࢚ࣆࢤࣀ࣒ẘᛶ㛵㐃㑇ఏᏊࡢ᥈⣴
36
⥴⥴ ゝ
࣊࢟ࢧࢡࣟࣟࣇ࢙ࣥ㸦HCP㸧ࡣ, 1960ᖺ௦▼㮯, Ὑࡸ⢝ရ࡞ࡢᢠ⳦ࡋ࡚⏝ࡉ
ࢀ࡚࠾ࡾ, 㜵࢝ࣅࡸ㎰⸆ࡋ࡚ࡶ⏝ࡉࢀ࡚࠸ࡓ [43]ࠋ1950 ᖺ௦ࡽ
1960
ᖺ௦ࡅ࡚3%ࡢ⃰ᗘ࡛ࡢ HCP
ࡢᮍ⇍ඣࡢ࠾㢼࿅࡛ࡢ⧞ࡾ㏉ࡋࡢ⏝ࡀ, ⬻ᖿ⥙ᵝయ࠾ࡅࡿ✵⬊ᙧᡂᛶ⬻㛵㐃ࡍࡿࡇࡸ [74], 㱎ṑ㢮ࡢ⬻ⓑ㉁ࡢ࣑࢚ࣜࣥ㠧ࡀ୰㛫࡛ゎ㞳ࡍࡿࡇ࡛࣑࢚ࣜ
ࣥࡀ✵⬊ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ [49]ࠋࡲࡓ, ࣛࢵࢺ
HCP
ࢆ៏ᛶ᭚㟢ࡉࡏࡿ, ⚄⤒⣽⬊ࡢ㍈⣴࠾࠸࡚⬺㧊࣑࢚ࣜࣥࡀ⧞ࡾ㏉ࡋ⾜ࢃࢀ, ᑡ࡞ࡃ࡞ࡗࡓ⚄⤒⥺⥔ࡀ㍈⣴ࡢ
⬺ⴠࢆㄏⓎࡉࡏࡿࡇࡸ [58], HCP ࢆ⏝࠸ࡓ࣐࢘ࢫⓎ㐩ᮇ᭚㟢ᐇ㦂࡛ࡶ, Ꮚື≀ࡢ⬻࡛࣑࢚
ࣜࣥࡢ✵⬊ࢆ☜ㄆࡉࢀ࡚࠸ࡿࡇࡽ [41], ⌧ᅾ࡛ࡣᆺⓗ࡞⚄⤒ẘ▱ࡽࢀ࡚࠸ࡿࠋ⚄
⤒᪂⏕㞀ᐖᛶ㛵ࡋ࡚ࡣ, HCP ࡢⓎ㐩ᮇ᭚㟢ࡼࡗ࡚, ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢ㢛⢏⣽⬊⣔
㆕ࡢศ୰ᮇࢆᶆⓗࡋࡓയᐖᛶࢆ♧ࡍࡇࡀぢฟࡉࢀ࡚࠾ࡾ, ࢥࣜࣥసືᛶ⚄⤒ཬࡧࢢࣝ
ࢱ࣑ࣥ㓟సືᛶ⚄⤒ࡢ㛵ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [36, 42]ࠋ
DNA
ࡢ㐣࣓ࢳࣝࢆྵࡴከᵝ࡞࢚ࣆࢤࣀ࣒ಟ㣭ኚືࡀ, ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࢆไᚚࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ [79]ࠋ⣽⬊ࡢ⾲⌧ᆺ⥔ᣢࡢࡓࡵࡢ⣽⬊グ᠈ࡋ࡚
DNA
ࡢ࣓ࢳࣝࡢືⓗᛶ㉁ࡀぢฟࡉࢀ࡚࠾ࡾ [16], DNA ࡢ࣓ࢳࣝ㑇ఏᏊⓎ⌧ㄪ⠇ࡢ㛵㐃ᛶࡣ, ┤᥋ⓗ
࡞㌿ㄪ⠇ᅉᏊࡢ
DNA
ࡢ⤖ྜ㜼ᐖࡸ, ࢡ࣐ࣟࢳࣥจ㞟ࡑࢀᘬࡁ⥆ࡃ㑇ఏᏊࢧࣞࣥࢫࢆㄏᑟࡍࡿ
HDAC
ࢆືဨࡍࡿ࣓ࢳࣝDNA
⤖ྜࢱࣥࣃࢡࢆࡋࡓ㛫᥋ⓗ࡞㌿ᢚไࡀぢฟ ࡉࢀ࡚࠾ࡾ, ࡑࡢࡼ࠺࡞DNA
ࡢ࣓ࢳࣝࡣࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢCpG
ࢧࢺ≉ඹ㏻ࡋ࡚☜ㄆࡉࢀ࡚࠸ࡿ [40]ࠋ⚄⤒᪂⏕࠾ࡅࡿ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࡣࡲࡔ࡛᫂࠶ࡿࡀ, ⎔ቃⓗ࡞
せᅉࡀ
DNA
ࡢ࣓ࢳࣝ◚⥢ࢆㄏⓎࡍࡿ࠸࠺◊✲ࡶ㐍ࡵࡽࢀ࡚࠾ࡾ [14], ⚄⤒Ⓨ㐩ᑐࡍࡿ
DNA
ࡢ࣓ࢳࣝࡢ㔜せᛶࡀ᫂☜ࡉࢀ࡚࠸ࡿࠋ࣐࢘ࢫ࠾࠸࡚Ⓨ㐩ᮇࡢ࣐ࣥ࢞ࣥ (Mn) ࡢ᭚㟢ࡼࡾ, type-1⚄⤒ᖿ⣽⬊ࢆྵࡴྍ㏫ᛶࡢᾏ㤿ࡢ⚄⤒᪂⏕㞀ᐖࡀㄏⓎࡉࢀࡿࡇࡀぢฟ ࡉࢀ࡚࠸ࡿࡀ [86], ṑ≧ᅇ࡛ࡢ
CpG
ࣛࣥࢻ࣐ࢡࣟࣞゎᯒࢆᐇࡋࡓ⤖ᯝ, type-1⚄⤒ᖿ⣽⬊ࢆྵࡴ㢛⢏⣽⬊⣔㆕࡛ࡢ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆ♧ࡍ㑇ఏᏊࡀྠᐃࡉࢀ࡚࠸ࡿ [86,
87]ࠋࡋࡋ,
➨2
❶࡛ぢฟࡋࡓࡼ࠺࡞, ⚄⤒᪂⏕ไᚚ⣔ࢆ୰ᚰࡋࡓ㞀ᐖᛶᑐࡍࡿ࢚ࣆࢤ37
ࣀ࣒ẘᛶࡢ㛵ࡘ࠸࡚ࡢሗ࿌ࡣࡲࡔ࡞࠸ࠋ
➨
3
❶࡛ࡣ, ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆࡋࡓ⚄⤒᪂⏕␗ᖖࡢゎ᫂ࢆ┠ⓗࡋ࡚, ࢤࣀ࣒ሗ ࡢ㇏ᐩ࡞࣐࢘ࢫࢆ⏝࠸࡚, ᪤⚄⤒᪂⏕㞀ᐖࢆぢฟࡋ࡚࠸ࡿHCP
ࡢⓎ㐩ᮇ᭚㟢ࢆ⾜࠸, ᾏ㤿 ṑ≧ᅇ࡛ࡢ㐣࣓ࢳࣝ㑇ఏᏊࡢゎᯒࢆ, ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒἲࢆ⏝࠸᳨࡚⣴ࡋࡓࠋࡉࡽ, ぢฟࡉࢀࡓ㐣࣓ࢳࣝ㑇ఏᏊࡘ࠸࡚ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢ㛵㐃ᛶࡘ࠸࡚ࡶ᳨ウ ࡋࡓࠋ
38
ᮦᮦᩱ࠾ࡼࡧ᪉ἲ
Ꮫ≀㉁ཬࡧ౪ヨື≀
HCP (CAS. No. 70-30-4)
ࡣMP Biomedicals
♫ࡼࡾ㉎ධࡋࡓࠋ24༉ࡢዷፎICR
࣐࢘ࢫࢆዷፎ1
᪥┠࡛SLC
ᰴᘧ♫ (ᯇ) ࡼࡾ㉎ධࡋࡓࠋዷፎ࣐࢘ࢫࡣศፔᚋ22
᪥ࡲ࡛, ᗘ23s3r
C,
‵ᗘ55s15%,
↷᫂ࢧࢡࣝ12
㛫᫂/12㛫ᬯ᮲௳࡛ಶู㣫⫱ࡋࡓࠋHCP
ᢞ㛤ጞࡲ࡛ࡣ㣫⫱ᮇ㛫ࢆ㏻ࡌ࡚, ⢊ᮎ
MF (࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗ,
ᮾி) ࠾ࡼࡧ㣧ᩱỈࡢ⮬⏤ᦤྲྀࡉࡏࡓࠋฟ⏕ᚋ
21
᪥ (ฟ⏕᪥ࢆ0
᪥ࡍࡿ) ௨㝆, Ꮚື≀ࡣ3㹼4
༉㸭ࢣ࣮ࢪ࡛, ⢊ᮎMF
࠾ࡼࡧ㣧ᩱỈࡢ⮬⏤ᦤྲྀୗ࡛㣫⫱ࡋࡓࠋ
ᐇ㦂ࢹࢨࣥ
Ⓨ㐩⚄⤒ẘᛶヨ㦂࢞ࢻࣛࣥグ㍕ࡢື≀ᩘཬࡧ᭚㟢ࢫࢣࢪ࣮ࣗࣝᚑ࠸, 㓄࣐࢘ࢫࢆ
ྛ⩌
12
༉ࡢ2
⩌ศࡅ, 0, 100 ppmࡢHCP
ࢆྵࡴ⢊ᮎMF
ࢆዷፎ6
᪥┠ࡽศፔᚋ21
᪥┠ࡲ࡛ᦤྲྀࡉࡏࡓࠋᮏᐇ㦂࡛ࡣฟ⏕ᚋ
3
᪥┠㛫ᘬࡁࢆ⾜࠸, ྛẕື≀8
༉ࡢᏊື≀࡞ࡿࡼ࠺㞝Ꮚື≀ࢆࣛࣥࢲ࣒ࡾᙜ࡚, ㊊ࡍࡿሙྜࡣ㞤Ꮚື≀ࢆ㏣ຍࡋ࡚Ꮚ≀ᩘࢆㄪᩚࡋࡓࠋ ᛶ࿘ᮇ࠾ࡅࡿࢫࢸࣟࢻ࣍ࣝࣔࣥࡢ࿘ᮇⓗኚືࡼࡾࢽ࣮ࣗࣟࣥ᪂⏕ࡀᙳ㡪ࢆཷࡅࡿࡇ
ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇࡽ[67], 㞝Ꮚື≀ࡢࡳࢆゎᯒᑐ㇟ࡋᐇ㦂ࢆ⾜ࡗࡓࠋฟ⏕ᚋ
21
᪥,ྛ⩌
10
༉ࡢ㞝Ꮚື≀ (1༉/ẕື≀) ࢆච⤌⧊Ꮫⓗ᳨ウࡢࡓࡵ, CO2/O
2㯞㓉ୗ࡛ị෭ࡋࡓ4%
PFA㸭0.1M
ࣜࣥ㓟ࣂࢵࣇ࣮ (pH 7.4) ࡼࡾ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿10 mL㸭ศ)ࠋ㑇ఏᏊ
Ⓨ⌧ゎᯒࡢࡓࡵ, 㞝Ꮚື≀ (2㹼3༉/ẕື≀) ࡣ, CO2
/O
2㯞㓉ୗ࡛ᨺ⾑Ṛࡉࡏࡓᚋ, ⬻ࢆ᥇ᮦࡋ ࡓࠋẕື≀ཬࡧ㞤Ꮚື≀ࡣ, ࡑࢀࡒࢀฟ⏘࣭ฟ⏕ᚋ21
᪥CO
2/O
2㯞㓉ୗ࡛ᨺ⾑ࡋࡓࠋṧࡾࡢᏊື≀ࡣ, ฟ⏕ᚋ
77
᪥┠ࡲ࡛HCP
ࢆྵࡲ࡞࠸㏻ᖖ㣫ᩱࡼࡾ㣫⫱ࡋࡓࠋฟ⏕ᚋ77
᪥, ච⤌⧊Ꮫⓗ᳨ウࡢࡓࡵ, ྛ⩌
10
༉ࡢ㞝Ꮚື≀ (1༉/ẕື≀) ࢆCO
2/O
2㯞㓉ୗ࡛ị෭ ࡋࡓ4% PFA㸭0.1M
ࣜࣥ㓟ࣂࢵࣇ࣮ (pH 7.4) ࡼࡾ℺ὶᅛᐃࢆ⾜ࡗࡓ (ὶ㏿10 mL㸭ศ)ࠋ
㑇ఏᏊⓎ⌧ゎᯒࡢࡓࡵ, 㞝Ꮚື≀ (2㹼3༉/ẕື≀) ࡣ, CO2/O
2㯞㓉ୗ࡛ᨺ⾑Ṛࡉࡏࡓᚋ, ⬻ࢆ᥇ᮦࡋࡓࠋື≀ᐇ㦂ィ⏬ࡣ, ᅜ❧Ꮫἲேᮾி㎰ᕤᏛࡢື≀ᐇ㦂⌮ጤဨᥦฟࡋ࡚ᢎ
39
ㄆࢆཷࡅ, ື≀㣫⫱, ⟶⌮࠶ࡗ࡚ࡣ, ᅜ❧Ꮫἲே ᮾி㎰ᕤᏛࡢᐇ㦂ྲྀᢅ࠸⌮つᐃ
ᚑࡗࡓࠋ
DNA
ཬཬࡧRNA
ᢳฟ࣓ࢳࣝゎᯒࡢࡓࡵ, ฟ⏕ᚋ
21
᪥┠ཬࡧ77
᪥┠ࡢ㞝Ꮚື≀ᾏ㤿ṑ≧ᅇ࠾ࡅࡿDNA
ཬࡧRNA
ࢆᢳฟࡋࡓࠋ4% PFA㸭0.1M ࣜࣥ㓟ࣂࢵࣇ࣮ (pH 7.4) ℺ὶᅛᐃࢆ⾜ࢃ࡞࠸㞝Ꮚື≀ࢆᑐ㇟ࡋ࡚, ⬻ᶆᮏࢆ࣓ࢱ࣮࢝ࣥᅛᐃᾮࡼࡾᅛᐃࡋࡓ [4] ࠋ࣓ࢱ࣮࢝ࣥᅛᐃ⬻ᶆᮏࢆ⏝
࠸࡚, ⬻ࡢ
bregma
ࡢᚋ᪉⣙-2.2 mm (⏕ᚋ21
᪥┠) ཬࡧ-2.8 mm (⏕ᚋ77
᪥┠) ࡢ2 mm
ཌษ∦ࡼࡾ⏕᳨ࢺࣞࣃࣥ (KAI ࣥࢲࢫࢺ࣮ࣜࢬᰴᘧ♫, ᒱ㜧) ࢆ⏝࠸࡚ᾏ㤿ṑ≧ᅇ㒊ศࢆ᥇
ྲྀࡋࡓࠋ0ཬࡧ
100 ppm
⩌ࡘ࠸࡚, All-prep DNA/RNA mini kit (Qiagen) ࢆ⏝࠸࡚DNA
ཬࡧtotal RNA
ࢆᢳฟࡋࡓ (N = 6/⩌)ࠋᢳฟࡋࡓDNA
ࡣ, ࣓ࢳࣝᇶ≉␗ⓗḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒ,࣓ࢳࣝ≉␗ⓗᐃ㔞
PCR
ゎᯒཬࡧࣃࣟࢩ࣮ࢡ࢚ࣥࢫゎᯒ⏝࠸ࡓࠋᢳฟࡋࡓRNA
ࡣ, ࣜࣝࢱ࣒
RT-PCR
ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ⏝࠸ࡓࠋ࣓ࢳࣝᇶ≉␗ⓗḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒ
⏕ᚋ
21
᪥┠ࡢᏊື≀ࡽᢳฟࡋࡓࢤࣀ࣒DNA
ࢆ㉸㡢Ἴ◚○⨨ (Bioruptor UCD-250;Cosmo Bio CO. Ltd., Tokyo, Japan)
ࢆ⏝࠸࡚᩿∦ࡋ, EpiXploreTMMethylated DNA Enrichment kit (Clontech Laboratories, Inc., Mountain View, Canada)ࢆ⏝࠸࡚ metyl-CpG binding domain 2 protein (MBD2) ࡼࡿ࣓ࢳࣝ DNA
ࡢ⃰⦰ࢆ⾜ࡗࡓࠋ⥆࠸࡚, DNA NEB®Next ChIP-Seq Library Prep Master Mix Set for Illumina
®(New England Biolabs, Inc., Ipswich, MA, USA)
ࢆ⏝࠸࡚࣓ࢳࣝ
DNA
ࣛࣈࣛࣜࡢస〇ࢆ⾜ࡗࡓࠋࡇࡢ࣓ࢳࣝDNA
ࣛࣈࣛࣜࢆ⏝࠸࡚, MiseqSequencing System (Illumina, Inc., San Diego, CA, USA)
ࡼࡿࢩ࣮ࢡ࢚ࣥࢫゎᯒࢆᐇࡋ, ᚓࡽࢀࡓࢹ࣮ࢱࢆ
Strand NGS next generation sequencing analysis software (Strand Genomics, Inc.,
San Francisco, CA)
ࢆ⏝࠸࡚ゎᯒࡋࡓࠋゎᯒ᮲௳ࡣ, ㌿㛤ጞⅬࡼࡾ1000
ሷᇶᑐୖὶࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࠾ࡅࡿ
CpG
ࢧࢺ࡛, ᑐ↷⩌ẚࡋ5
ಸ௨ୖࡢ࢝࢘ࣥࢺࡀ☜ㄆࡉࢀࡿ㑇ఏᏊࢆᢳฟࡍࡿࡼ࠺タᐃࡋࡓࠋ
40
㐣㐣࣓ࢳࣝೃ⿵㑇ఏᏊⓎ⌧ゎᯒฟ⏕ᚋ
21
᪥ཬࡧ77
᪥┠ࡢᏊື≀ࡢᾏ㤿ṑ≧ᅇ࠾ࡅࡿ࣓ࢳࣝᇶ≉␗ⓗḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡗ࡚㑅ฟࡉࢀࡓ࣓ࢳࣝೃ⿵㑇ఏᏊࡢ
mRNA
Ⓨ⌧㔞ࢆࣜࣝࢱ࣒RT-PCR
ࢆ⏝࠸࡚ゎᯒࡋࡓࠋᢳฟࡋࡓ
2 μg
ࡢtotal RNA
ࡽSuperScript
®III Reverse Transcriptase (Thermo Fisher Scientific) ࢆ⏝࠸࡚ cDNA
ࢆྜᡂࡋࡓࠋPCR ᛂࡣSYBR
®Green PCR Master Mix (Thermo Fisher Scientific) ࢆ⏝࠸, Step One Plus™ Real-time PCR System (Thermo Fisher Scientific)
࡚,
〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝᚑࡗ࡚ᐇࡋࡓࠋࣉ࣐࣮ࣛࡣPrimer Express software (Version 3.0; Thermo Fisher Scientific)
ࢆ⏝࠸࡚タィࡋ, Table 3-1♧ࡋࡓࠋྛ㑇ఏᏊࡢmRNA
Ⓨ⌧㔞ࡣ, ෆᅉᛶࢥࣥࢺ࣮ࣟࣝࡋ࡚hypoxanthine phosphoribosyl transferase (Hprt)
ࡲ ࡓࡣglyceraldehyde 3-phosphate dehydrogenase (Gapdh)
ࡢC
T್ࢆྠ⟬ฟࡋ, 0 ppm⩌ࡢΔΔC
T್ࢆ⟬ฟࡋ࡚ẚ㍑ࡋࡓ [50]ࠋ࣓ࢳࣝ≉␗ⓗᐃ㔞
PCR
ゎᯒ࣓ࢳࣝೃ⿵㑇ఏᏊࡢ୰ࡽ, mRNA Ⓨ⌧㔞ࡢୗ᪉ไᚚࡀ☜ㄆࡉࢀࡓ㑇ఏᏊࡘ࠸࡚, ࣓ ࢳࣝ≉␗ⓗᐃ㔞
PCR
ゎᯒࢆ⾜ࡗࡓࠋ⏕ᚋ21
᪥┠ࡢᏊື≀ࡽᢳฟࡋࡓࢤࣀ࣒DNA
ࢆ㉸㡢Ἴ◚○⨨ (Bioruptor UCD-250) ࢆ⏝࠸࡚᩿∦ࡋ, ⇕ࡼࡾኚᛶࡉࡏࡓࠋࡑࡢᚋ, ࣥࣉࢵࢺ
ࡋ࡚
20㸣ࡢ DNA
ࢆ-20ΥಖᏑࡋ, ṧࡾࢆEpiXplore
TMMethylated DNA Enrichment kit (Clontech Laboratories, Inc.)ࢆ⏝࠸࡚ metyl-CpG binding domain 2 protein (MBD2)
ࡼࡿ࣓ࢳࣝ
DNA
ࡢ⃰⦰ࢆ⾜ࡗࡓࠋDNA
㔞ࢆᥞ࠼ࡓࣥࣉࢵࢺࢧࣥࣉࣝཬࡧ⃰⦰ࢧࣥࣉࣝࢆ⏝࠸࡚, ࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢᶆⓗ
CpG
ࢧࢺࢆྵࡴቑᖜᛂࢆStepOnePlus Real-time PCR System (Thermo Fisher Scientific)
ࢆ⏝࠸࡚⾜࠸, ቑᖜᛂ᭤⥺ࡣPower SYBR
®Green PCR Master Mix (Thermo Fisher Scientific)
ࢆ⏝࠸᳨࡚ฟࡋࡓࠋTable 3-2
♧ࡍ࣓ࢳࣝ≉␗ⓗᐃ㔞PCR
⏝ࡢࣉ࣐࣮ࣛࡣ, Methyl Primer Express software Ver. 1.0 (Thermo Fisher Scientific) ཬࡧ
Primer Express software Ver. 3.0 (Supplementary Table 3; Thermo Fisher Scientific)
ࢆ⏝࠸࡚タィࡋࡓࠋᐃ㔞ࡣ, ẚ㍑
Ct
ἲࢆ⏝࠸࡚⾜࠸, ࣥࣉࢵࢺDNA
࣓ࢳࣝ⃰⦰DNA
ࡢCt
್ࡢᕪࡽ⟬ฟࡋࡓࠋ41
ࣃࣃࣟࢩ࣮ࢡ࢚ࣥࢫゎᯒ
CpG
ࢧࢺࡢ࣓ࢳࣝẚ⋡ࢆPyroMark Q24 pyrosequencing system (Qiagen)ࢆ⏝࠸࡚ゎᯒࡋ
ࡓࠋ⏕ᚋ21
᪥┠ཬࡧ7
᪥┠ࡢᢳฟࡋࡓࢤࣀ࣒DNA 10 ng
ࢆEpiTect
®Plus DNA Bisulfite kit
(Qiagen)ࢆ⏝࠸࡚ࣂࢧࣝࣇࢺኚࡋ,
ࢧ࣮࣐ࣝࢧࢡࣛ̿ࡼࡗ࡚ቑṪࡋࡓࠋࢩ࣮ࢡ࢚ࣥࢫᛂࡣ
PyroMark Gold Q24 reagents (Qiagen)
⏝࠸᳨࡚ฟࡋࡓࠋTable 3-3
♧ࡍࣃࣟࢩ࣮ࢡ࢚ࣥࢫゎᯒ⏝ࡢࣉ࣐࣮ࣛࡢタィཬࡧ
CpG
ࢧࢺࡢ࣓ࢳࣝẚ⋡ࡢゎᯒࡣPyrosequencing Assay Design software Ver. 2.0 (Supplementary Table 4; Qiagen)
ࢆ⏝࠸࡚⾜ࡗࡓࠋච⤌⧊Ꮫⓗゎᯒ
4% PFA㸭0.1M
ࣜࣥ㓟ࣂࢵࣇ࣮ (pH 7.4) ℺ὶᅛᐃࢆ⾜ࡗࡓฟ⏕ᚋ21
᪥ཬࡧ77
᪥ࡢ㞝Ꮚື≀ཪࡣ㞳ஙᚋ᭚㟢ື≀ࡽ⬻ࢆ᥇ྲྀࡋ
,
ྠࡌᅛᐃᾮࢆ⏝࠸୍࡚ኪᅛᐃࡋࡓࠋ⬻ࡢbregma
ࡢᚋ᪉⣙-3.0 mmཪࡣ-3.5 mmࡢ1
࢝ᡤ࡛ෙ≧㠃ࢆస〇ࡋ࡚, ࡉࡽ4% PFA㸭0.1M
ࣜࣥ㓟ࣂࢵࣇ࣮ (pH 7.4) ࣂࢵࣇ୍࣮࡛ኪᅛᐃࡋࡓࠋ๓ᚋࡢᑐ⛠㠃 (2 ษ㠃) ࡀⷧษ㠃
࡞ࡿࡼ࠺ࣃࣛࣇࣥໟᇙࡋ, 3 μmཌࡢ㐃⥆ษ∦ࢆస〇ࡋࡓࠋษ∦ࡣ
Table 3-4
♧ࡋࡓ᮲௳࡛௨ୗࡢྛศᏊᑐࡍࡿᢠయࢆ⏝࠸࡚චᰁⰍࢆ⾜ࡗࡓࠋゎᯒࡣ, 㐣࣓ࢳࣝ㑇ఏᏊࡋ
᳨࡚ฟࡉࢀࡓ
DLX4, DMRT1
ཬࡧPLCB4
ࡢ, GABAᛶᅾࢽ࣮ࣗࣟࣥᣦᶆ࡛࠶ࡿGAD67 [30],
ࢢࣝࢱ࣑ࣥ㓟ཷᐜయࡢࢧࣈࢱࣉ࡛࠶ࡿglutamate metabotropic receptor 1 [GRM1, 55]ཬࡧ
ࢩࢼࣉࢫྍረᛶᣦᶆ࡛࠶ࡿARC [27] ࡘ࠸࡚ᐇࡋࡓࠋࢩࢢࢼ᳨ࣝฟࡣ VECTASTAIN
טElite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA)
ࢆ⏝࠸࡚〇㐀ඖࡢࣉࣟࢺࢥ࣮ࣝᚑࡗ࡚ᐇࡋ, චᛂࡣ
DAB/H
2O
2ࢆ⏝࠸࡚ྍどࡋࡓᚋ, ࣐࣊ࢺ࢟ࢩࣜࣥࡼࡾᑐẚᰁ Ⰽࡋࡓࠋ⬺ࣃࣛࣇࣥࡣ0.3 v/v %㐣㓟Ỉ⣲Ỉࢆྵࡴ࣓ࢱࣀ࣮ࣝ⁐ᾮ(ᐊ , 30
ศ)ࢆ⏝࠸ࡓࠋ ඹⓎ⌧ゎᯒࡢࡓࡵ, ྠ୍ⷧษ㠃ࢆᣢࡘษ∦ࡼࡿゎᯒࢆᐇࡋࡓࠋࢩࢼࣉࢫ㛵㐃㑇ఏᏊⓎ⌧ゎᯒ
ฟ⏕ᚋ
21
᪥ཬࡧ77
᪥┠ࡢᏊື≀ࡢᾏ㤿ṑ≧ᅇ࠾ࡅࡿࢩࢼࣉࢫྍረᛶ㛵㐃㑇ఏᏊࡢ42
mRNA
Ⓨ⌧㔞ࢆࣜࣝࢱ࣒RT-PCR
ࢆ⏝࠸࡚ゎᯒࡋࡓࠋᢳฟࡋࡓ2 μg
ࡢtotal RNA
ࡽSuperScript
®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ඖࡢࣉࣟࢺࢥ࣮ࣝᚑࡗ࡚ᐇࡋ ࡓࠋࣉ࣐࣮ࣛࡣPrimer Express software (Version 3.0; Thermo Fisher Scientific)
ࢆ⏝࠸࡚タィࡋ,Table 3-1
♧ࡋࡓࠋྛ㑇ఏᏊࡢmRNA
Ⓨ⌧㔞ࡣ, ෆᅉᛶࢥࣥࢺ࣮ࣟࣝࡋ࡚hypoxanthine
phosphoribosyl transferase (Hprt)
ࡲࡓࡣglyceraldehyde 3-phosphate dehydrogenase (Gapdh)
ࡢC
T್ࢆྠ⟬ฟࡋ, 0 ppm⩌ࡢ
ΔΔC
T್ࢆ⟬ฟࡋ࡚ẚ㍑ࡋࡓ [50]ࠋ⤫⤫ィゎᯒ
㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝཬࡧච⤌⧊ᏛᰁⰍ࠾ࡅࡿ㝧ᛶ⣽⬊ࡢ࢝࢘ࣥࢺᩘࡣ⩌ᖹᆒཬࡧᶆ
‽೫ᕪࢆ⟬ฟࡋࡓࠋ⤫ィᏛⓗゎᯒࡣ, 㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝཬࡧ㝧ᛶ⣽⬊ᩘࡘ࠸࡚, 2⩌㛫ࡢ ẚ㍑࡛⾜࠸, ྛ⩌ࡢศᩓࢆ
F
᳨ᐃࡼࡾẚ㍑ࡋ, ➼ศᩓࡢሙྜࡣStudent
ࡢt
᳨ᐃ, ➼ศᩓࡢሙྜࡣ
Aspin-Welch
ࡢt
᳨ᐃࡼࡾ0 ppm
⩌ྛᢞ⩌ࡢ᳨ᐃࢆ⾜ࡗࡓࠋ43
⤖⤖ ᯝ
ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡿ㐣࣓ࢳࣝ㑇ఏᏊࡢ᥈⣴
ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࡢ⤖ᯝ, ㌿㛤ጞⅬࡼࡾ
1000 bp
ୖὶࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࡛, 5ಸ ௨ୖࡢ㐣࣓ࢳࣝࢩࢢࢼࣝࢆ᭷ࡍࡿ19
㑇ఏᏊࢆ≉ᐃࡋࡓ [Table 3-5]ࠋᾏ㤿ṑ≧ᅇ㑇ఏᏊⓎ⌧ゎᯒ
ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࡢ⤖ᯝ, 㐣࣓ࢳࣝࢆ♧ࡋࡓ19㑇ఏᏊࡢෆ, Dmrt1, Gsdma3, Mms19,
Vmn1r90, Adgrl3, Dlx4, Fign, Maz, Plcb4ཬࡧReps1ࡢⓎ⌧ࡀ,
⏕ᚋ21᪥┠ࡢᾏ㤿࡛ῶᑡࡋࡓ[Fig. 3-1]ࠋࡑࡢ, Cdkl4, Mafg, Mcur1, Nusap1, Rasal2, Spsb1, Tex14, Trim62ཬࡧVps37aࡢⓎ⌧
ኚືࡣ☜ㄆࡉࢀ࡞ࡗࡓ [Table 3-6]ࠋ
⏕ᚋ77᪥┠࡛ࡣ, Dlx4ࡢⓎ⌧ࡀῶᑡࡋ, FignཬࡧPlcb4ࡢⓎ⌧ࡀቑຍࡋࡓࠋࡑࡢ, Adgrl3,
Dmrt1, Gsdma3, Maz, Mms19, Reps1ཬࡧVmn1r90ࡢⓎ⌧ኚືࡣ☜ㄆࡉࢀ࡞ࡗࡓࠋ
࣓ࢳࣝ≉␗ⓗ
PCR
ἲࡼࡿ㐣࣓ࢳࣝࡢࣂࣜࢹ࣮ࢩࣙࣥゎᯒ㑇ఏᏊⓎ⌧ࡢῶᑡࡀ☜ㄆࡉࢀࡓ
10
㑇ఏᏊࡢෆ, Dlx4, Dmrt1, Plcb4ཬࡧReps1
ࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࡛ࡢ㐣࣓ࢳࣝࢆ☜ㄆࡋࡓ [Fig. 3-2]ࠋ
Adgrl3
ཬࡧVmn1r90
ࣉ࣐࣮ࣛタィࡀᅔ㞴࡛࠶ࡗࡓࡓࡵᐇࡋ࡞ࡗࡓࠋ
ࣃࣟࢩ࣮ࢡ࢚ࣥࢫἲࡼࡿ㐣࣓ࢳࣝሷᇶࡢ≉ᐃ
ࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࡛ࡢ㐣࣓ࢳࣝࡀ☜ㄆࡉࢀࡓ
Dlx4, Dmrt1, Plcb4
ཬࡧReps1
ࡘ࠸࡚, 㐣࣓ࢳࣝሷᇶࡢ≉ᐃࢆ⾜ࡗࡓࠋ⏕ᚋ
21
᪥┠࡛ࡣ, Dlx4ࡢ1
␒ࡢሷᇶ, Dmrt1ࡢ3
␒ཬࡧ5
␒ࡢ ሷᇶ, Plcb4ࡢ3
␒ࡢሷᇶࡢ㐣࣓ࢳࣝࡀ☜ㄆࡉࢀࡓ [Fig. 3-3ཬࡧ3-4]ࠋ
⏕ᚋ
77
᪥࡛ࡣ, Dlx4ࡢ9
␒ཬࡧ16
␒ࡢሷᇶ,Dmrt1
ࡢ1
␒ཬࡧ2
␒ࡢሷᇶࡢ㐣࣓ࢳࣝࡀ☜ㄆࡉࢀࡓࠋ
44
චච⤌⧊ᏛⓗᰁⰍἲࡼࡿศᕸゎᯒࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢ㐣࣓ࢳࣝࡀ≉ᐃࡉࢀࡓ
Dlx4, Dmrt1, Plcb4
ࡘ࠸࡚, ච⤌⧊ᏛⓗᰁⰍἲࡼࡿᾏ㤿ṑ≧ᅇ࡛ࡢศᏊ࡛ࣞ࣋ࣝࡢⓎ⌧ศᕸࢆゎᯒࡋࡓ⤖ᯝ, ࠸ࡎࢀࡢⓎ⌧ศᏊࡶ
ᾏ㤿ṑ≧ᅇ㛛㒊࡛Ⓨ⌧ࡋ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀࡓ [Fig. 3-5]ࠋࡲࡓ, ඹⓎ⌧ゎᯒ࡛ࡣ, ࠸ࡎࢀ
ࡢศᏊࡶ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥᣦᶆ࡛࠶ࡿGAD67
ඹⓎ⌧ࡍࡿ⣽⬊ࡀ☜ㄆࡉࢀࡓࠋ ࡉࡽ, ṑ≧ᅇ㛛࡛ࡢⓎ⌧⣽⬊ᩘࢆゎᯒࡋࡓ⤖ᯝ, ⏕ᚋ21
᪥┠࠾࠸࡚, DLX4, DMRT1,PLCB4
ཬࡧGAD67
Ⓨ⌧⣽⬊ᩘࡀᑐ↷⩌ẚࡋ, ᭷ពῶᑡࡋࡓ [Fig. 3-6]ࠋ⏕ᚋ77
᪥┠࡛ࡣ, ኚືࡣᅇࡋࡓࠋ
PLCB4
㛵ࡋ࡚ࡣ, GRM1ࡢඹⓎ⌧ࡀ☜ㄆࡉࢀࡓ [Fig.3-7]ࠋࡉࡽ, ⏕ᚋ21
᪥┠ࡢGCL
࡛
ARC
Ⓨ⌧⣽⬊ᩘࡀῶᑡࡋࡓࠋ⏕ᚋ77
᪥┠࡛ࡣ, ኚືࡣᅇࡋࡓࠋᾏ㤿ṑ≧ᅇࡢࢩࢼࣉࢫྍረᛶ㛵㐃㑇ఏᏊ
⏕ᚋ21᪥┠࡛ࡣ, Stx4a , Dlg4, Gabbr1ཬࡧGabra1ࡢⓎ⌧ࡀῶᑡࡋࡓ [Table 3-7]ࠋGabbr2,
Gabrb2, Homer1ཬࡧSypࡢⓎ⌧ࡣኚືࡋ࡞ࡗࡓࠋ⏕ᚋ77᪥┠࡛ࡣ, Gabbr2, Gabra1ཬࡧGabrb2
ࡢⓎ⌧ࡀቑຍ, Homer1ཬࡧStx4aࡢⓎ⌧ࡀῶᑡࡋࡓࠋDlg4, Gabbr1ཬࡧSypࡢⓎ⌧ࡣኚືࡋ࡞ࡗࡓࠋ
45
⪃⪃ ᐹ
ᮏ❶࡛ࡣ, HCPࢆⓎ㐩ᮇ᭚㟢ࡋࡓᏊື≀ࡢᾏ㤿ṑ≧ᅇࡽ
DNA
ࢆᢳฟࡋ, 㐣࣓ࢳࣝ㑇ఏ Ꮚࢆḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡗ᳨࡚⣴ࡋࡓ⤖ᯝ, ㌿㛤ጞⅬࡽ1000
ሷᇶᑐୖὶࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࡛㐣࣓ࢳࣝࢆㄏⓎࡋ࡚࠸ࡿ
19
㑇ఏᏊࢆぢฟࡋࡓࠋࡑࡢෆ, 㑇ఏᏊⓎ⌧ࡀపୗࡋ࡚࠸ࡿ㑇ఏᏊࡣ
10
㑇ఏᏊ࠶ࡾ, ࡉࡽ࣓ࢳࣝ≉␗ⓗPCR
ゎᯒཬࡧࣃࣟࢩ࣮ࢡ࢚ࣥࢫゎ ᯒࡼࡾ㐣࣓ࢳࣝሷᇶࡀ≉ᐃ࡛ࡁࡓࡢࡣ, Dlx4, Dmrt1, Plcb4ࡢ3
㑇ఏᏊ࡛࠶ࡗࡓࠋ㐣࣓ࢳࣝࡀㄆࡵࡽࢀࡓ㑇ఏᏊࡢච⤌⧊ᏛⓗᰁⰍἲࡼࡾⓎ⌧ศᕸゎᯒࢆ⾜ࡗࡓ⤖ᯝ,
DLX4, DMRT1, PLCB4
㝧ᛶ⣽⬊ࡣᾏ㤿ṑ≧ᅇ㛛㒊ᒁᅾࡋ, ࣑࣮ࣛษ∦ࢆ⏝࠸ࡓゎᯒࡼࡾ,ࡑࡢࢇࡀ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥᣦᶆ࡛࠶ࡿGAD67
ࢆඹⓎ⌧ࡋ࡚࠸ࡓࠋ࡚ࡢGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡀGAD67
ࢆⓎ⌧ࡋ࡚ࡣ࠸࡞࠸ࡢ࡛ [30], GAD67 ࢆඹⓎ⌧ࡋࡓDLX4,
DMRT1, PLCB4
㝧ᛶ⣽⬊ࡣGABA
ᛶᅾࢽ࣮ࣗࣟࣥᵝࡢ⣽⬊࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ⯆῝࠸ࡇ, ௨๓ࡢ
HCP
Ⓨ㐩ᮇ᭚㟢ࡢሗ࿌࡛ࡣ, ᾏ㤿ṑ≧ᅇ㛛㒊ࡢGABA
ᛶᅾࢽ࣮ࣗࣟࣥᣦᶆ࡛࠶ࡿ
RELN, PVALB, CALB2
Ⓨ⌧⣽⬊ࡣῶᩘࡋ࡚࠸࡞ࡗࡓࡀ [41], DLX4, DMRT1, PLCB4ཬࡧ
GAD67
㝧ᛶ⣽⬊ࡢ⏕ᚋ21
᪥┠࡛ῶᩘࡀࡳࡽࢀࡓࠋHCP
ࡣ, ࣑࢚ࣜࣥ㠧ࢆᶆⓗࡍࡿ࡛࠶ࡾ, Ⓨ㐩ᮇ᭚㟢ࡼࡗ࡚Ꮚື≀ࡢ⬻࡛࣑࢚ࣜࣥࡢ✵⬊ኚᛶࡀ☜ㄆࡉࢀ࡚࠸ࡿ [41]ࠋᾏ㤿ṑ≧ᅇࡢ㢛⢏⣽⬊ࡣ㍈⣴࣑࢚ࣜࣥࢆ᭷ࡋ࡚࠸࡞࠸ࡀ
[46], GABA
ᛶᅾࢽ࣮ࣗࣟࣥࢆጞࡵࡋࡓᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࢆㄪ⠇ࡍࡿእ㒊ࡽࡢ⚄⤒⣽⬊ࡣ㍈⣴࣑࢚ࣜࣥࢆ᭷ࡋ࡚࠸ࡿࡓࡵ [39], HCP ࡢ᭚㟢ࡣᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࢆㄪ⠇ࡍ
ࡿእ㒊ࡽࡢ⚄⤒⣽⬊ࡢ࣑࢚ࣜࣥ㠧ࢆᶆⓗࡋ, ⚄⤒᪂⏕㞀ᐖࢆㄏⓎࡉࡏࡿࡇࡀ᥎ᐹࡉࢀ
ࡿࠋࡇࢀࡲ࡛ࡶ, HCPࡢⓎ㐩ᮇ᭚㟢ࡀᾏ㤿୰㝸᰾⤒㊰ࡽ㢛⢏⣽⬊ࡸ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥᢞᑕࡍࡿࢥࣜࣥసືᛶ⚄⤒ࡢ࣑࢚ࣜࣥ㠧ࢆ✵⬊ࡋ, ⚄⤒ఏ㐩㏿ᗘࢆ㐜ࡃࡍࡿ࠸࠺
ࡇࡀ㆟ㄽࡉࢀ࡚࠸ࡓ[41]ࠋࢢࣝࢱ࣑ࣥ㓟సືᛶ⚄⤒ࡢᶵ⬟㞀ᐖࡶࡲࡓሗ࿌ࡉࢀ࡚࠸ࡿ [41]ࠋ ௨ୖࡼࡾ, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢㄪ⠇⚄⤒⣔ࡢയᐖࡣ, GABAᛶ
ᅾࢽ࣮ࣗࣟࣥࡢ
Dlx4, Dmrt1, Plcb4
㑇ఏᏊࡢ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࡼࡿⓎ⌧ῶᑡࡀ, ┤᥋ⱝ ࡋࡃࡣ㛫᥋ⓗ㛵ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ46
DLX4
ࡣ, distal-less homeobox genesࡢ୍ࡘ࡛࠶ࡾ, Dlx1/Dlx2ࡢ࣑࣮ࣗࢱࣥࢺ࣐࢘ࢫ࡛ࡣ, ⬻ ࡢᵝࠎ࡞㡿ᇦ࡛GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢῶᩘࡀ☜ㄆࡉࢀ࡚࠸ࡿ [66]ࠋࡇࡢῶᩘࡣ, ᮍ⇍࡞
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢ⤊⬻⓶㉁ࡽ⬻⓶㉁ࡢỈᖹ⛣ື㞀ᐖࡀㄏⓎࡉࢀࡿࡇࡀ⪃࠼ࡽࢀ࡚࠸ࡿ [7]ࠋᚑࡗ࡚, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆࡋࡓ
Dlx4
ࡢῶ ᑡࡀ, ᮍ⇍࡞GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢỈᖹ⛣ື㞀ᐖࢆㄏⓎࡉࡏࡓྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ୍᪉࡛, ⫾⏕ᖿ⣽⬊
Dlx4
㑇ఏᏊࢆⓎ⌧ࡉࡏࡿࡇ࡛GABA67
ศᏊࢆⓎ⌧ࡍࡿGABA
ᛶࢽ࣮ࣗࣟࣥᵝࡢ⣽⬊ࡀㄏᑟࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ [83]ࠋᮏ◊✲࡛ࡣ, HCP ࡢⓎ㐩ᮇ᭚㟢
ࡼࡾᾏ㤿ṑ≧ᅇ㛛ࡢ
GAD67
㝧ᛶGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡀῶᩘࡋ࡚࠸ࡿࠋᚑࡗ࡚, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿ
Dlx4
㑇ఏᏊࡢ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࡼࡿⓎ⌧ῶᑡࡀGABA
ᛶࢽ࣮ࣗࣟࣥࡢศࢆᢚไࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋṑ≧ᅇ㛛㒊ࡢ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡣᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࢆㄪ⠇ࡍࡿࢥࣜࣥసືᛶධຊࢆཷࡅ࡚࠾ࡾ [92], HCP ࡢⓎ㐩ᮇ᭚㟢ࡼࡾࢥࣜࣥస
ືᛶධຊཷᐜయ㑇ఏᏊࡢⓎ⌧ῶᑡࡶ☜ㄆࡋ࡚࠸ࡿ [41]ࠋDlx ࣇ࣑࣮ࣜ㑇ఏᏊࡢᶵ⬟ⓗ࡞⚄
⤒᪂⏕ࡢ㛵ಀࡘ࠸࡚, Dlx1/Dlx2ཬࡧ
Dlx5/Dlx6
ࡢ࣑࣮ࣗࢱࣥࢺ࣐࢘ࢫ࡛ࡣ, ഃ⬻ᐊୗᖏ࡛ࡢ⚄⤒᪂⏕࡛ࡢศ␗ᖖࡀ☜ㄆࡉࢀ࡚࠸ࡿ [66]ࠋDLX4 ࡢᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᶵ⬟ⓗ
࡞㛵ࡢሗ࿌ࡣ࡞࠸ࡀ, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿ
DLX4
㝧ᛶGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢῶᩘࡀ⚄⤒᪂⏕ࢆᢚไࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋṑ≧ᅇ㛛ࡢ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡀtype-2
๓ 㥑⣽⬊ࡢቑṪཬࡧศࢆไᚚࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ [84]ࠋᚑࡗ࡚, ௨๓ሗ࿌ࡉࢀ࡚࠸ࡿHCP
ࡢⓎ㐩ᮇ᭚㟢ࡼࡿtype-2a
ཬࡧtype-2b
๓㥑⣽⬊ࡢῶᩘࡣ, DLX4㝧ᛶGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢῶᩘ㉳ᅉࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ
PLCB4
ࡣ, ࣐࢘ࢫᑠ⬻࡛௦ㅰᆺࢢࣝࢱ࣑ࣥ㓟ཷᐜయ࡛࠶ࡿGRM1
ࡢᵝ࡞G
ࢱࣥࣃࢡඹᙺᆺཷᐜయࡢࢭ࢝ࣥࢻ࣓ࢵࢭࣥࢪ࣮ࣕࡋ࡚ࣇ࢛ࢫࣇࢳࢪࣝࣀࢩࢺ࣮ࣝࢽࣜࣥ㓟ࡽࢪࢩ ࢢࣜࢭ࣮ࣟࣝࣀࢩࢺ࣮ࣝ୕ࣜࣥ㓟ࡢ⏕ᡂ⤒㊰ࢆゐ፹ࡍࡿ [55]ࠋᙜ◊✲ᐊࡢࡇࢀࡲ࡛ࡢሗ
࿌࡛, HCPࡢⓎ㐩ᮇ᭚㟢ࡀᾏ㤿ṑ≧ᅇࡢ
Grm1
㑇ఏᏊࡢⓎ⌧ࢆῶᑡࡉࡏࡿࡇࢆ☜ㄆࡋ࡚࠸ࡿ[41]ࠋᮏ◊✲࡛ࡣ,
ṑ≧ᅇ㛛㒊ࡢPLCB4
Ⓨ⌧⣽⬊GRM1
ࡀඹⓎ⌧ࡍࡿࡇࢆ☜ㄆࡋ࡚࠾ࡾ,PLCB4
Ⓨ⌧⣽⬊ᩘࡀṑ≧ᅇ㛛㒊࡛ῶᑡࡋ࡚࠸ࡿࠋࡇࢀࡽࡢ⤖ᯝࡽ, HCP ࡢⓎ㐩ᮇ᭚㟢ࡀGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢGRM1-PLCB4
࢝ࢫࢣ࣮ࢻࢆᶆⓗࡋ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡓࠋࡉ47
ࡽ, ⸆⌮Ꮫⓗ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥⓎ⌧ࡋ࡚࠸ࡿཷᐜయࡢGRM1
ࡸGRM5
ࢆ㜼ᐖࡍࡿࡇ࡛, ࢩࢼࣉࢫྍረᛶࡢᢚไࢆࡋࡓ㢛⢏⣽⬊ࡢ㛗ᮇቑᙉࢆᾘኻࡉࡏࡿࡇࡀሗ࿌ࡉࢀ
࡚࠸ࡿ [28]ࠋᮏ◊✲࡛ࡣ, 㢛⢏⣽⬊ᒙ࡛ࡢ
ARC
Ⓨ⌧⣽⬊ࡀῶᩘࡋ࡚࠸ࡓࠋArc
ࡣ, IEGࡢ୍ࡘ࡛࠶ࡾ⚄⤒ྍረᛶࡢ᭱ึࡢẁ㝵࡛ᶵ⬟ࡍࡿ [27]ࠋࡲࡓ, 㞳ங
Dlg4, Gabbr1, Gabra1
ཬࡧStx4a
࠸ࡗࡓࢩࢼࣉࢫ㛵㐃㑇ఏᏊࡢⓎ⌧ῶᑡࡀ☜ㄆࡉࢀ࡚࠸ࡿࡇࡽ, HCPⓎ㐩ᮇ᭚㟢ࡼࡿࢩࢼࣉࢫྍረᛶࡢపୗࡀ♧၀ࡉࢀࡿࠋ௨ୖࡼࡾ, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡾ, GABAᛶᅾ ࢽ࣮ࣗࣟࣥࡢ
GRM1-PLCB4
ࡢᶵ⬟◚⥢ࡑࢀక࠺ࢩࢼࣉࢫྍረᛶࡢపୗࡀㄏⓎࡉࢀࡿࡇࡀ♧၀ࡉࢀࡓࠋ
DMRT1
ࡣ, DNA⤖ྜࢻ࣓࡛ࣥ࠶ࡿDM
ࢻ࣓ࣥࢆ᭷ࡍࡿࣇ࣑࣮ࣜࡢ୍ࡘ࡛࠶ࡾ, ⏕Ṫ⣽⬊ศࡸ᭷⣒ศཬࡧከ⬟ᛶ㛵ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ [82]ࠋ⫾⏕ᖿ⣽⬊
DMRT1
ࢆ㐣Ⓨ⌧ࡉࡏࡿࡇࡼࡾ, ⚄⤒ᵝࡢ⣽⬊ศࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ [89]ࠋࡋࡋ,
DMRT1
⚄⤒᪂⏕ࡢ㛵㐃ᛶࡘ࠸࡚ࡣ, ࡇࢀࡲ࡛ሗ࿌ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲⤖ᯝࡽ, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿ
DMRT1
ࡢGABA
ᛶᅾࢽ࣮࡛ࣗࣟࣥࡢ࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆࡋࡓⓎ⌧ῶᑡࡣ, GABAᛶᅾࢽ࣮ࣗࣟࣥࡢศᢚไ㛵㐃ࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ
48
ᑠᑠ ᣓ
➨
3
❶࡛ࡣ, ࢚ࣆࢤࣀ࣒ಟ㣭ࡢ୍ࡘ࡛࠶ࡿࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢ㐣࣓ࢳࣝ㉳ᅉࡋࡓ⚄⤒᪂⏕㞀ᐖ㛵ࢃࡿ㑇ఏᏊ᥈⣴ࢆ⾜ࡗࡓࠋ㓄ᚋࡢ㞤࣐࢘ࢫ
HCP
ࢆ0
ཬࡧ100 ppm
ࡢ⃰ᗘ࡛ዷፎ
6
᪥┠ࡽฟ⏘ᚋ21
᪥┠ࡢ㞳ஙࡲ࡛㣧Ỉᢞࡋ, 100 ppm᭚㟢Ꮚື≀ࡢᾏ㤿ṑ≧ᅇ࡛㐣࣓ࢳࣝࢆ♧ࡋࡓ㑇ఏᏊࡘ࠸࡚ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫゎᯒࢆ⏝࠸᳨࡚⣴ࡋࡓࠋ㑇ఏᏊⓎ⌧
ࡀୗ᪉ไᚚࡉࢀࡿ㐣࣓ࢳࣝ㑇ఏᏊࢆೃ⿵㑇ఏᏊࡋ, ࣓ࢳࣝ≉␗ⓗ
PCR
ἲཬࡧࣃࣟࢩ࣮ࢣࣥࢫἲࡼࡿࣂࣜࢹ࣮ࢩࣙࣥゎᯒࢆᐇࡋࡓࠋࡑࡢ⤖ᯝ, 㞳ங࠾࠸࡚㐣࣓ࢳࣝࡋ ࡓ
Dlx4, Dmrt1, Plcb4
ࡢ3
㑇ఏᏊࡀᚓࡽࢀ, ච⤌⧊Ꮫⓗゎᯒࡢ⤖ᯝ, ࡇࢀࡽࡢⓎ⌧ศᏊࡣ ṑ≧ᅇ㛛ࡢGABA
సືᛶᅾࢽ࣮ࣗࣟࣥඹᒁᅾࡍࡿࡇࢆぢฟࡋࡓࠋࡲࡓ, 㞳ங࡛ࡣDLX4, DMRT1
ཬࡧPLCB4
㝧ᛶ⣽⬊ᩘࡀῶᑡࡋࡓࡀ, ᡂ⇍ᚋ࡛ࡣᅇࡋࡓࠋࡉࡽ, 㞳ங࡛ࡣ
GCL
ࡢARC
㝧ᛶᡂ⇍㢛⢏⣽⬊ᩘࡀῶᑡࡋ࡚࠾ࡾ, ᾏ㤿ṑ≧ᅇࡢࢩࢼࣉࢫ㛵㐃㑇ఏᏊࡢⓎ⌧ࡀྍ㏫ⓗኚືࡋ࡚࠸ࡓࠋ௨ୖࡼࡾ, HCPࡢⓎ㐩ᮇ᭚㟢ࡼࡿ⚄⤒᪂⏕㞀ᐖ, DNAࡢ 㐣࣓ࢳࣝࡼࡿ㑇ఏᏊⓎ⌧ኚືࡣṑ≧ᅇ㛛ࡢ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥ⏕ࡌ, 㛫᥋ⓗ⚄⤒᪂⏕㞀ᐖࢆㄏⓎࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ࣓ࢳࣝ㛵㐃ࡋࡓⓎ⌧ኚࡣᅇࡋࡓࡀ, ⚄
⤒᪂⏕ᚋࡢࢩࢼࣉࢫྍረᛶࡢᙳ㡪ࡣᅇࡋ࡞ࡗࡓࡇࡽ㸪࢚ࣆࢤࣀ࣒ಟ㣭ኚືࡼࡿ
ྍ㏫ⓗ࡞ᙳ㡪ࡶ♧၀ࡉࢀࡓࠋ
49
⥲⥲ྜ⪃ᐹ
MNU
ࢆࡋࡓDNA
┤᥋ᦆയ㉳ᅉࡋࡓᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, ᭚㟢ᮇࢆၥࢃࡎ, type-1⚄⤒ᖿ⣽⬊ࡽ
type-3
๓㥑⣽⬊ࡲ࡛ࡢቑṪάᛶࢆక࠺㢛⢏⣽⬊⣔㆕ࢆᶆⓗࡋ ࡓ࣏ࢺ࣮ࢩࢫࢆయࡍࡿ㞀ᐖ࡛࠶ࡿࡇࢆぢฟࡋࡓࠋࡇࡢ⤖ᯝࡣ, MNUࡢDNA
യᐖస⏝㉳ᅉࡍࡿࡶࡢ࡛࠶ࡾ, type-1⚄⤒ᖿ⣽⬊ࡼࡾቑṪάᛶࡢ㧗࠸
type-2
ཬࡧtype-3
๓㥑⣽⬊ࡢ ᪉ࡀയᐖࢆཷࡅࡸࡍࡃ, യᐖࡀ✚ࡋࡓ⤖ᯝ, ࣏ࢺ࣮ࢩࢫ⮳ࡿྍ⬟ᛶࢆ♧ࡋࡓ [25]ࠋࡋࡋ, Ⓨ㐩ᮇ᭚㟢࡛ࡣ㞳ங
type-2
ཬࡧtype-3
๓㥑⣽⬊ࡀቑຍࡋࡓࠋࡇࡢ⤖ᯝࡣ, ⣽⬊യᐖᑐࡍࡿಖㆤཬࡧ௦ൾᛶࡢᛂࡀ, 㢛⢏⣽⬊⣔㆕ࡢቑṪάᛶࡀ㧗࠸Ⓨ㐩ᮇ࡛ࡳࡽࢀࡓ⪃࠼ࡽ
ࢀࡿࠋࡲࡓ, ࡑࡢቑṪάᛶࢆಁ㐍ࡍࡿᶵᗎࡋ࡚
GCL
ࡢᡂ⇍㢛⢏⣽⬊ࡢCOX2
Ⓨ⌧ࡢ㛵ࢆぢฟࡋࡓ [54, 61]ࠋ௨ୖࡼࡾ, Ⓨ㐩ᮇཬࡧ㞳ஙᚋ᭚㟢ࡢᙳ㡪ࡢ㐪࠸ࡣ, ᶆⓗ⣽⬊ࡢቑṪάᛶࡢ
⛬ᗘ౫Ꮡࡋࡓ
MNU
ࡢឤཷᛶࡢ㐪࠸ࢆᫎࡋࡓ⤖ᯝ࡛࠶ࡿุ᩿ࡉࢀࡓࠋ୍᪉࡛, VPA ࢆࡋࡓ࢚ࣆࢤࣀ࣒ಟ㣭ኚື㉳ᅉࡋࡓ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, ᭚㟢ᮇࢆ
ၥࢃࡎ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢ㞀ᐖࢆ♧ࡋࡓࡀ, Ⓨ㐩ᮇཬࡧ㞳ஙᚋ᭚㟢ࡢ㛫࡛㢛⢏⣽⬊⣔㆕ࡢᙳ㡪ࡀ␗࡞ࡗ࡚࠸ࡓࠋ༶ࡕ, Ⓨ㐩ᮇ᭚㟢࡛ࡣ᭚㟢┤ᚋ࡛࠶ࡿ㞳 ங, 㢛⢏⣽⬊⣔㆕ࡢኚືࡀࡳࡽࢀ࡞ࡗࡓࡢᑐࡋ, 㞳ஙᚋ᭚㟢࡛ࡣ᭚㟢ᚋ
type-1
⚄⤒ᖿ⣽⬊ࡢቑຍཬࡧ
type-2, type-3
๓㥑⣽⬊ཬࡧᮍ⇍㢛⢏⣽⬊ࡢῶᑡࡀࡳࡽࢀࡓࠋࡇࡢ⤖ᯝࡣ, 㞳ஙᚋ࡛ࡣ㢛⢏⣽⬊⣔㆕ࡢVPA
ཪࡣ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿGABA
ᛶᅾࢽ࣮ࣗࣟࣥᑐࡍࡿឤཷᛶࡀ␗࡞ࡿࡇࢆ♧၀ࡍࡿࠋ௨ୖࡼࡾ, ᭚㟢ᮇࢆၥࢃࡎ
VPA
ࡣ⚄⤒᪂⏕ไᚚ⣔స⏝ࡋ, ࢚ࣆࢤࣀ࣒ẘᛶࡢᶆⓗᛶࡣ᫂ࡽ࡛ࡣ࡞࠸ࡶࡢࡢ, 㢛⢏⣽⬊⣔㆕ࡢኚືᑐࡍࡿ
┤᥋࡞࠸ࡋ㛫᥋ⓗ࡞㛵ࡀ♧၀ࡉࢀࡓࠋ
MNU
ཬࡧVPA
ࡢ᳨ウ⤖ᯝࡽ, DNA┤᥋ᦆയㄏⓎ≀㉁࢚ࣆࢤࣀ࣒ಟ㣭ኚືㄏⓎ≀㉁ࡢⓎ㐩ᮇ᭚㟢ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ᑐࡍࡿᙳ㡪ࢆẚ㍑ࡍࡿ, ๓⪅࡛ࡣᗈ⠊࡞㢛⢏⣽
⬊⣔㆕ࡢศᕸኚືࡀࡳࡽࢀࡓࡢᑐࡋ, ᚋ⪅࡛ࡣ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢ㞀ᐖࡀ࡞స⏝࡛࠶ࡗࡓ⪃࠼ࡽࢀࡿࠋDNA┤᥋ᦆയ㛵ࡋ࡚, ᨺᑕ⥺↷ᑕࡼࡿᾏ 㤿࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖ࡛ࡣ, 㢛⢏⣽⬊⣔㆕࡛ࡢ┤᥋ⓗ࡞
DNA
യᐖࡀㄆࡵࡽࢀ࡚࠸ࡿࡇ50
ࡽ [52], MNUࡀ㢛⢏⣽⬊⣔㆕ࢆ┤᥋യᐖࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࠋ୍᪉࡛, ࢚ࣆࢤࣀ࣒ಟ㣭 ኚື㛵ࡋ࡚ࡶ, VPA ࡼࡿ㢛⢏⣽⬊⣔㆕࡛ࡢࢭࢳࣝࣄࢫࢺࣥࡢቑຍࡀㄆࡵࡽࢀ࡚࠸ࡿ
ࡇࡽ [31], 㢛⢏⣽⬊⣔㆕ࡢ┤᥋ⓗ࡞࢚ࣆࢤࣀ࣒ಟ㣭ኚືࢆண ࡋࡓࠋࡋࡋ, ᅇࡢ
VPA
Ⓨ㐩ᮇ᭚㟢ࡢ᳨ウ࡛ࡣ, ṑ≧ᅇ㛛ࡢ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢศ ᕸኚືࢆ☜ㄆࡋࡓࡇࡽ, VPAࡢ࢚ࣆࢤࣀ࣒ẘᛶࡀ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿGABA
ᛶᅾࢽ࣮ࣗࣟࣥࡶᶆⓗࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ
Ⓨ㐩ᮇ᭚㟢ࡢఇ⸆ᚋࡢᙳ㡪ࡋ࡚, MNU࡛ࡣᡂ⇍㢛⢏⣽⬊ࡀῶᑡ, VPA࡛ࡣᡂ⇍㢛⢏⣽⬊
ࡀቑຍࡋࡓࠋMNUࡣⓎ㐩ᮇࡢ᭚㟢ࡼࡾᑠ㢌࡞ࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ [3, 45, 65], ୍ ᪉࡛, VPAࡣⓎ㐩ᮇࡢ᭚㟢ࡼࡾ㢌㛵㐃ࡋࡓሗ࿌ࡀ࠶ࡿ [20, 47]ࠋ
MNU
࡛ࡣ, 㞳ஙࡽ⬻㔜㔞ࡢప್, SGZ 㛗ࡢ▷⦰ཬࡧṑ≧ᅇ㠃✚ࡢ⦰ᑠࡀࡳࡽࢀ࡚࠾ࡾ, ⫾⏕ᮇࡢ⬻Ⓨ㐩㐣⛬࡛
ࡢ
MNU
ࡢᙳ㡪ࡼࡾ, ᾏ㤿ᙧᡂࡢ๓ࡢẁ㝵࡛ᾏ㤿ࡢపᙧᡂࡀຓ㛗ࡉࢀ࡚࠸ࡿࠋࡇࡢࡇࡽ, ᾏ㤿௨እࡢ㒊࡛ࡶྠᵝపᙧᡂࡀ㒊ศⓗㄏⓎࡉࢀ, ⤖ᯝⓗᑠ㢌ᵝࡢᵓ㐀࡞ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋఇ⸆ᚋ㢛⢏⣽⬊⣔㆕ࡢᙳ㡪ࡀⓎ⌧ࡋࡓ⌮⏤ࡋ࡚ࡣ, DNAയᐖࡢ✚
ࡼࡿ
p21
Cip1/Waf1ࡢⓎ⌧ቑຍࡼࡿ⣽⬊࿘ᮇṆ⣽⬊ࡢቑຍࡀ⪃࠼ࡽࢀ, SGZ࡛ࡢቑṪάᛶࡢῶᑡඹ㢛⢏⣽⬊ᒙࡢ⳼ⷧࡀ㐍⾜ࡋࡓ⪃࠼ࡽࢀࡿࠋ୍᪉, VPA࡛ࡣ, 㞳ங⬻⓶㉁
ཬࡧᑠ⬻࠾࠸࡚ࡶṑ≧ᅇྠᵝ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡢῶᩘࡀࡳࡽࢀ࡚࠾ࡾ, ⫾⏕ᮇࡢ⬻Ⓨ㐩㐣⛬࡛ࡢ
VPA
ࡢᙳ㡪ࡼࡾ, ᾏ㤿ࡢGABA
ఏ㐩ᶵᵓࡢ㞀ᐖࡀㄏⓎࡉࢀ࡚࠸ࡿࠋࡋࡋ,VPA
᭚㟢୰ࡣVPA
ࡢస⏝ࡼࡾ, GABA⃰ᗘࡀಖᣢࡉࢀ࡚࠸ࡓࡓࡵ, 㢛⢏⣽⬊⣔㆕ࡢᙳ㡪 ࡣ㢧ᅾࡏࡎ, ఇ⸆ᚋGABA
ఏ㐩㞀ᐖࡀ㢧ᅾࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋᚑࡗ࡚, ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕⬻యࡢᵓ㐀ࡢⓎ㐩㛵ࡋ࡚ࡣ┤᥋ⓗ࡞㛵㐃ᛶࡣྰᐃ࡛ࡁࡿࠋࡋࡋ, ឤ ᰁࡼࡾᑠ㢌ࡢⓎሗ࿌ࡀ࠶ࡿࢪ࢝࢘ࣝࢫࢆឤᰁࡉࡏࡓ࣐࢘ࢫࡢ
SGZ
࠾࠸࡚ࡶቑṪά ᛶࡢపୗࡸ࣏ࢺ࣮ࢩࢫࡀ☜ㄆࡉࢀ࡚࠸ࡿࡇ[51], ཬࡧᡂ⇍㢛⢏⣽⬊ᩘࡢኚື⬻ᵓ㐀ࡢⓎ㐩ᗘྜ࠸┦㛵ᛶࡀ࠶ࡿࡇࡽ, ᡂ⇍㢛⢏⣽⬊ᩘࡀ⬻ᙧᡂ␗ᖖࡢᣦᶆ࡞ࡿྍ⬟ᛶࡀ⪃
࠼ࡽࢀࡓࠋ
Ⓨ㐩⚄⤒ẘᛶ≀㉁࡛࠶ࡿ
MNU
ཬࡧVPA
ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, Ⓨ㐩ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛ࡣ, ␗࡞ࡿࣃࢱ࣮ࣥࡢ㢛⢏⣽⬊⣔㆕ࡢኚືࡀ☜ㄆࡉࢀࡓࠋࡇࡢ⤖ᯝࡣ, Ⓨ
51
㐩ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛␗࡞ࡿࣃࢱ࣮ࣥࡢ㢛⢏⣽⬊⣔㆕ࡢኚືࡀ☜ㄆࡉࢀࡓⓎ㐩⚄⤒ẘᛶ≀
㉁ࡢࣉࣟࣆࣝࢳ࢜࢘ࣛࢩࣝྠᵝ࡛࠶ࡗࡓ [73]ࠋࡋࡋ, ࢡࣉࣜࢰࣥཬࡧࢢࣜࢩࢻ࣮ࣝࡢࡼ
࠺࡞᪤▱ࡢᡂ⇍⚄⤒ẘᛶ≀㉁࡛ࡣ, Ⓨ㐩ᮇ᭚㟢ཬࡧ㞳ஙᚋ᭚㟢࠾࠸࡚ྠ୍ࣃࢱ࣮࡛ࣥࡢ⚄
⤒᪂⏕ࡢᙳ㡪ࡀ☜ㄆࡉࢀ࡚࠸ࡿ [1, 2, 5, 6]ࠋⓎ㐩ᮇ᭚㟢ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ᑐࡍ
ࡿᙳ㡪ࡢᶆⓗᛶࢆࡑࢀࡒࢀ⪃࠼ࡿ, ࣉࣟࣆࣝࢳ࢜࢘ࣛࢩࣝཬࡧ
MNU
ࡣtype-1
⚄⤒ᖿ⣽⬊ࢆྵࡴ㢛⢏⣽⬊⣔㆕, VPAࡣṑ≧ᅇ㛛ࡢ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿ
GABA
ᛶᅾࢽ࣮࡛ࣗࣟࣥ࠶ࡿࡢᑐࡋ, ࢡࣉࣜࢰࣥཬࡧࢢࣜࢩࢻ࣮࡛ࣝࡣ㢛⢏⣽⬊⣔㆕ࡢศࡢᚋᮇࢆᶆⓗࡋ࡚࠸ࡿࠋ 㞳ஙᚋࡢᡂࡉࢀࡓ⬻ࡢᾏ㤿࡛ࡣ, type-1⚄⤒ᖿ⣽⬊ࢆྵࡴ㢛⢏⣽⬊⣔㆕ࡢቑṪάᛶࡣ㞳ங๓
ẚప࠸ࡓࡵ [72], ࣉࣟࣆࣝࢳ࢜࢘ࣛࢩࣝཬࡧ
MNU
࡛ࡣࡇࡢቑṪάᛶࡢ㐪࠸ࡼࡾⓎ㐩 ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛␗࡞ࡿࣃࢱ࣮ࣥࡢ㢛⢏⣽⬊⣔㆕ࡢኚື࡞ࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡲࡓ, ṑ≧ᅇ㛛ࡢ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࡣ, type-1⚄⤒ᖿ⣽⬊ᑐࡋ࡚ቑṪ࣭ศࢆಁ㐍ࡉࡏࡿാࡁࡀ࠶ࡿ [75]ࠋࡇࡢࡓࡵ, VPAࡶ
GABA
ᛶᅾࢽ࣮ࣗࣟࣥࢆࡋ࡚
type-1
⚄⤒ᖿ⣽⬊ࢆᶆⓗࡍࡿࡇࡽ, Ⓨ㐩ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛␗࡞ࡿࣃࢱ࣮ࣥࡢ㢛⢏⣽⬊⣔㆕ࡢኚື࡞ࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ୍᪉࡛, ᡂ⇍⚄⤒ẘᛶ≀㉁࡛࠶ࡿࢡࣉࣜ
ࢰࣥཬࡧࢢࣜࢩࢻ࣮࡛ࣝࡣ, 㢛⢏⣽⬊⣔㆕ࡢศࡢᚋᮇࢆᶆⓗࡍࡿࡓࡵ, ቑṪάᛶࡢᙳ㡪 ࡀపࡃ, Ⓨ㐩ᮇ᭚㟢ཬࡧ㞳ஙᚋ᭚㟢࠾࠸࡚ྠ୍ࣃࢱ࣮࡛ࣥࡢ⚄⤒᪂⏕㞀ᐖࢆㄏⓎࡋࡓ⪃
࠼ࡽࢀࡓࠋᚑࡗ࡚, ᭚㟢ᮇࡢ㐪࠸ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕࠼ࡿᙳ㡪ࡘ࠸࡚ࡣ,
type-1
⚄⤒ᖿ⣽⬊ࢆᶆⓗࡍࡿሙྜ࠾࠸࡚␗࡞ࡿࣃࢱ࣮ࣥࡢ⚄⤒᪂⏕㞀ᐖᛶࢆㄏⓎࡍࡿࡶࡢ᥎ᐹࡉࢀࡓࠋ
୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࠾ࡅࡿⓎ㐩⚄⤒ẘᛶࡢ᳨ฟᛶ㛵ࡋ࡚ࡣ, 㞳ஙᚋ᭚㟢࡛⚄⤒᪂⏕
㞀ᐖࢆㄏⓎࡍࡿ≀㉁ࡣ, Ⓨ㐩ᮇ᭚㟢࠾࠸࡚ࡶ⚄⤒᪂⏕㞀ᐖᛶࢆ♧ࡍྍ⬟ᛶࡀ㧗ࡃ, Ⓨ㐩⚄
⤒ẘᛶㄏⓎᛶࡋ࡚ᤊ࠼ࡿࡇࡀྍ⬟࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋᚑࡗ࡚, Ⓨ㐩⚄⤒ẘᛶ≀㉁࡛ࡣ
Ⓨ㐩ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛⚄⤒᪂⏕㞀ᐖᛶࡀ␗࡞ࡿࣃࢱ࣮ࣥࢆ♧ࡋࡓࡶࡢࡢ, 㢛⢏⣽⬊⣔㆕,
⚄⤒᪂⏕ไᚚ⣔ཬࡧ㛵㐃ศᏊཬࡧ㑇ఏᏊࡢኚື࡞ࡢᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕⎔ቃࡢᙳ㡪ࡣ ᑡ࡞ࡽࡎᏑᅾࡋ, ␗࡞ࡿࣃࢱ᳨࣮࡛ࣥฟ࡛ࡁࡿࡓࡵ, ⚄⤒᪂⏕㞀ᐖᛶࢆ㍈ࡋࡓ⌮Ⓨ⏕
᰿ࡊࡋࡓホ౯ࡣྍ⬟ุ᩿ࡉࢀࡓࠋⓎ㐩⚄⤒ẘᛶ≀㉁࡛ࡣ
type-1
⚄⤒ᖿ⣽⬊ࢆᶆⓗࡍࡿ52
ྰ࡛㞀ᐖࡢࣃࢱ࣮ࣥࡀ༊ูࡉࢀࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࡀ, 㞀ᐖࡢ⛬ᗘཬࡧ✀㢮㛵ࡋ࡚
ࡢࢹ࣮ࢱࡀࡲࡔࡲࡔ༑ศ࡛࠶ࡿ, Ⓨ㐩⚄⤒ẘᛶ≀㉁ᑐࡍࡿ⚄⤒᪂⏕ࡢάᛶẘᛶឤཷ
ᛶࡢ㛵ಀ, ྛ✀ไᚚ⣔ࡢᡂ⇍ᗘྜ࠸ࡼࡿឤཷᛶࡢ㐪࠸࡞, ᳨ウࡍࡁ㡯┠ࡀከ࠸ࠋ≉ᗎ ㄽ࡛㏙ࡓ⚄⤒ᖿ⣽⬊ẘᛶࡢᠱᛕࡍࡁⅬࡋ࡚, 㑇ఏᏊࡢᵓ㐀ࡸᶵ⬟ࡢ㞀ᐖࡀ⏕ࡌࡿࡇ
࡛, 㑇ఏᏊⓎ⌧ࣉࣟࢢ࣒ࣛᚑࡗࡓ⚄⤒ศᶵᵓࡢ◚⥢ࡀ⏕ࡌ, ⏕ᚋࡢ⚄⤒୰ᯡࡢ㧗ḟᶵ⬟
ᙧᡂᙳ㡪ࢆ࠼ࡿࡇࡀ⪃࠼ࡽࢀࡿࡓࡵ, ⌮Ⓨ⏕᰿ࡊࡋࡓホ౯グ᠈ࡸ⾜ື㞀ᐖᛶ
ࡢ㛵㐃ᛶࡘ࠸᳨࡚ドࡋ࡚࠸ࡃᚲせࡀ࠶ࡿࠋᚑࡗ࡚, ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࢆ⏝࠸ࡓⓎ㐩⚄
⤒ẘᛶホ౯ࡣ, ⌧ẁ㝵࡛ࡣ㧗ḟࡢⓎ㐩⚄⤒ẘᛶホ౯⣔ࡋ࡚ࡢ᭷⏝ᛶ㛵ࡍࡿ᳨ドࡣ༑ศ࡛
ࡣ࡞࠸ࡶࡢࡢ, ⚄⤒ẘᛶࡀᠱᛕࡉࢀࡿࡼ࠺࡞ྜ≀ࡢⓎ㐩⚄⤒ẘᛶホ౯ࢆ, ⌮Ⓨ⏕᰿ࡊ ࡋࡓ⡆᫆ⓗ࡞ࢫࢡ࣮ࣜࢽࣥࢢホ౯⣔ࡋ࡚, ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ᐇ⏝࡛ࡁࡿྍ⬟ᛶࡀ 㧗࠸⪃࠼ࡽࢀࡓࠋ
➨
3
❶࡛♧ࡋࡓHCP
ࢆࡋࡓᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖᑐࡍࡿ㐣࣓ࢳࣝไᚚ◚⥢ࡢ 㛵ࡢ᳨ウ࡛ࡣ, ṑ≧ᅇ㛛ࡢGABA
ᛶᅾࢽ࣮ࣗࣟࣥⓎ⌧ࡍࡿ㑇ఏᏊࡢࡳ࡛, DNAࡢ㐣࣓ࢳࣝࡼࡿ㑇ఏᏊⓎ⌧ࡢୗ᪉ไᚚࡀㄆࡵࡽࢀࡓࡇࡽ, HCP ࡼࡿ⚄⤒᪂⏕㒊࡛ࡢ࢚
ࣆࢤࣀ࣒ẘᛶࡣ⚄⤒᪂⏕ไᚚ⣔ാࡃྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋGABA ᛶᅾࢽ࣮ࣗࣟࣥ
ࡢᙳ㡪ࡣᅇࢆ♧ࡋࡓࡀ, ⚄⤒᪂⏕ᚋࡢࢩࢼࣉࢫྍረᛶࡶࡓࡽࡋࡓຠᯝࡣ㑇ఏᏊⓎ⌧ࡢほ
Ⅼࡽࡣྍ㏫ⓗ࡛࠶ࡗࡓࠋࡇࡢࡇࡣ, ࢚ࣆࢤࣀ࣒ኚືࡼࡿᙳ㡪ࡀ, 」ᩘࡢࢫࢸࢵࣉࡢ㞀 ᐖࡢ࢝ࢫࢣ࣮ࢻࢫࢵࢳࢆධࢀ, ࡑࡢ୍㒊ࡣᚋᡠࡾ࡛ࡁࡎྍ㏫ⓗ࡞ᙳ㡪ࢆṧࡋࡓྍ⬟
ᛶࢆ♧၀ࡍࡿࠋࡲࡓ, ࢚ࣆࢤࣀ࣑ࢡࢫࡢᐃ⩏ࡣ, DNAࡢሷᇶ㓄ิࡢኚ౫Ꮡࡋ࡞࠸㑇ఏᏊⓎ
⌧ࡢಟ㣭ࡉࢀ࡚࠸ࡿࡇࡽ [16], 㞳ஙᚋࡢ᭚㟢ᐇ㦂࠾࠸࡚ࡶ࢚ࣆࢤࣀ࣒ኚືࡀ⏕ࡌ
ࡿࡇࡀᐃࡉࢀࡿࠋࡇࢀࡲ࡛ࡢሗ࿌࡛ࡣ, ᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕࠾࠸࡚ࡣ⚄⤒ᖿ⣽⬊ࡀ
࢚ࣆࢤࣀ࣒ኚືಟ㣭ࢆཷࡅࡿࡇ࡛, ⚄⤒ᖿ⣽⬊ࡢ㐠ࢆኚ࠼, ⚄⤒᪂⏕㞀ᐖࡀ㉳ࡇࡿࡋ
࡚ࡁࡓ [14]ࠋࡋࡋ, ㏆ᖺ, ⚄⤒ᖿ⣽⬊ࡔࡅ࡛࡞ࡃ, ṇᖖ࡞⚄⤒᪂⏕㔜せ࡞ᙺࢆᯝࡓࡍ
ࡉࢀࡿ⬻⏤᮶⚄⤒ᡂ㛗ᅉᏊ
Bdnf
ࡢⓎ⌧ࡶ࢚ࣆࢤࣀ࣒ኚືࡢ㛵ࡀ☜ㄆࡉࢀ࡚࠸ࡿ [91 ]ࠋᮏ
◊✲࠾࠸࡚ࡶ, Ⓨ㐩ᮇ᭚㟢ᐇ㦂࠾࠸࡚, ⚄⤒᪂⏕ࡢไᚚࢆ⾜࠺
GABA
ᅾᛶࢽ࣮ࣗࣟࣥࡢ୍㐣ⓗ࡞࢚ࣆࢤࣀ࣒ኚືࡼࡿᙳ㡪ࢆ᭚㟢┤ᚋぢฟࡋࡓࠋࡇࡢࡇࡣ, 28 ᪥㛫ᢞ
53
ヨ㦂࠾࠸࡚ࡶྠᵝࡢኚືࢆ᳨ฟ࡛ࡁࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋtype-1 ⚄⤒ᖿ⣽⬊ࢆྵࡴ㢛⢏
⣽⬊⣔㆕ࡢቑṪάᛶࡀⓎ㐩ᮇཬࡧ㞳ஙᚋ࡛ࡣ␗࡞ࡿࡀ[72], ไᚚ⣔ᑐࡍࡿᙳ㡪ࡣⓎ㐩ᮇཬ ࡧ㞳ஙᚋ࡛ྠ୍࡛࠶ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ௨ୖࡼࡾ, ࢚ࣆࢤࣀ࣒ኚືಟ㣭ࡼࡿ㑇ఏᏊⓎ
⌧ไᚚᶵᵓࡢ◚⥢ࡢゎ᫂ࡀ, Ⓨ㐩⚄⤒ẘᛶࢆ᳨ฟࡍࡿⅭࡢゎᯒἲࡢᵓ⠏ࡢ୍ຓ࡞ࡿࡇࡀ ᮇᚅࡉࢀࡿࠋ
54
⤖⤖ ㄽ
ᮏ◊✲࡛ࡣ, MNUࢆࡋࡓ
DNA
┤᥋ᦆയ㉳ᅉࡋࡓᾏ㤿ࡢ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, ⚄⤒ᖿ⣽⬊ࢆྵࡴቑṪάᛶࢆక࠺㢛⢏⣽⬊⣔㆕ࢆᶆⓗࡋࡓ࣏ࢺ࣮ࢩࢫࢆయࡋ, VPA ࢆ
ࡋࡓ࢚ࣆࢤࣀ࣒ಟ㣭ኚື㉳ᅉࡋࡓ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, ⚄⤒᪂⏕ไᚚ⣔ࡢ㞀ᐖࢆ♧ࡋ ࡓࠋࡇࡢࡇࡽ, 㞀ᐖᶵᗎࡈᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᙳ㡪ࡀ␗࡞ࡿࡇࡀ♧၀ࡉࢀ, യᐖᶵᗎ᰿ᕪࡋࡓᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕㞀ᐖᛶ㛵ࡍࡿሗࡢᩚ⌮ࡢ㔜せᛶࡀぢฟࡉࢀࡓࠋ
MNU
ཬࡧVPA
ࡼࡿᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᙳ㡪ࡣ, Ⓨ㐩ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛ࡣ, ␗࡞ࡿࣃࢱ࣮ࣥࡢ㢛⢏⣽⬊⣔㆕ࡢኚືࡀ☜ㄆࡉࢀࡓࠋയᐖᶵᗎࡼࡾⓎ㐩ᮇ㞳ஙᚋࡢቑṪά ᛶࡢ㐪࠸ᑐࡍࡿឤཷᛶࡀ␗࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ, type-1⚄⤒ᖿ⣽⬊ࢆྵࡴ㢛⢏⣽⬊⣔㆕ࢆ
ᶆⓗࡍࡿሙྜ, ཬࡧ⚄⤒᪂⏕ไᚚ⣔ࢆᶆⓗࡍࡿሙྜ࡛ࡣ, Ⓨ㐩ᮇ᭚㟢ཬࡧ㞳ஙᚋ᭚㟢࡛
ࡢᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕ࡢᙳ㡪ࡀ␗࡞ࡿࣃࢱ࣮࡛ࣥ࠶ࡿྍ⬟ᛶࢆぢฟࡋࡓࠋ
Ⓨ㐩⚄⤒ẘᛶࡢ୍⯡ẘᛶヨ㦂ࡢᯟ⤌ࡳ࡛ࡢ᳨ฟᛶ㛵ࡋ࡚ࡣ, Ⓨ㐩⚄⤒ẘᛶ≀㉁࡛ࡶⓎ㐩 ᮇ᭚㟢㞳ஙᚋ᭚㟢࡛ࡣ⚄⤒᪂⏕㞀ᐖᛶࡀ␗࡞ࡿࣃࢱ࣮ࣥࢆ♧ࡋࡓࡶࡢࡢ, 㢛⢏⣽⬊⣔㆕,
⚄⤒᪂⏕ไᚚ⣔ཬࡧ㛵㐃ศᏊཬࡧ㑇ఏᏊࡢኚື࡞ࡢᾏ㤿࠾ࡅࡿ⚄⤒᪂⏕⎔ቃࡢᙳ㡪ࡣ ᑡ࡞ࡽࡎᏑᅾࡋ, ␗࡞ࡿࣃࢱ᳨࣮࡛ࣥฟ࡛ࡁࡿࡓࡵ, ⚄⤒᪂⏕㞀ᐖᛶࢆ㍈ࡋࡓ⌮Ⓨ⏕
᰿ࡊࡋࡓホ౯ࡣྍ⬟ุ᩿ࡉࢀࡓࠋᚋࡣ, ⌮Ⓨ⏕᰿ࡊࡋࡓホ౯グ᠈ࡸ⾜ື㞀ᐖᛶ
ࡢ㛵㐃ᛶࡘ࠸᳨࡚ドࡋ࡚࠸ࡃࡇ࡛, 㧗ḟࡢ⚄⤒ᶵ⬟㞀ᐖࡢண 㛵ࡋ࡚ࡶホ౯࡛ࡁࡿ
ࡼ࠺࡞ࡿࡇࡀᮃࡲࢀࡿࠋ
HCP
ࡼࡿ⚄⤒᪂⏕㒊࡛ࡢ࢚ࣆࢤࣀ࣒ẘᛶࡣ⚄⤒᪂⏕ไᚚ⣔ാࡃྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓ, VPAࡢ࡞ᶆⓗࡀṑ≧ᅇ㛛ࡢ⚄⤒᪂⏕ไᚚ⣔࡛࠶ࡿࡇࡽࡶ, ࢚ࣆࢤࣀ࣒ẘᛶ ࡀ
GABA
ᛶᅾࢽ࣮࡛ࣗࣟࣥᙳ㡪ࢆཬࡰࡍྍ⬟ᛶࡀぢฟࡉࢀࡓࠋไᚚ⣔ᑐࡍࡿᙳ㡪ࡣⓎ㐩 ᮇཬࡧ㞳ஙᚋ࡛ྠ୍࡛࠶ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࡇࡽ, ࢚ࣆࢤࣀ࣒ኚືಟ㣭ࡼࡿ㑇ఏᏊⓎ⌧ไᚚᶵᵓࡢ◚⥢ࡢゎ᫂ࡀ, ୍⯡ẘᛶヨ㦂࠾࠸࡚Ⓨ㐩⚄⤒ẘᛶࢆ᳨ฟࡍࡿⅭࡢゎᯒἲࡢ ᵓ⠏ࡢ୍ຓ࡞ࡿࡇࡀᮇᚅࡉࢀࡿࠋ
55
ㅰㅰ ㎡
ᮏ✏ࢆ⤊࠼ࡿ࠶ࡓࡾ, ᮏ◊✲ࡢ㏲⾜㝿ࡋ࡚⤊ጞᚚᣦᑟ, ᚚ㠴᧡ࢆ㈷ࡾࡲࡋࡓᒱ㜧Ꮫ
Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉࡞ࡽࡧᮾி㎰ᕤᏛᏛ㝔㎰Ꮫ◊✲㝔ື≀⏕⛉Ꮫ㒊㛛 ㇂
῟ ᩍᤵ, ᮾி㎰ᕤᏛᏛ㝔㎰Ꮫ◊✲㝔ື≀⏕⛉Ꮫ㒊㛛 ྜྷ⏣ ᩄ๎ ᩍᤵ῝ㅰ࠸
ࡓࡋࡲࡍࠋ
ᮏ✏సᡂ㝿ࡋ, ᠓⠜࡞ᚚᣦᑟ, ᚚຓゝࢆ㈷ࡾࡲࡋࡓ, ᮾி㎰ᕤᏛᏛ㝔㎰Ꮫ◊✲㝔ື
≀⏕⛉Ꮫ㒊㛛 ୗ⏣ ᐇ ᩍᤵ, ᖏᗈ␆⏘Ꮫᇶ♏⋇་Ꮫ◊✲㒊㛛ែண㜵Ꮫศ㔝 ྂᯘ
ᚿᏳ ᩍᤵ, ᒾᡭᏛ㎰Ꮫ㒊ඹྠ⋇་Ꮫ⛉ బ⸨ ὒ ᩍᤵ, ᒱ㜧Ꮫᛂ⏝⏕≀⛉Ꮫ㒊ᛂ
⏝⏕≀⛉Ꮫ⛉ ᰡ ᚨ☻ ᩍᤵㅽࢇ࡛῝ㅰࡢពࢆ⾲ࡋࡲࡍࠋ
◊✲㝿ࡋ, ᚚᣦᑟ࣭ᚚ༠ຊ㈷ࡾࡲࡋࡓ, ᫍ⸆⛉Ꮫඛ➃⏕⛉Ꮫ◊✲ࢭࣥࢱ࣮ ༑ᔒ
⚽ ࢭࣥࢱ࣮㛗, ᫍ⸆⛉Ꮫඛ➃⏕⛉Ꮫ◊✲ࢭࣥࢱ࣮ ሯ ࡲࡁ◊✲ဨ, ᰴᘧ♫
ᐩኈ⸆ရ➨◊✲ᡤ, ᐇ㦂ࡈ༠ຊୗࡉࡾࡲࡋࡓྠᩍᐊㅖẶࡢⓙᵝᚰࡽឤㅰ࠸ࡓࡋࡲࡍࠋ
56
ᘬᘬ⏝ᩥ⊩