複数の CYP inducerの併用投与によるラット肝発がんプロモーション作用の修飾に関する研究

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Title

複数の CYP inducerの併用投与によるラット肝発がんプロモ

_{ーション作用の修飾に関する研究( 本文(Fulltext) )}

Author(s)

盛田, 怜子

Report No.(Doctoral

Degree)

博士(獣医学) 甲第407号

Issue Date

2014-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/49030

※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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1

」ᩘࡢ

CYP inducer ࡢే⏝ᢞ୚࡟ࡼࡿ

ࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡢ

ಟ㣭࡟㛵ࡍࡿ◊✲

2013 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉

㸦ᮾி㎰ᕤ኱Ꮫ㸧

┒⏣ ᛭Ꮚ

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2 ┠ḟ ᗎㄽ 4 ➨1 ❶ Orphenadrine ࡢࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ᶵᗎ࡟㛵ࡍࡿ◊✲ ⥴ゝ 8 ᮦᩱཬࡧ᪉ἲ 10 ⤖ᯝ 17 ⪃ᐹ 21 ᑠᣓ 25 ➨2 ❶ Phenobarbital ཬࡧ orphenadrine ే⏝ᢞ୚࡟ࡼࡿࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస ⏝ಟ㣭ࡢ᳨ウ ⥴ゝ 27 ᮦᩱཬࡧ᪉ἲ 29 ⤖ᯝ 35 ⪃ᐹ 40 ᑠᣓ 43

➨3 ❶ Phenobarbital ཬࡧ piperonyl butoxide ే⏝ᢞ୚࡟ࡼࡿࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩ ࣙࣥస⏝ಟ㣭ࡢ᳨ウ ⥴ゝ 45

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3 ⤖ᯝ 52 ⪃ᐹ 56 ᑠᣓ 57 ⥲ྜ⪃ᐹ 58 ⤖ㄽ 63 ㅰ㎡ 65 ཧ⪃ᩥ⊩ 66 せ᪨ 74 Abstract 78 ᅗ⾲ 83

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4 ᗎ ᗎㄽ

୍㒊ࡢ໬Ꮫ≀㉁࡟ࡣ㸪ࡑࡢ⫢Ⓨࡀࢇ㐣⛬࡟άᛶ㓟⣲✀㸦ROS㸧⏘⏕࡟ࡼࡿ㓟໬ⓗ ࢫࢺࣞࢫࡀ㛵୚ࡍࡿࡶࡢࡀ࠶ࡿࠋࡑࡢ୰࡟ࡣ cytochrome P450㸦CYP㸧ࢆㄏᑟࡍࡿ≀㉁㸦CYP inducer㸧ࡶྵࡲࢀ࡚࠸ࡿࠋᐇ㝿࡟㸪piperonyl butoxide㸦PBO㸧ࡸ phenobarbital㸦PB㸧 ࡞࡝ࡢCYP inducer ࡢ࠸ࡃࡘ࠿ࡣ㸪ࣛࢵࢺ࡟ᑐࡋ࡚⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡋ㸪 ࡑࡢᶵᗎ࡟ࡣCYP ㄏᑟ࡟క࠺ ROS ⏘⏕࡟ࡼࡿ㓟໬ⓗࢫࢺࣞࢫࡢ㛵୚ࡀሗ࿌ࡉࢀ࡚࠸ࡿ 㹙28㸪15㹛ࠋලయⓗ࡟ࡣ㸪ࡇࢀࡽࡢ≀㉁࡛ࡣ㸪CYP ࡟ࡼࡿ⸆≀௦ㅰࡢ㐣⛬࡛ ROS ࡀ⏘⏕ ࡉࢀ㸪ࡑࡢROS ࡀ⣽⬊⭷ཬࡧ㑇ఏᏊࢆയᐖࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢ⓎࡀࢇࢆㄏⓎࡍࡿ࡜᥎ ᐹࡉࢀࡿࠋࡉࡽ࡟㸪」ᩘࡢⓎࡀࢇ≀㉁ࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪ࡑࡢⓎࡀࢇᛶࡀ┦ຍ࣭ ┦஌ⓗ࡟ቑᙉࡍࡿࡇ࡜ࡣ᪤࡟࠸ࡃࡘ࠿ࡢᩥ⊩࡛♧ࡉࢀ࡚࠸ࡿ㹙9㸪10㹛ࡇ࡜࠿ࡽ㸪ྠᵝ ࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ᶵᗎࢆᣢࡘCYP inducer ࢆ」ᩘྠ᫬࡟ᭀ㟢ࡉࢀࡿࡇ࡜࡟ ࡼࡾ㸪CYP ࡢ௦ㅰάᛶࡀቑຍࡋ㸪 ROS ⏘⏕ࡢቑຍ࡟ࡼࡾ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ ࡀቑᙉࡍࡿྍ⬟ᛶࡀ᥎ᐹࡉࢀࡿࠋࡇࢀࡲ࡛ࡢ◊✲࡛㸪CYP family 1 subfamily A (CYP1A) inducer ྠኈࡢే⏝ᢞ୚࡟ࡼࡗ࡚⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ᙳ㡪ࡀ࠶ࡿࡇ࡜ࡀ᫂ࡽ ࠿࡜࡞ࡗ࡚࠸ࡿ㹙12㹛ࠋᮏ◊✲࡛ࡣ㸪CYP inducer ࡢ୰࡛ࡶ CYP family 2 subfamily B 㸦CYP2B㸧ࢆㄏᑟࡍࡿ CYP2B inducer ࡟╔┠ࡋ㸪」ᩘࡢ CYP inducer ࢆࣛࢵࢺ࡟ྠ᫬ᢞ ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ᑐࡋ࡚࡝ࡢࡼ࠺࡞ಟ㣭స⏝ࡀぢฟࡉ ࢀࡿࡢ࠿࡟ࡘ࠸࡚ศᏊ⑓⌮Ꮫⓗ࡟ゎᯒࡋࡓࠋCYP2B inducer ࡜ࡋ࡚㸪ࣃ࣮࢟ࣥࢯࣥ⑓἞

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5

⒪⸆࡛࠶ࡿorphenadrine㸦ORPH㸧ཬࡧྥ⢭⚄⸆࡛࠶ࡿ PB㸪CYP1A/2B inducer ࡜ࡋ࡚ẅ ⹸ඹຊ๣࡛࠶ࡿPBO ࢆ౑⏝ࡋࡓࠋ

ࡲࡎ➨1 ❶࡛ࡣ㸪 ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚࠸࡞࠸ ORPH ࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡢ᭷↓ࢆ᳨ウࡋ㸪ࡉࡽ࡟ࡑࡢ࣓࢝ࢽࢬ࣒ࢆ㓟໬ⓗࢫࢺࣞ ࢫ࡟╔┠ࡋ࡚ゎᯒࡋࡓࠋࡲࡓ㸪ORPH ࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝㜈್ࢆ᳨ウࡋࡓࠋ CYP2B inducer ࡢࡦ࡜ࡘ࡛࠶ࡿ PB ࡣ㸪CAR ࡢ᰾ෆ⛣⾜࡜࣐࢖ࢡࣟࢯ࣮࣒ ROS ⏘⏕࡟క ࠺㓟໬DNA ᦆയ࡟ࡼࡾ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇ ࡢࡇ࡜࠿ࡽ㸪ORPH ࡀ PB ࡜ྠᵝࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆᣢࡘྍ⬟ᛶࡀ࠶ࡿ࡜ ᥎ㄽࡋ㸪ᐇ㦂ࢆ⾜ࡗࡓࠋ➨2 ❶࡛ࡣ㸪 PB ࡜ ORPH ࢆࣛࢵࢺ࡟ే⏝ᢞ୚ࡋ㸪⫢Ⓨࡀࢇࣉ ࣮ࣟࣔࢩࣙࣥ࡟ᑐࡍࡿಟ㣭స⏝ࢆ᳨ウࡋࡓࠋࣛࢵࢺ࡟࠾࠸࡚㸪⫢Ⓨࡀࢇ≀㉁ࢆప⏝㔞࡛ 」ᩘే⏝ᢞ୚ࡋࡓሙྜ㸪Ⓨࡀࢇ⏝㔞ࡼࡾࡶప࠸⏝㔞ࢆ⏝࠸࡚ࡶ㸪Ⓨࡀࢇస⏝ࡀⓎ⌧ࡍࡿ ྍ⬟ᛶࡀ♧၀ࡉࢀ࡚࠸ࡿ㹙9㹛ࠋࡶࡋ」ᩘࡢ໬Ꮫ≀㉁࡟ྠ᫬࡟ᭀ㟢ࡉࢀࡿࡇ࡜࡟ࡼࡾࡑࡢ Ⓨࡀࢇస⏝ࡀቑᙉࡍࡿ࡞ࡽࡤ㸪ࡑࡢࣜࢫࢡホ౯ࢆ෌⪃ࡍࡿᚲせࡀ࠶ࡿࠋᮏ◊✲࡟ࡼࡾ㸪」ᩘࡢ໬Ꮫ≀㉁ࡢే⏝ᢞ୚࡟ࡼࡿᙳ㡪ࢆ᳨ウࡍࡿࡇ࡜ࡣ㸪㣗ရ୰࡟ྵࡲࢀࡿ໬Ꮫ≀㉁ࡢ ࣜࢫࢡホ౯࡟࠾࠸࡚᭷┈࡞᝟ሗࢆᥦ౪࡛ࡁࡿࡶࡢ࡜⪃࠼ࡿࠋࡑࡇ࡛㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔ ࢩࣙࣥస⏝ࢆᣢࡘCYP2B inducer ࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ స⏝ࡀቑᙉࡉࢀࡿࡶࡢ࡜௬ᐃࡋ㸪ᐇ㦂ࢆ⾜ࡗࡓࠋࡉࡽ࡟㸪➨3 ❶࡛ࡣ㸪PB ࡜ PBO ࢆࣛ ࢵࢺ࡟ే⏝ᢞ୚ࡋ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ᑐࡍࡿಟ㣭స⏝ࢆ᳨ウࡋࡓࠋ➨2 ❶ ࡟࠾࠸࡚CYP2B inducer ࡛࠶ࡿ PB ࡜ ORPH ࢆే⏝ᢞ୚ࡋࡓሙྜ࡟㸪ࣛࢵࢺ⫢Ⓨࡀࢇࣉ

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6

࣮ࣟࣔࢩࣙࣥస⏝ࡀቑᙉࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࡓࡵ㸪CYP ࢆㄏᑟࡍࡿస⏝ࢆ᭷ ࡍࡿ⸆๣ࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀቑᙉࡍࡿྍ⬟ᛶࡀ ᥎ᐹࡉࢀࡿࠋࡑࡇ࡛㸪➨3 ❶࡛ࡣ㸪CYP2B inducer ࡜ CYP1A/2B inducer ࢆ⏝࠸࡚ే⏝ᢞ ୚ᐇ㦂ࢆ⾜࠸㸪␗✀ࡢ CYP ࢆㄏᑟࡍࡿ≀㉁ྠኈࡢే⏝ᢞ୚࡟࠾࠸࡚ࡶ⫢Ⓨࡀࢇࣉࣟࣔ ࣮ࢩࣙࣥస⏝ࡀቑᙉࡍࡿ࠿ྰ࠿ࢆ᳨ウࡍࡿࡓࡵᐇ㦂ࢆ⾜ࡗࡓࠋ ௨ୖࡢᐇ㦂࠿ࡽ㸪」ᩘࡢCYP inducer ࢆࣛࢵࢺ࡟ྠ᫬ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢Ⓨ ࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ᑐࡋ࡚࡝ࡢࡼ࠺࡞ಟ㣭స⏝ࡀぢ࠸ࡔࡉࢀࡿࡢ࠿࡟ࡘ࠸࡚᫂ ☜࡟ࡍࡿ࡜ඹ࡟㸪CYP ࢆㄏᑟࡍࡿ໬Ꮫ≀㉁ࡢ」ᩘᭀ㟢࡟ࡼࡿ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ ᶵᗎࢆゎᯒࡋ㸪ࣄࢺ࡟࠾ࡅࡿ⫢Ⓨࡀࢇࣜࢫࢡࡢྍ⬟ᛶ࡟ࡘ࠸࡚⪃ᐹࢆຍ࠼ࡓࠋ

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7 ➨ ➨1 ❶

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8 ⥴ ⥴ゝ ORPH ࡣ㸪ᢠࢥࣜࣥ๣࡛࠶ࡾ➽ᘱ⦆స⏝ࢆᣢࡘࡓࡵ㸪ࣃ࣮࢟ࣥࢯࣥ⑓἞⒪⸆࡜ࡋ ࡚⏝࠸ࡽࢀ࡚࠸ࡿ㹙2㹛ࠋᮏ๣ࡣ 1950 ᖺ௦࡟ᢎㄆࡉࢀࡓ་⸆ရ࡛࠶ࡾ㸪㑇ఏẘᛶヨ㦂ࡢ ⤖ᯝࡣ⌧ᅾ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠸ࠋ୍᪉㸪ORPH ࡣࣛࢵࢺࡢ⫢⮚࡟࠾࠸࡚⣽⬊ෆཷᐜ య࡛࠶ࡿconstitutive active/androstane receptor㸦CAR㸧ࢆ௓ࡋ࡚ CYP2B ࢆㄏᑟࡍࡿࡇ࡜ ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿ㹙29㹛ࡀ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ࡘ࠸࡚ࡣ᫂ࡽ࠿࡜࡞ࡗ ࡚࠸࡞࠸ࠋ

ྥ⢭⚄⸆࡛࠶ࡾ㸪CYP2B inducer ࡢࡦ࡜ࡘ࡛࠶ࡿ PB ࡣ㸪CAR ࡢ᰾ෆ⛣⾜࡜࣐࢖ ࢡࣟࢯ࣮࣒ROS ⏘⏕࡟క࠺㓟໬ DNA ᦆയ࡟ࡼࡾ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡍࡿ ࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝ࡟࠾࠸࡚㸪PB ࢆᢞ୚ࡍࡿ࡜ CYP2B ࡢmRNA ཬࡧ⺮ⓑ㉁ࡢⓎ⌧ࡀቑ࠼ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㹙21㸪39㹛ࠋࡲࡓ㸪PB ࡟ࡼ ࡿCAR ࡢ᰾ෆ⛣⾜ࡣ⫢Ⓨࡀࢇࢆಁ㐍ࡍࡿᵝࠎ࡞㑇ఏᏊࡢⓎ⌧࡟ᙳ㡪ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿ ࡜࡞ࡗ࡚࠸ࡿ㹙5㸪19㹛ࠋࡉࡽ࡟㸪࣐࢖ࢡࣟࢯ࣮࣒ ROS ⏘⏕ࡸ⬡㉁㐣㓟໬ࡢᣦᶆ࡛࠶ࡿ ࢳ ࢜ ࣂ ࣝ ࣅ ࢶ ࣮ ࣝ 㓟 ཯ ᛂ 㝧 ᛶ ≀ ㉁㸦TBARS 㸧 ཬ ࡧ DNA ᦆ യ ࡢ ᣦ ᶆ ࡛ ࠶ ࡿ 8-hydroxydeoxyguanosine㸦8-OHdG㸧ࡀ PB ࡟ࡼࡿ Cyp2b ࡢㄏᑟ࡟కࡗ࡚ቑຍࡍࡿࡇ࡜ࡀ ♧ࡉࢀ࡚࠸ࡿ㹙26㹛ࠋࡇࢀࡽࡢࡇ࡜࠿ࡽ㸪ORPH ࡀ PB ࡜ྠᵝࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ స⏝ࢆᣢࡘྍ⬟ᛶࡀ࠶ࡿ࡜᥎ㄽࡋࡓࠋ ᮏ◊✲࡛ࡣ㸪ࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝࢆ⏝࠸࡚㸪ORPHࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔ

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9

ࢩࣙࣥస⏝ࡢ᭷↓ࢆ᳨ウࡋ㸪ࡉࡽ࡟ࡑࡢ࣓࢝ࢽࢬ࣒࡟ࡘ࠸࡚㸪㑇ఏᏊⓎ⌧ゎᯒཬࡧ࣐࢖ ࢡࣟࢯ࣮࣒ROS⏘⏕ࡸTBARS㔞ཬࡧ8-OHdG㔞ࡢゎᯒࢆ⾜ࡗࡓࠋຍ࠼࡚㸪⫢⣽⬊ෆࡢ CARࡢᒁᅾཬࡧCAR㛵㐃㑇ఏᏊࡢⓎ⌧ࢆゎᯒࡋࡓࠋ

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10 ᮦ

ᮦᩱཬࡧ᪉ἲ

໬Ꮫ≀㉁

Orphenadrine citrate salt 㸦 ORPH 㸹 CAS No. 4682-36-4 㸪 ⣧ ᗘ 99% 㸧 ཬ ࡧ

N-diethylnitrosamine㸦DEN㸹CAS No. 55-18-5㸪⣧ᗘ99%㸧ࡣ㸪ࡑࢀࡒࢀSigma-Aldrich㸦࢔

࣓ࣜ࢝㸧ཬࡧᮾி໬ᡂᕤᴗ㸦ᮾி㸧࠿ࡽ㉎ධࡋࡓࠋ ౑⏝ື≀ ᐇ㦂1࡛⏝࠸ࡓ5㐌㱋ࡢ㞝ᛶF344ࣛࢵࢺࡣ㸪᪥ᮏSLCᰴᘧ఍♫㸦㟼ᒸ㸧ࡼࡾ㉎ධࡋ㸪 12᫬㛫᫂ᬯࢧ࢖ࢡࣝ㸪 ᗘ20㹼26Υ㸪‵ᗘ40~70%ࡢ᮲௳ୗ࡛3࡞࠸ࡋ4༉ࡎࡘᗋᩜࡁ㸦࣌ ࣃ࣮ࢡ࣮ࣜࣥ㸹᪥ᮏSLCᰴᘧ఍♫㸪㟼ᒸ㸧ࢆᩜ࠸ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢪ࡟࡚㣫⫱ࢆ⾜ࡗ ࡓࠋ⢊ᮎᇶ♏㣫ᩱ㸦࢚࢜ࣜࣥࢱࣝMF㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸪ᮾி㸧ཬࡧ㣧ᩱỈࡣ⮬ ⏤ᦤྲྀࡉࡏࡓࠋ1㐌㛫ࡢ᳨␿࣭㥆໬ᮇ㛫ࢆタࡅࡓᚋ㸪ᐇ㦂࡟౪ࡋࡓࠋ࡞࠾㸪ື≀ᐇ㦂ࡣ ᮾி㎰ᕤ኱Ꮫᐇ㦂ື≀೔⌮つᐃ࡟‽ᣐࡋ࡚⾜ࡗࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ᐇ㦂1 ORPHࡢࣛࢵࢺ࡟࠾ࡅࡿ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᳨ウࡍࡿࡓࡵ㸪௨ୗࡢᡭ 㡰࡟ᚑࡗ࡚㸪ࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇᐇ㦂㹙17㹛ࢆ⾜ࡗࡓࠋࡍ࡞ࢃࡕ㸪඲࡚ࡢື≀࡟ᑐࡋ

(12)

11 ࢖ࢽࢩ࢚࣮ࢱ࣮࡜ࡋ࡚DENࡢ༢ᅇ⭡⭍ෆᢞ୚㸦200 mg/kg య㔜㸧ࢆ⾜࠸㸪ࡑࡢ2㐌㛫ᚋ ࠿ࡽ㸪ᇶ♏㣫ᩱࡢࡳ㸦DEN༢⊂⩌㸪n = 12㸧㸪ࡶࡋࡃࡣ750 ppm㸦ORPHప⏝㔞⩌㸪n = 12㸧 ࡞࠸ࡋ1,500 ppm㸦ORPH㧗⏝㔞⩌㸪n = 12㸧ࡢORPHΰ㣵ᢞ୚ࢆ6㐌㛫⾜ࡗࡓࠋ⏝㔞タᐃ ࡟࠾࠸࡚ࡣ㸪㐣ཤࡢሗ࿌ࢆཧ↷ࡋࡓ㹙29㹛ࡀ㸪ORPH࡟ᦤ㣗ᚷ㑊ᛶࡀㄆࡵࡽࢀࡓࡓࡵ㸪 ᭱㧗⏝㔞ࡣሗ࿌࠿ࡽ⟬ᐃࡋࡓ⏝㔞ࡢ༙㔞࡜ࡋࡓࠋ⫢⣽⬊ࡢቑṪάᛶࢆ㧗ࡵࡿࡓࡵ㸪඲࡚ ࡢື≀࡟2/3㒊ศ⫢ษ㝖ࢆDENᢞ୚3㐌㛫ᚋ࡟ᐇ᪋ࡋࡓࠋᮏᐇ㦂࡛ࡣ㒊ศ⫢ษ㝖ࡢࡳࡢࢥ ࣥࢺ࣮ࣟࣝ⩌㸦n = 8㸧ࢆタࡅࡓࠋDEN༢⊂⩌ཬࡧORPHప⏝㔞⩌࡟࠾࠸࡚ྛ1༉ࡀ㒊ศ ⫢ษ㝖ࡢᢏ⾡ⓗ㐣ኻ࡟ࡼࡾṚஸࡋࡓࠋࡲࡓ㸪㒊ศ⫢ษ㝖2㐌㛫ᚋ࡟ࢥࣥࢺ࣮ࣟࣝ⩌ࡢ1 ༉ࡀṚஸࡋࡓࠋ1㐌㛫࡟୍ᗘ㸪య㔜 ᐃཬࡧᦤ㣵ᦤỈ㔞ࡢ ᐃࢆࢆ⾜ࡗࡓࠋᢞ୚ᮇ㛫⤊ ஢ᚋ㸪῝㯞㓉ୗ࡟࡚ᨺ⾑ẅࢆ⾜࠸㸪⫢⮚ࢆ᥇ྲྀᚋ㸪㔜㔞 ᐃࢆ⾜ࡗࡓࠋ⫢⮚ࡢ⤌⧊Ꮫⓗ ᳨⣴ཬࡧච␿⤌⧊໬Ꮫⓗ᳨⣴࡟ࡣ㸪ゎ๗᫬࡟ࢫࣛ࢖ࢫࡋࡓ⫢⤌⧊ࢆ4%ࣃ࣒ࣛ࣍ࣝ࢔ࣝ ࢹࣄࢻ⁐ᾮ࡟࡚ᅛᐃࢆ⾜࠸㸪ࡲࡓ㸪ṧࡾࡢ⫢ࢧࣥࣉࣝࡣ㑇ఏᏊⓎ⌧ཬࡧ⏕໬Ꮫⓗゎᯒ⏝ ࡜ࡋ࡚ᾮయ❅⣲࡟ᾐₕࡋ࡚෾⤖ࡋ-80Υ࡟࡚ಖᏑࡋࡓࠋ ㏣㏣ຍᐇ㦂 ᐇ㦂1ࡢゎᯒᚋ㸪ORPHࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝㜈್ࢆ᳨ウࡍࡿࡓࡵ㸪ᐇ 㦂1࡜ྠᵝ࡟ࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝࢆ⏝࠸࡚㏣ຍᐇ㦂ࢆ⾜ࡗࡓࠋࣉ࣮ࣟࣔࢱ࣮࡜ ࡋ࡚375 ppm ORPHࡢ6㐌㛫ΰ㣵ᢞ୚ࢆ⾜ࡗࡓࠋ⫢⮚ࢆ᥇ᮦࡋ㸪⫢๓ࡀࢇ⑓ኚࡢⓎ⏕࡟ࡘ ࠸࡚ච␿⤌⧊໬Ꮫⓗ࡟᳨⣴ࢆ⾜ࡗࡓࠋ

(13)

12 ⑓

⑓⌮⤌⧊Ꮫⓗゎᯒ࣭ච␿⤌⧊໬Ꮫⓗゎᯒ

ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻ࡛ᅛᐃᚋ㸪ᐃἲ࡟ᚑ࠸࢚ࢱࣀ࣮ࣝ⣔ิ࡛⬺Ỉࢆ⾜࠸㸪ࣃࣛ ࣇ࢕ࣥ࡟ໟᇙࡋࡓࠋ㐃⥆ษ∦ࢆస〇ࡋ㸪୍㒊ࡣH&E ᰁⰍࢆ⾜ࡗࡓࠋࡉࡽ࡟ glutathione

S-transferase placental form㸦GST-P㸧㸪proliferating cell nuclear antigen㸦PCNA㸧ཬࡧ CAR

ࡢච␿⤌⧊໬ᏛᰁⰍࢆ௨ୗࡢᡭ㡰࡛⾜ࡗࡓࠋጞࡵ࡟⬺ࣃࣛࣇ࢕ࣥฎ⌮ࢆࡋࡓษ∦ࢆ㸪 0.3%㐣㓟໬Ỉ⣲ࢆྵࡴ࣓ࢱࣀ࣮ࣝ࡟ࡼࡾᐊ ࡛ 30 ศ㛫ෆᅉᛶ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ࢆ ࡋ㸪ṇᖖ⾑Ύ࡟࡚ࣈࣟࢵ࢟ࣥࢢฎ⌮ࢆ⾜ࡗࡓࠋࡲࡓ㸪࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ᚋ㸪PCNA ᰁⰍ࡟㛵ࡋ࡚ࡣࢡ࢚ࣥ㓟⦆⾪ᾮ㸦pH 6.0㸧ୗ࡛ 60Υ㸪20 ศࡢ ᾎฎ⌮ࢆ⾜࠸ CAR ࡢᰁ Ⰽ࡟㛵ࡋ࡚ࡣࢡ࢚ࣥ㓟⦆⾪ᾮ㸦pH 6.0㸧ୗ࡛ 121Υ㸪100 ⛊ࡢ࣮࢜ࢺࢡ࣮ࣞࣈฎ⌮ࢆ⾜ ࡗࡓࠋࡑࡢᚋ㸪࢘ࢧࢠᢠGST-P ࣏ࣜࢡ࣮ࣟࢼࣝᢠయ㸦1,000 ಸᕼ㔘㸪ᰴᘧ఍♫་Ꮫ⏕≀ Ꮫ◊✲ᡤ㸪ឡ▱㸧㸪࣐࢘ࢫᢠPCNA ࣔࣀࢡ࣮ࣟࢼࣝᢠయ㸦500 ಸᕼ㔘㸪Dako㸪ࢹ࣐࣮ࣥ ࢡ㸧ཬࡧ࣐࢘ࢫᢠCAR1/2 ࣔࣀࢡ࣮ࣟࢼࣝᢠయ㸦50 ಸᕼ㔘㸪Santa Cruz Biotechnology㸪 ࢔࣓ࣜ࢝㸧ࢆࡑࢀࡒࢀ4Υ୍࡛ᬌ཯ᛂࡉࡏࡓࠋ୍ḟᢠయ཯ᛂᚋ㸪Vectastain Elite ABC kit 㸦Vector Laboratories㸪࢔࣓ࣜ࢝㸧ࢆ⏝࠸㸪3,3’-diaminobenzidine㸦DAB㸧࡟ࡼࡿⓎⰍᚋ㸪 ࣐࣊ࢺ࢟ࢩ࡛ࣜࣥ᰾ࡢᑐẚᰁⰍࢆ⾜ࡗࡓࠋ

GST-P 㝧ᛶ⫢⣽⬊ᕢ࡟ࡘ࠸࡚ࡣ㸪┤ᚄ 200 μm ௨ୖ࡛᫂ࡽ࠿࡞㝧ᛶ⫢⣽⬊ᕢࡢษ ∦㠃✚ᙜࡓࡾࡢಶᩘཬࡧ㠃✚⋡ࢆ⏬ീゎᯒࢯࣇࢺWinROOF㸦୕㇂ၟ஦㸪ᮾி㸧ࢆ⏝࠸ ࡚ゎᯒࡋࡓࠋPCNA 㝧ᛶ⫢⣽⬊࡟ࡘ࠸࡚ࡣ㸪1 ౛࡟ࡘࡁ↓సⅭ࡟㑅ࢇࡔ 10 ど㔝㸦1 ど㔝

(14)

13

࠶ࡓࡾ700㹼900 ⫢⣽⬊㸧࡟࡚ᙉ㝧ᛶ⫢⣽⬊ࡢ ᐃࢆ⾜࠸㸪ⓒศ⋡࡛⾲♧ࡋࡓࠋ

cDNA ࣐࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ

cDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟౑⏝ࡍࡿ total RNA ࢧࣥࣉࣝࡣ㸪RNeasy kit㸦Qiagen㸪ࢻ ࢖ࢶ㸧࡟ࡼࡾ㸪ࢥࣥࢺ࣮ࣟࣝ⩌㸪DEN ༢⊂⩌ཬࡧ ORPH 㧗⏝㔞⩌࠿ࡽ 1 ౛ࡎࡘᢳฟࡋ ࡓࠋᢳฟࡋࡓRNA ࢧࣥࣉࣝ 10 μg ࠿ࡽ㸪Superscript Double-Stranded cDNA Synthesis kit

㸦Life technologies㸪CA㸪࢔࣓ࣜ࢝㸧࡟ࡼࡾ஧ᮏ㙐DNA ࢆྜᡂࡋࡓࠋࡉࡽ࡟ Cy3 ࡟ࡼ

ࡾࣛ࣋ࣝ໬ࢆ⾜࠸㸪ࡑࡢ࠺ࡕ6 μg ࢆ࣐࢖ࢡࣟ࢔ࣞ࢖ Euk Expr 385K Catalog Arr㸹26,739 targets/microarray㸦Roche NimbleGen㸪ᮾி㸧࡟཯ᛂࡉࡏࡓࠋᚓࡽࢀࡓ⤖ᯝࡣ Robust Multiple Averege㸦RMA㸧normalization ἲ㹙16㹛࡟ࡼࡾᶆ‽໬ࢆ⾜ࡗࡓࠋ࡞࠾㸪ᚓࡽࢀࡓ⤖ᯝࡢ 㑇 ఏ Ꮚ ᝟ ሗ ࡟ ࡘ ࠸ ࡚ ࡣ 㸪 ⡿ ᅜ ᅜ ❧ ⏕ ≀ ᝟ ሗ ࢭ ࣥ ࢱ ࣮ ࡀ බ ⾲ ࡍ ࡿ ࢘ ࢙ ࣈ ࢧ ࢖ ࢺ 㸦http://www.ncbi.nlm.nih.gov/㸧࠿ࡽධᡭࡋࡓࠋ

Real-timereverse transcription polymerase chain reaction㸦RT-PCR㸧ゎᯒ

㑇ఏᏊⓎ⌧ゎᯒ࡟౑⏝ࡍࡿtotal RNA ࢧࣥࣉࣝࡣ㸪RNeasy kit㸦Qiagen㸧࡟ࡼࡾᢳ ฟࡋࡓࠋcDNA ྜᡂ࡬ࡢ㏫㌿෗཯ᛂࡣ 2 μg ࡢ total RNA ࠿ࡽ Thermoscript RT-PCR system 㸦Life Technologies㸧࡟࡚⾜ࡗࡓࠋᐃ㔞 real-time RT-PCR ࡣ PowerSYBR®_{Green PCR}

Master Mix 㸦Life Technologies㸧 ཬ ࡧ StepOnePlusTM_{Real-Time PCR System㸦}_Life

(15)

14

Technologies㸧࡟࡚స〇ࡋࡓࠋྛࣉࣛ࢖࣐࣮㓄ิࡣTable 1-1 ࡟グ㍕ࡋࡓࠋ㑇ఏᏊⓎ⌧ࡢ

ᐃ㔞໬ࡣ㸪ෆᅾᛶࢥࣥࢺ࣮ࣟࣝ㑇ఏᏊ㸦Actb㸧࡟ᑐࡋ࡚ 2-ΔΔCt_{ἲ࡟๎ࡾᶆ‽໬ࡋࡓ㹙24㹛ࠋ}

㐣

㐣㓟໬⬡㉁ ᐃ

⫢⮚࡟࠾ࡅࡿ㐣㓟໬⬡㉁ ᐃࡣ TBARS 㔞ࢆ ᐃࡋࡓࠋ⫢ TBARS 㔞ࡣ Ohkawa ࡽ㸦1979㸧ࡢ᪉ἲ࡟ᚑ࠸ 1 ⩌࠶ࡓࡾ 6 ౛࡟ࡘ࠸࡚ ᐃࢆ⾜ࡗࡓࠋ⫢ࢧࣥࣉࣝ㸦⣙ 50 mg㸧 ࢆ450 μL ࡢ 1.15% KCl ⦆⾪ᾮ୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋ⺮ⓑ⃰ ᗘ ᐃࡣBCA protein assay kit㸦Pierce, ࢔࣓ࣜ࢝㸧࡟࡚⾜ࡗࡓࠋᆒ㉁໬ࡋࡓ⫢࣍ࣔࢪࢿ ࣮ࢺ㸦18.0 mg protein㸧ࢆ 0.2 mL ࡢ 8.1% SDS ࡜ 3.0 mL ࡢ 0.4% thiobarbituric acid-10%㓑㓟⁐ᾮ࡜࡛95Υ㸪60 ศ㛫཯ᛂࡉࡏࡓᚋ㸪෭Ỉ࡛෭༷ࡋࡓࠋ཯ᛂᾮ࡟ 1 mL ࡢ⵨␃Ỉࢆຍ ࠼㸪2.0 mL ࡢ n-butanol-pyridine ΰྜᾮ㸦15:1㸧ࢆຍ࠼ࡓᚋ㸪2,100 ×g㸪10 ศ㛫㐲ᚰࡋࡓࠋ 532 nm ࡟࠾ࡅࡿ྾ගᗘࡣ㸪ศගගᗘィ SynergyTM_{HT Multi-Detection Microplate Reader}

㸦BioTek㸪࢔࣓ࣜ࢝㸧࡟ࡼࡾ ᐃࡋࡓࠋTBARS ᐃ್ࡣ 1,1,3,3-tetramethoxypropane ⏤ ᮶ࡢ malondialdehyde㸦MDA㸧㔞࡟࡚ᐃ㔞ࡋࡓࠋ

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢㄪ〇

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢㄪ〇ࡣ㸪Yoshihara ࡽ㸦2001㸧ࡢሗ࿌࡟ᇶ࡙ࡁⱝᖸࡢኚ᭦ ࢆຍ࠼⾜ࡗࡓࠋ⫢ࢧࣥࣉࣝ㸦⣙100㹼130 mg㸧ࢆ 3 ಸ㔞ࡢ⦆⾪ᾮ㸦50 mM Tris-HCl㸹pH 7.4㸪1.15% KCl㸪0.2 mM EDTA㸪0.1 mM DTT㸪0.1 mM Protease Inhibitor Cocktail㸪20%

(16)

15

glycerol㸧୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋḟ࡟ࡑࡢ࣍ࣔࢪࢿ࣮ࢺࢆ 700 ×g ࡛ 10 ศ㛫㐲ᚰࢆ⾜࠸㸪ୖΎࢆ 10,000 ×g ࡛ 20 ศ㛫㐲ᚰࡋࡓࠋᚓࡽࢀࡓୖΎࢆࡉ ࡽ࡟105,000 ×g ࡛ 90 ศ㛫㉸㐲ᚰࡋࡓᚋ㸪ỿ´ࢆ 20 mM Tris-HCl㸦pH 7.4㸧࡟ᠱ⃮ࡋࡓ ࡶࡢࢆ࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡜ࡋࡓࠋ⺮ⓑ⃰ᗘ ᐃࡣBCA protein assay kit㸦Pierce㸧࡟࡚ ⾜ࡗࡓࠋ

࣐

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢROS ⏘⏕⬟ ᐃ

NADPH ౫Ꮡᛶࡢ࣐࢖ࢡࣟࢯ࣮࣒ ROS ⏘⏕⬟ࡢ ᐃ࡟ࡘ࠸࡚ࡣ㸪Schlezinger ࡽ 㸦1999㸧࡟ᚑ࠸㸪2’7’-dichlorodihydrofluorescein diacetate㸦H2DCFDA㸧ࡀ ROS ࡟ࡼࡾ㓟

໬ࡉࢀ࡚⏕ࡌࡿ2’7’-dichlorofluorescein㸦DCF㸧ࢆ ᐃࡍࡿࡇ࡜࡛ ᐃࢆ⾜ࡗࡓࠋ1 ⩌࠶ ࡓࡾ6 ౛࡟ࡘ࠸࡚࣐࢖ࢡࣟࢯ࣮࣒⺮ⓑ㸦᭱⤊⃰ᗘ 0.2 mg/ml㸧࡟ 40 mM Tris buffer㸦pH 7.4㸧 ࡜5 μM H2DCFDA㸦Life Technologies㸧ࢆຍ࠼࡚96 ࢙࢘ࣝࣉ࣮ࣞࢺ࡟⛣ࡋ㸪37Υ࡛ 30

ศ㛫཯ᛂࡉࡏࡓࠋ཯ᛂ㛤ጞࡣnicotinamide adenine dinucleotide phosphate㸦NADPH㸹᭱⤊ ⃰ᗘ0.5 mM㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸧ࢆῧຍࡍࡿࡇ࡜࡟ࡼࡾ⾜࠸㸪⏕ᡂࡉࢀࡿ⺯ගࢆ ບ㉳Ἴ㛗 485 nm㸪528 nm ࡢ᮲௳࡛ 120 ศ㛫 5 ศࡈ࡜࡟㸪ศගගᗘィ SynergyTM HT Multi-Detection Microplate Reader㸦BioTek㸧࡟ࡼࡾ ᐃࡋࡓࠋ㝧ᛶᑐ↷࡟ H2O2㸦᭱⤊⃰

ᗘ0.1 mM㸧ࢆ㸪ヨ㦂⣔ࡢ᳨ド࡟ CYP 㜼ᐖ๣࡜ࡋ࡚▱ࡽࢀࡿ SKF-525A㸦᭱⤊⃰ᗘ 0.1 mM 㸹Tronto Reserch Chemicals㸪࢝ࢼࢲ㸧ࢆῧຍࡋࡓࠋࢹ࣮ࢱࡣ DEN ༢⊂⩌ࢆ 100%࡜ ࡋ࡚ᶆ‽໬ࡋࡓࠋ

(17)

16 㓟

㓟໬ⓗDNA യᐖ

⫢⮚࡟࠾ࡅࡿ㓟໬ⓗ DNA യᐖࡣ 8-OHdG 㔞ࢆᐃ㔞ࡋࡓࠋࡑࡢ⫢⮚୰ DNA ࡢ 8-OHdG ྵ㔞ࡢ ᐃࡣ㸪Umemura ࡽ㸦2006㸧ࡢ᪉ἲ࡟ᚑ࠸㸪DEN ༢⊂⩌ཬࡧ ORPH 㧗 ⏝㔞⩌ࡢྛ⩌6 ౛࡟ࡘ࠸࡚ ᐃࢆ⾜ࡗࡓࠋ0.3 g ࡢ⫢⮚ࢧࣥࣉࣝࢆ⏝࠸㸪ᢠ㓟໬๣࡜ࡋ ࡚ࣚ࢘໬ࢼࢺ࣒ࣜ࢘⁐ᾮࢆྵࡴDNA Extractor WB Kit㸦࿴ග⣧⸆ᕤᴗ㸧ࢆ⏝࠸࡚⣽⬊ᡂ ศࢆ⁐ゎࡉࡏࡓᚋ㸪᰾DNA ࢆ༢㞳ࡋࡓࠋ⣽⬊⁐ゎࡢࢫࢸࢵࣉ࡟࠾ࡅࡿ⮬ື㓟໬ࢆ㑊ࡅ ࡿࡓࡵ㸪⁐ゎᾮ࡟ࡣdeferoxamine mesylate ࢆῧຍࡋࡓࡶࡢࢆ౑⏝ࡋࡓ㹙13㹛ࠋࢾࢡࣞ࢔ ࣮ࢮ P1 ཬࡧ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮࢆ⏝࠸࡚ DNA ࢆࢹ࢜࢟ࢩࢾࢡࣞ࢜ࢳࢻ࡟ศゎ ࡋ㸪㟁Ẽ໬Ꮫ᳨ฟჾ௜ࡁ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ㸦HPLC-EDC㸹Coulochem II㸹ESA Biosciences㸪࢔࣓ࣜ࢝㸧࡟࡚ 8-OHdG 㔞㸦8-OHdG/105_{deoxyguanosine㸧ࢆ ᐃࡋࡓ㹙38㹛ࠋ}

⤫ィゎᯒ

඲࡚ࡢࢹ࣮ࢱࡣᖹᆒ್ཬࡧᶆ‽೫ᕪ್࡟࡚⾲グࡋࡓࠋศᩓࡢᆒ୍ᛶࡢ᳨ᐃࡣ㸪 Bartlett ᳨ᐃࢆ⾜ࡗࡓࠋᆒ୍ᛶࡀ࠶ࡿሙྜ࡟ࡣ Tukey ࡢከ㔜ẚ㍑᳨ᐃࢆ⾜࠸㸪ᆒ୍ᛶࡀ ࡞࠸ሙྜࡣSteel-Dwass ᳨ᐃࢆ⾜ࡗࡓࠋP ್ࡀ 0.05 ᮍ‶ࡢሙྜ㸪⤫ィᏛⓗ࡟᭷ព࡛࠶ࡿ ࡜ุ᩿ࡋࡓࠋ

(18)

17 ⤖ ⤖ᯝ ᐇ㦂1 య㔜㸪⫢㔜㔞㸪ᦤ㣵ᦤỈ㔞ཬࡧ᥎ᐃ⸆≀ᦤྲྀ㔞ヨ㦂ᮇ㛫୰㸪ORPH ᢞ୚⩌࡟࠾࠸࡚ ORPH ᢞ୚࡟㛵㐃ࡍࡿ⮫ᗋ⑕≧ཬࡧṚஸࡣㄆ ࡵࡽࢀ࡞࠿ࡗࡓࠋORPH 㧗⏝㔞⩌࡛ࡣ㸪DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚య㔜ቑຍᢚไࡀࡳࡽࢀ ࡓ㸦Fig. 1-1㸧ࠋᖹᆒᦤ㣵㔞ཬࡧᖹᆒᦤỈ㔞ࡣ ORPH 㧗⏝㔞⩌࡟࠾࠸࡚ࢥࣥࢺ࣮ࣟࣝ⩌࡜ ẚ㍑ࡋ࡚᭷ព࡟ᑡ࡞࠿ࡗࡓ㸦Table 1-2㸧ࠋ ๗᳨᫬ࡢయ㔜ࡣ⏝㔞౫Ꮡⓗ࡟ ORPH ᢞ୚⩌࡛ῶᑡࡋࡓࠋ⤯ᑐ⫢㔜㔞࡟ኚ໬ࡣ࡞ࡃ㸪 ┦ᑐ⫢㔜㔞ࡣࢥࣥࢺ࣮ࣟࣝ⩌ཬࡧDEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ ORPH ᢞ୚⩌࡟࠾࠸࡚᭷ព࡟ ቑຍࡋࡓࠋORPH ᦤྲྀ㔞ࡣ ORPH ప⏝㔞⩌࡛ 40.4 mg/kg య㔜㸪ORPH 㧗⏝㔞⩌࡛ 73.9 mg/kg య㔜࡛࠶ࡾ㸦Table 1-2㸧㸪࠾࠾ࡼࡑண᝿ࡋࡓ㏻ࡾࡢ㔞࡛࠶ࡗࡓࠋ ⑓⌮⤌⧊Ꮫⓗ᳨⣴ ⑓⌮⤌⧊Ꮫⓗ᳨⣴࡛ࡣ㸪ᑠⴥ୰ᚰᛶ⫢⣽⬊⫧኱ཬࡧ✵⬊ኚᛶࡀ ORPH ᢞ୚⩌࡛ ㄆࡵࡽࢀࡓ㸦Fig. 1-2㸧ࠋࡉࡽ࡟㸪ኚ␗⫢⣽⬊ᕢ㸦᫂⣽⬊ᛶཬࡧዲ㓟ᛶ㸧ࡀ DEN ༢⊂⩌ ཬࡧORPH ᢞ୚⩌࡛ㄆࡵࡽࢀࡓࠋ GST-P 㝧ᛶ⫢⣽⬊ᕢ㸪PCNA 㝧ᛶ⫢⣽⬊⋡ཬࡧ CAR Ⓨ⌧

(19)

18

ORPH ᢞ୚࡟ࡼࡾ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘཬࡧ㠃✚ࡀ ᭷ព࡟ቑຍࡋࡓ㸦Fig. 1-2㸪1-3㸧ࠋPCNA 㝧ᛶ⫢⣽⬊⋡ࡣ ORPH ᢞ୚⩌࡛ࢥࣥࢺ࣮ࣟࣝ⩌ ࡟ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓ㸦Fig. 1-2㸪1-3㸧ࠋຍ࠼࡚㸪ORPH ᢞ୚⩌ࡢ CAR ࡢ⣽⬊ෆⓎ

⌧ࡣ, DEN ༢⊂⩌࡛ࡣᙅ࠸࡞ࡀࡽࡶ᰾࡜⣽⬊㉁࡟ศᕸࡋ࡚࠸ࡓࡢ࡟ᑐࡋ࡚, ORPH ᢞ୚⩌ ࡛ࡣ᫂ࡽ࠿࡟⫢⣽⬊ࡢ᰾࡟ᒁᅾࡋ࡚࠸ࡓ㸦Fig. 2㸧ࠋ cDNA ࣐࣐࢖ࢡࣟ࢔ࣞ࢖ཬࡧreal-time RT-PCR ࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ cDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࢆ⾜࠸㸪ORPH ᢞ୚࡟ࡼࡾⓎ⌧ኚືࡋࡓ⫢⮚ࡢ㑇ఏᏊ ࣉࣟࣇ࢓࢖ࣝࢆồࡵ, ≉ᚩⓗ࡞㑇ఏᏊ࡟ࡘࡁⓎ⌧ࡢ᳨ドゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ORPH 㧗⏝㔞⩌࡟࠾࠸࡚ 2 ಸ௨ୖࡶࡋࡃࡣ 1/2 ಸ௨ୗ࡟Ⓨ⌧ኚືࡋࡓ ⫢⮚ࡢ㑇ఏᏊࡣ㸪392 ಶࡶࡋࡃࡣ 205 ಶ࡛࠶ࡗࡓ㸦ࢹ࣮ࢱࡣ┬␎㸧ࠋ㌿෗⏘≀㔞ࡀቑຍ ࡋࡓ㑇ఏᏊࡢ୰࡛㸪➨୍┦⸆≀௦ㅰ㓝⣲㑇ఏᏊ࡜ᢠ㓟໬㓝⣲㑇ఏᏊཬࡧCAR 㛵㐃㑇ఏ Ꮚ࡟ὀ┠ࡋ᳨ドゎᯒࢆ⾜ࡗࡓࠋTable 1-1 ࡟♧ࡋࡓ㑇ఏᏊ࡟㛵ࡋ࡚㸪ྛ⩌ 6 ౛ࡎࡘ real-time RT-PCR ゎᯒࢆ⾜ࡗࡓ㸦Table 1-3㸧ࠋ➨୍┦⸆≀௦ㅰ㓝⣲㑇ఏᏊ࡛ࡣ Cyp1a1 ࡀ ORPH 㧗 ⏝㔞⩌࡛㸪Cyp2b1/2 ࡀ ORPH ᢞ୚⩌࡛ DEN ༢⊂⩌࡟ẚ㍑ࡋ࡚᭷ព࡟Ⓨ⌧ࡀቑຍࡋࡓࠋ

ࡲࡓ㸪ᢠ㓟໬㓝⣲㑇ఏᏊ࡛࠶ࡿGpx2 ཬࡧ Gstm3 ࡢⓎ⌧ࡀ ORPH 㧗⏝㔞⩌࡛ DEN ༢⊂

⩌࡟ẚ㍑ࡋ࡚᭷ព࡟Ⓨ⌧ࡀቑຍࡋࡓࠋࡉࡽ࡟㸪CAR 㛵㐃㑇ఏᏊ࡛࠶ࡿ Mrp2 ࡢⓎ⌧ࡀ㸪 DEN ༢⊂⩌࡟ẚ㍑ࡋ࡚ ORPH 㧗⏝㔞⩌࡛㸪ࢥࣥࢺ࣮ࣟࣝ⩌࡟ẚ㍑ࡋ࡚ ORPH ᢞ୚⩌࡛ ᭷ព࡟ቑຍࡋࡓࠋCAR 㛵㐃㑇ఏᏊࡢࡦ࡜ࡘ࡛࠶ࡾ⣽⬊࿘ᮇไᚚ㑇ఏᏊ࡛࠶ࡿ Cyclin D1

(20)

19

ࡢⓎ⌧ࡶ DEN ༢⊂⩌ཬࡧ ORPH ᢞ୚⩌࡛ࢥࣥࢺ࣮ࣟࣝ⩌࡟ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ㸪 DEN ༢⊂⩌࡟ẚ㍑ࡋ࡚ ORPH 㧗⏝㔞⩌࡛᭷ព࡟ቑຍࡋࡓࠋCAR ཬࡧ CAR 㛵㐃㑇ఏᏊ

࡛࠶ࡿSlco1a4 ࡢⓎ⌧࡟᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋcDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡢ ⤖ᯝ㸪DEN ༢⊂⩌ཬࡧ ORPH 㧗⏝㔞⩌࡟࠾࠸࡚ࢥࣥࢺ࣮ࣟࣝ⩌࡜ࡢẚ㍑࡛⯆࿡῝࠸ኚ ືࡢ㐪࠸ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ ࣐ ࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢROS ⏘⏕⬟ ROS ⏘⏕ࡢ ᐃࢆࡍࡿࡓࡵ㸪⫢⮚ࡢ࣐࢖ࢡࣟࢯ࣮࣒࡟࠾ࡅࡿ NADPH ౫Ꮡⓗ࡞ ROS ⏘⏕⬟ࢆ ᐃࡋࡓ㸦Fig. 1-4㸧ࠋCYP ࡢࣔࣀ࢜࢟ࢩࢤࢼ࣮ࢮάᛶࡣ NADPH ࠿ࡽࡢ㑏 ඖຊࡀᚲせ࡛࠶ࡿࡇ࡜࠿ࡽ㸪NADPH Ꮡᅾୗ࣭㠀Ꮡᅾୗ࡟ ROS ⏘⏕⬟ࢆ ᐃࡋࡓࠋ NADPH ࢆῧຍࡋࡓ࡜ࡇࢁ㸪࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡟࠾ࡅࡿ ROS ⏘⏕ࡣ᫂ࡽ࠿࡟ቑຍࡋ㸪 ORPH ᢞ୚⩌࡛ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟㧗್࡛࠶ࡗࡓࠋH2O2ࢆῧຍࡋࡓ࡜ࡇࢁ㸪

࣐࢖ࢡࣟࢯ࣮࣒ROS ⏘⏕ࡣቑຍࡋ㸦ࢹ࣮ࢱࡣ┬␎㸧㸪CYP ࡢ㜼ᐖ๣࡛࠶ࡿ SKF-525A ࢆ ῧຍࡋࡓ࡜ࡇࢁROS ⏘⏕ࡢపୗࡀぢࡽࢀࡓ㸦Fig. 1-4㸧ࠋ

⣽⬊⭷ཬࡧDNA ࡟ᑐࡍࡿ㓟໬ⓗࢫࢺࣞࢫ

ROS ⏘⏕࡟ࡼࡿ⣽⬊࡬ࡢ㓟໬ⓗയᐖࢆ᳨⣴ࡍࡿࡓࡵ㸪⫢⮚୰ࡢ TBARS 㔞ཬࡧ 8-OHdG 㔞ࢆ ᐃࡋࡓ㸦Fig. 1-4㸧ࠋTBARS 㔞ࡣ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ ORPH 㧗⏝㔞⩌ ࡛᭷ព࡟ቑຍࡋࡓࠋ8-OHdG 㔞ࡶ ORPH 㧗⏝㔞⩌࡛ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ቑຍ

(21)

20 ࡀㄆࡵࡽࢀࡓ㸦Fig. 1-4㸧ࠋ

(22)

21 ⪃ ⪃ᐹ

CYP2B inducer ࡢࡦ࡜ࡘ࡛࠶ࡿ ORPH ࡣ㸪ࣄࢺࡢࣃ࣮࢟ࣥࢯࣥ⑓἞⒪⸆࡜ࡋ࡚ ౑⏝ࡉࢀ࡚࠸ࡿ㹙2㹛ࡀ㸪ື≀ᐇ㦂࡟࠾ࡅࡿ ORPH ࡢ㑇ఏẘᛶཬࡧⓎࡀࢇᛶ࡟ࡘ࠸࡚ࡢ ᝟ሗࡣ☜ㄆࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋᮏ◊✲࡟࠾࠸࡚ࡣ㸪DEN ࢆ࢖ࢽࢩ࢚࣮ࢱ࣮࡜ࡋ࡚⏝ ࠸ࡓࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝᐇ㦂ࢆ⾜࠸㸪ORPH ࡢᢞ୚࡟ࡼࡿ GST-P 㝧ᛶ⫢⣽⬊ᕢ ࡢಶᩘཬࡧ㠃✚࡜PCNA 㝧ᛶ⫢⣽⬊⋡ࡢቑຍࡀㄆࡵࡽࢀࡓࠋࡇࡢࡇ࡜࠿ࡽ㸪ORPH ࡣࣛ ࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡍࡿ࡜⪃࠼ࡽࢀࡓࠋࣄࢺ࡟࠾࠸࡚ ORPH ࡀࣃ࣮ ࢟ࣥࢯࣥ⑓ࡢ἞⒪⸆࡜ࡋ࡚⏝࠸ࡽࢀࡿ⃰ᗘࡣ200 mg/ே࡛࠶ࡾ㸪ࡇࢀࡣయ㔜ࢆ 60 kg ࡜ ௬ᐃࡋࡓሙྜ3.3 mg/kg య㔜࡟┦ᙜࡍࡿࠋࡇࢀࡣ㸪ᮏ◊✲࡛ࡢ ORPH ప⏝㔞⩌࡟࠾ࡅࡿ ORPH ࡢᦤྲྀ㔞㸦40.4 mg/kg య㔜/᪥㸧ࡢ 10 ศࡢ 1 ᮍ‶࡛࠶ࡿࠋࡲࡓ㸪㏣ຍᐇ㦂ࡢゎᯒ ࢆ⾜࠸ORPH ࡢ 375 ppm ΰ㣵ᢞ୚࡟࠾࠸࡚⫢Ⓨࡀࢇࡀಁ㐍ࡉࢀ࡞࠸ࡇ࡜ࢆ☜ㄆࡋࡓࠋ ࡋࡓࡀࡗ࡚㸪750 ppm ࡀ ORPH ࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝㜈್࡛࠶ࡿ࡜ồࡵࡽࢀࡓ 㸦Fig. 1-5㸧ࠋࡇࡢࡇ࡜࠿ࡽ㸪ࣄࢺࡢࣃ࣮࢟ࣥࢯࣥ⑓἞⒪࡟⏝࠸ࡽࢀࡿ⃰ᗘ࡛ࡣ⫢Ⓨࡀࢇ ࡣಁ㐍ࡉࢀ࡞࠸࡜⪃࠼ࡽࢀࡓࡀ㸪⫢Ⓨࡀࢇ࡟ᑐࡍࡿ㑇ఏẘᛶࡢ㛵୚࡟ࡘ࠸࡚ࡣ௒ᚋ⪃៖ ࡍࡿᚲせࡀ࠶ࡿࠋ CAR ࡢ᰾ෆ⛣⾜࡟ࡼࡾ CYP2B ࢆㄏᑟࡍࡿ໬Ꮫ≀㉁ࡣࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩ ࣙࣥస⏝ࢆᣢࡘࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ㹙5㹛ࠋChang㸦2009㸧ࡣ CAR ࢱࣥࣃࢡ㉁ࡀ⏕యෆ ࡢ␗≀ࢆࣜ࢞ࣥࢻ࡜ࡍࡿཷᐜయ࡛࠶ࡾCYP2B1 ࢆㄏᑟࡍࡿ࡜ሗ࿌ࡋ࡚࠸ࡿࠋCAR ࡣ⣽

(23)

22

⬊㉁࡟heat shock protein 90㸦HSP90㸧࡜ cytoplasmic CAR retention protein㸦CCRP㸧ࡢ」 ྜయ࡜ࡋ࡚ᏑᅾࡍࡿࠋCAR ࡀࣜ࢞ࣥࢻ࡜⤖ྜࡍࡿ࡜㸪HSP90 ࡜ CCRP ࡢ」ྜయ࠿ࡽ㞳 ࢀ㸪ࣜ࢞ࣥࢻ࡜⤖ྜࡋࡓCAR ࡣ᰾࡬⛣⾜ࡍࡿࠋ᰾ࡢ୰࡛ CAR ࡣ retinoid X receptor㸦RXR㸧 ࡜࣊ࢸࣟࢲ࢖࣐࣮ࢆᙧᡂ㹙14㹛ࡋࡓࡢࡕ㸪Cyp2b ࢆྵࡴ CAR ࡢᶆⓗ㑇ఏᏊࡢ DNA ᛂ⟅ ࢚࣓ࣞࣥࢺ࡟⤖ྜࡍࡿ㹙35㹛ࠋࡉࡽ࡟㸪CAR ࡣ Cyp2b ࡔࡅ࡛࡞ࡃゎẘ㸪⣽⬊ቑṪࡸ⭘⒆ ᙧᡂ࡟㛵୚ࡍࡿ㑇ఏᏊࢆไᚚࡋ࡚࠾ࡾ㹙3㹛㸪ࡑࡢ୰࡟ࡣ Cyclin D1 ࡢࡼ࠺࡟ CAR ࡟ࡼࡾ άᛶ໬ࡉࢀ⫢⣽⬊ቑṪࢆಁ㐍ࡍࡿᅉᏊࡶྵࡲࢀ࡚࠸ࡿ㹙23㹛ࠋࡑࢀࡽࡢᅉᏊࡢᙳ㡪࡛㸪 CAR ࡀ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿ㹙5㹛ࠋ

ORPH ࡟ࡼࡿ CYP2B ࡢㄏᑟ࡟࠾࠸࡚ࡶ CAR ࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ 㹙29㹛ࠋᮏ◊✲࡟࠾࠸࡚㸪ච␿⤌⧊໬Ꮫⓗ࡟ ORPH ᢞ୚࡟ࡼࡾ CAR ࡢ᰾ෆ⛣⾜ࡀቑຍ ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋmRNA ࡢⓎ⌧ࡣ㸪CAR ࡜ CAR 㛵㐃㑇ఏᏊࡢࡦ࡜ࡘ࡛࠶ࡿ

Slco1a4 ࡛ࡣ᭷ព࡞ኚືࢆ♧ࡉ࡞࠿ࡗࡓࡶࡢࡢ㸪CAR 㛵㐃㑇ఏᏊ࡛࠶ࡿ Mrp2 ཬࡧ Cyclin D1㹙23㸪25㹛ࡢ mRNA Ⓨ⌧ࡀ ORPH ᢞ୚⩌࡛ᑐ↷⩌ࡼࡾ᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋࡇࢀࡽ

ࡢ⤖ᯝ࠿ࡽ㸪ORPH ࡣࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ㸪ࡑ ࡢᶵᗎ࡟ࡣCAR ࡢ᰾ෆ⛣⾜ቑຍࡀ㛵୚ࡋ࡚࠸ࡿ࡜᥎ᐹࡉࢀࡿࠋ

CYP family ࡣ࣐࢖ࢡࣟࢯ࣮࣒ࡢ㓟໬๪⏘≀࡜ࡋ࡚ ROS ࢆ⏘⏕ࡍࡿࡓࡵ㸪CYP1A1 ࡸCYP1A2 ࡢቑຍࡣ㛫᥋ⓗ࡟ ROS ⏘⏕ࡢቑຍࢆㄏᑟࡍࡿ㹙31㸪33㹛ࠋࡋࡓࡀࡗ࡚㸪CYP1A inducer ࡣ ROS ࢆ⏘⏕ࡍࡿ≀㉁࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿࠋࡉࡽ࡟㸪PBO ࡸ β-naphthoflavone ࡞࡝ࡢCYP1A inducer ࡣࡑࡢ⫢⮚࡟࠾ࡅࡿ௦ㅰ㐣⛬࡛ ROS ࡀ⏘⏕ࡉࢀ㸪ࡑࢀ࡟క࠺㓟

(24)

23

໬ⓗࢫࢺࣞࢫ࡟ࡼࡾ⫢Ⓨࡀࢇస⏝ࢆ♧ࡍ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㹙6㸪28㹛ࠋྠᵝ࡟㸪CYP2B inducer ࡛࠶ࡿ PB ࡶࣛࢵࢺࡢ⫢⮚࡟࠾࠸࡚ࣄࢻࣟ࢟ࢩࣝࣛࢪ࢝ࣝࢆቑຍࡉࡏࡿࡇ࡜ࡀ▱ ࡽࢀ࡚࠸ࡿ㹙20㸪39㹛ࠋImaoka ࡽ㸦2004㸧ࡣ PB ࡟ࡼࡿ CYP2B1 ࡢㄏᑟࡣ ROS ⏘⏕ࢆቑ ຍࡉࡏ㸪DNA ࡢ㓟໬ࡀ⫢Ⓨࡀࢇ࢖ࢽࢩ࢚࣮ࢩࣙࣥࡸࣉ࣮ࣟࣔࢩࣙࣥ࡟㛵୚ࡋ࡚࠸ࡿ࡜ ሗ࿌ࡋ࡚࠸ࡿࠋᮏ◊✲࡛ࡣ㸪➨୍┦⸆≀௦ㅰ㓝⣲࡛࠶ࡿCyp1a1 ཬࡧ Cyp2b1/2 ࡀ ORPH

ᢞ୚࡟ࡼࡾ᭷ព࡟ቑຍࡋࡓࠋ࡞࠿࡛ࡶCyp2b1/2 ࡣ Cyp1a1 ࡼࡾࡶቑຍࡢ⛬ᗘࡀ኱ࡁࡃ㸪

ORPH ప⏝㔞⩌࡟࠾࠸࡚ࡶቑຍࡋࡓࠋࡇࡢ⤖ᯝ࠿ࡽ㸪ORPH ࡀ CYP2B inducer ࡛࠶ࡿࡇ ࡜ࡀ☜ㄆࡉࢀࡓࠋCYP2B inducer ࡣ⫢⮚࡟࠾ࡅࡿ௦ㅰ㐣⛬࡛ CYP2B ㄏᑟ࡟ࡼࡿ ROS ⏘ ⏕ࡢቑຍ࡜㸪ࡑࢀ࡟క࠺㓟໬ⓗࢫࢺࣞࢫ࡟ࡼࡗ࡚ࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ ♧ࡍࡇ࡜ࡀ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡿ㹙6㸪26㹛ࠋᮏ◊✲࡛ࡣ㸪ORPH ᢞ୚⩌࡛ ROS ⏘⏕ࡢቑ ຍࡀㄆࡵࡽࢀ㸪ORPH 㧗⏝㔞⩌࡛ TBARS 㔞ཬࡧ 8-OHdG 㔞ࡢቑຍࡀㄆࡵࡽࢀࡓࠋTBARS ࡣ⫢⣽⬊ࡢ⬡㉁㐣㓟໬ࡢᣦᶆ࡛࠶ࡿࡓࡵ㹙32㹛㸪ORPH 㧗⏝㔞⩌࡛ࡢ TBARS 㔞ࡢቑຍࡣ㸪㐣๫࡞ROS ࡢ⏘⏕ࢆಁࡋ㸪ࡑࡢ⤖ᯝ࡜ࡋ࡚⫢⣽⬊࡟㓟໬ⓗࢫࢺࣞࢫࡀㄏ㉳ࡉࢀࡓࡇ࡜ ࢆ♧၀ࡋ࡚࠸ࡿࠋ8-OHdG ࡣ㸪㓟໬ࡋࡓࢢ࢔ࢽࣥ࡜ࡋ࡚▱ࡽࢀ࡚࠾ࡾ㸪㓟໬ⓗ࡞⣽⬊യ ᐖࡢᣦᶆ࡛࠶ࡾ㹙18㹛㸪㓟໬ⓗ DNA ᦆയࡢᣦᶆࡢࡦ࡜ࡘ࡛࠶ࡿ㹙4㹛ࠋࡲࡓ㸪8-OHdG ࡢ ቑຍ࡟ࡼࡾㄏⓎࡉࢀࡿ㓟໬ⓗࢫࢺࣞࢫࡣ࣌ࣥࢱࢡࣟࣟࣇ࢙ࣀ࣮ࣝ࡟ࡼࡿ࣐࢘ࢫࡢ⫢Ⓨ ࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ㹙38㹛ࠋࡼࡗ࡚㸪 ORPH ࡟ࡼࡿࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟ࡣ㸪ROS ⏘⏕ࡢቑຍ࡟ࡼࡿ㓟໬ⓗࢫࢺࣞ ࢫࡀ㛵୚ࡋ࡚࠸ࡿ࡜᥎ᐹࡉࢀࡓࠋ

(25)

24 ࡉࡽ࡟㸪➨஧┦⸆≀௦ㅰ㓝⣲࡛࠶ࡾ㸪ᢠ㓟໬㓝⣲㑇ఏᏊ࡛࠶ࡿGpx2 ཬࡧ Gstm3 ࡢmRNA Ⓨ⌧ࡀ ORPH 㧗⏝㔞⩌࡛᭷ព࡟ቑຍࡋࡓࠋࡇࢀࡽࡢ㑇ఏᏊࡣ㓟໬ⓗࢫࢺࣞࢫ ࡟ᑐᛂࡋ࡚Nrf2 ࡟ࡼࡗ࡚ไᚚࡉࢀࡿ㹙1㹛ࠋGstm3 ࢆྵࡴ glutathione S-transferases 㸦GSTs㸧 ࡣࢢࣝࢱࢳ࢜ࣥ࡜」ྜయࢆᙧᡂࡍࡿࡇ࡜࡟ࡼࡾ␗≀ࢆゎẘࡍࡿ㹙37㹛ࠋGpx2 ࡣ H2O2ࢆ ῶᑡࡉࡏࡿglutathione peroxidase㸦GPX㸧family ࡟ྵࡲࢀࡿ㹙30㹛ࠋࡇࢀࡽࡢ㑇ఏᏊࡢⓎ ⌧ቑຍࡣORPH ᢞ୚࡟ࡼࡿ ROS ⏘⏕ࡀ Nrf2 㛵㐃㑇ఏᏊ࡟ࡼࡗ࡚ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࢆ ♧၀ࡋ࡚࠸ࡿࠋࡋ࠿ࡋ㸪ORPH 㧗⏝㔞⩌࡟࠾࠸࡚ TBARS ཬࡧ 8-OHdG ࡀቑຍࡋ࡚࠸ࡿ ࡇ࡜࠿ࡽ㸪Nrf2 㛵㐃ᢠ㓟໬㓝⣲࡟ࡼࡿᢠ㓟໬స⏝ࢆ ROS ࡢ㐣๫⏘⏕ࡀୖᅇࡾ㸪ORPH 㧗⏝㔞⩌࡛ࡣࣞࢻࢵࢡࢫࣂࣛࣥࢫࡢᔂࢀࡀ⏕ࡌ࡚࠸ࡿ࡜᥎ᐹࡉࢀࡿࠋ

ᮏ❶࡛ࡣ㸪ORPH ࡀࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜ ࡞ࡾ㸪ࡑࡢᶵᗎ࡟ࡣCAR ࡢάᛶ໬࡜㸪ᢠ㓟໬㓝⣲ࡢቑຍࢆୖᅇࡿ ROS ⏘⏕ࡢቑຍࡀ㛵 ୚ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

(26)

25 ᑠ ᑠᣓ

CYP2B inducer ࡛࠶ࡿ ORPH ࡢࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡢ᭷↓ࢆ㸪ࣛ ࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝࢆ⏝࠸᳨࡚ウࡋࡓࠋ6 㐌㱋㞝ᛶ F344 ࣛࢵࢺ࡟ DEN ࡛࢖ࢽࢩ ࢚࣮ࢩࣙࣥฎ⨨ࢆ᪋ࡋ㸪ࡑࡢ2 㐌㛫ᚋ࠿ࡽ 6 㐌㛫ࣉ࣮ࣟࣔࢱ࣮࡜ࡋ࡚ ORPH㸦0, 750, 1,500 ppm㸧ࡢΰ㣵ᢞ୚ࢆ⾜࠸㸪⫢⣽⬊ࡢቑṪάᛶࢆ㧗ࡵࡿࡓࡵ࡟ࣉ࣮ࣟࣔࢱ࣮ᢞ୚ 1 㐌㛫ᚋ ࡟ 2/3 㒊ศ⫢ษ㝖ࢆ᪋ࡋࡓࠋ⤖ᯝ㸪ORPH ᢞ୚࡟ࡼࡗ࡚⫢๓ࡀࢇ⑓ኚ࣐࣮࣮࡛࢝࠶ࡿ GST-P 㝧ᛶ⫢⣽⬊ᕢࡀቑຍࡋࡓࡓࡵ㸪ORPH ࡟ࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ࠶ ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡾ㸪㏣ຍᐇ㦂࡟ࡼࡗ࡚ࡑࡢస⏝㜈್ࡀ750 ppm ࡛࠶ࡿ࡜ồࡵࡽࢀࡓࠋ ᐇ㦂1 ࡢᮦᩱ࡛ゎᯒࢆ⾜࠸㸪ࡑࡢ࣓࢝ࢽࢬ࣒ゎᯒࢆ⾜ࡗࡓ⤖ᯝ, ➨୍┦⸆≀௦ㅰ㓝⣲࡛

࠶ࡿCyp2b1/2 ࡢ mRNA Ⓨ⌧ࡀቑຍࡋ㸪⣽⬊ෆཷᐜయ࡛࠶ࡿ CAR ࡢ᰾ෆ⛣⾜ࡢቑᙉࡀ

ච␿⤌⧊Ꮫⓗ࡟ㄆࡵࡽࢀࡓࠋࡲࡓ㸪CAR 㛵㐃㑇ఏᏊࡢ Mrp2 ࡢ mRNA Ⓨ⌧ࡀ ORPH ᢞ ୚࡟ࡼࡾቑຍࡋࡓࠋࡉࡽ࡟ROS ⏘⏕࡟ࡼࡾㄏᑟࡉࢀࡿ Gpx2 ࡸ Gstm3 ࡞࡝ࡢ Nrf2 㛵㐃 ᢠ㓟໬㓝⣲ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀ㸪࣐࢖ࢡࣟࢯ࣮࣒ROS ⏘⏕㸪⬡㉁㐣㓟໬ࡢᣦᶆ࡛ ࠶ࡿTBARS ཬࡧ DNA ᦆയࡢ࣐࣮࣮࡛࢝࠶ࡿ 8-OHdG ࡢቑຍࡶㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ ⤖ᯝ࠿ࡽ㸪ORPH ࡣࣛࢵࢺ࡟ᑐࡋ࡚⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ᭷ࡋ㸪ࡑࡢ࣓࢝ࢽࢬ ࣒࡟ࡣCAR ࡢ᰾ෆ⛣⾜࡜㓟໬ⓗࢫࢺࣞࢫࡀ㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

(27)

26 ➨ ➨2 ❶

Phenobarbital ཬࡧ orphenadrine ే⏝ᢞ୚࡟ࡼࡿࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝

(28)

27 ⥴ ⥴ゝ PBࡸPBO࡞࡝ࡢ࠸ࡃࡘ࠿ࡢCYP inducerࡣ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ♧ࡋ㸪 ࠿ࡘ㸪࣐࢖ࢡࣟࢯ࣮࣒⏤᮶ࡢROS⏘⏕ࡸ8-OHdG㸪TBARS࡞࡝ࡢ㓟໬ⓗࢫࢺࣞࢫᛂ⟅ࢆ ᘬࡁ㉳ࡇࡍࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚ࡁࡓ 㹙26㸪28㹛ࠋࡉࡽ࡟㸪➨1❶࡛ࡣ㸪CYP2Bࢆㄏᑟ ࡍࡿORPH࡟࠾࠸࡚ࡶ㓟໬ⓗࢫࢺࣞࢫࡀ㛵୚ࡋࡓࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ ࢆ♧ࡍࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ ࣛࢵࢺ࡟࠾࠸࡚㸪⫢Ⓨࡀࢇ≀㉁ࢆప⏝㔞࡛」ᩘే⏝ᢞ୚ࡋࡓሙྜ㸪୍⯡ⓗ࡞Ⓨࡀ ࢇ⏝㔞ࡼࡾࡶప࠸⏝㔞ࢆ⏝࠸࡚ࡶ㸪Ⓨࡀࢇస⏝ࡀⓎ⌧ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀ࡚࠸ࡿ㹙9㹛ࠋ ࡶࡋ」ᩘࡢ໬Ꮫ≀㉁࡟ྠ᫬࡟ᭀ㟢ࡉࢀࡿࡇ࡜࡟ࡼࡾࡑࡢⓎࡀࢇస⏝ࡀቑᙉࡍࡿ࡞ࡽࡤ㸪 ࡑࡢࣜࢫࢡホ౯ࢆ෌⪃ࡍࡿᚲせࡀ࠶ࡿࠋᮏ◊✲࡟ࡼࡾ㸪」ᩘࡢ໬Ꮫ≀㉁ࡢే⏝ᢞ୚࡟ࡼ ࡿᙳ㡪ࢆ᳨ウࡍࡿࡇ࡜ࡣ㸪㣗ရ୰࡟ྵࡲࢀࡿ໬Ꮫ≀㉁ࡢࣜࢫࢡホ౯࡟࠾࠸࡚᭷┈࡞᝟ሗ ࢆᥦ౪࡛ࡁࡿࡶࡢ࡜⪃࠼ࡿࠋࡑࡇ࡛㸪PB࡜ORPHࡢCYP2B inducerྠኈࢆే⏝ᢞ୚ࡍࡿ ࡇ࡜࡟ࡼࡾ㸪CYP2Bࡢㄏᑟࡀቑᙉࡋ㸪ࡑࢀ࡟క࠸ROS⏘⏕ࡀቑຍࡋ㸪ࡑࡢ⤖ᯝ࡜ࡋ࡚㓟 ໬ⓗࢫࢺࣞࢫࡢቑຍ࡟ࡼࡗ࡚⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀቑᙉࡉࢀࡿࡶࡢ࡜௬ᐃࡋ㸪 ௨ୗࡢᐇ㦂ࢆ⾜ࡗࡓࠋ ᮏ◊✲࡛ࡣ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ♧ࡍCYP2B inducer࡛࠶ࡿPBཬࡧ ORPHࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪ࡑࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝࡟࡝ࡢࡼ࠺࡞ಟ㣭 స⏝ࡀぢฟࡉࢀࡿࡢ࠿ࢆ᳨ウࡍࡿ࡜ඹ࡟㸪ࡑࡢಟ㣭స⏝ࡢⓎ⌧ᶵᗎࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ

(29)

28

ྛ✀ࡢ㑇ఏᏊⓎ⌧ࡢ௚㸪࣐࢖ࢡࣟࢯ࣮࣒ROS⏘⏕㸪TBARS➼㓟໬ⓗࢫࢺࣞࢫ࡟㛵୚ࡍ ࡿ⏕໬Ꮫⓗ࡞ᣦᶆ࡟↔Ⅼࢆ⤠ࡾ㸪ศᏊ⑓⌮Ꮫⓗ࡟ゎᯒࡋࡓࠋ

(30)

29 ᮦ

ᮦᩱཬࡧ᪉ἲ

໬Ꮫ≀㉁

PB sodium㸦CAS No. 57-30-7㸪⣧ᗘ98%㸧ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ 㸦኱㜰㸧࠿ࡽ㸪 ORPH㸦CAS No. 4682-36-4㸪⣧ᗘ99%㸧ࡣSigma-Aldrich㸦࢔࣓ࣜ࢝㸧࠿ࡽ㉎ධࡋࡓࠋDEN 㸦CAS No. 55-18-5㸪⣧ᗘ99%㸧ࡣᮾி໬ᡂᕤᴗ㸦ᮾி㸧࠿ࡽ㉎ධࡋࡓࠋ ౑⏝ື≀ ᐇ㦂2࡟⏝࠸ࡓ5㐌㱋ࡢ㞝ᛶF344ࣛࢵࢺࡣ㸪᪥ᮏSLCᰴᘧ఍♫㸦㟼ᒸ㸧ࡼࡾ㉎ධࡋ㸪 12᫬㛫᫂ᬯࢧ࢖ࢡࣝ㸪 ᗘ20㹼26Υ㸪‵ᗘ40~70%ࡢ᮲௳ୗ࡛3࡞࠸ࡋ4༉ࡎࡘᗋᩜࡁ㸦࣌ ࣃ࣮ࢡ࣮ࣜࣥ㸹᪥ᮏSLCᰴᘧ఍♫㸪㟼ᒸ㸧ࢆᩜ࠸ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢪ࡟࡚㣫⫱ࢆ⾜ࡗ ࡓࠋ⢊ᮎ㣫ᩱ㸦࢚࢜ࣜࣥࢱࣝMF㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸪ᮾி㸧ཬࡧ㣧ᩱỈࡣ⮬⏤ᦤ ྲྀࡉࡏࡓࠋ1㐌㛫ࡢ᳨␿࣭㥆໬ᮇ㛫ࢆタࡅࡓᚋ㸪ᐇ㦂࡟౪ࡋࡓࠋ࡞࠾㸪ື≀ᐇ㦂ࡣᮾி ㎰ᕤ኱Ꮫᐇ㦂ື≀೔⌮つᐃ࡟‽ᣐࡋ࡚⾜ࡗࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ᐇ㦂2 ௨ୗࡢᡭ㡰࡟ᚑࡗ࡚㸪ࣛࢵࢺ⫢Ⓨࡀࢇ஧ẁ㝵ࣔࢹࣝ࡟ࡼࡿᐇ㦂㹙17㹛ࢆ⾜ࡗࡓࠋ ࡍ࡞ࢃࡕ㸪඲࡚ࡢື≀࡟ᑐࡋDENࡢ༢ᅇ⭡⭍ෆᢞ୚㸦200 mg/kg య㔜㸧ࢆ⾜࠸㸪ࡑࡢ2

(31)

30 㐌㛫ᚋ࠿ࡽ㸪ᇶ♏㣫ᩱࡢࡳ㸦DEN༢⊂⩌㸪n =12㸧㸪PB㸦60 ppm㸹PBప⏝㔞⩌㸪n = 12 ࡞࠸ࡋ120 ppm㸹PB㧗⏝㔞⩌㸪n = 12㸧ࡢ㣧Ỉᢞ୚㸪ORPH㸦750 ppm㸹ORPHప⏝㔞⩌㸪 n = 12࡞࠸ࡋ1,500 ppm㸹ORPH㧗⏝㔞⩌㸪n = 12㸧ࡢΰ㣵ᢞ୚㸪ࡶࡋࡃࡣ60 ppm PBཬࡧ 750 ppm ORPHࡢే⏝ᢞ୚㸦ే⏝ᢞ୚⩌㸪n = 12㸧ࢆ6㐌㛫⾜ࡗࡓࠋ⏝㔞タᐃ࡟࠾࠸࡚ࡣ㸪 ప⏝㔞࡟⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ♧ࡍ㜈್ࢆ㸪㧗⏝㔞࡟ࡣࡑࢀࡒࢀࡢ㜈್ࡢ2ಸ 㔞ࢆ⏝࠸㸪ే⏝ᢞ୚࡟ࡣࡑࢀࡒࢀࡢప⏝㔞ࢆ⏝࠸ࡓࠋ㜈್タᐃࡣ㸪PB࡟ࡘ࠸࡚ࡣKitano ࡽ㸦1998㸧ࡢሗ࿌࡟ᇶ࡙ࡁ60 ppm㸪ORPHࡣ➨1❶ࡢ⤖ᯝ࠿ࡽ750 ppm࡜ࡋࡓࠋࡉࡽ࡟⫢ ⣽⬊ࡢቑṪάᛶࢆ㧗ࡵࡿࡓࡵ㸪඲࡚ࡢື≀࡟2/3㒊ศ⫢ษ㝖ࢆDENᢞ୚3㐌㛫ᚋ࡟ᐇ᪋ࡋ ࡓࠋORPHప⏝㔞⩌࡟࠾࠸࡚1༉㸪DEN༢⊂⩌࡟࠾࠸࡚2༉ࡀ㒊ศ⫢ษ㝖ࡢᢏ⾡ⓗ㐣ኻ࡟ ࡼࡾṚஸࡋࡓࠋᢞ୚ᮇ㛫⤊஢ᚋ㸪࢖ࢯࣇࣝࣛࣥ῝㯞㓉ୗ࡟࡚ᨺ⾑ẅࢆ⾜࠸㸪⫢⮚ࢆ᥇ྲྀ ᚋ㸪㔜㔞 ᐃࢆ⾜ࡗࡓࠋ⫢⮚ࡢ⤌⧊Ꮫⓗ᳨⣴ཬࡧච␿⤌⧊໬Ꮫⓗ᳨⣴࡟ࡣ㸪ゎ๗᫬࡟ࢫ ࣛ࢖ࢫࡋࡓ⫢⤌⧊ࢆ4%ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻ⁐ᾮ࡟࡚ᅛᐃࢆ⾜࠸㸪ࡲࡓ㸪ṧࡾࡢ⫢ࢧ ࣥࣉࣝࡣ㑇ఏᏊⓎ⌧ཬࡧ⏕໬Ꮫⓗゎᯒ⏝࡜ࡋ࡚ᾮయ❅⣲࡟ᾐₕࡋ࡚෾⤖ࡋ-80Υ࡟࡚ಖ Ꮡࡋࡓࠋ ⑓ ⑓⌮⤌⧊Ꮫⓗゎᯒ࣭ච␿⤌⧊໬Ꮫⓗゎᯒ ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻᅛᐃᚋ㸪ᐃἲ࡟ᚑ࠸࢚ࢱࣀ࣮ࣝ⣔ิ࡛⬺Ỉࢆ⾜࠸㸪ࣃࣛࣇ ࢕ࣥ࡟ໟᇙࡋࡓࠋ㐃⥆ษ∦ࢆస〇ࡋ㸪୍㒊ࡣH&E ᰁⰍࢆ⾜ࡗࡓࠋࡉࡽ࡟ GST-P㸪PCNA

(32)

31 ࡢච␿⤌⧊໬ᏛᰁⰍࢆ௨ୗࡢᡭ㡰࡛⾜ࡗࡓࠋጞࡵ࡟⬺ࣃࣛࣇ࢕ࣥฎ⌮ࢆࡋࡓษ∦ࢆ㸪 0.3%㐣㓟໬Ỉ⣲ࢆྵࡴ࣓ࢱࣀ࣮ࣝ࡟ࡼࡾᐊ ࡛ 30 ศ㛫ෆᅉᛶ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ࢆ ࡋ㸪ṇᖖ⾑Ύ࡟࡚ࣈࣟࢵ࢟ࣥࢢฎ⌮ࢆ⾜ࡗࡓࠋࡲࡓ㸪࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ᚋ㸪PCNA ࡟㛵ࡋ࡚ࡣࢡ࢚ࣥ㓟⦆⾪ᾮ㸦pH 6.0㸧ୗ࡛ 60Υ㸪20 ศࡢ ᾎࢆ⾜ࡗࡓࠋࡑࡢᚋ㸪࢘ࢧ ࢠᢠGST-P ࣏ࣜࢡ࣮ࣟࢼࣝᢠయ㸦1,000 ಸᕼ㔘㸪ᰴᘧ఍♫་Ꮫ⏕≀Ꮫ◊✲ᡤ㸪ឡ▱㸧ཬ ࡧ࣐࢘ࢫᢠPCNA ࣔࣀࢡ࣮ࣟࢼࣝᢠయ㸦500 ಸᕼ㔘㸪Dako㸪ࢹ࣐࣮ࣥࢡ㸧ࢆࡑࢀࡒࢀ 4Υ ୍ᬌ཯ᛂࡉࡏࡓࠋ୍ḟᢠయ཯ᛂᚋ㸪Vectastain Elite ABC kit㸦Vector Laboratories㸪࢔࣓ࣜ ࢝㸧ࢆ⏝࠸㸪DAB ࡟ࡼࡿⓎⰍᚋ㸪࣐࣊ࢺ࢟ࢩ࡛ࣜࣥ᰾ࡢᑐẚᰁⰍࢆ⾜ࡗࡓࠋ

GST-P 㝧ᛶ⫢⣽⬊ᕢ࡟ࡘ࠸࡚ࡣ㸪┤ᚄ 200 μm ௨ୖ࡛᫂ࡽ࠿࡞㝧ᛶ⫢⣽⬊ᕢࡢษ ∦㠃✚ᙜࡓࡾࡢಶᩘཬࡧ㠃✚⋡ࢆ⏬ീゎᯒࢯࣇࢺWinROOF㸦୕㇂ၟ஦㸪ᮾி㸧ࢆ⏝࠸ ࡚ゎᯒࡋࡓࠋPCNA 㝧ᛶ⫢⣽⬊࡟ࡘ࠸࡚ࡣ㸪1 ౛࡟ࡘࡁ↓సⅭ࡟㑅ࢇࡔ 10 ど㔝㸦1 ど㔝 ࠶ࡓࡾ700㹼900 ⫢⣽⬊㸧࡟࡚ᙉ㝧ᛶ⫢⣽⬊ࡢ ᐃࢆ⾜࠸㸪ⓒศ⋡࡛⾲♧ࡋࡓࠋ

Real-timereverse transcription polymerase chain reaction㸦㸦RT-PCR㸧ゎᯒ

㑇ఏᏊⓎ⌧ゎᯒ࡟౑⏝ࡍࡿtotal RNA ࢧࣥࣉࣝࡣ㸪RNeasy kit㸦Qiagen㸧࡟ࡼࡾᢳ ฟࡋࡓࠋcDNA ྜᡂ࡬ࡢ㏫㌿෗཯ᛂࡣ 2 μg ࡢ total RNA ࠿ࡽ Thermoscript RT-PCR system 㸦Life Technologies㸪CA㸪࢔࣓ࣜ࢝㸧࡟࡚⾜ࡗࡓࠋᐃ㔞real-time RT-PCR ࡣ PowerSYBR®

Green PCR Master Mix㸦Life Technologies㸧ཬࡧStepOnePlusTM_{Real-Time PCR System㸦}_Life

(33)

32

Technologies㸧࡟࡚స〇ࡋࡓࠋྛࣉࣛ࢖࣐࣮㓄ิࡣTable 2-1 ࡟グ㍕ࡋࡓࠋ㑇ఏᏊⓎ⌧ࡢ

ᐃ㔞໬ࡣ㸪ෆᅾᛶࢥࣥࢺ࣮ࣟࣝ㑇ఏᏊ 㸦Actb㸧࡟ᑐࡋ࡚ 2-ΔΔCt_{ἲ࡟๎ࡾᶆ‽໬ࡋࡓ㹙24㹛ࠋ}

㐣

㐣㓟໬⬡㉁ ᐃ

⫢⮚࡟࠾ࡅࡿ㐣㓟໬⬡㉁ ᐃࡣ TBARS 㔞ࢆ ᐃࡋࡓࠋ⫢ TBARS 㔞ࡣ Ohkawa ࡽ㸦1979㸧ࡢ᪉ἲ࡟ᚑ࠸ 1 ⩌࠶ࡓࡾ 6 ౛࡟ࡘ࠸࡚ ᐃࡋࡓࠋࡍ࡞ࢃࡕ㸪⫢ࢧࣥࣉࣝ㸦⣙ 50 mg㸧ࢆ 450 μl ࡢ 1.15% KCl ⦆⾪ᾮ୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋ ⺮ⓑ⃰ᗘ ᐃࡣBCA protein assay kit㸦Pierce, ࢔࣓ࣜ࢝㸧࡟࡚⾜ࡗࡓࠋᆒ㉁໬ࡋࡓ⫢࣍ ࣔࢪࢿ࣮ࢺ㸦17.8 mg protein㸧ࢆ 0.2 mL ࡢ 8.1% SDS ࡜ 3.0 mL ࡢ 0.4% thiobarbituric aci-10㸣㓑㓟⁐ᾮ࡜࡛ 95Υ㸪60 ศ㛫཯ᛂࡉࡏࡓᚋ㸪෭Ỉ࡛෭༷ࡋࡓࠋ཯ᛂᾮ࡟ 1 mL ࡢ ⵨␃Ỉࢆຍ࠼㸪2.0 mL ࡢ n-butanol-pyridine ΰྜᾮ㸦15:1㸧ࢆຍ࠼ࡓᚋ㸪2,100 ×g㸪10 ศ 㛫㐲ᚰࡋࡓࠋ532 nm ࡟࠾ࡅࡿ྾ගᗘࡣ㸪ศගගᗘィ SynergyTM_{HT Multi-Detection}

Microplate Reader 㸦 BioTek 㸪 ࢔ ࣓ ࣜ ࢝ 㸧 ࡟ ࡼ ࡾ ᐃ ࡋ ࡓ ࠋ TBARS ᐃ ್ ࡣ 1,1,3,3-tetramethoxypropane ⏤᮶ࡢ malondialdehyde㸦MDA㸧㔞࡟࡚ᐃ㔞ࡋࡓࠋ

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢㄪ〇ࡣ㸪Yoshihara ࡽ㸦2001㸧ࡢሗ࿌࡟ᇶ࡙ࡁⱝᖸࡢኚ᭦ ࢆຍ࠼⾜ࡗࡓࠋ⫢ࢧࣥࣉࣝ㸦⣙100㹼130 mg㸧ࢆ 3 ಸ㔞ࡢ⦆⾪ᾮ㸦50 mM Tris-HCl㸹pH 7.4㸪1.15% KCl㸪0.2 mM EDTA㸪0.1 mM DTT㸪0.1 mM Protease Inhibitor Cocktail㸪20%

(34)

33

glycerol㸧୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋḟ࡟ࡑࡢ࣍ࣔࢪࢿ࣮ࢺࢆ 700 ×g ࡛ 10 ศ㛫㐲ᚰࢆ⾜࠸㸪ୖΎࢆ 10,000 ×g ࡛ 20 ศ㛫㐲ᚰࡋࡓࠋᚓࡽࢀࡓୖΎࢆࡉ ࡽ࡟105,000 ×g㸪90 ศ㛫㉸㐲ᚰࡋࡓᚋ㸪ỿ´ࢆ 20 mM Tris-HCl㸦pH 7.4㸧࡟ᠱ⃮ࡋࡓࡶ ࡢࢆ࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡜ࡋࡓࠋ⺮ⓑ⃰ᗘ ᐃࡣBCA protein assay kit㸦Pierce㸧࡟࡚⾜ ࡗࡓࠋ

࣐

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢROS ⏘⏕⬟ ᐃ

ศ㛫཯ᛂࡉࡏࡓࠋ཯ᛂ㛤ጞࡣnicotinamide adenine dinucleotide phosphate㸦NADPH㸹᭱⤊ ⃰ᗘ0.5 mM㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸧ࢆῧຍࡍࡿࡇ࡜࡟ࡼࡾ⾜࠸㸪⏕ᡂࡉࢀࡿ⺯ගࢆ ບ㉳Ἴ㛗 485 nm㸪528 nm ࡢ᮲௳࡛ 120 ศ㛫 5 ศࡈ࡜࡟㸪ศගගᗘィ SynergyTM HT Multi-Detection Microplate Reader㸦BioTek㸧࡟ࡼࡾ ᐃࡋࡓࠋ㝧ᛶᑐ↷࡟ H2O2㸦᭱⤊⃰

ᗘ0.1 mM㸧ࢆ㸪ヨ㦂⣔ࡢ᳨ド࡟ CYP 㜼ᐖ๣࡜ࡋ࡚▱ࡽࢀࡿ SKF-525A㸦᭱⤊⃰ᗘ 0.1 mM 㸹Tronto Reserch Chemicals㸪࢝ࢼࢲ㸧ࢆῧຍࡋࡓࠋࢹ࣮ࢱࡣ DEN ༢⊂⩌ࢆ 100%࡜ ࡋ࡚ᶆ‽໬ࡋࡓࠋ

(35)

34 ⤫ ⤫ィゎᯒ ඲࡚ࡢࢹ࣮ࢱࡣᖹᆒ್ཬࡧᶆ‽೫ᕪ࡟࡚⾲グࡋࡓࠋศᩓࡢᆒ୍ᛶࡢ᳨ᐃࡣ㸪 Bartlett ᳨ᐃࢆ⾜ࡗࡓࠋᆒ୍ᛶࡀ࠶ࡿሙྜࡣ Dunnet ࡢከ㔜ẚ㍑᳨ᐃࢆ⾜࠸㸪ᆒ୍ᛶࡀ࡞ ࠸ሙྜ࡟ࡣ Steel ᳨ᐃࢆ⾜ࡗࡓࠋࡉࡽ࡟㸪ే⏝ᢞ୚࡟ࡼࡿಟ㣭స⏝ࢆホ౯ࡍࡿࡓࡵ㸪 Futakuchi ࡽ㸦1996㸧ࡸ Hasegawa ࡽ㸦1991㸧ࡀ᥎ዡࡍࡿ᪉ἲࢆ⏝࠸࡚⤫ィゎᯒࢆ⾜ࡗࡓࠋ P ್ࡀ 0.05 ᮍ‶ࡢሙྜ㸪⤫ィᏛⓗ࡟᭷ព࡛࠶ࡿ࡜ุ᩿ࡋࡓࠋ

(36)

35 ⤖ ⤖ᯝ ᐇ㦂2 య㔜㸪⫢㔜㔞㸪ᦤ㣵ᦤỈ㔞ཬࡧ᥎ᐃ⸆≀ᦤྲྀ㔞ヨ㦂ᮇ㛫ࢆ㏻ࡌ࡚㸪඲࡚ࡢᢞ୚⩌࡟ࡘ࠸࡚㸪⿕᳨≀㉁࡟㉳ᅉࡍࡿ⮫ᗋ⑕≧ཬࡧṚ ஸࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋORPH 㧗⏝㔞⩌࡛ࡣ㸪DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚య㔜ቑຍᢚไࡀ ࡳࡽࢀ㸪๗᳨᫬ࡢయ㔜ࡀDEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ῶᑡࡋ࡚࠸ࡓ㸦Table 2-2㸧ࠋᖹᆒ ᦤ㣵㔞ཬࡧᖹᆒᦤỈ㔞ࡣORPH 㧗⏝㔞⩌࡟࠾࠸࡚ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ᑡ࡞࠿ ࡗࡓ㸦Table 2-2㸧ࠋ ⤯ᑐ⫢㔜㔞ࡣ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ PB ᢞ୚⩌ࡀ㸪ORPH ప⏝㔞⩌ཬࡧ㧗⏝㔞 ⩌࡜ẚ㍑ࡋ࡚ే⏝ᢞ୚⩌ࡀ᭷ព࡟ቑຍࡋࡓࠋ┦ᑐ⫢㔜㔞ࡣ PB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌㸪 ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡟࠾࠸࡚ DEN ༢⊂⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋࡲࡓ㸪 ే⏝ᢞ୚⩌࡟࠾࠸࡚PB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌ཬࡧ ORPH ప⏝㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ ࡚࠸ࡓ㸦Table 2-2㸧ࠋPB ཬࡧ ORPH ᦤྲྀ㔞ࡣ Table 2-2 ࡟♧ࡍ㏻ࡾ࡛࠶ࡿࠋప⏝㔞⩌࡜ ే⏝ᢞ୚⩌ࡢ㛫࡟⸆≀ᦤྲྀ㔞ࡢ㐪࠸ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

⑓⌮⤌⧊Ꮫⓗゎᯒ㸪GST-P 㝧ᛶ⫢⣽⬊ᕢཬࡧ PCNA 㝧ᛶ⣽⬊

⑓⌮⤌⧊Ꮫⓗゎᯒ࡛ࡣ㸪PB ཬࡧ ORPH ᢞ୚⩌࡜ࡶ࡟⣽⬊㉁ࡢዲ㓟ᛶ໬ࢆక࠺⫢ ⣽⬊⫧኱ࡀㄆࡵࡽࢀࡓࠋ⫢⣽⬊✵⬊ኚᛶཬࡧኚ␗⫢⣽⬊ᕢ㸦᫂⣽⬊ᛶཬࡧዲ㓟ᛶ㸧ࡣ

(37)

36 PB 㧗⏝㔞⩌㸪ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡛ㄆࡵࡽࢀࡓ㸦Fig. 2-1㸧ࠋච␿⤌⧊໬Ꮫⓗ ゎᯒ࡟࠾࠸࡚㸪GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘࡣ PB 㧗⏝㔞⩌㸪ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ ୚⩌࡛ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ㸪ే⏝ᢞ୚⩌࡟࠾࠸࡚ PB ప⏝㔞⩌ཬࡧ ORPH ప⏝㔞⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋGST-P 㝧ᛶ⫢⣽⬊ᕢࡢ㠃✚ࡣే⏝ᢞ୚ ⩌࡟࠾࠸࡚DEN ༢⊂⩌㸪PB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌ཬࡧ ORPH ప⏝㔞⩌ࡼࡾࡶ᭷ព࡟ ቑຍࡋࡓ㸦Fig. 2-1㸪2-2A㸪2-2B㸧ࠋࡇࡢ⤖ᯝࡀ┦ຍ┦஌ⓗ࡛࠶ࡿ࠿ྰ࠿ࢆ᫂ࡽ࠿࡟ࡍࡿ ࡓࡵ࡟┦ຍࣔࢹࣝ࡟ࡼࡿ᳨ᐃࢆ⾜ࡗࡓࠋ␗✀┦ຍࣔࢹࣝࢆ⏝࠸ࡓ᳨ᐃࢆ⾜ࡗࡓ⤖ᯝ㸪 GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘཬࡧ㠃✚ࡣ㸪ే⏝ᢞ୚⩌ࡢṇ࿡್㸦DEN ༢⊂⩌ࡢ್ࢆᘬ࠸ ࡓ್㸹ಶᩘ㸸5.03 ಶ/cm2_{㸪㠃✚㸸0.66 mm}2_/cm2_{㸧ࡀప⏝㔞⩌ࡢṇ࿡್ࡢྜィ㸦ಶᩘ㸸1.55} ಶ/cm2_{㸪㠃✚㸸0.08 mm}2_/cm2_{㸧ࡼࡾࡶ᭷ព࡟኱ࡁࡃ㸦ಶᩘ㸸}_{p < 0.05㸪㠃✚㸸p < 0.01㸧㸪} ྠ✀┦ຍࣔࢹࣝࢆ⏝࠸ࡓ᳨ᐃ࡟࠾࠸࡚ࡶ GST-P 㝧ᛶ⫢⣽⬊ᕢࡢ㠃✚㸦1.04 ± 0.43 mm2_/cm2_{㸧ࡣ㸪ే⏝ᢞ୚⩌ࡀ㧗⏝㔞⩌ࡢᖹᆒ㸦0.65 ± 0.52 mm}2_/cm2_{㸧ࡼࡾࡶ᭷ព࡟኱ࡁ} ࠸㸦p < 0.05, t-test㸧ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦Fig. 2-2C㸪2-2D㸧ࠋ PCNA 㝧ᛶ⫢⣽⬊⋡ࡣ㸪PB 㧗⏝㔞⩌㸪ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡟࠾࠸࡚ DEN ༢⊂⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ㸪ే⏝ᢞ୚⩌࡟࠾࠸࡚ࡣ PB 㧗⏝㔞⩌ཬࡧ ORPH ప⏝ 㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ࡚࠸ࡓ㸦Table 2-2㸪Fig. 2-1㸧ࠋ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚ PCNA 㝧ᛶ⫢⣽⬊⋡ࡣే⏝ᢞ୚⩌࡟࠾ࡅࡿ᭷ព࡞ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡀ㸪ྠ✀┦ຍࣔࢹࣝ ࡟࠾࠸࡚ే⏝ᢞ୚⩌㸦0.51 ± 0.03%㸧ࡀ㧗⏝㔞⩌ࡢᖹᆒ㸦0.35 ± 0.01%㸧ࡼࡾࡶ᭷ព࡟ቑ ຍࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦p < 0.05, t-test㸧ࠋ

(38)

37 Real-time RT-PCR ࡟࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ

Table 2-1 ࡟♧ࡋࡓ㑇ఏᏊ࡟㛵ࡋ࡚㸪ྛ⩌ 6 ౛ࡎࡘ real-time RT-PCR ゎᯒࢆ⾜ࡗࡓ 㸦Table 2-3㸧ࠋ➨୍┦⸆≀௦ㅰ㓝⣲࡛࠶ࡿ Cyp1a1 ࡢ mRNA Ⓨ⌧ࡣ ORPH ᢞ୚⩌࡛㸪

Cyp2b1/2 ࡢ mRNA Ⓨ⌧ࡣ PB ཬࡧ ORPH ᢞ୚⩌࡛ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ

ࡓࠋࡲࡓ㸪ే⏝ᢞ୚⩌࡟࠾ࡅࡿCyp2b1/2 ࡢ mRNA Ⓨ⌧ࡣ PB ప⏝㔞⩌ཬࡧ ORPH ప⏝

㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋࡓࠋࡉࡽ࡟㸪␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚ే⏝ᢞ୚⩌ࡢ Cyp2b1/2 ࡢmRNA Ⓨ⌧ࡢṇ࿡್ࡀప⏝㔞⩌ࡢṇ࿡್ྜィࡼࡾࡶ᭷ព࡟ቑຍࡋ㸦ే⏝⩌ṇ࿡್㸸 77.91 ಸ㸪ప⏝㔞⩌ṇ࿡್ྜィ㸸51.74 ಸ㸪p < 0.01㸧ࠋྠ✀┦ຍࣔࢹ࡛ࣝࡣ᭷ព࡞ኚ໬ ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ ຍ࠼࡚㸪➨஧┦⸆≀௦ㅰ㓝⣲࡛࠶ࡾ㓟໬ⓗࢫࢺࣞࢫ࡟཯ᛂࡋ࡚ᢠ㓟໬ࡸゎẘ࡟ാ ࡃ㓝⣲㑇ఏᏊ࡛࠶ࡿGstm3 ࡸ Gpx2 ࡢ mRNA Ⓨ⌧ࡀ PB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌㸪ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡛DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓࠋే⏝ᢞ୚⩌࡟࠾࠸ ࡚ࡣGstm3 ࡀ PB ప⏝㔞⩌ཬࡧ ORPH ప⏝㔞⩌ࡼࡾࡶ㸪Gpx2 ࡀ ORPH ప⏝㔞⩌ࡼࡾࡶ ᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚Gstm3 ཬࡧ Gpx2 ࡢే⏝ᢞ୚⩌࡛ࡢ᭷ព ࡞ቑຍࡀㄆࡵࡽࢀ㸦Gstm3㸹ే⏝⩌ṇ࿡್㸸9.31 ಸ㸪ప⏝㔞⩌ṇ࿡್ྜィ㸸3.71 ಸ㸪p < 0.01㸧㸦Gpx2㸹ే⏝⩌ṇ࿡್㸸1.61 ಸ㸪ప⏝㔞⩌ṇ࿡್ྜィ㸸0.81 ಸ㸪p < 0.05㸧㸪ྠ✀ ┦ຍࣔࢹࣝ࡟࠾࠸࡚ࡶGstm3 ࡢే⏝ᢞ୚⩌࡛ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ㸦ే⏝ᢞ୚⩌㸸 10.33 ± 4.95 ಸ, 㧗⏝㔞⩌㸸7.53 ± 6.98 ಸ㸪p < 0.05㸪t-test㸧ࠋ

(39)

38 ࣐

࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡟࠾ࡅࡿROS ⏘⏕⬟

࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡟࠾ࡅࡿCYP άᛶ⏤᮶ࡢ ROS ⏘⏕ࢆ ᐃࡋࡓ㸦Table 2-4㸧ࠋ CYP ࡢࣔࣀ࢜࢟ࢩࢤࢼ࣮ࢮάᛶࡣ NADPH ࠿ࡽࡢ㑏ඖຊࡀᚲせ࡛࠶ࡿࡓࡵ㸪ROS ⏘⏕ ࢆNADPH Ꮡᅾୗ࣭㠀Ꮡᅾୗ࡟࡚ẚ㍑ࡋࡓࠋNADPH 㠀Ꮡᅾୗ࡛ࡣ㸪඲࡚ࡢ⩌࡟࠾࠸࡚ ROS ⏘⏕࡟ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡋ࠿ࡋ㸪NADPH ࢆῧຍࡋࡓ࡜ࡇࢁ㸪࣐࢖ࢡࣟࢯ࣮ ࣒⏬ศ࡟࠾ࡅࡿROS ⏘⏕ࡣ᫂ࡽ࠿࡟ቑຍࡋ㸪PB ཬࡧ ORPH ᢞ୚⩌࡛ DEN ༢⊂⩌࡜ẚ ㍑ࡋ࡚᭷ព࡟㧗್࡛࠶ࡗࡓࠋే⏝ᢞ୚⩌࡟࠾࠸࡚ࡣ㸪PB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌㸪ཬࡧ ORPH ప⏝㔞⩌࡟ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋH2O2 ࢆῧຍࡋࡓ࡜ࡇࢁ㸪࣐࢖ࢡࣟࢯ

࣮࣒ROS ⏘⏕ࡣቑຍࡋ㸦ࢹ࣮ࢱࡣ┬␎㸧㸪CYP ࡢ㜼ᐖ๣࡛࠶ࡿ SKF-525A ࢆῧຍࡋࡓ࡜ ࡇࢁ㸪ROS ⏘⏕ࡢపୗࡀぢࡽࢀࡓ㸦Table 2-4㸧ࠋ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚ే⏝ᢞ୚⩌ࡢ ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ㸦ే⏝⩌ṇ࿡್㸸117.91%㸪ప⏝㔞⩌ṇ࿡್ྜィ㸸110.84%㸪p < 0.05㸧ࡀ㸪ྠ✀┦ຍࣔࢹ࡛ࣝࡣ᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

⣽⬊⭷࡟ᑐࡍࡿ㓟໬ⓗࢫࢺࣞࢫ

ROS ⏘⏕࡟ࡼࡿ⣽⬊࡬ࡢ㓟໬ⓗ㞀ᐖࢆ᳨⣴ࡍࡿࡓࡵ㸪⫢⮚୰ࡢ TBARS 㔞ࢆ ᐃ ࡋࡓ㸦Table 2-4㸧ࠋTBARS 㔞ࡣ ORPH 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡟࠾࠸࡚ DEN ༢⊂⩌࡟ ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓࠋే⏝ᢞ୚⩌ࡣPB ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌㸪ཬࡧ ORPH ప⏝㔞

(40)

39

⩌࡟ẚ࡭࡚᭷ព࡟ቑຍࡋࡓࠋ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚ే⏝ᢞ୚⩌࡛ࡢ᭷ព࡞ቑຍࡀㄆࡵ ࡽࢀࡓ㸦ే⏝⩌ṇ࿡್㸸0.45 nmol MDA/mg protein㸪ప⏝㔞⩌ṇ࿡್ྜィ㸸0.07 nmol MDA/mg protein㸪p < 0.01㸧ࡀ㸪ྠ✀┦ຍࣔࢹ࡛ࣝࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

(41)

40 ⪃ ⪃ᐹ ᮏ◊✲࡛ࡣ㸪PB ࡜ ORPH ࡢప⏝㔞࡛ࡢే⏝ᢞ୚࡟ࡼࡾ GST-P 㝧ᛶ⫢⣽⬊ᕢࡀቑ ຍࡋࡓࡀ㸪ࡑࡢຠᯝࡀ┦ຍⓗ࡛࠶ࡿࡢ࠿㸪┦஌ⓗ࡛࠶ࡿࡢ࠿࡟ࡘ࠸࡚ࡣ㏻ᖖ⏝࠸ࡽࢀ࡚ ࠸ࡿ⤫ィᡭἲ࡛ࡣ᫂☜࡟ࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠸ࠋ」ᩘࡢⓎࡀࢇ≀㉁ࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ ࡼࡾ㸪ࡑࡢⓎࡀࢇᛶࡀ┦ຍ࣭┦஌ⓗ࡟ቑຍࡍࡿࡇ࡜ࡣࡍ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿ㹙9㹛ࠋ2-࢔ࢭࢳࣝ࢔࣑ࣀࣇࣝ࢜ࣞࣥࡸ࣋ࣥࢰ㹙a㹛ࣆࣞࣥ㸪N-࢚ࢳࣝ-N-ࣄࢻࣟ࢟ࢩ࢚ࢳࣝࢽࢺࣟ ࢯ࢔࣑ࣥ࡞࡝ࡢ⫢Ⓨࡀࢇ≀㉁ࢆప⏝㔞࡛ే⏝ᢞ୚ࡋࡓሙྜ㸪ࡑࢀࡒࢀ༢๣࡛ᢞ୚ࡋࡓሙ ྜ࡟ẚ࡭࡚㸪ࡑࡢⓎࡀࢇస⏝ࡀ┦஌ⓗ࡟ቑຍࡋࡓ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ㹙9㹛ࠋHasegawa ࡽ 㸦1994㸪1995㸧ࡢ◊✲࡛ࡣ㸪2 ✀㢮ࡢ┦ຍࣔࢹࣝࢆ⏝࠸࡚ゎᯒࢆ⾜ࡗ࡚࠸ࡿࠋࡍ࡞ࢃࡕ㸪 ␗✀┦ຍࣔࢹࣝ࡜ྠ✀┦ຍࣔࢹ࡛ࣝ࠶ࡿࠋ␗✀┦ຍࣔࢹࣝࡣ㸪DEN ༢⊂⩌ࡢ್࡛⿵ṇ ࡋࡓṇ࿡್㸦ྛ⩌ࡢ್࠿ࡽDEN ༢⊂⩌ࡢ್ࢆᕪࡋᘬ࠸ࡓ್㸧ࢆ⟬ฟࡋ㸪༢⊂⩌ࡢṇ࿡ ್ࡢྜィ࡜ే⏝ᢞ୚⩌ࡢṇ࿡್ࢆẚ㍑ࡍࡿࠋྠ✀┦ຍࣔࢹ࡛ࣝࡣ㸪㧗⏝㔞⩌ࡢᖹᆒ್࡜ ే⏝ᢞ୚⩌ࡢ್ࢆẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾ㸪⏝㔞౫Ꮡ᭤⥺ࢆ⪃៖ࡋ࡚ゎᯒࡋ࡚࠸ࡿࡶࡢ࡛࠶ ࡿࠋࡇࢀࡽࡢ┦ຍࣔࢹࣝ࡟࠾࠸࡚᭷ព࡞ቑຍࡀㄆࡵࡽࢀࢀࡤ㸪ࡑࡢຠᯝࡣ┦஌ⓗ࡛࠶ࡿ ࡜ゝ࠼ࡿࠋFutakuchi ࡽ㸦1996㸧ࡣ㸪4 ✀㢮ࡢ㹌㸫ࢽࢺࣟࢯ໬ྜ≀㸦nitrosomorpholine㸪 nitrosodimethylamine㸪nitrosodiethanolamine㸪nitroso-oxazolidine㸧࡟࠾࠸࡚㸪ࡑࢀࡒࢀ 1/4 㔞࡛ే⏝ᢞ୚ࡍࡿࡇ࡜࡛┦஌ຠᯝࡀ࠶ࡿ࠿ྰ࠿ࢆࣛࢵࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝࢆ⏝࠸ ᳨࡚ウࡋࡓࠋᙼࡽࡣ㸪DEN ༢⊂⩌ࡢ್࡛⿵ṇࡋࡓಶࠎࡢ໬ྜ≀ࡢṇ࿡್㸦ྛ⩌ࡢ್㸫

(42)

41 DEN ༢⊂⩌ࡢ್㸧ࢆ⟬ฟࡋࡓࠋ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚㸪ᙼࡽࡣಶࠎࡢ⸆๣ࢆ 1/4 㔞࡛ ᢞ୚ࡋࡓ㝿ࡢṇ࿡್ࡢྜィ್࡜㸪1/4 㔞࡛ 4 ✀㢮ࡢ⸆๣ࢆే⏝ᢞ୚ࡋࡓ㝿ࡢṇ࿡್ࢆẚ ㍑ࡋࡓࡀ㸪ࡑࢀࡽࡢ㛫࡛᫂ࡽ࠿࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡉࡽ࡟㸪ྠ✀┦ຍࣔࢹࣝ࡟࠾ ࠸࡚㸪1/4 㔞࡛ࡢే⏝ᢞ୚⩌ࡢ್ࡣ㸪1/1 㔞࡛ 4 ✀㢮ࡢ⸆๣ࢆᢞ୚ࡋࡓ㝿ࡢᖹᆒ್࡜࡯ࡰ ྠᵝ࡛࠶ࡗࡓࡀ㸪1/16 㔞࡛ࡢే⏝ᢞ୚⩌ࡢ್ࡣ㸪1/4 㔞ࡢಶࠎࡢ್ࡢᖹᆒ್࡟ẚ࡭᭷ព ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽ㸪ᙼࡽࡣే⏝ᢞ୚࡟ࡼࡿᙳ㡪ࡣ┦஌࡛ࡣ࡞ࡃ㸪ྠ ✀┦ຍⓗ࡛࠶ࡿ࡜ሗ࿌ࡋࡓࠋୖグࡢ⤫ィ᳨ᐃἲ࡟ᚑ࠸㸪ᮏ◊✲࡟࠾࠸࡚ࡶ᳨ᐃࢆ⾜ࡗࡓ ⤖ᯝ㸪PB ࡜ ORPH ࡢే⏝ᢞ୚࡟ࡼࡗ࡚ GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘཬࡧ㠃✚㸪ཬࡧ PCNA 㝧ᛶ⫢⣽⬊⋡࡟࠾࠸࡚┦஌ⓗ࡞ቑຍࡀ࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡇࡢࡇ࡜࠿ࡽ㸪PB ࡜ ORPH ࡢే⏝ᢞ୚࡟ࡼࡗ࡚ࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ┦஌ⓗ࡟ቑᙉࡋࡓࡇ ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓ㸪Cyp2b1/2 ࡸᢠ㓟໬㓝⣲㑇ఏᏊࡢ mRNA Ⓨ⌧ࡢే⏝ᢞ୚⩌࡛ࡢ ቑຍࡶ┦஌ⓗ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋࡉࡽ࡟㸪ቑຍࡢ⛬ᗘࡀᙅ࠸࡞ࡀࡽࡶ࣐࢖ࢡࣟࢯ࣮࣒ ROS ⏘⏕⬟ཬࡧ TBARS 㔞ࡀ␗✀┦ຍࣔࢹࣝ࡟࠾࠸࡚ే⏝ᢞ୚⩌࡛᭷ព࡟ቑຍࡋ㸪ే⏝ ᢞ୚࡟ࡼࡿ┦஌ⓗ࡞ᙳ㡪ࡀ࠶ࡿࡶࡢ࡜᥎ᐹࡉࢀࡓࠋPB ࡜ ORPH ࡢే⏝ᢞ୚࡟ࡼࡿࣛࢵ ࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡢ┦஌ⓗ࡞ቑᙉࡣ㸪ᢠ㓟໬㓝⣲࡟ࡼࡿᢠ㓟໬స⏝ࡼࡾࡶ CYP2B ㄏᑟቑຍ࡟ࡼࡿ ROS ࡢ㐣๫⏘⏕ࡀୖᅇࡾ㸪ࡑࡢࣞࢻࢵࢡࢫࣂࣛࣥࢫࡢᔂࢀ࡟ࡼ ࡾㄏ㉳ࡉࢀࡓࡶࡢ࡜᥎ᐹࡉࢀࡓࠋ

ᮏ❶࡛ࡣ㸪CYP2B inducer ྠኈ࡛࠶ࡿ PB ཬࡧ ORPH ࡢే⏝ᢞ୚ࢆ⾜࠺ࡇ࡜࡟ࡼ ࡾ㸪ࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ┦஌ⓗ࡟ቑᙉࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡑ

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42

ࡢᶵᗎ࡟ࡣCYP2B ㄏᑟࡢቑຍ࡟క࠺ ROS ⏘⏕ࡢቑຍ࡜㸪ࡑࢀ࡟ࡼࡿ㓟໬ⓗࢫࢺࣞࢫࡢ ቑຍࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ

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43 ᑠ ᑠᣓ ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ♧ࡍCYP2B inducer࡛࠶ࡿPBཬࡧORPHࢆే⏝ᢞ ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪ࡑࡢ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ࡟ᑐࡍࡿಟ㣭స⏝ࡢ᳨ウࢆ⾜࠸㸪㑇ఏ ᏊⓎ⌧ゎᯒཬࡧ࣐࢖ࢡࣟࢯ࣮࣒ROS⏘⏕⬟㸪TBARS➼㓟໬ⓗࢫࢺࣞࢫ࡟㛵୚ࡍࡿ⏕໬ Ꮫⓗ࡞ᣦᶆࢆ୰ᚰ࡟ゎᯒࡋࡓࠋ6㐌㱋㞝ᛶF344ࣛࢵࢺ࡟DEN࡛࢖ࢽࢩ࢚࣮ࢩࣙࣥฎ⨨ࢆ ᪋ࡋ㸪ࡑࡢ2㐌㛫ᚋ࠿ࡽ6㐌㛫࡟ࢃࡓࡾࣉ࣮ࣟࣔࢱ࣮࡜ࡋ࡚PB㸦60 ࡲࡓࡣ120 ppm㸧㣧 Ỉᢞ୚㸪ORPH㸦750ࡲࡓࡣ1,250 ppm㸧ΰ㣵ᢞ୚ࡶࡋࡃࡣ60 ppm PBཬࡧ750 ppm ORPH ࡢే⏝ᢞ୚ࢆ⾜࠸㸪ࣉ࣮ࣟࣔࢱ࣮ᢞ୚1㐌㛫ᚋ࡟ࡣ2/3㒊ศ⫢ษ㝖ࢆ᪋ࡋࡓࠋࡑࡢ⤖ᯝ㸪 GST-Pࡢ㝧ᛶ⫢⣽⬊ᕢࡢಶᩘཬࡧ㠃✚㸪ཬࡧPCNA㝧ᛶ⫢⣽⬊⋡ࡀే⏝ᢞ୚⩌࡛┦஌ⓗ ࡟ቑຍࡋ㸪Cyp2b1/2ࡢmRNAⓎ⌧ࡸ࣐࢖ࢡࣟࢯ࣮࣒ROS⏘⏕⬟㸪TBARS㔞ࡢ┦஌ⓗ࡞ቑ ຍࡶㄆࡵࡽࢀࡓࠋࡉࡽ࡟㸪཯ᛂᛶࡢኚ໬࡜ࡋ࡚ᢠ㓟໬㓝⣲㑇ఏᏊࡢmRNAⓎ⌧ࡢ┦஌ⓗ ࡞ቑຍࡶㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ㸪PB࡜ORPHࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾCYP2B ㄏᑟࡀቑຍࡋ㸪ࡑࢀ࡟క࠺ROS⏘⏕ࡢቑຍ࡟ࡼࡗ࡚㓟໬ⓗࢫࢺࣞࢫࡀቑຍࡋ㸪ࣛࢵࢺ⫢ Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ┦஌ⓗ࡟ቑᙉࡍࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ

(45)

44 ➨ ➨3 ❶

Phenobarbital ཬࡧ piperonyl butoxide ే⏝ᢞ୚࡟ࡼࡿࣛࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ

(46)

45 ⥴ ⥴ゝ

CYP1A/2B inducer ࡛࠶ࡿ PBO ࡣࣆࣞࢫࣟ࢖ࢻ⣔㎰⸆ࡢඹຊ๣࡛࠶ࡾ㸪ᐙᗞ⏝ẅ ⹸๣࡟ᗈࡃ฼⏝ࡉࢀ࡚࠸ࡿࠋTakahashi ࡽ㸦1994㸧ࡣ㸪1.2%ࡶࡋࡃࡣ 2.4% PBO ࢆࣛࢵࢺ ࡟2 ᖺ㛫ΰ㣵ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢⣽⬊⭘⒆ࡀㄏⓎࡉࢀࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋࣛࢵ ࢺ⫢஧ẁ㝵Ⓨࡀࢇࣔࢹࣝ࡟࠾࠸࡚㸪1 ࡞࠸ࡋ 2% PBO ࡢΰ㣵ᢞ୚ࡀࣛࢵࢺ࡟⫢Ⓨࡀࢇࣉ ࣮ࣟࣔࢩࣙࣥస⏝ࢆㄏⓎࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿ㹙28㹛ࠋࡉࡽ࡟ 0.25% PBO ࡀࣛ ࢵࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡢ㜈್࡛࠶ࡿࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠸ࡿ㹙27㹛ࠋ

➨2 ❶࡟࠾࠸࡚㸪CYP2B inducer ࡛࠶ࡿ PB ࡜ ORPH ࢆే⏝ᢞ୚ࡋࡓሙྜ㸪ࣛࢵ ࢺ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀ┦஌ⓗ࡟ቑᙉࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡾ㸪CYP ࢆㄏ ᑟࡍࡿస⏝ࢆ᭷ࡍࡿ⸆๣ࢆే⏝ᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ㸪⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࡀቑ ᙉࡍࡿྍ⬟ᛶࡀ᥎ᐹࡉࢀࡿࠋ

ࡑࡇ࡛㸪ᮏ◊✲࡛ࡣ㸪CYP2B inducer ࡜ CYP1A/2B inducer ࢆ⏝࠸࡚ే⏝ᢞ୚ᐇ㦂 ࢆ⾜࠸㸪␗✀ࡢ CYP ࢆㄏᑟࡍࡿ≀㉁ྠኈࡢే⏝ᢞ୚࡟࠾࠸࡚ࡶ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙ ࣥస⏝ࡀቑᙉࡍࡿ࠿ྰ࠿ࢆ᳨ウࡋࡓࠋే⏝ᢞ୚࡟ࡼࡿ⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥ࡟࠾ࡅࡿ ಟ㣭స⏝ࡢ᳨ウࢆ⾜࠸㸪㑇ఏᏊⓎ⌧ゎᯒཬࡧ࣐࢖ࢡࣟࢯ࣮࣒ROS ⏘⏕⬟㸪TBARS ➼㓟 ໬ⓗࢫࢺࣞࢫ࡟㛵୚ࡍࡿ⏕໬Ꮫⓗ࡞ᣦᶆ࡟↔Ⅼࢆ⤠ࡾ㸪ゎᯒࢆᐇ᪋ࡋࡓࠋ

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46 ᮦ

ᮦᩱཬࡧ᪉ἲ

໬Ꮫ≀㉁

PB㸦CAS No. 57-30-7㸪⣧ᗘ98%㸧ཬࡧPBO㸦CAS No. 51-03-6㸪⣧ᗘ90%㸧ࡣࡑࢀ ࡒࢀ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫㸦኱㜰㸧ཬࡧSigma-Aldrich㸦࢔࣓ࣜ࢝㸧࠿ࡽ㉎ධࡋࡓࠋDEN 㸦CAS No. 55-18-5㸪⣧ᗘ99%㸧ࡣᮾி໬ᡂᕤᴗ㸦ᮾி㸧࠿ࡽ㉎ධࡋࡓࠋ ౑⏝ື≀ ᐇ㦂3࡟⏝࠸ࡓ5㐌㱋ࡢ㞝ᛶF344ࣛࢵࢺࡣ㸪᪥ᮏSLCᰴᘧ఍♫㸦㟼ᒸ㸧ࡼࡾ㉎ධࡋ㸪 12᫬㛫᫂ᬯࢧ࢖ࢡࣝ㸪 ᗘ20㹼26Υ㸪‵ᗘ40~70%ࡢ᮲௳ୗ࡛3࡞࠸ࡋ4༉ࡎࡘᗋᩜࡁ㸦࣌ ࣃ࣮ࢡ࣮ࣜࣥ㸹᪥ᮏSLCᰴᘧ఍♫㸪㟼ᒸ㸧ࢆᩜ࠸ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢪ࡟࡚㣫⫱ࢆ⾜ࡗ ࡓࠋ⢊ᮎ㣫ᩱ㸦࢚࢜ࣜࣥࢱࣝMF㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸪ᮾி㸧ཬࡧ㣧ᩱỈࡣ⮬⏤ᦤ ྲྀࡉࡏࡓࠋ1㐌㛫ࡢ᳨␿࣭㥆໬ᮇ㛫ࢆタࡅࡓᚋ㸪ᐇ㦂࡟౪ࡋࡓࠋ࡞࠾㸪ື≀ᐇ㦂ࡣᮾி ㎰ᕤ኱Ꮫᐇ㦂ື≀೔⌮つᐃ࡟‽ᣐࡋ࡚⾜ࡗࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ᐇ㦂3 ௨ୗࡢᡭ㡰࡟ᚑࡗ࡚㸪ࣛࢵࢺ⫢Ⓨࡀࢇ஧ẁ㝵ࣔࢹࣝ࡟ࡼࡿᐇ㦂㹙17㹛ࢆ⾜ࡗࡓࠋ ࡍ࡞ࢃࡕ㸪඲࡚ࡢື≀࡟ᑐࡋDENࡢ༢ᅇ⭡⭍ෆᢞ୚㸦200 mg/kg య㔜㸧ࢆ⾜࠸㸪ࡑࡢ2

(48)

47 㐌㛫ᚋ࠿ࡽ㸪ᇶ♏㣫ᩱࡢࡳ㸦DEN༢⊂⩌㸪n =12㸧㸪PB㸦60 ppm㸹PBప⏝㔞⩌㸪n = 12 ࡞࠸ࡋ120 ppm㸹PB㧗⏝㔞⩌㸪n = 12㸧ࡢ㣧Ỉᢞ୚㸪PBO㸦1,250 ppm㸹PBOప⏝㔞⩌㸪n = 12࡞࠸ࡋ2,500 ppm㸹PBO㧗⏝㔞⩌㸪n = 12㸧ࡢΰ㣵ᢞ୚㸪ࡶࡋࡃࡣ60 ppm PBཬࡧ1,250 ppm PBOࡢే⏝ᢞ୚㸦ే⏝ᢞ୚⩌㸪n = 12㸧ࢆ6㐌㛫⾜ࡗࡓࠋ⏝㔞タᐃ࡟࠾࠸࡚ࡣ㸪ప⏝ 㔞࡟⫢Ⓨࡀࢇࣉ࣮ࣟࣔࢩࣙࣥస⏝ࢆ♧ࡍ㜈್ࢆ㸪㧗⏝㔞࡟ࡣࡑࢀࡒࢀࡢ㜈್ࡢ2ಸ㔞ࢆ ⏝࠸㸪ే⏝ᢞ୚࡟ࡣࡑࢀࡒࢀࡢప⏝㔞ࢆ⏝࠸ࡓࠋ㜈್タᐃࡣ㸪PB࡟ࡘ࠸࡚ࡣ➨2❶࡜ྠ ᵝ࡟Kitanoࡽ㸦1998㸧ࡢሗ࿌࡟ᇶ࡙࠸࡚60 ppm㸪PBOࡣ Mugurumaࡽ㸦2009㸧ࡢሗ࿌࡟ ᇶ࡙࠸࡚1,250 ppm࡜ࡋࡓࠋࡉࡽ࡟⫢⣽⬊ࡢቑṪάᛶࢆ㧗ࡵࡿࡓࡵ㸪඲࡚ࡢື≀࡟2/3㒊 ศ⫢ษ㝖ࢆDENᢞ୚3㐌㛫ᚋ࡟ᐇ᪋ࡋࡓࠋDEN༢⊂⩌࡟࠾࠸࡚1༉㸪PBప⏝㔞⩌࡟࠾࠸ ࡚1༉㸪PB㧗⏝㔞⩌࡟࠾࠸࡚4༉㸪PBOప⏝㔞⩌࡟࠾࠸࡚1༉㸪ࡉࡽ࡟ే⏝ᢞ୚⩌࡟࠾࠸ ࡚2༉ࡀ㒊ศ⫢ษ㝖ࡢᢏ⾡ⓗ㐣ኻ࡟ࡼࡾṚஸࡋࡓࠋᢞ୚ᮇ㛫⤊஢ᚋ㸪࢖ࢯࣇࣝࣛࣥ῝㯞㓉ୗ࡟࡚ᨺ⾑ẅࢆ⾜࠸㸪⫢⮚ࢆ᥇ྲྀᚋ㸪㔜㔞 ᐃࢆ⾜ࡗࡓࠋ⫢⮚ࡢ⤌⧊Ꮫⓗ᳨⣴ཬࡧච ␿⤌⧊໬Ꮫⓗ᳨⣴࡟ࡣ㸪ゎ๗᫬࡟ࢫࣛ࢖ࢫࡋࡓ⫢⤌⧊ࢆ4%ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻ⁐ᾮ ࡟࡚ᅛᐃࢆ⾜࠸㸪ࡲࡓ㸪ṧࡾࡢ⫢ࢧࣥࣉࣝࡣ㑇ఏᏊⓎ⌧ཬࡧ⏕໬Ꮫⓗゎᯒ⏝࡜ࡋ࡚ᾮయ ❅⣲࡟ᾐₕࡋ࡚෾⤖ࡋ-80Υ࡟࡚ಖᏑࡋࡓࠋ ⑓ ⑓⌮⤌⧊Ꮫⓗゎᯒ࣭ච␿⤌⧊໬Ꮫⓗゎᯒ ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻᅛᐃᚋ㸪ᐃἲ࡟ᚑ࠸࢚ࢱࣀ࣮ࣝ⣔ิ࡛⬺Ỉࢆ⾜࠸㸪ࣃࣛࣇ ࢕ࣥ࡟ໟᇙࡋࡓࠋ㐃⥆ษ∦ࢆస〇ࡋ㸪୍㒊ࡣH&E ᰁⰍࢆ⾜ࡗࡓࠋࡉࡽ࡟ GST-P㸪PCNA

(49)

48 ࡢච␿⤌⧊໬ᏛᰁⰍࢆ௨ୗࡢᡭ㡰࡛⾜ࡗࡓࠋጞࡵ࡟⬺ࣃࣛࣇ࢕ࣥฎ⌮ࢆࡋࡓษ∦ࢆ㸪 0.3%㐣㓟໬Ỉ⣲ࢆྵࡴ࣓ࢱࣀ࣮ࣝ࡟ࡼࡾᐊ ࡛ 30 ศ㛫ෆᅉᛶ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ࢆ ࡋ㸪ṇᖖ⾑Ύ࡟࡚ࣈࣟࢵ࢟ࣥࢢฎ⌮ࢆ⾜ࡗࡓࠋࡲࡓ㸪࣌ࣝ࢜࢟ࢩࢲ࣮ࢮฎ⌮ᚋ㸪PCNA ࡟㛵ࡋ࡚ࡣࢡ࢚ࣥ㓟⦆⾪ᾮ㸦pH 6.0㸧ୗ࡛ 60Υ㸪20 ศࡢ ᾎࢆ⾜ࡗࡓࠋࡑࡢᚋ㸪࢘ࢧ ࢠᢠGST-P ࣏ࣜࢡ࣮ࣟࢼࣝᢠయ㸦1,000 ಸᕼ㔘㸪ᰴᘧ఍♫་Ꮫ⏕≀Ꮫ◊✲ᡤ㸪ឡ▱㸧ཬ ࡧ࣐࢘ࢫᢠPCNA ࣔࣀࢡ࣮ࣟࢼࣝᢠయ㸦500 ಸᕼ㔘㸪Dako㸪ࢹ࣐࣮ࣥࢡ㸧ࢆࡑࢀࡒࢀ 4Υ ୍࡛ᬌ཯ᛂࡉࡏࡓࠋ୍ḟᢠయ཯ᛂᚋ㸪Vectastain Elite ABC kit㸦Vector Laboratories㸪࢔࣓ ࣜ࢝㸧ࢆ⏝࠸㸪DAB ࡟ࡼࡿⓎⰍᚋ㸪࣐࣊ࢺ࢟ࢩ࡛ࣜࣥ᰾ࡢᑐẚᰁⰍࢆ⾜ࡗࡓࠋ

GST-P 㝧ᛶ⫢⣽⬊ᕢ࡟ࡘ࠸࡚ࡣ㸪┤ᚄ 200 μm ௨ୖ࡛᫂ࡽ࠿࡞㝧ᛶ⫢⣽⬊ᕢࡢษ ∦㠃✚ᙜࡓࡾࡢಶᩘཬࡧ㠃✚⋡ࢆ⏬ീゎᯒࢯࣇࢺWinROOF㸦୕㇂ၟ஦㸪ᮾி㸧ࢆ⏝࠸ ࡚ゎᯒࡋࡓࠋPCNA 㝧ᛶ⫢⣽⬊࡟ࡘ࠸࡚ࡣ㸪1 ౛࡟ࡘࡁ↓సⅭ࡟㑅ࢇࡔ 10 ど㔝㸦1 ど㔝 ࠶ࡓࡾ700㹼900 ⫢⣽⬊㸧࡟࡚ᙉ㝧ᛶ⫢⣽⬊ࡢ ᐃࢆ⾜࠸㸪ⓒศ⋡࡛⾲♧ࡋࡓࠋ

Real-timereverse transcription polymerase chain reaction㸦㸦RT-PCR㸧ゎᯒ

㑇ఏᏊⓎ⌧ゎᯒ࡟౑⏝ࡍࡿtotal RNA ࢧࣥࣉࣝࡣ㸪RNeasy kit㸦Qiagen㸪ࢻ࢖ࢶ㸧 ࡟ࡼࡾᢳฟࡋࡓࠋcDNA ྜᡂ࡬ࡢ㏫㌿෗཯ᛂࡣ 2 μg ࡢ total RNA ࠿ࡽ Thermoscript RT-PCR system㸦Life Technologies㸪CA㸪࢔࣓ࣜ࢝㸧࡟࡚⾜ࡗࡓࠋᐃ㔞real-time RT-PCR ࡣPowerSYBR®_{Green PCR Master Mix㸦}_{Life Technologies}_㸧ཬࡧ_StepOnePlusTM_Real-Time

(50)

49

Express㸦Life Technologies㸧࡟࡚స〇ࡋࡓࠋྛࣉࣛ࢖࣐࣮㓄ิࡣTable 3-1 ࡟グ㍕ࡋࡓࠋ 㑇ఏᏊⓎ⌧ࡢᐃ㔞໬ࡣ㸪ෆᅾᛶࢥࣥࢺ࣮ࣟࣝ㑇ఏᏊ 㸦Actb㸧࡟ᑐࡋ࡚ 2-ΔΔCt_{ἲ࡟๎ࡾᶆ}

‽໬ࡋࡓ㹙24㹛ࠋ

㐣

㐣㓟໬⬡㉁ ᐃ

⫢⮚࡟࠾ࡅࡿ㐣㓟໬⬡㉁ ᐃࡣ TBARS 㔞ࢆ ᐃࡋࡓࠋ⫢ TBARS 㔞ࡣ Ohkawa ࡽ㸦1979㸧ࡢ᪉ἲ࡟ᚑ࠸ 1 ⩌࠶ࡓࡾ 6 ౛࡟ࡘ࠸࡚ ᐃࡋࡓࠋࡍ࡞ࢃࡕ㸪⫢ࢧࣥࣉࣝ㸦⣙ 50 mg㸧ࢆ 450 μl ࡢ 1.15% KCl ⦆⾪ᾮ୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋ ⺮ⓑ⃰ᗘ ᐃࡣBCA protein assay kit㸦Pierce, ࢔࣓ࣜ࢝㸧࡟࡚⾜ࡗࡓࠋᆒ㉁໬ࡋࡓ⫢࣍ ࣔࢪࢿ࣮ࢺ㸦17.8 mg protein㸧ࢆ 0.2 mL ࡢ 8.1% SDS ࡜ 3.0 mL ࡢ 0.4% thiobarbituric aci-10㸣㓑㓟⁐ᾮ࡜࡛ 95Υ㸪60 ศ㛫཯ᛂࡉࡏࡓᚋ㸪෭Ỉ࡛෭༷ࡋࡓࠋ཯ᛂᾮ࡟ 1 mL ࡢ ⵨␃Ỉࢆຍ࠼㸪2.0 mL ࡢ n-butanol-pyridine ΰྜᾮ㸦15:1㸧ࢆຍ࠼ࡓᚋ㸪2,100 ×g㸪10 ศ 㛫㐲ᚰࡋࡓࠋ532 nm ࡟࠾ࡅࡿ྾ගᗘࡣ㸪ศගගᗘィ SynergyTM_{HT Multi-Detection}

Microplate Reader 㸦 BioTek 㸪 ࢔ ࣓ ࣜ ࢝ 㸧 ࡟ ࡼ ࡾ ᐃ ࡋ ࡓ ࠋ TBARS ᐃ ್ ࡣ 1,1,3,3-tetramethoxypropane ⏤᮶ࡢ malondialdehyde㸦MDA㸧㔞࡟࡚ᐃ㔞ࡋࡓࠋ

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢㄪ〇ࡣ㸪Yoshihara ࡽ㸦2001㸧ࡢሗ࿌࡟ᇶ࡙ࡁⱝᖸࡢኚ᭦ ࢆຍ࠼⾜ࡗࡓࠋ⫢ࢧࣥࣉࣝ㸦⣙100㹼130 mg㸧ࢆ 3 ಸ㔞ࡢ⦆⾪ᾮ㸦50 mM Tris-HCl㸹pH

(51)

50

7.4㸪1.15% KCl㸪0.2 mM EDTA㸪0.1 mM DTT㸪0.1 mM Protease Inhibitor Cocktail㸪20% glycerol㸧୰࡛ TissueLyser㸦Qiagen㸧ࢆ⏝࠸࡚ᆒ㉁໬ࡋࡓࠋḟ࡟ࡑࡢ࣍ࣔࢪࢿ࣮ࢺࢆ 700 ×g ࡛ 10 ศ㛫㐲ᚰࢆ⾜࠸㸪ୖΎࢆ 10,000 ×g ࡛ 20 ศ㛫㐲ᚰࡋࡓࠋᚓࡽࢀࡓୖΎࢆࡉ ࡽ࡟105,000 ×g㸪90 ศ㛫㉸㐲ᚰࡋࡓᚋ㸪ỿ´ࢆ 20 mM Tris-HCl㸦pH 7.4㸧࡟ᠱ⃮ࡋࡓࡶ ࡢࢆ࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡜ࡋࡓࠋ⺮ⓑ⃰ᗘ ᐃࡣBCA protein assay kit㸦Pierce㸧࡟࡚⾜ ࡗࡓࠋ

࣐

࣐࢖ࢡࣟࢯ࣮࣒⏬ศࡢROS ⏘⏕ ᐃ

ศ㛫཯ᛂࡉࡏࡓࠋ཯ᛂ㛤ጞࡣnicotinamide adenine dinucleotide phosphate㸦NADPH㸹᭱⤊ ⃰ᗘ0.5 mM㸹࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ㸧ࢆῧຍࡍࡿࡇ࡜࡟ࡼࡾ⾜࠸㸪⏕ᡂࡉࢀࡿ⺯ගࢆ ບ㉳Ἴ㛗 485 nm㸪528 nm ࡢ᮲௳࡛ 120 ศ㛫 5 ศࡈ࡜࡟㸪ศගගᗘィ SynergyTM_HT

Multi-Detection Microplate Reader㸦BioTek㸧࡟ࡼࡾ ᐃࡋࡓࠋ㝧ᛶᑐ↷࡟ H2O2㸦᭱⤊⃰

ᗘ0.1 mM㸧ࢆ㸪ヨ㦂⣔ࡢ᳨ド࡟ CYP 㜼ᐖ๣࡜ࡋ࡚▱ࡽࢀࡿ SKF-525A㸦᭱⤊⃰ᗘ 0.1 mM 㸹Tronto Reserch Chemicals㸪࢝ࢼࢲ㸧ࢆῧຍࡋࡓࠋࢹ࣮ࢱࡣ DEN ༢⊂⩌ࢆ 100%࡜

(52)

51 ࡋ࡚ᶆ‽໬ࡋࡓࠋ ⤫ ⤫ィゎᯒ ඲࡚ࡢࢹ࣮ࢱࡣᖹᆒ್ཬࡧᶆ‽೫ᕪ࡟࡚⾲グࡋࡓࠋศᩓࡢᆒ୍ᛶࡢ᳨ᐃࡣ㸪 Bartlett ᳨ᐃࢆ⾜ࡗࡓࠋᆒ୍ᛶࡀ࠶ࡿሙྜ࡟ࡣ Dunnet ከ㔜ẚ㍑᳨ᐃࢆ⾜࠸㸪ᆒ୍ᛶࡀ࡞ ࠸ሙྜ࡟ࡣ Steel ᳨ᐃࢆ⾜ࡗࡓࠋࡉࡽ࡟㸪ే⏝ᢞ୚࡟ࡼࡿಟ㣭స⏝ࢆホ౯ࡍࡿࡓࡵ㸪 Futakuchi ࡽ㸦1996㸧ࡸ Hasegawa ࡽ㸦1991㸧ࡀ᥎ዡࡍࡿ᪉ἲࢆ⏝࠸࡚⤫ィゎᯒࢆ⾜ࡗࡓࠋ P ್ࡀ 0.05 ᮍ‶ࡢሙྜ㸪⤫ィᏛⓗ࡟᭷ព࡛࠶ࡿ࡜ุ᩿ࡋࡓࠋ

(53)

52 ⤖ ⤖ᯝ ᐇ㦂3 య㔜㸪⫢㔜㔞㸪ᦤ㣵ᦤỈ㔞ཬࡧ᥎ᐃ⸆≀ᦤྲྀ㔞ヨ㦂ᮇ㛫ࢆ㏻ࡌ࡚㸪඲࡚ࡢᢞ୚⩌࡟ࡘ࠸࡚㸪⿕᳨≀㉁࡟㉳ᅉࡍࡿ⮫ᗋ⑕≧ཬࡧṚ ஸࡣࡳࡽࢀ࡞࠿ࡗࡓࠋయ㔜㸪ᖹᆒᦤ㣵㔞ཬࡧᖹᆒᦤỈ㔞࡟࠾࠸࡚᭷ព࡞ኚືࡣㄆࡵࡽࢀ ࡞࠿ࡗࡓ㸦Table 3-2㸧ࠋ ⤯ᑐ⫢㔜㔞ࡣDEN ༢⊂⩌࡜ẚ㍑ࡋ࡚ PB 㧗⏝㔞⩌ཬࡧ PBO ᢞ୚⩌ࡀ᭷ព࡟ቑຍ ࡋ㸪ే⏝ᢞ୚⩌ࡀPB ప⏝㔞⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋ┦ᑐ⫢㔜㔞ࡣ PB ཬࡧ PBO ࡢᢞ୚࡟ࡼࡾ DEN ༢⊂⩌࡜ẚ࡭࡚᭷ព࡟ቑຍࡋ㸪ే⏝ᢞ୚⩌࡟࠾࠸࡚ࡣ PB ప⏝ 㔞⩌ཬࡧ㧗⏝㔞⩌㸪ཬࡧPBO ప⏝㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ࡚࠸ࡓ㸦Table 3-2㸧ࠋPB ཬ ࡧPBO ᦤྲྀ㔞ࡣ Table 3-2 ࡟♧ࡍ㏻ࡾ࡛࠶ࡿࠋప⏝㔞⩌࡜ే⏝ᢞ୚⩌ࡢ㛫࡟⸆≀ᦤྲྀ㔞 ࡢ㐪࠸ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ ⑓⌮⤌⧊Ꮫⓗゎᯒ㸪GST-P 㝧ᛶ⫢⣽⬊ᕢཬࡧ PCNA 㝧ᛶ⣽⬊ ⑓⌮⤌⧊Ꮫⓗゎᯒ࡛ࡣ㸪PB ཬࡧ PBO ᢞ୚⩌࡜ࡶ࡟⣽⬊㉁ࡢዲ㓟ᛶ໬ࢆక࠺⫢⣽ ⬊⫧኱ࡀㄆࡵࡽࢀࡓࠋ⫢⣽⬊✵⬊ኚᛶࡀPB ᢞ୚⩌࡛ㄆࡵࡽࢀ㸪ኚ␗⫢⣽⬊ᕢ㸦᫂⣽⬊ ᛶཬࡧዲ㓟ᛶ㸧ࡣPB 㧗⏝㔞⩌㸪PBO 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡛ㄆࡵࡽࢀࡓ㸦ࢹ࣮ࢱࡣ ┬␎㸧ࠋච␿⤌⧊໬Ꮫⓗゎᯒ࡟࠾࠸࡚㸪GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘࡣ PB 㧗⏝㔞⩌㸪PBO

(54)

53 㧗⏝㔞⩌ཬࡧే⏝ᢞ୚⩌࡛DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓࠋGST-P 㝧ᛶ⫢⣽⬊ ᕢࡢ㠃✚ࡣ PB 㧗⏝㔞⩌ཬࡧ PBO 㧗⏝㔞⩌࡛ DEN ༢⊂⩌࡟ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓ 㸦Fig. 3-1㸧ࠋࡇࡢ⤖ᯝࡀ┦ຍ┦஌ⓗ࡛࠶ࡿ࠿ྰ࠿ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟┦ຍࣔࢹࣝ࡟ࡼ ࡿ᳨ᐃࢆ⾜ࡗࡓࠋ␗✀┦ຍࣔࢹࣝࢆ⏝࠸ࡓ⤖ᯝ㸪GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘཬࡧ㠃✚࡟ ࠾࠸࡚ే⏝ᢞ୚⩌࡟࠾ࡅࡿ᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 3-2㸧ࠋྠ✀┦ຍࣔࢹࣝ ࢆ⏝࠸ࡓ᳨ᐃ࡟࠾࠸࡚㸪GST-P 㝧ᛶ⫢⣽⬊ᕢࡢಶᩘࡣే⏝ᢞ୚⩌࡟࠾ࡅࡿ᭷ព࡞ኚືࡣ ㄆࡵࡽࢀ࡞࠿ࡗࡓࡀ㸪㠃✚ࡣే⏝ᢞ୚⩌ࡀ㧗⏝㔞⩌ࡢᖹᆒࡼࡾࡶ᭷ព࡟ᑡ࡞࠸㸦ే⏝ᢞ ୚⩌㸸0.44 ± 0.15 mm2_/cm2_{, 㧗⏝㔞⩌㸸0.60 ± 0.26 mm}2_/cm2_㸪_{p < 0.05㸪t-test㸧ࡇ࡜ࡀ᫂ࡽ} ࠿࡜࡞ࡗࡓ㸦Fig. 3-2㸧ࠋ PCNA 㝧ᛶ⫢⣽⬊⋡ࡣ㸪ే⏝ᢞ୚⩌ෆ࡛ࡢࡤࡽࡘࡁࡀ኱ࡁ࠿ࡗࡓࡓࡵ᭷ព࡞ኚື ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Table 3-2㸧ࠋ Real-time RT-PCR ࡟࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ Table 3-1 ࡟♧ࡋࡓ㑇ఏᏊ࡟㛵ࡋ࡚㸪ྛ⩌ 6 ౛ࡎࡘ real-time RT-PCR ゎᯒࢆ⾜ࡗࡓ 㸦Table 3-3㸧ࠋ➨୍┦⸆≀௦ㅰ㓝⣲࡛࠶ࡿ Cyp1a1 ࡢ mRNA Ⓨ⌧ࡣ PBO ᢞ୚⩌࡛ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓࡀ㸪PBO 㧗⏝㔞⩌ࡼࡾࡣ᭷ព࡟పୗࡋࡓࠋ୍᪉㸪

Cyp2b1/2 ࡢ mRNA Ⓨ⌧ࡣే⏝ᢞ୚⩌࡛ PB ప⏝㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋࡓࠋࡇࢀࡽࡢ

⤖ᯝࢆ┦ຍࣔࢹ᳨࡛ࣝᐃࡋࡓ⤖ᯝ㸪␗✀ཬࡧྠ✀┦ຍࣔࢹࣝ࡜ࡶ࡟㸪Cyp1a1 ࡢ mRNA

(55)

54 ⩌ṇ࿡್ྜィ㸸7.75 ಸ㸪p < 0.05㸧㸦ే⏝ᢞ୚⩌㸸4.06 ± 2.18 ಸ㸪㧗⏝㔞⩌ᖹᆒ㸸14.81 ± 16.17 ಸ㸪p < 0.05㸪Welch’s t-test㸧㸪ే⏝ᢞ୚࡟ࡼࡿᙳ㡪ࡀᢚไⓗ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀ ࡓࠋCyp2b1/2 ࡢ mRNA Ⓨ⌧ࡣ␗✀ཬࡧྠ✀┦ຍࣔࢹࣝ࡟࠾ࡅࡿ᭷ព࡞ኚືࡣㄆࡵࡽࢀ ࡞࠿ࡗࡓࠋ ➨஧┦⸆≀௦ㅰ㓝⣲࡛࠶ࡾ㓟໬ⓗࢫࢺࣞࢫ࡟཯ᛂࡋ࡚ᢠ㓟໬ࡸゎẘ࡟ാࡃ㓝⣲ 㑇ఏᏊ࡛࠶ࡿGstm3 ࡢ mRNA Ⓨ⌧ࡀ PB ᢞ୚⩌࡟࠾࠸࡚ DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ ቑຍࡋࡓࠋࡲࡓ㸪Gpx2 ࡢ mRNA Ⓨ⌧ࡣ PB 㧗⏝㔞⩌㸪PBO ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌㸪ཬ ࡧే⏝ᢞ୚⩌࡛DEN ༢⊂⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍࡋࡓࠋࡉࡽ࡟㸪ูࡢᢠ㓟໬㓝⣲㑇ఏ

Ꮚ࡛࠶ࡿNqo1 ࡢ mRNA Ⓨ⌧ࡣ PBO ᢞ୚⩌࡟࠾࠸࡚ DEN ༢⊂⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋࡓࠋ

ຍ࠼࡚ే⏝ᢞ୚⩌࡟࠾࠸࡚ࡣGstm3 ࡀ PBO ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌ࡼࡾࡶ㸪Gpx2 ࡀ PB

ప⏝㔞⩌ཬࡧ㧗⏝㔞⩌ࡼࡾࡶ᭷ព࡟ቑຍࡋ࡚࠸ࡓࡀ㸪Nqo1 ࡟᭷ព࡞ኚືࡣㄆࡵࡽࢀ࡞

࠿ࡗࡓࠋ

࣐

࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡟࠾ࡅࡿROS ⏘⏕⬟

࣐࢖ࢡࣟࢯ࣮࣒⏬ศ࡟࠾ࡅࡿCYP άᛶ⏤᮶ࡢ ROS ⏘⏕ࢆ ᐃࡋࡓ㸦Table 3-4㸧ࠋ CYP ࡢࣔࣀ࢜࢟ࢩࢤࢼ࣮ࢮάᛶࡣ NADPH ࠿ࡽࡢ㑏ඖຊࡀᚲせ࡛࠶ࡿࡓࡵ㸪ROS ⏘⏕ ࢆNADPH Ꮡᅾୗ࣭㠀Ꮡᅾୗ࡟࡚ẚ㍑ࡋࡓࠋNADPH 㠀Ꮡᅾୗ࡛ࡣ㸪඲࡚ࡢ⩌࡟࠾࠸࡚ ROS ⏘⏕࡟ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡋ࠿ࡋ㸪NADPH ࢆῧຍࡋࡓ࡜ࡇࢁ㸪࣐࢖ࢡࣟࢯ࣮