DNA直接損傷とエピゲノム修飾変動に起因する海馬における神経新生の障害機序の比較に関する研究

Title

DNA直接損傷とエピゲノム修飾変動に起因する海馬におけ

_{る神経新生の障害機序の比較に関する研究( 本文(Fulltext) )}

Author(s)

渡邉, 洋祐

Report No.(Doctoral

Degree)

博士(獣医学) 甲第498号

Issue Date

2018-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/75221

※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。

DNA ┤᥋ᦆയ࡜࢚ࣆࢤࣀ࣒ಟ㣭ኚື࡟㉳ᅉࡍࡿ

ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕ࡢ㞀ᐖᶵᗎࡢẚ㍑࡟㛵ࡍࡿ

◊✲

2017 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉

㸦ᮾி㎰ᕤ኱Ꮫ㸧

Ώ㑓 ὒ♸

1 ᗎ ᗎ ㄽ ... 2 ➨1 ❶ ... 6 ⥴ ゝ ... 7 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 8 ⤖ ᯝ ... 13 ⪃ ᐹ ... 16 ᑠ ᣓ ... 19 ➨2 ❶ ... 20 ⥴ ゝ ... 21 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 22 ⤖ ᯝ ... 27 ⪃ ᐹ ... 30 ᑠ ᣓ ... 34 ➨3 ❶ ... 35 ⥴ ゝ ... 36 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 38 ⤖ ᯝ ... 43 ⪃ ᐹ ... 45 ᑠ ᣓ ... 48 ⥲ྜ⪃ᐹ ... 49 ⤖ ㄽ ... 54 ㅰ ㎡ ... 55 ᘬ⏝ᩥ⊩ ... 56 ᅗ ⾲ ... 63

2

ᗎ

ᗎ ㄽ

Ⓨ㐩⚄⤒ẘᛶヨ㦂ࡣ, ໬Ꮫ≀㉁࡞࡝ࡢ᭚㟢࡟ࡼࡿ⫾⏕ᮇࡲࡓࡣ⏕ᚋⓎ㐩ᮇࡢ⚄⤒⣔ࡢᵓ㐀 ཬࡧᶵ⬟࡟ᑐࡍࡿ᭷ᐖస⏝ࡢ᭷↓ࢆホ౯ࡍࡿヨ㦂࡜ᐃ⩏ࡉࢀࡿࠋ⌧⾜ࡢⓎ㐩⚄⤒ẘᛶヨ㦂ࡣ, ⤒῭༠ຊ㛤Ⓨᶵᵓ (OECD) ࡢ TG426 ཬࡧ⡿ᅜ⎔ቃಖㆤᒁ (EPA) ࡢ OPPTS 870.6300 ࢆᣦ㔪 ࡜ࡋ࡚ᐇ᪋ࡉࢀ࡚࠸ࡿࡀ (OECD, US EPA), ࡑࡢホ౯࡟ࡣ㛗ᮇ㛫࠿ࡘ኱つᶍ࡞ື≀ヨ㦂ࡀせ ồࡉࢀࡿࡇ࡜࠿ࡽ, ⑓⌮Ⓨ⏕ࡢゎ᫂࡟᰿ࡊࡋࡓ⡆౽࠿ࡘຠ⋡ⓗ࡞ホ౯⣔ࡢ☜❧ࡀồࡵࡽࢀ࡚ ࠸ࡿ [17]ࠋࡲࡓ, ✀ࠎࡢ⑌ᝈ࡟㢮ఝࡋࡓẘᛶ඙ೃ࣭ኚ໬ࢆᤊ࠼ࡿࡓࡵ࡟, ⾜ືᏛ࣭ᶵ⬟Ꮫⓗ࡞ ᳨ᰝࢆせồࡉࢀࡿࡇ࡜ࡀከࡃ, ࡇࢀࡀⓎ㐩⚄⤒ẘᛶࡢホ౯ࡢᐇ᪋ࢆࡼࡾ୍ᒙᅔ㞴࡟ࡉࡏ࡚࠸ ࡿഃ㠃ࡶ࠶ࡾ, ࡑࡢẘᛶホ౯᪉ἲ࡟ࡣᮍࡔㄢ㢟ࡀከ࠸ࠋ≉࡟, Ⓨ㐩ᮇࡢ⬻࡟࠾ࡅࡿ⚄⤒⣽⬊ࡢ ศ໬㐣⛬࡟ࡣ, ⚄⤒ᖿ⣽⬊ࡢ⮬ᕫ」〇࡟ጞࡲࡾ, ๓㥑⣽⬊ࡢቑṪ࡟ຍ࠼࡚, ㍈⣴ఙ㛗ࡸࢩࢼ ࣉࢫᙧᡂ, 㧊㠧ᙧᡂ࡞࡝ࡢศ໬࣭ᡂ⇍㐣⛬ࡶྵࡲࢀࡿࡓࡵ, ⚄⤒ẘᛶ≀㉁ࡢస⏝ⅬࡀከᩘᏑᅾ ࡍࡿ࡜⪃࠼ࡽࢀࡿࠋࡲࡓ, ࡇࢀࡽࡢ⚄⤒Ⓨ㐩㐣⛬࡜ᡂ⇍⚄⤒ࡢ⏕Ꮡ࣭⥔ᣢ㐣⛬ࡣඹ㏻ࡢศᏊ ᶵᗎ࡟ᚑࡗ࡚࠸ࡿሙྜࡀከࡃ [62], ᡂ⇍⚄⤒࡟ᑐࡍࡿ⚄⤒ẘᛶ≀㉁ࡣ, Ⓨ㐩⚄⤒ẘᛶࢆㄏⓎ ࡍࡿྍ⬟ᛶࡀ㧗࠸࡜⪃࠼ࡽࢀࡿࠋᚑࡗ࡚, ẘᛶᶆⓗࡢ␗࡞ࡿ⚄⤒ẘᛶ≀㉁ (㍈⣴യᐖ≀㉁ࡸ⚄ ⤒ఏ㐩㞀ᐖ≀㉁, 㧊㠧യᐖ≀㉁࡞࡝) ࡢⓎ㐩⚄⤒ẘᛶ࡜ࡑࡢẘᛶᶵᗎࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜࡟ ࡼࡾ, ࡑࢀࡒࢀࡢയᐖᶵᗎ࡟᰿ࡊࡋࡓⓎ㐩ᮇཬࡧᡂ⇍ᮇ࡛ࡢໟᣓⓗ࡞⚄⤒ẘᛶ᝟ሗࡀᚓࡽࢀ, ࡇࢀࡽࡢ᝟ሗࢆᩚ⌮ࡍࡿࡇ࡜࡟ࡼࡾ, ㎿㏿࠿ࡘ᳨ฟຊࡢ㧗࠸⡆౽࡞⚄⤒ẘᛶホ౯ἲࢆᵓ⠏ࡍ ࡿࡇ࡜ࡀྍ⬟࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋ ኱⬻㎶⦕⣔ࡢ୍㒊࡛࠶ࡿᾏ㤿ࡣグ᠈ࡸᏛ⩦࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠾ࡾ, ṑ≧ᅇ࡟ ࠶ࡿ㢛⢏⣽⬊ᒙୗᖏ (subgranular zone: SGZ㸧 ࡛ࡣ, ⏕ᚋ࡟ጞࡲࡿ㐣⛬࡜ࡋ࡚⚄⤒⣽⬊ࡀసࡾ ฟࡉࢀࡿ⚄⤒᪂⏕ࡀ⾜ࢃࢀ࡚࠸ࡿ [21, 42]ࠋࡑࡢ㐣⛬࡟ࡣ⚄⤒ᖿ⣽⬊ࡢ⮬ᕫ」〇, ๓㥑⣽⬊ࡢ ቑṪࡸศ໬, ⚄⤒✺㉳ᙧᡂࡸࢩࢼࣉࢫᙧᡂ࡞࡝ࡢ⚄⤒Ⓨ⏕ࡢྛẁ㝵ࡀྵࡲࢀ, ⚄⤒᪂⏕㛤ጞ ᫬ࡢ⏕ᚋⓎ㐩ᮇࡀ᭱ࡶάⓎ࡛࠶ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ [29, 42]ࠋࡑࡢࡓࡵ, SGZ ࡛ࡢࡇࢀࡽࡢ ྛẁ㝵ࢆᶆⓗ࡜ࡍࡿࡼ࠺࡞Ⓨ㐩⚄⤒ẘᛶ≀㉁ࡢⓎ㐩ᮇ࡛ࡢ᭚㟢࡟ࡼࡾ, ⏕ᚋࡢ⚄⤒᪂⏕ࡀ㞀 ᐖࢆཷࡅࡿྍ⬟ᛶࡀ࠶ࡿࠋࡉࡽ࡟, ᾏ㤿ṑ≧ᅇ㛛࡟Ꮡᅾࡍࡿ γ-aminobutyric acid (GABA) ᛶ௓

3 ᅾࢽ࣮ࣗࣟࣥࡣ, ᡂ⇍㢛⢏⣽⬊࡟ᑐࡍࡿᢚไᛶࡢධຊࢆ⾜࠺ࡔࡅ࡛࡞ࡃ, ቑṪ࣭ศ໬୰ࡢ㢛⢏ ⣽⬊⣔㆕࡟ᢞᑕࡋ࡚⚄⤒᪂⏕ࢆㄪ⠇ࡍࡿᶵ⬟ࢆᣢࡘ [56, 84]ࠋࡲࡓ, GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥ ࡟ຍ࠼SGZ ࡢእ㒊࠿ࡽࡢᵝࠎ࡞⚄⤒ࡶᾏ㤿ṑ≧ᅇࡢ⚄⤒࡟ᑐࡋࢩࢼࣉࢫࢆᙧᡂࡋධຊࢆ⾜ࡗ ࡚࠾ࡾ, ࢢࣝࢱ࣑ࣥ㓟సືᛶ⚄⤒ࡸ࢔ࢭࢳࣝࢥࣜࣥసືᛶ⚄⤒ࡣ㢛⢏⣽⬊ࡢቑṪཬࡧศ໬࡟ 㛵୚ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [12, 24]ࠋࡇࡢࡇ࡜࠿ࡽ, ⚄⤒᪂⏕࡟㛵୚ࡍࡿ඲࡚ࡢ⣽⬊ࡸධ ຊ࡞࡝ࡀẘᛶࡢᶆⓗ࡜࡞ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ≉࡟, ⮬ᕫ」〇⬟ࢆᣢࡘ type-1 ⚄⤒ᖿ⣽⬊, ቑṪ⬟ࢆᣢࡘྛ๓㥑⣽⬊, ✺㉳ఙᒎ࡞࡝ࡢ᭱⤊ศ໬ࢆ♧ࡍᮍ⇍㢛⢏⣽⬊, ⚄⤒᪂⏕ࡢไᚚ⣔ ධຊ࡛࠶ࡿGABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥ, ᡂ⇍㐣⛬࡟࠶ࡿ㢛⢏⣽⬊ࡢࢩࢼࣉࢫྍረᛶ࡬ࡢᙳ㡪ࡣ, ⚄⤒᪂⏕࡟┤᥋ⓗࡶࡋࡃࡣ㛫᥋ⓗ࡞㞀ᐖࢆ୚࠼ࡿྍ⬟ᛶࡀ㧗ࡃ, ࡇࢀࡽࡢ⣽⬊ศᕸࡢኚືࡣ Ⓨ㐩⚄⤒ẘᛶ࡜ࡋ࡚ホ౯࡛ࡁࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋᐇ㝿࡟, ᡤᒓࡍࡿ◊✲ᐊ࡛ࡣ, ᪤▱ࡢᡂ⇍ ⚄⤒ẘᛶ≀㉁࡛࠶ࡿࢡࣉࣜࢰࣥ (㧊㠧ᙧᡂയᐖ≀㉁) ࡜ࢢࣜࢩࢻ࣮ࣝ (㍈⣴ᮎ➃യᐖ≀㉁㸧 ࡟ࡘ࠸࡚ࣛࢵࢺⓎ㐩ᮇ᭚㟢ヨ㦂ࢆᐇ᪋ࡋ࡚, ᾏ㤿ࡢ⚄⤒᪂⏕࡟ᑐࡍࡿᙳ㡪᳨⣴ࢆ⾜ࡗࡓ⤖ᯝ, 㢛⢏⣽⬊⣔㆕ࡢ⣽⬊ศ໬ࡢᚋᮇࢆᶆⓗ࡜ࡋࡓᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖࡀㄏⓎࡉࢀࡿࡇ࡜ࢆ ぢฟࡋ࡚࠸ࡿ [2, 6]ࠋࡇࡢࡇ࡜࠿ࡽ, ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡟╔┠ࡋ, 㢛⢏⣽⬊⣔㆕ࡢ⣽⬊ศ ᕸཬࡧ㑇ఏᏊࡸࢱࣥࣃࢡ㉁ࡢⓎ⌧㔞ࡢኚືࢆホ౯ࡍࡿࡇ࡜࡛, Ⓨ㐩⚄⤒ẘᛶࢆᾏ㤿࡟࠾ࡅࡿ ⚄⤒᪂⏕㞀ᐖ࡟⨨ࡁ᥮࠼ࡿࡇ࡜ࡀ࡛ࡁ, ⑓⌮Ⓨ⏕࡟᰿ࡊࡋࡓ⡆᫆ホ౯⣔ࡢᵓ⠏࡟ࡘ࡞ࡀࡿྍ ⬟ᛶࡀ㧗࠸࡜⪃࠼ࡽࢀࡿࠋ ⚄⤒᪂⏕ࡣ⏕ᾭ࡟ࢃࡓࡾ⥅⥆ࡍࡿࡇ࡜࠿ࡽ, ᡂ⇍ື≀࡟ᑐࡍࡿⓎ㐩⚄⤒ẘᛶ≀㉁ࡢ᭚㟢࡟ ࡼࡗ࡚ࡶ㞀ᐖࢆཷࡅࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋᐇ㝿࡟, ๓㏙ࡋࡓࢡࣉࣜࢰࣥཬࡧࢢࣜࢩࢻ࣮ࣝ ࡟ࡘ࠸࡚, 㞳ஙᚋື≀ࢆ⏝࠸࡚ 28 ᪥㛫཯᚟ᙉไ⤒ཱྀᢞ୚ࡋࡓሙྜ࡟࠾࠸࡚ࡶ㢛⢏⣽⬊⣔㆕ࡢ ୰ᮇ࠿ࡽᚋᮇࡢയᐖࡀ☜ㄆࡉࢀ, Ⓨ㐩ᮇ᭚㟢࡟ࡼࡿᙳ㡪࡜ྠᵝࡢᶆⓗᛶࢆ♧ࡍࡇ࡜ࡀሗ࿌ࡉ ࢀ࡚࠸ࡿ [1, 5]ࠋࡇࢀࡽࡢ⤖ᯝࡣ, 㞳ஙᚋࡢື≀ࢆ⏝࠸ࡓヨ㦂࡟࠾࠸࡚ࡶ, ᾏ㤿࡟࠾ࡅࡿ⚄⤒ ᪂⏕㞀ᐖᛶࢆホ౯ࡍࡿࡇ࡜࡛Ⓨ㐩⚄⤒ẘᛶࢆண ࡛ࡁࡿྍ⬟ᛶࢆ♧၀ࡋ࡚࠾ࡾ, ࡇࢀ࡟ࡼࡾ ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ࡢⓎ㐩⚄⤒ẘᛶࡢ⡆᫆ண ⣔☜❧ࡢ㐨ࡀ㛤࠿ࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽ, ᢠ⏥≧⭢๣࡛࠶ࡾᵝࠎ࡞Ⓨ㐩㞀ᐖᛶࢆㄏⓎࡍࡿࣉࣟࣆࣝࢳ࢜࢘ࣛࢩࣝࢆ⏝࠸ࡓࣛࢵࢺⓎ㐩ᮇ ᭚㟢ヨ㦂࡛ࡣ, type-1 ⚄⤒ᖿ⣽⬊ࢆྵࡵࡓ㢛⢏⣽⬊⣔㆕ࢆᶆⓗ࡜ࡍࡿ⚄⤒᪂⏕㞀ᐖࡀㄏⓎࡉࢀ

4 ࡿࡢ࡟ᑐࡋ, 㞳ஙᚋࡢࣛࢵࢺࢆ⏝࠸ࡓ 28 ᪥㛫཯᚟ᙉไ⤒ཱྀᢞ୚ヨ㦂࡛ࡣ, type-1 ⚄⤒ᖿ⣽⬊࡬ ࡢᙳ㡪ࡣㄆࡵࡽ࡞࠿ࡗࡓ [73]ࠋᚑࡗ࡚, Ⓨ㐩㞀ᐖᛶࢆ᭷ࡍࡿ≀㉁࡟ࡼࡿ⚄⤒ᖿ⣽⬊ࢆᶆⓗ࡜ ࡋࡓᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖᛶࡣ, Ⓨ㐩ᮇ᭚㟢࡜㞳ஙᚋ᭚㟢ࡢヨ㦂㛫࡛␗࡞ࡿࣃࢱ࣮ࣥࢆ ♧ࡍྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ Ⓨ㐩㐣⛬࡛ࡢ⚄⤒ᖿ⣽⬊ẘᛶࡢᙳ㡪࡜ࡋ࡚ᠱᛕࡍ࡭ࡁⅬ࡜ࡋ࡚, 㑇ఏᏊࡢᵓ㐀ࡸᶵ⬟ࡢ㞀 ᐖࡀ⏕ࡌࡿࡇ࡜࡛, 㑇ఏᏊⓎ⌧ࣉࣟࢢ࣒ࣛ࡟ᚑࡗࡓ⚄⤒ศ໬ᶵᵓࡢ◚⥢ࡀ⏕ࡌ, ࡦ࠸࡚ࡣ⏕ ᚋࡢ⚄⤒୰ᯡࡢ㧗ḟᶵ⬟ᙧᡂ࡟ᙳ㡪ࢆ୚࠼ࡿࡇ࡜ࡀᣲࡆࡽࢀࡿࠋ㑇ఏᏊࡢᵓ㐀ࡸᶵ⬟ࡢ㞀ᐖ ࡣ, DNA ┤᥋ᦆയ࡟㉳ᅉࡍࡿ㑇ఏᏊኚ␗ࡸᰁⰍయ␗ᖖࡢ௚࡟, ㏆ᖺ, ࢚ࣆࢤࣀ࣒ẘᛶ࡟ࡼࡿ 㑇ఏᏊಟ㣭ࡸࣄࢫࢺࣥಟ㣭ࡢኚ໬ࡢ㛵୚ࡀὀ┠ࡉࢀ࡚࠾ࡾ [14, 40, 79], ࡇࢀࡽࡢ㞀ᐖࡀ⚄⤒ ᖿ⣽⬊࡟⏕ࡌࡿ࡜, ࡑࢀ࡟ࡼࡗ࡚⏕ࡌࡿ㑇ఏᏊࡸࣄࢫࢺࣥࡢኚ໬ࡣ, ⚄⤒ᖿ⣽⬊ࡢࡳ࡞ࡽࡎ, ፉ⣽⬊࡛࠶ࡿ⚄⤒⣽⬊⣔㆕࡟ࡶཷࡅ⥅ࡀࢀࡿᛶ㉁ࡀ࠶ࡿࠋ༶ࡕ, ⚄⤒⣔ࡢ DNA ࡟ᑐࡍࡿ┤᥋ 㞀ᐖᛶࡢ࠶ࡿ㑇ఏẘᛶ≀㉁࡜ DNA ࡟ࡣ┤᥋ᦆയࢆ୚࠼ࡎ࡟ࢤࣀ࣒࡟ಟ㣭ࢆ୚࠼ࡿ࢚ࣆࢤࣀ ࣒ẘᛶ≀㉁ࡣඹ࡟⚄⤒ᖿ⣽⬊ẘᛶ≀㉁࡛࠶ࡿ࡜⪃࠼ࡽࢀ, Ⓨ㐩⚄⤒ẘᛶࢆ♧ࡍ࡜ඹ࡟, ⏕ᚋ ࡢ᭚㟢࡟ࡼࡗ࡚ࡶ⚄⤒᪂⏕࡟ᙳ㡪ࢆ୚࠼ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽ, Ⓨ㐩ᮇ࡛ࡢࡇࢀࡽ ࡢᶵᗎ࡟ࡼࡿᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖ࡟ࡼࡿ⏕ᚋᙳ㡪࡟㛵ࡍࡿ◊✲ࡣஈࡋࡃ, ඹ࡟Ⓨ㐩⚄ ⤒ẘᛶ≀㉁࡜ࡋ࡚⪃࠼ࡓሙྜ, Ⓨ㐩ᮇ࡜ᡂ⇍ᚋ࡛ࡢ᭚㟢࡟࠾ࡅࡿ, ࡑࢀࡒࢀࡢᾏ㤿࡟࠾ࡅࡿ ⚄⤒᪂⏕࡟ᑐࡍࡿᙳ㡪ࡢ┦㐪࡟ࡘ࠸࡚ࡶ᫂ࡽ࠿࡟ࡣࡉࢀ࡚࠸࡞࠸ࠋ ᮏ◊✲࡛ࡣ, DNA ┤᥋ᦆയࡢ௦⾲≀㉁࡜ࡋ࡚࣓ࢳࣝࢽࢺࣟࢯᒀ⣲ (MNU), ࢚ࣆࢤࣀ࣒ẘᛶ ≀㉁ࡢ௦⾲࡜ࡋ࡚ࣄࢫࢺࣥࡢ࢔ࢭࢳࣝ໬ࢆㄏⓎࡍࡿࣂࣝࣉࣟ㓟 (VPA) ࢆ⏝࠸࡚, ࣛࢵࢺࡢ ዷፎᮇ㹼㞳ஙᮇࡲ࡛ࡢⓎ㐩ᮇ᭚㟢ࢆ⾜࠸, ⚄⤒ᖿ⣽⬊ࢆྵࡴ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆẚ㍑᳨࣭ウ ࡋࡓࠋࡑࡢ┠ⓗࡢࡓࡵ, SGZ ࡢ㢛⢏⣽⬊ᣦᶆ, ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕ࢆไᚚࡍࡿṑ≧ᅇ㛛ࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥᣦᶆཬࡧ㢛⢏⣽⬊ᒙ (granule cell layer: GCL) ࡢࢩࢼࣉࢫྍረᛶᣦᶆ ࡟ࡘ࠸࡚ච␿⤌⧊໬Ꮫⓗゎᯒ࡟ࡼࡾྛ⣽⬊ศᕸࢆ᳨ウࡋࡓ௚, ᾏ㤿࡟ධຊࡉࢀࡿᵝࠎ࡞ఏ㐩 ≀㉁ࡢྜᡂ㓝⣲, ཷᐜయཬࡧᛂ⟅㑇ఏᏊࡢⓎ⌧ゎᯒࢆ⾜࠸, ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢ㛵୚ ࡟ࡘ࠸᳨࡚ウࡋࡓࠋࡉࡽ࡟, ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ࡢ᳨ฟᛶࢆ᳨ウࡍࡿ┠ⓗ࡛, 㞳ஙᚋື≀

5 ᙉࡉࢆⓎ㐩ᮇ᭚㟢࡜ẚ㍑ࡋࡓࠋ ࡲࡓ, ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆ௓ࡋࡓⓎ㐩ᮇ࡛ࡢ⚄⤒᪂⏕␗ᖖࡀࡑࡢᚋࡢ⚄⤒᪂⏕࡟୚࠼ࡿ ࢖ࣥࣃࢡࢺࢆゎ᫂ࡍࡿࡇ࡜ࢆ┠ⓗ࡟, ࢤࣀ࣒᝟ሗࡢ㇏ᐩ࡞࣐࢘ࢫࢆ⏝࠸࡚, ᪤࡟⚄⤒᪂⏕㞀 ᐖࢆぢฟࡋ࡚࠸ࡿ࣊࢟ࢧࢡࣟࣟࣇ࢙ࣥ (HCP) ࡢⓎ㐩ᮇ᭚㟢ࢆ⾜࠸,ᾏ㤿ṑ≧ᅇ࡛ࡢࢤࣀ࣒ࡢ ࣓ࢳࣝ໬࡟↔Ⅼࢆ⤠ࡗࡓḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࢆᐇ᪋ࡋ࡚, ࢚ࣆࢤࣀ࣒ኚື࡟㉳ᅉࡋࡓ⚄ ⤒᪂⏕㞀ᐖࡢ᳨ウࡶ⾜ࡗࡓࠋ

6

➨

➨

1 ❶

MNU ࡢⓎ㐩ᮇཬࡧ㞳ஙᚋ᭚㟢࡟ࡼࡿ

⚄⤒᪂⏕࡟ཬࡰࡍᙳ㡪ࡢ᳨ウ

7

⥴

⥴ ゝ

࣓ࢳࣝࢽࢺࣟࢯᒀ⣲ (MNU) ࡣ, DNA ࢔ࣝ࢟ࣝ໬๣࡛࠶ࡾቑṪ⣽⬊࡟ᑐࡋẅ⣽⬊స⏝ࢆ᭷ ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ [35, 37]ࠋᐇ㦂ື≀࡛ࡣ, ẕື≀࡟ MNU ࢆ༢ᅇᢞ୚ࡍࡿ࡜, ࡑࡢ⬇ ⫾⏕Ꮚࡢ⫼ഃ⤊⬻⚄⤒ୖ⓶࡛ቑṪάᛶࢆ᭷ࡍࡿ⚄⤒ᖿ⣽⬊ཬࡧ๓㥑⣽⬊ࢆᶆⓗ࡜ࡋࡓ⣽⬊യ ᐖࡀ㉳ࡇࡾ, ࡑࡢ⤖ᯝ, ⏕ᚋࡢ⬻࡛ᑠ㢌⑕ࡀㄏⓎࡉࢀࡿࡇ࡜ࡀ࣐࢘ࢫཬࡧࣛࢵࢺ࡛ሗ࿌ࡉࢀ ࡚࠸ࡿ [3, 45, 65]ࠋࡇࢀࡽࡢ⤖ᯝࡣ, MNU ࡀⓎ㐩ᮇࡢ⚄⤒ᖿ⣽⬊ࢆᶆⓗ࡜ࡋࡓⓎ㐩⚄⤒ẘᛶ ࢆ᭷ࡍࡿ≀㉁࡛࠶ࡿࡇ࡜ࢆ♧၀ࡍࡿࠋࡲࡓ, ᙜ◊✲ᐊ࡟࠾ࡅࡿ㐣ཤࡢ᳨ウ࡛, MNU ࡢ࣐࢘ࢫ ዷፎ୰ᮇ༢ᅇᢞ୚ཬࡧࣛࢵࢺዷፎᚋᮇ▷ᮇ᭚㟢ࡢ⤖ᯝ, 㞳ஙᮇࡢᏊື≀ࡢᾏ㤿࡟࠾࠸࡚ᾏ㤿 ṑ≧ᅇ࡟࠾ࡅࡿ⚄⤒๓㥑⣽⬊ࡢῶᩘࢆ☜ㄆࡋ࡚࠾ࡾ, MNU ࡀᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖࢆㄏ Ⓨࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ [35, 81]ࠋࡋ࠿ࡋ࡞ࡀࡽ, ୧ヨ㦂࡟࠾࠸࡚ type-1 ⚄⤒ᖿ⣽⬊ࡢയᐖ ᛶࡣ☜ㄆࡉࢀ࡞࠿ࡗࡓࠋ

➨1 ❶࡛ࡣ, MNU ࢆ DNA ┤᥋ᦆയ≀㉁࡜ࡋ࡚⏝࠸࡚, OECD ࡢⓎ㐩⚄⤒ẘᛶヨ㦂࢞࢖ࢻࣛ

࢖ࣥ [63]ࡢ᪉ἲ࡟‽ᣐࡋࡓ, ዷፎᮇ㹼㞳ஙᮇࡲ࡛ࡢⓎ㐩ᮇ᭚㟢ᐇ㦂ࢆࣛࢵࢺ࡛⾜࠸, type-1 ⚄ ⤒ᖿ⣽⬊ࢆྵࡴᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ┠ⓗࡢࡓࡵ, SGZ ࡟࠾ࡅࡿ㢛 ⢏⣽⬊⣔㆕ᣦᶆ, ṑ≧ᅇ㛛࡟࠾ࡅࡿ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥᣦᶆཬࡧ GCL ࡢࢩࢼࣉࢫྍረᛶ ᣦᶆࡢච␿⤌⧊໬Ꮫⓗゎᯒࢆ⾜࠸, ྛ⣽⬊ࡢศᕸኚືࢆ☜ㄆࡋࡓࠋࡲࡓ, ᾏ㤿࡟ධຊࡉࢀࡿ ᵝࠎ࡞ఏ㐩≀㉁ࡢྜᡂ㓝⣲, ཷᐜయཬࡧᛂ⟅㑇ఏᏊࡢࣜ࢔ࣝࢱ࢖࣒㏫㌿෗࣏࣓࣮ࣜࣛࢮ㐃㙐 ཯ᛂ (RT-PCR) ゎᯒࢆ⾜࠸, ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢ㛵୚࡟ࡘ࠸᳨࡚ウࡋࡓࠋ ࡉࡽ࡟, 㞳ஙᚋື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖࡢ᳨ฟᛶࢆ᳨ウࡍࡿ┠ⓗ࡜ࡋ࡚, ⏕ᚋ5 㐌㱋ࡢࣛࢵࢺ࡟ᑐࡍࡿ MNU ࡢ 28 ᪥㛫཯᚟ᢞ୚ࢆ OECD ࡢ 28 ᪥㛫཯᚟ᢞ୚ヨ㦂࢞࢖ ࢻࣛ࢖ࣥ [64]࡟‽ᣐࡋ࡚⾜࠸, Ⓨ㐩ᮇ᭚㟢ヨ㦂࡜ྠᵝࡢゎᯒ࡟ࡼࡾᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ ࡢᙳ㡪࡟ࡘ࠸࡚ẚ㍑᳨࣭ウࡋࡓࠋ

8

ᮦ

ᮦᩱ࠾ࡼࡧ᪉ἲ

໬Ꮫ≀㉁ཬࡧ౪ヨື≀

MNU (CAS No. 684-93-5) ࡣ Sigma-Aldrich Corporation (St. Louis, MO, USA) ࡼࡾ㉎ධࡋࡓࠋ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ஺㓄ᚋ (ࣉࣛࢢ☜ㄆ᪥ࢆዷፎ 0 ᪥࡜ࡋ࡚㉳⟬㸧 ࡢ Sprague-Dawley ⣔ SPF ࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ఍♫ (ᶓ὾) ࠿ࡽ㉎ධࡋ࡚ᐇ㦂࡟౪ࡋࡓࠋື≀ࡣ ᗘ 23±2ºC, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓ ࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟ 1 ༉ࡎࡘ཰ᐜࡋ, ศፔᚋࡣẕᏊඹ࡟ྠ୍ࢣ࣮ࢪ࡟཰ᐜࡋࡓࠋẕື≀ ࡣዷፎ2 ᪥࠿ࡽศፔᚋ 21 ᪥ (ศፔ᪥ࢆ⏕ᚋ 0 ᪥࡜ࡋ࡚㉳⟬) ࡢ㞳ஙࡲ࡛, ᇶ♏㣫ᩱ CRF-1 (࢜ ࢚ࣜࣥࢱࣝ㓝ẕᕤᴗᰴᘧ఍♫㸪ᮾி) ࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ㣧ᩱỈࡣዷፎ 6 ᪥┠ࡢ MNU ᢞ୚ 㛤ጞࡲ࡛ࡣỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ㞳ஙᚋࡢᏊື≀ࡣࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟ 3 ࠿ࡽ 4 ༉ࡎࡘ཰ᐜࡋ, ᇶ♏㣫ᩱ CRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 4 㐌㱋ࡢ㞝ᛶ Sprague-Dawley ⣔ SPF ࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ ᰴᘧ఍♫࠿ࡽ㉎ධࡋ࡚ᐇ㦂࡟౪ࡋࡓࠋື≀ࡣ ᗘ 23±2°C, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓࣉࣛࢫࢳࢵࢡ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࢖ࣥࠖ (᪥ᮏᏛ⾡఍㆟, ᖹᡂ 18 ᖺ 6 ᭶ 1 ᪥) ࢆ㑂Ᏺࡋ࡚⾜ࡗࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ձⓎ㐩ᮇ᭚㟢ヨ㦂 ஺㓄ᚋࡢ㞤ࣛࢵࢺࢆ1 ⩌࠶ࡓࡾ 13 ༉ࡎࡘࡢ 4 ⩌࡟↓సⅭ࡟⩌ศࡅࡋ, ዷፎ 6 ᪥┠࠿ࡽศፔ ᚋ21 ᪥┠ࡢ㞳ங᫬ࡲ࡛, MNU ࢆ 0, 3, 10, 30 ppm ࡢ⃰ᗘ࡛⢭〇Ỉ࡟ΰࡌ, ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ ᡤᒓ◊✲ᐊ࡛௨ୗࡢ⏝㔞タᐃヨ㦂ࢆᐇ᪋ࡋ, ᢞ୚㔞ࡢタᐃ᰿ᣐ࡜ࡋࡓࠋ༶ࡕ, ⏝㔞タᐃヨ㦂࡟ ࠾࠸࡚, 50 ppm ࡢ MNU 㣧Ỉᢞ୚࡟ࡼࡾẕື≀ࡢయ㔜ቑຍᢚไ, ᦤ㣵㔞ཬࡧ㣧Ỉ㔞ࡢప್ࡀ☜

9 ㄆࡉࢀࡓࠋࡲࡓ, 10 ppm ⩌࡛ࡣẕື≀ࡢయ㔜ࡸᦤ㣵㔞ཬࡧ㣧Ỉ㔞ࡢኚືࡣ࡞࠿ࡗࡓࡇ࡜࠿ࡽ, OECD ࡢⓎ㐩⚄⤒ẘᛶヨ㦂࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ࠸, ẕື≀࡟㍍ᗘ࡞ẘᛶࡀฟࡿ⏝㔞࡜ࡋ࡚᭱㧗 ⏝㔞ࢆ30 ppm ࡜ࡋ, බẚ 3 ࡛㝖ࡋࡓ 3 ⏝㔞ࢆタᐃࡋࡓࠋ 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 ⭡㞝ྛ 8 ༉࡟࡞ࡿࡼ࠺↓సⅭ࡟㑅ᢤࡋ࡚Ꮚᩘࡢㄪᩚࢆ⾜ࡗࡓࠋ1 ⭡ࡢ㞝Ꮚື≀ ࡀ8 ༉࡟‶ࡓ࡞࠸ሙྜࡣ, 1 ⭡ 8 ༉࡜࡞ࡿࡼ࠺࡟㞤Ꮚື≀ࢆṧࡋ࡚ㄪᩚࡋࡓࠋᏊື≀ࡣ, య㔜 ࢆᤵஙᮇ㛫୰ࡣ㐌2 ᅇ, 㞳ஙᚋࡣయ㔜ཬࡧᦤ㣵㔞ࢆ㐌 1 ᅇࡢ㢖ᗘ࡛ ᐃࡋࡓࠋ ẕື≀ࡣ, ฟ⏘ᚋ 21 ᪥┠࡟ CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉ ࡏ๗᳨ࡋ⬻㔜㔞ࢆ ᐃࡋࡓࠋᏊື≀ࡣ, ⏕ᚋ 21 ᪥┠࡟↓సⅭ࡟㑅ᢤࡋࡓྛ⭡㞝ྛ 1–2 ༉ (ྛ ⩌11–12 ༉) ࡢ㞝Ꮚື≀ࡢ⬻ࢆච␿⤌⧊໬Ꮫⓗゎᯒ࡟⏝࠸ࡿࡓࡵ, 4% (w/v) ࣃ࣒ࣛ࣍ࣝ࢔ࣝ ࢹࣄࢻ (PFA)㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿ 10 mL/ศ)ࠋ ࡉࡽ࡟, ྛ⭡㞝ྛ 2–3 ༉ (ྛ⩌ 24–36 ༉) ࢆ, CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ ࡼࡾᏳᴦṚࡉࡏ๗᳨ࡋ, ⬻ࢆ㑇ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋྛ⭡ṧࡾࡢᏊື≀ࡣᇶ♏㣫ᩱ CRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏ࡚⥅⥆㣫⫱ࡋ㸪య㔜࠾ࡼࡧᦤ㣵㔞ࢆ㐌1 ᅇ ᐃࡋࡓࠋ⏕ᚋ 77 ᪥ ┠࡟↓సⅭ࡟㑅ᢤࡋࡓྛ⭡㞝ྛ1–2 ༉ (ྛ⩌ 11–12 ༉) ࡢ㞝Ꮚື≀ࡢ⬻ࢆච␿⤌⧊໬Ꮫⓗゎᯒ ࡟⏝࠸ࡿࡓࡵ, 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿ 35 mL/ศ)ࠋࡉࡽ࡟, ྛ⭡㞝ྛ 2–3 ༉ (ྛ⩌ 24–36 ༉) ࢆ, CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏ๗᳨ࡋ, ⬻ࢆ㑇ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋ ๗᳨᫬, ⬻㔜㔞ࢆ ᐃࡋࡓࠋ ᛶ࿘ᮇ࡟࠾ࡅࡿࢫࢸࣟ࢖ࢻ࣍ࣝࣔࣥࡢ࿘ᮇⓗኚື࡟ࡼࡾࢽ࣮ࣗࣟࣥ᪂⏕ࡀᙳ㡪ࢆཷࡅࡿࡇ࡜ ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽ [67], 㞤ࡢヨᩱࡣಖᏑࡢࡳ࡜ࡋ, ௨㝆ࡢゎᯒࡣᐇ᪋ࡋ࡞࠿ࡗࡓࠋ ໬Ꮫ≀㉁ࡢ᭚㟢᫬ᮇࡣ, OECD ࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ࠸, ╔ᗋ᫬ᮇ (ዷፎ 6 ᪥┠) ࠿ࡽ㞳ங᫬ (⏕ᚋ 21 ᪥┠) ࡜ࡋࡓࠋ๗᳨᫬ᮇࡣ, ᭚㟢ࡀ⤊஢ࡍࡿ㞳ங᫬Ⅼ, ࠾ࡼࡧᛶᡂ⇍ࡀ᏶஢ࡍࡿ᫬ᮇ (⏕ᚋ 77 ᪥┠) ࢆ┠Ᏻ࡟Ỵᐃࡋࡓࠋࡇࡢ 2 Ⅼ࡛ࡢ᳨ウࢆ⾜࠺ࡇ࡜࡛, Ⓨ㐩⚄⤒ẘᛶࡢྍ㏫ᛶ ࢆ᳨ウࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿࠋ ղ㞳ஙᚋᭀ㟢ヨ㦂

10 1 㐌㛫ࡢ㥆໬㣫⫱ᚋ, 5 㐌㱋ࡢື≀ࢆ 1 ⩌ᙜࡓࡾ 24㹼30 ༉ࡎࡘࡢ 3 ⩌࡟↓సⅭ࡟⩌ศࡅࡋ, 0, 5, 15 mg/kg ࡢ⏝㔞࡛ MNU ࢆ 28 ᪥㛫ᙉไ⤒ཱྀᢞ୚ࡋࡓࠋᡤᒓ◊✲ᐊ࡛⏝㔞タᐃヨ㦂ࢆᐇ᪋ࡋ, ᢞ୚㔞ࡢタᐃ᰿ᣐ࡜ࡋࡓࠋ༶ࡕ, ⏝㔞タᐃヨ㦂࡟࠾࠸࡚, 30 mg/kg ࡢ MNU ᙉไ⤒ཱྀᢞ୚࡟ࡼ ࡾయ㔜ῶᑡࡀ☜ㄆࡉࢀ, 15 mg/kg ࡛ࡣయ㔜ቑຍᢚไࡀ☜ㄆࡉࢀࡓࡇ࡜ࡼࡾ, ᭱㧗⏝㔞ࢆ 15 mg/kg ࡜ࡋ, බẚ 3 ࡛㝖ࡋࡓ 5 mg/kg ࢆప⏝㔞࡜ࡋ࡚タᐃࡋࡓࠋᢞ୚ᮇ㛫୰, య㔜ཬࡧᦤ㣵㔞 ࢆ㐌࡟1–2 ᅇ ᐃࡋࡓࠋ᭱⤊ᢞ୚⩣᪥࡟, ↓సⅭ࡟㑅ᢤࡋࡓྛ⩌ 11 ༉ࡢ⬻ࢆච␿⤌⧊໬Ꮫⓗ ゎᯒ࡟⏝࠸ࡿࡓࡵ, 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ㸦ὶ ㏿ 35 mL/ศ㸧ࠋṧࡾࡢື≀ࡣ CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏ ๗᳨ࡋ, ⬻ࢆ㑇ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋ๗᳨᫬, ⬻㔜㔞ࢆ ᐃࡋࡓࠋ ⬻ ⬻࡟࠾ࡅࡿච␿⤌⧊ᏛⓗᰁⰍཬࡧ࢔࣏ࢺ࣮ࢩࢫ᳨ฟ 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ℺ὶᅛᐃࢆ⾜ࡗࡓฟ⏕ᚋ 21 ᪥ཬࡧ 77 ᪥ࡢ㞝Ꮚ ື≀ཪࡣ㞳ஙᚋ᭚㟢ື≀࠿ࡽ⬻ࢆ᥇ྲྀࡋ, ྠࡌᅛᐃᾮࢆ⏝࠸୍࡚᫨ኪᅛᐃࡋࡓࠋ኱⬻ࡢ bregma ࡢᚋ᪉⣙-3.0 mm ཪࡣ-3.5 mm ࡢ 1 ࢝ᡤ࡛ෙ≧๭㠃ࢆస〇ࡋ࡚, ࡉࡽ࡟ 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࣂࢵࣇ࢓୍࣮࡛᫨ኪᅛᐃࡋࡓࠋ๓ᚋࡢᑐ⛠㠃 (2 ษ㠃) ࡀⷧษ㠃 ࡜࡞ࡿࡼ࠺࡟ࣃࣛࣇ࢕ࣥໟᇙࡋ, 3 μm ཌࡢ㐃⥆ษ∦ࢆస〇ࡋࡓࠋษ∦ࡣ Table 1-11 ࡟♧ࡋࡓ᮲ ௳࡛௨ୗࡢྛศᏊ࡟ᑐࡍࡿᢠయࢆ⏝࠸࡚ච␿ᰁⰍࢆ⾜ࡗࡓࠋ༶ࡕ, 㢛⢏⣽⬊ᒙ࡟࠾ࡅࡿ⚄⤒

᪂⏕ࡢศ໬ࢫࢸ࣮ࢪᣦᶆ࡛࠶ࡿglial fibrillary acidic protein (GFAP) [42], SRY box 2 (SOX2) [77],

T box brain 2 (TBR2) [53], doublecortin (DCX) [42, 53], Neuron specific Nuclea (NeuN) [42, 53], ௓ ᅾࢽ࣮ࣗࣟࣥࡢᣦᶆ࡛࠶ࡿ [24, 26], reelin (RELN), parvalbumin (PVALB), calbindin-D-28K (CALB1), calbindin-D-29K (Calretinin, CALB2), somatostatin (SST), ᡂ⇍ࢽ࣮ࣗࣟࣥࡢᣦᶆ࡛࠶

ࡿNeuN, ⣽⬊ቑṪάᛶࡢᣦᶆ࡛࠶ࡿ proliferating cell nuclear antigen (PCNA), ⣽⬊࿘ᮇᅉᏊ࡛

࠶ࡿcyclin-dependent kinase inhibitor 1A (p21) [68], immediate early gene ࡛࠶ࡿ activity-regulated

cytoskeleton-associated protein (ARC) ཬࡧ cyclooxygenase-2 (COX2) [27, 59, 61]࡟ࡘ࠸࡚ᐇ᪋ࡋ

ࡓࠋࢩࢢࢼ᳨ࣝฟࡣVECTASTAINט _{Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA,}

11

໬ࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࡋࡓࠋ⬺ࣃࣛࣇ࢕ࣥࡣ 0.3 v/v %㐣㓟໬Ỉ⣲Ỉࢆྵ ࡴ࣓ࢱࣀ࣮ࣝ⁐ᾮ (ᐊ , 30 ศ) ࢆ⏝࠸ࡓࠋᾏ㤿ṑ≧ᅇࡢ SGZ ࡟࠾ࡅࡿ࢔࣏ࢺ࣮ࢩࢫࡢホ౯ ࡟ࡘ࠸࡚ࡣ㸪TUNEL ἲ㸦Apop Tag® Peroxidase in Situ apoptosis detection kit㸪Millipore Corporation㸪Billerica㸪MA㸪USA㸧ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ᳨࡚ฟࢆ⾜ࡗࡓࠋ ᶆᮏࡣ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࢆ᪋ࡋࡓࠋ

ච

ච␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧTUNEL ᰁⰍ㝧ᛶ⣽⬊ࡢᐃ㔞ゎᯒ

ᾏ㤿ṑ≧ᅇSGZ/GCL ࡟࠾ࡅࡿ GFAP, SOX2, TBR2, DCX, NeuN, PCNA, p21Cip1/Waf1_{, TUNEL,}

ARC ཬࡧ COX2 㝧ᛶ⣽⬊ᩘ࡟ࡘ࠸࡚, ୧ഃ࡛࢝࢘ࣥࢺࡋ, ༢఩㛗ࡉᙜࡾࡢ㝧ᛶ⣽⬊ᩘࢆ⟬ฟ ࡋࡓ [Fig. 1-1]ࠋṑ≧ᅇ㛛࡟ศᕸࡍࡿ RELN, PVALB, CALB1, CALB2, SST, NeuN 㝧ᛶ⣽⬊࡟ࡘ ࠸࡚ࡣ, ୧ഃ࡛࢝࢘ࣥࢺࡋ, ṑ≧ᅇ㛛ࡢ༢఩㠃✚ᙜࡾࡢ㝧ᛶ⣽⬊ᩘࡢ᳨⣴ࢆ⾜ࡗࡓࠋࡇࢀࡽࡢ ゎᯒࡣ┣᳨ἲ࡛ᐇ᪋ࡋࡓࠋṑ≧ᅇ㛛࡟࠾ࡅࡿ࢔ࣥࣔࣥゅ CA3 㡿ᇦࡢ㢛⢏⣽⬊ࡣ࢝࢘ࣥࢺ࠿ࡽ 㝖እࡋࡓࠋᐃ㔞ⓗゎᯒࡢࡓࡵࡢ෗┿᧜ᙳࡣ200 ཬࡧ 400 ಸࡢಸ⋡࡛⾜࠸ゎᯒࡋࡓࠋ 㑇ఏᏊⓎ⌧Ⓨ⌧ゎᯒ ฟ⏕ᚋ21 ᪥┠ࡢᏊື≀ཬࡧ㞳ஙᚋ᭚㟢ື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ mRNA Ⓨ⌧㔞ࢆࣜ࢔ࣝ ࢱ࢖࣒ RT-PCR ࢆ⏝࠸࡚ゎᯒࡋࡓࠋ⬻ᶆᮏࢆ࣓ࢱ࣮࢝ࣥᅛᐃᾮ࡟ࡼࡾᅛᐃࡋࡓ [4]ࠋᑐ↷⩌ ཬࡧ㧗⏝㔞⩌ࡢ࣓ࢱ࣮࢝ࣥᅛᐃ⬻ᶆᮏࢆ⏝࠸࡚, ኱⬻ࡢ bregma ࡢᚋ᪉⣙-3.0 mm ཪࡣ-3.5 mm ࡢ2 mm ཌษ∦ࡼࡾ⏕᳨ࢺࣞࣃࣥ㸦KAI ࢖ࣥࢲࢫࢺ࣮ࣜࢬᰴᘧ఍♫㸪ᒱ㜧㸧ࢆ⏝࠸࡚ᾏ㤿ṑ

≧ᅇ㒊ศࢆ᥇ྲྀࡋ, QIAzol (Qiagen, Germany) ཬࡧ RNeasy Mini kit (Qiagen) ࢆ⏝࠸࡚ total

RNA ࢆᢳฟࡋࡓ (N = 6/⩌)ࠋ2 μg ࡢ total RNA ࠿ࡽ SuperScript® _{III Reverse Transcriptase (Thermo}

Fisher Scientific, Waltham, MA, USA) ࢆ⏝࠸࡚ cDNA ࢆྜᡂࡋࡓࠋPCR ཯ᛂࡣ SYBR®Green

PCR Master Mix (Thermo Fisher Scientific) ࢆ⏝࠸, Step One Plus™ Real-time PCR System (Thermo Fisher Scientific) ࡟࡚, 〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚ᐇ᪋ࡋࡓࠋࣉࣛ࢖࣐࣮ࡣ Primer Express software (Version 3.0; Thermo Fisher Scientific) ࢆ⏝࠸࡚タィࡋ, Table 1-16 ࡟♧ࡋ

12

transferase (Hprt1) ࡲࡓࡣ glyceraldehyde 3-phosphate dehydrogenase (Gapdh) ࡢ CT್ࢆྠ᫬࡟⟬

ฟࡋ, 0 ppm ⩌ཪࡣ 0 mg/kg ࡜ࡢ ΔΔCT್ࢆ⟬ฟࡋ࡚ẚ㍑ࡋࡓ [50]ࠋ ⤫ ⤫ィゎᯒ ྛ᭚㟢ᐇ㦂ࡢయ㔜࡞ࡽࡧ࡟⮚ჾ㔜㔞, ᦤ㣵㔞, ᦤỈ㔞, ච␿⤌⧊໬ᏛᰁⰍ, TUNEL ᰁⰍ࡟࠾ ࡅࡿ㝧ᛶ⣽⬊ࡢ࢝࢘ࣥࢺᩘ, 㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝࡣ⩌ᖹᆒཬࡧᶆ‽೫ᕪࢆ⟬ฟࡋࡓࠋⓎ㐩ᮇ ᭚㟢ᐇ㦂ࡢゎᯒ⤖ᯝ࡟ࡘ࠸࡚ࡣẕື≀ࡈ࡜࡟ᖹᆒ್ࢆ⟬ฟࡋ, ࡉࡽ࡟⩌ᖹᆒཬࡧᶆ‽೫ᕪࢆ ⟬ฟࡋࡓࠋ⤫ィᏛⓗゎᯒࡣ, య㔜, ᦤ㣵㔞, ᦤỈ㔞, ⮚ჾ㔜㔞, 㝧ᛶ⣽⬊ᩘ࡟ࡘ࠸࡚, ྛ⩌ࡢศ

ᩓࢆBartlett ࡢ᪉ἲ᳨࡛ᐃࡋ, ➼ศᩓࡢሙྜࡣ Dunnett, ୙➼ศᩓࡢሙྜࡣ Steel ࡢ᪉ἲ࡟ࡼࡾ

ᑐ↷⩌࡜ྛ⩌࡜ࡢ᳨ᐃࢆ⾜ࡗࡓࠋ2 ⩌㛫ࡢẚ㍑࡟࠾࠸࡚ࡣྛ⩌ࡢศᩓࢆ F ᳨ᐃ࡟ࡼࡾẚ㍑ࡋ,

➼ศᩓࡢሙྜࡣStudent ࡢ t ᳨ᐃ, ୙➼ศᩓࡢሙྜࡣ Aspin-Welch ࡢ t ᳨ᐃ࡟ࡼࡾᑐ↷⩌࡜ྛᢞ

୚⩌࡜ࡢ᳨ᐃࢆ⾜ࡗࡓࠋࡍ࡭࡚ࡢ⤫ィᏛⓗゎᯒࡣ࢚ࢡࢭࣝ⤫ィ2010 ࢯࣇࢺ࢙࢘࢔ (♫఍᝟ሗ

ࢧ࣮ࣅࢫᰴᘧ఍♫, ᮾி) ࢆ⏝࠸࡚⾜ࡗࡓࠋ

13

⤖

⤖ ᯝ

ື≀࡬ࡢᙳ㡪 Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶẕື≀ࡢṚஸࡣ࡞࠿ࡗࡓࠋ୍᪉࡛, 3 ཬ ࡧ10 ppm ⩌࡛ྛ 1 ౛ࡢᮍዷፎື≀ࡀࡳࡽࢀࡓࡓࡵ, ヨ㦂ࢹ࣮ࢱ࠿ࡽ㝖እࡋࡓ [Table 1-1]ࠋࡲ ࡓ, ᭚㟢⩌ࡢ╔ᗋ⑞ᩘ, ฟ⏕ᩘ, 㞝ᛶ⋡࡟㛵ࡋ࡚᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓࠋ⤒⏘ᚋ 4 ᪥┠࡟, 㞝 Ꮚື≀ᩘࡢㄪᩚࡢࡓࡵྛ⩌2 ౛ࡢẕື≀ࢆᐇ㦂࠿ࡽ㝖እࡋࡓ [Table 1-1]ࠋヨ㦂ᮇ㛫୰, ࠸ࡎࢀ ࡢ᭚㟢⩌࡛ࡶẕື≀ࡢయ㔜࡟᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 1-2]ࠋ⤒⏘ᚋ 7, 14, 21 ᪥┠࡟ 30 ppm ⩌࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-3]ࠋዷፎ 9, 13 ᪥┠࡟࠸ࡎࢀࡢ᭚㟢⩌࡛ ࡶ㣧Ỉ㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ௚, ዷፎ 7, 20 ᪥┠ཬࡧ⤒⏘ᚋ 1, 11, 14, 21 ᪥┠࡟ 30 ppm ⩌࡛ᦤỈ㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓࠋヨ㦂ᮇ㛫୰࠸ࡎࢀࡢ᭚㟢⩌࡛ࡶ␗ᖖ࡞Ṍ⾜ࡸ⾜ືࡣ ࡞࠿ࡗࡓࠋ⤒⏘ᚋ21 ᪥┠ࡢゎ๗᫬ࡣ, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶẕື≀ࡢయ㔜ཬࡧ⬻㔜㔞࡟᭷ព࡞ ኚ໬ࡣ࡞࠿ࡗࡓ [Table 1-8]ࠋ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ,ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶᏊື≀ࡢṚஸཬࡧ␗ᖖ࡞Ṍ⾜ࡸ⾜ືࡣ ࡞࠿ࡗࡓࠋ⏕ᚋ7, 70 ᪥┠࡟ 30 ppm ⩌࡛య㔜ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-4]ࠋ⏕ᚋ 35 ᪥┠࡟3, 10 ppm ⩌࡛, ⏕ᚋ 42 ᪥┠࡟ 3 ppm ⩌࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼࡾ㧗್ࢆ♧ࡋࡓ [Table 1-5]ࠋ ⏕ᚋ56, 63 ᪥┠࡟ 30 ppm ⩌࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓࠋ⏕ᚋ, 35, 42 ᪥┠࡟ 30 ppm ⩌࡛㣧Ỉ㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓࠋ⏕ᚋ21 ᪥┠ࡢゎ๗᫬ࡣ, 30 ppm ⩌࡛య㔜ࡀ, 10 ppm ⩌௨ୖ࡛⬻㔜㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-9]ࠋࡲࡓ⑓⌮⤌⧊Ꮫⓗኚ໬࡛ࡣ, SGZ ࡢ㛗 ࡉཬࡧᾏ㤿ṑ≧ᅇࡢ㠃✚ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-12]ࠋ⏕ᚋ 77 ᪥┠ࡢゎ๗᫬ࡣ, ࠸ ࡎࢀࡢ⩌࡛ࡶయ㔜࡟ኚ໬ࡣ࡞࠿ࡗࡓࠋ30 ppm ⩌࡛⬻㔜㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-9]ࠋࡲࡓ⑓⌮⤌⧊Ꮫⓗኚ໬࡛ࡣ, SGZ ࡢ㛗ࡉࡣ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓࡀ, 30 ppm ⩌࡛ᾏ㤿ṑ≧ᅇࡢ㠃✚ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-12]ࠋ 㞳ஙᚋᭀ㟢ヨ㦂࡛ࡣ, ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶື≀ࡢṚஸཬࡧ⫗║ⓗ࡞␗ᖖࡣ࡞࠿ࡗ ࡓࠋᢞ୚7 ᪥┠࠿ࡽ 28 ᪥┠࡟ 5 mg/kg ⩌௨ୖ࡛య㔜ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-6]ࠋ ᢞ୚3 ᪥┠࠿ࡽ 21 ᪥┠࡟ 5 mg/kg ⩌௨ୖ࡛, ᢞ୚ 28 ᪥┠࡟ 15 mg/kg ⩌࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼ

14 ࡾప್ࢆ♧ࡋࡓ [Table 1-7]ࠋᢞ୚ 14 ᪥┠࠿ࡽ 28 ᪥┠࡟ 15 mg/kg ⩌࡛㣧Ỉ㔞ࡀᑐ↷⩌ࡼࡾప ್ࢆ♧ࡋࡓࠋゎ๗᫬ࡣ, 5 mg/kg ⩌௨ୖ࡛య㔜ࡀ, 15 mg/kg ࡛⬻㔜㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 1-10]ࠋࡲࡓ, ⑓⌮⤌⧊Ꮫⓗኚ໬࡛ࡣ, SGZ ࡢ㛗ࡉཬࡧᾏ㤿ṑ≧ᅇࡢ㠃✚ࡣ࠸ࡎࢀࡢᢞ୚ ⩌࡛ࡶ᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 1-13]ࠋ ᭚ ᭚㟢ື≀ࡢSGZ ཬࡧ GCL ࡟࠾ࡅࡿ㢛⢏⣽⬊⣔㆕ࡢศᕸ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, 㞝Ꮚື≀ࡢฟ⏕ᚋ 21 ᪥࡟࠾࠸࡚ 30 ppm ⩌࡛, GFAP 㝧ᛶ⣽⬊ (type-1 ᖿ⣽⬊) ᩘࡀῶᑡཬࡧ SOX2 㝧ᛶ⣽⬊ (type-1 ᖿ⣽⬊㹼type-2b ๓㥑⣽⬊) ᩘ, TBR2 㝧ᛶ⣽⬊ (type-2b ๓㥑⣽⬊) ᩘཬࡧ DCX 㝧ᛶ⣽⬊ (type-2b ๓㥑⣽⬊㹼ᮍ⇍㢛⢏⣽⬊) ᩘࡀቑຍࡋࡓ [Fig. 1-2]ࠋ୍᪉࡛, NeuN 㝧ᛶ⣽⬊ (ᡂ⇍㢛⢏⣽⬊) ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓࠋฟ⏕ᚋ 77 ᪥࡟࠾࠸

࡚10 ཬࡧ 30 ppm ⩌࡛, NeuN 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓࠋ୍᪉࡛, GFAP 㝧ᛶ⣽⬊ᩘ, SOX2 㝧ᛶ⣽

⬊ᩘ, TBR2 㝧ᛶ⣽⬊ᩘཬࡧ DCX 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓࠋࡲࡓ, ฟ⏕ᚋ 77 ᪥࡟࠾ࡅࡿ NeuN 㝧ᛶ㢛⢏⣽⬊ᒙࡢཌࡉ, ཬࡧฟ⏕ᚋ 21 ᪥ཬࡧ 77 ᪥࡟࠾ࡅࡿ NeuN 㝧ᛶ㢛⢏⣽⬊ᒙࡢ㠃 ✚ࡀ30 ppm ⩌࡛ῶᑡࡋࡓ [Table 1-14]ࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 15 mg/kg ⩌࡛ GFAP 㝧ᛶ⣽⬊ᩘཬࡧ SOX2 㝧ᛶ⣽⬊ᩘࡀῶᑡ, 5 ཬࡧ 15 mg/kg ⩌࡛ TBR2 㝧ᛶ⣽⬊ᩘ, DCX 㝧ᛶ⣽⬊ᩘཬࡧ NeuN 㝧ᛶ⣽⬊ࡀῶᑡࡋࡓ [Fig. 1-3]ࠋ ࡲࡓ, NeuN 㝧ᛶ㢛⢏⣽⬊ᒙࡢཌࡉࡀ 5 ཬࡧ 15 mg/kg ⩌࡛ῶᑡࡋࡓ [Table 1-15]ࠋ ᭚㟢ື≀ࡢṑ≧ᅇ㛛࡟࠾ࡅࡿᡂ⇍ࢽ࣮ࣗࣟࣥཬࡧ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ฟ⏕ᚋ 21 ᪥ཬࡧ 77 ᪥࡟࠾࠸࡚, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, RELN 㝧ᛶ⣽ ⬊ᩘ, PVALB 㝧ᛶ⣽⬊ᩘ, CALB1 㝧ᛶ⣽⬊ᩘ, CALB2 㝧ᛶ⣽⬊ᩘཬࡧ NeuN 㝧ᛶ⣽⬊ᩘࡣኚ໬ ࡋ࡞࠿ࡗࡓ [Fig. 1-4]ࠋ

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, RELN 㝧ᛶ⣽⬊ᩘ, PVALB 㝧ᛶ⣽⬊ᩘ, CALB1 㝧ᛶ⣽⬊ᩘ, CALB2 㝧ᛶ⣽⬊ᩘཬࡧ NeuN 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓ [Fig. 1-5]ࠋ

15 ᭚

᭚㟢ື≀ࡢSGZ ࡟࠾ࡅࡿ⣽⬊ቑṪάᛶ, ⣽⬊࿘ᮇ೵Ṇཬࡧ࢔࣏ࢺ࣮ࢩࢫ

Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ฟ⏕ᚋ 21 ᪥࡟࠾࠸࡚ 30 ppm ⩌࡛, p21Cip1/Waf1_{㝧ᛶ⣽⬊ᩘཬࡧTUNEL}

㝧ᛶ⣽⬊ᩘࡀቑຍࡋࡓࠋ୍᪉࡛, PCNA 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓ [Fig. 1-6]ࠋฟ⏕ᚋ 77 ᪥

࡟࠾࠸࡚, PCNA 㝧ᛶ⣽⬊ᩘ, p21Cip1/Waf1_{㝧ᛶ⣽⬊ᩘཬࡧTUNEL 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓࠋ}

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 15 mg/kg ⩌࡛, TUNEL 㝧ᛶ⣽⬊ᩘࡀቑຍࡋࡓࠋp21Cip1/Waf1_{㝧ᛶ⣽⬊ᩘ}

ཬࡧTUNEL 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓ [Fig. 1-7]ࠋ

᭚㟢ື≀ࡢGCL ࡟࠾ࡅࡿࢩࢼࣉࢫྍረᛶ㛵㐃ศᏊ

Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ฟ⏕ᚋ 21 ᪥࡟࠾࠸࡚ 30 ppm ⩌࡛ COX2 㝧ᛶ⣽⬊ᩘࡀቑຍࡋࡓࠋ୍ ᪉࡛, ARC 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓ [Fig. 1-8]ࠋฟ⏕ᚋ 77 ᪥࡟࠾࠸࡚, ARC 㝧ᛶ⣽⬊ᩘཬ

ࡧCOX2 㝧ᛶ⣽⬊ᩘࡣኚ໬ࡋ࡞࠿ࡗࡓࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 5 ཬࡧ 15 mg/kg ⩌࡛ ARC 㝧ᛶ⣽⬊ᩘཬࡧ COX2 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋ ࡓ [Fig. 1-9]ࠋ ᭚㟢ື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ゎᯒ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, Ꮚື≀ࡢ⬻ࡀᑠࡉ࠿ࡗࡓࡓࡵᾏ㤿㒊ศࢆ 1 mm ࡢࣃࣥࢳ⏕᳨࡛ᅇ཰ ࡍࡿࡇ࡜ࡀฟ᮶࡞࠿ࡗࡓࡓࡵ, 㑇ఏᏊⓎ⌧ゎᯒࢆᐇ᪋ࡋ࡛ࡁ࡞࠿ࡗࡓࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 㢛⢏⣽⬊ࡢศ໬࣐࣮࣮࢝ࡢ Gfap ཬࡧ Dpsyl3, N-࣓ࢳࣝ-D-࢔ࢫࣃࣛࢠ ࣥ㓟 (NMDA) ᆺࣞࢭࣉࢱ࣮࡛࠶ࡿ Grin2a ࡢⓎ⌧ࡀῶᑡࡋࡓ [Table1-17]ࠋ௓ᅾࢽ࣮ࣗࣟࣥ㛵 㐃ศᏊࡢ Pvalb, ⚄⤒ᡂ㛗ᅉᏊ࡛࠶ࡿ Bdnf ࡢⓎ⌧, ࢔࣏ࢺ࣮ࢩࢫ࡟㛵㐃ࡍࡿศᏊࡢ Casp6, Casp8, Bax, Tnf, DNA ᦆയ㛵㐃ศᏊ࡛࠶ࡿ Tp53, Mdm2, Cdkn1a, DNA ಟ᚟㛵㐃ศᏊࡢ Mgmt ࡢ

Ⓨ⌧ࡀቑຍࡋࡓࠋࡑࡢ௚ࡢ௓ᅾࢽ࣮ࣗࣟࣥ, ࢢࣝࢱ࣑ࣥ㓟ࡶࡋࡃࡣ࢔ࢭࢳࣝࢥࣜࣥࣞࢭࣉࢱ ࣮࡟㛵㐃ࡍࡿ㑇ఏᏊ⩌࡟ኚ໬ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ

16

⪃

⪃ ᐹ

MNU ࡢⓎ㐩ᮇ᭚㟢࡟ࡼࡾ, Ꮚື≀ࡢ⬻㔜㔞ࡢప್ࡀ 30 ppm ᢞ୚⩌ࡢ⏕ᚋ 21 ᪥┠ཬࡧ⏕ᚋ 77 ᪥┠, ཬࡧ 10 ppm ᢞ୚⩌ࡢ⏕ᚋ 21 ᪥┠࡛☜ㄆࡉࢀࡓࠋࡇࢀࡽ⤖ᯝࡣ, 㐣ཤࡢሗ࿌࡛࠶ࡿ, MNU ࡢዷፎ 14 ᪥┠ࡢⓎ㐩ᮇ᭚㟢࡟ࡼࡗ࡚ㄏⓎࡍࡿᑠ㢌⑕ [45] ࢆ♧၀ࡍࡿࠋࡲࡓ, ௒ᅇࡢ MNU Ⓨ㐩ᮇ᭚㟢࡟ࡼࡾ, SGZ 㛗ࡢ▷⦰ཬࡧᾏ㤿ṑ≧ᅇࡢ㠃✚ࡢῶᑡࡀ, 30 ppm ᢞ୚⩌ࡢ⏕ᚋ 21 ᪥┠ཬࡧ 77 ᪥┠࡛☜ㄆࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ, 㐣ཤࡢሗ࿌࡛࠶ࡿ, MNU ࡢዷፎ 14 ᪥┠ ࡟10 mg/kg ࡢ MNU ࢆ༢ᅇ⭡⭍ෆᢞ୚࡟ࡼࡗ࡚ㄏⓎࡍࡿᾏ㤿ࡢపᙧᡂ [81] ࢆ♧၀ࡍࡿࠋ୍ ᪉࡛, ዷፎᮇ㹼㞳ஙᮇࡲ࡛ࡢᣢ⥆ⓗ࡞ MNU Ⓨ㐩ᮇ᭚㟢ࡣ, GCL ࡢᒙᵓ㐀ࡢཌࡉࢆῶᑡࡉࡏࡓ ࡀ, ዷፎᮇࡢ༢ᅇᢞ୚࡛ࡣ GCL ࡢཌࡉ࡟ኚືࡣ࡞࠿ࡗࡓ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ [81]ࠋࡇࢀࡽࡢ⤖ ᯝࡣ, ୍㐣ⓗ࡞ MNU Ⓨ㐩ᮇ᭚㟢ࡣ⤊⬻ࡢ⚄⤒ୖ⓶ࡢቑṪάᛶࢆ᭷ࡍࡿ⚄⤒⣽⬊ࢆᶆⓗ࡜ࡋ ᾏ㤿ࡢపᙧᡂࡢࡳࢆㄏⓎࡍࡿࡢ࡟ᑐࡋ, ᣢ⥆ⓗ࡞ MNU Ⓨ㐩ᮇ᭚㟢ࡣᾏ㤿ࡢ㢛⢏⣽⬊⣔㆕ࢆ ྵࡵࡓከࡃࡢቑṪᛶ⚄⤒⣽⬊ࡀᶆⓗ࡜࡞ࡾ, GCL ࡢཌࡳࢆῶᑡࡉࡏࡓ࡜⪃࠼ࡽࢀࡿࠋࡲࡓ, ௒ ᅇࡢ㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣGCL ࡢཌࡉࡣῶᑡࡋࡓࡀ, ᾏ㤿ṑ≧ᅇࡢ㠃✚ࡣኚືࡋ࡞࠿ࡗࡓࠋᚑ ࡗ࡚, ⏕ᚋࡢ MNU ᭚㟢ࡢᙳ㡪ࡣ, ᪤࡟ᙧᡂࡉࢀࡓᾏ㤿ṑ≧ᅇࡢ⚄⤒᪂⏕ [48] ࢆᶆⓗ࡜ࡍࡿࠋ ᑐ↷ⓗ࡟, ⫾⏕ᮇࡢ MNU ᭚㟢ࡢᙳ㡪ࡣᾏ㤿ࢆᙧᡂࡍࡿ⤊⬻ࡢ⚄⤒ୖ⓶ࡢቑṪάᛶࢆ᭷ࡍࡿ ⚄⤒⣽⬊ࢆᶆⓗ࡜ࡍࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡓࠋ

௒ᅇࡢMNU Ⓨ㐩ᮇ᭚㟢࡛ࡣ, ⏕ᚋ 21 ᪥┠࡟ 30 ppm ᢞ୚⩌࡛ SGZ ࡢ GFAP 㝧ᛶ type-1 ⚄

⤒ᖿ⣽⬊ࡢῶᩘཬࡧSOX2, TBR2, DCX 㝧ᛶ⣽⬊ࡢቑᩘࡀ☜ㄆࡉࢀࡓࠋࡲࡓ, TUNEL 㝧ᛶ࢔࣏ ࢺ࣮ࢩࢫ⣽⬊ࡢቑᩘࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽ, MNU ࡀ୺࡟ type-1 ⚄⤒ᖿ⣽⬊ࢆᶆⓗ࡜ࡋࡓ࢔࣏ ࢺ࣮ࢩࢫࢆㄏⓎࡋࡓࡶࡢ࡜᥎ᐹࡉࢀࡓࠋࡋ࠿ࡋ, type-1 ⚄⤒ᖿ⣽⬊ࡣ SGZ ࡛ࡢ๭ྜࡣᑡ࡞ࡃቑ Ṫᛶࡶ࡯࡜ࢇ࡝࡞࠸ࠋᚑࡗ࡚, ࡼࡾ㧗࠸ቑṪάᛶࢆ᭷ࡍࡿ type-2 ཬࡧ type-3 ๓㥑⣽⬊ࡢ᪉ࡀ MNU ࡢᶆⓗ࡜࡞ࡾ⣽⬊യᐖࢆㄏⓎࡍࡿ࡜⪃࠼ࡿࡢࡀ୍⯡ⓗ࡛࠶ࡿࠋ⪃࠼ࡽࢀࡿྍ⬟ᛶ࡜ࡋ࡚ ࡣ, SGZ ࡢ⏕Ꮡ⣽⬊ࡢቑṪάᛶࡀ MNU Ⓨ㐩ᮇ᭚㟢࡟ࡼࡾಁ㐍ࡉࢀࡿࡇ࡜࡛࠶ࡾ, ྠ᫬࡟ಁ㐍 ࡉࢀࡿቑṪ⣽⬊ࡢ⣽⬊Ṛࡀ┿ࡢቑṪάᛶࢆ㞃ⶸࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࠋᡂ⇍㢛⢏⣽⬊ ࡢCOX2 Ⓨ⌧ࡀ SGZ ๓㥑⣽⬊ࡢࣉࣟࢫࢱࢢࣛࣥࢪࣥ㹃/EP2 ࢆ௓ࡋ࡚ቑṪάᛶࢆಁ㐍ࡍࡿࡇ࡜

17 ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [54]ࠋࡉࡽ࡟, COX2 ࡢ KO ࣐࢘ࢫ࡛ࡣ, SGZ ࡢ DCX 㝧ᛶ⣽⬊ࡢῶᩘࡀሗ࿌ ࡉࢀ࡚࠸ࡿ [61]ࠋࡇࢀࡽࡢ⤖ᯝࡣ, ௒ᅇࡢ MNU Ⓨ㐩ᮇ᭚㟢࡟ࡼࡾ㞳ங᫬࡛ࡳࡽࢀࡓ GCL ࡢ COX2 㝧ᛶ⣽⬊ࡢቑຍࡀ, 㢛⢏⣽⬊⣔㆕ࡢ࢔࣏ࢺ࣮ࢩࢫࡢ௦ൾ཯ᛂ࡜ࡋ࡚ SGZ ⣽⬊ࡢቑṪά ᛶࢆಁ㐍ࡉࡏ࡚࠸ࡿྍ⬟ᛶࢆ♧၀ࡍࡿࠋ ᚑࡗ࡚, 㞳ங᫬࡟ PCNA 㝧ᛶ⣽⬊࡟᭷ព࡞ቑᩘࡣ࡞ ࠸ࡶࡢࡢ, SGZ ࡢ๓㥑⣽⬊ࡢቑṪάᛶࡀቑຍࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࠋ୍᪉, ⏕ᚋ 77 ᪥ ┠࡛ࡣ, 30 ppm ᢞ୚⩌࡛ GCL ࡢཌࡉࡢῶᑡࢆక࠸ NeuN 㝧ᛶᮍ⇍ཬࡧᡂ⇍㢛⢏⣽⬊ࡀ MNU ࡢఇ⸆ᚋ࡟ῶᩘࡋࡓࠋࡇࢀࡽࡢື≀࡛ࡣ, ⏕ᚋ 21 ᪥┠࡟ p21Cip1/Waf1_{㝧ᛶ⣽⬊ࡀቑᩘࡋ࡚࠸ࡿࠋ} p21Cip1/Waf1_{ࡣ, ࢧ࢖ࢡࣜࣥ౫Ꮡᛶࣜࣥ㓟໬㓝⣲㜼ᐖస⏝ࢆ᭷ࡋ, DNA യᐖᚋ࡟ G} 1/S ࢳ࢙ࢵࢡ࣏ ࢖ࣥࢺ࡛⣽⬊࿘ᮇ೵ṆࢆㄏⓎࡍࡿ [71]ࠋࡲࡓ, p21Cip1/Waf1_{ࡣ⣽⬊ࡢ㟼Ṇᮇࢆ⥔ᣢࡋ, ៏ᛶⓗ࡞} p21Cip1/Waf1ࡢάᛶ໬ࡣ୙ྍ㏫ⓗ࡞⣽⬊࿘ᮇࡢ೵Ṇཬࡧ⣽⬊⪁໬ࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀሗ࿌ࡉࢀ ࡚࠸ࡿ [67]ࠋ௨ୖࡼࡾ, ᡂ⇍᫬࡟ࡳࡽࢀࡓ NeuN 㝧ᛶᮍ⇍ཬࡧᡂ⇍㢛⢏⣽⬊ࡢῶᩘࡣ, 㞳ங᫬ ࡟ቑຍࡋࡓp21Cip1/Waf1㝧ᛶ⣽⬊ࡢDNA യᐖࡀ⵳✚ࡋ, G1/S ⣽⬊࿘ᮇ೵Ṇࡀಁ㐍ࡉࢀࡓ⤖ᯝ࡜ ⪃࠼ࡽࢀࡓࠋ

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 15 mg/kg ᢞ୚⩌࡛ GFAP 㝧ᛶཪࡣ SOX2 㝧ᛶ type-1 ⚄⤒ᖿ⣽⬊ཬࡧ type-2a ๓㥑⣽⬊ࡀ, 5 mg/kg ௨ୖࡢᢞ୚⩌࡛ TBR2, DCX, NeuN 㝧ᛶ type-2, type-3 ๓㥑⣽⬊ཬ ࡧᮍ⇍㢛⢏⣽⬊, ᡂ⇍㢛⢏⣽⬊ࡀ SGZ ཪࡣ GCL ࡛ῶᩘࡋࡓࠋ TUNEL 㝧ᛶ⣽⬊ࡶ 15 mg/kg ᢞ

୚⩌࡛ቑᩘࡋࡓࠋࡇࡢࡇ࡜࠿ࡽ, MNU ࡢᙳ㡪ࡣ type-2 ௨㝆ࡢ㢛⢏⣽⬊⣔㆕࡟ᑐࡍࡿ᪉ࡀ type-1

⚄⤒ᖿ⣽⬊ࡼࡾᙉࡃ 㞳ஙᚋࡢ MNU ࡢ᭚㟢ࡣቑṪάᛶࡢ㧗࠸⣽⬊ࢆᶆⓗࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ ࡓࠋྠᵝ࡟, ࢽࢺࣟࢯᒀ⣲໬ྜ≀࡛࠶ࡿ࢚ࢳࣝࢽࢺࣟࢯᒀ⣲ࡢ༢ᅇ㟼⬦ෆᢞ୚ࡢ⤖ᯝ, SGZ ࡛

ቑṪ⣽⬊ཬࡧDCX 㝧ᛶ๓㥑⣽⬊ࢆῶᩘࡉࡏࡿ [13]ࠋࡇࡢ⤖ᯝࡣ, MNU ࡢ㞳ஙᚋ᭚㟢ࡀቑṪ

άᛶࡢ㧗࠸⣽⬊࡟ᑐࡋ⣽⬊യᐖᛶࢆ᭷ࡍࡿࡇ࡜ࢆ♧၀ࡋ࡚࠾ࡾ, ⣽⬊ಖㆤࡸ௦ൾ཯ᛂࡀࡳࡽ

ࢀࡓMNU ࡢⓎ㐩ᮇ᭚㟢⤖ᯝ࡜ࡣ␗࡞ࡿࠋMNU ࡢ㞳ஙᚋ᭚㟢࡛ࡣ, p21Cip1/Waf1㝧ᛶ⣽⬊ࡢኚᩘ

ࡣ࡞ࡃ, 㑇ఏᏊⓎ⌧ゎᯒ࡟࠾࠸࡚ࡶ Cdkn1a ࡢኚືࡣ☜ㄆࡉࢀ࡞࠿ࡗࡓࠋࡇࡢ⤖ᯝࡣ, MNU ࡢ

ẅ⣽⬊స⏝࡟ࡼࡗ࡚ㄏⓎࡉࢀࡓ᭱኱㝈ࡢ㢛⢏⣽⬊⣔㆕ࡢῶᩘཬࡧCOX2 ࢆ௓ࡋࡓ⣽⬊ቑṪά

ᛶࡢᢚไ࡟ࡼࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ୍᪉࡛, MNU ࡢ㞳ஙᚋ᭚㟢࡛ࡣ Pvalb ཬࡧ Bdnf ࡢ㑇ఏᏊ Ⓨ⌧㔞ࡢቑຍࡀࡳࡽࢀࡓࠋ⬻⏤᮶⚄⤒ᰤ㣴ᅉᏊ (BDNF) ࡣ⚄⤒ࡢᡂ⇍ࢆಁ㐍ࡋ, PVALB 㝧ᛶ

18

GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࢆάᛶ໬ࡍࡿሗ࿌ࡀ࠶ࡿ [10, 32]ࠋࡉࡽ࡟, ṑ≧ᅇ㛛ࡢ PVALB 㝧ᛶ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡣ type-2 ๓㥑⣽⬊ࡢቑṪࢆಁ㐍ࡍࡿ [84]ࠋᚑࡗ࡚, Pvalb ཬࡧ Bdnf ࡢ 㑇ఏᏊⓎ⌧㔞ࡢቑຍࡣ, MNU ࡢ㞳ஙᚋ᭚㟢࡟ࡼࡿᙳ㡪࡟ᑐࡍࡿ௦ൾᛶࡢ཯ᛂࡢྍ⬟ᛶࡀ࠶ࡿ ࡀ, MNU ࡢẅ⣽⬊స⏝ࡸ COX2 ࢆ௓ࡋࡓቑṪάᛶࡢᢚไ࡟ࡼࡿᙳ㡪ࡀᙉ࠸ࡓࡵ, type-2 ๓㥑⣽ ⬊ࡶῶᩘࡋ࡚࠸ࡿࠋ Ⓨ㐩ᮇ᭚㟢ヨ㦂ཬࡧ㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ ඹ㏻ࡋ࡚ TUNEL 㝧ᛶ⣽⬊ࡀቑᩘࡋࡓࠋࡉࡽ࡟, 㞳ஙᚋ᭚㟢ヨ㦂ࡢᾏ㤿ṑ≧ᅇ࡛ࡣ㑇ఏᏊゎᯒࡢ⤖ᯝ, ࢝ࢫࣃ࣮ࢮ 6 ཬࡧ࢝ࢫࣃ࣮ࢮ 8 ࡸ Bax ཬࡧTnf ࡞࡝ࡢ࢔࣏ࢺ࣮ࢩࢫࢆ♧၀ࡍࡿ㑇ఏᏊⓎ⌧ࡀ, DNA യᐖᣦᶆ࡛࠶ࡿ Tp53 ࡜ඹ࡟ቑ ຍࡋࡓࠋ࢝ࢫࣃ࣮ࢮ8 ࡣ, እᅉ⤒㊰࡟ࡼࡗ࡚άᛶ໬ࡍࡿ࢖ࢽࢩ࢚࣮ࢱ࣮࡛࠶ࡾ, ࢝ࢫࣃ࣮ࢮ 6 ࡣ, ࣑ࢺࢥࣥࢻࣜ࢔⤒㊰ࡢୗὶࡢ࢔࣏ࢺ࣮ࢩࢫᐇ⾜ᅉᏊ࡜ࡋ࡚ᶵ⬟ࡍࡿ [88]ࠋTnf ࡸ Bax ࡢ 㑇ఏᏊⓎ⌧㔞ࡢቑຍࡣ, MNU ࡢ᭚㟢࡟ࡼࡾᵝࠎ࡞࢔࣏ࢺ࣮ࢩࢫ⤒㊰ࡀάᛶ໬ࡍࡿࡇ࡜ࢆ♧ ၀ࡋ࡚࠸ࡿࠋࡉࡽ࡟, ࡇࢀࡽࡢ⤖ᯝࡣ SGZ ࡛ࡢ DNA യᐖࡀ MNU ࡢ᭚㟢࡟ࡼࡗ࡚ㄏⓎࡉࢀ ࡓ཯ᛂ࡟ᑐࡋ, p53 ࡀάᛶ໬ࡋ, ࢔࣏ࢺ࣮ࢩࢫཪࡣ DNA ಟ᚟࡞࡝ࡢ⣽⬊㐠࿨ࢆᐃࡵ࡚࠸ࡿࠋ MNU ࡣ, O6-࣓ࢳࣝࢢ࢔ࢽࢪࣥሷᇶࢆྵࡴ࣑ࢫ࣌࢔࡟ࡼࡗ࡚࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿ [25]ࠋ ᮏᐇ㦂࡛ࡶ, O6_{-࣓ࢳࣝࢢ࢔ࢽࢪࣥࢆⓎ⌧ࡍࡿ㑇ఏᏊࡢ Mgmt ࡢⓎ⌧ቑຍࡀᾏ㤿ṑ≧ᅇ࡛ࡳࡽ} ࢀࡓࠋࡇࡢ⤖ᯝࡣ, MNU ࡀ O6-࣓ࢳࣝࢢ࢔ࢽࢪࣥ⵳✚ࢆㄏⓎࡉࡏࡓ⤖ᯝ, ࢔࣏ࢺ࣮ࢩࢫࡀ㉳ ࡇࡿࡇ࡜ࢆ♧၀ࡍࡿࠋⓎ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ⚄⤒᪂⏕࡟ᙳ㡪ࡢ࠶ࡿ㧗⏝㔞⩌࡛, ᑠ㢌⑕ࡢࡓ ࡵ1 mm ࡢ⏕᳨ࢺࣞࣃ࡛ࣥࡣᾏ㤿ṑ≧ᅇࡢࢧࣥࣉࣜࣥࢢࡀୖᡭࡃ࠸࠿ࡎ, 㑇ఏᏊゎᯒ⤖ᯝࡣ ᚓࡽࢀ࡚࠸࡞࠸ࡀ, TUNEL 㝧ᛶ⣽⬊ࡢቑᩘࡀࡳࡽࢀࡿࡇ࡜࠿ࡽ, 㞳ஙᚋ᭚㟢࡜ྠᵝࡢ㑇ఏᏊ Ⓨ⌧ኚືࡀ⏕ࡌࡓࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ

௨ୖࡼࡾ, MNU ࡛ࡣ᭚㟢᫬ᮇࢆၥࢃࡎ, DNA ࡢ┤᥋ᦆയ࡟㉳ᅉࡋࡓ type-1 ⚄⤒ᖿ⣽⬊ࢆྵ

ࡴSGZ ⣽⬊ࡢ࢔࣏ࢺ࣮ࢩࢫࢆ୺せ࣓࢝ࢽࢬ࣒࡜ࡋࡓ⚄⤒᪂⏕㞀ᐖࡀ♧၀ࡉࢀࡓࠋࡲࡓ, Ⓨ㐩

ᮇ᭚㟢࡟ࡼࡿ୍㐣ⓗ࡞๓㥑⣽⬊ࡢቑຍࡣ COX2 Ⓨ⌧࡟ࡼࡿቑṪάᛶಁ㐍࡟㉳ᅉࡍࡿࡶࡢ࡜⪃

࠼ࡽࢀ, Ⓨ㐩ᮇ≉᭷࡞཯ᛂ࡛࠶ࡿ࡜᥎ᐹࡉࢀࡿࡇ࡜࠿ࡽ, ᭚㟢᫬ᮇࡢ㐪࠸࡟ࡼࡿ⚄⤒᪂⏕㞀 ᐖࡢ཯ᛂࣃࢱ࣮ࣥࡢ㐪࠸࡟཯ᫎࡉࢀࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ

19

ᑠ

ᑠ ᣓ

➨1 ❶࡛ࡣ, MNU ࡢⓎ㐩ᮇ᭚㟢࡜㞳ஙᚋ᭚㟢ࢆ⾜ࡗࡓࠋⓎ㐩ᮇ᭚㟢࡛ࡣ, ஺㓄ᚋࡢ㞤ࣛࢵ ࢺ࡟0, 3, 10 ཬࡧ 30 ppm ࡢ⃰ᗘ࡛ዷፎ 6 ᪥┠࠿ࡽฟ⏘ᚋ 21 ᪥┠ࡢ㞳ங᫬ࡲ࡛ MNU ࢆ㣧Ỉᢞ ୚ࡋ, Ꮚື≀ࡢ㞳ங᫬ཬࡧᡂ⇍ᚋࡢ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝ, 㞳ங᫬࡟ 30 ppm

⩌࡛SGZ/GCL ࡢ GFAP 㝧ᛶ type-1 ⚄⤒ᖿ⣽⬊ᩘࡀῶᑡ, SOX2, TBR2, DCX 㝧ᛶ type-2/type-3

⚄⤒๓㥑⣽⬊ཬࡧᮍ⇍㢛⢏⣽⬊ᩘ, TUNEL 㝧ᛶ࢔࣏ࢺ࣮ࢩࢫ⣽⬊ᩘ, p21 㝧ᛶ⣽⬊࿘ᮇ೵Ṇ⣽ ⬊ᩘ, COX2 㝧ᛶᡂ⇍㢛⢏⣽⬊ᩘࡀቑຍࡋࡓࠋᡂ⇍ᚋ࡛ࡣ, GCL ࡢ NeuN 㝧ᛶᡂ⇍㢛⢏⣽⬊ᩘ ࡀῶᑡࡋࡓࠋḟ࡟, 5 㐌㱋ࡢࣛࢵࢺ࡟ 0, 5 ཬࡧ 15 mg/kg ࡢ MNU ࢆ 28 ᪥㛫཯᚟ᙉไ⤒ཱྀᢞ୚ ࡋ, 㞳ஙᚋ᭚㟢࡟ࡼࡿ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝ, 5 mg/kg ⩌࠿ࡽ SGZ/GCL ࡢ GFAP, SOX2, TBR2, DCX, NeuN, COX2, ARC 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋ, TUNEL 㝧ᛶ⣽⬊ᩘࡣቑຍ ࡋࡓࠋࡲࡓ, ṑ≧ᅇ࡛ࡣ NMDA ᆺࢢࣝࢱ࣑ࣥ㓟ཷᐜయࡢ㑇ఏᏊⓎ⌧ࡀῶᑡࡋࡓࠋ௨ୖࡼࡾ, MNU ࡛ࡣ᭚㟢᫬ᮇࢆၥࢃࡎ, DNA ࡢ┤᥋ᦆയ࡟㉳ᅉࡋࡓ SGZ ⣽⬊ࡢ࢔࣏ࢺ࣮ࢩࢫࢆ୺せ࣓ ࢝ࢽࢬ࣒࡜ࡋࡓ⚄⤒᪂⏕㞀ᐖࡀ♧၀ࡉࢀࡓࠋࡲࡓ, Ⓨ㐩ᮇ᭚㟢࡟ࡼࡿ୍㐣ⓗ࡞๓㥑⣽⬊ࡢቑ ຍࡣ COX2 Ⓨ⌧࡟ࡼࡿቑṪάᛶಁ㐍࡟㉳ᅉࡋ, Ⓨ㐩ᮇ≉᭷࡞཯ᛂ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࡇ࡜࠿ ࡽ, ᭚㟢᫬ᮇࡢ㐪࠸࡟ࡼࡿ⚄⤒᪂⏕㞀ᐖࡢ཯ᛂࣃࢱ࣮ࣥࡢ㐪࠸࡟཯ᫎࡉࢀࡿྍ⬟ᛶࡀ♧၀ࡉ ࢀࡓࠋ

20

➨

➨

2 ❶

VPA ࡢࣛࢵࢺⓎ㐩ᮇཬࡧ㞳ஙᚋ᭚㟢࡟ࡼࡿ

⚄⤒᪂⏕࡟ཬࡰࡍᙳ㡪ࡢ᳨ウ

21

⥴

⥴ ゝ

ࣂࣝࣉࣟ㓟㸦VPA㸧ࡣ, ᢠ࡚ࢇ࠿ࢇ⸆, ⢭⚄Ᏻᐃ⸆ཬࡧ㢌③⸆࡜ࡋ࡚౑⏝ࡉࢀ࡚࠸ࡿࠋ⫾⏕ ᮇ㸦≉࡟⚄⤒⟶㛢ሰࡢ㛫㸧ࡢVPA ᭚㟢ࡀ, ᑠ⬻ࢆྵࢇࡔᙧ࡛ࡢ␗ᖖ࡞⬻ᙧᡂཬࡧ⮬㛢⑕ᵝ⑕ ≧࡟㛵୚ࡍࡿࡇ࡜ࡀᐇ㦂ື≀ࡢ᭚㟢ᐇ㦂࡛᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ [34, 70]ࠋࣛࢵࢺࡢ⮬㛢⑕ࣔࢹ ࣝࡣ, ዷፎ 11 ᪥┠ࡢẕື≀࡟ 800 mg/kg ࡢ VPA ࢆᢞ୚ࡍࡿࡇ࡜࡛☜ㄆࡉࢀ࡚࠾ࡾ, ዷፎ 16 ᪥┠ࡢ⫾Ꮚ࡛ࡣ, ⓶㉁ࡢ⳼ⷧ໬ࡸ⚄⤒ࡢ⛣ື␗ᖖࡀሗ࿌ࡉࢀ࡚࠸ࡿ [47]ࠋࡉࡽ࡟, ዷፎ 12.5 ᪥┠࡟500 mg/kg ࡢ VPA ࢆᢞ୚ࡋࡓ⮬㛢⑕ࣔࢹࣝ౛࡛ࡣ, ⏕ᚋ 67 ᪥┠ࡢᏊື≀࡟࠾࠸࡚, ࢔ ࣥࣔࣥゅ, ᾏ㤿ྎ, ᾏ㤿ṑ≧ᅇࡢ⚄⤒⣽⬊ࡢᐦᗘࡀቑຍࡋ࡚࠸ࡿ [20]ࠋࡲࡓ, VPA ࡣࣄࢫࢺࣥ ⬺࢔ࢭࢳࣝ໬㓝⣲㸦HDAC㸧㜼ᐖ๣࡜ࡋ࡚ࡶ▱ࡽࢀ࡚࠾ࡾ, ᡂ⇍ࣛࢵࢺ࡟ 1 ᪥ 2 ᅇ, 300 mg/kg ࡢVPA ࢆ 14 ᪥㛫⭡⭍ෆᢞ୚ࡍࡿ࡜, ᾏ㤿ࡢ⚄⤒๓㥑⣽⬊ࡢࣄࢫࢺࣥࡢ࢔ࢭࢳࣝ໬࡟㛵㐃ࡋࡓ SGZ ࡢศ໬ಁ㐍స⏝ࡀㄏᑟࡉࢀࡿ [31]ࠋ௨ୖࡼࡾ, VPA ࡣⓎ㐩⚄⤒ẘᛶࢆ᭷ࡋ࡚࠾ࡾ, ᾏ㤿࡟ ࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖ≀㉁࡜ࡋ࡚᪤▱≀㉁࡛࠶ࡿࡀ, Ⓨ㐩ᮇࡢ⬻࡟ᣢ⥆ⓗ࡟᭚㟢ࡉࡏࡿ OECD ࢞࢖ࢻࣛ࢖ࣥࡢ᪉ἲ࡟‽ᣐࡋࡓⓎ㐩⚄⤒ẘᛶヨ㦂࡛ࡢᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢᙳ㡪ࡣࡲࡔ ☜ㄆࡉࢀ࡚࠸࡞࠸ࠋ ➨2 ❶࡛ࡣ, VPA ࢆ࢚ࣆࢤࣀ࣒ẘᛶ≀㉁ࡢ௦⾲࡜ࡋ࡚⏝࠸࡚, OECD ࡢⓎ㐩⚄⤒ẘᛶヨ㦂࢞ ࢖ࢻࣛ࢖ࣥ [63]ࡢ᪉ἲ࡟‽ᣐࡋࡓ, ዷፎᮇ㹼㞳ஙᮇࡲ࡛ࡢⓎ㐩ᮇ᭚㟢ᐇ㦂ࢆࣛࢵࢺ࡛⾜࠸, type-1 ⚄⤒ᖿ⣽⬊ࢆྵࡴᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ┠ⓗࡢࡓࡵ, SGZ ࡟ ࠾ࡅࡿ㢛⢏⣽⬊⣔㆕ᣦᶆ, ṑ≧ᅇ㛛࡟࠾ࡅࡿ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥᣦᶆཬࡧ GCL ࡢࢩࢼࣉ ࢫྍረᛶᣦᶆࡢච␿⤌⧊໬Ꮫⓗゎᯒࢆ⾜࠸, ྛ⣽⬊ࡢศᕸኚືࢆ☜ㄆࡋࡓࠋࡲࡓ, ᾏ㤿࡟ධຊ ࡉࢀࡿᵝࠎ࡞ఏ㐩≀㉁ࡢྜᡂ㓝⣲, ཷᐜయཬࡧᛂ⟅㑇ఏᏊࡢࣜ࢔ࣝࢱ࢖࣒ RT-PCR ゎᯒࢆ⾜ ࠸, ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡬ࡢ㛵୚࡟ࡘ࠸᳨࡚ウࡋࡓࠋ ࡉࡽ࡟, ➨ 1 ❶ྠᵝ࡟, 㞳ஙᚋື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖࡢ᳨ฟᛶࢆ᳨ウࡍ ࡿ┠ⓗ࡛, ⏕ᚋ 5 㐌㱋ࡢࣛࢵࢺ࡟ᑐࡍࡿ VPA ࡢ 28 ᪥㛫཯᚟ᢞ୚ࢆ OECD ࡢ 28 ᪥㛫཯᚟ᢞ୚ ヨ㦂࢞࢖ࢻࣛ࢖ࣥ [64]࡟‽ᣐࡋ࡚⾜࠸, Ⓨ㐩ᮇ᭚㟢ヨ㦂࡜ྠᵝࡢゎᯒ࡟ࡼࡾᾏ㤿࡟࠾ࡅࡿ⚄ ⤒᪂⏕࡬ࡢᙳ㡪࡟ࡘ࠸࡚ẚ㍑᳨࣭ウࡋࡓࠋ

22

ᮦ

ᮦᩱ࠾ࡼࡧ᪉ἲ

໬Ꮫ≀㉁ཬࡧ౪ヨື≀

VPA (CAS No. 99-66-1) ࡣ Sigma-Aldrich Corporation (St. Louis, MO, USA) ࡼࡾ㉎ධࡋࡓࠋⓎ 㐩ᮇ᭚㟢ᐇ㦂࡛ࡣ, ஺㓄ᚋ (ࣉࣛࢢ☜ㄆ᪥ࢆዷፎ 0 ᪥࡜ࡋ࡚㉳⟬) ࡢ Sprague-Dawley ⣔ SPF ࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ఍♫ (ᶓ὾) ࠿ࡽ㉎ධࡋ࡚ᐇ㦂࡟౪ࡋࡓࠋື≀ࡣ ᗘ 23±2ºC, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓ ࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟ 1 ༉ࡎࡘ཰ᐜࡋ, ศፔᚋࡣẕᏊඹ࡟ྠ୍ࢣ࣮ࢪ࡟཰ᐜࡋࡓࠋẕື≀ ࡣዷፎ2 ᪥࠿ࡽศፔᚋ 21 ᪥ (ศፔ᪥ࢆ⏕ᚋ 0 ᪥࡜ࡋ࡚㉳⟬) ࡢ㞳ஙࡲ࡛, ᇶ♏㣫ᩱ CRF-1 (࢜ ࢚ࣜࣥࢱࣝ㓝ẕᕤᴗᰴᘧ఍♫, ᮾி) ࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ㣧ᩱỈࡣዷፎ 6 ᪥┠ࡢ VPA ᢞ୚ 㛤ጞࡲ࡛ࡣỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ㞳ஙᚋࡢᏊື≀ࡣࣉࣛࢫࢳࢵࢡ〇ࢣ࣮ࢪ࡟ 3 ࠿ࡽ 4 ༉ࡎࡘ཰ᐜࡋ, ᇶ♏㣫ᩱ CRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ 㞳ஙᚋ᭚㟢ᐇ㦂࡛ࡣ, 4 㐌㱋ࡢ㞝ᛶ Sprague-Dawley ⣔ SPF ࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ ᰴᘧ఍♫࠿ࡽ㉎ධࡋ࡚ᐇ㦂࡟౪ࡋࡓࠋື≀ࡣ ᗘ 23±2°C, ┦ᑐ‵ᗘ 55±15%, ↷᫂ࢧ࢖ࢡࣝ 12 ᫬㛫᫂/12 ᫬㛫ᬯ᮲௳ࡢ㣫⫱ᐊ࡛, ᗋᩜࢆධࢀࡓࣉࣛࢫࢳࢵࢡ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࢖ࣥࠖ (᪥ᮏᏛ⾡఍㆟, ᖹᡂ 18 ᖺ 6 ᭶ 1 ᪥) ࢆ㑂Ᏺࡋࡓࠋ ᐇ㦂ࢹࢨ࢖ࣥ ձⓎ㐩ᮇ᭚㟢ヨ㦂 ஺㓄ᚋࡢ㞤ࣛࢵࢺࢆ1 ⩌࠶ࡓࡾ 12 ༉ࡎࡘࡢ 4 ⩌࡟↓సⅭ࡟⩌ศࡅࡋ, ዷፎ 6 ᪥┠࠿ࡽศፔ ᚋ21 ᪥┠ࡢ㞳ங᫬ࡲ࡛, VPA ࢆ 0, 667, 2000 ppm ࡢ⃰ᗘ࡛⢭〇Ỉ࡟ΰࡌ, ⮬⏤࡟ᦤྲྀࡉࡏࡓࠋ ᡤᒓ◊✲ᐊ࡛⏝㔞タᐃヨ㦂ࢆᐇ᪋ࡋ, ᢞ୚㔞ࡢタᐃ᰿ᣐ࡜ࡋࡓࠋ༶ࡕ, ⏝㔞タᐃヨ㦂࡟࠾࠸࡚, 2000 ppm ௨ୖࡢ VPA 㣧Ỉᢞ୚࡟ࡼࡾẕື≀ࡢయ㔜ቑຍᢚไࡀ☜ㄆࡉࢀ, 4000 ppm ⩌࡛ࡣᦤ

23 㣵㔞ཬࡧ㣧Ỉ㔞ࡢప್ࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽ, OECD ࡢⓎ㐩⚄⤒ẘᛶヨ㦂࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ ࠸, ẕື≀࡟㍍ᗘ࡞ẘᛶࡀฟࡿ⏝㔞࡜ࡋ࡚᭱㧗⏝㔞ࢆ 2000 ppm ࡜ࡋ㸪බẚ 3 ࡛㝖ࡋࡓ 3 ⏝㔞 ࢆタᐃࡋࡓࠋ 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 ⭡㞝ྛ 8 ༉࡟࡞ࡿࡼ࠺↓సⅭ࡟㑅ᢤࡋ࡚Ꮚᩘࡢㄪᩚࢆ⾜ࡗࡓࠋ1 ⭡ࡢ㞝Ꮚື≀ ࡀ8 ༉࡟‶ࡓ࡞࠸ሙྜࡣ, 1 ⭡ 8 ༉࡜࡞ࡿࡼ࠺࡟㞤Ꮚື≀ࢆṧࡋ࡚ㄪᩚࡋࡓࠋᏊື≀ࡣ, య㔜 ࢆᤵஙᮇ㛫୰ࡣ㐌2 ᅇ, 㞳ஙᚋࡣయ㔜ཬࡧᦤ㣵㔞ࢆ㐌 1 ᅇࡢ㢖ᗘ࡛ ᐃࡋࡓࠋ ẕື≀ࡣ, ฟ⏘ᚋ 21 ᪥┠࡟ CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉ ࡏ๗᳨ࡋ⬻㔜㔞ࢆ ᐃࡋࡓࠋᏊື≀ࡣ, ⏕ᚋ 21 ᪥┠࡟↓సⅭ࡟㑅ᢤࡋࡓྛ⭡㞝ྛ 1–2 ༉ (ྛ ⩌11–12 ༉) ࡢ㞝Ꮚື≀ࡢ⬻ࢆච␿⤌⧊໬Ꮫⓗゎᯒ࡟⏝࠸ࡿࡓࡵ, 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵ ࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿ 10 mL/ศ)ࠋࡉࡽ࡟, ྛ⭡㞝ྛ 2–3 ༉ (ྛ⩌ 24–36 ༉) ࢆ, CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏ๗᳨ࡋ, ⬻ࢆ㑇 ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋྛ⭡ṧࡾࡢᏊື≀ࡣᇶ♏㣫ᩱCRF-1 ཬࡧỈ㐨Ỉࢆ⮬⏤࡟ᦤྲྀࡉࡏ࡚ ⥅⥆㣫⫱ࡋ, య㔜࠾ࡼࡧᦤ㣵㔞ࢆ㐌 1 ᅇ ᐃࡋࡓࠋ⏕ᚋ 77 ᪥┠࡟↓సⅭ࡟㑅ᢤࡋࡓྛ⭡㞝ྛ 1–2 ༉㸦ྛ⩌ 11–12 ༉㸧ࡢ㞝Ꮚື≀ࡢ⬻ࢆච␿⤌⧊໬Ꮫⓗゎᯒ࡟⏝࠸ࡿࡓࡵ, 4% PFA㸭0.1M ࣜ ࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ㸦ὶ㏿ 35 mL/ศ㸧ࠋࡉࡽ࡟, ྛ⭡㞝ྛ 2–3 ༉㸦ྛ⩌24–36 ༉㸧ࢆ, CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉࡏ๗᳨ࡋ, ⬻ࢆ㑇ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋ ๗᳨᫬, ⬻㔜㔞ࢆ ᐃࡋࡓࠋᛶ࿘ᮇ࡟࠾ࡅࡿࢫࢸࣟ࢖ࢻ࣍ࣝ ࣔࣥࡢ࿘ᮇⓗኚື࡟ࡼࡾࢽ࣮ࣗࣟࣥ᪂⏕ࡀᙳ㡪ࢆཷࡅࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽ [67], 㞤ࡢヨᩱࡣಖᏑࡢࡳ࡜ࡋ, ௨㝆ࡢゎᯒࡣᐇ᪋ࡋ࡞࠿ࡗࡓࠋ ໬Ꮫ≀㉁ࡢ᭚㟢᫬ᮇࡣ, OECD ࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ࠸, ╔ᗋ᫬ᮇ (ዷፎ 6 ᪥┠) ࠿ࡽ㞳ங᫬ (⏕ᚋ 21 ᪥┠) ࡜ࡋࡓࠋ๗᳨᫬ᮇࡣ, ᭚㟢ࡀ⤊஢ࡍࡿ㞳ங᫬Ⅼ, ࠾ࡼࡧᛶᡂ⇍ࡀ᏶஢ࡍࡿ᫬ᮇ (⏕ᚋ 77 ᪥┠) ࢆ┠Ᏻ࡟Ỵᐃࡋࡓࠋࡇࡢ㸰Ⅼ࡛ࡢ᳨ウࢆ⾜࠺ࡇ࡜࡛, Ⓨ㐩⚄⤒ẘᛶࡢྍ㏫ᛶࢆ ᳨ウࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿࠋ ղ㞳ஙᚋᭀ㟢ヨ㦂

24 1 㐌㛫ࡢ㥆໬㣫⫱ᚋ, 5 㐌㱋ࡢື≀ࢆ 1 ⩌ᙜࡓࡾ 20㹼24 ༉ࡎࡘࡢ 4 ⩌࡟↓సⅭ࡟⩌ศࡅࡋ, 0, 200, 800, 900 mg/kg ࡢ⏝㔞࡛ VPA ࢆ 28 ᪥㛫ᙉไ⤒ཱྀᢞ୚ࡋࡓࠋᡤᒓ◊✲ᐊ࡛⏝㔞タᐃヨ㦂ࢆ ᐇ᪋ࡋ, ᢞ୚㔞ࡢタᐃ᰿ᣐ࡜ࡋࡓࠋ༶ࡕ, ⏝㔞タᐃヨ㦂࡟࠾࠸࡚, 1000 mg/kg ࡢ VPA ᙉไ⤒ཱྀ ᢞ୚࡟ࡼࡾᢞ୚ື≀ࡢṚஸࡀ☜ㄆࡉࢀࡓࡀ, ࡑࡢᚋ, ⏝㔞ኚ᭦ࡋࡓ 700 mg/kg ࡛ࡣṚஸࡣ☜ ㄆࡉࢀ࡞࠿ࡗࡓࡇ࡜࠿ࡽ, ᭱㧗⏝㔞ࢆ 900 mg/kg ࡜ࡋ࡚タᐃࡋࡓࠋᢞ୚ᮇ㛫୰, య㔜ཬࡧᦤ㣵 㔞ࢆ㐌࡟1–2 ᅇ ᐃࡋࡓࠋ᭱⤊ᢞ୚⩣᪥࡟, ↓సⅭ࡟㑅ᢤࡋࡓྛ⩌ 11 ༉ࡢ⬻ࢆච␿⤌⧊໬Ꮫ ⓗゎᯒ࡟⏝࠸ࡿࡓࡵ, 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࡟ࡼࡿ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿ 35 mL/ศ)ࠋṧࡾࡢື≀ࡣ CO2/O2㯞㓉ୗ࡛⭡኱ື⬦ษ᩿࡟ࡼࡿᨺ⾑⮴Ṛ࡟ࡼࡾᏳᴦṚࡉ ࡏ๗᳨ࡋ, ⬻ࢆ㑇ఏᏊⓎ⌧ゎᯒ࡟౪ࡋࡓࠋ๗᳨᫬, ⬻㔜㔞ࢆ ᐃࡋࡓࠋ ⬻ ⬻࡟࠾ࡅࡿච␿⤌⧊ᏛⓗᰁⰍཬࡧ࢔࣏ࢺ࣮ࢩࢫ᳨ฟ 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ℺ὶᅛᐃࢆ⾜ࡗࡓฟ⏕ᚋ 21 ᪥ཬࡧ 77 ᪥ࡢ㞝Ꮚ ື≀ཪࡣ㞳ஙᚋ᭚㟢ື≀࠿ࡽ⬻ࢆ᥇ྲྀࡋ, ྠࡌᅛᐃᾮࢆ⏝࠸୍࡚᫨ኪᅛᐃࡋࡓࠋ኱⬻ࡢ bregma ࡢᚋ᪉⣙-3.0 mm ཪࡣ-3.5 mm ࡢ 1 ࢝ᡤ࡛ෙ≧๭㠃ࢆస〇ࡋ࡚, ࡉࡽ࡟ 4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࢓࣮ (pH 7.4) ࣂࢵࣇ࢓୍࣮࡛᫨ኪᅛᐃࡋࡓࠋ๓ᚋࡢᑐ⛠㠃 (2 ษ㠃) ࡀⷧษ㠃 ࡜࡞ࡿࡼ࠺࡟ࣃࣛࣇ࢕ࣥໟᇙࡋ, 3 μm ཌࡢ㐃⥆ษ∦ࢆస〇ࡋࡓࠋษ∦ࡣ Table 2-11 ࡟♧ࡋࡓ᮲ ௳࡛௨ୗࡢྛศᏊ࡟ᑐࡍࡿᢠయࢆ⏝࠸࡚ච␿ᰁⰍࢆ⾜ࡗࡓࠋ㢛⢏⣽⬊ᒙ࡟࠾ࡅࡿ⚄⤒᪂⏕ࡢ

ศ໬ࢫࢸ࣮ࢪᣦᶆ࡛࠶ࡿglial fibrillary acidic protein (GFAP) [42], SRY box 2 (SOX2) [77], T box

brain 2 (TBR2) [53], Brain lipid-binding protein (BLBP), doublecortin (DCX) [42, 53], Neuron specific Nuclea (NeuN) [42, 53], ௓ ᅾࢽ ࣮ࣗ ࣟࣥ ࡢ ᣦ ᶆ࡛ ࠶ࡿ [24, 26], reelin (RELN), parvalbumin (PVALB), calbindin-D-28K (CALB1), calbindin-D-29K (Calretinin, CALB2), glutamate decarboxyrase, ᡂ⇍ࢽ࣮ࣗࣟࣥࡢᣦᶆ࡛࠶ࡿ NeuN, ⣽⬊ቑṪάᛶࡢᣦᶆ࡛࠶ࡿ proliferating cell nuclear antigen (PCNA), immediate early gene ࡛࠶ࡿ FBJ osteosarcoma oncogene (FOS), activity-regulated cytoskeleton-associated protein (ARC) ཬࡧ cyclooxygenase-2 (COX2) [27, 59, 61]

࡟ࡘ࠸࡚ᐇ᪋ࡋࡓࠋࢩࢢࢼ᳨ࣝฟࡣ VECTASTAINט _{Elite ABC Kit (Vector Laboratories Inc.,}

25

DAB/H2O2ࢆ⏝࠸࡚ྍど໬ࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࡋࡓࠋ⬺ࣃࣛࣇ࢕ࣥࡣ 0.3

v/v %㐣㓟໬Ỉ⣲Ỉࢆྵࡴ࣓ࢱࣀ࣮ࣝ⁐ᾮ (ᐊ , 30 ศ) ࢆ⏝࠸ࡓࠋᾏ㤿ṑ≧ᅇࡢ SGZ ࡟࠾ࡅ

ࡿ࢔࣏ࢺ࣮ࢩࢫࡢホ౯࡟ࡘ࠸࡚ࡣ㸪TUNEL ἲ (Apop Tag® Peroxidase in Situ apoptosis detection

kit㸪Millipore Corporation㸪Billerica㸪MA㸪USA) ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ᳨࡚ ฟࢆ⾜ࡗࡓࠋᶆᮏࡣ࣐࣊ࢺ࢟ࢩࣜࣥ࡟ࡼࡾᑐẚᰁⰍࢆ᪋ࡋࡓࠋ

ච

ච␿⤌⧊໬ᏛᰁⰍ࠾ࡼࡧTUNEL ᰁⰍ㝧ᛶ⣽⬊ࡢᐃ㔞ゎᯒ

ᾏ㤿ṑ≧ᅇSGZ/GCL ࡟࠾ࡅࡿ GFAP, SOX2, TBR2, DCX, NeuN, PCNA, TUNEL, ARC ཬࡧ

COX2 㝧ᛶ⣽⬊ᩘ࡟ࡘ࠸࡚, ୧ഃ࡛࢝࢘ࣥࢺࡋ, ༢఩㛗ࡉᙜࡾࡢ㝧ᛶ⣽⬊ᩘࢆ⟬ฟࡋࡓ [Fig. 1-1]ࠋṑ≧ᅇ㛛࡟ศᕸࡍࡿ RELN, PVALB, CALB1, CALB2, GAD67, NeuN 㝧ᛶ⣽⬊࡟ࡘ࠸࡚ࡣ, ୧ഃ࡛࢝࢘ࣥࢺࡋ, ṑ≧ᅇ㛛ࡢ༢఩㠃✚ᙜࡾࡢ㝧ᛶ⣽⬊ᩘࡢ᳨⣴ࢆ⾜ࡗࡓࠋࡇࢀࡽࡢゎᯒࡣ ┣᳨ἲ࡛ᐇ᪋ࡋࡓࠋṑ≧ᅇ㛛࡟࠾ࡅࡿ࢔ࣥࣔࣥゅ CA3 㡿ᇦࡢ㢛⢏⣽⬊ࡣ࢝࢘ࣥࢺ࠿ࡽ㝖እࡋ ࡓࠋᐃ㔞ⓗゎᯒࡢࡓࡵࡢ෗┿᧜ᙳࡣ200 ཬࡧ 400 ಸࡢಸ⋡࡛⾜࠸ゎᯒࡋࡓࠋ 㑇ఏᏊⓎ⌧Ⓨ⌧ゎᯒ ฟ⏕ᚋ21 ᪥ࡢᏊື≀ཬࡧ㞳ஙᚋ᭚㟢ື≀ࡢᾏ㤿ṑ≧ᅇ࡟࠾ࡅࡿ mRNA Ⓨ⌧㔞ࢆࣜ࢔ࣝࢱ ࢖࣒㏫㌿෗࣏࣓࣮ࣜࣛࢮ㐃㙐཯ᛂ (RT-PCR) ࢆ⏝࠸࡚ゎᯒࡋࡓࠋ⬻ᶆᮏࢆ࣓ࢱ࣮࢝ࣥᅛᐃᾮ ࡟ࡼࡾᅛᐃࡋࡓ [4]ࠋᑐ↷⩌ཬࡧ㧗⏝㔞⩌ࡢ࣓ࢱ࣮࢝ࣥᅛᐃ⬻ᶆᮏࢆ⏝࠸࡚, ኱⬻ࡢ bregma ࡢᚋ᪉⣙-3.0 mm ཪࡣ-3.5 mm ࡢ 2 mm ཌษ∦ࡼࡾ⏕᳨ࢺࣞࣃࣥ㸦KAI ࢖ࣥࢲࢫࢺ࣮ࣜࢬᰴᘧ ఍♫, ᒱ㜧㸧ࢆ⏝࠸࡚ᾏ㤿ṑ≧ᅇ㒊ศࢆ᥇ྲྀࡋ, QIAzol (Qiagen, Germany) ཬࡧ RNeasy Mini kit

(Qiagen) ࢆ⏝࠸࡚ total RNA ࢆᢳฟࡋࡓ (N = 6/⩌)ࠋ2 μg ࡢ total RNA ࠿ࡽ SuperScript® _III

Reverse Transcriptase (Thermo Fisher Scientific) ࢆ⏝࠸࡚ cDNA ࢆྜᡂࡋࡓࠋPCR ཯ᛂࡣ

SYBR®_{Green PCR Master Mix (Thermo Fisher Scientific) ࢆ⏝࠸, Step One Plus™ Real-time PCR}

System (Thermo Fisher Scientific) ࡟࡚, 〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚ᐇ᪋ࡋࡓࠋࣉࣛ࢖࣐࣮

ࡣPrimer Express software (Version 3.0; Thermo Fisher Scientific) ࢆ⏝࠸࡚タィࡋ, Table 2-13 ࡟

26

transferase (Hprt1) ࡲࡓࡣ glyceraldehyde 3-phosphate dehydrogenase (Gapdh) ࡢ CT್ࢆྠ᫬࡟⟬

ฟࡋ, 0 ppm ⩌ཪࡣ 0 mg/kg ⩌࡜ࡢ ΔΔCT್ࢆ⟬ฟࡋ࡚ẚ㍑ࡋࡓ [50]ࠋ ⤫ ⤫ィゎᯒ ྛ᭚㟢ヨ㦂ࡢయ㔜࡞ࡽࡧ࡟⮚ჾ㔜㔞, ᦤ㣵㔞, ᦤỈ㔞, ච␿⤌⧊໬ᏛᰁⰍ, TUNEL ᰁⰍ࡟࠾ ࡅࡿ㝧ᛶ⣽⬊ࡢ࢝࢘ࣥࢺᩘ, 㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝࡣ⩌ᖹᆒཬࡧᶆ‽೫ᕪࢆ⟬ฟࡋࡓࠋⓎ㐩ᮇ ᭚㟢ヨ㦂ࡢゎᯒ⤖ᯝ࡟ࡘ࠸࡚ࡣẕື≀ࡈ࡜࡟ᖹᆒ್ࢆ⟬ฟࡋ, ࡉࡽ࡟⩌ᖹᆒཬࡧᶆ‽೫ᕪࢆ ⟬ฟࡋࡓࠋ⤫ィᏛⓗゎᯒࡣ, య㔜, ᦤ㣵㔞, ᦤỈ㔞, ⮚ჾ㔜㔞, 㝧ᛶ⣽⬊ᩘ, ྛ⩌ࡢศᩓࢆ Bartlett ࡢ᪉ἲ᳨࡛ᐃࡋ, ➼ศᩓࡢሙྜࡣ Dunnett, ୙➼ศᩓࡢሙྜࡣ Steel ࡢ᪉ἲ࡟ࡼࡾᑐ↷⩌

࡜ྛ⩌࡜ࡢ᳨ᐃࢆ⾜ࡗࡓࠋ2 ⩌㛫ࡢẚ㍑࡟࠾࠸࡚ࡣྛ⩌ࡢศᩓࢆ F ᳨ᐃ࡟ࡼࡾẚ㍑ࡋ, ➼ศᩓ

ࡢሙྜࡣStudent ࡢ t ᳨ᐃ, ୙➼ศᩓࡢሙྜࡣ Aspin-Welch ࡢ t ᳨ᐃ࡟ࡼࡾᑐ↷⩌࡜ྛᢞ୚⩌࡜

ࡢ᳨ᐃࢆ⾜ࡗࡓࠋࡍ࡭࡚ࡢ⤫ィᏛⓗゎᯒࡣ࢚ࢡࢭࣝ⤫ィ2010 ࢯࣇࢺ࢙࢘࢔ (♫఍᝟ሗࢧ࣮ࣅ

27

⤖

⤖ ᯝ

ື≀࡬ࡢᙳ㡪 Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶẕື≀ࡢṚஸࡣ࡞࠿ࡗࡓࠋ୍᪉࡛, 0 ཬ ࡧ2000 ppm ⩌࡛ྛ 1 ౛ࡢᮍዷፎື≀ࡀࡳࡽࢀࡓࡓࡵ, ヨ㦂ࢹ࣮ࢱ࠿ࡽ㝖እࡋࡓ [Table 2-1]ࠋ ࡲࡓ, ᭚㟢⩌ࡢ╔ᗋ⑞ᩘ, ฟ⏕ᩘ, 㞝ᛶ⋡࡟㛵ࡋ࡚᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓࠋヨ㦂ᮇ㛫୰, ࠸ࡎ ࢀࡢ᭚㟢⩌࡛ࡶẕື≀ࡢయ㔜࡟᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 2-2]ࠋ⤒⏘ᚋ 2 ᪥┠࡟ 667 ppm ⩌௨ୖ࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 2-3]ࠋ⤒⏘ᚋ 2 ᪥┠ཬࡧ 14 ᪥┠࠿ࡽ 20 ᪥ ┠࡟2000 ppm ⩌࡛㣧Ỉ㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓࠋヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ᭚㟢⩌࡛ࡶ␗ᖖ ࡞Ṍ⾜ࡸ⾜ືࡣ࡞࠿ࡗࡓࠋ⤒⏘ᚋ21 ᪥┠ࡢゎ๗᫬ࡣ, ࠸ࡎࢀࡢ᭚㟢⩌࡛ࡶẕື≀ࡢయ㔜ཬࡧ ⬻㔜㔞࡟᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 2-8]ࠋ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶᏊື≀ࡢṚஸཬࡧ␗ᖖ࡞Ṍ⾜ࡸ⾜ືࡣ ࡞࠿ࡗࡓࠋࡲࡓ, ࠸ࡎࢀࡢ᭚㟢⩌࡛ࡶᏊື≀ࡢయ㔜, ᦤ㣵㔞, 㣧Ỉ㔞࡟᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 2-4 ཬࡧ Table 2-5]ࠋ⏕ᚋ 21 ᪥┠ཬࡧ 77 ᪥┠ࡢゎ๗᫬ࡣ, ࠸ࡎࢀࡢ᭚㟢⩌࡛ࡶẕື≀ࡢ య㔜ཬࡧ⬻㔜㔞࡟᭷ព࡞ኚ໬ࡣ࡞࠿ࡗࡓ [Table 2-9]ࠋ 㞳ஙᚋᭀ㟢ヨ㦂࡛ࡣ, ヨ㦂ᮇ㛫୰࡟࠸ࡎࢀࡢ⩌࡛ࡶື≀ࡢṚஸཬࡧ⫗║ⓗ࡞␗ᖖࡣ࡞࠿ࡗ ࡓࠋᢞ୚6 ᪥┠࡟ 900 mg/kg ⩌࡛, ᢞ୚ 10 ᪥┠࠿ࡽ 28 ᪥┠࡟ 800 mg/kg ⩌௨ୖ࡛య㔜ࡀᑐ↷ ⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 2-6]ࠋᢞ୚ 1 ᪥┠࡟ 900 mg/kg ⩌࡛ᦤ㣵㔞ࡀᑐ↷⩌ࡼࡾ㧗್ࢆ♧ ࡋ, ᢞ୚ 21 ᪥┠࡟ 900 mg/kg ⩌࡛, ᢞ୚ 4 ᪥┠࠿ࡽ 11 ᪥┠࡟ 800 mg/kg ⩌௨ୖ࡛ప್ࢆ♧ࡋ ࡓ [Table 2-7]ࠋᢞ୚ 1 ᪥┠࡟ 800 mg/kg ⩌࡛㣧Ỉ㔞ࡀᑐ↷⩌ࡼࡾ㧗್ࢆ♧ࡋࡓࠋゎ๗᫬ࡣ, 800 mg/kg ⩌௨ୖ࡛య㔜ཬࡧ⬻㔜㔞ࡀᑐ↷⩌ࡼࡾప್ࢆ♧ࡋࡓ [Table 2-10]ࠋ ᭚㟢ື≀ࡢSGZ ཬࡧ GCL ࡟࠾ࡅࡿ㢛⢏⣽⬊⣔㆕ࡢศᕸ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ⏕ᚋ 21 ᪥┠࡟㢛⢏⣽⬊⣔㆕ࡢศᕸኚືࡣ࡞࠿ࡗࡓ [Fig. 2-1]ࠋ୍᪉࡛, ⏕ᚋ77 ᪥┠࡟ 2000 ppm ⩌࡛ NeuN 㝧ᛶ⣽⬊ࡀቑᩘࡋࡓࠋࡲࡓ, ฟ⏕ᚋ 77 ᪥࡟࠾ࡅࡿ NeuN 㝧ᛶ㢛⢏⣽⬊ᒙࡢཌࡉࡀ667 ppm ⩌௨ୖቑຍࡋ, NeuN 㝧ᛶ㢛⢏⣽⬊ᒙࡢ㠃✚ࡀ 2000 ppm ⩌࡛

28 ቑຍࡋࡓ [Table 2-12]ࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 900 mg/kg ⩌࡛ GFAP 㝧ᛶ⣽⬊ࡀቑᩘ, DCX 㝧ᛶ⣽⬊ࡀῶᩘࡋࡓ [Fig. 2-2]ࠋ ᭚ ᭚㟢ື≀ࡢṑ≧ᅇ㛛࡟࠾ࡅࡿᡂ⇍ࢽ࣮ࣗࣟࣥཬࡧ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ⏕ᚋ 21 ᪥࡟, Ӎ667 ppm ⩌࡛ RELN 㝧ᛶ⣽⬊ࡢῶᩘࡀ, 2000 ppm ⩌࡛ PVALB ཬࡧ GAD67 㝧ᛶ⣽⬊ࡀῶᩘࡋࡓ [Fig. 2-3]ࠋ⏕ᚋ 77 ᪥࡟ࡣ, 2000 ppm ⩌࡛ GAD67 㝧 ᛶ⣽⬊ࡀῶᩘࡋࡓࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 900 mg/kg ⩌࡛ NeuN 㝧ᛶ⣽⬊ࡀῶᩘࡋࡓ [Fig. 2-4]ࠋ࠸ࡎࢀࡢᢞ୚⩌ ࡛ࡶ, ௓ᅾࢽ࣮ࣗࣟࣥࡢศᕸኚືࡣ࡞࠿ࡗࡓࠋ ᭚㟢ື≀ࡢSGZ ࡟࠾ࡅࡿ⣽⬊ቑṪάᛶཬࡧ࢔࣏ࢺ࣮ࢩࢫ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ⏕ᚋ 21 ᪥࡟, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, ⣽⬊ቑṪάᛶཬࡧ࢔࣏ࢺ࣮ࢩࢫ ࡢศᕸኚືࡣ࡞࠿ࡗࡓ [Fig. 2-5]ࠋฟ⏕ᚋ 77 ᪥࡟, Ӎ667 ppm ⩌࡛ PCNA 㝧ᛶ⣽⬊ࡀቑᩘࡋࡓࠋ TUNEL 㝧ᛶ⣽⬊ࡢศᕸኚືࡣ࡞࠿ࡗࡓࠋ 㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, ⣽⬊ቑṪάᛶཬࡧ࢔࣏ࢺ࣮ࢩࢫࡢศᕸኚືࡣ ࡞࠿ࡗࡓ [Fig. 2-6]ࠋ ᭚㟢ື≀ࡢGCL ࡟࠾ࡅࡿࢩࢼࣉࢫྍረᛶ㛵㐃ศᏊ Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ⏕ᚋ 21 ᪥࡟, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, ࢩࢼࣉࢫྍረᛶ㛵㐃ศᏊࡢศᕸ ኚືࡣ࡞࠿ࡗࡓ [Fig. 2-7]ࠋฟ⏕ᚋ 77 ᪥࡟, Ӎ667 ppm ⩌࡛ ARC 㝧ᛶ⣽⬊ᩘཬࡧ COX2 㝧ᛶ ⣽⬊ࡀቑᩘࡋࡓࠋFOS 㝧ᛶ⣽⬊ࡢศᕸኚືࡣ࡞࠿ࡗࡓࠋ

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, ࠸ࡎࢀࡢᢞ୚⩌࡛ࡶ, ࢩࢼࣉࢫྍረᛶ㛵㐃ศᏊࡢศᕸኚືࡣ࡞࠿ࡗ ࡓ [Fig. 2-8]ࠋ

29

Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, ௓ᅾࢽ࣮ࣗࣟࣥᣦᶆࡢ Reln ཬࡧ Gad1 ࡢⓎ⌧ࡀቑຍࡋ, Pvalb ࡢⓎ⌧ ࡀῶᑡࡋࡓ௚, NMDA ᆺཷᐜయࡢࢧࣈࢱ࢖ࣉ࡛࠶ࡿ Grin2d ࡢⓎ⌧ࡀῶᑡࡋࡓ [Table 2-14]ࠋ

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 㢛⢏⣽⬊ࡢศ໬ᣦᶆࡢ Dpsyl3 ࡢⓎ⌧ࡀῶᑡ, ௓ᅾࢽ࣮ࣗࣟࣥᣦᶆࡢ

Pvalb ࡢⓎ⌧ࡀῶᑡ, ᖿ⣽⬊ཷᐜయࡢ Kit ࡢⓎ⌧ࡀቑຍ, ࢽࢥࢳࣥᛶ࢔ࢭࢳࣝࢥࣜࣥཷᐜయࢧ

ࣈࢱ࢖ࣉࡢChrnb2 ࡢⓎ⌧ࡀῶᑡ, AMPA ᆺཷᐜయࢧࣈࢱ࢖ࣉࡢ Gria1 ࡢⓎ⌧ࡀቑຍࡋ, Gria3

30

⪃

⪃ ᐹ

Ⓨ㐩ᮇ᭚㟢ヨ㦂࡛ࡣ, 2000 ppm ᢞ୚⩌࡛㞳ங᫬࡟ṑ≧ᅇ㛛࡛ RELN, PVALB, GAD67 㝧ᛶ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡀῶᩘࡋࡓࠋRELN 㝧ᛶ⣽⬊ࡣ 667 ppm ᢞ୚⩌࡛ࡶῶᩘࡋࡓࠋࢡࣉ ࣜࢰࣥࡸࢢࣜࢩࢻ࣮ࣝࡢⓎ㐩ᮇ᭚㟢ࡣ, SGZ ࡢ㢛⢏⣽⬊ࢆᶆⓗ࡜ࡋࡓ⚄⤒᪂⏕㞀ᐖࢆㄏⓎࡋ,

ࡑࢀ࡟⥆࠸࡚ṑ≧ᅇ㛛ࡢGABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥᩘࡀኚືࡍࡿࡇ࡜ࡀぢฟࡉࢀ࡚࠸ࡿ [2, 6]ࠋ

ࡋ࠿ࡋ, ௒ᅇࡢ VPA ࡢⓎ㐩ᮇ᭚㟢⤖ᯝ࡛ࡣ, ᡂ⇍ᚋ࡟ SGZ/GCL ࡢ PCNA 㝧ᛶቑṪ⣽⬊, NeuN 㝧ᛶᡂ⇍㢛⢏⣽⬊, ཬࡧ ARC ཬࡧ COX2 㝧ᛶ⣽⬊ࡀ 667 ppm ௨ୖࡢ᭚㟢⩌࡛ቑᩘࡋ࡚࠸ࡿࠋ ࡇࢀࡽࡢ⤖ᯝ࠿ࡽ, VPA ࡢⓎ㐩ᮇ᭚㟢ࡣ᭚㟢⤊஢᫬࡛࠶ࡿ㞳ங᫬࡟ࡣ୺࡟ GABA ᛶ௓ᅾࢽࣗ ࣮ࣟࣥࢆᶆⓗ࡜ࡋ, ఇ⸆ᚋࡢᡂ⇍᫬࡟㐜Ⓨⓗ࡞ᙳ㡪࡜ࡋ࡚㢛⢏⣽⬊⣔㆕࡟ᙳ㡪ࢆ⏕ࡌࡉࡏࡿ ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ㢛⢏⣽⬊⣔㆕࡬ࡢ㐜Ⓨⓗ࡞ᙳ㡪࡟ࡘ࠸࡚ࡣ௨ୗࡢሗ࿌ࡀ࠶ࡿࠋ ࢝ࣝࢩ࢘ ࣒஺᥮ᅉᏊ࡛ࢩࢼࣉࢫྍረᛶ࡟ᐤ୚ࡍࡿGrf1 㑇ఏᏊࢆᶆⓗ࡜ࡍࡿ࡜, ࣐࢘ࢫᾏ㤿ṑ≧ᅇࡢ๓ 㥑⣽⬊ཬࡧᮍ⇍㢛⢏⣽⬊ࡢῶᩘࡀ1 ࠿᭶㱋࡛ࡣࡳࡽࢀࡎ, 2 ࠿᭶㱋࡛ࡳࡽࢀࡿ [18]ࠋࡲࡓ, ᐇ 㦂ⓗ࡟㞳ங᫬࡟ẕぶ࡜㞳ࢀࡿࡇ࡜࡟ࡼࡿࢫࢺࣞࢫࡀ, ࢚ࣆࢪ࢙ࢿࢸ࢕ࢵࢡ࡞ไᚚࢆ௓ࡋ࡚ᡂ ⇍ᚋࡢᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡟ᙳ㡪ࡍࡿࡇ࡜ࡀぢฟࡉࢀ࡚࠸ࡿ [80]ࠋࡇࡢࡼ࠺࡞ሗ࿌ࡀ࠶ࡿ ࡇ࡜࠿ࡽ, VPA ࡢⓎ㐩ᮇ᭚㟢ࡀ㢛⢏⣽⬊⣔㆕࡬ࡢ┤᥋ⓗ࡞࢚ࣆࢪ࢙ࢿࢸ࢕ࢵࢡ࡞ไᚚࢆ௓ࡋ ࡚ᾏ㤿࡟࠾ࡅࡿ⚄⤒᪂⏕࡟ᙳ㡪ࢆ୚࠼࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ ⮬㛢⑕ᝈ⪅ࡢ኱⬻⓶㉁࡛GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥධຊࡢῶᑡࡀࡳࡽࢀ࡚࠸ࡿ [85]ࠋ⮬㛢⑕

ࢫ࣌ࢡࢺ࣒ࣛ㞀ᐖࡢ࣑࣮ࣗࢱࣥࢺື≀, ౛࠼ࡤ Fragile X, Rett ⑕ೃ⩌, Angelman ⑕ೃ⩌, Dravet ⑕ೃ⩌, ࡸ VPA ㄏⓎ⮬㛢⑕ࣔࢹࣝື≀࡛ࡣ, ኱⬻⓶㉁, ᡥ᱈య, ᾏ㤿࡛ GAD67 ཬࡧ PVALB 㝧ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢῶᩘࡀ☜ㄆࡉࢀ࡚࠸ࡿ [15]ࠋ ࡲࡓ, VPA ㄏⓎࣔࢹ࡛ࣝࡣ, ᑠつᶍᢚไ ᛶࢩࢼࣉࢫᚋ㟁ὶࡢ㢖ᗘࡢపୗ, ᢚไᛶࢩࢼࣉࢫᚋ㟁ὶࢆ㟁Ẽⓗ࡟ㄏⓎࡍࡿῶ⾶᫬㛫ࡢపୗ, ኱⬻⓶㉁ 2/3 ᒙ࡟࠾ࡅࡿ⣽⬊ࡢධຊཬࡧฟຊࡢపୗࡶࡳࡽࢀࡿ [8]ࠋGABA సື⣔ࡢയᐖࡣ, ឤ᝟, ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࡸ♫఍ᛶ࡜࠸ࡗࡓ⮬㛢⑕ࢫ࣌ࢡࢺ࣒ࣛ㞀ᐖࡢ᰾࡜࡞ࡿࢩࢼࣉࢫࡢ ⯆ዧ/ᢚไࡢ๭ྜࢆቑຍࡉࡏࡿ [8]ࠋRELN 㝧ᛶ⣽⬊࡟㛵ࡋ࡚ࡶ, ⮬㛢⑕ᝈ⪅ࡢ኱⬻⓶㉁࡛ῶᩘ ࡋ࡚࠸ࡿࡇ࡜ [11], ᶵ⬟㞀ᐖࡀ⮬㛢⑕࡛ࡳࡽࢀࡿᵓ㐀ཬࡧ⾜ື␗ᖖ࡟ᐤ୚ࡍࡿྍ⬟ᛶࡀሗ࿌

31 ࡉࢀ࡚࠸ࡿ [23]ࠋࡇࢀࡽࡢሗ࿌ࡣ, ௒ᅇࡢ VPA Ⓨ㐩ᮇ᭚㟢࡛☜ㄆࡉࢀࡓṑ≧ᅇ㛛㒊ࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢῶᩘࢆᨭᣢࡍࡿ⤖ᯝ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ ௒ᅇࡢVPA Ⓨ㐩ᮇ᭚㟢࡛ࡣ, 㞳ங᫬࡟ṑ≧ᅇ㛛ࡢ RELN 㝧ᛶ⣽⬊ࡢῶᩘ࡜ᑐ↷ⓗ࡟, ᾏ㤿 ṑ≧ᅇ࡛Reln 㑇ఏᏊࡢⓎ⌧ࡀቑຍࡋ࡚࠸ࡓࠋRELN ࡣ, ᾏ㤿ṑ≧ᅇ࡛, 㢛⢏⣽⬊ࡢ⛣ືࢆㄪ⠇ ࡋ, ṇᖖ࡞㢛⢏⣽⬊⣔㆕ࡢ⤫ไ࡟㛵୚ࡍࡿ [26]ࠋRELN ࡢධຊࡣ, Vldlr ࡸ Dab1 ࡀⓎ⌧ࡍࡿཷ ᐜయࢆ௓ࡋ࡚ఏ㐩ࡉࢀ, ⮬㛢⑕ᝈ⪅࡛ࡣ Vldlr 㑇ఏᏊࡢⓎ⌧ቑຍࡸ Dab1 㑇ఏᏊࡢⓎ⌧ῶᑡࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ [23]ࠋࡋ࠿ࡋ, ௒ᅇࡢ VPA Ⓨ㐩ᮇ᭚㟢࡛ࡣ Vldlr ࡸ Dab1 㑇ఏᏊࡢⓎ⌧ኚືࡣ ☜ㄆࡉࢀ࡞࠿ࡗࡓࠋ୍᪉࡛, VPA ᭚㟢ࡋࡓ࣐࢘ࢫࡢ๓⬻࡛ࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢ࣓ࢳࣝ໬ῶᑡ࡟ ࡼࡾ Reln 㑇ఏᏊࡢⓎ⌧ࡀቑຍࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [19]ࠋ௒ᅇࡢ VPA Ⓨ㐩ᮇ᭚㟢ࡢ⤖ ᯝ࡛ࡣ, ṑ≧ᅇ㛛ࡢ RELN 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋ, Reln 㑇ఏᏊⓎ⌧ࡀቑຍࡋ࡚࠸ࡿࡇ࡜࠿ࡽ, ῶ ᩘࡋࡓRELN 㝧ᛶ⣽⬊ࡢ Reln 㑇ఏᏊⓎ⌧ࡀቑຍࡋ, 㢛⢏⣽⬊⣔㆕ࡢ⛣ືࡸ⤫ไᶵ⬟ࢆಖᣢࡋ ࡚࠸ࡓࡇ࡜ࡀ⪃࠼ࡽࢀࡓࠋࡲࡓ, RELN ྠᵝ࡟, 㞳ங᫬࡟ṑ≧ᅇ㛛ࡢ GAD67 㝧ᛶ⣽⬊ࡢῶᩘ ࡜ᑐ↷ⓗ࡟, ᾏ㤿ṑ≧ᅇ࡛ Gad1 㑇ఏᏊࡢⓎ⌧ࡀቑຍࡋ࡚࠸ࡓࠋGAD67 ࡣ, GABA ࡢྜᡂ㓝⣲ ࡛࠶ࡾ, GABA ࡣṑ≧ᅇ㛛ࡢ௓ᅾࢽ࣮ࣗࣟࣥ㛫ࡢࢩࢢࢼࣝఏ㐩ࢆᢸࡗ࡚࠾ࡾ, GABA 㔞ࡣ GAD67 ࡢᏳᐃᛶཬࡧⓎ⌧ㄪ⠇࡟ࡼࡗ࡚, ⢭ᐦ࡟ไᚚࡉࢀ࡚࠸ࡿ [69]ࠋ୍᪉࡛, Reln 㑇ఏᏊ࡜ ྠᵝ࡟, VPA ᭚㟢ࡋࡓ࣐࢘ࢫࡢ๓⬻࡛ࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢ࣓ࢳࣝ໬ῶᑡ࡟ࡼࡾ Gad1 㑇ఏᏊࡢ Ⓨ⌧ࡀቑຍࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [19]ࠋᚑࡗ࡚, VPA ࡢⓎ㐩ᮇ᭚㟢࡟ࡼࡾ Gad1 㑇ఏᏊࡢ Ⓨ⌧ࡀቑຍࡋ, GABA ⏘⏕㔞ࡀቑ࠼ࡓࡓࡵ, GAD67 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓ࡜⪃࠼ࡽࢀࡓࠋ HDAC2 ࡣ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥ࡟Ⓨ⌧ࡋ, Ⓨ㐩ᮇࡢᑠ⬻࡛ࢽ࣮ࣗࣟࣥࡢ⛣ືཬࡧᡂ⇍ࢆ ಁ㐍ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [90]ࠋࡲࡓ,ከࡃࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡀ⫾⏕ᮇ࡟኱⬻ ᇶᗏ᰾࡛⏘⏕ࡉࢀ, ᾏ㤿ṑ≧ᅇ࡬ྥ࠿ࡗ࡚⛣ືࡍࡿ [60]ࠋ௒ᅇࡢ᳨ウ࡛ࡣ, VPA ࡢ HDAC ࡢ άᛶࢆ☜ㄆࡋ࡚࠸࡞࠸ࡀ, VPA ࡣ HDAC 㜼ᐖస⏝ࢆ᭷ࡋ࡚࠸ࡿࡇ࡜࠿ࡽ [31], HDAC άᛶࡢ㜼

ᐖࡀⓎ㐩ᮇࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢᾏ㤿࡬ࡢ⛣ືࢆ㞀ᐖࡋ, ࡑࡢ⤖ᯝ, 㞳ஙᚋࡢᾏ㤿ṑ

≧ᅇ࡛GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥᩘࡀῶᑡࡋ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ

㞳ஙᚋ᭚㟢ヨ㦂࡛ࡣ, 900 mg/kg ᭚㟢⩌࡛ SGZ/GCL ࡢ DCX 㝧ᛶ㢛⢏⣽⬊ࡀῶᩘࡋࡓࠋࡉࡽ ࡟, ᾏ㤿ṑ≧ᅇ࡛ࡣᮍ⇍㢛⢏⣽⬊ࡢᣦᶆ࡛࠶ࡿ Dpsyl3 [44] ࡢ㑇ఏᏊⓎ⌧ࡀῶᑡࡋ࡚࠸ࡓࡇ࡜

32 ࠿ࡽ, type-2 ཬࡧ type-3 ๓㥑⣽⬊࡜ᮍ⇍㢛⢏⣽⬊࡬ࡢศ໬ࡀᶆⓗ࡜࡞ࡗ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀ ࡓࠋ୍᪉࡛, ᾏ㤿ṑ≧ᅇ࡛ࡣ Chrnb2 ࡢ㑇ఏᏊⓎ⌧ࡀῶᑡࡋ࡚࠸ࡓࠋᾏ㤿ṑ≧ᅇ㛛ࡢ GABA ᛶ ௓ᅾࢽ࣮ࣗࣟࣥࡣ, ṑ≧ᅇእ㒊࠿ࡽࡢ࢔ࢭࢳࣝࢥࣜࣥసືᛶཬࡧࢢࣝࢱ࣑ࣥ㓟సືᛶධຊࢆ ཷࡅ࡚࠸ࡿ [12, 24]ࠋ⬻ⓑ㉁ࡢ࣑࢚ࣜࣥ࡟✵⬊໬ࢆㄏⓎࡉࡏࡿ HCP ࢆ࣐࢘ࢫⓎ㐩ᮇ᭚㟢ࡉࡏ ࡿࡇ࡜࡛, ᾏ㤿ṑ≧ᅇ㛛㒊ࡢ CHRNB2 㝧ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢῶᩘ࡟㛵㐃ࡋࡓ SGZ ࡢ୰ᮇቑṪ ๓㥑⣽⬊ࡢῶᩘࡀሗ࿌ࡉࢀ࡚࠸ࡿ [36]ࠋGABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡀ CHRNB2 ࢆⓎ⌧ࡋ࡚࠸ࡿ ࡇ࡜ࢆ⪃៖ࡍࡿ࡜ [76], VPA ࡢ㞳ஙᚋ᭚㟢ࡣ CHRNB2 㝧ᛶ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡶᶆⓗࡍ ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡉࡽ࡟, ṑ≧ᅇ㛛㒊ࡢ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢᶵ⬟㞀ᐖࡀ୰ᮇ๓㥑 ⣽⬊ࢆയᐖࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾ [84], GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢ Chrnb2 ࡢ㑇ఏᏊⓎ⌧ ῶᑡࡀ୰ᮇ࠿ࡽᚋᮇ࡟࠿ࡅࡓ㢛⢏⣽⬊ࡢῶᩘࢆㄏⓎࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋᡈ࠸ࡣ, ᾏ㤿 ṑ≧ᅇ㛛㒊ࡢNeuN 㝧ᛶᡂ⇍ࢽ࣮ࣗࣟࣥࡢῶᩘࡣ, CHRNB2 㝧ᛶ GABA ᛶ௓ᅾࢽ࣮ࣗࣟࣥࡢ ῶᩘࡢྍ⬟ᛶࡶ⪃࠼ࡽࢀࡓࠋ୍᪉࡛, ṑ≧ᅇ㛛࡛ࡣ PVLB 㝧ᛶ⣽⬊ࡢῶᩘࡣ࡞࠸ࡀ, ᾏ㤿ṑ≧ ᅇࡢPvalb ࡢ㑇ఏᏊⓎ⌧ࡣῶᑡࡋ࡚࠸ࡓࠋ ᚑࡗ࡚, GABA ᛶ௓ᅾࢽ࣮࡛ࣗࣟࣥࡢ Pvalb 㑇ఏ

ᏊࡢⓎ⌧ῶᑡࡀPVALB 㝧ᛶࢽ࣮ࣗࣟࣥࡢᶵ⬟ࢆᢚไࡋ, ୰ᮇ࠿ࡽᚋᮇ࡟࠿ࡅࡓ㢛⢏⣽⬊ࡢῶ

ᩘࢆㄏⓎࡍࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ

ࡲࡓ, VPA ࡢ㞳ஙᚋ᭚㟢࡟ࡼࡾ 900 mg/kg ᭚㟢⩌࡛ SGZ ࡢ DCX 㝧ᛶ㢛⢏⣽⬊ࡀῶᩘࡋࡓࡢ

࡟ᑐࡋGFAP 㝧ᛶ type-1 ⚄⤒ᖿ⣽⬊ࡀቑᩘࡋࡓࠋṑ≧ᅇ࡛ࡣ, AMPA ᆺࢢࣝࢱ࣑ࣥ㓟ཷᐜయࢆ

ࢥ࣮ࢻࡍࡿGria1 ࡢ㑇ఏᏊⓎ⌧ࡀቑຍࡋ࡚࠸ࡓࠋAMPA ཷᐜయࡣ, ࢢࣝࢱ࣑ࣥ㓟ཷᐜయࣇ࢓࣑

࣮ࣜࡢ୍ࡘ࡜ࡋ࡚▱ࡽࢀ࡚࠾ࡾ, ⯆ዧᛶࡢ⚄⤒ఏ㐩ཷᐜయ࡛࠶ࡾ, ᵝࠎ࡞ṇᖖ⚄⤒άື࡟࠾ ࠸࡚άᛶ໬ࡉࢀࡿ [57]ࠋ៏ᛶⓗ࡞ AMPA ཷᐜయࡢసື⸆ᢞ୚ࡀ㢛⢏⣽⬊⣔㆕ࡢቑṪάᛶࢆቑ ຍࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [9]ࠋᚑࡗ࡚, VPA 㞳ஙᚋ᭚㟢ࡀ, SGZ ࡬ࡢ GRIA1 ࢆ௓ࡋࡓධຊ

࡟ࡼࡿ GFAP 㝧ᛶ type-1 ⚄⤒ᖿ⣽⬊ࡢቑṪάᛶࢆቑຍࡉࡏࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡉࡽ࡟,

VPA ࡢ㞳ஙᚋ᭚㟢࡟ࡼࡾṑ≧ᅇ࡛ࡣ Kit 㑇ఏᏊࡢⓎ⌧ࡶቑຍࡋࡓࠋ࣐࢘ࢫࡢ⬻࡛ࡣ, KIT ࡣ type-1 ⚄⤒ᖿ⣽⬊ཬࡧ type-2 ๓㥑⣽⬊࡟Ⓨ⌧ࡋ⣽⬊ࡢศ໬ࡸቑṪࢆ㧗ࡵ࡚࠸ࡿ [78]ࠋࡲࡓ, ࣛ

ࢵࢺࡢ⬻࡛ࡶⓎ⌧ࡀሗ࿌ࡉࢀ࡚࠾ࡾSCF ࡢᢞ୚࡛ SGZ ࡢቑṪάᛶࢆቑຍࡉࡏࡿࡇ࡜ࡀሗ࿌ࡉ

33 VPA ࡢ㞳ஙᚋ᭚㟢ࡀ SGZ ࡢ⚄⤒ᖿ⣽⬊ࡢ⮬ᕫቑṪࢆቑຍࡉࡏࡿ࡜⪃࠼ࡽࢀࡿࠋSGZ ࡢ PCNA 㝧ᛶ⣽⬊ࡢቑᩘࡣ☜ㄆ࡛ࡁ࡞࠿ࡗࡓࡀ, SGZ ࡛ࡢ type-1 ⚄⤒ᖿ⣽⬊ᩘࡣ๓㥑⣽⬊࡟ẚ࡭ᑡ࡞ ࠸ࡓࡵ, PCNA 㝧ᛶ type-1 ⚄⤒ᖿ⣽⬊ࡢቑᩘࡀࡳࡽࢀ࡞࠿ࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ୍᪉࡛, VPA ࡢ㞳ஙᚋ᭚㟢࡟ࡼࡾṑ≧ᅇ࡛ࡣ Btg2 㑇ఏᏊࡢⓎ⌧ࡣῶᑡࡋࡓࠋBTG2 ࡣ, ⚄⤒ⱆ⣽⬊ࡢ ᭱ᚋࡢ࢔ࢩ࣓ࣥࢺࣜࢵࢡ࡞᭷⣒ศ⿣ࢆㄏᑟࡍࡿ [33]ࠋ᭱㏆ࡢሗ࿌࡛ࡣ, Btg2 ࡢ㑇ఏᏊḞᦆ࣐ ࢘ࢫ࡛ࡣ, ഃ⬻ᐊୗᖏࡢ GFAP 㝧ᛶ⚄⤒ᖿ⣽⬊ࡀቑᩘࡋ, DCX 㝧ᛶ⣽⬊ࡀῶᩘࡋ࡚࠾ࡾ, BTG2 ࡀቑṪᛶ๓㥑⣽⬊ࡢ⣽⬊࿘ᮇ࠿ࡽࡢ⬺༷࡜ศ໬ࢆㄏᑟࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [22]ࠋᚑࡗ ࡚, VPA ࡢ㞳ங᫬᭚㟢࡟ࡼࡿ Btg2 ࡢ㑇ఏᏊⓎ⌧ࡢῶᑡࡣ, SGZ ࡢ GFAP 㝧ᛶ⣽⬊ࢆቑຍࡉࡏ, DCX 㝧ᛶ⣽⬊ࢆῶᑡࡉࡏࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ ௨ୖࡼࡾ, VPA ࡣ᭚㟢᫬ᮇࢆၥࢃࡎ, ⚄⤒᪂⏕ไᚚ⣔ࡢ㞀ᐖᛶࢆ♧ࡋࡓࡀ, Ⓨ㐩ᮇ᭚㟢ࡢ㞳 ங᫬࡛ࡣ㢛⢏⣽⬊⣔㆕࡟ࡣᙳ㡪ࢆ୚࠼ࡎ, Ⓨ㐩ᮇ᭚㟢ࡢᡂ⇍ᚋཬࡧ㞳ஙᚋ᭚㟢ࡢ཯ᛂᛶ࡜␗ ࡞ࡗ࡚࠸ࡓࠋࡇࡢࡇ࡜࠿ࡽ, Ⓨ㐩ᮇ࡜㞳ஙᚋ࡛ࡣ VPA ᭚㟢࡟㉳ᅉࡋࡓไᚚ⣔ධຊࡢኚື࡟ᑐ ࡍࡿ㢛⢏⣽⬊⣔㆕ࡢឤཷᛶࡀ␗࡞ࡿࡶࡢࡢ, ᭚㟢᫬ᮇࢆၥࢃࡎ VPA ࡣ⚄⤒᪂⏕ไᚚ⣔࡟୺࡟ స⏝ࡋ, 㢛⢏⣽⬊⣔㆕ࡢኚື࡟ᑐࡋ࡚࢚ࣆࢤࣀ࣒ẘᛶࡀ㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

34

ᑠ

ᑠ ᣓ

➨2 ❶࡛ࡣ, VPA ࡟ࡼࡿ⚄⤒᪂⏕㞀ᐖࢆ➨ 1 ❶࡜ྠᵝ࡟᳨ウࡋࡓࠋⓎ㐩ᮇ᭚㟢࡛ࡣ, 0, 667 ཬࡧ2000 ppm ࡢ⃰ᗘ࡛ẕື≀࡟ᑐࡋ࡚㣧Ỉᢞ୚ࡋ, Ꮚື≀ࡢ㞳ங᫬ཬࡧᡂ⇍ᚋࡢ⚄⤒᪂⏕࡬ ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝ, 㞳ங᫬࡟ SGZ/GCL ࡢ㢛⢏⣽⬊⣔㆕ࡣኚືࡋ࡞࠿ࡗࡓࡀ, 667 ppm ⩌࠿ࡽṑ≧ᅇ㛛㒊ࡢ RELN, GAD67, PVALB 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓࠋࡲࡓ, ṑ≧ᅇࡢ㑇ఏ ᏊⓎ⌧࡜ࡋ࡚, GABA ྜᡂ㓝⣲ࡀⓎ⌧ቑຍ, NMDA ᆺࢢࣝࢱ࣑ࣥ㓟ཷᐜయࡀⓎ⌧ῶᑡࡋࡓࠋᡂ ⇍ᚋ࡛ࡣ, 2000 ppm ⩌࡛ SGZ/GCL ࡢ PCNA 㝧ᛶቑṪ⣽⬊ᩘ, NeuN, ARC, COX2 㝧ᛶ⣽⬊ᩘࡀ ቑຍࡋ, GAD67 㝧ᛶ⣽⬊ᩘࡢῶᩘཬࡧ NMDA ᆺࢢࣝࢱ࣑ࣥ㓟ཷᐜయࡢ㑇ఏᏊⓎ⌧ࡢῶᑡࡣ㞳 ங᫬࠿ࡽ⥅⥆ࡋࡓࠋḟ࡟, 5 㐌㱋ࡢࣛࢵࢺ࡟ 0, 200 ཬࡧ 900 mg/kg ࡢ VPA ࢆ 28 ᪥㛫ᙉไ⤒ཱྀ ᢞ୚ࡋ, 㞳ஙᚋ᭚㟢࡟ࡼࡿ⚄⤒᪂⏕࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝ, 900 mg/kg ⩌࡛ SGZ/GCL ࡛GFAP 㝧ᛶ⣽⬊ᩘࡀቑຍ, DCX 㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓ௚, ṑ≧ᅇ㛛ࡢ NeuN 㝧ᛶ⣽⬊ᩘࡀῶ ᑡࡋࡓࠋࡲࡓ, Pvalb ཬࡧࢽࢥࢳࣥᛶ࢔ࢭࢳࣝࢥࣜࣥཷᐜయ㑇ఏᏊⓎ⌧ࡶῶᑡࡋࡓࠋ௨ୖࡼࡾ, VPA ࡣ᭚㟢᫬ᮇࢆၥࢃࡎ, ࣄࢫࢺࣥࡢ࢔ࢭࢳࣝ໬ࢆ୺ᶵᗎ࡜ࡍࡿ⚄⤒᪂⏕ไᚚ⣔ࡢ㞀ᐖࢆ♧ ࡋࡓࡀ, Ⓨ㐩ᮇ᭚㟢ࡢ㞳ங᫬࡛ࡣ㢛⢏⣽⬊⣔㆕࡟ࡣᙳ㡪ࢆ୚࠼ࡎ, Ⓨ㐩ᮇ᭚㟢ࡢᡂ⇍ᚋཬࡧ 㞳ஙᚋ᭚㟢ࡢ཯ᛂᛶ࡜␗࡞ࡗ࡚࠸ࡓࠋࡇࡢࡇ࡜࠿ࡽ, Ⓨ㐩ᮇ࡜㞳ஙᚋ࡛ࡣ VPA ᭚㟢࡟㉳ᅉࡋ ࡓไᚚ⣔ධຊࡢኚື࡟ᑐࡍࡿ㢛⢏⣽⬊⣔㆕ࡢឤཷᛶࡀ␗࡞ࡿࡶࡢࡢ, ᭚㟢᫬ᮇࢆၥࢃࡎ VPA ࡣ⚄⤒᪂⏕ไᚚ⣔࡟୺࡟స⏝ࡋ, 㢛⢏⣽⬊⣔㆕ࡢኚື࡟ᑐࡋ࡚࢚ࣆࢤࣀ࣒ẘᛶࡀ㛵୚ࡋ࡚࠸ ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

35

➨

➨

3 ❶

HCP ࡢ࣐࢘ࢫⓎ㐩ᮇ᭚㟢࡟ࡼࡿ

࢚ࣆࢤࣀ࣒ẘᛶ㛵㐃㑇ఏᏊࡢ᥈⣴