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(2) ┠ḟ ➨ 1 ❶ ᗎㄽ ........................................................................................................................ 2 ➨ 2 ❶ ᐇ㦂ᮦᩱ ................................................................................................................. 7 ➨ 1 ⠇ ヨ⸆ .................................................................................................................... 7 ➨ 2 ⠇ ヨ⸆ㄪ〇 ........................................................................................................... 11 ➨ 3 ⠇ ᶵჾ .................................................................................................................. 13 ➨ 4 ⠇ ⣽⬊ .................................................................................................................. 15 ➨ 3 ❶ ᐇ㦂᪉ἲ ............................................................................................................... 16 ➨ 1 ⠇ ⣽⬊ᇵ㣴 ........................................................................................................... 16 ➨ 2 ⠇ ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢㄪ〇 .................................................................................. 16 ➨ 3 ⠇ RT-PCR ἲࡼࡿྛ✀㑇ఏᏊࡢⓎ⌧᳨ウ ......................................................... 16 ➨ 4 ⠇ WST assay ࡼࡿ⣽⬊ẘᛶࡢ᳨ウ................................................................... 19 ➨ 5 ⠇ quantitativeRT-PCR ἲࡼࡿྛ✀㑇ఏᏊࡢⓎ⌧ኚືࡢゎᯒ ........................ 20 ➨ 6 ⠇ G93ASOD1 ኚ␗యⓎ⌧⣽⬊ࡢస〇 ................................................................. 20 ➨ 7 ⠇ HSP70 㑇ఏᏊⓎ⌧ࡢ᳨ウ ................................................................................ 23 ➨ 8 ⠇ BDNF ࡢാࡁᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 .............................................. 23 ➨ 9 ⠇ VEGF Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪.................................................. 25 ➨ 10 ⠇ ⤫ィⓗゎᯒ ...................................................................................................... 27 ➨ 4 ❶ ᐇ㦂⤖ᯝ ............................................................................................................... 28 ➨ 1 ⠇ ྛ⚄⤒⣔⣽⬊࠾ࡅࡿ⚄⤒ಖㆤ㛵㐃㑇ఏᏊࡢⓎ⌧᳨ウ .................................... 28 ➨ 2 ⠇ ࢥ࣮ࣄ࣮㇋ᢳฟᾮཬࡧࢥ࣮ࣄ࣮ᡂศࡢ⚄⤒⣽⬊ᑐࡍࡿẘᛶ...................... 28 ➨ 3 ⠇ SOD1G93A Ⓨ⌧ SH-SY5Y ⣽⬊ࡢస〇ཬࡧኚ␗యⓎ⌧ࡢᙳ㡪 ....................... 28 ➨ 4 ⠇ HSP70 Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ................................................ 29 ➨ 5 ⠇ BDNF ࡢാࡁᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 .............................................. 30 ➨ 6 ⠇ VEGF Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪.................................................. 32 ➨ 7 ⠇ AD 㛵㐃㑇ఏᏊⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ..................................... 35 ➨ 5 ❶ ⪃ᐹ ...................................................................................................................... 61 ➨ 1 ⠇ SOD1G93A Ⓨ⌧⣽⬊ཬࡧ HSP70 㑇ఏᏊⓎ⌧ኚࡘ࠸࡚ ........................... 61 ➨ 2 ⠇ BDNF ࡢാࡁᑐࡍࡿᙳ㡪ࡘ࠸࡚................................................................ 62 ➨ 3 ⠇ VEGF Ⓨ⌧ᑐࡍࡿᙳ㡪ࡘ࠸࡚ ................................................................... 63 ➨ 4 ⠇ AD 㛵㐃㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࡘ࠸࡚....................................................... 65 ➨ 5 ⠇ ⥲ᣓ .................................................................................................................. 66 ➨ 6 ❶ ཧ⪃ᩥ⊩ ............................................................................................................... 67 ➨ 7 ❶ ㅰ㎡ ...................................................................................................................... 81 . 1.
(3) ➨ 1 ❶ ᗎㄽ ⚄⤒ኚᛶᝈࡣࠊ࠶ࡿ≉ᐃࡢ⚄⤒⣽⬊⩌ࡀᚎࠎ㞀ᐖࢆཷࡅࠊ⬺ⴠࡋ࡚ࡋࡲ࠺Ẽ࡛࠶ ࡿࠋࣝࢶࣁ࣐࣮㸦Alzheimer’s disease; AD㸧ࡸࣃ࣮࢟ࣥࢯࣥ㸦Parkinson’s disease; PD㸧ࠊ➽ⴎ⦰ᛶഃ⣴◳㸦Amyotrophic Lateral Sclerosis; ALS㸧࡞ࡀ௦⾲ⓗ࡞⚄⤒ኚ ᛶᝈࡋ࡚▱ࡽࢀ࡚࠸ࡿࠋከࡃࡢ⚄⤒ኚᛶᝈࡣᐙ᪘ᛶ㸦Familial㸧ᏙⓎᛶ㸦Sporadic㸧 ࡀ࠶ࡾࠊ࠸ࡎࢀࡶ␗ᖖࢱࣥࣃࢡࡢ✚ࡀ☜ㄆࡉࢀ࡚࠸ࡿࡶࡢࡢࠊ᫂☜࡞ཎᅉࡣ᫂ࡽ࡞ࡗ ࡚࠾ࡽࡎࠊࡲࡓ⒪ἲࡶ☜❧ࡉࢀ࡚࠸࡞࠸ࠋ ୍᪉ࠊࢥ࣮ࣄ࣮ࡣୡ⏺୰࡛ឡ㣧ࡉࢀ࡚࠸ࡿ㣧ᩱࡢ୍ࡘ࡛࠶ࡿࠋࢥ࣮ࣄ࣮ࡣࢥ࣮ࣄ࣮㇋ࢆ 200㹼220°C ࡛ 15㹼20 ศ↦ࡋࠊᤂ࠸ࡓ⢊ᮎࢆᢳฟࡋࡓ㣧ᩱࡢࡇ࡛ࠊࡇࡢ↦ࡢ㐣⛬࡛ࠊ ࢥ࣮ࣄ࣮㇋୰ࡢᡂศࡀຍỈศゎࢆྵࡵࡓᵝࠎ࡞Ꮫᛂࡼࡾࠊࡑࡢᡂศ㔞ࡀኚࡍࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Fig.1㸧[1]ࠋࡇࡢᡂศࡢ௦⾲ⓗ࡞ࡶࡢࡋ࡚ࠊ⢾㢮ࡸ⬡㉁ࠊ࣑ࣀ㓟ࠊ ࢱࣥࣃࢡ㉁ࡢࣝ࢝ࣟࢻࡢ୍ࡘࡋ࡚▱ࡽࢀࡿ࢝ࣇ࢙ࣥࡸࢺࣜࢦࢿࣜࣥࠊ࣏ࣜࣇ ࢙ࣀ࣮࡛ࣝ࠶ࡿࢡࣟࣟࢤࣥ㓟ࠊࢡࣟࣟࢤࣥ㓟ࡀຍỈศゎࡉࢀ࡚⏕ᡂࡉࢀࡿࠊࢥ࣮ࣄ࣮ࡢໝ࠸ ࡢඖ࡛࠶ࡿ࢝ࣇ࢙㓟ࠊࢪࢸ࡛ࣝ࣌ࣥ࠶ࡿ࣮࢙࣮࢝࢘࢜ࣝࡸ࢝ࣇ࢙ࢫࢺ࣮ࣝࡀྵࡲࢀ࡚࠸ࡿࠋ. 2.
(4) ࢥ࣮ࣄ࣮ࡣ㏆ᖺᵝࠎ࡞⚄⤒ኚᛶᝈᑐࡋ࡚ண㜵ຠᯝࢆᣢࡘࡇࡀᏛ◊✲࡞ࡽ᫂ ࡽ࡞ࡗ࡚࠸ࡿ[2-6]ࠋ1 ᪥ 3㹼5 ᮼࡢࢥ࣮ࣄ࣮ࡢᦤྲྀࡀ AD ࡢࣜࢫࢡࢆ 65%పୗࡉࡏࡿࡇ [4]ࠊ1 ᪥ 800 mL ࡢࢥ࣮ࣄ࣮ᦤྲྀࡀ PD ࡢࣜࢫࢡࢆ 80%పୗࡉࡏࡿࡇ[5]ࠊALS ࡢᝈ⪅ ࡀᖖேẚ࡚ࢥ࣮ࣄ࣮ࢆᦤྲྀࡍࡿᶵࡀᑡ࡞࠸ࡇ[6]ࠊ࡞ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋ ࡋࠊࢥ࣮ࣄ࣮ࡢ⚄⤒ಖㆤຠᯝࡢヲ⣽࡞ศᏊ࣓࢝ࢽࢬ࣒ࡣ᫂ࡽ࡞ࡗ࡚࠸࡞࠸ࠋ Brain derived neurotrophic factor㸦BDNF㸧ࡸ nerve growth factor㸦NGF㸧ࠊglial cellline derived neurotrophic factor㸦GDNF㸧࡞ࡢ⚄⤒ᰤ㣴ᅉᏊࡣᵝࠎ࡞⚄⤒ኚᛶᝈᑐ ࡋ࡚ᝈࡢⓎࡢᢚไࡸᘏຠᯝࡀ࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[2,3,7]ࠋ≉ NGF ࡣ⬻ෆ ࡛ᾏ㤿ࠊGDNF ࡣࢩࣗ࣡ࣥ⣽⬊ࡸࢫࢺࣟࢧࢺࡰ㝈ᒁࡋࡓⓎ⌧ࢆࡋ࡚࠸ࡿࡢᑐ ࡋࠊBDNF ࡣᾏ㤿ࠊ⬻⓶㉁࡛㧗࠸Ⓨ⌧ࢆ♧ࡍࠊ⬻ෆ࡛ᗈࡃⓎ⌧ࡋ࡚࠾ࡾ[8]ࠊࡑࡢാ ࡁࡣ⚄⤒ᰤ㣴ᅉᏊࡢ୰࡛ࡶ≉㔜せ࡞ࡶࡢ࡞ࡗ࡚࠸ࡿࠋBDNF ࡣࡑࡢ㧗ぶᛶࡢཷᐜయ ࡛࠶ࡿ tropomyosin-related kinase type B㸦TrkB㸧⤖ྜࡋࠊTrkB ࢆࣜࣥ㓟ࡍࡿࡇ ࡛ᰤ㣴ຠᯝࢆⓎࡍࡿ[8]ࠋTrkB ࡣ⣙ 800 ಶࡢ࣑ࣀ㓟ࡽ࡞ࡿ 145 kDa ࡢศᏊ࡛ࠊཷᐜ యᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ࡛࠶ࡾࠊ⣽⬊ෆࢳࣟࢩࣥ࢟ࢼ࣮ࢮࢻ࣓ࣥࢆᣢࡘ[8]ࠋ㔞యࡢࣜ ࢞ࣥࢻࡀ⤖ྜࡍࡿࡇ࡛ TrkB ࡶ㔞యࡋࠊ⣽⬊ෆࢻ࣓ࣥࡢࢳࣟࢩࣥṧᇶࡀ⮬ᕫࣜࣥ㓟 ࡍࡿࡇ࡛⣽⬊ෆࢩࢢࢼࣝࡀఏ㐩ࡍࡿ㸦Fig.2㸧[8]ࠋTrkB ࡀࣜࣥ㓟ࡉࢀࠊ⣽⬊ෆࢩ ࢢ ࢼ ࣝ ࡀ ఏ 㐩 ࡍ ࡿ ࠊ Mitogen-activated Protein Kinase 㸦 MAPK 㸧 ⤒ ㊰ ࡸ Phosphatidylinositol-3 kinase㸦PI3K㸧/Protein kinase B㸦Akt㸧⤒㊰࡞ࡢ⤒㊰ࡀάᛶ ࡉࢀࠊ㌿ᅉᏊ cAMP response element binding protein㸦CREB㸧ࡀ㌿άᛶࡍࡿ㸦Fig.2㸧 [8]ࠋࡇࢀࡽࡢࢩࢢࢼࣝࡀάᛶࡉࢀࡿࡇࡼࡾࠊBDNF ⮬㌟ࡢ㑇ఏᏊⓎ⌧ࡀㄏᑟࡉࢀࠊ ࡲࡓ㛗ᮇቑᙉ㸦Long-term potentiation㸧ࡸ⚄⤒✺㉳ࡢఙ㛗࡞ࡀㄏᑟࡉࢀࡿ[8]ࠋ BDNF ⚄⤒ኚᛶᝈࡢ㛵㐃ࡘ࡚ࡣࠊᵝࠎ࡞ሗ࿌ࡀ࡞ࡉࢀ࡚࠸ࡿࠋࣝࢶࣁ࣐࣮ ࣔࢹ࣐ࣝ࢘ࢫ࠾࠸࡚ࠊTrkB ࢩࢢࢼࣝࡢ㜼ᐖࡼࡾ✵㛫グ᠈ຊࡀపୗࡍࡿ୍᪉࡛ࠊTrkB ࢆ 㧗Ⓨ⌧ࡉࡏࡿ✵㛫グ᠈ຊࡀᅇࡍࡿࡇ[9]ࠊࡲࡓ࣑ࣟࢻEࡢ✚ࠊẘᛶᑐࡋ࡚ᾏ㤿 ࡢ⚄⤒ࢆಖㆤࠊ⒵ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[10]ࠋࡲࡓࠊBDNF ࡀ㯮㉁ࡢࢻࣃ࣑ࣥసືᛶ. 3.
(5) ⚄⤒ࡢ⏕Ꮡࢆ⥔ᣢࡍࡿࡇࡸ[11,12]ࠊ6-ࣄࢻࣟ࢟ࢩࢻࣃ࣑ࣥࡼࡿ⣽⬊Ṛࡽಖㆤࡍࡿࡇ [13]ࠊࣃ࣮࢟ࣥࢯࣥᝈ⪅࡛㯮㉁ࡢ BDNF mRNA /ࢱࣥࣃࢡⓎ⌧ࡀపୗࡋ࡚࠸ࡿࡇ [14,15]ࠊ࡞ࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿࠋALS ࣔࢹࣝື≀࠾࠸࡚ࡣࠊBDNF ࢆቑຍࡉࡏࡿࡇ ࡸ[16]ࠊTrkB ࡢኚ␗యࡢⓎ⌧ࢆపୗࡉࡏࡿࡇࡼࡾ[17]ࠊALS ࡢⓎࡀ㐜ᘏࡍࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡇࡢࡼ࠺ࠊBDNF ⚄⤒ኚᛶᝈࡣ῝࠸㛵ࢃࡾࡀ࠶ࡿࡇࡀ᫂ࡽ ࡞ࡗ࡚࠸ࡿࠋ. ୍᪉ࠊVascular endothelial growth factor㸦VEGF㸧ࡣ⾑⟶ෆ⓶ቑṪᅉᏊࡋ࡚▱ࡽࢀ࡚ ࠾ࡾࠊ⾑⟶᪂⏕ࡸ⣽⬊ቑṪࢆࢩࢢࢼࣝࡍࡿࢱࣥࣃࢡ࡛࠶ࡾࠊ㏆ᖺࠊ⚄⤒ಖㆤຠᯝࢆ᭷ࡍࡿࡇ ࡀሗ࿌ࡉࢀࡓ[18-20]ࠋVEGF ࡣ VEGF receptor㸦VEGFR㸧⤖ྜࡍࡿࡇ࡛ࢩࢢࢼࣝ ࢆఏ㐩ࡍࡿ[21]ࠋVEGFR VEGF ࡀ⤖ྜࡍࡿࢲ࣐࣮ࡀᙧᡂࡉࢀࠊ⣽⬊ෆࢻ࣓ࣥࡢ ࢳࣟࢩࣥṧᇶࡀ⮬ᕫࣜࣥ㓟ࡍࡿࡇ࡛⣽⬊ෆࢩࢢࢼࣝࡀఏ㐩ࡍࡿ[21-23]ࠋ⣽⬊ෆࢩ. 4.
(6) ࢢࢼࣝࡀఏ㐩ࡍࡿ MAPK ⤒㊰ࡸ PI3K/Akt ⤒㊰ࡀάᛶࡉࢀࠊ⣽⬊ࡢ⏕Ꮡࡸ DNA ྜᡂ ࡀಁ㐍ࡉࢀࡿ[24]ࠋ VEGF ࡢⓎ⌧ࡣ㌿ᅉᏊ࡛࠶ࡿప㓟⣲ᛂ⟅ᅉᏊ㸦hypoxia inducible factor; HIF㸧ࡼࡾ ไᚚࡉࢀ࡚࠸ࡿ[25,26]ࠋHIF ࡣ HIF-1D HIF-1E࠸࠺ࡘࡢࢧࣈࣘࢽࢵࢺࡽᵓᡂࡉࢀ ࡚࠾ࡾࠊHIF-1DࡢⓎ⌧ࡣ reactive oxygen species㸦ROS㸧ࡸᢠ㓟ࡢ㔞ࡼࡾไᚚࡉࢀ ࡚࠸ࡿ[27]ࠋHIF-1Dࡣ㏻ᖖࠊࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒⣔ࢆࡋ࡚ศゎࡉࢀࡿࡇࡼࡾࠊ Ⓨ⌧㔞ࡀᢚไࡉࢀ࡚࠸ࡿ[27,28]ࠋࡍ࡞ࢃࡕࠊHIF-1Dࡣ 402 ␒┠ 564 ␒┠ࡢࣉࣟࣜࣥࡀࣄ ࢻࣟ࢟ࢩࣝࡉࢀࡿࡇ࡛ von Hippel-Lindau protein㸦pVHL㸧ࢆྵࡴ E3 ࣘࣅ࢟ࢳࣥࣜ ࣮࢞ࢮࡼࡾࣘࣅ࢟ࢳࣥࢆཷࡅࠊࣉࣟࢸࢯ࣮࣒౫Ꮡⓗศゎࡉࢀࡿ㸦Fig.3㸧[29-32]ࠋ ࣉࣟࣜࣥࣄࢻࣟ࢟ࢩࣝࡣ㓟⣲ࢆ⿵ᅉᏊࡋ࡚せồࡍࡿ prolyl hydroxylase domain protein 2㸦PHD2㸧ࡼࡾ⾜ࢃࢀࡿ[33,34]ࠋࡋࡋࠊప㓟⣲≧ែ࡞ࡿࡇࡢ PHD2 ࡢά ᛶࡀపୗࡋࠊHIF-1Dࡢࣉࣟࣜࣥࣄࢻࣟ࢟ࢩࣝࡀᢚไࡉࢀࡿ[27,35]ࠋࣉࣟࣜࣥࣄࢻࣟ࢟ࢩ ࣝࢆཷࡅ࡞ࡗࡓ HIF-1Dࡣ᰾ෆ⛣⾜ࡋࠊHIF-1E࣊ࢸࣟࢲ࣐࣮ࢆᙧᡂࠊDNA ୖࡢ Hypoxia Responsive Element㸦HRE㸧⤖ྜࡍࡿࡇ࡛ࢱ࣮ࢤࢵࢺ㑇ఏᏊࡢ㌿ࢆάᛶ ࡍࡿ㸦Fig.3㸧[36]ࠋ. 5.
(7) VEGF ⚄⤒ኚᛶᝈࡢ㛵㐃ࡘ࠸࡚ࡶࠊᵝࠎ࡞ሗ࿌ࡀ࡞ࡉࢀ࡚࠸ࡿࠋVEGF Ḟᦆ࣐࢘ ࢫࡀ ALS 㓞ఝࡋࡓ㐠ືࢽ࣮ࣗࣟࣥ㞀ᐖࢆᘬࡁ㉳ࡇࡍࡇࡸ[37,38]ࠊVEGF ࢆ ALS ࣔࢹ ࣝࣛࢵࢺࡢ⬻ᐊ┤᥋ᢞࡍࡿࡇ࡛ ALS Ⓨᢚไࡸ⏕Ꮡࡢᘏ㛗ാࡃࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ[39-41]ࠋࡲࡓࠊVEGF Ⓨ⌧㛵ࡍࡿ㑇ఏᏊࡢኚ␗ᆺࡀ ALS Ⓨࣜࢫࢡ῝ࡃ㛵㐃ࡋ ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓ[37,42]ࠋࡲࡓࠊVEGF ࡣࢻࣃ࣑ࣥసືᛶ⚄⤒ಖㆤຠᯝࡀ࠶ࡾࠊ 6-hydroxydopamine㸦6-OHDA㸧ㄏᑟᛶࡢ⚄⤒⣽⬊Ṛࢆᢚไࡍࡿࡇࡸ[43]ࠊVEGF ศἪ⣽ ⬊ࢆ⛣᳜ࡍࡿࡇ࡛ࠊ⾜ືᏛࠊ⌮Ꮫࡢ୧㠃࡛ࣃ࣮࢟ࣥࢯࣥࡢண㜵ാࡃࡇࡀሗ࿌ࡉࢀ ࡚࠸ࡿ[44,45]ࠋ ᮏ◊✲࡛ࡣࠊࢥ࣮ࣄ࣮ࡼࡿ⚄⤒ಖㆤຠᯝࡢ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ┠ᣦࡋࠊࣄࢺ⚄⤒ⱆ⣽⬊ ⭘ SH-SY5Y ࢆ⏝࠸࡚◊✲ࢆ⾜ࡗࡓࠋ. 6.
(8) ➨ 2 ❶ ᐇ㦂ᮦᩱ ➨ 1 ⠇ ヨ⸆ 1) ⣽⬊ᇵ㣴 Dulbecco’s Modified Eagle’s Medium (DMEM). ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Ham’s F12 Medium. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Minimum Essential Medium (MEM). ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Minimum Essential Medium Alpha (MEMD). ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Fetal Bovine Serum (FBS). GIBCO. Non-essential amino acid (NEAA). GIBCO. Penicillin-streptomycin liquid. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. 0.25% trypsin-1 mM EDTA solution. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Blasticidin. INVIVOGEN. Puromycin. INVIVOGEN. Trypan blue stain 0.4%. GIBCO. All-trans retinoic acid. ග⣧⸆ᕤᴗᰴᘧ♫. Fugene 6. PROMEGA. Polybrene. . SIGMA-ALDRICH. 2) ヨᩱࠊヨ⸆ ࢥ࣮ࣄ࣮(ࣁ࢘ࢫࣈࣞࣥࢻ). ࢫࢱ࣮ࣂࢵࢡࢫࢥ࣮ࣄ࣮ࢪࣕࣃࣥ. ࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮. . ࢿࢫࣞࢪࣕࣃࣥ. ⏕㇋. ࡸ࡞⌆⍄ᗑ. Caffeine. . SIGMA-ALDRICH. Chlorogenic acid. . SIGMA-ALDRICH. Caffeic acid. . SIGMA-ALDRICH. Trigonelline hydrochloride. . SIGMA-ALDRICH. Kahweol. . LKT laboratory, Inc. Cafestol. . LKT laboratory, Inc. Pyrogallol. . SIGMA-ALDRICH. Pyrocatechol. . SIGMA-ALDRICH. Hydroxyhydroquinone. . SIGMA-ALDRICH. Gallic acid. ᮾிᡂᕤᴗᰴᘧ♫. Baicalein. . SIGMA-ALDRICH. Paraquat. . SIGMA-ALDRICH. Brain derived neurotrophic factor (BDNF). . Deferoxamine mesylate salt. . 7. PEPROTECH. SIGMA-ALDRICH.
(9) Echinomycin. Enzo Life Science. U0126. ග⣧⸆ᕤᴗᰴᘧ♫. SP600125. ග⣧⸆ᕤᴗᰴᘧ♫. LY294002. Cayman Chemical. SB202190. . SIGMA-ALDRICH. Wortmannin. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Dehydroxymethylepoxyquinomicin (DHMEQ) ᠕⩏ሿᏛ⌮ᕤᏛ㒊ᱵἑ୍ኵᩍᤵࡼࡾᣏཷ MG132. Enzo Life Science. Cycloheximide (CHX). ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. 1-methyl-4-phenylpyridinium (MPP+). . Dimethyl sulfoxide. ග⣧⸆ᕤᴗᰴᘧ♫. SIGMA-ALDRICH. 3) ࢱࣥࣃࢡᢳฟ 2-amino-2-hydroxynethl-1,3-popanediol 999 (Tris). ග⣧⸆ᕤᴗᰴᘧ♫. EDTA. ග⣧⸆ᕤᴗᰴᘧ♫. Sodium Chloride (NaCl). ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Nonited P-40. . Protease inhibitor. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Phosphatase inhibitor. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. 6×SDS buffer. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. SIGMA-ALDRICH. 4) ࢱࣥࣃࢡᐃ㔞 Bradford. BIO-RAD. Albumin standard. Thermo. 5) Immunoblotting Acrylamide. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. N,N-methylene bis. ග⣧⸆ᕤᴗᰴᘧ♫. Hydrochloric acid. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Sodium dodecylsulfate (SDS). ග⣧⸆ᕤᴗᰴᘧ♫. N,N,N’,N’,-tetramethyl-ethylenediamine (TEMED). ග⣧⸆ᕤᴗᰴᘧ♫. Ammonium persulfate (APS). BIO-RAD. Glycine. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. Precision Plus Protein Dual Color. BIO-RAD. Methanol. 㛵ᮾᏛᰴᘧ♫. ࢫ࣒࣑࢟ࣝࢡ. 㞷༳࣓ࢢ࣑ࣝࢡ. 8.
(10) Bovine serum albumin. . SIGMA-ALDRICH. Tween20. 㛵ᮾᏛᰴᘧ♫. ECL. . GE Healthcare. 6) ᢠయ Anti TrkB antibody. Cell Signaling Technology. Anti p-TrkB (Y516) antibody. Cell Signaling Technology. Anti p-TrkB (Y816) antibody. Cell Signaling Technology. Anti Akt antibody. Santa Cruz Biotechnology. Anti pAkt (S473) antibody. Cell Signaling Technology. Anti p44/42 MAPK antibody. Cell Signaling Technology. Anti p-p44/42 MAPK (T202/Y204) antibody. Cell Signaling Technology. Anti CREB antibody. Cell Signaling Technology. Anti p-CREB (S133) antibody. Capital Biosciences. Anti p38 MAPK antibody. Cell Signaling Technology. Anti p-p38 MAPK (T180/Y182) antibody. Cell Signaling Technology. Anti SAPK/JNK antibody. Cell Signaling Technology. Anti p-SAPK/JNK (T183/Y185) antibody. Cell Signaling Technology. Anti NF-NB antibody. Cell Signaling Technology. Anti p-NF-NB (S536) antibody. Cell Signaling Technology. Anti Nrf2 antibody. Santa Cruz Biotechnology. Anti HIF-1D antibody. Cell Signaling Technology. Anti hydroxy-HIF-1D (P564) antibody. Cell Signaling Technology. Anti PHD2 antibody. Cell Signaling Technology. Anti HSP70 antibody. R&D Systems. Anti Cu/Zn SOD antibody. StressMarq Biosciences Inc.. Anti BACE1 antibody. abcam. Anti Flag antibody. . SIGMA-ALDRICH. Anti E-actin antibody. Santa Cruz Biotechnology. Anti Lamin B antibody. Santa Cruz Biotechnology. Anti rabbit IgG HRP linked antibody. . GE Healthcare. Anti mouse IgG HRP linked antibody. . GE Healthcare. Anti goat IgG HRP linked antibody. . Dako-Japan. 7) RNA ᢳฟ ISOGEN. ග⣧⸆ᕤᴗᰴᘧ♫. Chloroform. 㛵ᮾᏛᰴᘧ♫. 9.
(11) Isopropyl alcohol. 㛵ᮾᏛᰴᘧ♫. Ethanol. 㛵ᮾᏛᰴᘧ♫. ⁛⳦Ỉ. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. 8) ㏫㌿ᛂ ReverTra Ace (100 U/PM). TOYOBO. Random Primer (25 pmol/PM). TOYOBO. dNTP mixture. TOYOBO. RNase inhibitor. TOYOBO. 5×RT buffer. TOYOBO. 9) real-time PCR FastStart Universal SYBR Green Master mix. . SsoFast Evagreen Supermix. BIO-RAD. 10) SOD άᛶ. Roche. ᐃ. SOD Assay Kit. ྠோᏛ◊✲ᡤ. 11) Flow cytometry Anti-Human TrkB/NTRK2 Antibody (PE). Sino Biological Inc.. Mouse IgG1, kappa monoclonal (PE/Cy5®)-Isotype Control. abcam. Human BDNF-Biotin. . alomone labs. FITC-Streptavidin. . Invitrogen. 12) ELISA Human VEGF-A Platinum ELISA Kit 13) PHD άᛶ. eBioscience. ᐃ. [5-14C]-D-ketoglutaric acid. Moravek Biochemicals. dithiothreitol㸦DTT㸧. . SIGMA-ALDRICH. ammonium ferrous sulfate. . SIGMA-ALDRICH. ascorbic acid. ග⣧⸆ᕤᴗᰴᘧ♫. catalase. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. (Pro-Pro-Gly)10 × 9 H2O peptide. ࣌ࣉࢳࢻ◊✲ᡤ. a-ketoglutaric acid. . Succinate. ࢼ࢝ࣛࢸࢫࢡᰴᘧ♫. 2,4-dinitrophenylhydrazine. ᮾிᡂᕤᴗᰴᘧ♫. 10. SIGMA-ALDRICH.
(12) HClO4. 㛵ᮾᏛᰴᘧ♫. ➨ 2 ⠇ ヨ⸆ㄪ〇 1) medium ࣭DMEM : Ham’s F-12( 1:1 )ᇵᆅࡢㄪ〇㸦⥅௦⏝㸧 DMEM (high glucose). 300 mL. Ham’s F-12. 300 mL. FBS. 100 mL (15%). NEAA. 7 mL. Penicillin-Streptomycin liquid. 7 mL (100 units/mL). ࣭DMEM : Ham’s F-12( 1:1)ᇵᆅࡢㄪᩚ㸦ศ⏝㸧 DMEM (high glucose). 500 mL. Ham’s F-12. 500 mL. NEAA. 10 mL. Penicillin-Streptomycin liquid. 10 mL (100 units/mL). All-trans retinoic acid. 100 μL (10 μM). ࣭DMEM ᇵᆅࡢㄪ〇 DMEM (high glucose). 500 mL. FBS. 50 mL (10%). Penicillin-Streptomycin liquid. 5 mL (100 units/mL). ࣭MEM ᇵᆅࡢㄪ〇 MEM. 500 mL. FBS. 50 mL (10%). NEAA. 10 mL. Penicillin-Streptomycin liquid. 5 mL (100 units/mL). ࣭MEMDᇵᆅࡢㄪ〇 MEMD. 500 mL. FBS. 50 mL (10%). Penicillin-Streptomycin liquid. 5 mL (100 units/mL). 11.
(13) 2) PBS(-) 1D&O. . J. .&O. . J. 1D+32. J. .+32. J . . ⢭〇Ỉ . XSWRP/. 3) Tris-EDTA buffer Tris-HCl (pH 8.0). 10 mM. EDTA. 1 mM. 4) 1×TAE Tris. 4.8 g. Acetic acid. 1.14 mL. EDTA࣭3Na࣭H2O. 0.41 g. ⢭〇Ỉ. up to 1000 mL. 6) ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ ࣭⃰⦰ࢤࣝ㸦࣑ࢽࢤࣝ 2 ᯛศ㸧 1 M Tris-HCl pH 6.8. 1.25 mL. 30% Acrylamide. 0.84 mL. ⢭〇Ỉ. 2.8 mL. 10% SDS. 50 PL. 10% APS. 50 PL. TEMED. 10 PL. ࣭ศ㞳ࢤࣝ㸦࣑ࢽࢤࣝ 2 ᯛศ㸧 7.5%. 10%. 12%. 15%. 1 M Tris-HCl pH 8.7. 4.84 mL. 4.84 mL. 4.84 mL. 4.84 mL. 30% Acrylamide. 2.5 mL. 3.3 mL. 4 mL. 5 mL. ⢭〇Ỉ. 2.5 mL. 1.7 mL. 1 mL. 0 mL. 10% SDS. 100 PL. 100 PL. 100 PL. 100 PL. 10% APS. 50 PL. 50 PL. 50 PL. 50 PL. TEMED. 10 PL. 10 PL. 10 PL. 10 PL. 12.
(14) 7) ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㟁ẼὋືὋື buffer Tris. 1.52 g. Glycine. 7.2 g. SDS. 0.5 g. ⢭〇Ỉ. up to 500 mL. 8) transfer buffer Glycine. 7.21 g. Tris. 1.51 g. Methanol. 100 mL. ⢭〇Ỉ. up to 500 mL. 9) T-TBS Tris. 6.1 g. NaCl. 8.1 g. Tween 20. 250 PL. ⢭〇Ỉ. up to 500 mL. ➨ 3 ⠇ ᶵჾ 1) CO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮ BHA-11. ESPEC. 2) 㐲ᚰᶵ Optima TM TLX Ultracentrifuge. BECKMAN. MX-300. T OM Y. CPW-80X. . HITACHI. 3) ㉸㡢Ἴ◚○ᶵ VP-050 4) ྾ගᗘ. TAITEC ᐃჾ. Biospec Nano. . Infinite M200 pro. SHIMAZU TECAN. 13.
(15) 5) Thermal cycler PTC-100 Programmable Thermal Controller. . MJ RESEARCH, INC. TaKaRa PCR Thermal Cycler Dice® Gradient. TaKaRa. 6) ࢤࣝᙳ⨨ Omega Lum G. aplegen. Alpha Imager® Mini. Alpha Innotech. 7) real-time PCR CFX96 Real-Time System. BIO-RAD. 8) ࢥ࣮ࣝࢱ࣮࢝࢘ࣥࢱ࣮ Beckman Coulter Vi-CELL. Beckman coulter. 9) Flow cytometer BD LSR II. ᪥ᮏ࣋ࢡࢯࣥࢹࢵ࢟ࣥࢯࣥ♫. 10) Immunoblotting ࣭Ὃື⨨ Mini-PROTEIN Tetra system. BIO-RAD. ࣭Ὃື㟁※ ࣃ࣮࣡ࣃࢵࢡ. TM. Basic. BIO-RAD. ࣃ࣮࣡ࣃࢵࢡ. TM. HC. BIO-RAD. ࣭⮬ື⌧ീᶵ KODAK X-O mat 1000 Processor. KODAK. 11) ┦ᕪ㢧ᚤ㙾 ECLIPSE Ti-U. . Nikon. 12) ⺯ග㢧ᚤ㙾 HS ࣮࢜ࣝࣥ࣡ࣥ⺯ග㢧ᚤ㙾. KEYENCE. 13) ᾮయࢩࣥࢳ࣮ࣞࢩࣙࣥ࢝࢘ࣥࢱ࣮ LSC-3500. ALOKA. LSC-6101. ALOKA. 14.
(16) ➨ 4 ⠇ ⣽⬊ SH-SY5Y. DS Pharma Biomedical Co. Ltd. IMR-32. ⌮Ꮫ◊✲ᡤ. NB-1. ⌮Ꮫ◊✲ᡤ. HEK293T. ⌮Ꮫ◊✲ᡤ. 15.
(17) ➨ 3 ❶ ᐇ㦂᪉ἲ ➨ 1 ⠇ ⣽⬊ᇵ㣴 SH-SY5Y ⣽⬊ࡣ 15% FBSࠊ100 units/mL Penicillin-100 Pg/mL Streptomycinࠊ1% NEAA ࢆྵࡴ DMEM/Ham’s F-12 ᇵᆅ (1:1)࡛ 37°Cࠊ&2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮࡛ᇵ㣴ࡋࡓࠋ ࡲࡓ SH-SY5Y ⣽⬊ࡣࠊ10 PM ࡢ All-trans retinoic acid (ATRA)่⃭ࡼࡾศࡋࡓࠋ HEK293T ⣽⬊ࠊNB-1 ⣽⬊ࠊIMR-32 ⣽⬊ࡣࡑࢀࡒࢀ 10% FBSࠊ100 units/mL Penicillin100 Pg/mL Streptomycin ྵ᭷ DMEMࠊMEMDࠊMEM ࡛ᇵ㣴ࡋࡓࠋ⣽⬊ࡣ 1.0×105 ಶ/mL ࡛✀ࡋࠊࡑࢀࡒࢀ 10 cm dish ࡣ 10 mLࠊ6 cm dish ࡣ 4 mLࠊ3.5 cm dish ࡣ 1.5 mLࠊ6 well plate ࡣ 2 mLࠊ12 well plate ࡣ 1 mLࠊ24 well plate ࡣ 500 PLࠊ96 well plate ࡣ 100 PL ࡢ medium ࡛ᇵ㣴ࡋࡓࠋ. ➨ 2 ⠇ ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢㄪ〇 ࢥ࣮ࣄ࣮⢊ (ࢥࣟࣥࣅ⏘ࠊࣛࣅ࢝✀)8 g ࢆ 95°C ࡢ⢭〇Ỉ P/ ࡼࡾࢻࣜࢵࣉᘧ ࡛ᢳฟࡋࡓᚋࠊ PP ࡢࣇࣝࢱ࣮ࢆ⏝࠸࡚ℐ㐣⁛⳦ࢆ⾜ࡗࡓࠋࡇࢀࢆ YY ࡋ࡚ ⏝࠸ࡓࠋ ➨ 3 ⠇ RT-PCR ἲࡼࡿྛ✀㑇ఏᏊࡢⓎ⌧᳨ウ ➨ 1 㡯 ⣽⬊ࡽࡢ RNA ᢳฟ 3 ᪥㛫ᇵ㣴ࡋࡓ⣽⬊ࢆ PBS (-)࡛ wash ࡋࠊISOGEN 1 mL ࢆຍ࠼࡚⣽⬊ࢆᠱ⃮ࠊ1.5 mL tube ⛣ࡋࡓࠊࢧࣥࣉࣝ 1 mL ᑐࡋ࡚ chloroform 200 PL ࢆຍ࠼ vortex ᚋࠊᐊ. ࡛2ศ. 㟼⨨ࡋࠊ12,000 gࠊ4°C ࡛ 15 ศ㐲ᚰࡋࡓࠋୖΎ 500 PL ࢆูࡢ 1.5 mL tube ⛣ࡋࠊࡑࡇ 500 PL ࡢ isopropanol ࢆຍ࠼ vortex ᚋࠊᐊ. ࡛ 10 ศ㟼⨨ࡋࠊ12,000 gࠊ4°C ࡛ 15 ศ㐲. ᚰࡋࡓࠋ࣌ࣞࢵࢺࢆṧࡋ࡚ୖΎࢆᤞ࡚ࠊ1 mL ࡢ 70% Ethanol ࢆຍ࠼㌿ಽΰᚋࠊ12,000. gࠊ4°C ࡛ 5 ศ㐲ᚰࡋࡓࠋୖΎࢆᤞ࡚ࠊࡉࡽ 12,000 gࠊ4°C ࡛ 3 ศ㐲ᚰࡋࠊ࣌ࣞࢵࢺࢆ ⇱ࡉࡏࡓᚋࠊ⁛⳦Ỉ 30 PL ⁐ゎࡉࡏࠊ-80°C ࡛ಖᏑࡋࡓࠋᢳฟࡋࡓ RNA 2 PL ࢆ⏝. 16.
(18) ࠸࡚ 260 nm ࡛ࡢ྾ගᗘࢆ. ᐃࡋࠊRNA 㔞ࢆồࡵࡓࠋ. ➨ 2 㡯 total RNA ࡽ cDNA ࡢྜᡂ (Reverse Transcription) ➨ 3 ❶ࠊ➨ 3 ⠇ࠊ➨ 1 㡯ࡼࡾᢳฟࡋࡓ RNA 1 Pg ┦ᙜ㔞ࢆ Random primer 1 PLࠊ5×RT buffer 4 PLࠊ40 units/PL RNase inhibitor 0.2 PLࠊ10 mM dNTP mixture 2 PLࠊReverTra Ace 1 PL (㔞 20 PL)ࢆ⏝࠸࡚ࠊ30°C ࡛ 10 ศࠊ42°C ࡛ 60 ศࠊ90°C ࡛ 5 ศᛂࡉࡏࠊ㏫ ㌿ᛂࢆ⾜ࡗࡓࠋ. ➨ 3 㡯 PCR ἲࡼࡿ㑇ఏᏊⓎ⌧ࡢ᳨ウ ➨ 3 ❶ࠊ➨ 3 ⠇ࠊ➨ 2 㡯ࡼࡾㄪ〇ࡋࡓ cDNA ⁐ᾮࢆ 1 PL ࢸࣥࣉ࣮ࣞࢺࡋࠊGo Taq Green Master mix 10 PLࠊ50 PM primer (Forward) 0.1 PLࠊ50 PM primer (Reverse) 0.1 PL (㔞 20 PL)ࢆ⏝࠸࡚ PCR ࢆ⾜ࡗࡓࠋPCR ࡣ 95°C 2 ศ (1 cycle)ࠊ95°C 1 ศࠊ58°C 1 ศࠊ72°C 1 ศ (35 cycle)ࠊ72°C 5 ศ (1 cycle)ࡢ᮲௳࡛⾜ࡗࡓࠋᛂ⤊ᚋࠊPCR ⏘≀ࢆ 1%࣮࢞ࣟࢫࢤ࡛ࣝ 100 Vࠊ30 ศὋືࠊ10 ng/mL etidium bromide ࡛ᰁⰍᚋࠊ⣸እග ࡚┿ᙳࡋࡓࠋ ྛ㑇ఏᏊࡢቑᖜࡣ௨ୗࡢ primer ࢆ⏝࠸ࡓࠋ Table. 1 Primer 㓄ิ (F: forward R: reverse 5’ to 3’) Target mRNA. 㻌. 5' to 3'. 18s rRNA. F. TGGTTGCAAAGCTGAAACTTAAAG. (90 bp). R. AGTCAAATTAAGCCGCAGGC. BDNF. F. GAAGGCTGCAGGGGCATAGAC. (135 bp). R. TACACAAGAAGTGTCTATCCTTAT. TrkB. F. ACGATGGTGCAAACCCAAAT. (127 bp). R. CCGGTTTTATCAGTGACGTCTGT. 17.
(19) A2AR. F. GAATCCCTTCATCTACGCCTACC. (136 bp). R. GCCATGAGCTGCCAAGACC. SOD1. F. CTCTCAGGAGACCATTGCATCAT. (134 bp). R. CCAATTACACCACAAGCCAAACG. DNMT3A. F. GCCACCTCTTCGCTCCGCTG. (239 bp). R. GATGATGTCCAACCCTTTTCGCAA. VEGF. F. AGGCCAGCACATAGGAGAGA. (133 bp). R. TTTCTTGCGCTTTCGTTTTT. HSP70. F. AACACCGTGTTTGACGCGAA. (183 bp). R. GGTCAGCACCATGGACGAGA. ARC. F. CCTCCCAGGGGAGAGTAGAA. (237 bp). R. TTTGTGGGAACCTTGAGACC. c-Fos. F. ATGATGTTCTCGGGCTTCAA. (188 bp). R. GTGGGAATGAAGTTGGCACT. EGR1. F. AGCTGGAGGAGATGATGCTG. (246 bp). R. GCACCTTCTCGTTGTTCAGA. HIF-1D. F. CATCAGCTATTTGCGTGTGAGGA. (83 bp). R. AGCAATTCATCTGTGCTTTCATGTC. APP. F. TGCTTAGTTGGTGAGTTTGTAAGTGAT. (110 bp). R. ACGGTGTGCCAGTGAAGATG. BACE1. F. CACCACCAACCTTCGTTTGC. (173 bp). R. TTAGGTAGAGTGAGATGACTGGGAAA. BACE2. F. CAGAAGCTACAGATTCTCGTTGACA. (121 bp). R. AGCCCTTGGAGCGGTATGT. 18.
(20) IDE. F. GTCCTGTTGTTGGAGAGTTCCCATGTCA. (173 bp). R. GGGGAATCTTCAGAGTTTTGCAGCCAT. PHD2. F. CGACCTGATACGCCACTGTA. (182 bp). R. CCTTGGCATCCCAGTCTTTA. VEGFR1. F. AAGCAAACCACACTGGCTTC. (172 bp). R. ACGAGCTCCCTTCCTTCAGT. VEGFR2. F. CCTCTGTGGGTTTGCCTAGT. (160 bp). R. CTTTGCTCACTGCCACTCTG. VEGFR3. F. GAGAGCACCGAGGTCATTGT. (165 bp). R. CAGTGCCTTTCCATCCTTGT. HK2. F. ATCCCTGAGGACATCATGCGA. (81 bp). R. CTTATCCATGAAGTTAGCCAGGCA. GLUT1. F. GGGCAAGTCCTTTGAGATGC. (111 bp). R. AGGGCTGTGGGTGACACTTCA. GDNF. F. GCTGAGCAGTGACTCAAATATG. (120 bp). R. AGCACTGCCATTTGTTTATCTG. NGF. F. ATGGCATGCTGGACCCAAGCTC. (159 bp). R. GGTTCCGCCTGTATGCCGATCA. ➨ 4 ⠇ WST assay ࡼࡿ⣽⬊ẘᛶࡢ᳨ウ 3 ᪥㛫ᇵ㣴ࡋࡓ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆࡑࢀࡒࢀ 10%(v/v)ࡲ࡛ࡢ⃰ᗘ࡛ࠊࡲࡓࢥ࣮ࣄ ࣮ࡢᡂศ࡛࠶ࡿ࢝ࣇ࢙ࣥࠊ࢝ࣇ࢙㓟ࠊࢡࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥࢆࡑࢀࡒࢀ 100 PM ࡲ࡛ࡢ⃰ᗘ࡛ῧຍࡋࠊ24 㛫ᚋࠊPBS(-)࡛ washࠊ10%WST-8 ྵ᭷ medium ࢆຍ࠼ࡓࠋ 37°Cࠊ5% CO2 ࣮ࣥ࢟ࣗ࣋ࢱ࣮࡛ 1.5 㛫ᇵ㣴ࡋࠊ450 nm ࡢ྾ගᗘࢆ. 19. ᐃࡋࡓࠋ.
(21) ➨ 5 ⠇ quantitativeRT-PCR ἲࡼࡿྛ✀㑇ఏᏊࡢⓎ⌧ኚືࡢゎᯒ ➨ 1 㡯 ⣽⬊ࡽࡢ RNA ᢳฟ 3 ᪥㛫ᇵ㣴ࡋࡓ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆῧຍࡋࠊ24 㛫ࡲ࡛ࡢ௵ពࡢ㛫ᚋ PBS(-) ࡛ washࠊ1 mL ࡢ ISOGEN ࡛⣽⬊ࢆᅇࡋࡓࠋ௨ୗ➨ 3 ❶ࠊ➨ 3 ⠇ྠᵝࡢ᪉ἲ࡛ RNA ࢆᢳฟࠊRT ࡼࡾ cDNA ࢆᚓࡓࠋ. ➨ 2 㡯 real-time PCR ࡼࡿⓎ⌧ኚືࡢ᳨ウ ➨ 3 ❶ࠊ➨ 5 ⠇ࠊ➨ 1 㡯ࡢ㏻ࡾㄪ〇ࡋࡓ cDNA ⁐ᾮࢆ 10 ಸᕼ㔘ࡋࡓࠋ2 PL ࢆࢸࣥ ࣉ࣮ࣞࢺࡋࠊSYBR Green master mix 5 PLࠊ50 PM primer (Forward) 0.025 PLࠊ50 PM primer (Reverse) 0.025 PL (㔞 10 PL)ࢆ⏝࠸࡚ PCR ࢆ⾜ࡗࡓࠋPCR ࡣ 95°C 10 ศ (1 cycle)ࠊ95°C 15 ⛊ࠊ60°C 1 ศ (40 cycle)ࡢ᮲௳࡛⾜ࡗࡓࠋྛ㑇ఏᏊࡢቑᖜࡣ Table 1 ࡢ primer ࢆ⏝࠸ࡓࠋྛ㑇ఏᏊⓎ⌧㔞ࡢ⿵ṇࡣ 18s rRNA ࡛⾜ࡗࡓࠋ. ➨ 6 ⠇ G93ASOD1 ኚ␗యⓎ⌧⣽⬊ࡢస〇 ➨ 1 㡯 ࣉࣛࢫ࣑ࢻࡢస〇 ࣞࢺࣟ࢘ࣝࢫⓎ⌧࣋ࢡࢱ࣮MSCV-Puro ཬࡧ MSCV-Puro-N-Flag ࢆ⏝࠸ࡓࠋSOD1 ࢡ ࣮ࣟࣥ cDNA ࡣ⌮Ꮫ◊✲ᡤࡼࡾ㉎ධࡋࡓࠋࡲࡎึࡵࠊไ㝈㓝⣲ㄆ㆑ࢧࢺࢆ⤌ࡳ㎸ࡴ ࡓࡵࠊ௨ୗࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆࣉ࣐࣮ࣛࡋ࡚⏝࠸࡚ PCR ࢆ⾜࠸ࠊTA cloning ࢆ⤒ ࡚ࠊ⭠⳦ᙧ㉁ᑟධࡋࡓࠋ. EcoRI-SOD1-A 5’-CCCGAATTCATGGCGACGAAGGCCGTGTG-3’. XhoI-SOD1-B 5’-CCCCTCGAGTTATTGGGCGATCCCAATTAC-3’. XhoI-C-Flag-SOD1-B 5’-CCCCTCGAGTCACTTGTCATCGTCGTCCTTGTAGTCTTGGGCGATCCCAATTAC-3’. 20.
(22) ᚓࡽࢀࡓࣉࣛࢫ࣑ࢻࡢ㓄ิࢆไ㝈㓝⣲ฎ⌮ࠊSequence ゎᯒࡼࡾ☜ㄆᚋࠊࡑࢀࡒࢀࡢࣉࣛ ࢫ࣑ࢻࢆࢸࣥࣉ࣮ࣞࢺࡋ࡚ Site-Directed Mutagenesis Kit ࢆ⏝࠸࡚ mutagenesis PCR ࡼࡾ SOD1G93A ኚ␗యࢆస〇ࡋࡓࠋ௨ୗ♧ࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆࣉ࣐࣮ࣛࡋ ࡚⏝࠸ࡓࠋ 5’- GACTGCTGACAAAGATGCTGTGGCCGATGTGTC-3’ (sense) 5’- GACACATCGGCCACAGCATCTTTGTCAGCAGTC-3’ (antisense). ➨ 2 㡯 ࣞࢺࣟ࢘ࣝࢫࡢㄪ〇 HEK293T ⣽⬊㸦0.3×105 cells/mLࢆ 10 cm dish ✀ࡋࠊ2 ✀㢮ࡢ࣊ࣝࣃ࣮࣋ࢡࢱ࣮ 㸦pE-Eco helper, pGP helper㸧ࢆྛ 1.3 Pgࠊࣞࢺࣟ࢘ࣝࢫⓎ⌧࣋ࢡࢱ࣮ࢆ 1.4 Pg ࡢィ 4.0 Pg ࢆ 12 PL ࡢ Fugene 6 ࢆ⏝࠸࡚ࢺࣛࣥࢫࣇ࢙ࢡࢺࡋࡓࠋ16 㛫ᇵ㣴ᚋࠊᇵᆅࢆ㝖ࡁࠊ ᪂ࡋ࠸ᇵᆅࢆ 4 mL ຍ࠼ࡓࠋ6 㛫ᚋࣞࢺࣟ࢘ࣝࢫࢆྵࡴᇵ㣴ୖΎࢆᅇࡋࠊ᭦᪂ࡋ ࠸ᇵ㣴ᇵᆅࢆຍ࠼ࡓࠋࡇࡢ᧯సࢆ᭦ 2 ᪥㛫⧞ࡾ㏉ࡋࠊᅇࡋࡓࣞࢺࣟ࢘ࣝࢫྵ᭷ᇵ㣴 ᇵᆅࢆ 0.45 Pm ࡢࣇࣝࢱ࣮࡛ℐ㐣ࡋࠊ-80°C ࡛ಖᏑࡋࡓࠋ. ➨ 3 㡯 ࣞࢺࣟ࢘ࣝࢫឤᰁ ➨ 3 ❶ࠊ➨ 6 ⠇ࠊ➨ 2 㡯࡛ᅇࡋࡓࣞࢺࣟ࢘ࣝࢫྵ᭷ᇵ㣴ᇵᆅ࣏ࣜࣈࣞࣥࢆ 10 Pg/mL ࡞ࡿࡼ࠺ΰࡋࠊSH-SY5Y ⣽⬊㸦0.3×105 cells/mLຍ࠼ࡓࠋ⩣᪥᪂ࡋ࠸ᇵ㣴ᇵᆅ ࡋࠊ᭦ 2 ᪥㛫ᇵ㣴ᚋࠊࢭࣞࢡࢩࣙࣥࢆ⾜ࡗࡓࠋEcotropic receptor Ⓨ⌧⣽⬊ࡢస〇 ࡣ 5 Pg/mL ࡢ Blasticidin ࢆ⏝࠸ࠊSOD1G93A Ⓨ⌧⣽⬊ࡢస〇ࡣ 2 Pg/mL ࡢ Puromycin ࢆ⏝࠸ࡓࠋ. ➨ 4 㡯 immunoblot ἲࡼࡿⓎ⌧☜ㄆ ⣽⬊ࢆ PBS(-)࡛ wash ᚋࠊLysis buffer㸦50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% Nonidet P-40, phosphatase inhibitor cocktail, protease inhibitor. 21.
(23) cocktail㸧ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ⣽⬊⁐ゎᾮࢆ °Cࠊ15,000 r.p.m.࡛ 5 ศ㛫㐲ᚰศ㞳ࡋࠊ ୖΎࢆ⏝࠸࡚ Bradford ἲࡼࡾࢱࣥࣃࢡᐃ㔞ࢆ⾜ࡗࡓࠋࢱࣥࣃࢡ㔞ࢆ୍ᐃࡑࢁ࠼ࠊSDS ࢧࣥࣉࣝ buffer ࢆຍ࠼ࠊ100°C ࡛ 10 ศ㛫ຍ⇕ࡋࡓࠋࡑࢀࡒࢀࡢࢱࣥࣃࢡ 10 Pg ࢆ࣏ࣜࢡ ࣑ࣜࣝࢻࢤࣝ㟁ẼὋື࡛ศ㞳ᚋࠊ500 mA ࡢᐃ㟁ὶ࡛ 90 ศ㛫㏻㟁ࡋࠊPVDF ⭷㌿ࡋ ࡓࠋ㌿ࡋࡓ PVDF ⭷ࢆ 5%ࢫ࣒࣑࢟ࣝࢡ/T-TBS㸦0.05% Tween 2 ྵ᭷ TBS㸧ࢆ⏝࠸࡚ 1 㛫ࣈࣟࢵ࢟ࣥࢢࢆ⾜ࡗࡓࠋᢠయ㸦1:1000㸧ࢆຍ࠼ࡓ 5%ࢫ࣒࣑࢟ࣝࢡ/T-TBS PVDF ⭷ ࢆධࢀࠊ4°C ୍࡛ᬌ㟼⨨ࡋࡓࠋḟ PVDF ⭷ࢆ T-TBS ࡛ 10 ศ㛫Ὑίࡋࠊࡇࢀࢆ 3 ᅇ⧞ࡾ ㏉ࡋࡓࠋ2 ḟᢠయ㸦1:3000㸧ࢆຍ࠼ࡓ 5%ࢫ࣒࣑࢟ࣝࢡ/T-TBS PVDF ⭷ࢆᾐࡋࠊᐊ. ࡛. 1 㛫㟼⨨ࡋࡓࠋࡑࡢᚋ PVDF ⭷ࢆ T-TBS ࡛ 5 ศ㛫Ὑίࡋࠊࡇࢀࢆ 5 ᅇ⧞ࡾ㏉ࡋࡓࠋECL ᛂᾮࢆ⏝࠸࡚┠ⓗࡢࢱࣥࣃࢡࢆྍどࡋࡓࠋ. ➨ 5 㡯 ࢺࣜࣃࣥࣈ࣮ࣝᰁⰍࡼࡿ⣽⬊ቑṪࡢ᳨ウ ⣽⬊ࢆ 0.25×105 cells/mL ࡛✀ࡋࠊ24ࠊ48ࠊ72ࠊ96 㛫ᚋ PBS(-)࡛⣽⬊ࢆ washࠊ 200 PL ࡢ 0.25%ࢺࣜࣉࢩ࣭ࣥEDTA ࡛⣽⬊ࢆࡀࡋࡓࠋᐊ ࢆ 350 PL ຍ࠼ࠊࢺࣜࣃࣥࣈ࣮ࣝᰁⰍࡼࡾ⏕Ꮡ⣽⬊ᩘࢆ. ➨ 6 㡯 SOD1 άᛶࡢ. ࡛ 5 ศᨺ⨨ࡋࠊᇵ㣴⏝ࡢ medium ᐃࡋࡓࠋ. ᐃ. ⣽⬊ࢆ PBS(-)࡛ 2 ᅇ wash ࡋࠊࢭࣝࢫࢡ࣮ࣞࣃ࣮࡛ࡀࡋ࡚ᅇࡋࡓࠋ1,000 g ࡛ 5 ศ㐲 ᚰࡋࠊୖΎࢆྲྀࡾ㝖࠸ࡓᚋࠊ࣍ࣔࢪࢼࢨ࣮ࡼࡾ⣽⬊⭷ࢆ◚○ࡋࡓࠋ᪂ࡓ PBS(-)ࢆ 300 PL ຍ࠼࡚⣽⬊ࢆᠱ⃮ࡋࠊ4°Cࠊ10,000 g ࡛ 15 ศ㛫㐲ᚰࠊୖΎࢆࢧࣥࣉࣝࡋ࡚⏝࠸ࡓࠋ SOD1 ࡢάᛶ. ᐃࡣ SOD1 Assay Kit ࢆ⏝࠸࡚⾜ࡗࡓࠋࡲࡎୖΎࢆ⏝࠸࡚ 5 ಸࡢᕼ㔘⣔ิ. ࢆస〇ࡋࠊࢧࣥࣉࣝࡋࡓࠋࡑࢀࡒࢀࡢࢧࣥࣉࣝ 20 PL ᑐࡋ࡚ WST working solution 200 PLࠊEnzyme working solution 20 PL ࢆຍ࠼ࠊ37°C ࡛ 20 ศᛂࡉࡏࠊ450 nm ࡢ྾ ගᗘࢆ. ᐃࡋࡓࠋࡲࡓࠊbradford ἲࡼࡾࢱࣥࣃࢡ⃰ᗘࢆ⟬ฟࡋࠊࢱࣥࣃࢡ㔞ᙜࡓࡾࡢ SOD1. άᛶ⿵ṇࡋࡓࠋ. 22.
(24) ➨ 7 㡯 ࣃࣛࢥ࣮ࢺࡼࡿ㓟ࢫࢺࣞࢫᑐࡍࡿ⣽⬊ቑṪࡢ᳨ウ ⣽⬊ࢆ 0.5×105 cells/mL ࡛✀ࡋࠊ⩣᪥ࣃࣛࢥ࣮ࢺࢆ 50, 75, 100, 150, 200 PM ࡛ῧຍࡋ ࡓࠋ72 㛫ᚋ PBS(-)࡛ washࠊ௨ୗ➨ 3 ❶ࠊ➨ 6 ⠇ࠊ➨ 5 㡯ྠᵝࡢ᪉ἲ࡛⏕Ꮡ⣽⬊ᩘ ࡘ࠸࡚. ᐃࡋࡓࠋ. ➨ 7 ⠇ HSP70 㑇ఏᏊⓎ⌧ࡢ᳨ウ 0.5×105 cells/mL ࡛✀ࡋࡓ SH-SY5Y ⣽⬊ࢆ 3 ᪥㛫ᇵ㣴ࡋࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ 㸦2.0%(v/v)㸧ࠊࡲࡓࡣࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊࢥ࣮ࣄ࣮ᡂศ㸦100 PM㸧ࠊ ↦ᗘࡢ␗࡞ࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊ࢝ࢸࢥ࣮ࣝ㢮㸦㹼50 PM㸧ࢆῧຍࡋࡓࠋ8 㛫ᚋ PBS(-)࡛ wash ᚋࠊRNA ᢳฟࠊRT ࡼࡾ cDNA ࢆྜᡂࡋࠊreal-time PCR ࡼࡾ Ⓨ⌧ኚືࢆ᳨ウࡋࡓࠋ. ➨ 8 ⠇ BDNF ࡢാࡁᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ➨ 1 㡯 BDNF ࡼࡿ TrkB ࡢࣜࣥ㓟 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ BDNF ࢆ 5ࠊ10ࠊ20 ng/mL ࡛ῧຍࡋࠊ5ࠊ10ࠊ15 ศ ᚋ 150 PL ࡢ Lysis Buffer㸦50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% Nonidet P-40, phosphatase inhibitor cocktail, protease inhibitor cocktail㸧ࢆ ຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ௨ୗ bradford ἲࡼࡾࢱࣥࣃࢡ㔞ࢆࡑࢁ࠼ࠊ50 Pg ࢆ⏝࠸࡚ Immunoblot ἲࡼࡾ TrkB ࡢࣜࣥ㓟ࡘ࠸᳨࡚ウࡋࡓࠋ. ➨ 2 㡯 BDNF/TrkB ࢩࢢࢼࣝᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡲࡓࡣࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆ 2.0%(v/v)࡛ῧຍࡋࠊ2 㛫ᚋ BDNF㸦20 ng/mL㸧ࢆῧຍࠊ0㹼120 ศᚋ 150 PL ࡢ Lysis Buffer ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ⃰ᗘ౫Ꮡᛶཬࡧᡂศࡼࡿᙳ㡪ࢆ᳨ウࡍࡿ㝿ࡣࠊࡑࢀ ࡒࢀ 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ῧຍࡋࠊ2 㛫ᚋ BDNF㸦20 ng/mL㸧ࢆῧຍࠊ. 23.
(25) 15 ศᚋ 150 PL ࡢ Lysis Buffer ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋCREB ࡘ࠸᳨࡚ウࡍࡿ㝿ࡣࠊ 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆῧຍࡋࠊ 2 㛫ᚋ BDNF 㸦20 ng/mL㸧 ࢆῧຍࠊ120 ศᚋ 150 PL ࡢ Lysis Buffer ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ௨ୗ bradford ἲ ࡼࡾࢱࣥࣃࢡ㔞ࢆࡑࢁ࠼ࠊTrkB ࡢ᳨ฟࡣ 50 Pg ࡢࢱࣥࣃࢡࢆ⏝࠸࡚ࠊࡢࢩࢢࢼࣝᅉᏊ ࡢ᳨ฟࡣ 10 Pg ࡢࢱࣥࣃࢡࢆ⏝࠸࡚ Immunoblot ἲࢆ⾜ࡗࡓࠋྛࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟 యࡘ࠸᳨࡚ฟࡍࡿ㝿ࡣ 1%BSA/T-TBS ࢆ⏝࠸࡚ࣈࣟࢵ࢟ࣥࢢࢆ⾜ࡗࡓࠋ. ➨ 3 㡯 BDNF 㑇ఏᏊⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ➨ 3 ❶ࠊ➨ 8 ⠇ࠊ➨ 2 㡯ྠᵝ่⃭ࢆ⾜࠸ࠊBDNF ῧຍᚋ 4 㛫ᚋ⣽⬊ࢆ PBS(-)࡛ washࠊRNA ᢳฟࠊRT ࡼࡾ cDNA ࢆྜᡂࡋࠊreal-time PCR ࡼࡾⓎ⌧ኚືࢆ᳨ウࡋࡓࠋ LY294002㸦10 PM㸧ࡢῧຍࡣ BDNF ῧຍࡢ 1 㛫๓⾜ࡗࡓࠋ. ➨ 4 㡯 ⭷⾲㠃ୖࡢ TrkB Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆ 2.0%(v/v)࡛ῧຍࡋࠊ2 㛫ᚋ ⣽⬊ࢆ PBS(-)࡛ washࠊ500 PL ࡢ 0.25%ࢺࣜࣉࢩ࣭ࣥEDTA ࢆ⏝࠸࡚⣽⬊ࢆᅇࡋࡓࠋᅇ ࡋࡓ⣽⬊ PE ᶆ㆑ࡉࢀࡓᢠ TrkB ᢠయᾮࠊࡲࡓࡣ isotype control ࡋ࡚ PE ᶆ㆑ᢠ࣐ ࢘ࢫ IgG1 ᢠయᾮࢆຍ࠼ࠊon ice ࡛ 20 ศ㛫ᛂࡉࡏࡓࠋᛂ⤊ᚋ⣽⬊ࢆᅇࠊPBS(-)࡛ 2 ᅇ wash ࡋࠊࣇ࣮ࣟࢧࢺ࣓࣮ࢱ࣮ࢆ⏝࠸࡚ 1×104 ಶࡢ⣽⬊ࡘ࠸࡚ゎᯒࡋࡓࠋ ➨ 5 㡯 BDNF-TrkB ⤖ྜ⬟ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 6 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆ 2.0%(v/v)࡛ῧຍࡋࠊ2 㛫ᚋ 10 ng/mL ࡢ human BDNF-Biotin ࢆῧຍࡋࠊon ice ࡛ 45 ศ㛫ᛂࡉࡏࡓࠋIce cold PBS(-) ࡛ wash ᚋࠊStreptavidin-FITC㸦1:2000㸧ࢆຍ࠼ࠊịୖ࡛ 1 㛫ᛂࡉࡏࡓࠋᛂ⤊ᚋ ice cold PBS(-)࡛ 2 ᅇ wash ࡋࠊ⺯ග㢧ᚤ㙾࡛ほᐹࠊࡲࡓࡣࣇ࣮ࣟࢧࢺ࣓࣮ࢱ࣮ࢆ⏝࠸࡚ 1×104 ಶࡢ⣽⬊ࡘ࠸࡚ゎᯒࡋࡓࠋ. 24.
(26) ➨ 6 㡯 BDNF ࡼࡿ⚄⤒✺㉳ࡢఙ㛗ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢຠᯝ 4 ᪥㛫ศࡉࡏࡓ SH-SY5Y ⣽⬊ࢥ࣮ࣄ࣮㇋ᢳฟᾮࢆ 0㹼2.0%(v/v)࡛ῧຍࡋࠊ2 㛫ᚋ BDNF㸦50 ng/mL㸧ࢆῧຍࠊ48 㛫ᚋ㢧ᚤ㙾ࢆ⏝࠸࡚ྛ⃰ᗘ 4 ᡤࡎࡘ┿ࢆᙳࠊ ࡚ࡢ┿ࡘ࠸࡚ࠊ┿ෆࡢ࡚ࡢ⚄⤒✺㉳ࡢ㛗ࡉࡘ࠸࡚ Image J ࢆ⏝࠸࡚. ᐃࡋࡓࠋ. ➨ 9 ⠇ VEGF Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ➨ 1 㡯 VEGF 㑇ఏᏊⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 0.5×105 cells/mL ࡛✀ࡋࡓ SH-SY5Y ⣽⬊ࢆ 3 ᪥㛫ᇵ㣴ࡋࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ 㸦2.0%(v/v)㸧ࠊࡲࡓࡣࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊࢥ࣮ࣄ࣮ᡂศ㸦100 PM㸧ࠊ ↦ᗘࡢ␗࡞ࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊ࢝ࢸࢥ࣮ࣝ㢮㸦㹼50 PM㸧ࢆῧຍࡋࡓࠋDFX 㸦100 PM㸧ࡣ࣏ࢪࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚⏝࠸ࡓࠋῧຍᚋ 8 㛫ᚋ⣽⬊ࢆ PBS(-)࡛ washࠊRNA ᢳฟࠊRT ࡼࡾ cDNA ࢆྜᡂࡋࠊreal-time PCR ࡼࡾⓎ⌧ኚືࢆ᳨ウࡋࡓࠋ ࢚࢟ࣀ࣐ࢩࣥ㸦5 nM㸧ࠊU0126㸦5 PM㸧ࠊSB202190㸦10 PM㸧ࡢῧຍࡣࢥ࣮ࣄ࣮㇋ᢳฟ ᾮῧຍࡢ 1 㛫๓⾜ࡗࡓࠋ. ➨ 2 㡯 VEGF ศἪ㔞ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 0.5×105 cells/mL ࡛✀ࡋࡓ SH-SY5Y ⣽⬊ࢆ 3 ᪥㛫ᇵ㣴ࡋࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦㹼 2.0%(v/v)㸧ࢆῧຍࡋࡓࠋ12 㛫ᚋࠊᇵ㣴ୖΎࢆᅇࡋࠊHuman VEGF-A Platinum ELISA ࢟ࢵࢺࢆ⏝࠸࡚ ELISA ἲࡼࡾ VEGF ศἪ㔞ࢆ᳨ウࡋࡓࠋࡲࡎࢳ࣮ࣗࣈࢆ 2 ᅇὙίࡋࠊ Sample Diluent ࢆ 50 PL ࡎࡘຍ࠼ࠊࡑࡇࢧࣥࣉࣝࢆ 50 PL ࡎࡘຍ࠼ࡓࠋᐊ. ࡛ 2 㛫. ┞ࡋࠊwash buffer ࡛ 6 ᅇὙίࡋࡓࠋḟ Biotin-Conjugate ࢆ 100 PL ࡎࡘຍ࠼ࠊᐊ. ࡛1. 㛫┞ࡋࡓࠋWash buffer ࡛ 6 ᅇὙίᚋࠊStreptavidin-HRP ࢆ 100 PL ࡎࡘຍ࠼ࠊᐊ ࡛ 1 㛫┞ࡋࡓࠋWash buffer ࡛ 6 ᅇὙίᚋࠊTMB Substrate Solution ࢆ 100 PL ࡎࡘ ຍ࠼ࠊ㐽ගࡋ࡚ 30 ศᐊ ࢆ. ࡛ᛂࡋࠊStop Solution ࢆ 100 PL ࡎࡘຍ࠼ࠊ450 nm ࡢ྾ගᗘ. ᐃࡋࡓࠋ. 25.
(27) ➨ 3 㡯 HIF-1DࢱࣥࣃࢡⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 0.5×105 cells/mL ࡛✀ࡋࡓ SH-SY5Y ⣽⬊ࢆ 3 ᪥㛫ᇵ㣴ࡋࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ 㸦2.0%(v/v)㸧ࠊࡲࡓࡣࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊࢥ࣮ࣄ࣮ᡂศ㸦100 PM㸧ࠊ ↦ᗘࡢ␗࡞ࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦2.0%(v/v)㸧ࠊ᭷ᶵ⁐፹ศ⏬ᢳฟ≀㸦2.0%(v/v)┦ᙜ㸧ࠊSep Pak C18 ศ⏬ᢳฟ≀㸦2.0%(v/v)┦ᙜ㸧ࠊ࢝ࢸࢥ࣮ࣝ㢮㸦㹼50 PM㸧࡛ῧຍࡋࡓࠋDFX㸦100 PM㸧ࡣ࣏ࢪࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚⏝࠸ࡓࠋῧຍᚋ 4 㛫ᚋ 150 PL ࡢ Lysis Buffer ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࡓࠋࡲࡓࢩࢢࢼࣝᅉᏊࡢⓎ⌧ኚືࡢ᳨ウࡣࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮῧຍ ᚋ 0㹼180 ศᚋࡢࢧࣥࣉࣝࢆ⏝࠸ࡓࠋ᰾ࢱࣥࣃࢡࡢᢳฟࡣ⣽⬊ࢆ PBS(-)࡛ᅇᚋࠊ400 PL ࡢ A buffer㸦10 mM HEPES-KOH(pH 7.8), 10 mM KCl, 0.1 mM EDTA(pH 8.0), 0.1% NP40, phosphatase inhibitor cocktail, protease inhibitor cocktail㸧ࢆຍ࠼࡚⣽⬊ࢆ⁐ゎࡋࠊ 5,000 r.p.m.࡛ 2 ศ㛫㐲ᚰศ㞳ࡋࠊ࣌ࣞࢵࢺࢆ 150 PL ࡢ Lysis Buffer ࡛⁐ゎࡋࡓࠋ᭦㉸ 㡢Ἴ◚○ࡼࡾ࣌ࣞࢵࢺࢆ◚○ࡋࠊ°Cࠊ15,000 r.p.m.࡛ 5 ศ㛫㐲ᚰศ㞳ࡋࠊୖΎࢆ᰾ࢱࣥ ࣃࢡࡋ࡚ᅇࡋࡓࠋᅇᚋࠊbradford ἲࡼࡾࢱࣥࣃࢡ㔞ࢆࡑࢁ࠼ࠊ10 Pg ࡢࢱࣥࣃࢡࢆ ⏝࠸࡚ Immunoblot ἲࢆ⾜ࡗࡓࠋྛࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟యࡘ࠸᳨࡚ฟࡍࡿ㝿ࡣ 1%BSA/T-TBS ࢆ⏝࠸࡚ࣈࣟࢵ࢟ࣥࢢࢆ⾜ࡗࡓࠋU0126㸦5 PM㸧ࠊSB202190㸦10 PM㸧ࠊ SP200125㸦10 PM㸧ࠊwortmannin㸦10 nM㸧ࠊLY294002㸦10 PM㸧ࠊDHMEQ㸦10 Pg/mL㸧 ࡢῧຍࡣࢥ࣮ࣄ࣮㇋ᢳฟᾮῧຍࡢ 1 㛫๓⾜ࡗࡓࠋ. ➨ 4 㡯 HIF-1Dࡢࣄࢻࣟ࢟ࢩࣝᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 0.5×105 cells/mL ࡛✀ࡋࡓ SH-SY5Y ⣽⬊ࢆ 3 ᪥㛫ᇵ㣴ࡋࠊMG132㸦10 PM㸧ࢆῧຍ ࡋࡓࠋ1 㛫ᚋࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸦0㹼2.0%(v/v)㸧ࡲࡓࡣ PG㸦50 PM㸧ࠊCTC㸦10 PM㸧ࠊ HHQ㸦25 PM㸧ࢆῧຍࡋࡓࠋῧຍᚋ 4 㛫ᚋࡲ࡛ 150 PL ࡢ Lysis Buffer ࢆຍ࠼࡚⣽⬊ ࢆ⁐ゎࡋࠊbradford ἲࡼࡾࢱࣥࣃࢡ㔞ࢆࡑࢁ࠼ࠊ10 Pg ࡢࢱࣥࣃࢡࢆ⏝࠸࡚ Immunoblot ἲࢆ⾜ࡗࡓࠋ. 26.
(28) ➨ 5 㡯 PHD άᛶᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 PHD ࡢάᛶࡣ[5-14C]-D-ࢣࢺࢢࣝࢱࣝ㓟ࡽ⏕ᡂࡉࢀࡿ[1-14C]ࢥࣁࢡ㓟ࡢ㔞ࢆᐃ㔞ࡍࡿ ࡇ࡛. ᐃࡋࡓࠋṧᏑࡋࡓ[5-14C]-D-ࢣࢺࢢࣝࢱࣝ㓟ࡣࢪࢽࢺࣟࣇ࢙ࢽࣝࣄࢻࣛࢰࣥࡋ࡚. ỿẊࡉࡏࠊྲྀࡾ㝖࠸ࡓࠋSH-SY5Y ⣽⬊ࢆ Lysis Buffer ࡛⁐ゎࡋࠊbradford ἲࡼࡾࢱࣥࣃ ࢡ㔞ࢆࡑࢁ࠼ࡓࠋ4 Pg ࡢࢱࣥࣃࢡ㉁ࢆ 0.5 mM DTTࠊ50 PM ◲㓟ࣥࣔࢽ࣒࢘㕲(ϩ)ࠊ1 mM ࢫࢥࣝࣅࣥ㓟ࠊ2 mg/mL BSAࠊ0.4 mg/mL ࢝ࢱ࣮ࣛࢮࠊ0.3 mM [5-14C]-D-ࢣࢺࢢ ࣝࢱࣝ㓟(1700 Bq)ࠊ40 mM Tris-HCl(pH 7.5)ࠊ50 PM (Pro-Pro-Gly)10×9H2O peptide ࢆྵ ࡴᛂᾮ㸦᭱⤊ 25PL㸧࡛ 37°Cࠊ10 ศᛂࡉࡏࡓࠋᛂ⤊ᚋࠊ20 mM D-ࢣࢺࢢࣝࢱࣝ 㓟-20 mM ࢥࣁࢡ㓟⁐ᾮࢆ 25 PLࠊ0.16 M 2,4-ࢪࢽࢺࣟࣇ࢙ࢽࣝࣄࢻࣛࢪࣥ㸦30% HClO4㸧 ⁐ᾮࢆ 25 PL ࡎࡘຍ࠼ࠊᐊ ຍ࠼ࠊᐊ. ࡛ 30 ศ㛫ᛂࡉࡏࡓࠋࡉࡽ 1 M D-ࢣࢺࢢࣝࢱࣝ㓟ࢆ 50 PL. ࡛ 30 ศ㛫ᛂࡉࡏࠊ3,000 g ࡛ 5 ศ㐲ᚰࡋࠊୖΎ 100 PL ୰ࡢᨺᑕάᛶࢆࠊᾮయ. ࢩࣥࢳ࣮ࣞࢩࣙࣥ࢝࢘ࣥࢱ࣮ࢆ⏝࠸࡚. ᐃࡋࡓ[46,47]ࠋ. ➨ 10 ⠇ ⤫ィⓗゎᯒ Student’s t ᳨ᐃࡼࡾ⤫ィⓗゎᯒࢆ⾜࠸ࠊp<0.05 ࢆ᭷ព࡞ኚࡋࡓࠋ. 27.
(29) ➨ 4 ❶ ᐇ㦂⤖ᯝ ➨ 1 ⠇ ྛ⚄⤒⣔⣽⬊࠾ࡅࡿ⚄⤒ಖㆤ㛵㐃㑇ఏᏊࡢⓎ⌧᳨ウ IMR-32ࠊSH-SY5YࠊNB-1 ࡢࡑࢀࡒࢀࡢ⚄⤒⣽⬊࠾࠸࡚ࠊBDNFࠊTrkBࠊA2ARࠊSOD1ࠊ TDP43ࠊFUS ࡢ㑇ఏᏊⓎ⌧ࡘ࠸᳨࡚ウࡋࡓࠋIMR-32 ࡣ TDP43 ࡢⓎ⌧ࡀపࡃࠊNB-1 ࡣ TrkB ࡢⓎ⌧ࡀపࡗࡓ㸦Fig.4㸧ࠋSH-SY5Y ࡣࡍ࡚ࡢ㑇ఏᏊࡢⓎ⌧ࡀ☜ㄆ࡛ࡁࡓࠋ. ➨ 2 ⠇ ࢥ࣮ࣄ࣮㇋ᢳฟᾮཬࡧࢥ࣮ࣄ࣮ᡂศࡢ⚄⤒⣽⬊ᑐࡍࡿẘᛶ IMR-32ࠊSH-SY5YࠊNB-1 ࡢࡑࢀࡒࢀࡢ⚄⤒⣽⬊ࡘ࠸࡚ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮཬࡧࢥ࣮ ࣄ࣮ᡂศ࡛࠶ࡿ࢝ࣇ࢙ࣥࠊ࢝ࣇ࢙㓟ࠊࢡࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥࡢ 24 㛫᭚㟢࡛ࡢ ẘᛶࡘ࠸࡚ࠊWST assay ࡼࡾ᳨ウࡋࡓࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡘ࠸࡚ࡣࠊIMR-32 ࡛ 1.25%(v/v)ࡽࠊSH-SY5Y ⣽⬊࡛ࡣ 5%(v/v)ࡽࠊNB-1 ⣽⬊࡛ࡣ 2.5%(v/v)ࡽẘᛶࡀぢ ࡽࢀࡓ㸦Fig.5A, C, E㸧ࠋ୍᪉ࢥ࣮ࣄ࣮ᡂศࡘ࠸࡚ࡣࠊࢥ࣮ࣄ࣮୰ྵࡲࢀࡿࡼࡾࡶ㧗࠸ ⃰ᗘ㸦100 PM㸧࡛ࡶẘᛶࡣぢࡽࢀ࡞ࡗࡓ㸦Fig.5B, D, F㸧 ࠋ. ➨ 3 ⠇ SOD1G93A Ⓨ⌧ SH-SY5Y ⣽⬊ࡢస〇ཬࡧኚ␗యⓎ⌧ࡢᙳ㡪 ALS ࡢཎᅉ㑇ఏᏊࡋ࡚ SOD1 㑇ఏᏊࡢ G93A ኚ␗ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[48]ࠋࡑࡇ࡛ࠊ SOD1G93A ࢆⓎ⌧ࡋࡓ ALS ࡢࣔࢹࣝ⣽⬊ࢆస〇ࡍࡿࡇࡋࡓࠋኚ␗యࢆⓎ⌧ࡉࡏࡿ⣽ ⬊ࡋ࡚ࡣࠊBDNFࠊTrkBࠊA2AR ࡀⓎ⌧ࡋ࡚࠾ࡾ㸦Fig.4㸧ࠊࡲࡓูࡢ ALS ཎᅉ㑇ఏᏊ ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ FUS ࡢኚ␗యⓎ⌧⣽⬊ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[49]SH-SY5Y ⣽⬊ࢆ⏝࠸ࡓࠋ ࢥࣥࢺ࣮ࣟࣝ⣽⬊ࠊtag ࡞ࡋࡢ⣽⬊㸦SOD1WTࠊSOD1G93A㸧ࠊN ᮎ Flag tag ࢆࡘࡅࡓ ⣽⬊㸦SOD1WT-N-FlagࠊSOD1G93A-N-Flag㸧ࠊC ᮎ Flag tag ࢆࡘࡅࡓ⣽⬊㸦SOD1WTC-FlagࠊSOD1G93A-C-Flag㸧ࡢ 7 ✀㢮ࡢ⣽⬊ࢆస〇ࡋࡓࠋࡇࡢ 7 ✀㢮ࡢ⣽⬊ࡘ࠸࡚⣽⬊ ቑṪࢆ᳨ウࡋࡓࡇࢁࠊC-Flag ࡢ 2 ✀㢮ࡢ⣽⬊࡛ቑṪࡀపୗࡋࠊࡲࡓ N-Flag ࡢ 2 ✀㢮ࡢ ⣽⬊࡛ࡶⱝᖸࡢቑṪᢚไࡀ☜ㄆࡉࢀࡓ㸦Fig.6B㸧ࠋSOD1G93A ࡢኚ␗࡛ࡣ SOD άᛶࡀኚ ࡋ࡞࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ[50]ࠊࢥࣥࢺ࣮ࣟࣝࡢ⣽⬊ẚ࡚ SOD1 ࢆᑟධࡋࡓ⣽⬊. 28.
(30) ࡛ࡣኚ␗ࡢ᭷↓ࢃࡽࡎࠊάᛶࡢୖ᪼ࡀ☜ㄆ࡛ࡁࡓ㸦Fig.6C㸧ࠋࡇࢀࡲ࡛ࡢ⤖ᯝࢆ㋃ࡲ ࠼ࠊᚋࡢ᳨ウࡣ tag ࡞ࡋࡢ⣽⬊ࢆ⏝࠸ࡿࡇࡋࡓࠋḟ SOD1 ኚ␗యⓎ⌧⣽⬊࡛ࡣ 㓟ࢫࢺࣞࢫ⪏ᛶࡀపୗࡍࡿሗ࿌ࡉࢀ࡚࠸ࡿࡓࡵ[51]ࠊࣃࣛࢥ࣮ࢺឤཷᛶࡘ࠸᳨࡚ウࡋ ࡓࠋࡋࡋࠊࣃࣛࢥ࣮ࢺឤཷᛶࡣኚࡀぢࡽࢀ࡞ࡗࡓ㸦Fig.6D㸧ࠋࡑࡢ୍᪉࡛ࠊศᏊࢩ ࡛ࣕ࣌ࣟࣥ࠶ࡿ HSP70 ࡢ㑇ఏᏊⓎ⌧ࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊSOD1G93A ࡛ SOD1WT ẚ࡚᭷ព࡞Ⓨ⌧ࡢୖ᪼ࡀぢࡽࢀࡓ㸦Fig.6E㸧ࠋࡲࡓࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ่⃭ࡼࡿ HSP70 㑇ఏᏊⓎ⌧ኚࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾⓎ⌧ࡣୖ᪼ࡋࠊࡑࡢୖ᪼ ࡣ SOD1G93A ࡛ࡼࡾࡁࡗࡓ㸦Fig.6F㸧ࠋ. ➨ 4 ⠇ HSP70 Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ๓⠇ࡢ⤖ᯝࡽࠊSH-SY5Y ⣽⬊࠾࠸࡚ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ HSP70 㑇ఏᏊⓎ⌧ࡀ ㄏᑟࡉࢀࡿࡇࡀࢃࡗࡓࠋࡑࡇ࡛ࠊHSP70 Ⓨ⌧ㄏᑟࡢάᛶᡂศࡘ࠸᳨࡚ウࡋࡓࠋࡲࡎࠊ ࢥ࣮ࣄ࣮㇋ᢳฟᾮ(2.0%(v/v))่⃭ᚋࡢ⤒ኚࢆㄪࡓࡇࢁࠊ่⃭ᚋ 6-9 㛫ࢆࣆ࣮ࢡ Ⓨ⌧ࡀୖ᪼ࡋࠊ15 㛫ᚋ௨㝆࡛ࡣඖࡢࣞ࣋ࣝࡲ࡛ᡠࡗࡓ㸦Fig.7A㸧ࠋࡑࡇ࡛ࠊ௨ᚋࡢ HSP70 㑇ఏᏊⓎ⌧ࡢ᳨ウࡣ่⃭ᚋ 8 㛫ᚋ࡛⾜࠺ࡇࡋࡓࠋḟࠊࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ ࡛ࡶྠᵝࡢኚࡀぢࡽࢀࡿ᳨ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ࡣ⏝㔞౫Ꮡⓗ࡞Ⓨ⌧ㄏ ᑟࡀぢࡽࢀࡓࡢᑐࡋࠊࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ࡣࢇኚࡀぢࡽࢀࡎࠊࢥ࣮ࣄ࣮ ㇋ᢳฟᾮࡼࡿⓎ⌧ㄏᑟࡣ࢝ࣇ࢙ࣥ࡞ࠊࢹ࢝ࣇ࢙ฎ⌮ࡼࡾྲྀࡾ㝖ࢀࡿᡂศࡀᚲ せ࡛࠶ࡿࡇࡀࢃࡗࡓ㸦Fig.7B㸧ࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿㄏᑟࡀࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳ ฟᾮ୰ࡢᡂศ࢝ࣇ࢙ࣥࡀ┦స⏝ࡍࡿࡇࡼࡿྍ⬟ᛶࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊ ࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ༢⊂ࡢ่⃭ࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ㸩࢝ࣇ࢙ࣥࡢ่⃭ ࡛ㄏᑟࡢࡁࡉࢇኚࡣぢࡽࢀࡎ࢝ࣇ࢙ࣥࡀࡢᡂศ┦స⏝ࡍࡿࡇࡼ ࡿኚ࡛ࡣ࡞࠸ࡇࡀ᫂ࡽ࡞ࡗࡓ㸦Fig.7C㸧ࠋḟࠊࢥ࣮ࣄ࣮ᡂศ࡛࠶ࡿ࢝ࣇ࢙ࣥࠊ ࢝ࣇ࢙㓟ࠊࢡࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥࡼࡿᙳ㡪ࢆぢࡓࡇࢁࠊ࢝ࣇ࢙㓟ࢺࣜࢦࢿࣜࣥ ࡢ่⃭࡛ㄏᑟࡀぢࡽࢀࡓࡀࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ⛬ࡁ࡞ኚࡣぢࡽࢀࡎࠊ⏝࠸ࡓ⃰ᗘ㸦100. 29.
(31) PM㸧ࡣࢥ࣮ࣄ࣮2.0%(v/v)௨ୖ┦ᙜࡍࡿࡢ࡛ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿⓎ⌧ㄏᑟࡣࡇࢀࡽࡢ ᡂศࡢ௨እࡢᡂศࡼࡿࡶࡢ࡛࠶ࡿ⪃࠼ࡽࢀࡿ㸦Fig.7D㸧ࠋࡑࡇ࡛ HSP70 ㄏᑟάᛶࢆᣢ ࡘᡂศࡘ࠸࡚᭦᳨ウࡍࡿࡓࡵࠊ↦ᗘࡢ␗࡞ࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿᙳ㡪ࢆ᳨ウࡋ ࡓࠋࡑࡢ⤖ᯝࠊ⏕㇋ᢳฟᾮࡢ่⃭࡛ࡣኚࡣぢࡽࢀࡎࠊ↦ࡋࡓࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ࡣⓎ⌧ ㄏᑟࡀぢࡽࢀࡓࡇࡽࠊHSP70 Ⓨ⌧ㄏᑟᡂศࡣ↦ࡼࡾ⏕ᡂࡉࢀࡿᡂศ࡛࠶ࡿ⪃࠼ ࡽࢀࡿ㸦Fig.7E㸧ࠋ࢝ࢸࢥ࣮ࣝ㢮࡛࠶ࡿࣆ࣮ࣟ࢞ࣟࣝ㸦PG㸧ࡸࣆࣟ࢝ࢸࢥ࣮ࣝ㸦CTC㸧ࠊࣄ ࢻࣟ࢟ࢩࣄࢻࣟ࢟ࣀࣥ㸦HHQ㸧ࡣ↦ࡼࡾࢡࣟࣟࢤࣥ㓟ࡀຍỈศゎࡉࢀ࡚⏕ᡂࡍࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[52]ࠋࡑࡇ࡛ࡇࢀࡽࡢᡂศࡼࡿⓎ⌧ኚࢆ᳨ウࡋࡓࡇࢁࠊHHQ ࡢ่ ⃭࡛ᖜⓎ⌧ࡀㄏᑟࡉࢀࡓ㸦Fig.7F㸧ࠋࡇࡢ⤖ᯝࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ୰ࡢ HSP70 ㄏ ᑟάᛶᡂศࡣ HHQ ࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ. ➨ 5 ⠇ BDNF ࡢാࡁᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ➨ 1 㡯 ATRA ࡼࡿ SH-SY5Y ⣽⬊ࡢศ ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ⚄⤒ಖㆤຠᯝࡘ࠸࡚ࠊBDNF ᑐࡍࡿᙳ㡪ࡼࡿྍ⬟ᛶࡘ ࠸᳨࡚ウࡋࡓࠋᮍศࡢ SH-SY5Y ⣽⬊ࡣ BDNF ࡢཷᐜయ࡛࠶ࡿ TrkB ࡀ࠶ࡲࡾⓎ⌧ࡋ࡚ ࠸࡞࠸ࡓࡵࠊATRA ࡼࡾศࡉࡏࠊTrkB ࡢⓎ⌧ࢆㄏᑟࡋࡓ[53]ࠋATRA ่⃭ࡼࡾࠊᇵ 㣴᪥ᩘ౫Ꮡⓗ TrkB ࡢ㑇ఏᏊࠊࢱࣥࣃࢡࡢⓎ⌧ࡀඹୖ᪼ࡋ࡚࠸ࡿࡇࡀ☜ㄆ࡛ࡁࡓ 㸦Fig.8A, B㸧ࠋࡲࡓࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡣศᑐࡋ࡚ࡣᙳ㡪ࢆ࠼࡞ࡗࡓ㸦Fig.8C㸧ࠋ. ➨ 2 㡯 BDNF ࡼࡿ TrkB ࢩࢢࢼࣝࡢάᛶᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ḟ BDNF ࡼࡿ TrkB ࢩࢢࢼࣝࡢάᛶᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪ࢆ᳨ウࡋࡓࠋ ࡲࡎࠊBDNF ⃰ᗘཬࡧ᭚㟢㛫ࡘ࠸࡚ỴࡵࡿࡓࡵࠊBDNF ࡼࡿ TrkB ࡢࣜࣥ㓟ࡘ ࠸᳨࡚ウࡋࡓࠋࡑࡢ⤖ᯝࠊBDNF ࡢ 20ng/mL ࡲ࡛ࡢ⃰ᗘ౫Ꮡⓗࠊࡲࡓ 15 ศࡲ࡛ࡢ㛫౫ Ꮡⓗ࡞ TrkB ࡢࣜࣥ㓟ࡀ☜ㄆࡉࢀ㸦Fig.8D㸧ࠊ௨ᚋࡢᐇ㦂ࡣ 20 ng/mL ࡢ BDNF15 ศ᭚㟢 ࡛⾜࠺ࡇࡋࡓࠋࡇࡢ᮲௳࡛ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪ࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊTrkB ࡢ. 30.
(32) ࣜࣥ㓟ࡣ 516 ␒┠ࡢࢳࣟࢩࣥ㸦Y516㸧ࠊ816 ␒┠ࡢࢳࣟࢩࣥ㸦Y816㸧ࡶᢚไࡉࢀࡓ 㸦Fig.9A㸧ࠋࡲࡓ BDNF ࡼࡿ Akt ࡢࣜࣥ㓟ࡣ TrkB ྠᵝᢚไࡉࢀࡓࡢᑐࡋࠊERK1/2 ࡢࣜࣥ㓟ࡣࢥ࣮ࣄ࣮㇋ᢳฟᾮ༢⊂࡛ቑຍࡋࡓ㸦Fig.9A㸧ࠋྠᵝࡢᙳ㡪ࢆࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮ ㇋ᢳฟᾮࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ⛬ࡁࡃࡣ࡞࠸ࡶࡢࡢࠊTrkBࠊAkt ࡢ ࣜࣥ㓟ࡢᢚไࠊཬࡧ ERK1/2 ࡢࣜࣥ㓟ࡢቑࡀ☜ㄆ࡛ࡁࡓ㸦Fig.9B㸧ࠋࡲࡓࠊࢥ࣮ࣄ࣮ ㇋ᢳฟᾮࡼࡿኚࡣ࠸ࡎࢀࡶࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗ࡛࠶ࡿࡇࡀ☜ㄆ࡛ࡁࡓ㸦Fig.9C㸧ࠋࢥ ࣮ࣄ࣮㇋ᢳฟᾮ୰ࡢάᛶᡂศࡘ࠸᳨࡚ウࡍࡿࡓࡵࠊࢥ࣮ࣄ࣮ᡂศ࡛࠶ࡿ࢝ࣇ࢙ࣥࠊ࢝ ࣇ࢙㓟ࠊࢡࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥࡘ࠸࡚ྠᵝࡢ᳨ウࢆ⾜ࡗࡓࡀࠊࡇࢀࡽࡢᡂศ࡛ࡣ BDNF ࡼࡿ TrkB ࡢࣜࣥ㓟ࡣᙳ㡪ࡀぢࡽࢀࡎࠊࡇࢀࡽࡢᡂศࡼࡿάᛶ࡛ࡣ࡞࠸ࡇ ࡀ♧ࡉࢀࡓ㸦Fig.9E㸧ࠋ BDNF/TrkB ࢩࢢࢼࣝࡢάᛶࡼࡾ BDNF ࡢ㑇ఏᏊⓎ⌧ࡀୖ᪼ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ[54]ࠋࡑࡇ࡛ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ BDNF 㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࠋ ࡲࡎ BDNF ่⃭ࡼࡿ BDNF 㑇ఏᏊⓎ⌧ࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊ่⃭ᚋ 4 㛫ࢆࣆ࣮ ࢡⓎ⌧ࡀୖ᪼ࡋࡓ㸦Fig.10A㸧ࠋࡇࡢ BDNF ࡼࡿ BDNF Ⓨ⌧ㄏᑟࡣࠊࢥ࣮ࣄ࣮㇋ᢳฟ ᾮཬࡧࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ᢚไࡉࢀ㸦Fig.10B㸧ࠊࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗ࡛࠶ࡾ 㸦Fig.10C㸧ࠊࢥ࣮ࣄ࣮ᡂศࡣⓎ⌧ᢚไάᛶࡣ࡞࠸ࡇࡀࢃࡗࡓࠋ㸦Fig.10D㸧ࡇࢀࡽ ࡣ TrkB ࡢࣜࣥ㓟ᑐࡍࡿኚྠᵝ࡛࠶ࡗࡓࠋࡲࡓࠊ↦ࡼࡿᙳ㡪ࢆぢࡓࡇࢁࠊ⏕ ㇋ࡣᢚไຠᯝࡣ࡞ࡃࠊ↦ࡼࡾ⏕ᡂࡉࢀࡿᡂศࡀᢚไάᛶࢆᣢࡘࡇࡀ♧ࡉࢀࡓ 㸦Fig.10E㸧ࠋBDNF ࡢ㑇ఏᏊⓎ⌧ไᚚࡣࠊERK1/2/CREB ࡢࢩࢢࢼࣝ Akt ࡢࢩࢢࢼࣝ ࡢࡘࡀሗ࿌ࡉࢀ࡚࠸ࡿ[8]ࠋᮏ◊✲ࡢ⤖ᯝࡼࡾࠊBDNF/TrkB ࢩࢢࢼࣝࡢୗὶࡢ㌿ᅉᏊ ࡛࠶ࡿ CREB ࡢࣜࣥ㓟ࡣࢥ࣮ࣄ࣮㇋ᢳฟᾮ༢⊂ࡢῧຍ࡛ಁ㐍ࡉࢀࡿࡇࡀ♧ࡉࢀࡓ 㸦Fig.9D㸧ࠋPI3K/Akt ࡢ㜼ᐖ࡛࠶ࡿ LY294002 ࡛ฎ⌮ࡋࡓࡇࢁࢥ࣮ࣄ࣮㇋ᢳฟᾮྠᵝ BDNF ࡼࡿⓎ⌧ㄏᑟࡀᢚไࡉࢀࡓ㸦Fig.10F㸧ࠋ ḟࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ BDNF/TrkB ࢩࢢࢼࣝᢚไࡢ࣓࢝ࢽࢬ࣒ࡘ࠸᳨࡚ウࡍ ࡿࡓࡵࠊ⭷⾲㠃ୖࡢ TrkB Ⓨ⌧ࡢኚࡘ࠸᳨࡚ウࡋࡓࡀࠊTrkB ࣏ࢪࢸࣈ࡞⣽⬊ࡢྜ. 31.
(33) ࡣࢥ࣮ࣄ࣮㇋ᢳฟᾮῧຍࡢ᭷↓࡛ᕪࡣぢࡽࢀ࡞ࡗࡓ㸦Fig.11㸧ࠋࡑࡇ࡛ࠊࢥ࣮ࣄ࣮㇋ᢳฟ ᾮࡀ BDNF TrkB ࡢ⤖ྜᙳ㡪ࢆ࠼࡚࠸ࡿྍ⬟ᛶࡘ࠸᳨࡚ウࡋࡓࡀࠊBDNF-TrkB ࡢ⤖ྜࡶࢥ࣮ࣄ࣮㇋ᢳฟᾮࡣࡁ࡞ᙳ㡪ࢆ࠼࡞ࡗࡓ㸦Fig.12A, B㸧ࠋ SH-SY5Y ⣽⬊ࡣ BDNF ࡢ่⃭ࡼࡾࠊ⚄⤒✺㉳ࢆఙ㛗ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[8]ࠋ ࡑࡇ࡛ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ⚄⤒✺㉳ࡢఙ㛗ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊ⚄⤒✺㉳ ࡢఙ㛗ࡣࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗᢚไࡉࢀࡓ㸦Fig.13㸧ࠋ. ➨ 6 ⠇ VEGF Ⓨ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ➨ 1 㡯ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ VEGF Ⓨ⌧ࡢᙳ㡪ཬࡧࡑࡢ࣓࢝ࢽࢬ࣒ゎᯒ ࢥ࣮ࣄ࣮ࡼࡿ⚄⤒ಖㆤຠᯝࡢ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽࡍࡿࡓࡵࠊVEGF ࡢⓎ⌧ᑐࡍࡿ ᙳ㡪ࡘ࠸᳨࡚ウࡋࡓࠋࡲࡎ VEGF 㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮ ῧຍᚋ 6-9 㛫ᚋࢆࣆ࣮ࢡ VEGF 㑇ఏᏊⓎ⌧㔞ࡀୖ᪼ࡋࠊ24 㛫ᚋࡣඖࡢⓎ⌧ࣞ࣋ࣝ ᡠࡗࡓ㸦Fig.14A㸧ࠋ௨ᚋࡢ VEGF 㑇ఏᏊⓎ⌧ࡢ᳨ウࡣ࡚ࢥ࣮ࣄ࣮㇋ᢳฟᾮ่⃭ᚋ 8 㛫ᚋ⾜ࡗࡓࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿⓎ⌧ㄏᑟࡣࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗ࡛࠶ࡗࡓࡀࠊࢹ࢝ࣇ ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ࡣⓎ⌧ㄏᑟࡣࢇぢࡽࢀ࡞ࡗࡓ㸦Fig.14B㸧ࠋࡉࡽ VEGF ࡢ ศἪ㔞ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗቑຍࡍࡿࡇࡀ♧ࡉࢀࡓ 㸦Fig.14C㸧ࠋ ḟࠊVEGF 㑇ఏᏊⓎ⌧ㄏᑟࡢ࣓࢝ࢽࢬ࣒ࢆ᳨ウࡍࡿࡓࡵࠊVEGF 㑇ఏᏊࡢ㌿ᅉᏊ ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ HIF-1DࡢⓎ⌧ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡣῧຍᚋ⣙ 60 ศᚋࡽ HIF-1DࢱࣥࣃࢡⓎ⌧ࢆㄏᑟࡋࠊ3 㛫ᚋࡀࣆ࣮ࢡࡔࡗࡓ㸦Fig.15A, B㸧ࠋHIF1Dࡢㄏᑟࡣࢥ࣮ࣄ࣮⏝㔞౫Ꮡⓗ࡛࠶ࡗࡓࡢᑐࡋࠊࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢῧຍ࡛ࡣ ࢥ࣮ࣄ࣮㇋ᢳฟᾮῧຍ⛬ࡁ࡞ㄏᑟࡣぢࡽࢀ࡞ࡗࡓ㸦Fig.15C㸧ࠋHIF-1DࡣࡑࡢⓎ⌧ࡀୖ ᪼ࡍࡿ᰾ෆ⛣⾜ࠊᶆⓗ㑇ఏᏊࡢ㌿ࢆάᛶࡍࡿ[36]ࠋࡑࡇ࡛᰾ෆࡢάᛶᆺ HIF-1D ࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮῧຍᚋ 30 ศᚋࡽⓎ⌧ࡢୖ᪼ࡀぢࡽࢀࡓ 㸦Fig.15D㸧ࠋ୍᪉ࠊHIF-1Dࡢ㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊᙳ㡪ࡣぢࡽࢀࡎࠊ. 32.
(34) ࢥ࣮ࣄ࣮ࡼࡿ HIF-1DⓎ⌧ㄏᑟࡀ㌿ᚋㄪ⠇ࡼࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸦Fig.15E㸧ࠋHIF1Dࡢ DNA ⤖ྜ㜼ᐖ࡛࠶ࡿ࢚࢟ࣀ࣐ࢩࣥࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊࢥ࣮ࣄ࣮㇋ᢳ ฟᾮࡼࡿⓎ⌧ㄏᑟࡣ࢚࢟ࣀ࣐ࢩࣥࡢฎ⌮ࡼࡾᢚไࡉࢀࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ VEGF Ⓨ⌧ㄏᑟࡀ HIF-1Dࡼࡿࡇࡀ♧ࡉࢀࡓ㸦Fig.15F㸧ࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ HIF1Dࡀάᛶࡉࢀ࡚࠸ࡿࡇࢆ᭦☜ㄆࡍࡿࡓࡵࠊHIF-1Dࢆ㌿ᅉᏊࡍࡿ Glucose transporter 1㸦GLUT1㸧ཬࡧ Hexokinase 2㸦HK2㸧ࡢ㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋ ࡓࡇࢁࠊVEGF ྠᵝ 6 㛫ࢆࣆ࣮ࢡࡋࡓⓎ⌧ㄏᑟࡀぢࡽࢀ㸦Fig.16A, B㸧ࠊࡲࡓࡑࡢㄏ ᑟࡣ࢚࢟ࣀ࣐ࢩࣥࡢฎ⌮ࡼࡾᢚไࡉࢀࡓ㸦Fig.16C, D㸧ࠋ. ➨ 2 㡯 HIF-1D/VEGF ㄏᑟάᛶࢆᣢࡘࢥ࣮ࣄ࣮ᡂศࡢྠᐃ ࢥ࣮ࣄ࣮㇋ᢳฟᾮ୰ࡢ HIF-1Dࡢㄏᑟάᛶᡂศࢆ᳨ウࡍࡿࡓࡵࠊࡲࡎ࢝ࣇ࢙ࣥ༢⊂࡛ࡢ ຠᯝࢆ᳨ウࡋࡓࡀࠊኚࡣぢࡽࢀ࡞ࡗࡓ㸦Fig.17A, B㸧ࠋࡑࡇ࡛ࠊ࢝ࣇ࢙ࣥࡀࢹ࢝ࣇ࢙ ࢥ࣮ࣄ࣮㇋ᢳฟᾮ୰ࡢࡢᡂศ┦స⏝ࡋ࡚ຠᯝࢆⓎࡋ࡚࠸ࡿྍ⬟ᛶࡘ࠸᳨࡚ウࡋ ࡓࠋࡋࡋࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳฟᾮ࢝ࣇ࢙ࣥࢆඹῧຍࡋ࡚ࡶࢹ࢝ࣇ࢙ࢥ࣮ࣄ࣮㇋ᢳ ฟᾮ༢⊂ࡢῧຍࡁ࡞ኚࡣぢࡽࢀࡎࠊ࢝ࣇ࢙ࣥࡀࡢᡂศ┦స⏝ࡋ࡚࠸ࡿࡢ࡛ ࡣ࡞࠸♧ࡉࢀࡓ㸦Fig.17A, B㸧ࠋḟࠊࢥ࣮ࣄ࣮ᡂศ࡛࠶ࡿ࢝ࣇ࢙ࣥࠊ࢝ࣇ࢙㓟ࠊࢡ ࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥࠊ࣮࢙࣮࢝࢘࢜ࣝࠊ࢝ࣇ࢙ࢫࢺ࣮ࣝࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࡀࠊ࠸ ࡎࢀࡢᡂศࡶ HIF-1Dㄏᑟάᛶࢆᣢࡓ࡞ࡗࡓ㸦Fig.17C-E㸧ࠋάᛶᡂศࡘ࠸࡚᭦᳨ウࡍ ࡿࡓࡵࠊࢥ࣮ࣄ࣮㇋ࡢ↦ࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࠋ⏕㇋ᢳฟᾮ࡛ࡣኚࡀぢࡽࢀ࡞ࡗࡓࡀࠊ ↦ࡋࡓࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢῧຍࡼࡾࠊⓎ⌧ࡀㄏᑟࡉࢀࡓ㸦Fig.18B, C㸧ࠋḟࠊάᛶࡀ ☜ㄆ࡛ࡁࡓ῝↦ࡾࢥ࣮ࣄ࣮㇋ᢳฟᾮࡘ࠸࡚ࠊ➼㔞ࡢ࣊࢟ࢧࣥࠊ㓑㓟࢚ࢳࣝࠊ1-ࣈࢱࣀ࣮ࣝ ࡛㡰ḟศ⏬ࡋࠊࡢศ⏬άᛶᡂศᅇࡉࢀࡿࢆ᳨ウࡋࡓ㸦Fig.19A㸧ࠋάᛶᡂศࡣ㓑㓟 ࢚ࢳࣝᅇࡉࢀࡓ㸦Fig.19B㸧ࠋ⥆࠸࡚ࠊࡇࡢ㓑㓟࢚ࢳࣝ⏬ศࢆࠊ୰ᛶ࣒࡛࢝ࣛ࠶ࡿ Sep Pak C18 ࢆ⏝࠸࡚᭦ 3 ࡘࡢ⏬ศศ⏬ࡋࡓ㸦Fig.20A㸧ࠋࡑࡢ⤖ᯝࠊάᛶᡂศࡣ 20% ࣓ࢱࣀ࣮࡛ࣝᅇࡉࢀࡿ R2 ⏬ศᅇࡉࢀࡓ㸦Fig.20B㸧ࠋHPLC ศᯒࡼࡾࡇࡢ⏬ศࡣ. 33.
(35) ࢝ࣇ࢙ࣥࠊཬࡧࣆࣟ࢝ࢸࢥ࣮ࣝࡀྵࡲࢀ࡚࠸ࡿࡇࡀࢃࡗࡓ㸦Fig.20C㸧ࠋ࢝ࣇ࢙ࣥࡣ ༢⊂࡛ࡣ HIF-1Dㄏᑟάᛶࢆ♧ࡉ࡞ࡗࡓࡢ࡛㸦Fig.17D㸧ࠊࣆࣟ࢝ࢸࢥ࣮ࣝࡀάᛶᡂศ࡛࠶ ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ࠊࣆࣟ࢝ࢸࢥ࣮ࣝ㸦CTC㸧ཬࡧ࢝ࢸࢥ࣮ࣝ㢮࡛࠶ࡿࣆࣟ࢞ࣟ ࣮ࣝ㸦PG㸧ࠊࣄࢻࣟ࢟ࢩࣄࢻࣟ࢟ࣀࣥ㸦HHQ㸧ࡼࡿᙳ㡪ࡘ࠸᳨࡚ウࡋࡓ㸦Fig.21A㸧ࠋ PG ཬࡧ CTC ࡢ่⃭࡛ࡣ VEGF 㑇ఏᏊⓎ⌧ࠊHIF-1DࢱࣥࣃࢡⓎ⌧ࡶୖ᪼ࡋࡓࡀࠊHHQ ࡢ่⃭࡛ࡣ HIF-1DࡢࢱࣥࣃࢡⓎ⌧ࡣኚࡀぢࡽࢀࡎࠊVEGF 㑇ఏᏊⓎ⌧ࡢࡳㄏᑟࡀぢࡽ ࢀࡓ㸦Fig.21B,C㸧ࠋࡲࡓࠊHPLC ศᯒࡼࡾ࢝ࢸࢥ࣮ࣝ㢮ࡀྵࡲࢀ࡚࠸࡞࠸ࡗࡓⰼ⋤ࡢ࣊ ࣝࢩࢥ࣮ࣄ࣮ࡢ่⃭࡛ࡣࠊHIF-1DࡢⓎ⌧ㄏᑟࡀぢࡽࢀ࡞ࡗࡓ㸦Fig.21D㸧ࠋ. ➨ 3 㡯 ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ HIF-1Dㄏᑟ࣓࢝ࢽࢬ࣒ࡢゎᯒ ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ HIF-1Dㄏᑟ࣓࢝ࢽࢬ࣒ࡘ࠸᳨࡚ウࡍࡿࡓࡵࠊࡲࡎྛ✀ࢩࢢࢼ ࣝఏ㐩⣔ࡢ㜼ᐖࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ HIF-1Dࡢㄏᑟࡣ. ERK1/2 ࡢ㜼ᐖ U0126ࠊp38 ࡢ㜼ᐖ SB202190 ࡢฎ⌮ࡼࡾⱝᖸᢚไࡉࢀࠊNFNB ࡢ㜼ᐖ࡛࠶ࡿ DHMEQ ࡢฎ⌮ࡼࡾᢚไࡉࢀࡓ㸦Fig.22A㸧ࠋU0126 ཬࡧ SB202190 ࡣ VEGF ࡢⓎ⌧ㄏᑟࡶᢚไࡋ㸦Fig.22B㸧ࠊ᭦ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡣ ERK1/2 ཬࡧ p38 ࡢࣜࣥ㓟ࢆಁ㐍ࡋࡓࡇࡽ㸦Fig.22C㸧ࠊࡇࢀࡽࡢࢩࢢࢼࣝࢆࡋ࡚ HIF1Dࢆㄏᑟࡋ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋ୍᪉ࠊNFNB ࡢࣜࣥ㓟ᑐࡋ࡚ࡣᙳ㡪ࡀぢࡽࢀ࡞ ࡗࡓ㸦Fig.22C㸧ࠋ HIF-1Dࡣ㏻ᖖ㓟⣲ศᅽ࡛ࡣ 402 ␒┠ 564 ␒┠ࡢࣉࣟࣜࣥṧᇶࡀࣄࢻࣟ࢟ࢩࣝࢆཷ ࡅࡿࡇࡼࡾࣘࣅ࢟ࢳࣥࢆཷࡅࠊࣉࣟࢸࢯ࣮࣒౫Ꮡⓗ࡞ศゎࢆཷࡅ࡚࠸ࡿࠋࡑࡇ࡛ࠊ ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿࠊHIF-1Dࡢࣄࢻࣟ࢟ࢩࣝᑐࡍࡿຠᯝࡘ࠸᳨࡚ウࡋࡓࠋࣉࣟ ࢸࢯ࣮࣒㜼ᐖ࡛࠶ࡿ MG132 ࡢฎ⌮ࡼࡾࣄࢻࣟ࢟ࢩࣝ HIF-1Dࡀ᳨ฟࡉࢀࠊࢥ࣮ࣄ ࣮㇋ᢳฟᾮࡼࡾ⏝㔞౫Ꮡⓗῶᑡࡋࡓ㸦Fig.23A㸧ࠋMG132 ฎ⌮ࡼࡾ㛫౫Ꮡⓗ࡞ࣄࢻ ࣟ࢟ࢩࣝ HIF-1DࡢⓎ⌧ୖ᪼ࡀ☜ㄆ࡛ࡁࡓࡀࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ࡛ฎ⌮ࡍࡿࣄࢻࣟ࢟ࢩ ࣝ HIF-1DࡢⓎ⌧ࡣኚࡋ࡞ࡗࡓ㸦Fig.23B㸧ࠋࡲࡓࠊࣆࣟ࢝ࢸࢥ࣮ࣝࡶ HIF-1Dࡢࣄࢻ. 34.
(36) ࣟ࢟ࢩࣝࢆ㜼ᐖࡋࡓ㸦Fig.23C㸧ࠋࡋࡋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡣ⣽⬊ࡢ PHD2 Ⓨ⌧ࡣᙳ㡪 ࡋ࡞ࡗࡓ㸦Fig.23D㸧ࠋ᭦ in vitro ࡛ࡢ PHD 㓝⣲άᛶࡶᙳ㡪ࢆ࠼࡞ࡗࡓ㸦Fig.23E㸧ࠋ ࡑࡇ࡛ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢῧຍࡢࠊPHD2 ᛂࡢ⿵㓝⣲࡛࠶ࡿ Fe2+ࡸࢫࢥࣝࣅࣥ㓟 ᑐࡍࡿຠᯝࢆ᳨ウࡋࡓ[55]ࠋࡑࡢ⤖ᯝࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ HIF-1DࡢⓎ⌧ㄏᑟࡣ Fe2+ࠊ ࢫࢥࣝࣅࣥ㓟ࠊ࢝ࢱ࣮ࣛࢮ࡞ࡢ⿵࡛ᢚไࡉࢀࡓ㸦Fig.24A, C, E㸧ࠋࣆࣟ࢝ࢸࢥ࣮ࣝ ࡼࡿ HIF-1Dࡢㄏᑟࡶࠊྠᵝᢚไࡉࢀࡓ㸦Fig.24B, D, F㸧ࠋ. ➨ 7 ⠇ AD 㛵㐃㑇ఏᏊⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪 ᪥ᖖⓗ࡞ࢥ࣮ࣄ࣮ࡢᦤྲྀࡼࡾࠊAD ࡢࣜࢫࢡࡀపୗࡍࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ[4]ࠋࡑࡇ࡛ࠊ SH-SY5Y ⣽⬊࠾ࡅࡿ AD 㛵㐃㑇ఏᏊࡋ࡚▱ࡽࢀࡿE-ࢭࢡࣞࢱ࣮ࢮ㸦beta-site APP cleaving enzyme1; BACE1㸧ࡸ࣑ࣟࢻ๓㥑ࢱࣥࣃࢡ㸦amyloid precursor protein; APP㸧ࠊ ࣥࢩࣗࣜࣥศゎ㓝⣲㸦insulin degrading enzyme; IDE㸧ࠊࡑࡋ࡚⚄⤒ᰤ㣴ᅉᏊ࡛࠶ࡿ NGFࠊ TrkBࠊBDNF ࡢ㑇ఏᏊⓎ⌧ᑐࡍࡿࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢᙳ㡪ࢆ᳨ウࡋࡓࠋBACE1 ࡸ APP ࡣࡁ࡞ኚࡣぢࡽࢀ࡞ࡗࡓࡀ㸦Fig.25A, B㸧ࠊIDE ཬࡧ NGF ࡢ㑇ఏᏊⓎ⌧ࡣ่⃭ᚋ 6㹼12 㛫ࢆࣆ࣮ࢡㄏᑟࡉࢀ㸦Fig.25C, D㸧ࠊBDNF ࡸ TrkB ࡘ࠸࡚ࡶ่⃭ᚋ 15㹼21 㛫࡛ㄏᑟഴྥࡀぢࡽࢀࡓ㸦Fig.25E, F㸧ࠋBACE1 ࡘ࠸࡚ࡣ㑇ఏᏊⓎ⌧ࡣኚࡀぢࡽࢀ ࡞ࡗࡓ୍᪉࡛ࠊࢱࣥࣃࢡⓎ⌧ࡣῧຍᚋ 12 㛫ࢆࣆ࣮ࢡῶᑡࡋࡓ㸦Fig.26㸧ࠋ. 35.
(37) 36.
(38) 37.
(39) 38.
(40) 39.
(41) 40.
(42) 41.
(43) 42.
(44) 43.
(45) 44.
(46) 45.
(47) 46.
(48) 47.
(49) 48.
(50) 49.
(51) 50.
(52) 51.
(53) 52.
(54) 53.
(55) 54.
(56) 55.
(57) 56.
(58) 57.
(59) 58.
(60) 59.
(61) 60.
(62) ➨ 5 ❶ ⪃ᐹ ➨ 1 ⠇ SOD1G93A Ⓨ⌧⣽⬊ཬࡧ HSP70 㑇ఏᏊⓎ⌧ኚࡘ࠸࡚ ALS ࡢཎᅉ㑇ఏᏊࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ SOD1 㑇ఏᏊࡢ G93A ኚ␗యࡢⓎ⌧⣽⬊ࢆゎᯒ ࡋࡓࠋSOD1 ࡢኚ␗ࡘ࠸࡚ࡣάᛶࡀపୗࡍࡿኚ␗ኚࡋ࡞࠸ኚ␗ࡀሗ࿌ࡉࢀ࡚࠾ࡾ [50]ࠊG93A ࡢኚ␗ࡣάᛶࡣᙳ㡪ࡀ࡞࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋᮏ◊✲ࡢ⤖ᯝ࡛ࡶᑟධࡋ ࡓ SOD1 άᛶࡀ࠶ࡿࡇࡀ☜ㄆ࡛ࡁࡓ㸦Fig.6C㸧ࠋ୍᪉ࠊSOD1G93A ኚ␗యⓎ⌧⣽⬊࡛ ࡣ㓟ࢫࢺࣞࢫࡢឤཷᛶࡀୖ᪼ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ[51]ࠊࣃࣛࢥ࣮ࢺࡢឤཷᛶࡘ ࠸࡚ࡣኚࡀぢࡽࢀ࡞ࡗࡓࡇࡽࠊᮏ◊✲࡛ࡣ㓟ࢫࢺࣞࢫ⪏ᛶࡢపୗࡣぢࡽࢀ࡞ ࡗࡓ㸦Fig.6D㸧ࠋࣃࣛࢥ࣮ࢺࡣ SOD1 ࡢᇶ㉁࡛࠶ࡿࢫ࣮ࣃ࣮࢜࢟ࢩࢻࢽ࢜ࣥࣛࢪ࢝ࣝࢆⓎ ⏕ࡉࡏࡿࡇ࡛㓟ࢫࢺࣞࢫࢆ࠼ࡿࡀࠊᮏ◊✲࡛ G93A ኚ␗࡛ࡣάᛶኚࡀ࡞࠸ࡇ ࡀࢃࡗ࡚࠾ࡾࠊࣃࣛࢥ࣮ࢺࡼࡿ㓟ࢫࢺࣞࢫ࡛ࡣᙳ㡪ࡀฟ࡞ࡗࡓࡇࡀ⪃࠼ࡽࢀࡿࠋ ሗ࿌ࡉࢀ࡚࠸ࡿ㓟ࢫࢺࣞࢫ⪏ᛶࡢపୗࡣࠊH2O2 ࡼࡿ㓟ࢫࢺࣞࢫࡢ⪏ᛶ࡛࠶ࡿࡓࡵࠊ ࡢ㓟ࢫࢺࣞࢫ࡛ᙳ㡪ࡀฟࡿࠊ᭦᳨ウࡋ࡚࠸ࡃᚲせࡀ࠶ࡿࠋ ୍᪉ࠊG93A ⣽⬊࡛ࠊHSP70 㑇ఏᏊࡢⓎ⌧ୖ᪼ࡀ☜ㄆ࡛ࡁࠊ᭦ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢῧ ຍࡼࡾ HSP70 㑇ఏᏊⓎ⌧ࡀㄏᑟࡉࢀࡿࡇࠊG93A ⣽⬊࡛ࡣࡑࡢㄏᑟࡀࡼࡾᙉ࠸ࡇࢆ ぢฟࡋࡓ㸦Fig.6E,F㸧ࠋHSP70 ࡣศᏊࢩࣕ࣌ࣟࣥࡋ࡚ࡢᶵ⬟ࢆᣢࡕࠊኚᛶࡋࡓࢱࣥࣃࢡ㉁ ࡸࣇ࢛࣮ࣝࢹࣥࢢẁ㝵࡛ၥ㢟ࡀ⏕ࡌࠊᶵ⬟࡛ࡁ࡞࠸ࢱࣥࣃࢡ㉁⤖ྜࡋࠊࢱࣥࣃࢡࡢಟ ࡸኚᛶᢚไാࡃ[56]ࠋࡲࡓࠊಟࡀྍ⬟࡞ࢱࣥࣃࢡ㉁㛵ࡋ࡚ࡣࣘࣅ࢟ࢳࣥࢆࡋࠊ ࣉࣟࢸࢯ࣮࣒ࡼࡿศゎᑟࡃࠋALS ࡸ ADࠊPD ࡣ␗ᖖࢱࣥࣃࢡࡀ✚ࡍࡿࣇ࢛࣮ࣝ ࢹࣥࢢ࡛࠶ࡿ⪃࠼ࡽࢀ࡚࠸ࡿ࠾ࡾࠊHSP70 ࡇࢀࡽࡢᝈࡘ࠸࡚ࡶࡇࢀࡲ࡛ሗ ࿌ࡀࡉࢀ࡚࠸ࡿࠋHSP70 ࢆ⚄⤒⣽⬊㧗Ⓨ⌧ࡉࡏࡿ AEࡼࡿẘᛶࡀ㍍ῶࡉࢀࡿࡇࡸ [57]ࠊAD ࣔࢹ࣐ࣝ࢘ࢫ࠾࠸࡚ࠊHSP70 ࡢ㧗Ⓨ⌧ࡼࡾㄆ▱ᶵ⬟㞀ᐖࡢపୗࡀᨵၿࡉࢀࠊ AEࣞ࣋ࣝཬࡧ⚄⤒⣽⬊ࡢ⬺ⴠࡀపୗࡍࡿࡇ[58]ࠊHSP70 ࡼࡾD-ࢩࢾࢡࣞࣥࡼࡿ⚄ ⤒ẘᛶࡀ㍍ῶࡉࢀࡿࡇ[59]ࠊHSP70 ࡢ㐣Ⓨ⌧ࡼࡾ ALS ࣔࢹ࣐ࣝ࢘ࢫࡀᘏࡋࠊึ௦ ᇵ㣴㐠ືࢽ࣮ࣗࣟࣥ࠾ࡅࡿࢱࣥࣃࢡจ㞟ࡀῶᑡࡍࡿࡇ[60]ࠊ࡞ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࢥ. 61.
(63) ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ HSP70 Ⓨ⌧ࡀୖ᪼ࡋࡓࡇࡽࠊHSP70 ࡢୖ᪼ࢆࡋ࡚ AD ࡸ PDࠊ ALS ࡞ࡢࣇ࢛࣮ࣝࢹࣥࢢᑐࡋ࡚ண㜵ຠᯝࢆⓎࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ ᚋࠊࢥ࣮ࣄ࣮ᡂศࡼࡿ HSP70 ࡢ㑇ఏᏊⓎ⌧ࡢゎᯒࡢࡳ࡛ࡣ࡞ࡃࠊࢱࣥࣃࢡⓎ⌧ࡸࢩࣕ࣌ ࣟࣥࡢάᛶࡘ࠸࡚ࠊࡲࡓࡑࡢㄏᑟάᛶࢆᣢࡘᡂศࡘ࠸᳨࡚ウࡋ࡚࠸ࡃࡇࡀᚲせ࡛࠶ ࡿࠋ. ➨ 2 ⠇ BDNF ࡢാࡁᑐࡍࡿᙳ㡪ࡘ࠸࡚ ᮏ◊✲ࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡀ TrkB ࡢࣜࣥ㓟ࡢᢚไࢆࡋ࡚ BDNF/TrkB ࢩࢢࢼ ࣝࢆㄪ⠇ࡍࡿࡇࡀ♧ࡉࢀࡓ㸦Fig.9A,C㸧ࠋࡑࡢ୍᪉࡛ࠊ㏆ᖺ⚄⤒ಖㆤຠᯝࢆ᭷ࡍࡿ࠸ࢃ ࢀ࡚࠸ࡿ࢝ࣇ࢙ࣥࡸࢡࣟࣟࢤࣥ㓟ࠊࢺࣜࢦࢿࣜࣥ࡞ࡣ BDNF/TrkB ࢩࢢࢼࣝᙳ㡪ࢆ ࠼࡞ࡗࡓ㸦Fig.9E㸧[61,62]ࠋࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ BDNF/TrkB ࢩࢢࢼࣝᢚไࡢ࣓ ࢝ࢽࢬ࣒ࡘ࠸࡚ゎ᫂ࡍࡿࡣࠊࡼࡾヲ⣽࡞ᡂศࡢྠᐃࡀᚲせ࡛࠶ࡿࠋ TrkB ࡢࣜࣥ㓟ᢚไࡢ࣓࢝ࢽࢬ࣒ࡋ࡚ࡣࠊ⭷⾲㠃ୖࡢ TrkB Ⓨ⌧ࡸ BDNF-TrkB ࡢ⤖ ྜࡣᙳ㡪ࡀぢࡽࢀ࡞ࡗࡓࡇࡽ㸦Fig.11,12㸧ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ୰ࡢᡂศࡀձ⣽⬊ෆ ࡛ TrkB ࡢ⮬ᕫࣜࣥ㓟ࢆᢚไࡍࡿࠊղ࣍ࢫࣇࢱ࣮ࢮάᛶࢆୖ᪼ࡉࡏࡿࠊ࡞ࡢྍ⬟ᛶࡀ ⪃࠼ࡽࢀࡿࠋ࣍ࢫࣇࢱ࣮ࢮάᛶ㛵ࡋ࡚ࡣࠊProtein-tyrosine Phosphatase 1B㸦PTP1B㸧 ࡀ BDNF-TrkB ࢩࢢࢼࣝࡢάᛶࢆไᚚࡋࠊ⚄⤒✺㉳ࡢఙ㛗ࢆᢚไࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ ࡿ[63]ࠋࢥ࣮ࣄ࣮ᡂศࡼࡾࠊࡇࡢ㓝⣲άᛶࡀୖ᪼ࡋ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࠊᚋゎᯒࡍ ࡿᚲせࡀ࠶ࡿࠋ ࡇࢀࡲ࡛ BDNF ࡢ㌿ᅉᏊࡋ࡚ࠊERK1/2 ࢩࢢࢼࣝࢆࡋࡓ CREB ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [64]ࠋࡋࡋࠊᮏ◊✲ࡢ⤖ᯝࡽࠊBDNF ࡼࡿ CREB ࡢࣜࣥ㓟ࡣࢥ࣮ࣄ࣮㇋ᢳฟᾮ ࡼࡾᢚไࡉࢀࡎࠊࡲࡓ BDNF 㠀Ꮡᅾୗ࡛ࡶࢥ࣮ࣄ࣮㇋ᢳฟᾮ༢⊂࡛ࣜࣥ㓟ࡀಁ㐍ࡉࢀࡿ ࡇࡀࢃࡗࡓ㸦Fig.9D㸧ࠋࡇࡢ⤖ᯝࡼࡾࠊᮏ◊✲࡛ぢࡽࢀࡓ BDNF 㑇ఏᏊⓎ⌧ࡢไᚚࡣ CREB ௨እࡢ㌿ᅉᏊࡀ㛵ࡋ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋBDNF ࡢ㌿ᅉᏊࡋ࡚ࡣ CREB ࡢ Neuron-restrictive silencer factor 㸦NRSF㸧ࡸࢢࣝࢥࢥࣝࢳࢥࢻཷᐜయ. 62.
(64) 㸦GR㸧࡞ࡢྍ⬟ᛶࡶ BDNF 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ⤖ྜྍ⬟࡞㌿ᅉᏊࡋ࡚♧ ࡉࢀ࡚࠾ࡾࠊࡇࢀࡽࡢ㌿ᅉᏊࡀࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾᙳ㡪ࢆཷࡅ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ ࡽࢀࡿࠋ ᮏ◊✲ࡼࡾࠊࢥ࣮ࣄ࣮୰ࡢάᛶᡂศࡀ྾ࡉࢀ⬻฿㐩ࡍࡿ௬ᐃࡍࡿࠊ᪥ᖖⓗ࡞ࢥ࣮ ࣄ࣮ࡢᦤྲྀࡼࡾ BDNF/TrkB ࢩࢢࢼࣝࡀᢚไࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡇ ࡢࡇࡣࢥ࣮ࣄ࣮ࡀ⚄⤒ಖㆤస⏝ࢆ♧ࡍ࠸࠺Ꮫ◊✲ࡣ┦ࡍࡿ⤖ᯝ࡛࠶ࡾࠊࢥ࣮ࣄ ࣮ࡢస⏝Ⅼࡀ BDNF/TrkB ࢩࢢࢼࣝ௨እ࠶ࡿ⪃࠼ࡽࢀࡿࠋ. ➨ 3 ⠇ VEGF Ⓨ⌧ᑐࡍࡿᙳ㡪ࡘ࠸࡚ ᮏ◊✲ࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ HIF-1Dࡢάᛶࢆࡋ࡚ VEGF Ⓨ⌧ࡀㄏᑟࡉࢀ ࡿࡇ㸦Fig.14A,C㸧ࠊࡲࡓࡑࢀࡀࣉࣟࣜࣥࣄࢻࣟ࢟ࢩࣝࡢᢚไࡼࡿࡇࡀ♧ࡉࢀࡓ 㸦Fig.23A㸧ࠋࡉࡽࡑࡢάᛶᡂศࡘ࠸࡚࢝ࢸࢥ࣮ࣝ㢮࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸦Fig.21 B,C,23C㸧ࠋ࢝ࢸࢥ࣮ࣝ 㢮ࡣᮏ◊✲ࡢ HPLC ศ ᯒ⤖ᯝࡽࡶ↦ࡼ ࡾᢳฟᾮ୰ࡢ⃰ᗘࡀୖ ᪼ࡍࡿࡇࡀ☜ㄆࡉࢀ ࡚࠾ࡾ㸦Fig.19A㸧ࠊ↦ ࡼࡾࢡࣟࣟࢤࣥ㓟 ࡽຍỈศゎࡋ࡚⏕ᡂࡍ ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ ࡿ㸦Fig.27㸧[52]ࠋࡲࡓࠊ ⰼ⋤࣊ࣝࢩࢥ࣮ࣄ࣮࡛ࡣㄏᑟࡀぢࡽࢀ࡞ࡗࡓࡀ㸦Fig.21D㸧ࠊᙜヱࢥ࣮ࣄ࣮ࡣࢡࣟࣟࢤ ࣥ㓟ࡢຠᯝࢆᙉࡃⓎࡉࡏࡿࡓࡵ HHQ ࢆάᛶⅣฎ⌮ࡼࡾ㝖ཤࡋ࡚࠾ࡾࠊࡇࡢ㐣⛬࡛ ࡢ࢝ࢸࢥ࣮ࣝ㢮ࡶྲྀࡾ㝖ࢀ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋࢹ࢝ࣇ࢙ฎ⌮ࡣࢫࢫᘧỈᢳฟ. 63.
(65) ἲ࠸࠺᪉ἲ࡛⾜ࢃࢀ࡚࠸ࡿࡀࠊࡇࡢ㐣⛬࡛ࡶ࢝ࢸࢥ࣮ࣝ㢮ࡀྲྀࡾ㝖ࢀࡿࡇࡀ HPLC ศᯒࡼࡾ☜ㄆࡉࢀࡓ㸦Fig.20D㸧ࠋ௨ୖࡢ⤖ᯝࡽࠊ࢝ࢸࢥ࣮ࣝ㢮ࡀάᛶᡂศ࡛࠶ࡿࡇ ࡀᙉࡃ♧၀ࡉࢀࡓࠋ࢝ࢸࢥ࣮ࣝࡸࣆ࣮ࣟ࢞ࣟࣝࡣ⾑ᾮ⬻㛵㛛ࢆ㏻㐣ࡋࠊ⬻ෆ฿㐩ࡍࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠾ࡾ[65]ࠊࡇࢀࡽࡢ࢝ࢸࢥ࣮ࣝ㢮ࡀ⬻ෆ⛣⾜ࡋࠊ⚄⤒ಖㆤຠᯝࢆⓎࡋ࡚࠸ ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ HIF-1Dㄏᑟ࣓࢝ࢽࢬ࣒ࡘ࠸࡚ࡣࠊࣉࣟࣜࣥࣄࢻࣟ࢟ࢩࣝࡀᢚไࡉࢀࡿࡇࡣ♧ࡋࡓ ࡀࠊPHD2 ࡣᙳ㡪ࡀ࡞ࡃࠊ⿵ᅉᏊࡢ⿵ࡼࡾⓎ⌧ㄏᑟࡀᢚไࡉࢀࡓࡇࡽ㸦Fig.24AF㸧ࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡀ⿵ᅉᏊᙳ㡪ࢆ࠼࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋROS ࡣ Fe2+ࠊ ࢫࢥࣝࣅࣥ㓟ࠊ࢝ࢱ࣮ࣛࢮࡢ㝖ཤാࡃ≀㉁࡛࠶ࡿࡇࠊࡲࡓࢥ࣮ࣄ࣮[66]ࡸ࢝ࢸࢥ࣮ࣝ㢮 [67]ࡼࡿ ROS ࡢ⏘⏕ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇࡽࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡸ࢝ࢸࢥ࣮ࣝࡢῧຍ ࡼࡾ ROS ࡀ⏘⏕ࡋࠊPHD2 ࡢάᛶࢆ㜼ᐖࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࠋ ࡲࡓࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿ HIF-1DࡢㄏᑟࡣࠊERK1/2 ࡸ p38 ࡞ࡢ MAPK ⣔ࡀ 㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡶ♧ࡉࢀࡓ㸦Fig.22A,B㸧ࠋMAPK ࡼࡿ HIF-1D㌿άᛶࡘ࠸࡚ ࡣࠊࡇࢀࡲ࡛ in vitro ࡛ࠊERK1/2 ࡸ p38 ࡼࡾ HIF-1Dࡢࣜࣥ㓟ࡀஹ㐍ࡍࡿࡇࡸ[68,69]ࠊ ERK1/2 ࡸ p38 ࡢ㜼ᐖࡼࡾ HIF-1Dไᚚࡉࢀࡓ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢⓎ⌧ࡀపୗࡍࡿࡇ [70]ࠊ࡞ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋERK1/2 ࡸ p38 ࡢ㜼ᐖࡼࡾ VEGF 㑇ఏᏊⓎ⌧ࡢㄏᑟࡣ ᢚไࡉࢀࡓࡀࠊࡑࡢᢚไࡣ࢚࢟ࣀ࣐ࢩࣥࢆฎ⌮ࡋࡓࡼࡾࡶᑠࡉࡃࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮ ࡼࡿㄏᑟࡣࡇࢀࡽࡢࢩࢢࢼࣝࠊ๓㏙ࡢ⿵ᅉᏊࡢ㜼ᐖࡢ୧᪉ࡀ㛵ࡋ࡚࠸ࡿ⪃࠼ࡽࢀ ࡿࠋDHMEQ ࡢฎ⌮ࡼࡾ HIF-1DࡢⓎ⌧ㄏᑟࡀᢚไࡉࢀࡓࡢᑐࡋࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢ ῧຍ࡛ࡣ NFNB ࡢࣜࣥ㓟ࡣ☜ㄆ࡛ࡁ࡞ࡗࡓ㸦Fig.22A,C㸧ࠋDHMEQ ࡣ NFNB ࡢ᰾⛣ ⾜ࡢ㜼ᐖ࡛࠶ࡿࡀࠊ㏆ᖺࠊNrf2 ࡢάᛶാࡃࡇࡶሗ࿌ࡉࢀ࡚࠾ࡾ[71]ࠊNFNB ࡣ ูࡢ⤒㊰࡛ࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡿⓎ⌧ㄏᑟࢆᢚไࡋࡓࡇࡀ⪃࠼ࡽࢀࡿࠋ MPP+ㄏᑟᛶࡢ PD ࣔࢹࣝ⣽⬊࠾࠸࡚ࠊHIF-1Dࡢάᛶࡼࡾ⚄⤒⣽⬊Ṛࡀᢚไࡉࢀ ࡿࡇࡸ[72,73]ࠊ㐠ືࡼࡾ୍ⓗప㓟⣲≧ែࢆᘬࡁ㉳ࡇࡍࡇ࡛⚄⤒ಖㆤຠᯝࡀⓎ ࡉࢀࡿࡇࡶሗ࿌ࡉࢀ࡚࠾ࡾ[74]ࠊVEGF ࡢࡳ࡞ࡽࡎࠊHIF-1Dࡶ⚄⤒ኚᛶᝈࡢ⒪ࢱ࣮. 64.
(66) ࢤࢵࢺ࡞ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋ ᮏ◊✲ࡼࡾ᪥ᖖⓗ࡞ࢥ࣮ࣄ࣮ࡢᦤྲྀࡼࡿ⚄⤒ಖㆤస⏝ࡀ VEGF ࡸ HIF-1DࡢⓎ⌧ㄏᑟࠊ άᛶࡼࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࡇࡽࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮྵࡲࢀࡿ HIF-1Dάᛶᡂ ศ࡛࠶ࡿ࢝ࢸࢥ࣮ࣝ㢮ࡘ࠸࡚ᚋヲ⣽◊✲ࡍࡿࡇ࡛ࠊALS ࡸ PD ࡢண㜵ཬࡧ⒪ ࡘ࡞ࡀࡗ࡚࠸ࡃࡇࡀᮇᚅ࡛ࡁࡿࠋ. ➨ 4 ⠇ AD 㛵㐃㑇ఏᏊⓎ⌧ᑐࡍࡿᙳ㡪ࡘ࠸࡚ AD ࡢཎᅉࡢ 1 ࡘ⪃࠼ࡽࢀ࡚࠸ࡿ AEࡣࠊAPP ࡀE-ࢭࢡࣞࢱ࣮ࢮཬࡧJ-ࢭࢡࣞࢱ࣮ࢮ ࡼࡾษ᩿ࡉࢀࡿࡇ࡛⏕ࡌࠊ⏕ᡂࡉࢀࡓ AEࡣ IDE ࡸࢿࣉࣜࣛࢩࣥ(NEP)࡞ࡼࡾศゎ ࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[75,76]ࠋᮏ◊✲ࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ IDE ࡢ㑇ఏᏊ Ⓨ⌧ࡀୖ᪼ࡋ㸦Fig.25C㸧ࠊBACE1 ࡢࢱࣥࣃࢡⓎ⌧ࡀῶᑡࡍࡿࡇࡀ♧ࡉࢀࡓ㸦Fig.26㸧ࠋ ࡲࡓࠊNEP ࡢ㑇ఏᏊⓎ⌧ࡣ HIF-1Dࡼࡾไᚚࡉࢀ࡚࠸ࡿࡇࡶሗ࿌ࡉࢀ࡚࠾ࡾ>@ࠊࢥ࣮ ࣄ࣮㇋ᢳฟᾮࡼࡾⓎ⌧ࡀୖ᪼ࡋ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋࡇࢀࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡢ ῧຍࡼࡾ AEࡢ⏘⏕ࡀᢚไࡉࢀࠊศゎࡀಁ㐍ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋᚋᐇ㝿ࡢ AEศ Ἢ㔞ࠊ≉ẘᛶࡀᙉ࠸ࡉࢀࡿ AE42 ࡢศἪ㔞ᑐࡍࡿᙳ㡪ࢆ᳨ウࡋ࡚࠸ࡃᚲせࡀ࠶ࡿࠋ ࢥ࣮ࣄ࣮ࡼࡿ⚄⤒ಖㆤస⏝ࡣ VEGF ࡸ NGF ࡞ࡀ༢⊂࡛ࡣ࡞ࡃࠊ༠ㄪⓗാࡃྍ⬟ ᛶࡶ⪃࠼ࡽࢀࡿࠋᐇ㝿ࠊALS ࣔࢹ࣐ࣝ࢘ࢫ࠾࠸࡚ࠊGDNF VEGF ࢆࡶศἪࡉࡏࡿࡇ ࡛ࠊGDNF ࡸ VEGF ࢆ༢⊂࡛ศἪࡉࡏࡓࡼࡾࡶຠᯝࡀࡁ࠸ࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ [78]ࠊࢥ࣮ࣄ࣮ᦤྲྀࡼࡾࠊᵝࠎ࡞ᅉᏊࡀ༠ㄪⓗస⏝ࡍࡿࡇ࡛ࡼࡾᙉ࠸⚄⤒ಖㆤຠᯝࡀ Ⓨࡉࢀ࡚࠸ࡿࡇࡶ⪃࠼ࡽࢀࡿࠋࡇࡢⅬࡘ࠸࡚ࡼࡾ᫂☜㆟ㄽࡍࡿࡣࠊࡑࢀࡒࢀࡢ㑇 ఏᏊ࣭ࢱࣥࣃࢡࡀࡢࡼ࠺࡞ᡂศࡼࡗ࡚ࠊࡢࡼ࠺࡞࣓࢝ࢽࢬ࣒࡛Ⓨ⌧ไᚚࡉࢀ࡚࠸ࡿ ࢆヲ⣽◊✲ࡋ࡚࠸ࡃᚲせࡀ࠶ࡿࠋ. 65.
(67) ➨ 5 ⠇ ⥲ᣓ ᮏ◊✲ࡼࡾࠊࢥ࣮ࣄ࣮㇋ᢳฟᾮࡼࡾ SH-SY5Y ⣽⬊࠾࠸࡚ᵝࠎ࡞㑇ఏᏊࠊࢱࣥࣃࢡⓎ ⌧ࡀኚືࡍࡿࡇࡀ♧ࡉࢀࡓࠋࡑࢀࡒࢀࡢⓎ⌧ኚື㛵ࢃࡿࢥ࣮ࣄ࣮ᡂศࡸࡑࡢ࣓࢝ࢽࢬ ࣒ࡘ࠸࡚ゎ᫂ࡋ࡚࠸ࡃࡇ࡛ࠊࢥ࣮ࣄ࣮ࡼࡿ⚄⤒ಖㆤຠᯝࡘ࠸࡚ศᏊ࡛ࣞ࣋ࣝド᫂ ࡋࠊᘏ࠸࡚ࡣ⚄⤒ኚᛶᝈࡢண㜵ࡸ⒪ࡘ࡞ࡀࡗ࡚࠸ࡃࡇࡀᮇᚅ࡛ࡁࡿࠋ. 66.
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