvi va l ( % ) ! !" !"" # $ " " &" ' " &! " &! ' " &% " &% ' 89:2 "æ!%"6+89:2 0123+%., 521%/!æ #$0123 #$ S u rvi va l ( % ) 100 10 1 0 0.05 0.1 0.15 0.2 0.25 4NQO (ng/ml) Wild-Type DOX(-) Wild-Type DOX(+)
SOD2-/-/hSOD2-HA DOX(-)
SOD2-/-/hSOD2-HA DOX(+)
Fig. 4-8 SOD2 ᯜῤࡡⷾវུᛮ
ࡐࡿࡑࡿࡡ⃨ᗐࡡ MMSࠉ4NQO ྱ࣒᭯ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ࡚ 7 ࠾ࡼ 14 㛣ᇰ㣬ࡊࠉ⏍࠻࡙ࡀࡒࢤࣞࢼ࣭ࢅ ῼᏽࡊࡒࠊᅒ୯ࡡࣁ࣭ࡢᵾ‵೩ᕣࢅ♟ࡌࠊ
4.4.2 ╀┶Ⰵฦమ㛣ஹᥦ (SCE) ࡡῼᏽ
ᙔ◂✪ᐄࡷࡡ◂✪ࢡ࣭ࣜࣈࡡሒ࿈ࡻࡽࠉSCE ࡢ┞⇆⤄ᥦ࠻ᶭᵋ (HRR) ࡞ࡻࡖ࡙ᙟᠺࡈࡿࠉ ╀┶Ⰵฦమ㛣ࡡcross over type ࡡHRR ࡞ࡻࡽᙟᠺࡈࡿࡾࡆ࡛♟ࡈࡿ࡙࠽ࡽࠉDNA ഭ࡞ࡻ ࡖ࡙ࡐࡡ㢎ᗐஸ㐅ࡌࡾࠊࡆࡡ SCE 㢎ᗐࢅᣞᵾ࡞ࠉSOD2 ࡡᾐ࡞ࡻࡖ࡙㉫ࡆࡾ࡛ῼࡈࡿࡾ 㐛ᗐࡡ㓗ࢪࢹࣝࢪࢣࢿ࣑DNA ࡞ഭࢅ࠻ࡾ࠾࠹࠾ࢅ᳠ゞࡊࡒࠊࡐࡡ⤎ᯕࠉࢺ࢞ࢨࢦ ࢠࣛࣤ῟ຊ5 ┘ࡡSOD2 ᮪௲◒ቪᰬ࡚ࡵࠉ㔕⏍ᰬ࡛⇆⛤ᗐࡡSCE 㢎ᗐࡊ࠾びᐳࡈࡿ࠾ ࡖࡒࡆ࡛࠾ࡼࠉSOD2 ࡡᾐ࡞ࡻࡖ࡙ᑛࡂ࡛ࡵSCE ࡚᳠ฝྊ⬗ࢣࢿ࣑ DNA ࡡഭ㉫ࡆ ࡖ࡙࠷࠷ࡆ࡛♟ြࡈࡿࡒ(Fig. 4-9)ࠊ
Fig. 4-9 ⮤ⓆⓏ╀┶Ⰵฦమஹᥦ㢎ᗐ
BrdU ࢅ 2 ⣵⬂࿔(16.5 㛣) ཱིࡽ㎰ࡱࡎࡒ⣵⬂ࢅࢤࣜࢬ࣐ࢺᏋᅹୖ࡚ 2 㛣ᇰ㣬ᚃࠉࣈࣝࣂ࣭ࣚࢹࢅష⿿ࡊࠉฦฌ⌦
4.4.3 DNA ྙᠺ㜴ᐐ࡞ᑊࡌࡾវུᛮ
ฝⰾ㓕ザ࡞࠽࠷࡙ࠉsod1◒ቪᰬࡢࠉDNA ྙᠺ㜴ᐐ࡚࠵ࡾhydroxy urea ࡞ᑊࡊ࡙㢟ⴥវུ ᛮࢅ♟ࡌࡆ࡛▩ࡼࡿ࡙࠷ࡾ(Carter et al.. 2005)ࠊࡐࡆ࡚ࠉ㓗ࢪࢹࣝࢪDNA 々⿿ࠉDNA ㅨ ࡞㛭ࡊ࡙࠷ࡾྊ⬗ᛮࢅ⩻࠻ࠉDNA ྙᠺ㜴ᐐ࡚࠵ࡾhydroxy urea ࠽ࡻࡦaphidicoline ࡞ᑊࡌࡾ វུᛮࢅㄢࡒࠊአࡆ࡛࡞ࠉSOD2 ࡡᾐ࡞ࡻࡖ࡙ࠉHU ࠽ࡻࡦaphidicoline ࡞ᑊࡊ࡙⪇ᛮ ࢅ♟ࡌࡆ࡛᪺ࡼ࠾࡞ࡖࡒ(Fig. 4-10)ࠊ ! !" !"" # $ " % ! " ! % &' "æ!(")*&' +,-(.!æ/012 +,-(.!æ #$/012 #$ Wild-Type DOX(-) Wild-Type DOX(+) SOD2-/-/hSOD2-HA DOX(-)
SOD2-/-/hSOD2-HA DOX(+)
Wild-Type DOX(-) Wild-Type DOX(+) SOD2-/-/hSOD2-HA DOX-)
SOD2-/-/hSOD2-HA DOX(+)
! !" !"" # $ " % ! " ! % &' "æ!(")*&' +,-(.!æ/012 +,-(.!æ #$/012 #$ 1 10 100 S u rvi va l ( % ) 10 mM HU treatment (hrs) 0 5 10 15 ! !" !"" # $ " ( 3% % æ 3% !" 45& "æ!(!"*45& +,-(.!æ/012 +,-(.!æ #$/012 #$ 0 2.5 5 7.5 10 5 !M Aphidicoline treatment (hrs) 1 10 100 S u rvi va l( % ) Fig. 4-10 SOD2 ᯜῤࡡⷾវུᛮ ⣵⬂ࢅ HU ࠵ࡾ࠷ࡢ Aphidicoline ࡚ᅒ࡞♟ࡊࡒ㛣ฌ⌦ࡊࡒᚃࠉⷾࢅὑὯࡊࠉ⣵⬂ࢅ ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ࡚ 7 ࠾ࡼ 14 㛣ᇰ㣬ࡊ࡙⏍࠻࡙ࡀࡒࢤࣞࢼ࣭ࢅᩐ࠻ࡒࠊᅒ୯ࡡࣁ ࣭ࡢᵾ‵೩ᕣࢅ♟ࡌࠊ
➠ 5 ⟿ ⣵⬂࿔ࡡ㐅⾔ 4.5.1 ⣵⬂࿔ฦᕱࡡን
SOD2 ࡡⓆ⌟ೳで࡞ࡻࡖ࡙ࠉ⣵⬂⑵る⬗ୖࡌࡾࡆ࡛᪺ࡼ࠾࡛ࡖࡒࠊࡐࡆ࡚ࠉࡆࡡ⣵⬂ ⑵る⬗ୖࡡⅴᅄࢅ᥀ࡾࡒࡴ࡞ࠉSOD2ᯜῤᚃ࡛ᯜῤ๑࡚⣵⬂࿔ࡡฦᕱࢅセ㍉᳠ゞࡊࡒࠊᑊᩐ ⑵る࡞࠵ࡾ⣵⬂ࢅBrdU (bromodeoxyuridine) ࡡྱ᭯ᇰᆀ࡚10 ฦᇰ㣬ࡊࠉࡐࡡᚃBrdU Flow Kit ࡞࡙⿼ᰶࡊࠉBrdU ࡡཱིࡽ㎰ࡲ㔖࡛DNA ྱ㔖ࢅflow cytometry ࡞ࡻࡽῼᏽࡌࡾࡆ࡛࡚⣵⬂࿔ ࡡฦᕱࢅㄢࡒࠊࡐࡡ⤎ᯕࠉ㔕⏍ᰬ࡛ SOD2 ᯜῤ๑ࡡ SOD2 ᮪௲◒ቪᰬࡢ⇆ᵕࡡ⣵⬂࿔ฦ ᕱࢅ♟ࡊࡒࠉSOD2 ᯜῤᚃࡢࠉG1ࡡ⣵⬂⩄⑵ຊࡊࠉS ࡡ⣵⬂⩄΅ᑛࡊࡒ(Fig. 4-11)ࠊ Fig. 4-11 細胞周期分布 各細胞BrdUの取り込み量とDNA含有量をフローサ イトメトリーで測定した。
4.6.2 ⇆ㄢᚃࡡ⣵⬂࿔ࡡ㐅⾔ ⣵⬂࿔ฦᕱࡡゆᯊ࠾ࡼࠉSOD2 ࡡⓆ⌟ೳで࡞ࡻࡖ࡙ࠉG1࠾ࡼ S ࡡ⛛⾔㐔ᘇࡌࡾྊ ⬗ᛮ♟ြࡈࡿࡒࠊࡐࡆ࡚ࠉSOD2 ᯜῤୖ࡚ᮇᙔ࡞G1࠾ࡼS ࡡ⛛⾔ࡡ㐔ᘇずࡼࡿࡾ࠾ ࠹࠾ࢅ᪺ࡼ࠾࡞ࡌࡾࡒࡴ࡞ࠉ᚜ᑚ⟮㔔ྙ㜴ᐐ࡚࠵ࡾnocodazol ࡚4 㛣ฌ⌦ࡊG2/M ࡞⣵ ⬂ࢅㄇᑙࡊࠉᇰᆀࢅὑὯᚃࠉ᭞࡞mimosine ࡚8 㛣ฌ⌦ࡊࠉG1࡞⇆ㄢࡈࡎࡒࠊࡐࡡᚃᇰᆀࢅ ὑὯࡊࠉ⣵⬂࿔ࢅ්㐅⾔ࡈࡎࡒ࡛ࡀࡡᵕᏄࢅCycle TESTTM
PLUS DNA Reagent Kit ࢅ⏕࠷࡙び
ᐳࡊࡒࠊࡐࡡ⤎ᯕࠉࡷࡢࡽSOD2 ᯜῤୖ࡚ࡢG1࠾ࡼS ࡡ⛛⾔ࡡ㐔ᘇびᐳࡈࡿࡒࠊࡱࡒࠉ ࡐࡡᚃࡡ⣵⬂࿔ࡡ㐅⾔ࢅびᐳࡊࡒ⤎ᯕࠉSࡡ㐅⾔⮤మࡵ㐔ᘇびᐳࡈࡿࡒ(Fig. 4-12)ࠊ Fig. 4-12 SOD2 ᯜῤࡡ⣵⬂࿔ࡡ㐔ᘇ (1) ⣵⬂ࢅG1࡞⇆ㄢᚃࠉⷾࢅὑὯࡊ࡙㝎ࡀࠉ⣵⬂࿔ࢅ㐅⾔ࡈࡎࡒࠊ2 㛣ࡇ࡛࡞⣵⬂ࢅᅂࡊࠉCycle TEST TMPLUS DNA Reagent Kitࢅ⏕࠷ࡒࣆ࣭ࣞࢦࢹ࣒ࢹ࣭࡚ࣛῼᏽࡊࡒࠊ
ࡊ࠾ࡊࡼࠉnocodazol-mimosine block ࡚ࡢᏰධ࡞G1࡞⇆ㄢࡈࡎࡾࡆ࡛࡚ࡀࡍࠉࡆࡡ⤎
ᯕࡡಘ㢏ᛮࡢ࠷࡛⩻࠻ࡼࡿࡒࠊࡊ࠾ࡊࠉࡆࡿ௧୕㛏㛣mimosine ࢅฌ⌦ࡌࡾ࡛ほ⣵⬂ኣࡂ
ࡾࡆ࡛ࡈࡿࡒࠊࡐࡆ࡚ࠉnocodazol ༟≺࡚⣵⬂ࢅ G2/M ࡞⇆ㄢࡊࠉࡐࡡᚃᇰᆀ࠾ࡼ
nocodazol ࢅ㝎ࡀࠉ⣵⬂࿔ࢅ㐅⾔ࡈࡎࡒࠊࡐࡡ⤎ᯕࠉnocodazol-mimosine block ࡛⇆ᵕࡡ⤎ᯕ ᚋࡼࡿࡒ(Fig. 4-13)ࠊ
Fig. 4-14 SOD2 ᯜῤࡡ⣵⬂࿔ࡡ㐔ᘇ (2)
⣵⬂ࢅG2/M ࡞⇆ㄢᚃࠉⷾࢅὑὯࡊ࡙㝎ࡀࠉ⣵⬂࿔ࢅ㐅⾔ࡈࡎࡒࠊ2 㛣ࡇ࡛࡞⣵⬂ࢅᅂࡊࠉCycle TEST
TMPLUS DNA
4.6.3 DNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹᶭᵋ
DNA 々⿿ᶭᵋ࡞రࡼ࠾ࡡ㝸ᐐ⏍ࡋࡒሔྙࠉDNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹὩᛮࡌࡾࠊࡆ ࡡᶭᵋࡢࠉձ DNA ಞᚗ࡞ᚪこ㛣ࡓࡄ DNA 々⿿ࢅೳでࡈࡎࡾࠊղ ࡵࡊࡐࡿྊ⬗ ሔྙࡢ࣎ࢹ࣭ࢨࢪࢅㄇᑙࡌࡾࡆ࡛࡞ࡻࡖ࡙ࢣࢿ࣑Ꮽᏽᛮࡡ⥌ᣚ࡞㛭ࡊ࡙࠷ࡾࠊ
SOD2 ᯜῤ࡞㉫ࡆࡾS ࡡ㐅⾔ࡡ㐔ᘇࠉDNA 々⿿ᶭᵋ࡞రࡼ࠾ࡡ㝸ᐐ㉫ࡆࡽࠉDNA 々 ⿿ࢲ࢘ࢴࢠ࣎ࣤࢹὩᛮࡈࡿࡒ⤎ᯕ࡚࠵ࡾྊ⬗ᛮࢅ⩻࠻ࠉDNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹᅄᏄ ࡡୌࡗ࡚࠵ࡾࠉChk-1 ࡡὩᛮ(Ser 345 ࡡࣛࣤ㓗)ࢅSOD2 ᯜῤ࡞᳠ฝࡊࡒࠊࡐࡡ⤎ᯕࠉSOD2
ᯜῤࡊ࡙ࡵࠉChk-1 ࡡὩᛮࡢධࡂ᳠ฝࡈࡿࡍ(over expose ࡊ࡙ࡵ࡛ࢆንࡢ᳠ฝࡈࡿ࠾ ࡖࡒ)ࠉS ࡡ㐅⾔ࡡ㐔ᘇࡡⅴᅄDNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹࡡὩᛮ࡞ࡻࡾࡵࡡ࡚ࡢ࠷ࡆ࡛ ♟ြࡈࡿࡒ(Fig. 4-14)ࠊ Fig. 4-14Chk-1 ࡡࣛࣤ㓗ࡡ᳠ฝ SOD2 ᮪௲◒ቪᰬ࡞ࢺ࢞ࢨࢦࢠࣛࣤࢅ῟ຊᚃࠉ1 ࠾ࡼ5 ┘ࡡ⣵⬂ࢅᅂࡊࠉSDS-PAGE ࡚ࢰࣤࣂࢠ㈹ࢅᒈ㛜ᚃࠉࣃࢹࣛ ࣤ㓗Chk1 ᢘమ࡚᳠ฝࡊࡒࠊpositive controlࡢࠉHU (10 mM) ࢅ1.5 hrฌ⌦ࡊࡒ㔕⏍ᰬࢅ⏕࠷ࡒࠊ
4.6.4 DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐ
SOD2 ᯜῤ࡞㉫ࡆࡾS 㐅⾔ࡡ㐔ᘇࡡⅴᅄ࡛ࡊ࡙ࠉࡵ࠹ୌࡗ⩻࠻ࡼࡿࡾࡡDNA 々⿿ࣆ࢚ ࣭ࢠࡡ㐅⾔㏷ᗐࡡୖ࡚࠵ࡾࠊ々⿿ࣆ࢚࣭ࢠࡡᏭᏽᛮୖࡌࡾ࡛ࠉ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐࡡ ୖ㉫ࡀࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊ
ࡐࡆ࡚ࠉ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐࢅῼᏽࡌࡾࡒࡴ࡞ࠉDNA fiber assay ࢅ⾔ࡖࡒࠊ(ࡆࡡassay ࡢࠉ
ᙔ◂✪ᐄ ⚽⏛࡞ࡻࡽ⾔ࢂࡿࡒࠊ) ࡱࡍࠉᵾㆉሲᇱ࡛ࡾCldUࠉIdU ࢅࡐࡿࡑࡿ▯㛣ࡍࡗDNA ࡞ཱིࡽ㎰ࡱࡎࠉDNA ࢅࢪࣚࢺ࢝ࣚࢪ୕࡚ఘ㛏ࡈࡎࡒᚃࠉࡐࡿࡑࡿࡡᵾㆉሲᇱᢘమ࡚ඞⰅ ࡊࠉCldUࠉIdU ཱིࡽ㎰ࡱࡿࡒDNA ࡡ㛏ࡈࢅῼᏽࡊࡒࠊࡐࡡ⤎ᯕࠉ㔕⏍ᰬ࡛セ㍉ࡊ࡙ࠉSOD2 ᮪௲◒ቪᰬࡢSOD2 ᯜῤ๑࡚ࡵ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐࡡୖびᐳࡈࡿࡒࠉSOD2 ᯜῤᚃ࡛ ࡡᕣࡢࡐࡿびᐳࡈࡿࡍࠉ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐࡡୖSOD2 ᯜῤ࡞㉫ࡆࡾ S 㐅⾔ ࡡ㐔ᘇࡡⅴᅄ࡚ࡢ࠷ࡆ࡛♟ြࡈࡿࡒࠊ
Fig. 4-15 DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ᗐ
⣵⬂࡞CldUࠉIdU ࢅཱིࡽ㎰ࡱࡎࡒᚃDNA ࢅఘ㛏ࡈࡎࠉᵾㆉሲᇱᢘమ࡚ⰅࡊࠉCldUࠉIdU ཱིࡽ㎰ࡱࡿࡒDNA ࡡ㛏 ࡈࢅῼᏽࡊࡒࠊRAD17-/-ࡢPositive control࡚࠵ࡾࠊ
Wild Type
SOD2-/-/hSOD2-HA DOX(-)
SOD2-/-/hSOD2-HA DOX(+)
mean± standard deviation (um/min) genotype CldU IdU 1.77 ± 0.79 1.47 ± 0.80 1.31 ± 0.52 0.62 ± 0.21 0.48 ± 0.19 0.46 ± 0.26
➠ 5 ❮ ⩻ᐳ
Ὡᛮ㓗⣪⛸ (ROS) ࡛ࠉࡐࡿࢅᾐཡࡌࡾᢘ㓗㓕⣪ࡢ⩹ࠉ♼⤊⑄ᝀࡷ⒬ࠉ⏍Ὡ⩞ៈࡡⓆ ࠉኣ⛸ኣᵕ⌟㇗࡞㛭ࡌࡾࡆ࡛ฦ࠾ࡽࠉROS ࡷᢘ㓗㓕⣪ࡡ⣵⬂හ࡞࠽ࡄࡾᙲ㝎ࠍ ࡞᪺ࡼ࠾࡞ࡽࡗࡗ࠵ࡾࠊࡆࡡࡻ࠹≟Ἓࡡ࠾࡚ࠉᙔ◂✪ᐄ࡚⢥ງⓏ࡞◂✪㐅ࡴࡼࡿ࡙࠷ࡾ RecQ ࣉࣛ࢜Ѹࢭࡡฝⰾ㓕ザ࣓࣌ࣞࢡ࡚࠵ࡾSgs1 ࡛Sod1 ࡡ㐿ఎᏄ㔔◒ቪᰬྙᠺ⮬ほ࡞ࡾ ࡆ࡛᪺ࡼ࠾࡛ࡖࡒ(Tong et al., 2001)ࠊࡐࡆ࡚ᙔ◂✪ᐄ࡚ࡢࠉᢘ㓗㓕⣪ࡡࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ ࡷ⣵⬂࿔㐅⾔ࡡᐞࢅゆᯊࡌࡾࣈࣞࢩ࢘ࢠࢹⓆ㊂ࡊࠉࡆࡡࣈࣞࢩ࢘ࢠࢹࡡୌ❻࡛ࡊ࡙ࠉᮇ ◂✪࡚ࡢDT40 SOD2 ᮪௲◒ቪᰬࢅష⿿ࡊࠉᢘ㓗㓕⣪SOD2 Ḗ࡞Ⓠ⏍ࡌࡾ㓗ࢪࢹࣝࢪ ࢣࢿ࣑Ꮽᏽᛮ࠽ࡻࡦ⣵⬂࿔ࡡ㐅⾔࡞ࡡࡻ࠹ᙫ㡢ࢅ࠻ࡾࡡ࠾ࠉゆᯊࢅ⾔ࡖࡒࠊᮇ◂✪࡚ࡢ SOD2 ᮪௲◒ቪᰬࢅ⏕࠷࡙ࠉSOD2 ࡡᯜῤ⣵⬂࿔ࡡ㐅⾔ࡷ࣎ࢹ࣭ࢨࢪ➴࡞ᙫ㡢ࢅ࠻ࡾࡆ ࡛ࢅ♟ࡌ⤎ᯕࢅᚋࡾࡆ࡛࡚ࡀࡒࠊᮇ❮࡚ࡢࠉࡆࡿࡼࡡ⤎ᯕࢅ୯ᚨ࡞⩻ᐳࡌࡾࠊ SOD2 ࡡⓆ⌟ೳで࡞ࡻࡖ࡙⤊Ⓩ࡞⾪⌟ᆵ㢟ⴥ࡞ࡾ SOD2 ᮪௲◒ቪᰬࡡᇰ㣬⣌࡞ࢺ࢞ࢨࢦࢠࣛࣤࢅ῟ຊࡊ࡙SOD2 ࡡⓆ⌟ࢅೳでࡈࡎࡾ࡛ࠉ⣵⬂ ⑵る⬗ࡢ⤊Ⓩ࡞ୖࡊࡒ(Fig. 4-2)ࠊࡱࡒࠉࢺ࢞ࢨࢦࢠࣛࣤᏋᅹୖ࡚1 ࠾ࡼ5 㛣ᇰ㣬ࡊࡒ ⣵⬂ࡡ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ࡞࠽ࡄࡾࢤࣞࢼ࣭ᙟᠺ⋙ࢅ゛ῼࡊࡒ⤎ᯕࠉࢤࣞࢼ࣭ᙟᠺ⋙ࡡ⤊Ⓩ ୖびᐳࡈࡿࡒ(Fig. 4-3)ࠊࡊ࠾ࡊࡼࠉ⮬ほ࠵ࡾ࠷ࡢ⣵⬂⑵るࡡᏰධೳで࡛࠷ࡖࡒࠉSod2 ࢿࢴࢠࢹ࣏ࢪ⏜ᮮ⣵⬂࡚びᐳࡈࡿࡾ⾪⌟ᆵ࡛セ㍉ࡌࡾ࡛ࠉSOD2 ᮪௲◒ቪᰬࡡ⾪⌟ᆵࡢ አ࡞ᘽ࠷ࡵࡡ࡚࠵ࡖࡒࠊSOD2ࡡⓆ⌟㔖ࡢ⤄⧂㛣࡚ኬࡀᕣ࠵ࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾࠉࢼ ࣠ࢹࣛࡡこ⤄⧂⏜ᮮࡡ⣵⬂ࡡ SOD2 Ⓠ⌟㔖ࢅセ㍉ࡊࡒሔྙࠉࣛࣤࣂ⌣ࡡⓆ⌟㔖ࡢᖲᆍ Ⓩ࡚࠵ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠽ࡽ(Kong et al., 2003)ࠉB ⣵⬂⏜ᮮ࡚࠵ࡾDT40⣵⬂ࡵRT-PCR ࡡ⤎ ᯕ࠾ࡼࠉSOD2 ࡡⓆ⌟㔖࠷࡛࠷࠹ࡆ࡛ࡢ⩻࠻࡞ࡂ࠷ࠊࡊࡒࡖ࡙ࠉDT40 ⣵⬂࡚ࡢSOD2 ࢪ࣭ࣂ࣭࢛࢞ࢨࢺࡡ㝎ཡ࡞ᚪこ࡚࠵ࡾ࡛࠷࠹ྊ⬗ᛮࡢ࠷ࠊࡳࡊࢀࠉSod2 ࢿࢴࢠࢹ࣏ ࢪ⏜ᮮࡡ♼⤊⣵⬂ࡢ㏳ᖏࡡᇰ㣬᮪௲࡚ࡢ⮬ほࡓࠉ㓗⣪ୖ࡚ࡢ⏍Ꮛྊ⬗࡚࠵ࡾ࡛࠷࠹ሒ࿈ࡷࠉ Sod2 ࢿࢴࢠࢹ࣏ࢪ㛣࡚ࡵࠉ㐿ఎⓏ⫴ᬊ࡞ࡻࡖ࡙ᖲᆍᑋ࡞ኬࡀᕣびᐳࡈࡿࡾࡆ࡛࠾ ࡼࠉ⎌⑧Ⓩࠉ㐿ఎⓏこᅄኬࡀ࠷࡛⩻࠻ࡼࡿࡾࠊᇰ㣬⣵⬂ࡢ㓗⣪ฦᅸ20 %ࡡ᮪௲࡚࠵ࡽࠉమ හ࡚ࡢ1ࠤ2 %ࡡ㓗⣪⃨ᗐ࡛⩻࠻ࡼࡿࡾࡆ࡛࠾ࡼࠉ㓗⣪⃨ᗐኬࡀࡂᙫ㡢ࡌࡾྊ⬗ᛮ࠵ࡾࠊࡱ ࡒࠉࢿࢴࢠࢹ࣏ࢪࡡ⾪⌟ᆵࡢ࡞࣐ࢹࢤࣤࢺࣛࡡᶭ⬗ኬࡀᙲࢅᯕࡒࡌ⤄⧂࡞౪Ꮛ ࡊ࡙࠷ࡾྊ⬗ᛮ㧏࠷࡛⩻࠻ࡼࡿࡾࠊ SOD2 ࡢ࣎ࢹ࣭ࢨࢪᢒโᶭᵋ࡞├㛭ࡊ࡙࠷ࡾࢂࡄ࡚ࡢ࠷ DT40 ࡡ࣐ࢹࢤࣤࢺࣛᑻᅹᆵࢲ࢛ࣝࢺ࢞ࢨࣤࠉTRX2 ࡡDT40 ᮪௲◒ቪᰬࡡゆᯊ࡞ࡻࡾ࡛ࠉ TRX2 ࡢࡐࡡᢘ㓗ష⏕࡛ࡢื࡞ࠉᢘ࣎ࢹ࣭ࢨࢪష⏕ࢅ᭯ࡌࡾBcl-xl ࢅࡊࠉ࣎ࢹ࣭ࢨ ࢪࢅᢒโࡌࡾᶭ⬗ࢅ᭯ࡌࡾࡆ࡛♟ြࡈࡿࡒ(TRX2 ࡢMAPKK ࡡ1 ⛸࡚࠵ࡾ ASK-1( apoptosis signal-regulating kinase-1)࡛⤎ྙࡊࠉࡐࡡᶭ⬗ࢅᢒโࡊ࡙࠷ࡾ࡛࠷࠹ሒ࿈࠵ࡾ) (Tanaka et al. 2002ࠉ Wang et al., 2006)ࠊ࡚ࡢ⇆ᵕ࡞࣐ࢹࢤࣤࢺ࡚ࣛᶭ⬗ࡌࡾᢘ㓗㓕⣪࡚࠵ࡾSOD2 ࡵࠉTRX2 ࡡ ࡻ࠹ᢘ࣎ࢹ࣭ࢨࢪష⏕࠵ࡾࡡࡓࢀ࠹࠾ࠊ SOD2 Ⓠ⌟ೳで├ᚃࡢࠉ⣵⬂⑵る⬗࠽ࡻࡦࣂࣚࢤ࣭ࢹវུᛮ࡞࡛ࢆንࡢびᐳࡈࡿࡍࠉᩐ ࠾ࡄ࡙⾪⌟ᆵᚆࠍ࡞㢟ⴥ࡞ࡖࡒࡆ࡛ࠉSOD2 ࡢࣂࣚࢤ࣭ࢹ࡞ࡻࡖ࡙ㄇᑙࡈࡿࡾ࣎ࢹ࣭ࢨ ࢪࡢᢒโࡌࡾࡵࡡࡡ(Fig. 4-4ࠉ4-5)ࠉH2O2ࡷDNA ഭᐐ࡚࠵ࡾ࢙ࢹ࣎ࢨࢺ࡞ࡻࡖ࡙ㄇᑙࡈࡿࡾ ࣎ࢹ࣭ࢨࢪࡢᢒโࡊ࠾ࡖࡒࡆ࡛࠾ࡼ(Fig. 4-5)ࠉSOD2TRX2 ࡡࡻ࠹࡞ࠉᢘ㓗ష⏕࡛ื࡞ ࣎ࢹ࣭ࢨࢪࢅᢒโࡌࡾᶭ⬗ࢅ᭯ࡌࡾྊ⬗ᛮࡢᮇ◂✪ࡡ⤎ᯕ࠾ࡼࠉ࠷࡛⩻࠻ࡼࡿࡾࠊࡊ࠾ࡊ ࡼSOD2 ࢨࢹࢠ࣑ࣞ c ࡡ࣐ࢹࢤࣤࢺࣛ࠾ࡼࡡᨲฝࢅᢒโࡌࡾ(Fujimura et al., 1999)ࠉ࣎ ࢹ࣭ࢨࢪ࡞ᑊࡊࠉᢒโⓏ࡞ᶭ⬗ࡌࡾ࡛࠷ࡖࡒሒ࿈ࡵᏋᅹࡌࡾࡆ࡛ࠉᮇ◂✪࡞࠽࠷࡙ SOD2 ᮪௲
◒ቪᰬ࡚ࡢhSOD2 Ⓠ⌟࡞࠽࠷࡙ࡵ࣎ࢹ࣭ࢨࢪஸ㐅ࡊ࡙࠷ࡾࡆ࡛ (ࢺ࢞ࢨࢦࢠࣛࣤ↋῟ ຊ࡚ࡵ SOD2 ᮪௲◒ቪᰬࡡ⣵⬂⑵る⬗㔕⏍ᰬ࡞セ࡙ୖࡊ࡙࠷ࡾⅴᅄ࡛⩻࠻ࡼࡿࡾࠊࣃࢹ SOD2 ࡛ࢼ࣠ࢹࣛ SOD2 ࡢ࣐ࢹࢤࣤࢺࣛ⛛⾔ࢨࢡࢻࣜࡡ┞⇆ᛮ࠷ࡒࡴ࡞Ᏸධ࡞ࡢ┞ࡊ ࡀࡿ࡙࠷࠷ྊ⬗ᛮ㧏࠷) ࠾ࡼ(Fig. 4-6)ࠉ࣐ࢹࢤࣤࢺࣛහࡡ㐛ࢪ࣭ࣂ࣭࢛࢞ࢨࢺ㛭 ࡌࡾ࣎ࢹ࣭ࢨࢪὩᛮ⤊㊨Ꮛᅹࡊࠉࡆࡡ⤊㊨࡞㛭ࡊ࡙࠷ࡾྊ⬗ᛮࡢᤖ࡙ࡀࡿ࠷ࠊ SOD2 ࡢࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ࡞㢟ⴥᙫ㡢ࢅ→ࡈ࠷ SOD2 రࡼ࠾ࡡᙟ࡚ࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ࡞㛭ࡊ࡙࠷ࡾ࠾࠹࠾᳠ゞࡌࡾࡒࡴ࡞ࠉSOD2 ᮪௲ ◒ቪᰬ࠾ࡼSOD2 ࡡⓆ⌟ࢅೳでᚃ࡞ࠉDNA ഭᐐ࡞ᑊࡌࡾវུᛮࢅびᐳࡊࡒ࡛ࡆࢀࠉ㔕⏍ᰬ࡛ ⇆⛤ᗐࡡវུᛮࢅ♟ࡊࡒ(Fig. 4-8)ࠊࡱࡒࠉDNA ഭᐐࡡᣞᵾ࡛ࡊ࡙ࠉDNA ን␏ム㥺࡞ࡵ⏕ ࡈࡿ࡙࠷ࡾ╀┶Ⰵฦమஹᥦࢅࢺ࢞ࢨࢦࢠࣛࣤ῟ຊᚃ 5 ┘ࡡ SOD2 ᮪௲◒ቪᰬࢅ⏕࠷࡙ῼ ᏽࡊࡒ⤎ᯕࠉࡐࡡ㢎ᗐࡢ㔕⏍ᰬ࡛⇆⛤ᗐ࡚࠵ࡾࡆ࡛ฦ࠾ࡖࡒ(Fig. 4-9)ࠊࡆࡡ⤎ᯕ࠾ࡼࠉ SOD2ࠉࡱࡒࡢSOD2 Ḗ࡞ࡻࡖ࡙Ⓠ⏍ࡌࡾ࡛⩻࠻ࡼࡿࡾ㓗ࢪࢹࣝࢪࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ࡞㢟 ⴥᙫ㡢ࢅ࠻࡙࠷ࡾྊ⬗ᛮࡢ࠷࡛⩻࠻ࡼࡿࡒࠊୌ᪁ࠉᙔ◂✪ᐄࡡ୕࡞ࡻࡖ࡙ష⿿ࡈࡿࡒ DT40 SOD1 ᮪௲◒ቪᰬࡢࠉSOD1 ࡡⓆ⌟㔖΅ᑛ࡛භ࡞SCE 㢎ᗐ୕᪴ࡊࠉ᭞࡞Ⓠ⌟ೳでࡌࡾ
࡛Ⰵమ␏ᖏびᐳࡈࡿࡒ偆୕ ᮅⓆ⾪ࢸ࣭ࢰ假ࠊࡱࡒࠉฝⰾ㓕ザ࡞࠽࠷࡙ࡵ sod1 ◒ቪᰬࡡ
unequal-SCR (sister chromatid recombination) ୕᪴ࡌࡾࡆ࡛᪺ࡼ࠾࡞ࡖ࡙࠷ࡾࠉsod2 ◒ቪ
ᰬ࡚ࡢ㔕⏍ᰬ࡛⇆⛤ᗐࡓࡖࡒ偆㧏⏛༛ㄵ假ࠊ ㏣࡞ࠉSod2 ࢿࢴࢠࢹ⏜ᮮ⦼⥌ⰾ⣵⬂ (MEF) ࡢ
㢟ⴥⰅమ␏ᖏ⏍ࡋ࡙࠷ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Samper et al., 2003)ࠉSod1 ࢿࢴࢠࢹ ࣏ࢪ࡚ࡢࡐࡡࡻ࠹ሒ࿈ࡢࡂࠉධమⓏ⾪⌟ᆵࡵ Sod2 ࡞セ࡙ᘽ࠷ࠊࡆࡡࡻ࠹࡞ࠉSOD1 ඁన࡞ᶭ⬗ࡊ࡙࠷ࡾ⛸࡛ SOD2 ඁన࡞ᶭ⬗ࡊ࡙࠷ࡾ⛸Ꮛᅹࡌࡾ࡛⩻࠻ࡼࡿࡾࠊᮇ◂✪࠽ ࡻࡦࠉDT40 SOD1 ᮪௲◒ቪᰬࡡ⤎ᯕ࠾ࡼDT40 ⣵⬂࡚ࡢࠉࢣࢿ࣑Ꮽᏽᛮ⥌ᣚ࡞㛭ࡌࡾࡡࡢ
࡞SOD1 ࡚࠵ࡽࠉSOD2 ࡡ㈁⊡ᗐࡢセ㍉Ⓩ࠷࡛⩻࠻ࡼࡿࡾࠊ SOD2 ࡢ⣵⬂࿔ࡡ㐅⾔࡞㛭ࡌࡾ SOD2 Ⓠ⌟ೳで࡞ࡻࡾ⣵⬂⑵る⬗ࡡୖࡡⅴᅄࢅ᥀ࡾࡒࡴࠉ⣵⬂࿔ࡡฦᕱࢅゆᯊࡊࡒ࡛ࡆࢀࠉ G1 ࡡ⣵⬂ᩐ⑵ຊࡊ࡙࠷ࡒ(Fig. 4-11)ࠊ᭞࡞ࠉG1 ࡞⇆ㄢᚃࠉ⣵⬂࿔ࢅ㐅⾔ࡈࡎࡒ࡛ࡆࢀࠉ G1 ࠾ࡼS ࡡ㐅⾔࡞㐔ࡿびᐳࡈࡿࡒ(Fig. 4-12ࠉ4-13)ࠊࡆࡡ⤎ᯕࡢࠉฝⰾ㓕ザ࡞࠽࠷࡙sod2 ◒ቪᰬࡡ G1 ᘇ㛏ࡈࡿࡾࡆ࡛ࠉࡱࡒࠉHeLa ⣵⬂࡞࠽࠷࡙ࠉ⣵⬂⑵るࡊࡒ⤎ᯕ㏳ᖏࡼ⑵ るೳでࡊ࡙G1 ࡞✒ࡌࡾࡻ࠹᮪௲࡚ࡵࠉSOD2 ࢅ㐛Ⓠ⌟ࡈࡎࡾ࡛⣵⬂⑵る⬗ࢅಕࡗࡆ ࡛࡚ࡀࡾ࡛࠷࠹ሒ࿈࡛➚ྙࡌࡾࡵࡡ࡚࠵ࡾ(Sarsour et al,. 2005)ࠊ ࡱࡒࠉSOD2 Ⓠ⌟ೳで࡞ࡻࡖ࡙S ࡡ㐅⾔⮤మ࡞ࡵ㐔ᘇびᐳࡈࡿࡒ(Fig. 4-12ࠉ4-13)ࠊࡆࡡ⤎ ᯕ࠾ࡼࠉ
ձ SOD2 Ⓠ⌟ೳで࡞ࡻࡖ࡙⏍ࡋࡒ㓗ࢪࢹࣝࢪDNA ࡞SCE ࢅ㉫ࡆࡈࡎ࠷ࡻ࠹ഭᐐࢅຊ
࠻ࡒࠉࡱࡒࡢDNA 々⿿࡞ᙫ㡢࠵ࡽࠉDNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹⓆິࡊࡒࠊ
ղ SOD2 Ⓠ⌟ೳで࡞ࡻࡖ࡙⏍ࡋࡒ㓗ࢪࢹࣝࢪ࡞ࡻࡖ࡙ࠉDNA 々⿿ࡡ㐅⾔㏷ᗐ⮤మୖࡊࡒࠊ ճ SOD2 Ⓠ⌟ೳで࡞ࡻࡖ࡙ࠉరࡼ࠾ࡡ⌦⏜࡚⣵⬂࿔ࢅ㐅⾔ࡈࡎࡾࢦࢠࣛࣤࡷ CDK (cyclin dependent kinase) ࡡὩ (ࢦࢠࣛࣤࡡ1 ࡗ࡚࠵ࡾcyclin D1 ࡢ㓗ࢪࢹࣝࢪ࡞ࡻࡖ࡙Ⓠ⌟㔖 ΅ᑛࡌࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾ)㉫ࡆࡖ࡙ࡊࡱ࠷ࠉ々⿿㛜╂㉫ࡆࡽ࡞ࡂࡂࡖ࡙࠷ࡾࠊ
௧୕ࡡ௫ㄕ⩻࠻ࡼࡿࡒࠊࡊ࠾ࡊࡼࠉSOD2 Ⓠ⌟ೳでᚃ࡞DNA 々⿿ࢲ࢘ࢴࢠ࣎ࣤࢹࡡ Ὡᛮ࡞㛭ࡌࡾCHK-1 ࡡSer345 ࡡࣛࣤ㓗びᐳࡈࡿࡾࡆ࡛ࡢࡂ(Fig. 4-14)ࠉձࡡ௫ㄕࢅ❟ チࡌࡾࡻ࠹チᣈࡢᚋࡼࡿ࠾ࡖࡒࠊࡱࡒࠉDNA fiber Ἢ࡞ࡻࡖ࡙DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㏷ ᗐࢅῼᏽࡊࡒ࡛ࡆࢀࠉSOD2 ࡡⓆ⌟ࢅೳでࡈࡎ࡙ࡵࠉ࡛ࢆᕣࡢびᐳࡈࡿ࠾ࡖࡒ(Fig. 4-15)ࠊ ࡐࡡࡒࡴࠉղࡡ௫ㄕࡵྫྷᏽࡈࡿࡾࠊ᭩ᚃ࡞ࠉճࡡ௫ㄕ࡚࠵ࡾࠉࡆࡿࢅチ᪺ࡌࡾࡆ࡛ࡢᅏ㞬࡚ࡢ
࠵ࡾࠉG1࠾ࡼ S ࡡ⛛⾔ࡵ㐔ᘇࡊ࡙࠷ࡾࡆ࡛࠾ࡼࠉG1/S ⛛⾔࡞㛭ࡌࡾࢦࢠࣛࣤࡷ
CDK (cyclin AࠉDࠉE ࡷCdk2ࠉ4ࠉ6)ࡡὩᛮ┭ࡅࡼࡿ࡙࠷ࡾྊ⬗ᛮ㧏࠷࡛⩻࠻ࡼࡿࡾࠊSOD2 ࡡᯜῤDNA 々⿿㜴ᐐ࡚࠵ࡾHU ࡷaphidicoline ࡞ᑊࡊ࡙⪇ᛮࢅ♟ࡌ⌦⏜ࡵ(Fig. 4-10)ࠉ々⿿ 㛜╂㉫ࡀ࡞ࡂࡂࡽࠉDNA 々⿿ࣆ࢚࣭ࢠᙟᠺࡈࡿࡾᩐᑛࡂࡾࡆ࡛࡚ࠉHU ࡷ aphidicoline ࡞ࡻࡾDNA 々⿿ࣆ࢚࣭ࢠࡡᓻቪ㉫ࡀ࡞ࡂࡂࡖࡒࡒࡴ࡚ࡢ࠷࠾࡛᥆ῼ࡚ࡀࡾࠊ ࡱࡒ᭩㎾ࠉHeLa ⣵⬂࡞ࠉᢘ㓗∸㈹࡚࠵ࡽࡱࡒ㓗ಀ㐅∸㈹࡚ࡵ࠵ࡾ N-acetyl cysteine ࢅฌ⌦
ࡊࡒ࡛ࡆࢀࠉࢪ࣭ࣂ࣭࢛࢞ࢨࢺࡡ⑵ຊ࡛භ࡞G1࡞ೳでࡊࠉࡐࡡⅴᅄcyclin D1ࡡ΅ᑛ࡞ࡻࡾ
ࡵࡡ࡚࠵ࡾࡆ࡛ሒ࿈ࡈࡿࡒ(Menon et al., 2007)ࠊࡈࡼ࡞ࠉࡆࡡcyclin D1 ࡢࠉH2O2ๆ⃥࡞ࡻࡖ࡙
ࡐࡡฦゆᢒโࡈࡿࡾࡆ࡛ࠉ⏍Ꮛಀ㐅ᅄᏄ AKT ࡵ H2O2ๆ⃥࡞ࡻࡖ࡙Ὡᛮࡈࡿࡾࡆ࡛▩ࡼ
ࡿ࡙࠷ࡾ(Murata et al., 2003)ࠊࡆࡡAKT ࡢࠉcyclin D1ࡡฦゆࢅಀ㐅ࡌࡾglycogen synthase kinase-3 ș࡛ࡣࡿࡾ࢞ࢻ࣭ࢭࢅᢒโࡌࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾ(Diehl et al., 1998)ࠊࡱࡒࠉࡆࡡcyclin D1 ࡷ
AKT ὩᛮࡌࡾG1࡞ࡢࠉSOD2 ࡡὩᛮ᭩ࡵ㧏ࡂࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ(Oberley et al.,
1995)ࠊ௧୕ࡡࡆ࡛࠾ࡼSOD2 ࢪ࣭ࣂ࣭࢛࢞ࢨࢺࡡ㝎ཡࡓࡄ࡚ࡂࠉH2O2⏍ᠺࢅࡊ࡙ࠉG1 ࠾ࡼS ࡡ⛛⾔ࡷS ࡡ㐅⾔ࢅಀ㐅ࡊ࡙࠷ࡾࡆ࡛⩻࠻ࡼࡿࡾࠊࡗࡱࡽࠉᮇ◂✪࡚SOD2 ࡡ ᯜῤㄇⓆࡊࡒ⾪⌟ᆵࡢࠉ㓗ࢪࢹࣝࢪࢅⓆ⏍ࡓࡄ࡚ࡢࡂࠉH2O2ࡡ΅ᑛࠉcyclin D1 ࡡὩᛮ ࢅ΅ᘽࡈࡎࡒࡒࡴ࡚࠵ࡾྊ⬗ᛮ⩻࠻ࡼࡿࡾࠊࡆࡡ௫ㄕࢅチ᪺ࡌࡾࡒࡴ࡞ࡢࠉSOD2 ᯜῤ H2O2ࢅ΅ᑛࡈࡎࠉcyclin D1 ࡡⓆ⌟㔖ୖࢅᘤࡀ㉫ࡆࡌ࠾ࢅ᳠チࡊࡄࡿࡣࡼ࠷ࠉよᛍ ࡼࢼ࣠ࢹࣛࡡcyclin D1 ࢅヾㆉࡌࡾᢘమᏋᅹࡎࡍࠉ⌟Ⅴ࡚ࡢDT40 ࡡ⣌࡚ࡡチ᪺ࡢᅏ㞬࡚ ࠵ࡾࠊࡊ࠾ࡊࡼࠉᮇ◂✪࡚ష⿿ࡊࡒSOD2᮪௲◒ቪᰬࡡ⾪⌟ᆵࡡ㧏➴┷ᰶ⏍∸⏜ᮮSOD2 ◒ቪᰬࡻࡽࡵ࣏ࣜࢺ࡚࠵ࡾࡆ࡛ᖶ࠷ࡊࠉᮇ◂✪࡞ࡻࡽSOD2 ࡞ࡻࡾH2O2⏍ᠺ⣵⬂⑵るࢅ ಀ㐅ࡌࡾྊ⬗ᛮิࡴ࡙♟ࡈࡿࡒࠊ
᭩ᚃ࡞
ᮇ◂✪࡚ࡢࠉSOD2 ࢅtet-off system ࡞ࡻࡖ࡙ᯜῤࡈࡎࡾࡆ࡛࡚ࠉSOD2 Ḗ࡞ࡻࡾ⣵⬂࿔㐅 ⾔ࡡᙫ㡢々ᩐࡡ⾪⌟ᆵࢅᚋࡾࡆ࡛࡚ࡀࡒࠊࡊ࠾ࡊࡼࠉᙔิࡡ┘Ⓩ࡚࠵ࡖࡒ SOD2 ᶭ⬗Ḗࢣࢿ࣑Ꮽᏽ⥌ᣚ࡞→ࡌᙫ㡢ࢅ᪺☔࡞ࡌࡾࡆ࡛ࡢ࡚ࡀ࠾ࡖࡒࠊ⌟ᅹࠉᙔ◂✪ᐄ࡚ SOD1ࠉPRDX1 ᮪௲◒ቪᰬࢅ⏕࠷ࡒゆᯊ⾔ࢂࡿ࡙࠽ࡽࠉᚃࠉࡆࡡࣈࣞࢩ࢘ࢠࢹࡡ㐅⾔ࡡⓆᒈ ࢅᙽࡌࡾ࡛࡛ࡵ࡞ࠉSOD2 ࡡࢣࢿ࣑Ꮽᏽᛮ⥌ᣚࡷ⣵⬂ᶭ⬗ࡡ⥌ᣚ࡞࠽ࡄࡾᙲ᪺☔࡞ࡖ࡙ ࠷ࡂࡆ࡛ࢅᙽࡌࡾࠊ
➠ 6 ❮ ᐁ㥺ᮞᩩ࠽ࡻࡦ᪁Ἢ
➠ 1 ⟿ ⣵⬂ᇰ㣬 1.1 ⏕ᰬ ᮇ◂✪࡚⏕ࡊࡒDT40 ⣵⬂ᰬࢅ௧ୖ࡞♟ࡊࡒࠊ Wild-type SOD2 -/-/hSOD2-HA (neo/ecogpt/hyg) 1.2 ᇰᆀ→ࡦムⷾ (1) RPMI ᇰᆀ RPMI#1640 ᇰᆀ (ラ⿿ⷾ) 10.2 gࠉHEPES (⇆ொᏕ◂✪ᡜ) 2.4 gࠉࣅࣜࣄࣤ 㓗ࢻࢹ࣑ࣛ (WAKO) 0.1 g ࢅ⬲࢛ࣤラ 1 L ࡞⁈ゆࡊࠉ㧏ᅸⵠポ⁓Ⳟ (121Υ,20 min) ࡊ࡙ 4Υ࡚ಕᏋࡊࡒࠊ⏕࡞ࡢ 10 % NaHCO3࡚ pH 7.2 ࡞ࡾ ࡻ࠹࡞ㄢᩒࡊࠉࡈࡼ࡞᭩⤂⃨ᗐ 2 mM L(+)-ࢡࣜࢰ࣐ࣤ (WAKO)ࠉ 10 %㟸ິ ῥࡲࡡ∭⫶ௗ⾉ΰ࡛ 1 %㟸ິῥࡲࡡࢼ࣠ࢹࣛ⾉ΰࠉ0.1 g/L ◪㓗࢜ࢻ࣏ࢨࣤ (᪺⿿ⳣ) ࡞ࡾࡻ࠹࡞ᇰᆀࢅㄢᩒࡊࡒࠊ (2) 10 % NaHCO3 10 % NaHCO3 (WAKO) 10 g ࢅ⬲࢛ࣤラ 100 mL ࡞ᠩ⃦ࡊ㧏ᅸⵠポ⁓Ⳟࡊࡒࠊ (3) L(+)-ࢡࣜࢰ࣐ࣤ 2.94 g ࡡ L(+)-ࢡࣜࢰ࣐ࣤ (WAKO) ࢅ⏍⌦㣏ሲラ࡞⁈࠾ࡊࠉࢀ㐛⁓Ⳟࡊࡒࠊ (4)∭⫶ௗ⾉ΰ (fetal calf serum ; FCS)Flow ♣⿿ࡱࡒࡢ Bioserum ♣⿿ࡡࡵࡡࢅ 56Υ࡚ 30 min 㟸ິࡊࡒᚃࠉ4Υ࡚ಕ Ꮛࡊࡒࠊ
(5) ࢼ࣠ࢹࣛ⾉ΰ (chicken serum ; CS)
Sigma ♣⿿ࡡࡵࡡࢅ FCS ࡛⇆ᵕ࡞㟸ິࡊࠉ4Υ࡚ಕᏋࡊࡒࠊ (6) PBS (phosphate-buffered saline)
NaCl (WAKO) 8.0 gࠉCaCl2 (WAKO) 0.2 gࠉNa2HPO4࣬12H2O (WAKO) 2.9 gࠉ
KH2PO4 (WAKO) 0.2 g ࢅ⬲࢛ࣤラ 1 L ࡞⼝ゆࡊࡒࠊ (7) ࣒ࢲࣜࢬ࣭ࣜࣞࢪᇰᆀ 216 mL MilliQ ラ࡛ 7.5 g ࣒ࢲࣜࢬ࣭ࣜࣞࢪ (Fluka) ࢅࡐࡿࡑࡿ 500 mL ࡡ࣒ ࢸ࣑⎴࡞ථࡿࠉ㧏ᅸⵠポ⁓Ⳟࡊࡒࠊࡐࡿࡑࡿ⇍࠷࠹ࡔ࡞ࠉMilliQ ラࢅ࣒ࢲ ࣜࢬ࣭ࣜࣞࢪࡡථࡖࡒ⎴࡞ຊ࠻ࠉ⣪᪡ࡂⰃࡂ᧘ᢶࡊࡒࠊ50Υ⛤ᗐࡱ̶࡚ࡴࡒࡼ 2 DMEM/F-12 ᇰᆀ(Gibco) ࢅ 216 mL ຊ࠻ࠉ4Υ࡚ୌᬄ᧘ᢶࡊࡒࠊ⩛ 75 mL 㟸 ິῥࡲ FCS ࡛ 7.5 mL 㟸ິῥࡲ CS ࢅຊ࠻ࠉ4Υ࡚ಕᏋࡊࡒࠊ (8) 2DMEM/F-12 ᇰᆀ
1 L ฦࡡ 2DMEM/F-12 (Gibco)࠽ࡻࡦࠉNaHCO3 (WAKO) 1.2 g ࢅ 500 mL ࡡ⬲
࢛ࣤラ࡞⼝ゆࡊࠉ㌱㐛⁓Ⳟࡊࡒᚃࠉ4Υ࡚ಕᏋࡊࡒࠊ ➠ 2 ⟿ ࢰ࣭ࢣࢴࢷࣤࢡ࣊ࢠࢰ࣭ࡡషᠺ 2.1 DT40 ⣵⬂࠾ࡼࡡࢣࢿ࣑ DNA ࡡฝ 2.1.1 ᮞᩩ࠽ࡻࡦムⷾ (1) Easy-DNATM kit (invitrogen) (2) PBS (3) ethanol (WAKO) (4) chloroform (ࢻ࢜ࣚ)
2.1.2 ᐁ㥺᪁Ἢ ⣑ 1107 ࡡ⣵⬂ࢅᅂࡊࠉ200 ȣL ࡡ PBS ࡞ᠩ⃦ࡊࡒࠊࡐࡡᚃࠉ350 ȣL ࡡ solution A ࢅຊ࠻ࠉ65 Υ࡚ 10 ฦ㐛⇍ࡊࡒࠊࡐࡡᚃࠉ150 ȣL ࡡ solution B ࡛ 500 ȣL ࡡ chlolroform ࢅຊ࠻ࠉ᧘ᢶࡊࠉ14,000 rpm ࡚ 20 minࠉ㐪ᚨࡊࡒࠊ⏍ᠺ ࡊࡒ୕ΰ 500 ȣL ࡞ 100% ethanol ࢅ 800 ȣL ຊ࠻ࠉ30 minࠉレ୕࡚㟴⨠ࡊࡒࠊ 14,000 rpm ࡚ 15 min 㐪ᚨࡊࠉỷ⃞ࡊࡒࢣࢿ࣑ DNA ࢅ 80% ethanol ࡚ὑὯࡊࡒࠊ ࢣࢿ࣑ DNA ࢅஜ⇩ࡈࡎࡒᚃࠉRNase ථࡽࡡ 100 ȣL TE ࡞⁈ゆࡈࡎࡒࠊ
2.2 PCR ࡞ࡻࡾ chicken SOD2 ࡡ genomic DNA ࢠ࣭ࣞࢼࣤࢡ 2.2.1 ᮞᩩ࠽ࡻࡦムⷾ
(1) LA Taq PCR Kit ; 10LA PCR bufferࠉ25 mM MgCl2ࠉ2.5 mM dNTPࠉLA Taq
polymerase (TaKaRa) Advantage GC2-PCR Kit ; GC-Melt (2) ⏕ࡊࡒࣈ࣏ࣚ-
Primer name Oligonucleotide
ch-SOD2 fw2 AAGCAGGGCTGCGCTGGTAGCACCG
ch-SOD2 rv2 TCCATCTACTTTCTGCAAGATTCATATCTTGAAG
(3) PCR ࣈࣞࢡ࣑ࣚ
94 Υ 2 minࠉ(94 Υ 30 secࠉ58Υ 30 secࠉ72Υ 6 min )35ࠉ72Υ 5 minࠉ12Υ forever
2.2 ᐁ㥺᪁Ἢ
ࢸ࣭ࢰ࣭࣊ࢪ୕ࡡࢼ࣠ࢹࣛࢣࢿ࣑㒼า࡞ᇱࡘࡀࠉgene-specific primer ࢅ⏕࠷࡙ DT40 ࢣࢿ࣑ DNA ࢅ㗢ᆵ࡛ࡊࠉlonger-PCR ࢅ⾔ࡖࡒࠊ࣭࢝ࣞࢪࢣࣜ࡞㞹ポὃ ິࡊࠉ⣑ 6 kbp ࡡࣁࣤࢺࢅᅂࡊ࡙ࠉpGEM-T easy vector (Promega) ࡞⤄ࡲ㎰ࡲࠉ ࣈࣚࢪ࣐ࢺ DNA ࢅㄢ⿿ࡊࠉሲᇱ㒼าࢅ☔ヾࡊࡒࠊ 2.3 ⷾ⪇ᛮ࣏࣭࣭࢜㐿ఎᏄථ⏕ BamHϨsite ࡡᑙථ 2.3.1 ᮞᩩ࠽ࡻࡦムⷾ (1) LA Taq PCR Kit (⇆୕) (2) ⏕ࡊࡒ primer L arm ⏕
Primer name Oligonucleotide
ch-SOD2 fw2 AAGCAGGGCTGCGCTGGTAGCACCG
SOD2 rv41016 BamH1
GGATCCCCTGAGGATGCAGAAGATAAATAAATAG
R arm ⏕
Primer name Oligonucleotide
SOD2fw4258 BamH1
GGATCCGCTTATGAGTGCTCATTGGG
(3) PCR ࣈࣞࢡ࣑ࣚ L armࠉR arm ⏕භ࡞ࠉ
94 Υ 2 minࠉ(94 Υ 30 secࠉ56Υ 30 secࠉ72Υ 3 min )30ࠉ72Υ 10 min
2.3.2 ᐁ㥺᪁Ἢ
2,2 ࡚షᠺࡊࡒ vector ࢅ template ࡛ࡊ࡙ࡐࡿࡑࡿ୕エࡡ primer ࢅ⏕࠷࡙ PCR ࢅ ⾔࠷ࠉᚋࡼࡿࡒ band ࢅᅂࡊ࡙ࠉpGEM-T easy vector (Promega) ࡞⤄ࡲ㎰ࡲࠉࣈ ࣚࢪ࣐ࢺ DNA ࢅㄢ⟿ࡊ࡙ࠉࢨ࣭ࢠ࢙ࣤࢪゆᯊࢅ⾔ࡖࡒࠊᚋࡼࡿࡒ偐ࡗࡡࣈࣚࢪ ࣐ࢺ DNA ࢅࠉࡐࡿࡑࡿ BamHϨ࡛ NdeϨ࡚ฌ⌦ࡊࠉ୦⩽ࢅ ligation ࡈࡎࡒࠊ
2.4 ࢰ࣭ࢣࢴࢷࣤࢡ࣊ࢠࢰѸࡡషᠺ 2.4.1 ᮞᩩ࠽ࡻࡦムⷾ
(1) 2.3 ࡚షᠺࡊࡒ vector
(2) ࣏࣭࣭࢜ (ࢼ࣠ࢹࣛș-actin promoter ୖὮ࡞ histidinolࠉblastidinࠉpuromycinࠉ ecogptࠉhygromycinࠉneomycinࠉbleomycin ⪇ᛮ㐿ఎᏄࢅ⤄ࡲ㎰ࢆࡓࡵࡡ)
2.5 chicken SOD2 ࢰ࣭ࢣࢴࢷࣤࢡ࣊ࢠࢰ࣭ࡡషᠺ
2.6 DNA ࢨ࣭ࢠ࢙ࣤࢪゆᯊ
DNA ࡡሲᇱ㒼าࢅ☔ヾࡌࡾࡒࡴࠉSanger ࡼࡡ dideoxy Ἢ࡞ᚉ࠷ࠉ௧ୖࡡᡥ㡨
࡚ DNA ࢨѸࢠ࢙ࣤࢪࢅ⾔ࡖࡒࠊTaq DyeDeoxyTM
Terminator Cycle Sequencing Kit ࢅ⏕ ࠷ ࡙⾔ ࡖࡒ ࠊ ࣈ ࣐ࣝ ࢴ ࢠࢪ ࡛ ࡊ ࡙ࠉ5 TACS bufferࠉdNTP mixtureࠉ
DyeDeoxyTM
Terminatorsࠉ࠽ࡻࡦ Ampli Taq ࢅΊࡊࠉࡆࡿ࡞㗢ᆵ DNA 0.25 ȣgࠉ primer 1.6 Pmol ࢅຊ࠻࡙ධ㔖ࢅ 10 ȣL ࡛ࡊࠉThemal Cycler ࡚௧ୖࡡཬᚺࢅ⾔ࡖ ࡒࠊཬᚺ⤂ᚃࠉethanol ỷ⃞࡞ࡻࡽ DNA ࢅᅂࠉloading buffer (TSR ; template suppression reagent) ࡞⁈ゆࡊࡒࠊABI PRISMR 310 Genetic Analyzer (Applied Biosystem) ࡞ࢦࣤࣈࣜࢅࢬࢴࢹࡊ࡙ࠉᏋࡡࣈࣞࢡ࣑ࣚ࡞ࡻࡽ࢞ࣔࣅ࣭ࣚࣛ㞹 ポὃິࢅ⾔࠷ࠉ⺧කⰅ⣪ࢅㄖࡲཱིࡖࡒᚃࠉࢸ࣭ࢰゆᯊࢅ⾔ࡖࡒࠊ
➠ 3 ⟿ human SOD2 Tet-off system ᑙථ⏕࣊ࢠࢰѸࡡష⿿ 3.1 HeLa ⣵⬂ࡡ total RNA ࡡฝ
3.1.1 ᮞᩩ࠽ࡻࡦムⷾ
(1) TRIzol Reagent (Gibco BLK) 3.1.2 ᐁ㥺᪁Ἢ
レ̶ PBS ࡚ὑὯࡊࡒ 5ࠤ10106
ࡡ⣵⬂ࡡ࣋ࣝࢴࢹ࡞ TRIzol Reagent 1 mL ࢅ ຊ࠻࡙ྊ⁈ࡊࠉᐄῺ࡚ 5 min 㟴⨠ࡌࡾࠊ200 ȣL ࡡ chloroform ࢅຊ࠻ࡻࡂΊ
ࡊࠉᐄῺ࡚ 3 min 㟴⨠ᚃࠉ14,000 rpmࠉ4Υ࡚ 15 min 㐪ᚨࡊࡒࠊࡐࡡ୕ΰ࡞ 2-propanol
(WAKO)ࢅ 500 ȣL ຊ࠻Ίࡊࠉ10 min ᐄῺ࡞㟴⨠ᚃࠉ14,000 rpmࠉ4Υ࡚ 10min
㐪ᚨࡊࠉỷ㜾ࡊࡒ RNA ࢅ 75% ethanol ࡚ὑὯࡊࡒࠊRNA ࣋ࣝࢴࢹࢅ㢴ஜࡊࠉDEPC
