Effect of CD146 positive stem cells from human exfoliated deciduous teeth on bone regeneration

概要

Ꮫ఩ㄽᩥ඲ᩥせ⣙

Effect of CD146 positive stem cells from human
exfoliated deciduous teeth on bone regeneration
㸦CD146 ཇ੓ࡋ๖͗ࣄࢺஙṑṑ㧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊ࡢ
㦵෌⏕⬟࡟ཬࡰࡍᙳ㡪㸧  

୺ᣦᑟᩍဨ㸸㇂ᮏ ᖾኴ㑻ᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ṑ⛉▹ṇᏛ㸧
๪ᣦᑟᩍဨ㸸ᰘ ⚽ᶞᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ṑ㧊⏕≀Ꮫ㸧
๪ᣦᑟᩍဨ㸸ᅧᯇ ு෸ᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ṑ⛉▹ṇᏛ㸧

ຊṊ ⯟኱
㸦་ṑ⸆ಖ೺Ꮫ◊✲⛉ ་ṑ⸆Ꮫᑓᨷ㸧

࠙⫼ᬒ࠾ࡼࡧ┠ⓗࠚ
 ཱྀ၁⿣ཱྀ࣭⵹⿣㸦CLP㸧ࡣࠊ㢌⵹㢡㢦㠃㡿ᇦ࡟࠾࠸࡚᭱ࡶ㧗࠸Ⓨ⑕⋡ࢆ♧ࡍඛኳᛶ⑌ᝈ࡛࠶ࡿࠋ
㢡⿣ࢆ᭷ࡍࡿᝈ⪅࡟࠾࠸࡚ࠊ㢡⿣㒊࡬ࡢ⭠㦵ᾏ⥥㦵⛣᳜ࡀᗈࡃ⾜ࢃࢀ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⭠
㦵᥇ྲྀ᫬ࡢእ⛉ⓗ౵くࡣࠊᝈ⪅࡟࡜ࡗ࡚኱ࡁ࡞㈇ᢸ࡜࡞ࡾࠊ⭠㦵᥇ྲྀᚋࡢ⑊③➼ࡢၥ㢟ࡀక࠺ࠋ
ࡑࡢࡓࡵࠊప౵く࡛㢡⿣㒊㦵෌⏕ࢆ㐩ᡂࡋ࠺ࡿᡭẁ࡜ࡋ࡚ࠊ㛫ⴥ⣔ᖿ⣽⬊ (MSCs) ࢆ⏝࠸ࡓ㦵
෌⏕་⒪ࡢᛂ⏝ࡀᮇᚅࡉࢀ࡚࠸ࡿࠋᡃࠎࡢ◊✲ࢢ࣮ࣝࣉ࡛ࡣࠊ⭠㦵᥇ྲྀ࡟క࠺౵くࢆపῶࡋ࡞ࡀ
ࡽ㦵෌⏕ࢆ㐩ᡂࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ௨๓ࡼࡾṑ㧊⏤᮶ MSCs ࡟╔┠ࡋࠊ᳨ドࢆ㔜ࡡ࡚ࡁࡓࠋࡇࢀࡲ
࡛ࡢ᳨ウࡼࡾࠊ㢌⵹㦵㦵Ḟᦆච␿୙඲࣐࢘ࢫ࡟࠾࠸࡚ࠊࣄࢺஙṑṑ㧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊ (SHED)
⛣᳜ࡣࠊ㦵㧊⏤᮶ MSCs (BMSCs) ⛣᳜࡜ྠ⛬ᗘࡢ㦵෌⏕⬟ࢆ᭷ࡍࡿࡇ࡜ࢆゎ᫂ࡋࡓࠋ
௨ୖࡢ⤖ᯝࡼࡾࠊSHED ࡢ㢡⿣㒊㦵෌⏕἞⒪࡟ᑐࡍࡿ᭷ຠ࡞⣽⬊※࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡿࡶࡢ
ࡢࠊṑ㧊⤌⧊࡟࠾ࡅࡿ࡝ࡢ⣽⬊ᡂศࡀ㦵෌⏕ㄏᑟ࡟㛵୚ࡋ࡚࠸ࡿ࠿࡟ࡘ࠸࡚ࠊゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ
 
㏆ᖺࠊ෌⏕་⒪ศ㔝࡟࠾࠸࡚ࠊMSCs ࡢ⾲㠃ᢠཎ࡟㛵ࡍࡿ◊✲ࡀ┒ࢇ࡟⾜ࢃࢀ࡚࠸ࡿࠋMSCs
ࡀ᭷ࡍࡿ⾲㠃ᢠཎࡢ୍ࡘ࡛࠶ࡿ CD146 ࡣ MSCs ࡢ㦵෌⏕⬟࡟ᙳ㡪ࢆཬࡰࡍྍ⬟ᛶࡀ♧၀ࡉࢀ࡚
࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊSHED ࡟ྵࡲࢀࡿ CD146 㝧ᛶ⣽⬊ࡀ㦵෌⏕࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸࡚ࡣ᫂ࡽ
࠿࡜࡞ࡗ࡚࠸࡞࠸ࠋ௨ୖࡢ⫼ᬒࡼࡾࠊᮏ◊✲࡛ࡣࠊSHED ࡟࠾ࡅࡿ㦵෌⏕࡟᭷⏝࡞⣽⬊㞟ᅋࢆ
ゎ᫂ࡍࡿࡓࡵࠊSHED ࡢ⾲㠃ᢠཎ CD146 ࡀ㦵෌⏕⬟࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸᳨࡚ウࡍࡿࡇ࡜࡜ࡋࡓࠋ

࠙ヨᩱ࠾ࡼࡧ᪉ἲࠚ
ᐇ㦂 1: ᙜ㝔࡟᮶㝔ࡉࢀࡓᝈ⪅ࡢᢤཤஙṑṑ㧊࠿ࡽ SHED ࢆ༢㞳࣭ᇵ㣴ᚋࠊࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ
࣮ࣜࢆ⾜࠸ࠊCD146 ࠾ࡼࡧ MSCs ࣐࣮࣮࢝࡟ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋࡲࡓࠊCD146 ࡟ᇶ࡙࠸࡚ࢭ
ࣝࢯ࣮ࢸ࢕ࣥࢢࢆ⾜࠸ࠊCD146+SHEDࠊCD146-SHED ࢆ༢㞳࣭ᇵ㣴ࡋࡓࠋḟ࡟ࠊච␿୙඲࣐࢘ࢫ
(BALB/c-nu) ࡢ㢌⵹㦵࡟ 5 mm ࡢ㦵Ḟᦆࢆస〇ࡋࠊ࢔ࢸࣟࢥ࣮ࣛࢤࣥࢆᢸయ࡜ࡋ࡚ࠊࢭࣝࢯ࣮ࢸ
࢕ࣥࢢࢆ⾜ࡗ࡚࠸࡞࠸ SHEDࠊCD146+SHEDࠊ࠾ࡼࡧ CD146-SHED ࢆ BALB/c-nu ࡢ㦵Ḟᦆ㒊࡬
⛣᳜ࡋࡓࠋ⛣᳜ᚋ 0ࠊ4ࠊ8 㐌㛫ᚋ࡟ μCT ࢆ᧜ᙳࡋࠊ⛣᳜㒊఩ࡢ୕ḟඖ⏬ീᵓ⠏࡜㦵෌⏕⋡ࡢ⟬
ฟࢆ⾜ࡗࡓࠋࡲࡓࠊ⛣᳜ 8 㐌㛫ᚋ࡟⛣᳜㒊఩ࡢ⤌⧊ษ∦ࢆస〇ࡋࠊ࣐࣊ࢺ࢟ࢩ࢚ࣜࣥ࢜ࢪࣥ
(H&E) ᰁⰍ࠾ࡼࡧ࣐ࢵࢯࣥࢺࣜࢡ࣮࣒ࣟ (MT) ᰁⰍࢆ⾜࠸ࠊᡂ⇍㦵ࢆホ౯ࡋࡓࠋࡉࡽ࡟ࠊ㦵ᙧ
ᡂᅉᏊ࡛࠶ࡿ ALP ࡢⓎ⌧࡟ࡘ࠸࡚ච␿⤌⧊ᰁⰍࢆ⾜ࡗࡓࠋࡑࡋ࡚ࠊVEGF ࠾ࡼࡧ CD31 ࡢⓎ⌧
࡟ࡘ࠸࡚ච␿⤌⧊ᰁⰍࢆ⾜࠸ࠊ⤌⧊ෆࡢ⾑⟶᪂⏕࡟ࡘ࠸࡚ࡶ᳨ウࡋࡓࠋ
ᐇ㦂 2: CD146 ࡀ⣽⬊ቑṪ⬟࡟ཬࡰࡍᙳ㡪ࢆㄪ࡭ࡿࡓࡵࠊSHEDࠊCD146+SHEDࠊCD146-SHED
ࢆᇵ㣴ࡋࠊBrdU assay ࠾ࡼࡧಸຍ᫬㛫ࡢ⟬ฟࢆ⾜ࡗࡓࠋࡲࡓࠊ㦵ศ໬⬟࡟ࡘ࠸࡚ẚ㍑ࡍࡿࡓࡵࠊ
㦵ศ໬ㄏᑟ㛤ጞ 21 ᪥┠࡟ ALP ᰁⰍ࡟࡚ホ౯ࡍࡿ࡜࡜ࡶ࡟ࠊALP ࢱࣥࣃࢡ㉁Ⓨ⌧㔞࡟ࡘ࠸᳨࡚
ウࡋࡓࠋࡑࡋ࡚ࠊ㦵ศ໬ㄏᑟ㛤ጞ 28 ᪥┠࡟࠾࠸࡚ࠊAlizarin red ᰁⰍࢆ⾜࠸ࠊ▼⅊໬ỿ╔≀ࡢⓎ
⌧࡟ࡘ࠸࡚ホ౯ࢆ⾜ࡗࡓࠋࡲࡓࠊ㦵ศ໬ㄏᑟ㛤ጞ 0ࠊ21 ࠾ࡼࡧ 28 ᪥┠࡟ࠊRT-PCR ࢆ⾜࠸ࠊ㦵
ศ໬࣐࣮࣮࡛࢝࠶ࡿ ALPࠊBMP-2ࠊ࠾ࡼࡧ OCN ࡢ㑇ఏᏊⓎ⌧࡟ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋࡉࡽ࡟ࠊ
⾑⟶᪂⏕⬟ࢆẚ㍑ࡍࡿࡓࡵࠊSHEDࠊCD146+SHEDࠊCD146-SHED ࢆࣄࢺ⮖ᖏ㟼⬦ෆ⓶⣽⬊
(HUVEC) ࡜ඹᇵ㣴ࡋࠊ⟶⭍ᙧᡂ⬟࡟ࡘ࠸᳨࡚ウࡋࡓࠋࡲࡓࠊSHEDࠊCD146+SHEDࠊCD146-SHED

࡟ࡘ࠸࡚ RT-PCR ࢆ⾜࠸ࠊ⾑⟶᪂⏕࣐࣮࣮࡛࢝࠶ࡿ VEGFࠊVEGFR2ࠊbFGFࠊCD31 ࡢ㑇ఏᏊⓎ
⌧࡟ࡘ࠸࡚ゎᯒࡋࡓࠋ

࠙⤖ᯝࠚ
ᐇ㦂 1㸸ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜゎᯒ࡟࠾࠸࡚ࠊ⾲㠃ᢠཎ CD146 ࢆ᭷ࡍࡿ⣽⬊ࡣࠊ࣊ࢸࣟ࡞ SHED
⣽⬊㞟ᅋ඲యࡢ⣙ 70.9%࡛࠶ࡗࡓࠋࡲࡓࠊMSCs ࣏ࢪࢸ࢕ࣈ࣐࣮࣮࡛࢝࠶ࡿ CD73ࠊCD90ࠊ࠾ࡼ
ࡧ CD105 ࡢ⾲㠃ᢠཎⓎ⌧⋡ࡣࠊ100㸣࡟㏆࠸್ࢆ♧ࡋࡓࠋື≀࡟ࡼࡿ᳨ウ࡛ࡣࠊ⛣᳜ 8 㐌㛫ᚋࡢ
CT ࡟࠾࠸࡚ࠊCD146+⩌ࡣࠊSHED ࠾ࡼࡧ CD146-SHED ࡢ⩌࡜ẚ㍑ࡋ࡚ࠊ᭷ព࡟㧗࠸㦵෌⏕⋡ࢆ
♧ࡋࡓࠋH&E ᰁⰍ࠾ࡼࡧ MT ᰁⰍ࡟ࡼࡿホ౯࡛ࡣࠊCD146+⩌࡟࠾࠸࡚ࠊⴭࡋ࠸㦵෌⏕⤌⧊ീࡀ
ほᐹࡉࢀࡓࠋVEGF-AࠊALP ච␿ᰁⰍ࡟࠾࠸࡚ࠊ⛣᳜㒊఩ࡢ࠺ࡕࠊⓎ⌧㡿ᇦࡀ♧ࡍ๭ྜࡣ CD146+
⩌࡛᭱ࡶ㧗࠸್ࢆ♧ࡋࡓࠋࡲࡓࠊCD31 ච␿ᰁⰍ࡟࠾࠸࡚ࠊ㝧ᛶ⾑⟶ᩘࢆẚ㍑ࡋࡓ࡜ࡇࢁࠊCD146+
⩌࡜ SHED ⩌ࡣࠊCD146-⩌࡜ẚ㍑ࡋ࡚ࠊ᭷ព࡞⾑⟶ᩘࡢቑຍࡀㄆࡵࡽࢀࡓࠋ
ᐇ㦂 2㸸SHEDࠊCD146+SHEDࠊCD146-SHED ࡟࠾࠸࡚ࠊ⣽⬊ቑṪ⬟ࡢ᭷ព࡞ᕪࢆㄆࡵ࡞࠿ࡗ
ࡓࠋALP ᰁⰍ࠾ࡼࡧ Alizarin red ᰁⰍ࡛ࡣࠊCD146+SHED ⩌ࡣ SHED ⩌ࠊCD146-SHED ⩌࡜ẚ㍑
ࡋ࡚ࠊ㧗࠸㦵ศ໬⬟ࢆ♧ࡋࡓࠋ㦵ศ໬࣐࣮࣮࢝ࡢ㑇ఏᏊⓎ⌧ࡣࠊ0 ᪥┠࡟ࡣ୕⩌㛫࡟᭷ព࡞ᕪࢆ
ㄆࡵ࡞࠿ࡗࡓࡶࡢࡢࠊ㦵ศ໬ㄏᑟ㛤ጞ 21 ࠾ࡼࡧ 28 ᪥┠࡟࠾࠸࡚ࠊCD146+SHED ⩌ࡣࠊSHED ⩌
࠾ࡼࡧ CD146-SHED ⩌࡜ẚ㍑ࡋ࡚ࠊALPࠊBMP-2ࠊOCN ࡢ᭷ព࡞㑇ఏᏊⓎ⌧ࡢቑຍࡀㄆࡵࡽࢀ
ࡓࠋHUVEC ࡜ࡢඹᇵ㣴࡟ࡼࡿ⟶⭍ᙧᡂࡢ᳨ウ࡛ࡣࠊCD146+SHED ⩌࠾ࡼࡧ SHED ⩌ࡣࠊ
CD146-SHED ⩌࡜ẚ㍑ࡋ࡚ࠊ᭷ព࡞⟶⭍ᙧᡂࡢቑຍࡀㄆࡵࡽࢀࡓࠋࡉࡽ࡟ࠊCD146+SHED ⩌ࡣࠊ
SHED ⩌࠾ࡼࡧ CD146-SHED ⩌࡜ẚ㍑ࡋ࡚ࠊVEGFࠊVEGFR2ࠊbFGFࠊCD31 ࡢ᭷ព࡞㑇ఏᏊⓎ
⌧ࡢቑຍࡀㄆࡵࡽࢀࡓࠋ

࠙⪃ᐹࠚ
 ᮏ◊✲࡛ࡣࠊin vivoࠊin vitro ࡢᐇ㦂࡟࠾࠸࡚ࠊCD146+SHED ࡀ SHEDࠊCD146-SHED ࡜ẚ㍑
ࡋ࡚ࠊඃࢀࡓ⾑⟶᪂⏕⬟࠾ࡼࡧ㦵෌⏕⬟ࢆ᭷ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋᮏ◊✲ࡢ⤖ᯝ࡜
CD146+BMSCs ࡞࡝࡟㛵ࡍࡿ㐣ཤࡢሗ࿌࠿ࡽࠊCD146+SHED ࡣࠊ
ࠕ࣌ࣜࢧ࢖ࢺ࡜㠀ᖖ࡟㏆࠸ᛶ㉁
ࢆ᭷ࡋ࡚࠸ࡿࠖࠊࠕCD146 ࡟ࡼࡗ࡚ࠊVEGF ࡸ PDGF ࡞࡝࡟ࡼࡿ⾑⟶᪂⏕స⏝ࡀಁ㐍ࡉࢀࡿࠖࠊ
ࠕCD146 ࡀ VEGFR2 ࡢࢥࣞࢭࣉࢱ࣮࡜ࡋ࡚స⏝ࡋ࡚࠸ࡿࡓࡵࠊVEGF ࡜ VEGFR2 ࡢ⤖ྜ࡟
ࡼࡾ bFGFࠊBMP-2ࠊRunx2ࠊOsterix ࡢⓎ⌧ࡀಁ㐍ࡉࢀࡿ࡜࠸ࡗࡓ⤒㊰ࡀࡉࡽ࡟ቑᙉࡉࢀࡿࠖ
࡞࡝ࡢᛶ㉁ࢆ᭷ࡋࠊࡇࢀ࡟ࡼࡾࠊ⾑⟶᪂⏕࠾ࡼࡧ㦵෌⏕ࡀಁ㐍ࡉࢀࡓࡢ࡛ࡣ࡞࠸࠿࡜⪃ᐹࡍࡿࠋ
ࡋ࠿ࡋ࡞ࡀࡽࠊᮏ◊✲࡟࠾࠸࡚ࠊࢩࢢࢼࣝ⤒㊰࡟ࡘ࠸࡚ࡢ᳨ウ࡞࡝ࡣ⾜ࡗ࡚࠸࡞࠸ࡓࡵࠊ௒ᚋࡢ
᳨ウㄢ㢟࡛࠶ࡿࠋ
࠙⤖ㄽࠚ
௨ୖࡢ⤖ᯝ࠿ࡽࠊCD146+ SHED ࡣࠊSHED ࠾ࡼࡧ CD146- SHED ࡜ẚ㍑ࡋ࡚ࠊ㧗࠸㦵ศ໬⬟ࠊ
⾑⟶᪂⏕⬟ࢆ᭷ࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋCD146 ࢆⓎ⌧ࡍࡿ SHED ࡣࠊ㦵෌⏕἞⒪࡟᭷⏝࡞⣽⬊㞟
ᅋ࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ