グリシドールを例としたニューロン発達指標の確立による神経毒性評価に関する研究

Title

グリシドールを例としたニューロン発達指標の確立による

_{神経毒性評価に関する研究( 本文(Fulltext) )}

Author(s)

赤根, 弘敏

Report No.(Doctoral

Degree)

博士(獣医学) 乙第128号

Issue Date

2014-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/49049

※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。

1

ࢢࣜࢩࢻ࣮ࣝࢆ౛࡜ࡋࡓࢽ࣮ࣗࣟࣥⓎ㐩ᣦᶆࡢ

☜❧࡟ࡼࡿ⚄⤒ẘᛶホ౯࡟㛵ࡍࡿ◊✲

2013 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉

㉥᰿ᘯᩄ

2 ࠙┠ḟࠚ ᗎㄽ ---4 ➨1 ❶ ࣛࢵࢺࡢ⬻㒊఩≉␗ⓗ࡞⥙⨶ⓗゎᯒࢆྍ⬟࡜ࡍࡿ࣓ࢱ࣮࢝ࣥ඲⬻ᅛ ᐃἲࡢ᳨ウ ---7 ㅖゝ ---8 ᮦᩱཬࡧ᪉ἲ ---10 ⤖ᯝ ---15 ⪃ᐹ ---18 ᑠᣓ ---20 ➨2 ❶ ࢢࣜࢩࢻ࣮ࣝࡢࣛࢵࢺẕື≀ᭀ㟢࡟ࡼࡿẕື≀࡜Ꮚື≀ࡢ⚄⤒⣔࡟ᑐ ࡍࡿᙳ㡪 ---21 ㅖゝ ---22 ᮦᩱཬࡧ᪉ἲ ---24 ⤖ᯝ ---31 ⪃ᐹ ---35 ᑠᣓ ---39 ➨3 ❶ ࢢࣜࢩࢻ࣮ࣝࡢࣛࢵࢺ࡬ࡢ 28 ᪥㛫཯᚟ᢞ୚࡟ࡼࡿ⚄⤒⣔࡟ᑐࡍࡿᙳ 㡪 ---40 ㅖゝ ---41 ᮦᩱཬࡧ᪉ἲ ---42 ⤖ᯝ ---48 ⪃ᐹ ---52 ᑠᣓ ---56

3 ⥲ྜ⪃ᐹ ---57 ⤖ㄽ ---61 ㅰ㎡ ---63 ཧ⪃ᩥ⊩ ---64 せ᪨ ---76 Abstract ---79 ᅗ⾲ ---83

4

ᗎ

ᗎ ㄽ

ᡂ⇍ື≀࡟⌧ࢀࡿ⚄⤒ẘᛶࡢከࡃࡣ⚄⤒⑓⌮Ꮫⓗ, ᶵ⬟Ꮫⓗホ౯࡟ࡼࡗ᳨࡚ฟࡀྍ⬟࡛ ࠶ࡿࡀ, ከᵝ࡞ ᐃ㡯┠࡟࠾ࡅࡿ⥲ྜⓗホ౯ࢆせࡋ, ẘᛶࡢุ᩿ࡀᅔ㞴࡞ሙྜࡀ࠶ࡿࠋࡲࡓ, Ⓨ㐩⚄⤒ẘᛶࡣᮍ▱࡞Ⅼࡀከࡃ, ≉࡟ࢽ࣮ࣗࣟࣥࡸࢢࣜ࢔ࡢศ໬࡟୚࠼ࡿᙳ㡪ࡣ୙ྍ㏫ⓗ ᙳ㡪ࡶᠱᛕࡉࢀࡿࡓࡵ, ẘᛶホ౯ୖࡢ࢚ࣥࢻ࣏࢖ࣥࢺ࡜ࡋ࡚㔜せᛶࢆቑࡋ࡚࠸ࡿࠋⓎ㐩ᮇ⚄ ⤒ẘᛶヨ㦂ࡣ㧗ḟヨ㦂࡟ᒓࡋ, ࡑࡢ⌧⾜ࡢ࢞࢖ࢻࣛ࢖ࣥࡢ୍ࡘ࡛࠶ࡿ⤒῭༠ຊ㛤Ⓨᶵᵓ 㸦OECD㸧ࡢ TG426㸦60㸧࡛ࡣ, ࡑࡢホ౯࡟ከࡃࡢ᫬㛫࡜ື≀ࡀᚲせ࡞኱つᶍ࡞ヨ㦂ࡢᐇ᪋ ࡀせồࡉࢀࡿࡓࡵ, ᑠつᶍ࡞ヨ㦂᳨࡛ฟྍ⬟࡞ࡼࡾ㎿㏿࡞ࢫࢡ࣮ࣜࢽࣥࢢホ౯ࢩࢫࢸ࣒ࡢ ᵓ⠏ࡀồࡵࡽࢀ࡚࠸ࡿศ㔝ࡢ୍ࡘ࡛࠶ࡿࠋ୍᪉, ⚄⤒✺㉳ᙧᡂ, ࢩࢼࣉࢫᙧᡂࡸ㧊㠧ᙧᡂ࡞ ࡝ࡢ⚄⤒⣔ࡢⓎ㐩㐣⛬࡜⚄⤒ࡢ⏕Ꮡ࣭⥔ᣢࡣྠ➼ࡢᶵᗎࢆ฼⏝ࡍࡿሙྜࡀከࡃ, ᡂ⇍ᮇࡢ⚄ ⤒ẘᛶ≀㉁ࡀⓎ㐩ᮇ࡟ᙳ㡪ࢆࡶࡓࡽࡍྍ⬟ᛶࡀ㧗࠸ࠋ≉࡟, ᡂ⇍ᚋ࡟ࡶࢽ࣮ࣗࣟࣥ᪂⏕ࢆ⥆ ࡅࡿ㒊఩ࡣ, Ⓨ㐩ᮇࡢࡳ࡞ࡽࡎᡂ⇍ᚋࡢᭀ㟢࡟ࡼࡗ࡚ࡶ⚄⤒Ⓨ㐩࡟ᙳ㡪ࢆཬࡰࡍ≀㉁ࡢᶆ ⓗ࡜࡞ࡿྍ⬟ᛶࡀ᝿ᐃࡉࢀࡿࠋࡇࢀࡽࡢ⬻㒊఩࡟╔┠ࡋ࡚, 㑇ఏᏊࡸࡓࢇࡥࡃ㉁ࡢⓎ⌧㔞ࡸ ศᕸࡢኚືࢆホ౯ࡍࡿࡇ࡜࡛, ᡂ⇍ᮇ⚄⤒ẘᛶ≀㉁࡟ࡼࡿࡇࢀࡲ࡛ぢ㏨ࡉࢀ࡚࠸ࡓ㧗ឤཷ ᛶࡢస⏝Ⅼࡸ, ᡂ⇍ື≀ࢆ⏝࠸ࡓⓎ㐩⚄⤒ẘᛶ᳨ฟࡢྍ⬟ᛶࢆ᫂ࡽ࠿࡟࡛ࡁࡿ࡜⪃࠼ࡽࢀ ࡿࠋ ᾏ㤿ṑ≧ᅇࡣᏛ⩦ࡸグ᠈, ࣔࢳ࣮࣋ࢩࣙࣥ࡞࡝ࡢ㧗ḟ⬻ᶵ⬟࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᢸ࠺ࠋ ࡲࡓ, ᾏ㤿ṑ≧ᅇࡣ㢛⢏⣽⬊ᒙୗᖏ㸦subgranular zone㸸SGZ㸧࡟࠾࠸࡚⏕ᾭ࡟ࢃࡓࡗ࡚ࢽࣗ ࣮ࣟࣥ᪂⏕ࡀ⾜ࢃࢀࡿ㒊఩࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿ㸦16, 34㸧ࠋSGZ ࡛ࡢࢽ࣮ࣗࣟࣥ᪂⏕ࡢ㐣⛬

࡛ࡣ, ⚄⤒ᖿ⣽⬊࠿ࡽ type-1 ⚄⤒๓㥑⣽⬊ࢆ⏕ࡌ, type-2a, type-2b, type-3 ๓㥑⣽⬊, ᮍ⇍㢛 ⢏⣽⬊ࡢ㡰࡛ศ໬ࡋ, ᭱⤊ⓗ࡟ᡂ⇍ࡋࡓ㢛⢏⣽⬊࡜࡞ࡿ㸦26, 34, 35㸧ࠋࡇࢀࡽ୍㐃ࡢศ໬, ᡂ ⇍㐣⛬ࡣ, ⚄⤒ᖿ⣽⬊ࡢ⮬ᕫ」〇, ๓㥑⣽⬊ࡢቑṪࡸศ໬, ⚄⤒✺㉳ᙧᡂࡸࢩࢼࣉࢫᙧᡂ࡞

5 ࢆᶆⓗ࡜ࡍࡿ⚄⤒ẘᛶ≀㉁ࡣ, ྠᵝࡢᶵᗎ࡟ࡼࡾ, ࢽ࣮ࣗࣟࣥ᪂⏕㒊఩࡛Ⓨ㐩୰ࡢ⚄⤒ࢆᶆ ⓗ࡜ࡍࡿẘᛶ≀㉁࡟ࡶ࡞ࡾ࠺ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ ᾏ㤿ṑ≧ᅇ㛛࡟Ꮡᅾࡍࡿ௓ᅾࢽ࣮ࣗࣟࣥࡣ, ᡂ⇍୰ࡢ㢛⢏⣽⬊࡟ᢞᑕࡋ࡚ࢽ࣮ࣗࣟࣥ᪂ ⏕ࢆㄪ⠇ࡍࡿᶵ⬟ࢆ᭷ࡍࡿ㸦13, 51㸧ࠋᾏ㤿ṑ≧ᅇ㛛ࡢ γ-aminobutyric acid㸦GABA㸧ᛶ௓ᅾ ࢽ࣮ࣗࣟࣥ࡟ࡣ࠸ࡃࡘ࠿ࡢࢧࣈ࣏ࣆ࣮ࣗࣞࢩࣙࣥࡀᏑᅾࡋ࡚࠾ࡾ㸦27㸧, GABA ᛶ௓ᅾࢽࣗ ࣮ࣟࣥࡀศἪࡍࡿreelin ࡣ, ศ໬㐣⛬࡟࠾ࡅࡿ㢛⢏⣽⬊ࡢᡂ⇍ࡸ⛣ື࡟㛵ࡍࡿ㔜せ࡞ᙺ๭ࢆ ᢸ࠺㸦10, 21, 47, 72㸧ࠋࡋࡓࡀࡗ࡚, ᾏ㤿ṑ≧ᅇ㛛࡟࠾ࡅࡿ௓ᅾࢽ࣮ࣗࣟࣥ࡜ࡑࡢ㛵㐃ศᏊࡢ ᳨⣴ࡣ, SGZ ࡟࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕ࡢホ౯࡟㔜せ࡞᝟ሗࢆ୚࠼ࡿࡶࡢ࡜ᮇᚅࡉࢀࡿࠋ௨ୖ ࡼࡾ, SGZ ࡟࠾ࡅࡿ⚄⤒๓㥑⣽⬊ࡢศᕸࢆᣦᶆ࡜ࡋࡓࢽ࣮ࣗࣟࣥ᪂⏕ࡢ᳨⣴ࡸṑ≧ᅇ㛛࡟ ࠾ࡅࡿ௓ᅾࢽ࣮ࣗࣟࣥࡢ᳨⣴ࡣ, ⚄⤒㞀ᐖࢆ᳨ฟࡍࡿࡓࡵࡢ᭷⏝࡞࢚ࣥࢻ࣏࢖ࣥࢺ࡜ࡋ࡚ ᮇᚅࡉࢀࡿࠋ ࣐࢖ࢡࣟ࢔ࣞ࢖ࢆ⏝࠸ࡓ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣜࣥࢢࡣ, ⑌ᝈࡸ໬Ꮫ≀㉁ࡢᭀ㟢࡟ࡼࡿ ẘᛶⓎ⌧ࡢᶵᗎ࡟㛵㐃ࡋࡓ⤌⧊≉␗ⓗኚ໬࡟ࡘ࠸࡚ࡢ⥙⨶ⓗ࡞᝟ሗ, ࡉࡽ࡟ẘᛶホ౯࡟㐺 ⏝࡛ࡁࡿ᪂ࡋ࠸⣽⬊࣐࣮࣮࢝ࢆ⋓ᚓࡍࡿᡭẁ࡜࡞ࡾ࠺ࡿࠋ୍᪉, ୰ᯡ⚄⤒⤌⧊ࡣゎ๗Ꮫⓗ࡞ ᵓᡂࡀ⢭⦓࡛㒊఩≉␗ᛶࡀ㧗࠸ࡓࡵ, 㑇ఏᏊⓎ⌧ゎᯒࢆ฼⏝ࡋࡓ໬Ꮫ≀㉁ࡢホ౯࡟ࡣ, ゎ๗ Ꮫⓗᵓ㐀, ⣽⬊ศᕸ࠶ࡿ࠸ࡣᶵ⬟࡟ᇶ࡙࠸ࡓ≉ᐃ⬻㒊఩ࡈ࡜ࡢホ౯ࡀᚲせ࡛࠶ࡿࠋࡋ࠿ࡋ, ẘᛶヨ㦂࡟࠾ࡅࡿື≀ゎ๗ࡢసᴗ᫬㛫ෆ࡛ࡣ⬻ࡢ≉ᐃ㡿ᇦࡢ᥇ᮦ࡟ᅔ㞴ࢆక࠺ࡓࡵ, 㐺ษ ࡞᥇ᮦ᪉ἲࡢ㛤Ⓨࡀᚲせ࡜࡞ࡿࠋ ᮏ◊✲࡛ࡣ, 28 ᪥㛫཯᚟ᢞ୚ヨ㦂ࡢᯟ⤌ࡳ࡟࠾ࡅࡿⓎ㐩⚄⤒ẘᛶࡢ᳨ฟࢆど㔝࡟ධࢀ, ⚄ ⤒Ⓨ㐩ᣦᶆࢆ฼⏝ࡋࡓ඲ࡃ᪂つࡢ⚄⤒ẘᛶホ౯ᡭἲࡢ☜❧ࢆ┠ᣦࡍࠋࡑࡢࡓࡵ࡟, ᡂ⇍ᚋࡶ ⥅⥆ࡋ࡚⚄⤒ࡢⓎ㐩࣭ᡂ⇍㐣⛬ࡢほᐹࡀྍ⬟࡞, ᾏ㤿࡟࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕࡟╔┠ࡋ, ࣔ ࢹࣝ≀㉁࡜ࡋ࡚ࡆࡗṑ㢮࡬ࡢᢞ୚࡟ࡼࡗ࡚⚄⤒࡬ࡢᙳ㡪ࡀሗ࿌ࡉࢀ࡚࠸ࡿࢢࣜࢩࢻ࣮ࣝࢆ ⏝࠸, ᡂ⇍ཬࡧⓎ㐩ᮇࡢ⚄⤒࡟ᑐࡍࡿẘᛶᶆⓗᛶࢆ᫂ࡽ࠿࡟ࡍࡿࠋࡉࡽ࡟ࡣ⬻࡟࠾ࡅࡿᙳ㡪 ࢆᤊ࠼ࡿࡓࡵ࡟, ኱⬻࡜ᑠ⬻ࡢ␗࡞ࡿ⬻㒊఩࡛ࡢ㑇ఏᏊࡢ⥙⨶ⓗⓎ⌧ࣉࣟࣇ࢓࢖ࣜࣥࢢࢆ ㏻ࡌ࡚, Ⓨ㐩ᮇ࡜ᡂ⇍ື≀ࡢ཯ᛂᛶࢆ᳨⣴ࡍࡿࠋ

6 ➨1 ❶࡛ࡣ, ྛ⬻㒊఩≉␗ⓗ࡞⥙⨶ⓗ㑇ఏᏊཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟㐺࠺⤌⧊᥇ᮦᡭἲࡢ☜ ❧ࢆ┠ᣦࡋࡓࠋࣛࢵࢺࡢ඲⬻ࢆ࣓ࢱ࣮࢝ࣥᅛᐃࡋ, ࣃࣥࢳ⏕᳨ࢹࣂ࢖ࢫࢆ⏝࠸ࡓ⡆౽࡞᥇ᮦ ࢆ⾜࠸, ⏕య㧗ศᏊࡢရ㉁ཬࡧⓎ⌧ࢹ࣮ࢱࡢᮍᅛᐃ⤌⧊࡟ᑐࡍࡿᛅᐇᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ ➨2 ❶࡛ࡣ, ࢢࣜࢩࢻ࣮ࣝࡢࣛࢵࢺẕື≀ࢆ௓ࡋࡓⓎ㐩ᮇᭀ㟢ࢆ⾜࠸, ẕື≀࡟ᑐࡍࡿ⚄⤒ ẘᛶཬࡧ⚄⤒ẘ࡟ᑐࡍࡿឤཷᛶࡀࡼࡾ㧗࠸࡜⪃࠼ࡽࢀࡿᏊື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿࢽࣗ ࣮ࣟࣥ᪂⏕࡬ࡢᙳ㡪ࢆ᳨⣴ࡋ, ࡑࢀࡽࡢᶆⓗᛶ࡟ᑐࡍࡿẘᛶⓎ⌧ᶵᗎࡢඹ㏻ᛶࢆ᳨ウࡋࡓࠋ ࡲࡓ, ➨ 1 ❶࡟᳨࡚ウࡋࡓ⬻㒊఩≉␗ⓗ࡞⬻⤌⧊᥇ᮦ᪉ἲࢆ⏝࠸࡚, Ꮚື≀ࡢᾏ㤿ṑ≧ᅇ, ⬻ᱱ, ᖏ≧ᅇཬࡧᑠ⬻⓶㉁࡟ࡘ࠸࡚Ⓨ㐩ᮇ⚄⤒ẘᛶ࡟࠿࠿ࡿ㑇ఏᏊⓎ⌧ኚືࣉࣟࣇ࢓࢖ࣝ ࡢ⋓ᚓࢆ⾜ࡗࡓࠋ➨3 ❶࡛ࡣ, ࢢࣜࢩࢻ࣮ࣝࡢࣛࢵࢺࢆ⏝࠸ࡓ 28 ᪥㛫཯᚟ᢞ୚ヨ㦂ࢆ⾜࠸, ᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋ, ➨ 2 ❶࡟࠾ࡅࡿᏊື≀࡬ࡢᙳ㡪࡜ẚ ㍑ࡋࡓࠋࡇࢀ࡟ࡼࡗ࡚⚄⤒Ⓨ㐩ᣦᶆࢆ฼⏝ࡋࡓᡂ⇍ཬࡧⓎ㐩ᮇ⚄⤒ẘᛶࡢ᳨ฟᛶࡢ᭷↓࡟ࡘ ࠸᳨࡚ウࡋࡓࠋࡲࡓ, ᾏ㤿ṑ≧ᅇ, ⬻ᱱ, ᖏ≧ᅇཬࡧᑠ⬻⓶㉁࡟ࡘ࠸࡚, ⚄⤒ẘᛶࣉࣟࣇ࢓࢖ ࣝࢆ⋓ᚓࡋ, ච␿⤌⧊໬Ꮫⓗ᳨⣴࡟ࡼࡿⓎ⌧ศᕸゎᯒࢆ⾜࠸, ཯ᛂᣦᶆࡢ᳨ウࢆ⾜ࡗࡓࠋ

7

➨

➨

1 ❶

8

⥴

⥴ ゝ

㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆⓎ㐩ᮇ࡜ᡂ⇍ᮇࡢ⚄⤒ẘᛶホ౯࡟฼⏝ࡍࡿ๓ᥦ᮲௳࡜ࡋ࡚, ᶆⓗ࡜ࡍࡿ⬻㒊఩ࡢⓎ⌧ࢹ࣮ࢱࡢࡤࡽࡘࡁࢆ᭱ᑠ㝈࡟ᢚ࠼ࡓ⤌⧊㒊఩≉␗ⓗ࡞᥇ᮦࢆྍ⬟ ࡟ࡍࡿ᪉ἲࢆྲྀࡾධࢀࡿᚲせࡀ࠶ࡿࠋ୰ᯡ⚄⤒⤌⧊ࡣゎ๗Ꮫⓗ࡞ᵓᡂࡀ⢭⦓࡛࠶ࡾ, ࢽ࣮ࣗ ࣟࣥࡸࢢࣜ࢔⣽⬊ศᕸࡢ㒊఩≉ᛶࡀ㧗࠸ࠋࡑࡢࡓࡵ, ⥙⨶ⓗᡭἲ࡟ࡼࡿ㑇ఏᏊཬࡧࡓࢇࡥࡃ ㉁ゎᯒࡢࡓࡵ࡟ࡣྛ⬻㒊఩ࢆಶู࡟᥇ᮦࡍࡿᚲせࡀ࠶ࡿࠋ୍᪉, ࡆࡗṑ㢮ࢆ⏝࠸ࡓື≀ヨ㦂 ࡢゎ๗᫬࡟࠾ࡅࡿ」ᩘࡢ⬻㒊఩ࡢᡭసᴗ࡛ࡢ᥇ᮦࡣ᫬㛫ࢆせࡋ, ゎ๗ᚋ࡟ࡳࡽࢀࡿ RNA ➼ ࡢ⏕య㧗ศᏊࡢศゎࡣ, ศᏊ⏕≀Ꮫⓗゎᯒࢹ࣮ࢱࡢ㉁ࡢపୗࢆࡶࡓࡽࡍࠋࡑࢀࢆゎỴࡍࡿ᪉ ἲ࡜ࡋ࡚඲⬻ᅛᐃἲࡢ฼⏝ࡀᣲࡆࡽࢀࡿࠋ඲⬻ᅛᐃ࡟ࡼࡾゎ๗ᚋࡢ᥇ᮦࡢᐇ᪋ࡀྍ⬟࡜࡞ࡿ ࡶࡢࡢ, 㧗⢭ᗘ࡞ゎᯒࢆᐇ᪋ࡍࡿࡓࡵ࡟ࡣ DNA, RNA ཬࡧࡓࢇࡥࡃ㉁➼ࡢ⏕య㧗ศᏊࡢရ ㉁ࢆಖᣢࡋࡓࡲࡲ⤌⧊ࢆᅛᐃࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓ, ㏆ᖺ≉ᐃ⬻㒊఩ࡢ㑇ఏᏊཬࡧࡓࢇࡥࡃ ㉁ゎᯒ࡟࣐࢖ࢡࣟࢲ࢖ࢭࢡࢩࣙࣥᢏ⾡ࡀ฼⏝ࡉࢀ࡚࠸ࡿ㸦5, 63, 95㸧ࠋࡋ࠿ࡋ, ࡑࡢᐇ᪋࡟ࡣ సᴗ࡟᫬㛫࡜ປຊࢆせࡍࡿࠋ㐺ษ࡞᫬㛫ෆ࡛ࡢ୍㈏ࡋࡓ᥇ᮦࡣ, ᡭసᴗ࡟ࡼࡿ᥇ᮦࡀ⡆౽࡛ ࠶ࡾ, ࡑࡢࡓࡵ࡟ࡶ⬻⤌⧊ᅛᐃࡢ฼⏝ࡀ㐺ࡍࡿ࡜⪃࠼ࡽࢀࡿࠋࡇࢀࡽࡢࡇ࡜ࡼࡾ, ⬻㒊఩≉ ␗ⓗ࡞㧗⢭ᗘࡢ⥙⨶ⓗศᏊゎᯒࡢࡓࡵ࡟ࡣ, ᢳฟศᏊࡢရ㉁ࢆ㧗ࡃಖᣢ࡛ࡁࡿᅛᐃἲࢆ฼ ⏝ࡋࡓ, ⡆౽࠿ࡘṇ☜࡞᥇ᮦ᪉ἲࡢ☜❧ࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ ࣓ࢱ࣮࢝ࣥᅛᐃἲࡣ, ᭷ᶵ⁐፹ࢆ⏝࠸ࡓࡓࢇࡥࡃ㉁ỿẊࢆᅛᐃཎ⌮࡜ࡋ࡚࠸ࡿ㸦54, 71㸧ࠋ ᅛᐃᚋࡢࣃࣛࣇ࢕ࣥໟᇙ⤌⧊ࢆ⏝࠸ࡓ⤌⧊Ꮫⓗホ౯࡟࠾࠸࡚⤌⧊ᙧែࡢಖᣢࡀྍ⬟࡛࠶ࡾ 㸦12, 83㸧, ච␿⤌⧊໬Ꮫⓗゎᯒ࡟࠾࠸࡚ࡶ, ࣒࣍ࣝ࢔ࣝࢹࣄࢻࢆ฼⏝ࡋࡓᯫᶫᅛᐃᾮ࡜ẚ㍑ ࡋ࡚ᢠཎᛶࡢ⥔ᣢ࡟ࡼࡗ࡚ඃࢀࡓ⤖ᯝࡀᚓࡽࢀࡿ㸦2, 64, 74㸧ࠋᅛᐃᚋࡢ᥇ᮦ࡛ࡶ, DNA, RNA ཬࡧࡓࢇࡥࡃ㉁ࡢရ㉁ࢆ㧗ࡃಖᣢ࡛ࡁࡿࡓࡵ, ඲⬻ᅛᐃ࡟ࡼࡿ≉ᐃ⬻㒊఩ࡢ᥇ᮦ࡟฼⏝࡛ ࡁࡿ࡜⪃࠼ࡽࢀࡿࠋᐇ㝿࡟࣓ࢱ࣮࢝ࣥᅛᐃἲࢆ฼⏝ࡋࡓࣃࣛࣇ࢕ࣥໟᇙ⤌⧊࡛ࡢࢤࣀ࣒ DNA ࡢ㓄ิゎᯒ, ㌿෗⏘≀ࡢ real-time RT-PCR ࡸ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ, ࡓࢇࡥࡃ㉁ศᏊࡢ࢘ ࢙ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢゎᯒἲࡀ☜❧ࡉࢀ, ⥙⨶ⓗゎᯒ࡟฼⏝ࡉࢀ࡚࠸ࡿ㸦79, 80, 86, 92㸧ࠋ

9 ᮏ❶ࡣ, ྛ⬻㒊఩≉␗ⓗ࡞⥙⨶ⓗ㑇ఏᏊཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟㐺࠺⤌⧊᥇ᮦᡭἲࡢ☜❧ ࡢࡓࡵ࡟, ࣛࢵࢺࡢ඲⬻ࡢ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࠿ࡽࣃࣥࢳ⏕᳨ࢹࣂ࢖ࢫࢆ⏝࠸࡚≉ᐃ⬻㒊 ఩ࢆ᥇ᮦࡋ, ᢳฟࡋࡓ total RNA ཬࡧ࣏ࣜ࣌ࣉࢳࢻࡢရ㉁࡞ࡽࡧ࡟Ⓨ⌧ࢹ࣮ࢱࡢᛅᐇᛶࢆホ ౯ࡋࡓࠋࡑࡢࡓࡵ࡟, ᮍᅛᐃ⤌⧊ཬࡧࡋࡤࡋࡤศᏊ⏕≀Ꮫⓗゎᯒ࡟฼⏝ࡉࢀ࡚࠸ࡿ࢔ࢭࢺࣥ ᅛᐃ⤌⧊㸦20, 96㸧࡜ࡢẚ㍑ࢆ⾜ࡗࡓࠋゎᯒ࡟ࡣ, ໬Ꮫ≀㉁ࡢ୍⯡ẘᛶヨ㦂࡛ࡢᢞ୚⤊஢᫬ ᮇ࡜ࡋ࡚, 28 ᪥㛫཯᚟ᢞ୚ẘᛶヨ㦂࡟࠾ࡅࡿ 9 㐌㱋㸦⏕ᚋ 63 ᪥㱋㸧ື≀ཬࡧⓎ㐩ᮇ⚄⤒ẘ ᛶヨ㦂࡟࠾ࡅࡿ⏕ᚋ21 ᪥㱋ື≀ࢆ⏝࠸ࡓࠋࡇࢀ࡟ࡼࡗ࡚඲⬻ࡢ࣓ࢱ࣮࢝ࣥᅛᐃἲࢆ⏝࠸ࡓ ≉ᐃ⬻㒊఩࡟࠾ࡅࡿ⥙⨶ⓗⓎ⌧ゎᯒࡢ᭷⏝ᛶࡢ᭷↓ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ

10

ᮦ

ᮦᩱཬࡧ᪉ἲ

౪ヨື≀ཬࡧᐇ㦂ࢹࢨ࢖ࣥ ዷፎ2 ᪥㱋㸦ࣉࣛࢢ☜ㄆ᪥ࢆዷፎ 0 ᪥࡜ࡋ࡚㉳⟬㸧ࡢ Sprague-Dawley ⣔ SPF ࣛࢵࢺཬࡧ 7 㐌㱋ࡢྠ⣔⤫ࡢ㞝ᛶࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ఍♫㸦⚄ዉᕝ㸧࠿ࡽ㉎ධࡋᐇ㦂 ࡟౪ࡋࡓࠋື≀ࡣ ᗘ23±2°C, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ 㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟཰ᐜࡋࡓࠋዷፎື≀ࡣ 1 ༉ࡎࡘ, ศፔᚋࡣ ẕᏊඹ࡟ྠ୍ࢣ࣮ࢪ࡟, ࡲࡓ 7 㐌㱋ࡢ㞝ື≀ࡣ 3 ࠿ࡽ 4 ༉ࡎࡘ཰ᐜࡋࡓࠋື≀ࡣᇶ♏㣫ᩱ CRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ ᐇ㦂1 7 㐌㱋ࡢ㞝ᛶࣛࢵࢺࢆ⏝࠸, 1 㐌㛫ࡢ㥆໬㣫⫱ᚋ, 8 㐌㱋ࡢື≀࡟⢭〇Ỉࢆ 5 ml/kg ࡟࡚ 7 ᪥ 㛫ᙉไ⤒ཱྀᢞ୚ࡋࡓࠋᢞ୚㛤ጞ᪥ཬࡧ๗᳨᪥࡟య㔜ࢆ ᐃࡋࡓࠋ᭱⤊ᢞ୚⩣᪥࡟, 9 㐌㱋㸦⏕ ᚋ63 ᪥㱋㸧ࡢື≀ࢆ 3 ⩌㸦ᮍᅛᐃ⩌㸸4 ༉, ࣓ࢱ࣮࢝ࣥᅛᐃ⩌ཬࡧ࢔ࢭࢺࣥᅛᐃ⩌㸸ྛ 8 ༉㸧࡟↓సⅭ࡟⩌ศࡅࡋ, ඲౛ࢆ๗᳨ࡋ࡚㑇ఏᏊⓎ⌧ゎᯒཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟౪ࡋࡓࠋ඲ ࡚ࡢື≀ࡣCO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏࡓࠋ ᐇ㦂2 ዷፎࣛࢵࢺࢆ⏝࠸ࡓࠋศፔᚋ4 ᪥࡟, 1 ⭡㞝ྛ 8 ༉࡟࡞ࡿࡼ࠺↓సⅭ࡟㑅ᢤࡋ࡚Ꮚᩘࡢㄪ ᩚࢆ⾜ࡗࡓࠋ1 ⭡ࡢ㞝Ꮚື≀ࡀ 8 ༉࡟‶ࡓ࡞࠸ሙྜࡣ, 1 ⭡ 8 ༉࡜࡞ࡿࡼ࠺࡟㞤Ꮚື≀ࢆṧࡋ ࡚ㄪᩚࡋࡓࠋ⏕ᚋ21 ᪥࡟, 㞝Ꮚື≀ 20 ༉㸦1 ⭡ྛ 2 ༉㸧ࢆ↓సⅭ࡟㑅ᢤࡋ, 3 ⩌㸦ᮍᅛᐃ⩌㸸 8 ༉, ࣓ࢱ࣮࢝ࣥᅛᐃ⩌ཬࡧ࢔ࢭࢺࣥᅛᐃ⩌㸸ྛ 16 ༉, ྛ⩌ 1 ⭡ྛ 1-2 ༉㸧࡟⩌ศࡅࡋ, ๗ ᳨ࡋ࡚㑇ఏᏊⓎ⌧ゎᯒཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟౪ࡋࡓࠋ඲࡚ࡢື≀ࡣCO2/O2㯞㓉ୗ࡛⭡኱ື ⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏࡓࠋ ඲࡚ࡢື≀ᐇ㦂ィ⏬ࡣᐇ㦂᪋タ㸦ᮾி㎰ᕤ኱Ꮫ㸧ࡢື≀ᐇ㦂ጤဨ఍ࡢᢎㄆࢆཷࡅ, ඲࡚ࡢ ື≀ᐇ㦂᧯సࡣࠕື≀ᐇ㦂ࡢ㐺ṇ࡞ᐇ᪋࡟ྥࡅࡓ࢞࢖ࢻࣛ࢖ࣥࠖ㸦᪥ᮏᏛ⾡఍㆟, ᖹᡂ 18 ᖺ 6 ᭶ 1 ᪥㸧ࢆ㑂Ᏺࡋࡓࠋ

11 ⤌ ⤌⧊ࡢᅛᐃཬࡧ≉ᐃ⬻㒊఩ࡢ᥇ྲྀ ࣓ࢱ࣮࢝ࣥᾮࡣ౑⏝๓࡟, ࣓ࢱࣀ࣮ࣝ, ࢡ࣒ࣟࣟ࣍ࣝཬࡧ㓑㓟ࢆ 6:3:1 ࡢ⤌ᡂ࡛ΰ࿴ࡋ࡚ ㄪ〇ࡋࡓࠋ࣓ࢱ࣮࢝ࣥᾮཬࡧ࢔ࢭࢺࣥࡣ౑⏝᫬ࡲ࡛4°C ࡟࡚ಖᏑࡋࡓࠋ๗᳨᫬, ᅛᐃ⩌ࡣ඲ ⬻ࢆ࣓ࢱ࣮࢝ࣥᾮ࠶ࡿ࠸ࡣ࢔ࢭࢺࣥ࡟ᾐₕࡋ, 4°C ࡛ 5 ᫬㛫᣺┞ࡋ࡚ᅛᐃࡋࡓࠋࡑࡢᚋ, 99.5%࢚ࢱࣀ࣮ࣝ࡟࡚ 1 ᫬㛫࠾ࡁ࡟ 3 ᅇ⬺Ỉࡋ, 4°C ୍࡛ᬌ᣺┞ࡋࡓࠋᮍᅛᐃ⩌ࡣ, ඲⬻᥇ྲྀ ᚋ, ị෭ࡋࡓ⏕⌮㣗ሷᾮ୰࡛┤ࡕ࡟ࢧࣥࣉࣜࣥࢢࢆ⾜ࡗࡓࠋ ኱⬻ࡣ⬻㗪ᆺ㸦ࣈࣞ࢖࣐ࣥࢺࣜࢵࢡࢫ㸸ᐊ⏫ᶵᲔᰴᘧ఍♫, ᮾி㸧ࢆ⏝࠸࡚ཌࡉ 2 mm ࡢ ෙ≧᩿ࢫࣛ࢖ࢫࢆస〇ࡋ, ᑠ⬻ࡣཌࡉ 2 mm ࡢ▮≧᩿ࢫࣛ࢖ࢫࢆస〇ࡋࡓࠋ1 mm ᚄࡢ⏕᳨ ࢺࣞࣃࣥ㸦࢝࢖࢖ࣥࢲࢫࢺ࣮ࣜࢬᰴᘧ఍♫, ᒱ㜧㸧ࢆ⏝࠸࡚, ᖏ≧ᅇ, ᑠ⬻⓶㉁, ᾏ㤿ṑ≧ᅇ ཬࡧ⬻ᱱ⤌⧊ࡢ4 㒊఩ࢆࡃࡾᢤ࠸࡚᥇ྲྀࡋࡓࠋᮍᅛᐃ⩌ࡣ 3 mm ᚄࡢ⏕᳨ࢺࣞࣃࣥࢆ⏝࠸࡚, ྠᵝࡢ㒊ศ࠿ࡽᖏ≧ᅇཬࡧᑠ⬻⓶㉁⤌⧊ࢆࡃࡾᢤ࠸࡚᥇ྲྀࡋࡓࠋ᥇ྲྀࡋࡓࢧࣥࣉࣝࡢ࠺ࡕ, ᅛᐃ⤌⧊ࡣ99.5%࢚ࢱࣀ࣮ࣝ࡟ᾐₕࡋ, -80°C ࡟࡚ಖᏑࡋࡓࠋᮍᅛᐃ⤌⧊ࡢ RNA ゎᯒ⏝ࢧࣥ

ࣉࣝࡣRNAlaterTM㸦Ambion, Inc., Austin, TX, USA㸧࡟ᾐₕࡋ࡚ 4°C ࡟࡚ 24 ᫬㛫ಖᏑᚋ, -80°C ࡟ಖᏑࡋࡓࠋᮍᅛᐃ⤌⧊ࡢࡓࢇࡥࡃ㉁ゎᯒ⏝ࢧࣥࣉࣝࡣᾮయ❅⣲࡟࡚෾⤖ࡋ, ┤ࡕ࡟-80°C ࡟ಖᏑࡋࡓࠋ

RNA ᢳฟཬࡧ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ

᥇ྲྀࡋࡓ⤌⧊ࢧࣥࣉࣝ࠿ࡽ, QIAzol㸦Qiagen, Valencia, CA, USA㸧ཬࡧ RNeasy kit㸦Qiagen㸧 ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚total RNA ࢆᢳฟࡋ, NanoDrop ND-1000㸦Thermo Fisher Scientific, Wilmington, DE, USA㸧ࢆ⏝࠸࡚⃰ᗘࢆ ᐃࡋࡓࠋ⏕ᚋ 63 ᪥㱋ࡢື≀ࡣྛ⩌ 4 ༉, 21 ᪥㱋ࡢື≀ࡣྛ⩌ 8 ༉࡟ࡘ࠸࡚, Agilent 2100 Bioanalyzer㸦Agilent Technologies, Inc., Santa Clara, CA, USA㸧ࢆ⏝࠸࡚ RNA Integrity Number㸦RIN㸧ࢆồࡵ, RNA ࡢရ㉁ࢆホ౯ࡋ ࡓࠋ

⏕ᚋ63 ᪥㱋ཬࡧ 21 ᪥㱋ࡢື≀࡜ࡶྛ⬻㒊఩ 3 ౛࡟ࡘ࠸࡚, Agilent Rat Oligo arrays㸦Agilent Technologies, Inc.㸧ࢆ⏝࠸࡚, ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࢆ⾜ࡗࡓࠋࢧࣥࣉࣝࡢฎ⌮ཬࡧ࣐࢖ࢡ

12

ࣟ࢔ࣞ࢖ᐇ㦂ࡣ〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚⾜ࡗࡓࠋࢥࣥࢺ࣮ࣟࣝRNA㸦Agilent

One-Color RNA Spike-In kit㸧࡟ 100 ng ࡢ total RNA ࢆຍ࠼, Agilent Low Input Quick Amp Labeling kit (one-color) ࢆ⏝࠸࡚ Cy3 ࣛ࣋ࣝ cRNA ࢆస〇ᚋ, RNeasy mini kit㸦Qiagen㸧ࢆ⏝ ࠸࡚⢭〇, in situ hybridization kit㸦Agilent Technologies, Inc.㸧ࢆ⏝࠸࡚᩿∦໬ࡋ, ࣁ࢖ࣈࣜࢲ ࢖ࢮ࣮ࢩࣙࣥࢆ⾜ࡗࡓࠋG4900DA scanner㸦Agilent Technologies, Inc.㸧ཬࡧ Agilent Feature Extraction software㸦version 11.7.1.1㸧ࢆ⏝࠸࡚㑇ఏᏊⓎ⌧ࢹ࣮ࢱࢆྲྀᚓࡋࡓࠋ࣐࢖ࢡࣟ࢔ࣞ ࢖ࢹ࣮ࢱࡢゎᯒࡣGene Spring GX㸦version 11.5.1㸧software㸦Agilent Technologies, Inc.㸧ࢆ⏝ ࠸࡚⾜ࡗࡓࠋ

࣏

࣏ࣜ࣌ࣉࢳࢻᢳฟ, SDS-PAGE, 2D-DIGE ཬࡧ MS ゎᯒ

⏕ᚋ63 ᪥㱋ࡢື≀ࡢྛᅛᐃ⩌ 4 ༉ࡢྛ⬻㒊఩࡟ࡘ࠸࡚, lysis buffer (4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate, 2 M thiourea, 8 M urea, 10 mM Tris-HCl; pH 8.8) ୰࡛, ᥇ྲྀࡋࡓ⤌⧊ࢆ࣍ࣔࢪࢼ࢖ࢬࡋ࡚࣏ࣜ࣌ࣉࢳࢻࢆᢳฟࡋ, ゎᯒ࡟౪ ࡋࡓࠋ

ᢳฟࡋࡓ࣏ࣜ࣌ࣉࢳࢻ࡟ࡘ࠸࡚Hoefer Mighty Small Gel System㸦GE Healthcare, Little Chalfont, UK㸧ࢆ⏝࠸࡚, SDS ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ㟁ẼὋື㸦SDS-PAGE㸧ࢆ⾜ࡗࡓࠋ5 μg ࡢ࣏ࣜ࣌ࣉࢳࢻࢆ⏝࠸࡚Cy5 ࣛ࣋ࣝࡋ, SDS buffer㸦2% SDS, 20% glycerol, 0.01% bromphenol blue, 50 mM Tris-HCl, 0.1 M dithiothreitol, pH 6.8㸧࡟ຍ࠼࡚, 15-25% gradient acrylamide gel㸦ࢥ ࢫࣔࣂ࢖࢜ᰴᘧ఍♫, ᮾி㸧࡟࢔ࣉࣛ࢖ࡋࡓࠋࢤࣝࡢ࢖࣓࣮ࢪゎᯒࡣ Typhoon㸦GE Healthcare㸧 ཬࡧImage Quant TL v2005㸦GE Healthcare㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋ

ᢳฟࡋࡓ࣏ࣜ࣌ࣉࢳࢻࢆ⏝࠸࡚஧ḟඖࢹ࢕ࣇ࢓ࣞࣥࢫࢤࣝ㟁ẼὋື㸦2D-DIGE㸧ࢆ⾜ࡗࡓࠋ 〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚Cy2, Cy3 ཬࡧ Cy5㸦GE Healthcare㸧ࣛ࣋ࣝࡋ, Pharmalytes pH 3-10㸦GE Healthcare㸧࡜ΰྜࡋ࡚ᅛᐃ໬ pH ໙㓄ࢫࢺࣜࢵࣉࢤࣝ㸦pH 3-10; 24cm㸧࡟࢔ࣉࣛ ࢖ࡋࡓࠋ࢖࣓࣮ࢪゎᯒࡣTyphoon 9400㸦GE Healthcare㸧ཬࡧ Decyder® software Ver7.5㸦GE Healthcare㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋ

13

600 μg ࡢ࣏ࣜ࣌ࣉࢳࢻࢆ⏝࠸࡚ mass spectrometry㸦MS㸧ゎᯒࡢࡓࡵࡢ 2D-DIGE ࢆ⾜ࡗࡓࠋ ┠ⓗࡢࢫ࣏ࢵࢺࢆ automated spot picker㸦GE Healthcare㸧ࢆ౑⏝ࡋ࡚ 2 ḟඖࢤࣝ࠿ࡽ᥇ྲྀࡋ, 12.5 ng/μl trypsin㸦Promega Corporation, Madison, WI, USA㸧୰࡛࣌ࣉࢳࢻࢆ⁐ฟࡋ, 0.1 v/v% ࢠ㓟࡟⁐ゎࡉࡏࡓࠋMS ゎᯒࡣ LC-MS/MS ࡟ࡼࡗ࡚ᐇ᪋ࡋࡓࠋ࣌ࣉࢳࢻࡣ automated MS-to-MS/MS ࢫ࢖ࢵࢳࣉࣟࢺࢥ࣮ࣝࢆᦚ㍕ࡋࡓ tandem mass spectrometric sequencing ࡛ศᯒ ࡋࡓࠋࢹ࣮ࢱ࣮࣋ࢫ᳨⣴ࡣMASCOT Deamon㸦Matrix Science, London, UK㸧㸦15, 48, 66, 69㸧 ࢆ౑⏝ࡋࡓࠋ

α-࢝࢝ࢮ࢖ࣥࡢ⁐ゎᛶศᯒ

」ᩘࡢࣜࣥ㓟໬ࣃࢱ࣮ࣥࢆᣢࡕ, ஧ḟඖ㟁ẼὋື࡟ࡼࡗ࡚」ᩘࡢࢫ࣏ࢵࢺ࡟ศ㞳ࡍࡿࡓ

ࢇࡥࡃ㉁࡛࠶ࡿα-࢝ࢮ࢖ࣥ࡟ࡘ࠸࡚, ྛᅛᐃᾮཬࡧ࣓ࢱ࣮࢝ࣥᾮࡢᵓᡂᾮ࡬ࡢ⁐ゎᛶࢆホ

౯ࡋࡓࠋ5 mg ࡢ α-࢝ࢮ࢖ࣥ㸦α-casein from bovine milk; Sigma-Aldrich Co., St. Louis, MO, USA㸧

ࢆ500 μl ࡢ 50 mM 㔜Ⅳ㓟࢔ࣥࣔࢽ࣒࢘࡟⁐ゎࡉࡏࡓࠋ⁐ゎࡉࡏࡓ α-࢝ࢮ࢖ࣥ⁐ᾮ㸦1 mg/100 μl㸧ࢆ஝⇱ࡉࡏ, 600 μl ࡢ࣓ࢱ࣮࢝ࣥᾮ, ࣓ࢱࣀ࣮ࣝཬࡧࢡ࣒ࣟࣟ࣍ࣝΰྜᾮ㸦6㸸3㸧, ࢔ࢭ ࢺࣥ, ࢡ࣒ࣟࣟ࣍ࣝ࠶ࡿ࠸ࡣ࣓ࢱࣀ࣮ࣝ࡟ 4°C ࡛ 5 ᫬㛫⁐ゎࡋࡓࠋ20,000×g, 2 ᫬㛫, 4°C ࡛ 㐲ᚰᚋ, ỿẊ≀࡜ࡋ࡚㠀⁐ゎ α-࢝ࢮ࢖ࣥࢆ, ୖΎࢆ஝⇱ࡉࡏ࡚⁐ゎ α-࢝ࢮ࢖ࣥࢆ᥇ྲྀࡋ, ྛ 㔜㔞࠿ࡽ⁐ゎᛶࢆ ᐃࡋࡓࠋᚓࡽࢀࡓ⁐ゎཬࡧ㠀⁐ゎα-࢝ࢮ࢖ࣥࢆ⏝࠸࡚, ୖ㏙ࡢ࡜࠾ࡾ 2D-DIGE ゎᯒ࡟౪ࡋࡓࠋ ⤫ィゎᯒ ᮍᅛᐃ⤌⧊ࡢᖏ≧ᅇཬࡧᑠ⬻⓶㉁㛫ࡢRIN ࡢẚ㍑ࡣ, F ᳨ᐃࢆ⏝࠸࡚ศᩓศᯒࢆ⾜ࡗࡓᚋ, Student ࡢ t ᳨ᐃ㸦➼ศᩓ㸧ࡶࡋࡃࡣ Aspin-Welch ࡢ t ᳨ᐃ㸦୙➼ศᩓ㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋྛ ᅛᐃ⤌⧊ࡢྛ⬻㒊఩㛫ࡲࡓࡣᖏ≧ᅇཬࡧᑠ⬻⓶㉁࡟࠾ࡅࡿᮍᅛᐃཬࡧྛᅛᐃ㛫ࡢRIN ࡢẚ

㍑ࡣ, Bartlett ᳨ᐃࢆ⏝࠸ࡓศᩓศᯒࢆ⾜ࡗࡓᚋ࡟, Tukey ᳨ᐃ㸦➼ศᩓ㸧ࡶࡋࡃࡣ Steel-Dwass

14

15

⤖

⤖ ᯝ

Total RNA ࡢရ㉁

ᮍᅛᐃ⬻⤌⧊㸦ᖏ≧ᅇཬࡧᑠ⬻⓶㉁㸧ཬࡧ࣓ࢱ࣮࢝ࣥ࠶ࡿ࠸ࡣ࢔ࢭࢺࣥᅛᐃ⤌⧊㸦ᖏ≧ᅇ, ᑠ⬻⓶㉁, ᾏ㤿ṑ≧ᅇཬࡧ⬻ᱱ㸧ࡢྛ㒊఩࠿ࡽᢳฟࡉࢀࡓ total RNA ࡟ࡘ࠸࡚ RIN ್ࢆẚ㍑

ࡋࡓࠋᮍᅛᐃ⤌⧊࡟࠾࠸࡚, ᑠ⬻⓶㉁ࡢ total RNA ࡢ RIN ್ࡣᖏ≧ᅇࡢࡑࢀࡼࡾࡶ㧗್ࢆ♧

ࡋࡓࠋ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡢtotal RNA ࡢ RIN ್ࡣ⏕ᚋ 63 ᪥㱋ཬࡧ 21 ᪥㱋ඹ࡟ྛ⬻㒊఩࡛

ྠ⛬ᗘࡢ್ࢆ♧ࡋࡓࠋ࢔ࢭࢺࣥᅛᐃ⤌⧊࡛ࡣ, ᑠ⬻⓶㉁ࡢ total RNA ࡢ RIN ್ࡣᖏ≧ᅇࡲࡓ

ࡣ⬻ᱱ࡜ẚ㍑ࡋ࡚⏕ᚋ63 ᪥㱋࡛᭷ព࡞㧗್ࢆ♧ࡋࡓࠋ⏕ᚋ 21 ᪥㱋࡛ࡣ, ᑠ⬻⓶㉁ࡢ total

RNA ࡢ RIN ್ࡣ⬻ᱱ࡜ẚ㍑ࡋ࡚᭷ព࡞㧗್ࢆ♧ࡋࡓ㸦Fig. 1-1A㸧ࠋ

ᖏ≧ᅇࡢtotal RNA ࡢ RIN ್ࡢᖹᆒ್ࡣ, ⏕ᚋ 63 ᪥㱋࡟࠾࠸࡚ᮍᅛᐃཬࡧ࣓ࢱ࣮࢝ࣥᅛ

ᐃ⤌⧊࡛ྠ⛬ᗘࡢ್ࢆ♧ࡋࡓࡀ, ⏕ᚋ 21 ᪥㱋࡟࠾࠸࡚ᮍᅛᐃ⤌⧊ࡣ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡼ

ࡾࡶ᭷ព࡞㧗್ࢆ♧ࡋࡓࠋᮍᅛᐃᖏ≧ᅇ⤌⧊ࡢtotal RNA ࡢ RIN ್ࡢᖹᆒ್ࡣ, ⏕ᚋ 63 ᪥

㱋ཬࡧ21 ᪥㱋ඹ࡟࢔ࢭࢺࣥᅛᐃ⤌⧊ࡼࡾࡶ᭷ព࡞㧗್ࢆ♧ࡋࡓࠋ࣓ࢱ࣮࢝ࣥᅛᐃᖏ≧ᅇ⤌

⧊ࡢtotal RNA ࡢ RIN ್ࡢᖹᆒ್ࡣ⏕ᚋ 63 ᪥㱋࡟࠾࠸࡚࢔ࢭࢺࣥᅛᐃ⤌⧊ࡼࡾࡶ᭷ព࡞㧗

್ࢆ♧ࡋࡓࠋࡉࡽ࡟⏕ᚋ21 ᪥㱋࡟࠾࠸࡚࣓ࢱ࣮࢝ࣥཬࡧ࢔ࢭࢺࣥᅛᐃᖏ≧ᅇ⤌⧊࡟ࡣኚ໬

ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋᑠ⬻⓶㉁ࡢtotal RNA ࡢ RIN ್ࡢᖹᆒ್ࡣ࣓ࢱ࣮࢝ࣥࡲࡓࡣ࢔ࢭࢺࣥ

ᅛᐃ⤌⧊ࡼࡾࡶᮍᅛᐃ⤌⧊ࡢ᪉ࡀ⏕ᚋ63 ᪥㱋ཬࡧ 21 ᪥㱋ඹ࡟㧗್ࢆ♧ࡋࡓࠋRIN ್ࡢᖹᆒ ್ࡣ⏕ᚋ63 ᪥㱋ཬࡧ 21 ᪥㱋ඹ࡟࣓ࢱ࣮࢝ࣥཬࡧ࢔ࢭࢺࣥᅛᐃᑠ⬻⓶㉁⤌⧊࡟࠾࠸࡚ኚ໬ࡣ ㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 1-1B㸧ࠋ ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡢࡤࡽࡘࡁ ⏕ᚋ63 ᪥㱋࡟࠾ࡅࡿྠࡌᅛᐃ⤌⧊ෆࡢྛື≀ࡢ⬻ 4 㒊఩ࡢ࣐࢖ࢡࣟ࢔ࣞ࢖㑇ఏᏊⓎ⌧ࢹ ࣮ࢱࡢᩓᕸᅗࡢ௦⾲౛ࢆFig. 1-2 ࡟♧ࡋࡓࠋ඲࡚ࡢ⬻㒊఩࡟࠾࠸࡚࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡜࢔ ࢭࢺࣥᅛᐃ⤌⧊࡛mRNA Ⓨ⌧ࢹ࣮ࢱࡢ㧗࠸┦㛵ࡀㄆࡵࡽࢀࡓࠋ⏕ᚋ 63 ཬࡧ 21 ᪥㱋ࡢྛ⬻

16 㒊఩ࡢ࣓ࢱ࣮࢝ࣥ࡜࢔ࢭࢺࣥᅛᐃ⤌⧊࡟࠾ࡅࡿỴᐃಀᩘࡢᖹᆒ್࡜ࡑࡢࡤࡽࡘࡁࡢᣦᶆ࡛ ࠶ࡿኚືಀᩘࢆẚ㍑ࡋࡓ㸦Table 1-1㸧ࠋྛ⬻㒊఩࡟࠾࠸࡚࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡣ࢔ࢭࢺࣥᅛ ᐃ⤌⧊ࡼࡾ㧗࠸Ỵᐃಀᩘࢆ♧ࡋࡓࠋኚືಀᩘࡶࡲࡓ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡣ࢔ࢭࢺࣥᅛᐃ⤌⧊ ࡜ẚ㍑ࡋ࡚ᖖ࡟ప್࡛࠶ࡗࡓࠋ ࣏ ࣏ࣜ࣌ࣉࢳࢻࡢရ㉁ ⏕ᚋ63 ᪥㱋࡟࠾࠸࡚, ᖏ≧ᅇཬࡧᑠ⬻⓶㉁ࡢᮍᅛᐃ⤌⧊, ࣓ࢱ࣮࢝ࣥᅛᐃཬࡧ࢔ࢭࢺࣥ ᅛᐃ⤌⧊㛫࡛, ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ୰ࡢ࣏ࣜ࣌ࣉࢳࢻࣂࣥࢻࡢࣃࢱ࣮ࣥཬࡧࡑࢀࡽࡢ ࢩࢢࢼࣝᙉᗘࡣྠ⛬ᗘ࡛࠶ࡗࡓ㸦Fig. 1-3㸧ࠋ 2D-DIGE ࡟ࡼࡿࡓࢇࡥࡃ㉁Ⓨ⌧ゎᯒ ⏕ᚋ63 ᪥ࡢ⤌⧊ࢆ⏝࠸࡚2D-DIGE ゎᯒࢆ⾜ࡗࡓ⤖ᯝ, ᖏ≧ᅇཬࡧᑠ⬻⓶㉁ࡢᮍᅛᐃ⤌ ⧊, ࣓ࢱ࣮࢝ࣥᅛᐃཬࡧ࢔ࢭࢺࣥᅛᐃ⤌⧊㛫࡛, ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ୰࡛ࡢ࣏ࣜ࣌ࣉࢳ ࢻࢫ࣏ࢵࢺࡢࣃࢱ࣮ࣥ࡟ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 1-4㸧ࠋ஧ḟඖࢤࣝ୰࡛ㄆࡵࡽࢀࡓ࠾ࡼ ࡑ2,100㹼2,300 ࡢ࣏ࣜ࣌ࣉࢳࢻࢫ࣏ࢵࢺ࡟ࡘ࠸࡚, ࡯ࡰ඲࡚ࡢࢫ࣏ࢵࢺᐜ✚࡛ᮍᅛᐃཬࡧྛ ᅛᐃ⤌⧊㛫࡟ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 1-4㸧ࠋ ᮍᅛᐃ⤌⧊ཬࡧྛᅛᐃ⤌⧊㛫ࡢ࣏ࣜ࣌ࣉࢳࢻⓎ⌧㔞ࡢᕪࢆ, ᮍᅛᐃ⤌⧊࡜࣓ࢱ࣮࢝ࣥᅛ ᐃ࠶ࡿ࠸ࡣ࢔ࢭࢺࣥᅛᐃ⤌⧊ࡢࢫ࣏ࢵࢺᐜ✚ࡢẚ࡟ࡼࡗ࡚ホ౯ࡋࡓࠋᖏ≧ᅇ࡛ࡣ, ᮍᅛᐃ⤌ ⧊࡜ẚ㍑ࡋ࡚, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡛ࡣ 7 ࣏ࣜ࣌ࣉࢳࢻ, ࢔ࢭࢺࣥᅛᐃ⤌⧊࡛ࡣ 10 ࣏ࣜ࣌ࣉ ࢳࢻ࡛᭷ព࡞ኚືࡀㄆࡵࡽࢀࡓ㸦Table 1-2㸧ࠋᑠ⬻⓶㉁࡛ࡣ, ᮍᅛᐃ⤌⧊࡜ẚ㍑ࡋ࡚, ࣓ࢱ࢝ ࣮ࣥᅛᐃ⤌⧊࡛ࡣ1 ࣏ࣜ࣌ࣉࢳࢻ, ࢔ࢭࢺࣥᅛᐃ⤌⧊࡛ࡣ 3 ࣏ࣜ࣌ࣉࢳࢻ࡛᭷ព࡞ᕪࡀㄆࡵ ࡽࢀࡓ㸦Table 1-3㸧ࠋᖏ≧ᅇཬࡧᑠ⬻⓶㉁࡜ࡶ, ࣓ࢱ࣮࢝ࣥᅛᐃ࡟ࡼࡿⓎ⌧㔞ࡢኚືࡣ࢔ࢭ ࢺࣥᅛᐃࡼࡾࡶᑠࡉ࠿ࡗࡓࠋ࠸ࡃࡘ࠿ࡢ࣏ࣜ࣌ࣉࢳࢻࢫ࣏ࢵࢺࢆ㑅ᢥࡋ࡚MS ゎᯒࢆ⾜ࡗࡓ

⤖ᯝ, Phosphoglycerate mutase 1 ཬࡧ Triosephosphate isomerase ࡟ࡘ࠸࡚, 2D-DIGE ࡟࠾࠸࡚➼

17 ࢺࣥᅛᐃ⤌⧊࡟࠾࠸࡚, ศ㞳ࡋࡓ 3 ࢫ࣏ࢵࢺࡢ࠺ࡕࡢ 1 ࡘ࡛, ᮍᅛᐃ⤌⧊࡜ẚ㍑ࡋ࡚ࢫ࣏ࢵ ࢺᐜ✚ࡢప್ࡀࡳࡽࢀࡓ㸦Fig. 1-5, 1-6㸧ࠋ୍᪉, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡛ࡣ, ศ㞳ࡉࢀࡓ 3 ࢫ࣏ ࢵࢺᐜ✚ࡣ඲࡚ᮍᅛᐃ⤌⧊࡜ྠ➼࡛࠶ࡗࡓࠋ ࣏ ࣏ࣜ࣌ࣉࢳࢻࡢᅛᐃᾮ࡬ࡢ⁐ゎᛶ ྛᅛᐃᾮཬࡧ࣓ࢱ࣮࢝ࣥᾮࡢᵓᡂ⁐፹࡬ࡢα-࢝ࢮ࢖ࣥࡢ⁐ゎᛶࢆ ᐃࡋࡓ⤖ᯝ, ࣓ࢱ࢝ ࣮ࣥᾮ࡬ࡢ⁐ゎᛶࡣᴟࡵ࡚ప್࡛࠶ࡗࡓ㸦Table 4㸧ࠋ2D-DIGE ࡟࠾࠸࡚, ࣜࣥ㓟໬ࣃࢱ࣮ࣥ ࡟ᛂࡌࡓ➼㟁Ⅼࡢ㐪࠸࡟ࡼࡗ࡚6 ࡘ࡟ศ㞳ࡉࢀࡓ α-࢝ࢮ࢖ࣥࡢ࣏ࣜ࣌ࣉࢳࢻࢫ࣏ࢵࢺ㸦Fig. 1-7㸧࡟ࡘ࠸࡚, ࢔ࢭࢺ࡛ࣥࡣ 1 ࡘࡢࢫ࣏ࢵࢺ㸦ࢫ࣏ࢵࢺ␒ྕ 3㸧࡛௚ࡢ 5 ࡘࡢ࣏ࣜ࣌ࣉࢳࢻ ࡜ẚ㍑ࡋ࡚, ⁐ゎᛶ࡟ᕪࡀࡳࡽࢀࡓࠋ6 ࡘࡢࢫ࣏ࢵࢺࡢ࣓ࢱ࣮࢝ࣥᾮ࡬ࡢ⁐ゎᛶ࡟ᕪࡣࡳࡽ ࢀ࡞࠿ࡗࡓ㸦Fig. 1-7, 1-8㸧ࠋ

18

⪃

⪃ ᐹ

ᅛᐃ⤌⧊୰࡛ࡢ⥙⨶ⓗ࡞㑇ఏᏊ࡞࠸ࡋศᏊゎᯒ࡟ࡣ, ᢳฟࡋࡓศᏊࡢရ㉁ࡢಖᣢ㸦18, 84㸧 ࡜ඹ࡟, Ⓨ⌧ࢹ࣮ࢱࡢᮍᅛᐃ⤌⧊࡟ᑐࡍࡿᛅᐇᛶࡢ㧗ࡉࡀ㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ࣒࣍ࣝ ࢔ࣝࢹࣄࢻࢆ⏝࠸ࡓᅛᐃ⤌⧊࠿ࡽᢳฟࡋࡓtotal RNA ࡢရ㉁ࡣ୍⯡࡟పࡃ㸦6, 17㸧, ᭷ᶵ⁐ ፹ࢆ⏝࠸ࡓࡓࢇࡥࡃ㉁ỿẊࢆཎ⌮࡜ࡋࡓᅛᐃ࡛ࡣ, ࡼࡾ㧗࠸ total RNA ࡢရ㉁ࡀಖᣢ࡛ࡁࡿ 㸦20, 80, 83㸧ࠋ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡛ࡣ, RIN ್ࡣ 7.0 ௨ୖࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㸦29, 90㸧ࠋᮏ❶࡛ࡣ, ࣓ࢱ࣮࢝ࣥཬࡧ࢔ࢭࢺࣥᅛᐃ⤌⧊࠿ࡽᢳฟࡋࡓ total RNA ࡢ RIN ್ࡢᖹᆒ

್ࡣ࠸ࡎࢀࡶ8.0 ௨ୖ࡛࠶ࡗࡓࡀ, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡀࡼࡾ㧗್ࢆ♧ࡋࡓࠋࡉࡽ࡟, ࢔ࢭ ࢺࣥᅛᐃ⤌⧊ࡢRIN ್ࡣ, 㒊఩㛫࡛᭷ព࡞ᕪࡀࡳࡽࢀࡓࡀ, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡛ࡢྛ㒊఩ ࡟࠾ࡅࡿRIN ್ࡣྠ⛬ᗘ࡛࠶ࡗࡓࠋࡇࡢࡇ࡜ࡼࡾ, ࣓ࢱ࣮࢝ࣥᅛᐃࡣྛ⬻㒊఩㛫࡛ࡢⓎ⌧㔞 ࡢẚ㍑࡟ࡶ᭷⏝࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱ࡟㛵ࡍࡿ࣓ࢱ࣮࢝ࣥཬࡧ࢔ࢭࢺࣥᅛᐃ⤌⧊㛫࡛ࡢẚ㍑࡟࠾࠸࡚, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡛࢔ࢭࢺࣥᅛᐃ⤌⧊࡟ẚ࡭࡚, ⏕ᚋ 63 ᪥㱋ཬࡧ 21 ᪥㱋ඹ࡟᳨⣴ࡋࡓ඲ ࡚ࡢ⬻㒊఩࡟࠾࠸࡚, ಶయ㛫ࢹ࣮ࢱࡢᩓᕸᅗ࡟࠾ࡅࡿ㧗࠸┦㛵ᛶࡀᚓࡽࢀࡓࠋࡉࡽ࡟, ࣓ࢱ ࣮࢝ࣥᅛᐃ⤌⧊࡛ࡢಶయ㛫ࡢⓎ⌧ࢹ࣮ࢱࡢࡤࡽࡘࡁࡢపࡉࡣ, total RNA ࡢࡼࡾ㧗࠸ RIN ್

࡛♧ࡉࢀࡿᢳฟࡋࡓRNA ࡢရ㉁ࡢ㧗ࡉࢆ཯ᫎࡋ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋ≉ᐃ⬻㒊఩࡟࠾ ࡅࡿ㑇ఏᏊⓎ⌧ゎᯒ࡟ࡣ, ࣐࢖ࢡࣟࢲ࢖ࢭࢡࢩࣙࣥᢏ⾡ࢆ⏝࠸ࡓ᥇ᮦࡀ᥎ዡࡉࢀࡿ㸦5㸧ࡀ, ᮏ◊✲࡟࠾ࡅࡿࣃࣥࢳ⏕᳨ࢹࣂ࢖ࢫࢆ⏝࠸ࡓᡭసᴗ࡟ࡼࡿ⡆౽࡞᥇ᮦ࡟ࡼࡗ࡚ࡶಶయ㛫࡛ ࡢࢹ࣮ࢱࡢᑠࡉ࠸ࡤࡽࡘࡁࡀᐇ⌧࡛ࡁࡓࠋ௨ୖࡢࡇ࡜ࡼࡾ, ࣓ࢱ࣮࢝ࣥᅛᐃ⬻࠿ࡽࡢᮏ᥇ᮦ ᪉ἲࡣࢧࣥࣉࣝ㛫ࡢࡤࡽࡘࡁࡢᑠࡉ࠸, ࣁ࢖ࢫ࣮ࣝࣉࢵࢺ࡞ࢹ࣮ࢱ཰㞟࡟᭷⏝࡛࠶ࡿ࡜♧ ၀ࡉࢀࡓࠋ ᢳฟࡋࡓࡓࢇࡥࡃ㉁ࡢ 2D-DIGE ゎᯒ࡟࠾࠸࡚, ࣐࣍ࣝࣜࣥᅛᐃ⤌⧊ࡣᮍᅛᐃ⤌⧊࡜ẚ㍑ ࡋ, ࢫ࣏ࢵࢺᐜ✚ࡢῶᑡࡸࢫ࣏ࢵࢺ఩⨨ࡢࢩࣇࢺࢆᘬࡁ㉳ࡇࡍ㸦88㸧ࠋᮏ❶࡟࠾࠸࡚, SDS-PAGE ࡟ࡼࡿ࣏ࣜ࣌ࣉࢳࢻࣂࣥࢻࡢࣃࢱ࣮ࣥࡸࡑࡢᙉᗘ࡟, ᮍᅛᐃ⤌⧊, ࣓ࢱ࣮࢝ࣥᅛ

19 ᐃཬࡧ࢔ࢭࢺࣥᅛᐃ⤌⧊㛫࡛᫂ࡽ࠿࡞ᕪࡣࡳࡽࢀࡎ, ࡝ࡕࡽࡢᅛᐃࡶ࣏ࣜ࣌ࣉࢳࢻࡢရ㉁ ࡟ᙳ㡪ࢆ୚࠼࡞࠿ࡗࡓࠋ2D-DIGE ゎᯒ࡟࠾࠸࡚, ࠾ࡼࡑ 2,100㹼2,300 ࡢ࣏ࣜ࣌ࣉࢳࢻࡢ࡯࡜ ࢇ࡝඲࡚࡛, ࢫ࣏ࢵࢺᐜ✚ཬࡧࢫ࣏ࢵࢺ఩⨨࡟ᮍᅛᐃ⤌⧊, ࣓ࢱ࣮࢝ࣥᅛᐃཬࡧ࢔ࢭࢺࣥᅛ ᐃ⤌⧊㛫࡛᫂ࡽ࠿࡞ᕪࡣࡳࡽࢀࡎ, ࡝ࡕࡽࡢᅛᐃࡶࣉࣟࢸ࣮࣒࢜ゎᯒࡀ㐺ᛂྍ⬟࡛࠶ࡿࡇ ࡜ࡀ♧၀ࡉࢀࡓࠋᑡᩘࡢ࣏ࣜ࣌ࣉࢳࢻ࡟ࡘ࠸࡚ᮍᅛᐃ⤌⧊࡜ᅛᐃ⤌⧊㛫࡛ࢫ࣏ࢵࢺᐜ✚ࡢᕪ ࡀࡳࡽࢀࡓࡀ, ᙳ㡪ࢆཷࡅࡓ࣏ࣜ࣌ࣉࢳࢻࡢᩘࡣ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊ࡢ᪉ࡀ࢔ࢭࢺࣥᅛᐃ ⤌⧊࡜ẚ㍑ࡋ࡚ᑡ࡞࠿ࡗࡓࠋࡉࡽ࡟, ࢔ࢭࢺࣥᅛᐃ⤌⧊࡛ࡣ, ⩻ヂᚋಟ㣭ࡢᙳ㡪࡛ࢫ࣏ࢵࢺ ࡀ3 ࡘ࡟ศ㞳ࡍࡿ 2 ࡘࡢ࣏ࣜ࣌ࣉࢳࢻ࡟࠾࠸࡚, ඹ࡟ 3 ࡘࡢ࠺ࡕࡢ 1 ࢫ࣏ࢵࢺ࡛ࢫ࣏ࢵࢺᐜ ✚ࡢኚືࡀࡳࡽࢀࡓࡀ, ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࡛ࡣࡑࢀࡽࡢࢫ࣏ࢵࢺᐜ✚࡟ᙳ㡪ࡣࡳࡽࢀ࡞ ࠿ࡗࡓࠋ ⤌⧊ᅛᐃᾮ୰࡟⤌⧊ࡢ࣏ࣜ࣌ࣉࢳࢻࡀ⁐ฟࡍࡿྍ⬟ᛶ࡟㛵ࡋ࡚, 2D-DIGE ࡟࠾࠸࡚ࣜࣥ 㓟໬ࡢࣃࢱ࣮ࣥࡢ㐪࠸࡟ࡼࡗ࡚ࢫ࣏ࢵࢺࡢศ㞳ࡀㄆࡵࡽࢀࡿࡓࢇࡥࡃ㉁ࡢ௦⾲࡜ࡋ࡚㑅ᢥ ࡋࡓ α-࢝ࢮ࢖ࣥ࡟ࡘ࠸࡚, ࢔ࢭࢺࣥཬࡧ࣓ࢱ࣮࢝ࣥᾮࡢᵓᡂ⁐፹࡬ࡢ㍍ᗘ࡞⁐ゎᛶࡀ☜ㄆ ࡉࢀࡓࡀ, ࣓ࢱ࣮࢝ࣥᾮ࡬ࡢ⁐ゎࡣ࡯࡜ࢇ࡝ㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ⁐ゎཬࡧ㠀⁐ゎ α-࢝ࢮ࢖ࣥ ࡢ2D-DIGE ࡟ࡼࡗ࡚ศ㞳ࡋࡓ 6 ࡘࡢࢫ࣏ࢵࢺࡢ࠺ࡕࡢ 1 ࡘ࡛, ௚ࡢ 5 ࢫ࣏ࢵࢺ࡜ẚ㍑ࡋ࡚ ࢔ࢭࢺࣥ࡬ࡢ⁐ゎᛶ࡟ᕪࡀࡳࡽࢀࡓࠋࡇࡢࡇ࡜ࡼࡾ, ࢔ࢭࢺࣥᅛᐃ࡟ࡼࡾ࣏ࣜ࣌ࣉࢳࢻࡢⓎ ⌧ࣞ࣋ࣝࡀࣜࣥ㓟໬ࡢ≧ἣ࡟ࡼࡗ࡚ᙳ㡪ࢆཷࡅࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

20

ᑠ

ᑠ ᣓ

㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆⓎ㐩ᮇ࡜ᡂ⇍ᮇࡢ⚄⤒ẘᛶホ౯࡟฼⏝ࡍࡿ๓ᥦ᮲௳࡜ࡋ࡚, ᶆⓗ࡜ࡍࡿ⬻㒊఩ࡢࡤࡽࡘࡁࡢᑡ࡞࠸㑅ᢥⓗ࡞᥇ᮦࢆྍ⬟࡟ࡍࡿ᪉ἲࢆྲྀࡾධࢀࡿᚲせࡀ ࠶ࡿࠋᮏ❶࡛ࡣྛ⬻⤌⧊㒊఩≉␗ⓗ࡞⥙⨶ⓗ㑇ఏᏊཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟㐺࠺⤌⧊᥇ᮦᡭἲ ☜❧ࡢࡓࡵ࡟, ࣛࢵࢺࡢ඲⬻ࡢ࣓ࢱ࣮࢝ࣥᅛᐃ⤌⧊࠿ࡽࣃࣥࢳ⏕᳨ࢹࣂ࢖ࢫࢆ⏝࠸࡚≉ᐃ ⬻㒊఩ࢆ᥇ᮦࡋ, ᢳฟࡋࡓ total RNA ཬࡧ࣏ࣜ࣌ࣉࢳࢻࡢရ㉁ࡸⓎ⌧ࢹ࣮ࢱࡢᛅᐇᛶࢆホ౯ ࡋࡓࠋ ࡑࡢ⤖ᯝ, ࣓ࢱ࣮࢝ࣥᅛᐃ⬻࠿ࡽᢳฟࡋࡓ totalRNA ཬࡧ࣏ࣜ࣌ࣉࢳࢻࡣ, ⏕ᚋ 63 ᪥㱋ཬ ࡧ 21 ᪥㱋ඹ࡟ရ㉁ཬࡧⓎ⌧ࢹ࣮ࢱࡢᛅᐇᛶ࡟㛵ࡋ࡚, ⥙⨶ⓗⓎ⌧ゎᯒ࡟㐺࠺⛬ᗘࢆಖᣢࡋ

࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ࣓ࢱ࣮࢝ࣥᅛᐃ⬻࠿ࡽᢳฟࡋࡓtotal RNA ࡢ RIN ್ࡣ, ྛ⬻

㒊఩㛫ࡢẚ㍑࡟࠾࠸࡚ࡶྠ⛬ᗘ࡛࠶ࡾ, ࣓ࢱ࣮࢝ࣥ඲⬻ᅛᐃἲࡣᅛᐃ࡟ࡼࡿ RNA ࡢಖᏑ࡟ 㛵ࡋ࡚㎿㏿࡛ຠᯝⓗ࡞᪉ἲ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋࡲࡓ, ࣏ࣜ࣌ࣉࢳࢻࡢ࣓ࢱ࣮࢝ࣥᾮ࡬ࡢ⁐ ฟᛶࡣ㠀ᖖ࡟పࡃ, Ⓨ⌧ࢹ࣮ࢱ࡬ࡢᅛᐃ࡟ࡼࡿᙳ㡪ࡣ࡯࡜ࢇ࡝࡞࠸࡜⪃࠼ࡽࢀࡓࠋ ௨ୖࡼࡾ, ࣓ࢱ࣮࢝ࣥ඲⬻ᅛᐃἲࡣ, ࡆࡗṑ㢮ࢆ⏝࠸ࡓẘᛶヨ㦂ࡢᯟ⤌ࡳ࡟࠾ࡅࡿ≉ᐃ⬻ 㒊఩ࡢ⥙⨶ⓗ࡞㑇ఏᏊཬࡧࡓࢇࡥࡃ㉁ゎᯒ࡟࠾࠸࡚, ⏕ᚋ 63 ᪥㱋㸦9 㐌㱋㸧ཬࡧ 21 ᪥㱋㸦3 㐌㱋㸧ඹ࡟, ᭷⏝࡛࠶ࡿ࡜ุ᩿ࡉࢀࡓࠋ

21

➨

➨

2 ❶

22

⥴

⥴ ゝ

ᮏ❶࡛ࡣ⚄⤒ẘᛶ≀㉁࡛࠶ࡿࢢࣜࢩࢻ࣮ࣝࢆࣔࢹࣝ≀㉁࡜ࡋ࡚, ᡂ⇍ື≀ࡢ⚄⤒࡟ᑐࡍ ࡿ㞀ᐖ࡜, ⚄⤒ẘ࡟ᑐࡍࡿឤཷᛶࡀࡼࡾ㧗࠸࡜⪃࠼ࡽࢀࡿⓎ㐩ᮇࡢࢽ࣮ࣗࣟࣥ᪂⏕࡟ᑐࡍ ࡿᙳ㡪ࢆ᳨⣴ࡋ, ࡑࢀࡽࡢᶆⓗᛶ࡟ᑐࡍࡿẘᛶⓎ⌧ᶵᗎࡢඹ㏻ᛶ࡟ࡘ࠸᳨࡚ウࡍࡿࡇ࡜ࢆ ┠ⓗ࡜ࡋࡓࠋ ࢢࣜࢩࢻ࣮ࣝࡣ࢚࣏࢟ࢩࢻ࢔ࣝࢥ࣮ࣝࡢ୍ࡘ࡛࠶ࡾ, ᐊ ࡛ࡸࡸ⢓ᛶࡢ࠶ࡿᾮయ࡛࠶ࡿࠋ ་⸆ရࡸ໬Ꮫ≀㉁ࡢ〇㐀࡟౑⏝ࡉࢀ, ࣅࢽ࣏࣐࣮ࣝࣜࡢ〇㐀࡟࠾ࡅࡿᏳᐃ๣࡜ࡋ࡚, ࡲࡓἜ ࡢῧຍ๣࡜ࡋ࡚ࡶ⏝࠸ࡽࢀࡿ㸦59㸧ࠋࢢࣜࢩࢻ࣮ࣝࡣⓎࡀࢇᛶ㸦28㸧ࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚ ࠸ࡿࠋࣛࢵࢺཬࡧ࣐࢘ࢫࡢࢢࣜࢩࢻ࣮ࣝ13 㐌㛫཯᚟⤒ཱྀᢞ୚ẘᛶヨ㦂࡟࠾࠸࡚ᑠ⬻ࡢቯṚ ࡀሗ࿌ࡀࡉࢀ࡚࠸ࡿ㸦59㸧ࡀ, ⚄⤒ẘᛶⓎ⌧ᶵᗎࡣᮍࡔ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࠋࢢࣜࢩࢻ࣮ ࣝࡣ㣗⏝Ἔࡸஙඣ⏝࣑ࣝࢡ࡟ྵࡲࢀࡿࢢࣜࢩࢻ࣮ࣝ⬡⫫㓟࢚ࢫࢸࣝ࠿ࡽᾘ໬⟶࡛ࡢຍỈศ ゎ࡟ࡼࡗ࡚⏕ᡂࡉࢀࡿྍ⬟ᛶࡀ࠶ࡾ㸦1, 4㸧, 㣗ရ࠿ࡽࡢᭀ㟢ࡣ㏆ᖺୡ⏺ⓗ㛵ᚰ஦࡜࡞ࡗ࡚ ࠸ࡿࡇ࡜࠿ࡽࡶ, ᡂேཬࡧⓎ㐩ᮇࡢ⚄⤒ẘᛶ࡟ࡘ࠸࡚᭦࡞ࡿ᝟ሗࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ ➨2 ❶࡛ࡣ, ࢢࣜࢩࢻ࣮ࣝࢆẕື≀࡟ዷፎ࣭ᤵஙᮇ࡟ᭀ㟢ࡋ, ẕື≀࡟࠾ࡅࡿ⚄⤒ẘᛶᶵ ᗎࢆ᫂ࡽ࠿࡟ࡍࡿ࡜ඹ࡟, Ꮚື≀ࡢᾏ㤿ṑ≧ᅇ࡬ࡢᙳ㡪࡜ࡑࡢྍ㏫ᛶࡢ᭷↓࡟ࡘ࠸᳨࡚⣴ ࡋࡓࠋࡲࡓ, Ꮚື≀ࡢ⬻࡟࠾ࡅࡿᙳ㡪ࢆᤊ࠼ࡿࡓࡵ࡟, ➨ 1 ❶࡟࡚☜❧ࡋࡓ᥇ᮦ᪉ἲࢆ⏝࠸ ࡚ྛ⬻㒊఩࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࡢ⋓ᚓࢆ⾜ࡗࡓࠋᏊື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ ホ౯࡛ࡣ, SGZ ࡢ⣽⬊ቑṪ࡜࢔࣏ࢺ࣮ࢩࢫ࡞ࡽࡧ࡟㢛⢏⣽⬊⣔㆕ࡢ᳨⣴࡜ṑ≧ᅇ㛛࡟࠾ࡅ ࡿ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸࡢ᳨⣴ࢆ, ᭀ㟢᫬࡜ᡂ⇍ᚋ࡟ච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡾ⾜ࡗࡓࠋࡉ ࡽ࡟, ⏥≧⭢࣍ࣝࣔࣥࡣࢽ࣮ࣗࣟࣥ᪂⏕ࢆྵࡴ⬻ࡢⓎ㐩࡟୙ྍḞ࡛࠶ࡾ, ẘᛶヨ㦂࡛ࡋࡤࡋ ࡤࡑࡢ⾑୰ࣞ࣋ࣝࡢኚືࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࡇ࡛, ࢢࣜࢩࢻ࣮ࣝࡢ⬻࡬ࡢᙳ㡪࡟ᑐࡍࡿ⏥≧ ⭢ᶵ⬟ࡢ㛵୚ࡢ᭷↓ࢆ᳨⣴ࡍࡿࡓࡵ, ẕཬࡧᏊື≀ࡢ⾑Ύ୰⏥≧⭢㛵㐃࣍ࣝࣔࣥࡢ ᐃࢆ ⾜ࡗࡓࠋ ໬Ꮫ≀㉁ࡢ⚄⤒ẘᛶホ౯࡟ࡣ, ྍ⬟࡞㝈ࡾከࡃࡢ⬻㒊఩ࢆゎᯒࡍࡿࡇ࡜ࡀᮃࡲࡋ࠸ࡀ, ࣐

23

࢖ࢡࣟ࢔ࣞ࢖ࢆ⏝࠸ࡓ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣜࣥࢢ࡟ࡣゎ๗Ꮫⓗᵓ㐀, ⣽⬊ศᕸ࠶ࡿ࠸ࡣ

ᶵ⬟࡟ᇶ࡙࠸ࡓ௦⾲ⓗ࡞⬻㒊఩ࢆ㑅ᢥࡍࡿᚲせࡀ࠶ࡿࠋࡑࡢࡓࡵ, ኱⬻⓶㉁ࡢ௦⾲࡜ࡋ࡚ᖏ

≧ᅇ, ࢽ࣮ࣗࣟࣥ᪂⏕㒊఩ࡢ௦⾲࡜ࡋ࡚ᾏ㤿ṑ≧ᅇ, ࡲࡓ኱⬻ⓑ㉁ࡢ௦⾲࡜ࡋ࡚⬻ᱱ, ᑠ⬻ ࡢ௦⾲࡜ࡋ࡚ᑠ⬻⓶㉁ࢆ㑅ᢥࡋࡓࠋ

24

ᮦ

ᮦᩱཬࡧ᪉ἲ

໬Ꮫ≀㉁ ࢢࣜࢩࢻ࣮ࣝ㸦CAS No. 556-52-5㸧ࡣ, ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫㸦ᮾி㸧ࡼࡾ㉎ධࡋ, ᐇ㦂 ࡟౪ࡋࡓࠋ ౪ヨື≀ ዷፎ2 ᪥㱋㸦ࣉࣛࢢ☜ㄆ᪥ࢆዷፎ 0 ᪥࡜ࡋ࡚㉳⟬㸧ࡢ Sprague-Dawley ⣔ SPF ࣛࢵࢺࢆ᪥ ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ఍♫࠿ࡽ㉎ධࡋ࡚ᐇ㦂࡟౪ࡋࡓࠋື≀ࡣ ᗘ23±2ºC, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓࣉࣛࢫࢳࢵࢡ〇 ࢣ࣮ࢪ࡟1 ༉ࡎࡘ཰ᐜࡋ, ศፔᚋࡣẕᏊඹ࡟ྠ୍ࢣ࣮ࢪ࡟཰ᐜࡋࡓࠋẕື≀ࡣዷፎ 2 ᪥࠿ࡽ ศፔᚋ21 ᪥㸦ศፔ᪥ࢆ⏕ᚋ 0 ᪥࡜ࡋ࡚㉳⟬㸧ࡢ㞳ஙࡲ࡛, ᇶ♏㣫ᩱ CRF-1 ࢆ⮬⏤࡟ᦤྲྀࡉ ࡏࡓࠋ㣧ᩱỈࡣዷፎ2 ᪥࠿ࡽ 6 ᪥ࡲ࡛ࡣỈ㐨Ỉࢆ୚࠼, ዷፎ 6 ᪥࠿ࡽศፔᚋ 21 ᪥ࡲ࡛ࡣᚋ ㏙ࡢ⿕㦂㣧ᩱỈࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ㞳ஙᚋࡢᏊື≀ࡣࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟3 ࠿ࡽ 4 ༉ࡎࡘ཰ᐜࡋ, ᇶ♏㣫ᩱ CRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ ඲࡚ࡢື≀ᐇ㦂ィ⏬ࡣᐇ㦂᪋タ㸦ᮾி㎰ᕤ኱Ꮫ㸧ࡢື≀ᐇ㦂ጤဨ఍ࡢᢎㄆࢆཷࡅ, ඲࡚ࡢ ື≀ᐇ㦂᧯సࡣࠕື≀ᐇ㦂ࡢ㐺ṇ࡞ᐇ᪋࡟ྥࡅࡓ࢞࢖ࢻࣛ࢖ࣥࠖ㸦᪥ᮏᏛ⾡఍㆟, ᖹᡂ 18 ᖺ 6 ᭶ 1 ᪥㸧ࢆ㑂Ᏺࡋࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ࢢࣜࢩࢻ࣮ࣝࡣẕື≀ࡢ㣧ᩱỈࢆ௓ࡋ࡚Ꮚື≀࡟ᭀ㟢ࡉࡏࡓࠋNTP ࡟ࡼࡿࢢࣜࢩࢻ࣮ࣝ ࡢ13 㐌㛫཯᚟⤒ཱྀᢞ୚ヨ㦂ࡢሗ࿌㸦59㸧ࢆཧ⪃࡟, ẕື≀ࡢ㍍ᚤ࡞య㔜ῶᑡࡀᘬࡁ㉳ࡇࡉ ࢀࡿ࡜ண᝿ࡉࢀࡿ1,000 ppm ࢆ㧗⏝㔞࡟タᐃࡋ, ୰ཬࡧప⏝㔞ࡣ 1,000 ppm ࢆබẚ⣙ 3 ࡛㝖 ࡋ࡚300 ཬࡧ 100 ppm ࡟タᐃࡋࡓࠋ ዷፎࣛࢵࢺࢆ1 ⩌ᙜࡓࡾ 12 ༉ࡎࡘࡢ 4 ⩌࡟↓సⅭ࡟⩌ศࡅࡋ, ࢢࣜࢩࢻ࣮ࣝࢆ 0㸦↓ฎ⨨

25 ᑐ↷⩌㸧, 100, 300 ཬࡧ 1,000 ppm ࡢ⃰ᗘ࡛⢭〇Ỉ࡟ΰࡌ࡚ዷፎ 6 ᪥࠿ࡽศፔᚋ 21 ᪥㸦㞳ங ᫬㸧ࡲ࡛⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ ⚄⤒Ꮫⓗ␗ᖖࡢホ౯ࡢࡓࡵ, ẕື≀ࡢṌ⾜␗ᖖࢆḟࡢ 4 ࡘࡢ࢝ࢸࢦ࣮ࣜ࡟ᇶ࡙ࡁࢫࢥ࢔௜ ࡅࡋࡓࠋࢢ࣮ࣞࢻ0㸸ṇᖖṌ⾜, ࢢ࣮ࣞࢻ 1㸸㍍ᗘࡢṌ⾜␗ᖖ㸦㍍ᗘࡢᘬࡁࡎࡾṌ⾜㸧, ࢢࣞ ࣮ࢻ2㸸୰➼ᗘࡢṌ⾜␗ᖖ㸦୰➼ᗘࡢᘬࡁࡎࡾṌ⾜㸧, ࢢ࣮ࣞࢻ 3㸸㔜ᗘࡢṌ⾜␗ᖖ㸦య㔜 ࢆᨭ࠼ࡽࢀࡎ, ᅄ⫥ࡀᗈࡀࡗࡓ≧ែࢆྵࡴ㸧ࠋホ౯ࡣࣉࣞ࢟ࢩ࢞ࣛࢫ〇࣎ࢵࢡࢫ㸦90 cm × 90 cm × 20 cm㸧ෆ࡛ 3 ศ㛫, 1 ༉ࡎࡘ┣᳨ୗ࡛ᐇ᪋ࡋࡓࠋ ẕື≀ࡣ, ᢞ୚ᮇ㛫୰, య㔜ࢆ㐌 2-3 ᅇ, ᦤ㣵㔞ཬࡧᦤỈ㔞ࢆ㐌 2 ᅇࡢ㢖ᗘ࡛ ᐃࡋࡓࠋ ⏕ᚋ4 ᪥࡟, 1 ⭡㞝ྛ 6 ༉, 㞤ྛ 2 ༉࡟࡞ࡿࡼ࠺↓సⅭ࡟㑅ᢤࡋ࡚Ꮚᩘࡢㄪᩚࢆ⾜ࡗࡓࠋ1 ⭡ ࡢ㞝Ꮚື≀ࡀ6 ༉࡟‶ࡓ࡞࠸ሙྜࡣ, 1 ⭡ 8 ༉࡜࡞ࡿࡼ࠺࡟㞤Ꮚື≀ࢆṧࡋ࡚ㄪᩚࡋࡓࠋᏊ ື≀ࡣ, య㔜ࢆᤵஙᮇ㛫୰ࡣ㐌 2 ᅇ, 㞳ஙᚋࡣ㐌 1 ᅇ, ᦤ㣵㔞ࢆ㞳ஙᚋ࡟㐌 1 ᅇࡢ㢖ᗘ࡛  ᐃࡋࡓࠋ ศፔᚋ21 ᪥࡟ẕື≀඲౛ࢆ๗᳨ࡋ, ⑓⌮⤌⧊Ꮫⓗゎᯒ, ච␿⤌⧊໬Ꮫⓗゎᯒཬࡧ⾑Ύ୰ ࣍ࣝࣔࣥ⃰ᗘ ᐃ࡟౪ࡋࡓࠋᏊື≀ࡣ⏕ᚋ21 ᪥࡟ྛ⭡㞝ྛ 2-3 ༉㸦ྛ⩌ 30-33 ༉㸧, 㞤ྛ 1 ༉㸦ྛ⩌11 ༉㸧ࢆ↓సⅭ࡟㑅ᢤࡋ࡚๗᳨ࡋ, 㑇ఏᏊⓎ⌧ゎᯒ, ⑓⌮⤌⧊Ꮫⓗゎᯒ, ච␿⤌⧊ ໬Ꮫⓗゎᯒ, ࢔࣏ࢺ࣮ࢩࢫࡢ᳨⣴ཬࡧ⾑Ύ୰࣍ࣝࣔࣥ⃰ᗘ ᐃ࡟౪ࡋࡓࠋṧࡾࡢᏊື≀ࡣ⥅ ⥆㣫⫱ࡋ࡚⏕ᚋ77 ᪥࡟ྛ⭡㞝ྛ 2-3 ༉㸦ྛ⩌ 31-33 ༉㸧, 㞤ྛ 1 ༉㸦ྛ⩌ 11 ༉㸧ࢆ๗᳨ࡋ, ⑓⌮⤌⧊Ꮫⓗゎᯒ, ච␿⤌⧊໬Ꮫⓗゎᯒཬࡧ࢔࣏ࢺ࣮ࢩࢫࡢ᳨⣴࡟౪ࡋࡓࠋ ᛶ࿘ᮇ࡟࠾ࡅࡿࢫࢸࣟ࢖ࢻ࣍ࣝࣔࣥࡢ࿘ᮇⓗኚື࡟ࡼࡾࢽ࣮ࣗࣟࣥ᪂⏕ࡀᙳ㡪ࢆཷࡅࡿ ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽ㸦67㸧, 㞤ࡢ⏕ᚋ 21 ཬࡧ 77 ᪥ࡢヨᩱࡣಖᏑࡢࡳ࡜ࡋ, ௨㝆 ࡢゎᯒࡣᐇ᪋ࡋ࡞࠿ࡗࡓࠋẕື≀ཬࡧᏊື≀ࡢ⏕ᚋ21 ཬࡧ 77 ᪥ࡢ࠸ࡎࢀࡢ๗᳨࡟࠾࠸࡚ࡶ, ⬻㔜㔞ࢆ ᐃࡋࡓࠋ඲࡚ࡢື≀ࡣCO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦ Ṛࡉࡏࡓࠋ ⾑ ⾑Ύ୰࣍ࣝࣔࣥ⃰ᗘ ᐃ

26

඲࡚ࡢẕື≀ཬࡧ⏕ᚋ21 ᪥ࡢྛ⭡ 1 ༉ࡢ㞝Ꮚື≀࡟ࡘ࠸࡚ CO2/O2㯞㓉ୗ࡛㛤⭡ࡋ, ⭡኱

ື⬦࠿ࡽ᥇⾑ࡋࡓࠋ㐲ᚰศ㞳㸦3,000 r.p.m.×10 ศ㸧࡟ࡼࡾ⾑Ύヨᩱࢆ᥇ྲྀࡋ, -80°C ࡛෾⤖ಖ

Ꮡࡋࡓࠋthyroid-stimulating hormone㸦TSH㸧, triiodothyronine㸦T3㸧 ཬࡧ thyroxine㸦T4㸧ࢆrodent

ELISA test kit㸦Endocrine Technologies, Newark, CA, USA㸧ཬࡧ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ 㸦FLUOstar Optima, BMG Labtechnologies, Durham, NC, USA㸧ࢆ⏝࠸࡚ ᐃࡋࡓࠋ

㑇 㑇ఏᏊⓎ⌧ゎᯒ⏝≉ᐃ⬻㒊఩ࡢ᥇ྲྀ ๗᳨᫬, 0㸦ᑐ↷⩌㸧, 300 ཬࡧ 1,000 ppm ⩌ࡢྛ⭡ 1 ༉ࡢ㞝Ꮚື≀࡟ࡘ࠸࡚, ᥇ྲྀࡋࡓ඲⬻ ࢆ࣓ࢱ࣮࢝ࣥᾮ࡟ᾐₕࡋ, 4°C ࡛ 5 ᫬㛫᣺┞ࡋ࡚ᅛᐃࡋࡓࠋࡑࡢᚋ, 99.5%࢚ࢱࣀ࣮ࣝ࡟࡚ 1 ᫬㛫࠾ࡁ࡟3 ᅇ⬺Ỉࡋ, 4°C ୍࡛ᬌ᣺┞ࡋࡓࠋ ኱⬻ࡣ⬻㗪ᆺ㸦ࣈࣞ࢖࣐ࣥࢺࣜࢵࢡࢫ㸸ᐊ⏫ᶵᲔᰴᘧ఍♫, ᮾி㸧ࢆ⏝࠸࡚ཌࡉ 2 mm ࡢ ෙ≧᩿ࢫࣛ࢖ࢫࢆస〇ࡋ, ᑠ⬻ࡣཌࡉ 2 mm ࡢ▮≧᩿ࢫࣛ࢖ࢫࢆస〇ࡋࡓࠋ1 mm ᚄࡢ⏕᳨ ࢺࣞࣃࣥ㸦࢝࢖࢖ࣥࢲࢫࢺ࣮ࣜࢬᰴᘧ఍♫, ᒱ㜧㸧ࢆ⏝࠸࡚, ᖏ≧ᅇ, ᑠ⬻⓶㉁, ᾏ㤿ṑ≧ᅇ ཬࡧ⬻ᱱ⤌⧊ࡢ4 㒊఩ࢆࡃࡾᢤ࠸࡚᥇ྲྀࡋࡓ㸦Fig. 2-1㸧ࠋ᥇ྲྀࡋࡓࢧࣥࣉࣝࡣ, 99.5%࢚ࢱࣀ ࣮ࣝ࡟ᾐₕࡋ, -80°C ࡟࡚ಖᏑࡋࡓࠋ ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ ⏕ᚋ21 ᪥㱋ࡢᏊື≀ࡢ࣓ࢱ࣮࢝ࣥᅛᐃ⬻࠿ࡽ᥇ྲྀࡋࡓྛ⬻㒊఩ࡢ⤌⧊ࢧࣥࣉࣝ࠿ࡽ,

QIAzol㸦Qiagen, Valencia, CA, USA㸧ཬࡧ RNeasy Mini kit㸦Qiagen㸧ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟ ࢺࢥ࣮ࣝ࡟ᚑࡗ࡚total RNA ࢆᢳฟࡋ, NanoDrop ND-1000㸦Thermo Scientific, Wilmington, DE, USA㸧ࢆ⏝࠸࡚⃰ᗘࢆ ᐃࡋࡓࠋ0㸦ᑐ↷⩌㸧, 300 ཬࡧ 1,000 ppm ⩌ࡢྛ⩌ 3 ༉㸦ྛ⭡㞝ྛ 1 ༉㸧ࡢྛ⬻㒊఩࡟ࡘ࠸࡚, 61,657 ࣉ࣮ࣟࣈࢆྵࡴ Agilent Whole Rat Genome array Toxplus 60Kx8㸦Agilent Technologies, Inc., Santa Clara, CA, USA㸧ࢆ⏝࠸࡚⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࢆ⾜ ࡗࡓࠋࢧࣥࣉࣝࡢฎ⌮ཬࡧ࣐࢖ࢡࣟ࢔ࣞ࢖ᐇ㦂ࡣ〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚⾜ࡗࡓࠋࢥ ࣥࢺ࣮ࣟࣝRNA㸦Agilent One-Color RNA Spike-In Kit㸧࡟ྛ⤌⧊ࡼࡾᢳฟࡋࡓ 200 ng ࡢ total

27

RNA ࢆຍ࠼, Agilent Low Input Quick Amp Labeling kit㸦one-color㸧ࢆ⏝࠸࡚ Cy3 ࣛ࣋ࣝ cRNA ࢆస〇ᚋ, RNeasy mini kit㸦Qiagen㸧ࢆ⏝࠸࡚⢭〇, in situ hybridization kit㸦Agilent Technologies, Inc.㸧ࢆ⏝࠸࡚᩿∦໬ࡋ, ࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࢆ⾜ࡗࡓࠋG2505C scanner㸦Agilent Technologies, Inc.㸧ཬࡧ Agilent Feature Extraction software㸦version 11.7.1.1㸧ࢆ⏝࠸࡚㑇ఏᏊ

Ⓨ⌧ࢹ࣮ࢱࢆྲྀᚓࡋࡓࠋ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡢゎᯒࡣGene Spring GX㸦version 11.5.1㸧

software㸦Agilent Technologies, Inc.㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋ

Real-time RT-PCR ゎゎᯒ

0㸦ᑐ↷⩌㸧ཬࡧ 1,000 ppm ⩌ࡢ⏕ᚋ 21 ᪥㱋ࡢᏊື≀ࡢྛ 6 ༉㸦ྛ⭡㞝ྛ 1 ༉㸧ࡢᾏ㤿ṑ ≧ᅇ, ⬻ᱱཬࡧᖏ≧ᅇ࡟ࡘ࠸࡚, 1 μg ࡢ total RNA ࡼࡾ SuperScript™ III Reverse Transcriptase 㸦Invitrogen Corp., Carlsbad, CA, USA㸧ࢆ⏝࠸࡚ cDNA ࢆྜᡂࡋࡓࠋTable 2-1 ࡟♧ࡋࡓ㑇ఏ Ꮚ࡟ࡘ࠸࡚, mRNA Ⓨ⌧ࡢᐃ㔞ゎᯒࢆᐇ᪋ࡋࡓࠋPCR ཯ᛂࡣ SYBR® Green PCR Master Mix 㸦Applied Biosystems Inc., Foster City, CA, USA㸧ࢆ⏝࠸, Prism 7000 Sequence Detection System 㸦Applied Biosystems Inc.㸧࡟࡚, 〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚ᐇ᪋ࡋࡓࠋ⏝࠸ࡓࣉࣛ࢖࣐ ࣮㸦Table 2-1㸧ࡣ, Primer Express software㸦Version 3.0; Applied Biosystems Japan Ltd.㸧ࢆ⏝࠸

࡚タィࡋࡓࠋྛ㑇ఏᏊࡢmRNA Ⓨ⌧㔞ࡣᑐ↷⩌࡛ࡢⓎ⌧್࡟ᑐࡍࡿ┦ᑐ್࡜ࡋ࡚ồࡵ, ෆ ᅉᛶࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚Hprt ཬࡧ Gapdh 㑇ఏᏊࡢ᳨㔞⥺ࢆồࡵ, 2–ΔΔCT method㸦43㸧࡟࡚ ⟬ฟࡋࡓࠋ ⑓⌮⤌⧊Ꮫⓗゎᯒ ๗᳨᫬, ྛ⩌ 11 ༉ࡢẕື≀ࡼࡾ⬻, ୕ཫ⚄⤒, ᆘ㦵⚄⤒ཬࡧ⬨㧊ࢆ᥇ྲྀࡋ, ⑓⌮⤌⧊Ꮫⓗ ゎᯒ࡟౪ࡋࡓࠋᆘ㦵⚄⤒ࡣ, ᥇ྲྀ๓ 3 ศ㛫ࣈ࢔ࣥᾮࢆᾐₕࡉࡏ࡚ in situ ᅛᐃࢆ⾜ࡗࡓࠋ⬨㧊 ࡣ㢁㒊, ⬚㒊ཬࡧ⭜㒊࡟ࡘ࠸࡚, ᳝㦵࡜୍ᣓࡋ࡚᥇ྲྀࡋࡓࠋ᥇ྲྀࡋࡓ⤌⧊ࡣࣈ࢔ࣥᾮ࡟ᾐₕ ࡋ, 4°C ୍࡛ᬌ᣺┞ࡋ࡚ᅛᐃࡋ, ๭㠃ࢆస〇ࡋࡓࠋ⬨㧊ࡣ, ᅛᐃᚋࣉࣛࣥ-ࢡࣜࣗࢡࣟᾮ࡟ᾐ ₕࡋ, ᐊ ࡛ 48 ᫬㛫᣺┞ࡋ࡚⬺⅊ࡋࡓᚋ, 5%◲㓟ࢼࢺ࣒ࣜ࢘ᾮ࡟ᾐₕࡋ, ᐊ ࡛ 48 ᫬㛫᣺

28 ┞ࡋ࡚୰࿴ࡋࡓࠋᏊື≀ࡣ⏕ᚋ21 ᪥ཬࡧ 77 ᪥ࡢྛࢫࢸ࣮ࢪྛ⩌ 11 ༉㸦ྛ⭡ 1 ༉㸧ࡢ⬻ࢆ ᥇ྲྀࡋ, ẕື≀࡜ྠᵝ࡟ࣈ࢔ࣥᾮ࡛ᅛᐃࡋ, ๭㠃ࢆస〇ࡋࡓࠋ኱⬻ࡣ⏕ᚋ 21 ᪥ࡢᏊື≀ࡣ Bregma ࡢᚋ᪉⣙-3.0 mm ࡢ 1 ࢝ᡤ࡛, ẕື≀࡜⏕ᚋ 77 ᪥ࡢᏊື≀ࡣ Bregma ࡢᚋ᪉⣙-3.5 mm ࡢ1 ࢝ᡤ࡛ෙ≧᩿๭㠃ࢆస〇ࡋ, ᑠ⬻ཬࡧᘏ㧊ࡣ࠸ࡎࢀࡢື≀ࡶ▮≧᩿๭㠃ࢆస〇ࡋࡓࠋ⤌ ⧊ࡣࣃࣛࣇ࢕ࣥໟᇙࡋ, 3 μm ཌࡢษ∦ࢆస〇ࡋࡓࠋษ∦ࡣ࣐࣊ࢺ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥᰁⰍࢆ ⾜ࡗࡓࠋ ච ච␿⤌⧊໬Ꮫⓗゎᯒཬࡧ࢔࣏ࢺ࣮ࢩࢫࡢ᳨⣴ ྛ⩌11 ༉ࡢẕື≀ཬࡧ⏕ᚋ 21 ᪥࡞ࡽࡧ࡟ 77 ᪥ࡢྛࢫࢸ࣮ࢪྛ⩌ 11 ༉ࡢ㞝Ꮚື≀㸦ྛ⭡ 1 ༉ࡢ㞝㸧ࡢ⤌⧊ษ∦ࢆ⏝࠸࡚, ゎᯒࢆ⾜ࡗࡓࠋ ྛࢫࢸ࣮ࢪࡢᏊື≀ࡢ⬻⤌⧊ษ∦࡟ࡘ࠸࡚ࡣ, Table 2-2 ࡟♧ࡋࡓᢠయࢆ⏝࠸࡚ච␿⤌⧊໬ ᏛⓗᰁⰍࢆ⾜ࡗࡓࠋẕື≀ࡢ⬻⤌⧊ษ∦࡟ࡘ࠸࡚ࡣᢠneurofilament-L㸦NF-L㸧ᢠయ㸦Table 2-2㸧 ࢆ⏝࠸࡚ච␿⤌⧊໬ᏛⓗᰁⰍࢆ⾜ࡗࡓࠋࡉࡽ࡟, ẕື≀ࡢ୕ཫ⚄⤒⤌⧊ษ∦࡟ࡘ࠸࡚, ᢠ phosphorylated neurofilament-H㸦p-NF-H㸧ᢠయ㸦Table 2-2㸧ࢆ⏝࠸࡚ච␿⤌⧊໬ᏛⓗᰁⰍࢆ ⾜ࡗࡓࠋྛ୍ḟᢠయࡣ୍ᬌ㸦4°C㸧཯ᛂࡉࡏ, 3,3’-diaminobenzidine/H2O2ⓎⰍࢆ⏝࠸࡚ABC

ἲ㸦Vector Laboratories Inc., Burlingame, CA, U.S.A.㸧࡟ࡼࡿච␿ᰁⰍࢆ⾜ࡗࡓࠋ࡞࠾, ࢡ࢚ࣥ 㓟⦆⾪ᾮ㸦10 mM, pH 6.0㸧ࢆ⏝࠸࡚, T box brain 2䠄Tbr2㸧࡟ࡘ࠸࡚ࡣ࣮࢜ࢺࢡ࣮ࣞࣈฎ⌮ 㸦121°C, 10 ศ㸧, calbindin-D-28K㸦Calb1㸧, calretinin㸦Calb2㸧, parvalbumin㸦Pvalb㸧ཬࡧ beta III tubulin ࡟ࡘ࠸࡚ࡣ࣐࢖ࢡ࢙࣮ࣟ࢘ࣈฎ⌮㸦90 °C, 10 ศ㸧࡟ࡼࡿᢠཎࡢ㈿ά໬ฎ⌮ࢆ⾜ࡗ ࡓࠋ

ࡲࡓ, ⏕ᚋ 21 ཬࡧ 77 ᪥ࡢᏊື≀ࡢ⬻࡟ࡘ࠸࡚ᢠ neuron-specific nuclear protein㸦NeuN㸧ᢠ య࡜ᢠreelin ᢠయ࡟ࡼࡿ஧㔜ᰁⰍࢆᐇ᪋ࡋ, reelin ࡣ 3,3’-diaminobenzidine/H2O2ⓎⰍࢆ⏝࠸࡚

ABC ἲ㸦Elite ABC kit, Vector Laboratories Inc.㸧࡟ࡼࡾ, NeuN ࡣ Vector Red Alkaline Phosphate Substrate Kit I㸦Vector Laboratories Inc.㸧ࡢⓎⰍࢆ⏝࠸࡚ ABC ἲ㸦ABC-AP kit, Vector

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ࡋ, Calb2 ࡣ 3,3’-diaminobenzidine/H2O2ⓎⰍ, reelin ࡣ Vector Red Alkaline Phosphate Substrate

Kit I ࡢⓎⰍࢆ⏝࠸࡚ྠᵝ࡟⾜ࡗࡓࠋ

ᾏ㤿ṑ≧ᅇࡢSGZ ࡟࠾ࡅࡿ࢔࣏ࢺ࣮ࢩࢫࡢホ౯࡟ࡘ࠸࡚ࡣ, cleaved caspase-3 ᢠయࢆ⏝࠸

ࡓච␿⤌⧊Ꮫⓗゎᯒ࡟ຍ࠼࡚, TUNEL ἲ㸦Apop Tag® in situ apoptosis detection kit, Millipore Corporation㸧ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ᳨࡚ฟࢆ⾜ࡗࡓࠋ

ᶆᮏࡣ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࢆ᪋ࡋࡓࠋ

ࡇࢀࡽࡢᏊື≀ゎᯒࡣ, ྠࡌಶయ㸦ྛࢫࢸ࣮ࢪྛ⩌ 11 ༉ࡢ㞝Ꮚື≀, ྛ⭡ 1 ༉ࡢ㞝㸧ࡢᶆ

ᮏࢆ⏝࠸࡚ᐇ᪋ࡋࡓࠋ

ᾏ㤿ṑ≧ᅇSGZ ࡟࠾࠸࡚, 㢛⢏⣽⬊ࡢ⣔㆕࡛࠶ࡿ dihydropyrimidinase-like 3㸦TUC-4㸧, doublecortin䠄DCX㸧, Tbr2, paired box 6䠄Pax6㸧ཬࡧ glial fibrillary acidic protein㸦GFAP㸧㝧ᛶ ⣽⬊ᩘ, ࢔࣏ࢺ࣮ࢩࢫࡢᣦᶆ࡛࠶ࡿ cleaved caspase-3 ཬࡧ TUNEL 㝧ᛶ⣽⬊ᩘ, ቑṪ⣽⬊ࡢᣦ ᶆ࡛࠶ࡿproliferating cell nuclear antigen㸦PCNA㸧㝧ᛶ⣽⬊ᩘࡢ᳨⣴ࢆᕥྑ࡟ࡘ࠸࡚⾜࠸, SGZ

ࡢ㛗ࡉࢆ⏝࠸࡚ࣀ࣮࣐ࣛ࢖ࢬࡋࡓ㸦Fig. 2-2㸧ࠋᾏ㤿ṑ≧ᅇ㛛࡟࠾࠸࡚, GABA ᛶ௓ᅾࢽ࣮ࣗ

ࣟࣥ࡟Ⓨ⌧ࡍࡿศᏊࡢreelin, Calb1, Calb2 ཬࡧ Pvalb ࡞ࡽࡧ࡟ᡂ⇍ࢽ࣮ࣗࣟࣥࡢᣦᶆ࡛࠶ࡿ NeuN ࡟ࡘ࠸࡚㝧ᛶ⣽⬊ᩘࡢ᳨⣴ࢆᕥྑ࡟ࡘ࠸࡚⾜࠸, ṑ≧ᅇ㛛ࡢ㠃✚ࢆ⏝࠸࡚ࣀ࣮࣐ࣛ࢖ ࢬࡋࡓ㸦Fig. 2-2㸧ࠋ ⤫ ⤫ィゎᯒ ẕື≀ࡢయ㔜, ᦤ㣵㔞, ᦤỈ㔞, ⬻㔜㔞, ච␿⤌⧊໬Ꮫⓗゎᯒཬࡧ࣍ࣝࣔࣥ ᐃࡢࢹ࣮ࢱ ࡞ࡽࡧ࡟Ꮚື≀ࡢreal-time RT-PCR, ච␿⤌⧊໬Ꮫⓗゎᯒ, TUNEL ゎᯒཬࡧ࣍ࣝࣔࣥ ᐃࡢ ࢹ࣮ࢱࡣྛಶయࡢᩘ್ࢆ⤫ィゎᯒ࡟⏝࠸ࡓࠋᏊື≀ࡢయ㔜, ᦤ㣵㔞ཬࡧ⬻㔜㔞ࡢࢹ࣮ࢱࡣྛ ⭡㸦ẕື≀㸧ࡢᖹᆒ್ࢆ⤫ィゎᯒ࡟⏝࠸ࡓࠋ య㔜, ᦤ㣵㔞, ᦤỈ㔞, ⬻㔜㔞, ච␿⤌⧊໬Ꮫⓗゎᯒཬࡧ࣍ࣝࣔࣥ ᐃࡢࢹ࣮ࢱࡣ, Bartlett ἲ࡟ࡼࡾྛ⩌ࡢ➼ศᩓᛶࡢ᳨ᐃࢆ⾜࠸, ➼ศᩓࡢሙྜࡣ Dunnett ἲ, ୙➼ศᩓࡢሙྜࡣ Steel ἲࢆ⏝࠸࡚ᑐ↷⩌࡜ྛᢞ୚⩌ࡢᕪࢆ᳨ᐃࡋࡓࠋreal-time RT-PCR ゎᯒ࡟࠾ࡅࡿ 2 ⩌㛫ࡢẚ㍑

30

ࡣ, F ᳨ᐃ࡟ࡼࡾྛ⩌ࡢ➼ศᩓᛶࡢ᳨ᐃࢆ⾜࠸, ➼ศᩓࡢሙྜࡣ Student ࡢ t ᳨ᐃ, ୙➼ศᩓ

ࡢሙྜࡣAspin-Welch ࡢ t ᳨ᐃࢆ⏝࠸࡚ᑐ↷⩌࡜ᢞ୚⩌ࡢᕪࢆ᳨ᐃࡋࡓࠋ࣐࢖ࢡࣟ࢔ࣞ࢖ゎ

ᯒࡢࢹ࣮ࢱࡣWelch ࡢ t ᳨ᐃࢆ⏝࠸࡚ᑐ↷⩌࡜ᢞ୚⩌ࡢᕪࢆ᳨ᐃࡋࡓࠋṌ⾜␗ᖖࡢࢫࢥ࢔ཬ

31

⤖

⤖ ᯝ

ẕື≀ࡢ୍⯡≧ែ, య㔜, ᦤ㣵㔞, ᦤỈ㔞ཬࡧ⬻㔜㔞 ๗᳨᫬ࡢ╔ᗋ⑞ࡢゎᯒ࡟࠾࠸࡚ྛ⩌1 ༉࡛㠀ዷፎື≀ࡀࡳࡽࢀ, ࡇࢀࡽࡢẕື≀ࡣホ౯࠿ ࡽ㝖࠸ࡓࠋ╔ᗋ⑞ᩘࡣᑐ↷⩌࡜ᢞ୚⩌ࡢ㛫࡟ᕪࡣࡳࡽࢀࡎ, ⏕ᏑᏊື≀ᩘཬࡧᏊື≀ࡢ㞤㞝 ẚ࡟ࢢࣜࢩࢻ࣮ࣝᢞ୚࡟ࡼࡿᙳ㡪ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Table 2-3㸧ࠋ1,000 ppm ⩌࡛, ศፔᚋ 11 ᪥࠿ࡽ 21 ᪥ࡲ࡛Ṍ⾜␗ᖖ㸦ࡦࡁࡎࡾṌ⾜㸧ࡀࡳࡽࢀࡓ㸦Table 2-4㸧ࠋศፔᚋ 14 ᪥௨㝆, ᑐ ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ᕪࡀㄆࡵࡽࢀ, ศፔᚋ 21 ᪥࡟ࡣ 1,000 ppm ⩌ࡢ඲౛࡛Ṍ⾜␗ᖖࡀࡳ ࡽࢀࡓࠋ0, 100 ཬࡧ 300 ppm ⩌࡛ࡣ␗ᖖࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋయ㔜ࡣ, 300 ppm ⩌࡛ศፔᚋ 21 ᪥࡟, 1,000 ppm ⩌࡛ዷፎ 10 ᪥࠿ࡽศፔᚋ 21 ᪥ࡲ࡛ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋ ࡓ㸦Fig. 2-3㸧ࠋᦤ㣵㔞ࡣ, 100 ppm ⩌࡛ศፔᚋ 2 ᪥࡟, 300 ppm ⩌࡛ศፔᚋ 2 ᪥࠿ࡽ 14 ᪥ࡲ࡛ ࡞ࡽࡧ࡟21 ᪥࡟, 1,000 ppm ⩌࡛ዷፎ 8 ᪥࠿ࡽศፔᚋ 21 ᪥ࡲ࡛ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ప ್ࢆ♧ࡋࡓ㸦Fig. 2-3㸧ࠋᦤỈ㔞ࡣ, 300 ppm ⩌࡛ዷፎ 16 ᪥࠿ࡽศፔᚋ 21 ᪥ࡲ࡛, 1,000 ppm ⩌ ࡛ዷፎ8 ᪥࠿ࡽศፔᚋ 21 ᪥ࡲ࡛ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Fig. 2-3㸧ࠋ⬻㔜㔞 ࡣ, 1,000 ppm ⩌࡛ᑐ↷⩌࡜ẚ㍑ࡋ࡚⤯ᑐ㔜㔞ࡢ᭷ព࡞ప್ཬࡧ┦ᑐ㔜㔞ࡢ᭷ព࡞㧗್ࡀㄆ ࡵࡽࢀࡓ㸦Table 2-5㸧ࠋ Ꮚື≀ࡢ୍⯡≧ែ, య㔜, ᦤ㣵㔞ཬࡧ⬻㔜㔞 ⏕ᚋ77 ᪥ࡲ࡛ࡢほᐹᮇ㛫ࢆ㏻ࡌ࡚඲౛୍࡛⯡≧ែ࡟␗ᖖࡣࡳࡽࢀ࡞࠿ࡗࡓࠋయ㔜ࡣ, 㞝 ࡛ࡣ300 ppm ⩌࡛⏕ᚋ 11 ᪥࠿ࡽ 35 ᪥ࡲ࡛, 1,000 ppm ⩌࡛⏕ᚋ 4 ᪥࠿ࡽ 77 ᪥ࡲ࡛ᑐ↷⩌࡜ ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Table 2-6㸧ࠋ㞤࡛ࡣ 1,000 ppm ⩌࡛⏕ᚋ 4 ᪥࠿ࡽ 77 ᪥ࡲ࡛ᑐ ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋࡓ㸦Table 2-6㸧ࠋᦤ㣵㔞ࡣ, 㞝࡛ࡣ 300 ppm ⩌࡛⏕ᚋ 35 ᪥ ࠿ࡽ49 ᪥ࡲ࡛, 1,000 ppm ⩌࡛⏕ᚋ 28 ᪥࠿ࡽ 77 ᪥ࡲ࡛ᑐ↷⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ప್ࢆ♧ࡋ ࡓ㸦Table 2-7㸧ࠋ㞤࡛ࡣྛᢞ୚⩌㛫࡟᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Table 2-7㸧ࠋ⬻㔜㔞ࡣ, 1,000 ppm ⩌࡛ࡣ, ⏕ᚋ 21 ᪥࡟㞤㞝ࡢ⤯ᑐ㔜㔞ࡢ᭷ព࡞ప್ཬࡧ┦ᑐ㔜㔞ࡢ᭷ព࡞㧗್ࡀㄆࡵࡽ

32 ࢀࡓ㸦Table 2-8㸧ࠋ⏕ᚋ 77 ᪥࡟ྠᵝࡢኚ໬ࡀ㞝ࡢ⤯ᑐཬࡧ┦ᑐ㔜㔞࡞ࡽࡧ࡟㞤ࡢ⤯ᑐ㔜㔞࡛ ㄆࡵࡽࢀࡓࠋ300 ppm ⩌࡛ࡣ, 㞝ࡢ┦ᑐ㔜㔞ࡢ㧗್ཬࡧ㞤ࡢ⤯ᑐ㔜㔞ࡢప್ࡀㄆࡵࡽࢀࡓࡀ, ⏕ᚋ77 ᪥࡛ࡣᑐ↷⩌࡜ẚ㍑ࡋ࡚ᕪࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Table 2-8㸧ࠋ ⾑ ⾑Ύ୰⏥≧⭢㛵㐃࣍ࣝࣔࣥ⃰ᗘ ẕື≀࡛ࡣTSH ࡢ᭷ព࡞㧗್ࡀ 1,000 ppm ⩌࡛, TSH, T3ཬࡧT4ࡢ᭷ព࡞㧗್ࡀ300 ppm ⩌࡛ㄆࡵࡽࢀࡓࠋT3ཬࡧT4࡟1,000 ppm ⩌࡜ᑐ↷⩌ࡢ㛫࡛ᕪࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Table 2-9㸧ࠋ ⏕ᚋ21 ᪥ࡢᏊື≀࡛ࡣ, T3ࡢ᭷ព࡞㧗್ࡀ1,000 ppm ⩌࡛ㄆࡵࡽࢀࡓ㸦Table 2-9㸧ࠋ ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ⤖ᯝࢆTable 2-10㹼2-17 ࡟♧ࡋࡓࠋ ᾏ㤿ṑ≧ᅇ࡛ࡣ, ㍈⣴ᙧᡂཬࡧ⚄⤒✺㉳ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ኚື㸦Frzb, Fzd6,

Cntn3, Nfasc, Ncam2 ཬࡧ Mag ➼㸧, 㧊㠧ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ኚື㸦Mal, Opalin, Mrf ཬࡧUgt8 ➼㸧, ⣽⬊ቑṪ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ኚື㸦Aurkb, Mcm10, Cdkn1 ཬࡧ Ndc80

➼㸧ࡀ1,000 ppm ⩌࡛ㄆࡵࡽࢀ, ࡯࡜ࢇ࡝ࡢ㑇ఏᏊࡣⓎ⌧ῶᑡࢆ♧ࡋࡓࠋࡇࢀࡽࡢ࠺ࡕ, Nfasc,

Mal, Mrf, Ugt8, Aurkb ཬࡧ Ndc80 ࡣ 300 ppm ⩌࡛ࡶྠᵝࡢኚືࡀࡳࡽࢀࡓࠋ

⬻ᱱ࡛ࡣ, 㧊㠧ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࡢῶᑡ㸦Kl, Igf2 ཬࡧ Igfbp2㸧ࡀ 1,000 ppm ⩌࡛ࡢࡳㄆࡵࡽࢀࡓࠋ ᖏ≧ᅇ࡛ࡣ, ㍈⣴ᙧᡂཬࡧ⚄⤒✺㉳ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࡢቑຍ㸦Cntn3, Efnb3, Tnc ཬࡧ Cd44㸧ࡀ 1,000 ppm ⩌࡛ㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ࠺ࡕ Tnc ࡣ 300 ppm ⩌࡛ࡶྠᵝࡢኚ ືࡀࡳࡽࢀࡓࠋ ᑠ⬻⓶㉁࡛ࡣ, ㍈⣴ᙧᡂཬࡧ⚄⤒✺㉳ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ㸦Cntn3㸧ࡢⓎ⌧㔞ࡢቑຍཬࡧ ࢩࢼࣉࢫఏ㐩࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࡢῶᑡ㸦Thbs2 ཬࡧ Ccl2㸧ࡀ 1,000 ppm ⩌࡛ࡢࡳㄆ ࡵࡽࢀࡓࠋ

33 Real-time RT-PCR ゎゎᯒ

࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡟ࡼࡿ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆ᳨ドࡍࡿࡓࡵ, ྛ⬻㒊఩࡟࠾࠸࡚

1,000 ppm ⩌࡛Ⓨ⌧ኚືࡀࡳࡽࢀࡓ㑇ఏᏊࡢ୰࠿ࡽࢽ࣮ࣗࣟࣥࡢⓎ㐩ࡸྍረᛶ, 㧊㠧ᙧᡂ࡟ 㛵㐃ࡍࡿࡶࡢࢆ࠸ࡃࡘ࠿㑅ᢥࡋ, real-time RT-PCR ࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒࢆᐇ᪋ࡋࡓࠋᾏ㤿ṑ ≧ᅇ࡟࠾ࡅࡿMag, Ncam2, Mal ཬࡧ Cdkn1 ཬࡧᖏ≧ᅇ࡟࠾ࡅࡿ Cd44, Efnb3 ཬࡧ Nr4a2 ࡛࣐

࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱ࡜ྠᵝࡢ᭷ព࡞ኚືࢆ♧ࡋࡓࠋࡑࡢ௚ࡢ㑇ఏᏊ࡟ࡣⓎ⌧㔞࡟᭷ព࡞ᕪࡣ ㄆࡵࡽࢀ࡞࠿ࡗࡓࡀ, ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱ࡜ྠᵝࡢቑຍ࠶ࡿ࠸ࡣῶᑡࡢഴྥࡀࡳࡽࢀࡓ 㸦Table 2-18㸧ࠋ ࡲࡓ, ࢔࣏ࢺ࣮ࢩࢫཬࡧ⣽⬊ቑṪ࣐࣮࣮࢝࡟࠾࠸࡚ࡣ, ࠸ࡎࢀࡶⓎ⌧㔞ࡢኚືࡣࡳࡽࢀ࡞ ࠿ࡗࡓ㸦Table 2-19㸧ࠋ ⑓⌮⤌⧊Ꮫⓗ᳨ᰝ ẕື≀ 1,000 ppm ⩌࡟࠾࠸࡚, ୕ཫ⚄⤒ࡢ⚄⤒⠇⣽⬊࡛୰ᚰᛶⰍ㉁⼥ゎ, ᆘ㦵⚄⤒࡛㍈⣴ኚᛶࡀ ࡳࡽࢀ㸦Fig. 2-4㸧, ࠸ࡎࢀࡶ⑓ኚࡢ⛬ᗘࡣᑐ↷⩌࡜ẚ㍑ࡋ࡚⤫ィᏛⓗ࡟᭷ព࡛࠶ࡗࡓ㸦Table 2-20㸧ࠋ100 ཬࡧ 300 ppm ⩌࡛ࡣࡇࢀࡽࡢኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ 1,000 ppm ⩌ࡢ඲౛࡟, ᑠ⬻ࡢ㢛⢏⣽⬊ᒙཬࡧᘏ㧊⫼⣴࡛ NF-L 㝧ᛶࡢࢫࣇ࢙ࣟ࢖ࢻᙧᡂ ࡀㄆࡵࡽࢀࡓ㸦Fig. 2-5㸧ࠋ1,000 ppm ⩌࡛ p-NF-H 㝧ᛶ⚄⤒⠇⣽⬊ࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀ ࡓ㸦Fig. 2-6㸧ࠋ100 ཬࡧ 300 ppm ⩌࡛ࡣࢫࣇ࢙ࣟ࢖ࢻᙧᡂཬࡧ p-NF-H 㝧ᛶ⚄⤒⠇⣽⬊ࡢኚ ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ Ꮚື≀

ᢠNF-L ᢠయཬࡧᢠ beta III tubulin ᢠయࢆ⏝࠸ࡓච␿ᰁⰍ࡟࠾࠸࡚, ᾏ㤿ṑ≧ᅇ, ⬻ᱱ, ᖏ

≧ᅇ, ᑠ⬻⓶㉁ཬࡧᘏ㧊࡛ࡢ⚄⤒⣽⬊య, ⚄⤒✺㉳ཬࡧ㍈⣴ࡢᙧែ࡟, ᑐ↷⩌࡜ẚ㍑ࡋ࡚

34

SGZ ࡟࡟࠾ࡅࡿච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡿ㢛⢏⣽⬊ࡢศ໬⣔㆕ࡢ᳨⣴

⏕ᚋ21 ᪥࡛ࡣ, 1,000 ppm ⩌࡛ TUC-4 㝧ᛶ⣽⬊ᩘࡢ᭷ព࡞ῶᑡࡀㄆࡵࡽࢀࡓࠋGFAP, Pax6, Tbr2 ཬࡧ DCX 㝧ᛶ⣽⬊ᩘ࡟ኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Fig. 2-7㸧ࠋ⏕ᚋ 77 ᪥࡛ࡣ, GFAP, Pax6, Tbr2, DCX ཬࡧ TUC-4 㝧ᛶ⣽⬊ᩘࡢ࠸ࡎࢀ࡟࠾࠸࡚ࡶᑐ↷⩌࡜ᢞ୚⩌ࡢ㛫࡟ᕪࡣࡳࡽࢀ࡞ ࠿ࡗࡓ㸦Fig. 2-7㸧ࠋ

ᾏ㤿ṑ≧ᅇ㛛࡟࠾ࡅࡿච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡿࢽ࣮ࣗࣟࣥศᕸࡢ᳨⣴

⏕ᚋ21 ᪥࡛ࡣ 1,000 ppm ⩌࡛ NeuN, reelin ཬࡧ Calb2 㝧ᛶ⣽⬊ᩘࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀ ࡓ㸦Fig. 2-8㸧ࠋCalb1 ཬࡧ Pvalb 㝧ᛶ⣽⬊ᩘ࡟ኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ⏕ᚋ 77 ᪥࡛ࡣ, 300 ཬ ࡧ1,000 ppm ⩌࡛ NeuN ཬࡧ reelin 㝧ᛶ⣽⬊ᩘ, ࡲࡓ, 1,000 ppm ⩌࡛ Calb2 㝧ᛶ⣽⬊ᩘࡢ᭷

ព࡞ቑຍࡀㄆࡵࡽࢀࡓࠋCalb1 ཬࡧ Pvalb 㝧ᛶ⣽⬊ᩘ࡟ኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Fig. 2-8㸧ࠋ

ࡉࡽ࡟, ࡇࢀࡽࡢኚືࢆࡼࡾヲ⣽࡟᳨⣴ࡍࡿࡓࡵ࡟ᐇ᪋ࡋࡓ NeuN ࡜ reelin ࡢ஧㔜ᰁⰍ࡛

ࡣ, 1,000 ppm ⩌࡛⏕ᚋ 21 ཬࡧ 77 ᪥࡟ reelin 㝧ᛶ/NeuN 㝜ᛶ࠿ࡽᙅ㝧ᛶ⣽⬊ᩘࡢ᭷ព࡞ቑຍ ࡀㄆࡵࡽࢀࡓࠋreelin 㝧ᛶ/NeuN 㝧ᛶ⣽⬊ᩘࡣ࠸ࡎࢀࡢࢫࢸ࣮ࢪ࡛ࡶኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓ 㸦Fig. 2-9㸧ࠋࡲࡓ, reelin ࡜ Calb2 ࡢ஧㔜ᰁⰍ࡛ࡣ 1,000 ppm ⩌࡛⏕ᚋ 21 ཬࡧ 77 ᪥࡟ reelin 㝜ᛶ/Calb2 㝧ᛶ⣽⬊ᩘཬࡧ reelin 㝧ᛶ/Calb2 㝜ᛶ⣽⬊ᩘࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓࡀ, reelin

㝧ᛶ/Calb2 㝧ᛶ⣽⬊ᩘࡣ࠸ࡎࢀࡢࢫࢸ࣮ࢪ࡛ࡶኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Fig. 2-9㸧ࠋ

SGZ ࡟࠾ࡅࡿ࢔࣏ࢺ࣮ࢩࢫཬࡧ⣽⬊ቑṪࡢ᳨⣴

⏕ᚋ21 ཬࡧ 77 ᪥ඹ, TUNEL 㝧ᛶ࢔࣏ࢺ࣮ࢩࢫ⣽⬊, cleaved caspase-3 㝧ᛶ⣽⬊ཬࡧ PCNA 㝧ᛶ⣽⬊ᩘ࡟ᑐ↷⩌࡜ᢞ୚⩌ࡢ㛫࡛ᕪࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦Fig. 2-10㸧ࠋ

35

⪃

⪃ ᐹ

ᮏᐇ㦂࡟࠾ࡅࡿࢢࣜࢩࢻ࣮ࣝࡢ㣧Ỉῧຍ࡟ࡼࡿᦤỈ㔞࠿ࡽ⟬ฟࡉࢀࡿᦤྲྀ㔞ࡣ, 100 ppm ࡛18.5 mg/kg body weight/day, 300 ppm ࡛ 48.8 mg/kg body weight/day ཬࡧ 1,000 ppm ࡛ 108.8 mg/kg body weight/day ࡛࠶ࡗࡓࠋࢢࣜࢩࢻ࣮࢚ࣝࢫࢸࣝ㢮࠿ࡽࡢᾘ໬࡟ࡼࡗ࡚ 100%ࡢຠ⋡

࡛ࢢࣜࢩࢻ࣮ࣝࡀ⏕ᡂࡍࡿሙྜࢆ᝿ᐃࡋ࡚⟬ฟࡋࡓ, ᡂே࡟࠾ࡅࡿࢢࣜࢩࢻ࣮ࣝࡢ᥎ᐃ 1 ᪥

ᦤྲྀ㔞ࡣ, 1.33 μg/kg/day㸦1 ᪥᭱኱ 80g ࡢ⬡㉁ࢆᦤྲྀࡋࡓሙྜ㸧࡜ࡉࢀࡿ㸦1㸧ࠋᮏᐇ㦂࡛ࡢ

NOAEL ࡣẕື≀࡛ࡣ 300 ppm㸦48.8 mg/kg body weight/day㸧, Ꮚື≀࡛ࡣ 100 ppm㸦18.5 mg/kg body weight/day㸧࡛࠶ࡾ, ᡂே࡛ࡢ᥎ᐃ 1 ᪥ᦤྲྀ㔞ࡢࡑࢀࡒࢀ⣙ 35,000 ཬࡧ 15,000 ಸ࡛࠶ࡿࠋ ࡋ࠿ࡋ, ᪥ᮏ࡟࠾࠸࡚㛤Ⓨࡉࢀࡓࢪ࢔ࢩࣝࢢࣜࢭ࣮ࣟࣝࢆ㧗⃰ᗘ࡛ྵࡴ㣗⏝Ἔ࡟ࡣ, ௚ࡢ⢭ 〇Ἔ࡟ẚ࡭࡚12㹼40 ಸࡢࢢࣜࢩࢻ࣮ࣝ⬡⫫㓟࢚ࢫࢸࣝࡀྵࡲࢀ࡚࠾ࡾ㸦52㸧, ࣄࢺ࡬ࡢࣜ ࢫࢡࡢቑຍࡀ♧၀ࡉࢀࡿࠋ ẕື≀ࡢᘏ㧊⫼⣴ࡲࡓᑠ⬻࡛ࡳࡽࢀࡓࢫࣇ࢙ࣟ࢖ࢻᙧᡂࡣ㍈⣴㞀ᐖࢆ♧၀ࡍࡿኚ໬࡜⪃ ࠼ࡽࢀࡓࠋ㍈⣴ࡢ㞀ᐖࡣᮎᲈ⚄⤒࡛ࡶㄆࡵࡽࢀࡓࠋ୰ᯡཬࡧᮎᲈ⚄⤒⣔࡟࠾࠸࡚㐲఩㍈⣴ኚ ᛶࢆㄏⓎࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ࢔ࢡࣜࣝ࢔࣑ࢻ, ࢚ࢳࣞࣥ࢜࢟ࢧ࢖ࢻ, n-࣊࢟ࢧࣥ࠶ࡿ࠸ ࡣγ-ࢪࢣࢺࣥࡢᭀ㟢࡛ࡶ, ྠᵝࡢኚ໬ࡀࡳࡽࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦22, 39, 44, 45㸧ࠋᮏ 㞀ᐖࡢᙧែⓗ≉ᚩ࡜ࡋ࡚, NF ࢆ⵳✚ࡋࡓ㍈⣴ࡢ⭘⬽࡜ࡑࡢ⤖ᯝ㐲఩ᮎ➃࠿ࡽ⏕ࡌࡿ㍈⣴ኚ ᛶࡀᣲࡆࡽࢀࡿ㸦44, 45, 46, 82㸧ࠋ࢔ࢡࣜࣝ࢔࣑ࢻࡸ࢚ࢳࣞࣥ࢜࢟ࢧ࢖ࢻࡣ NF ࡟௜ຍࡉࢀࡿ ࡇ࡜࡛NF ࢆ‶ࡓࡋࡓ㍈⣴ࡢ⭘⬽ࢆㄏⓎࡍࡿ㸦22㸧ࠋ࢚ࢳࣞࣥ࢜࢟ࢧ࢖ࢻཬࡧࢢࣜࢩࢻ࣮ࣝ ࡣඹ࡟࢚࣏࢟ࢩࢻᵓ㐀ࢆᣢࡘࡓࡵ, ࡇࢀࡽࡢ≀㉁ࡣྠᵝࡢᶵᗎ࡛ NF ࡢᶵ⬟࡟ᙳ㡪ࢆཬࡰࡍ ࡇ࡜ࡀ᥎ ࡉࢀࡿࠋࡲࡓ, NF ࡢࣜࣥ㓟໬␗ᖖࡣ, ࢔ࣝࢶࣁ࢖࣐࣮⑓ࡸ➽ⴎ⦰ᛶഃ⣴◳໬⑕ࡢ ࡼ࠺࡞⚄⤒ኚᛶ⑌ᝈ࡛ㄆࡵࡽࢀࡿ㸦41㸧ࠋࡋࡓࡀࡗ࡚, ẕື≀ࡢ୕ཫ⚄⤒࡛ㄆࡵࡽࢀࡓ p-NF-H 㝧ᛶ⚄⤒⠇⣽⬊ࡢῶᑡཬࡧ୰ᚰᛶⰍ㉁⼥ゎ࠿ࡽࡶ, ࢢࣜࢩࢻ࣮ࣝ࡟ࡼࡿᮎᲈ⚄⤒࡟ ᑐࡍࡿ⚄⤒㞀ᐖᛶࡀ♧၀ࡉࢀࡓࠋ ࢔ࢡࣜࣝ࢔࣑ࢻࡢࣛࢵࢺ࡬ࡢⓎ㐩ᮇᭀ㟢ᐇ㦂࡟࠾࠸࡚, ẕື≀࡟⚄⤒ẘᛶࢆ♧ࡍ⏝㔞ࡢ

36 ᭀ㟢࡟ࡼࡗ࡚ࡶᏊື≀࡟ᑐࡋ࡚᫂ࡽ࠿࡞㍈⣴㞀ᐖࡣࡳࡽࢀ࡚࠸࡞࠸ࡀ㸦87㸧, Ꮚື≀ࡢ SGZ ࡛ᮍ⇍㢛⢏⣽⬊ࢆᶆⓗ࡜ࡋࡓࢽ࣮ࣗࣟࣥ᪂⏕ࡢᚋᮇ㐣⛬ࡢ㞀ᐖࡀ᳨ฟࡉࢀ࡚࠸ࡿ㸦61, 62㸧ࠋ ᮏ❶࡟࠾ࡅࡿࢢࣜࢩࢻ࣮ࣝࡢⓎ㐩ᮇᭀ㟢ࡣ, ⏕ᚋ 21 ᪥ࡢ 1,000 ppm ⩌࡛ᾏ㤿ṑ≧ᅇ SGZ ࡢ TUC-4 ࡢⓎ⌧ศᕸ࡟ᙳ㡪ࢆཬࡰࡍࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋTUC-4 ࡣᮍ⇍㢛⢏⣽⬊࡛Ⓨ⌧ࡍࡿࡇ ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽ㸦35㸧, ᮍ⇍㢛⢏⣽⬊ࡢῶᑡࡀ♧၀ࡉࢀࡓࠋGFAP ࡣ type-1 ⚄⤒ ᖿ⣽⬊࡛, Pax6 ࡣ type-1 ⚄⤒ᖿ⣽⬊࠿ࡽ type-2a ๓㥑⣽⬊ࡲ࡛࡛, Tbr2 ࡣ type-2b ๓㥑⣽⬊࡛,

ࡲࡓ, DCX ࡣ type-2b ๓㥑⣽⬊࠿ࡽᮍ⇍㢛⢏⣽⬊ࡲ࡛࡛Ⓨ⌧ࡍࡿศᏊ࡛࠶ࡾ㸦26, 34㸧, ࡇࢀ ࡽ๓ᮇࢽ࣮ࣗࣟࣥ᪂⏕ࡢᣦᶆࡢኚືࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋᮍ⇍㢛⢏⣽⬊ࡣ᪤࡟⚄⤒✺㉳ᡂ㛗 ෇㗹ཬࡧᇶᗏᶞ≧✺㉳ࢆ᭷ࡋ࡚࠸ࡿ㸦73㸧ࠋࡲࡓ, ⚄⤒ࡢⓎ㐩㐣⛬࡟࠾ࡅࡿࢽ࣮ࣗࣟࣥ⛣ື ࢆไᚚࡍࡿศᏊᶵᗎࡣ, ㍈⣴࢞࢖ࢲࣥࢫࡸ⚄⤒✺㉳ᙧᡂ࡜㢮ఝᛶࡀㄆࡵࡽࢀ࡚࠸ࡿ㸦58㸧ࠋ ௨ୖࡢࡇ࡜࠿ࡽ, ᡂ⇍ື≀࡛ࡢ㍈⣴㞀ᐖᛶࢆ⪃៖ࡍࡿ࡜, ࢢࣜࢩࢻ࣮ࣝࡀⓎ㐩⚄⤒ẘ࡜ࡋ࡚ ᪂⏕⚄⤒ᮎ➃࡟┤᥋ᙳ㡪ࡍࡿࡇ࡜࡛ᮍ⇍㢛⢏⣽⬊ࡢศ໬ࢆ㞀ᐖࡍࡿࡇ࡜ࡀ᥎ ࡉࢀࡓࠋࡋ࠿ ࡋ࡞ࡀࡽ, ᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿࡇࢀࡽࡢኚ໬ࡣ⏕ᚋ 77 ᪥࡛ࡣㄆࡵࡽࢀࡎ, ྍ㏫ᛶࡢኚ໬࡛ ࠶ࡗࡓࠋ ୍᪉, ᾏ㤿ṑ≧ᅇ㛛࡛ࡣ࢔ࢡࣜࣝ࢔࣑ࢻࡸᢠ⏥≧⭢๣ࡢⓎ㐩ᮇᭀ㟢ᐇ㦂ࡢሗ࿌㸦62, 76, 81㸧࡜ྠᵝ, reelin 㝧ᛶ⣽⬊, Calb2 㝧ᛶ⣽⬊ཬࡧ NeuN 㝧ᛶ⣽⬊ࡢቑຍࡀ⏕ᚋ 21 ᪥ࡢ 1,000 ppm ⩌࡛☜ㄆࡉࢀࡓࠋṑ≧ᅇ㛛ࡢ Calb2 㝧ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡣ, 㢛⢏⣽⬊࡟ᢞᑕࡍࡿ௚ࡢ GABA ᛶࢽ࣮ࣗࣟࣥ࡜┦஫࡟㐃⤡ࡋ࡚άᛶࢆไᚚࡍࡿࡇ࡜࡛, ᾏ㤿඲యࡢάᛶࢆㄪᩚࡍࡿ 㸦23㸧ࠋࡲࡓ, reelin ࡣ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥ࠿ࡽศἪࡉࢀ, ᾏ㤿ṑ≧ᅇ࡟࠾࠸࡚⏕ᚋࡢࢽ ࣮ࣗࣟࣥ᪂⏕ࢆㄪ⠇ࡍࡿᙺ๭ࢆᢸࡗ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦10, 21, 47, 72㸧ࠋࡋࡓࡀࡗ ࡚, GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥ࡟࠾ࡅࡿ, 㢛⢏⣽⬊ࡢ᪂⏕ࡢ㞀ᐖ࡜ࡑࢀ࡟ᘬࡁ⥆ࡃ⚄⤒⛣ື㞀 ᐖࢆ཯ᫎࡍࡿኚືࢆ᳨ฟࡋࡓࡶࡢ࡜⪃࠼ࡽࢀࡿࠋࡉࡽ࡟, reelin 㝧ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡣ NeuN 㝜ᛶࡲࡓࡣᙅ㝧ᛶ࡛࠶ࡗࡓࠋNeuN ࡢపⓎ⌧ࡣᮍ⇍࡞ࢽ࣮࡛ࣗࣟࣥ࠶ࡿࡇ࡜ࢆ♧ࡋ࡚࠾ࡾ 㸦78㸧, TUC-4 㝧ᛶᮍ⇍㢛⢏⣽⬊ࡢῶᑡࢆ཯ᫎࡋ࡚ reelin ࢆⓎ⌧ࡍࡿᮍ⇍ࢽ࣮ࣗࣟࣥࡢቑຍ ࢆᘬࡁ㉳ࡇࡍࡶࡢ࡜᥎ ࡉࢀࡓࠋᮍ⇍㢛⢏⣽⬊ᩘࡢኚືࡣ⏕ᚋ21 ᪥ࡢࡳ࡛࠶ࡗࡓࡀ, ௓ᅾ

37 ࢽ࣮ࣗࣟࣥࡢኚືࡣᡂ⇍ࡲ࡛⥆ࡃࡶࡢ࡛࠶ࡿࡇ࡜ࡀ☜ㄆࡉࢀ, ࢽ࣮ࣗࣟࣥ᪂⏕ࡸ⛣ື㞀ᐖ ࡢ⥅⥆ᛶࢆ♧ࡍࡶࡢ࡜᥎ ࡉࢀࡓࠋ ⏥≧⭢㛵㐃࣍ࣝࣔࣥࡣ⬻ࡢⓎ㐩࡟㔜せ࡛࠶ࡿ㸦3㸧ࠋᮏ❶࡛ࡣ, T3ࡢቑຍࡀ⏕ᚋ21 ᪥ࡢ 1,000 ppm ࡢᏊື≀࡛ࡳࡽࢀࡓࠋᡂ⇍ࣛࢵࢺ࡛ࡢ⏥≧⭢ᶵ⬟ஹ㐍ࡣࢩࢼࣉࢫྍረᛶࡸグ᠈㞀ᐖࢆ ᘬࡁ㉳ࡇࡍࡇ࡜࠿ࡽ㸦89㸧, ࢽ࣮ࣗࣟࣥ᪂⏕࡟ࡶᙳ㡪ࢆཬࡰࡍྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ1,000 ppm ⩌ࡢᏊື≀࡟࠾࠸࡚ࡶ, ࢽ࣮ࣗࣟࣥ᪂⏕࡟ᙳ㡪ࢆ୚࠼ࡿ⏥≧⭢࣍ࣝࣔࣥࡢ␗ᖖࡀㄏⓎ ࡉࢀࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࡀ, ẕື≀࡛ࡣ T3ཬࡧT4⃰ᗘ࡬ࡢᙳ㡪࡞ࡋ࡟㍈⣴㞀ᐖࡀㄏⓎࡉ ࢀࡓࡇ࡜࠿ࡽࡶ, ࢢࣜࢩࢻ࣮ࣝ⮬యࡢ㍈⣴㞀ᐖᛶࡀᏊື≀࡟࠾ࡅࡿ᪂⏕⚄⤒ᮎ➃ࡢ㞀ᐖᛶ ࢆ௓ࡋࡓࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖࢆㄏⓎࡋࡓ࡜᥎ ࡉࢀࡿࠋ ୍᪉, ⏕ᚋ 21 ᪥ࡢᏊື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ゎᯒ࡟࠾࠸࡚, 1,000 ppm ⩌࡛ ୺࡟Ncam2, Nfasc, Mrf, Mal ཬࡧ Ugt8 ࡞࡝⚄⤒ྍረᛶ, 㧊㠧ᙧᡂ࡟㛵୚ࡍࡿ㑇ఏᏊ⩌㸦14, 36, 37, 77, 94㸧ࡢⓎ⌧㔞ῶᑡࡀ☜ㄆࡉࢀࡓࠋࡇࢀࡽࡣ, ᾏ㤿ṑ≧ᅇ SGZ ࡛ࡢࢽ࣮ࣗࣟࣥ᪂⏕ࡢ ᚋᮇ࡟࠾ࡅࡿ㍈⣴ࡸ⚄⤒✺㉳ࡢఙᙇάᛶࢆ᭷ࡍࡿᮍ⇍㢛⢏⣽⬊ࡢῶᑡ࡜㛵㐃ࡋࡓ, ⚄⤒✺ ㉳ࡢᡂ㛗ࡸࡑࢀ࡟⥆ࡃ⚄⤒ᅇ㊰ᙧᡂࡢᢚไࢆ♧၀ࡍࡿኚື࡜⪃࠼ࡽࢀࡿࠋࡲࡓ, ᾏ㤿ṑ≧ᅇ ࡟࠾ࡅࡿච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡗ࡚PCNA 㝧ᛶቑṪ⣽⬊࡟ኚືࡣࡳࡽࢀ࡞࠿ࡗࡓࡀ, 㑇 ఏᏊⓎ⌧ゎᯒ࡟ࡼࡗ࡚1,000 ppm ⩌࡛ Aurkb, Ndc80 ཬࡧ Cdk1 ࡞࡝ࡢ⣽⬊ቑṪ㛵㐃㑇ఏᏊ㸦9, 75㸧ࡢⓎ⌧㔞ῶᑡࡀ☜ㄆࡉࢀ, ⣽⬊࿘ᮇࡢᢚไࡀ♧၀ࡉࢀࡓࠋ⣽⬊ቑṪάᛶ㜼ᐖࡢᶵᗎࡣ᫂ ࡽ࠿࡛࡞࠸ࡀ, ࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖࢆ཯ᫎࡋࡓኚື࡛࠶ࡿ࡜᥎ ࡉࢀࡿࠋᖏ≧ᅇ࡛ࡣ Tnc, Cd44 ཬࡧ Efnb3 ࡞࡝ࡢ㍈⣴ᙧᡂ㛵㐃㑇ఏᏊ㸦31, 42, 53㸧ࡢⓎ⌧㔞ቑຍࡀ, ᑠ⬻⓶㉁࡛ࡣ Ccl2 ཬࡧThbs2 ࡞࡝ࡢࢩࢼࣉࢫ㛵㐃㑇ఏᏊ㸦7, 100㸧ࡢⓎ⌧㔞ῶᑡࡀࡳࡽࢀࡓࠋࡇࢀࡽࡶࢢࣜࢩ ࢻ࣮ࣝᭀ㟢࡟ࡼࡿ⚄⤒ྍረᛶ࡬ࡢᙳ㡪ࢆ཯ᫎࡍࡿࡶࡢ࡜♧၀ࡉࢀࡓࠋࡲࡓ, ⬻ᱱ࡛ࡣ, Kl, Igf2 ཬࡧ Igfbp2 ࡞࡝ࡢ㧊㠧ᙧᡂ࡟㛵ࢃࡿ㑇ఏᏊ㸦8, 38㸧ࡢⓎ⌧㔞ῶᑡࡀࡳࡽࢀ, ㍈⣴ᙧᡂ, ࢩ ࢼࣉࢫᙧᡂ࡬ࡢᙳ㡪ࢆ཯ᫎࡋࡓ㧊㠧㛵㐃㑇ఏᏊࡢኚື࡜ࡋ᳨࡚ฟࡉࢀࡓࡶࡢ࡜♧၀ࡉࢀࡓࠋ ྛ⬻㒊఩࡟࠾ࡅࡿⓎ⌧ኚື㑇ఏᏊࡢ࠺ࡕ, Cntn3 ࡣ 1,000 ppm ⩌࡛ 4 ࡘࡢ⬻㒊఩࡟ඹ㏻ࡋ࡚ୖ ᪉ไᚚࡉࢀࡓࠋࡇࢀࡣ⚄⤒᥋╔ศᏊ࡜ࡋ࡚▱ࡽࢀ, ⚄⤒✺㉳ᡂ㛗άᛶࢆ᭷ࡋ, ṑ≧ᅇ㢛⢏⣽

38

⬊, ᑠ⬻ࣉ࢚ࣝ࢟ࣥ⣽⬊ཬࡧ኱⬻⓶㉁ࡢ⾲ᒙ࡟ศᕸࡍࡿࢽ࣮ࣗࣟࣥ࡞࡝࡛Ⓨ⌧ࡋ࡚࠾ࡾ

㸦97㸧, ࢢࣜࢩࢻ࣮ࣝⓎ㐩ᮇᭀ㟢࡟ࡼࡿẘᛶࢆ⌧ࡍᶆⓗ㑇ఏᏊ࡜ࡋ࡚࠶ࡆࡽࢀࡿ࡜⪃࠼ࡽࢀ

ࡓࠋࡇࢀࡽࡢᾏ㤿ṑ≧ᅇ࡛ኚືࡀࡳࡽࢀࡓ㑇ఏᏊ⩌ࡢ࠺ࡕ, Nfasc, Mrf, Mal, Aukb ཬࡧ Ndc80

࡞࡝ከࡃࡢ㑇ఏᏊࡣ300 ppm ⩌࡛ࡶྠᵝࡢኚືࡀࡳࡽࢀࡓࡀ, ௚ࡢ 3 㒊఩࡛ࡣ࡯࡜ࢇ࡝ࡀ

1,000 ppm ⩌ࡢࡳࡢኚື࡛࠶ࡗࡓࠋᾏ㤿ṑ≧ᅇࡣᡂ⇍ᚋࡲ࡛ࢽ࣮ࣗࣟࣥ᪂⏕ࢆ⥆ࡅࡿࡇ࡜࠿

ࡽ, 4 㒊఩ࡢ୰࡛㑇ఏᏊⓎ⌧ゎᯒ࡟ࡼࡿⓎ㐩⚄⤒ẘᛶᙳ㡪ࡢ᳨ฟ࡟㛵ࡋ࡚᭱ࡶឤཷᛶࡢ㧗࠸

㒊఩࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋᮏᐇ㦂࡟࠾࠸࡚, 1,000 ppm ⩌ࡢẕື≀ࡢ୰ᯡ⚄⤒࡛ࡣᑠ⬻

ཬࡧᘏ㧊⫼⣴࡛ࢫࣇ࢙ࣟ࢖ࢻᙧᡂࡀぢࡽࢀࡓࡀ, Ꮚື≀ࡢ⬻࡛ࡣ, NF-L ཬࡧ beta III tubulin ࡢච␿ᰁⰍ࡟ࡼࡿᙧែⓗホ౯࡛ࡣ㍈⣴ཬࡧ⚄⤒✺㉳ࡢ㞀ᐖࡣ᫂ࡽ࠿࡛࡞࠿ࡗࡓࠋࡋ࠿ࡋ, ≉ ᐃ⬻㒊఩࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ゎᯒ࡟ࡼࡗ࡚⋓ᚓࡋࡓ㍈⣴ᙧᡂࡸࢩࢼࣉࢫᙧᡂ࡟㛵㐃ࡍࡿ㑇

39

ᑠ

ᑠ ᣓ

➨2 ❶࡛ࡣ, ࢢࣜࢩࢻ࣮ࣝࢆࣔࢹࣝ≀㉁࡜ࡋ࡚⏝࠸࡚, ᡂ⇍ື≀ࡢ⚄⤒㞀ᐖཬࡧᏊື≀ࡢ ᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿࢽ࣮ࣗࣟࣥ᪂⏕࡬ࡢᙳ㡪࡟ࡘ࠸᳨࡚⣴ࡋ, ࡑࢀࡽࡢᶆⓗᛶ࡟ᑐࡍࡿẘ ᛶⓎ⌧ᶵᗎࡢඹ㏻ᛶࢆ᳨ウࡋࡓࠋࡲࡓ, Ꮚື≀ࡢྛ⬻㒊఩࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝ ࢆ⋓ᚓࡋࡓࠋ ࢢࣜࢩࢻ࣮ࣝࡢࣛࢵࢺࢆ⏝࠸ࡓⓎ㐩ᮇᭀ㟢࡛, 108.8 mg/kg body weight/day ࡟┦ᙜࡍࡿ 1,000 ppm ࡢࣞ࣋ࣝ࡟࠾࠸࡚, ẕື≀ࡢ୰ᯡཬࡧᮎᲈ⚄⤒⣔࡟㍈⣴㞀ᐖࢆㄏⓎࡍࡿࡇ࡜ࢆぢ ฟࡋࡓࠋࡲࡓ, ྠ⏝㔞࡟࠾࠸࡚, ྍ㏫ⓗ࡞ࡀࡽᏊື≀ࡢᾏ㤿ṑ≧ᅇ SGZ ࡛ࡢศ໬ᚋᮇ࡟࠾ࡅ ࡿ⚄⤒✺㉳ᙧᡂࢆᶆⓗ࡜ࡋࡓ, ࢽ࣮ࣗࣟࣥ᪂⏕ࡢᚋᮇ㐣⛬࡬ࡢᙳ㡪ࢆぢฟࡋࡓࠋ Ꮚື≀ࡢ⬻㒊఩ูࡢ㑇ఏᏊⓎ⌧ゎᯒ࡛ࡣ, ୺࡟㍈⣴ᙧᡂ, ⚄⤒✺㉳ᙧᡂ, 㧊㠧ᙧᡂཬࡧࢩ ࢼࣉࢫ㛵㐃㑇ఏᏊࡢⓎ⌧㔞ࡢኚືࡀホ౯࡟⏝࠸ࡓ⬻4 㒊఩࡛☜ㄆࡉࢀ, ᾏ㤿ṑ≧ᅇ SGZ ࡛ ぢฟࡋࡓ⚄⤒✺㉳ᙧᡂ࡬ࡢᶆⓗᛶ࡜㛵㐃ࡋࡓ⚄⤒ᅇ㊰ࡢྍረᛶ࡬ࡢᙳ㡪ࢆ♧ࡍ㑇ఏᏊࢡࣛ ࢫࢱ࣮࡜ࡋ᳨࡚ฟࡉࢀࡓࡶࡢ࡜⪃࠼ࡽࢀࡓࠋࡲࡓ, ᾏ㤿ṑ≧ᅇ࡛ࡣ 300 ppm ⩌࡟࠾࠸࡚ࡶ 1,000 ppm ⩌࡜ྠᵝࡢ㑇ఏᏊⓎ⌧ኚືࡀ☜ㄆࡉࢀࡓࡀ, ௚ࡢ 3 㒊఩࡛ࡣ࡯࡜ࢇ࡝ࡢ㑇ఏᏊࡀ 1,000 ppm ⩌ࡢࡳࡢኚື࡛࠶ࡗࡓࠋࡇࡢࡇ࡜ࡣ, ᡂ⇍ᚋࡶࢽ࣮ࣗࣟࣥ᪂⏕ࢆ⥆ࡅࡿᾏ㤿ṑ≧ ᅇࡀ, ᮏ≀㉁ࡢ⚄⤒ẘᛶࡢᶆⓗ࡜ࡋ࡚᭱ࡶឤཷᛶࡢ㧗࠸㒊఩࡛࠶ࡿࡇ࡜ࡀ᥎ ࡉࢀࡓࠋ ୖ㏙ࡢࡼ࠺࡟, ᮏ◊✲࡟ࡼࡾࢢࣜࢩࢻ࣮ࣝ࡟ࡼࡿᡂ⇍ື≀࡟࠾ࡅࡿ㍈⣴ኚᛶ࡜Ⓨ㐩୰ື ≀ࡢࢽ࣮ࣗࣟࣥ᪂⏕ࡢᚋᮇ㐣⛬ࢆᶆⓗ࡜ࡋࡓⓎ㐩⚄⤒ẘᛶࡀぢฟࡉࢀ, ྜࢃࡏ࡚Ꮚື≀ࡢ ⬻࡟࠾ࡅࡿ⚄⤒ᅇ㊰ࡢྍረᛶ࡬ࡢᙳ㡪ࢆ♧ࡍ㑇ఏᏊࢡࣛࢫࢱ࣮ࡢ⋓ᚓ࡟ࡼࡾ, ࢢࣜࢩࢻ࣮ ࣝࡢẘᛶᶵᗎ࡜ࡋ࡚, ᡂ⇍ࡋࡓ㍈⣴࡜Ⓨ㐩୰ࡢ⚄⤒✺㉳࡟ᑐࡋ࡚ඹ㏻ࡢᶆⓗᛶࢆᣢࡘࡇ࡜ ࡀ᥎ ࡉࢀࡓࠋ

40

➨

➨

3 ❶

41

⥴

⥴ ゝ

➨2 ❶࡛ࡣ, ࢢࣜࢩࢻ࣮ࣝࡢẘᛶᶵᗎ࡜ࡋ࡚, ᡂ⇍ື≀ࡢ⚄⤒⣔࡜Ⓨ㐩ᮇࡢື≀ࡢࢽ࣮ࣗ ࣟࣥ᪂⏕࡟࠾ࡅࡿᗂⱝ࡞᪂⏕⚄⤒ᮎ➃࡟ᑐࡋ࡚, ඹ㏻ࡢᶆⓗᛶࢆᣢࡗࡓ㞀ᐖᛶࡀ♧၀ࡉࢀ ࡓࠋ ࢽ࣮ࣗࣟࣥ᪂⏕ࡣᡂ⇍ື≀ࡢᾏ㤿ṑ≧ᅇSGZ ࡛ࡶࡳࡽࢀ, Ⓨ㐩⚄⤒ẘᛶ≀㉁ࡢᶆⓗ࡜ࡶ ࡞ࡾ࠺ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋᢠ⏥≧⭢๣࡜ࡋ࡚▱ࡽࢀࡿ࣓ࢳ࣐ࢰ࣮ࣝࡣ, ࣛࢵࢺⓎ㐩ᮇᭀ㟢 ࡟࠾࠸࡚, ᾏ㤿ṑ≧ᅇ SGZ ࡛ࡢ type-1 ⚄⤒ᖿ⣽⬊࠿ࡽᮍ⇍㢛⢏⣽⬊ࡲ࡛ࡢᗈ࠸⠊ᅖࡢ㢛⢏ ⣽⬊⣔㆕ࡢῶᑡཬࡧࡑࢀࢆ཯ᫎࡋࡓṑ≧ᅇ㛛ࡢGABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥศᕸࡢኚື࡟ࡼࡗ ࡚ࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖᛶࡀぢฟࡉࢀ࡚࠸ࡿ㸦81㸧ࠋࡉࡽ࡟ᡂ⇍ࣛࢵࢺ࡬ࡢ࣓ࢳ࣐ࢰ࣮ࣝࡢᢞ ୚࡟ࡼࡗ࡚ࡶ, ྠᵝࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸኚືࡀㄆࡵࡽࢀ࡚࠸ࡿࡇ࡜ࡼࡾ㸦81㸧, Ⓨ㐩ᮇཬࡧᡂ⇍ᮇࡢື≀࡟࠾࠸࡚ྠᵝࡢࢽ࣮ࣗࣟࣥ᪂⏕㞀ᐖࢆㄏⓎࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡿࠋ ࡑࡢࡓࡵ, SGZ ࡟࠾ࡅࡿ㢛⢏⣽⬊⣔㆕ࢆᣦᶆ࡜ࡋࡓࢽ࣮ࣗࣟࣥ᪂⏕ࡢ᳨⣴ࡸṑ≧ᅇ㛛࡟࠾ ࡅࡿ௓ᅾࢽ࣮ࣗࣟࣥࡢ᳨⣴࡟ࡼࡗ࡚, ᡂ⇍ື≀ࢆ⏝࠸ࡓ▷ᮇ୍⯡ẘᛶヨ㦂࡟࠾࠸࡚Ⓨ㐩ᮇ ཬࡧᡂ⇍ᮇࡢ⚄⤒࡬ࡢᙳ㡪ࢆඹ࡟ホ౯࡛ࡁࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ ᮏ❶࡛ࡣ, ᡂ⇍ື≀࡟ᑐࡍࡿ㍈⣴㞀ᐖᛶ≀㉁࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࢢࣜࢩࢻ࣮ࣝ ࡟ࡼࡿ, ᡂ⇍ື≀ࡢࢽ࣮ࣗࣟࣥ᪂⏕࡬ࡢᙳ㡪ࢆ☜ㄆࡋ, Ꮚື≀࡛ࡳࡽࢀࡓᙳ㡪࡜ẚ㍑ࡋࡓࠋ ࡇࡢ┠ⓗࡢࡓࡵ࡟, ࢢࣜࢩࢻ࣮ࣝࡢ 28 ᪥㛫ᙉไ⤒ཱྀᢞ୚ᚋ࡟, ᾏ㤿ṑ≧ᅇ SGZ ࡢ⣽⬊ቑṪ ࡜࢔࣏ࢺ࣮ࢩࢫ࡞ࡽࡧ࡟㢛⢏⣽⬊⣔㆕ࡢ᳨⣴࡜ṑ≧ᅇ㛛࡟࠾ࡅࡿ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸࡢ ᳨⣴ࢆ, ච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡾᐇ᪋ࡋࡓࠋࡉࡽ࡟, ㍈⣴㞀ᐖ≀㉁࡟ࡼࡿᡂ⇍ື≀ࡢ⬻࡬ ࡢᙳ㡪ࢆᤕࡽ࠼ࡿࡓࡵ࡟, ᖏ≧ᅇ, ᾏ㤿ṑ≧ᅇ, ⬻ᱱཬࡧᑠ⬻⓶㉁࡟࠾ࡅࡿ⥙⨶ⓗ㑇ఏᏊⓎ ⌧ࣉࣟࣇ࢓࢖ࣝࡢ⋓ᚓࢆ⾜ࡗࡓࠋࡲࡓ, ⋓ᚓࡋࡓⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆඖ࡟, Ⓨ⌧ኚືศᏊࡢ Ⓨ⌧⣽⬊ศᕸࢆච␿⤌⧊໬Ꮫⓗゎᯒࡼࡗ᳨࡚⣴ࡋࡓࠋ