無血清培養系を用いた口腔癌由来細胞株の樹立ならびに機能解析
概要
Ꮫ ㄽ ᩥ
↓⾑Ύᇵ㣴⣔ࢆ⏝࠸ࡓཱྀ⭍⒴⏤᮶⣽⬊ᰴࡢ
ᶞ❧࡞ࡽࡧᶵ⬟ゎᯒ
Ꮫ⏦ㄳ⪅ బ⸨ ᡂ⣖
ᗈᓥᏛᏛ㝔་ṑ⸆ಖᏛ◊✲⛉་ṑ⸆Ꮫᑓᨷ
2019 ᖺᗘ
ᣦᑟᩍဨ㸸ᒸᮏ ဴ ᩍᤵ
ศᏊཱྀ⭍་Ꮫ࣭㢡㢦㠃እ⛉Ꮫ
ㅰ㎡
ᮏ◊✲㝿ࡋࠊᚚ᠓⠜࡞ࡿᚚᣦᑟ࡞ࡽࡧᚚᰯ㜀ࢆ㈷ࡾࡲࡋࡓᗈᓥᏛᏛ㝔་⣔⛉Ꮫ◊
✲⛉ ศᏊཱྀ⭍་Ꮫ࣭㢡㢦㠃እ⛉Ꮫ ᒸᮏဴᩍᤵ῝ཌ࡞ࡿㅰពࢆ⾲ࡋࡲࡍࠋࡲࡓࠊᮏᩥస
ᡂୖࠊ⤊ጞᚚ᠓ษ࡞ࡿᚚᣦᑟࠊᚚᰯ㜀ࢆ㈷ࡾࡲࡋࡓᮏᏛ ⏕యᮦᩱᏛㅮᗙ ຍ⸨ຌ୍ᩍᤵࠊ㢡
㢦㠃ゎ๗Ꮫ ᑎᒣ㝯ྖᩍᤵ῝ㅰ⮴ࡋࡲࡍࠋᮏㄽᩥసᡂ㝿ࡋᚚຓゝࠊᚚᰯ㜀ࢆ㈷ࡾࡲࡋࡓࠊ
ᮏᏛṑ⛉ᨺᑕ⥺Ꮫ ᰠᮏ┤ஓᩍᤵࠊᮏᏛබ⾗ཱྀ⭍ಖᏛ ኴ⏣⪔ྖᩍᤵࠊᮏᏛศᏊཱྀ⭍་Ꮫ࣭
㢡㢦㠃እ⛉Ꮫ ㇂ⱱᩍᤵឤㅰࡢពࢆ⾲ࡋࡲࡍࠋᑦࠊᮏ◊✲ࢆ㐍ࡵࡿ㝿ࡋࠊ⤊ጞᚚ᠓
ษ࡞ࡿᚚᣦᑟᚚ㓄៖ࢆ㈷ࡾࡲࡋࡓᮏᏛ་⣔⛉Ꮫ◊✲⛉ ศᏊཱྀ⭍་Ꮫ࣭㢡㢦㠃እ⛉Ꮫ ℈⏣
Ꮚ༤ኈᚰࡽឤㅰࡢពࢆ⾲ࡋࡲࡍࠋࡉࡽࠊከ࡞ࡿᚚᨭࠊᚚ༠ຊࢆ㡬ࡁࡲࡋࡓᮏᏛ་
ṑ⸆ಖᏛ◊✲⛉ ศᏊཱྀ⭍་Ꮫ࣭㢡㢦㠃እ⛉Ꮫᩍ⫋ဨྛឤㅰࡋࡲࡍࠋ᭱ᚋࠊᖖ⮬ศ
ࢆᨭ࠼࡚ࡃࢀࡓᐙ᪘ཬࡧぶ᪘ᚰࡼࡾឤㅰ࠸ࡓࡋࡲࡍࠋ
┠ḟ
␎グ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭1
➨ 1 ❶ ⥴ゝ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭4
➨ 2 ❶ ཱྀ⭍⒴⏤᮶⣽⬊ᰴࡢᶞ❧࡞ࡽࡧᶵ⬟ゎᯒ
➨ 1 ⠇ ᐇ㦂ᮦᩱཬࡧ᪉ἲ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭7
(1) ᇵ㣴ᮦᩱཬࡧᇵ㣴᪉ἲ
(2) ⣽⬊ᰴࡢᶵ⬟ゎᯒ
1) ձ ⣽⬊ቑṪ⬟ࡢ᳨ウ
ղ ONEC-TS ࡢ⣽⬊᥋╔⬟ࡢ᳨ウ
2) ᰁⰍయゎᯒ
3) ࢾ࣮ࢻ࣐࢘ࢫ⫼㒊⓶ୗ࡛ࡢ㐀⭘⒆⬟ࡢ᳨ウ
4) ࣐ࢥࣉࣛࢬ࣐ឤᰁヨ㦂
5) Short tandem repeat (STR) ゎᯒ
6) ⺯ගචᰁⰍἲࢆ⏝࠸ࡓྛ⣽⬊࠾ࡅࡿ EGFR ࡢⓎ⌧᳨ウ
7) EGF ཬࡧᢠ EGFR ᢠయࡢ⣽⬊ቑṪཬࡰࡍᙳ㡪
8) ONEC ࠾ࡅࡿୖ⓶࣭⚄⤒⣔࣐࣮࣮࢝ཬࡧ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢⓎ⌧
ゎᯒ
ձ ⺯ගචᰁⰍἲࡼࡿୖ⓶⣔ཬࡧ⚄⤒⣔࣐࣮࣮࢝ࡢⓎ⌧᳨ウ
ղ ච⤌⧊ᰁⰍἲࡼࡿ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢⓎ⌧᳨ウ
ճ Western blot ἲࡼࡿ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢⓎ⌧᳨ウ
9) Transcriptome ゎᯒ
10) ྛ⣽⬊ᰴࡢᢠ⒴ᑐࡍࡿឤཷᛶヨ㦂
11) PonatinibࠊEverolimusࠊSunitinib ࡢྛ⣽⬊ᰴࡢቑṪཬࡧࢩࢢࢼ
ࣝఏ㐩ཬࡰࡍᙳ㡪
12) qRT-PCR ἲཬࡧ Western blot ἲࢆ⏝࠸ࡓྛ⣽⬊ᰴ࠾ࡅࡿ FGFR
ࡢⓎ⌧᳨ウ
➨ 2 ⠇ ⤖ᯝ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭21
1) ᶞ❧⣽⬊ᰴࡢ┦ᕪ㢧ᚤ㙾ീ
2) ձ ྛ⣽⬊ᰴࡢ⣽⬊ቑṪ⬟ࡢ᳨ウ⤖ᯝ
ղ ONEC-TS ࡢ⣽⬊᥋╔⬟ࡢ᳨ウ⤖ᯝ
3) ᰁⰍయゎᯒ⤖ᯝ
4) ࢾ࣮ࢻ࣐࢘ࢫ⫼㒊⓶ୗ࡛ࡢ㐀⭘⒆⬟ࡢ᳨ウ⤖ᯝ
5) ࣐ࢥࣉࣛࢬ࣐ឤᰁヨ㦂⤖ᯝ
6)
STR ゎᯒ⤖ᯝ
7) ⺯ගචᰁⰍἲࢆ⏝࠸ࡓྛ⣽⬊࠾ࡅࡿ EGFR ࡢⓎ⌧᳨ウ⤖ᯝ
8) EGF ཬࡧᢠ EGFR ᢠయࡢ⣽⬊ቑṪཬࡰࡍᙳ㡪ࡢ᳨ウ⤖ᯝ
9) ONEC ࠾ࡅࡿୖ⓶࣭⚄⤒⣔࣐࣮࣮࢝ཬࡧ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢⓎ⌧
ゎᯒ⤖ᯝ
ձ ⺯ගචᰁⰍἲࡼࡿ ONEC-TS ࠾ࡅࡿୖ⓶⣔ཬࡧ⚄⤒⣔࣐࣮
࣮࢝ࡢⓎ⌧᳨ウ⤖ᯝ
ղ ච⤌⧊ᰁⰍἲࡼࡿ ONEC-TS ࠾ࡅࡿ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢ
Ⓨ⌧᳨ウ⤖ᯝ
ճ Western blot ἲࡼࡿ ONEC-TS ࠾ࡅࡿ⚄⤒ෆศἪ࣐࣮࣮࢝ࡢ
Ⓨ⌧᳨ウ⤖ᯝ
10) Transcriptome ゎᯒ⤖ᯝ
11) ONEC ࡢᢠ⒴ᑐࡍࡿឤཷᛶヨ㦂⤖ᯝ
12) PonatinibࠊEverolimusࠊSunitinib ࡢྛ⣽⬊ᰴࡢቑṪཬࡧࢩࢢࢼࣝఏ
㐩ཬࡰࡍᙳ㡪ࡢ᳨ウ⤖ᯝ
13) qRT-PCR ἲཬࡧ Western blot ἲࢆ⏝࠸ࡓྛ⣽⬊ᰴ࠾ࡅࡿ FGFR ࡢ
Ⓨ⌧᳨ウ
➨ 3 ❶ ⪃ᐹ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭28
➨ 4 ❶ ⥲ᣓ ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭38
ཧ⪃ᩥ⊩
ᅗ⾲
࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭ 40
࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭50
␎グ
ABP
Alcian blue and PAS stain
BSA
Bovine Serum Albumin
CD56
CD56 molecule
CDDP
cis-diamminedichloro-platinum(II)
cDNA
Complementary deoxyribonucleic acid
CT
Computed tomography
DAB
Diaminobenzidine
DAPI 4’, 6-diamidino- 2-phenylindole
DMEM
Dulbecco’s modified Eagle’s medium
DT
Doubling time
EDTA
Ethylene Diamine Tetra Acetic acid
EGF
Epidermal growth factor
EGFR
Epidermal growth factor receptor
FGFR Fibroblast growth factor receptor
H-E
Hematoxylin and eosin stain
HRP
Horseradish peroxidase
kDa
Kilo Dalton
1
pKRT
Pan keratin
mRNA
Messenger RNA
NET
Neuroendocrine tumor
NGFR
Nerve growth factor receptor
OASCC
Oral adenosquamous cell carcinoma
ONEC
Oral neuroendocrine carcinoma
OSCC
Oral squamous cell carcinoma
PBS
Phosphate-buffered saline
PCR
Polymerase chain reaction
PET-CT
Positron emission tomography
PVDF
Polyvinylidene difluoride
qRT-PCR
quantitative Real time protein chain reaction
RPMI
Roswell park memorial institute media
SCC
Squamous cell carcinoma
SDS
Sodium dodecyl sulfate
STR
Short tandem repeat
TBS
Tris-buffered saline
Tris
Tris(hydroxyl methyl) amino methane
2
TritonX-100
Octylphenol ethoxylate
Tween20
Polysorbate 20
VP-16
Etoposid
5-FU
5-Fluorouracil
3
➨ 1 ❶ ⥴ゝ
GeyࡼࡾࣄࢺᏊᐑ㢕⒴ࡽୡ⏺ึࡢ⒴⣽⬊ᰴ࡛࠶ࡿHeLa⣽⬊ࡀᶞ❧ࡉࢀ࡚௨᮶ (Gey, et
al., 1952)ࠊከࡃࡢ⒴⣽⬊ᰴࡀᶞ❧ࡉࢀ࡚ࡁࡓ (Eagle, 1955; Rangan, 1972)ࠋཱྀ⭍㡿ᇦ࠾࠸࡚ࠊ
Mooreࡽࡼࡿႃ㢌⒴⏤᮶H.Ep3⣽⬊ᰴࡢᶞ❧ (Moore, et al., 1955)ࠊᇼ㉺ࡽࡼࡿṑ⫗⒴⏤᮶
Ca9-22⣽⬊ᰴ (ᇼ㉺, 1974)ࠊᐑෆࡽࡢHo-1-u1⣽⬊ᰴ࠾ࡼࡧHo-1-n1⣽⬊ᰴ (ᐑෆ et al., 1985;
Miyauchi, et al., 1988) ࢆࡣࡌࡵࡋ࡚ࠊከࡃࡢ⣽⬊ᰴࡢᶞ❧ࡀሗ࿌ࡀࡉࢀ࡚࠸ࡿࠋ
ᚑ᮶ࠊࡇࢀࡽ⣽⬊ࡢᶞ❧◊✲ࡸ⣽⬊ᰴࢆ⏝࠸ࡓᶵ⬟ゎᯒ࡞ࡢศᏊ࣭⏕≀Ꮫⓗ◊✲ࡣࠊ⾑Ύ
ῧຍ᮲௳࡛⾜ࢃࢀ࡚ࡁࡓ (Easty, et al., 1981)ࠋ⾑Ύ୰ࡣࠊ✀ࠎࡢ⺮ⓑࠊ⬡㉁ࡸ⏕⌮άᛶ≀
㉁ࠊࡉࡽᮍ▱ࡢᅉᏊࡀᏑᅾࡋ࡚࠾ࡾࠊ⣽⬊ᮏ᮶ࡢᰤ㣴せồᛶࡸቑṪᅉᏊせồᛶࢆṇ☜᳨ウ
ࡍࡿࡇࡣ㞴ࡋ࠸ࠋࡉࡽࠊ⣽⬊ࡀ⏘⏕࣭ศἪࡍࡿ✀ࠎࡢᅉᏊࢆ⣽⬊ෆศἪᏛⓗ᫂ࡽࡍ
ࡿࡇࡣᅔ㞴࡛࠶ࡿࠋ
⒴⣽⬊ᰴࡢᶞ❧ࡣ㠀ᖖᅔ㞴࡛ (Eun, et al., 2005)ࠊ㛗ᮇᇵ㣴㐣⛬࡛⣽⬊ࡀ⣽⳦ࡸ┿⳦࡞
ởᰁࡋࡓࡾ (Rebecca, et al., 2014)ࠊ⥺⥔ⱆ⣽⬊ࡢ㐣ቑṪࠊࡉࡽ⒴⣽⬊⮬㌟ࡢቑṪ⬟ࡢప
ୗ࡞ࡼࡿࡇࡀ࡞せᅉࡋ࡚ᣲࡆࡽࢀࡿ (Ganjibakhsh, et al., 2017)ࠋ୍᪉ࠊSato ࡽࡣ
ᇵᆅ୰ຍ࠼ࡿ⾑Ύࡢᶵ⬟ࡣࠊ⣽⬊ࡢቑṪ࣭ศࡗ࡚ᚲせ࡞࣍ࣝࣔࣥࡸ⣽⬊ቑṪᅉᏊࢆ౪
⤥ࡍࡿࡇ࡛࠶ࡿࡢ௬ㄝᇶ࡙ࡁࠊ↓⾑Ύᇵ㣴ἲࢆ㛤Ⓨࡋࡓ (Hayashi and Sato, 1976;
Barnes and Sato, 1980)ࠋ↓⾑Ύᇵ㣴ἲࡣࠊᇶ♏ᰤ㣴ᇵᆅࣥࢫࣜࣥࡸࢺࣛࣥࢫࣇ࢙ࣜࣥ࡞
ࡢ᪤▱ࡢ࣍ࣝࣔࣥࠊ⤖ྜ⺮ⓑࡸ⣽⬊ቑṪᅉᏊ࡞ࢆຍ࠼ࡓ↓⾑Ύᇵᆅࢆ⏝࠸࡚ࠊṇᖖཬࡧ⒴
4
⣽⬊ࡢቑṪ࣭⥔ᣢࢆྍ⬟ࡍࡿ᪉ἲ࡛࠶ࡾࠊ⌧ᅾࡲ࡛⾑⟶ෆ⓶⣽⬊௨እࡢ⣽⬊ࡣ࡚↓⾑Ύ
ᇵ㣴ἲ࡛ᇵ㣴ࡀྍ⬟࡛࠶ࡿ (Sato, et al, 2002)ࠋ
㏻ᖖࡢ⏕య࠾࠸࡚⾑Ύ┤᥋᥋ࡋ࡚࠸ࡿ⣽⬊ࡣࠊ⾑⟶ෆ⓶⣽⬊௨እᏑᅾࡋ࡞࠸ࠋࡘࡲ
ࡾࠊࢇࡢ⣽⬊ࡣ⾑ΎᡂศࢆࡑࡢቑṪ࣭ศࡢ⥔ᣢᚲせࡋ࡚࠸࡞࠸ࡇࢆពࡋ࡚࠸
ࡿࠋ࠼ࡤࠊཱྀ⭍⢓⭷ࡸ⓶ࡢୖ⓶⤌⧊࣭⣽⬊ࡣࠊ㛫㉁⤌⧊ࡽᇶᗏ⭷ࢆ⤒⏤ࡋ࡚౪⤥ࡉࢀࡿ
ᰤ㣴ᅉᏊ࡛ቑṪ࣭ศࡀ⥔ᣢࡉࢀ࡚࠸ࡿࠋ⏕య࡛ࡣୖ⓶⣽⬊ࡣ㠀ᖖάⓎቑṪࡋ࡚࠸ࡿࡀࠊ
㛫㉁⤌⧊ࡢ⥺⥔ⱆ⣽⬊ࡣࢇቑṪࡋ࡚࠸࡞࠸ࠋᚑ᮶ࡢ⾑Ύῧຍᇵ㣴ἲཱྀ࡛⭍⢓⭷⤌⧊ࢆᇵ
㣴ࡍࡿ⥺⥔ⱆ⣽⬊ࡣ㧗࠸ቑṪ⬟ࢆ♧ࡍࡀࠊୖ⓶⣽⬊ࡣࢇቑṪ࡛ࡁ࡞࠸ᐇࡣࠊ⾑Ύῧ
ຍᇵ㣴ἲࡀ⏕యᶵ⬟ࢆᫎࡋ࡚࠸࡞࠸ࡇࢆ♧ࡍࡼ࠸࡛࠶ࡿࠋ
↓⾑Ύᇵ㣴ୗ࡛ࡣࠊᇵ㣴ึᮇࡣ⥺⥔ⱆ⣽⬊ࡢቑṪࢆㄆࡵࡿࡶࡢࡢࠊ㏿ࡸࡑࡢቑṪࢆ
Ṇࡍࡿࠋࡇࢀᑐࡋࠊ⭘⒆⣽⬊ࡣ↓⾑Ύᇵ㣴ୗ࠾࠸࡚ࡶቑṪࡀ⥔ᣢࡉࢀࡿࡓࡵࠊ⥺⥔ⱆ⣽⬊
ࡢΰධ࡞ࡋ⭘⒆⣽⬊ᰴࡢ༢㞳ࡀᐜ᫆࡞ࡿⅬࢆ᭷ࡋ࡚࠸ࡿࠋࡇࡢࡼ࠺↓⾑Ύᇵᆅࢆ⏝࠸
ࡿࡇࡼࡾࠊ⾑Ύྵࡲࢀࡿᵝࠎ࡞⺮ⓑ➼ࡢᙳ㡪࡞ࡋࠊ⭘⒆⣽⬊ࡢቑṪ࣭ศ➼ࡢヲ⣽࡞
࣓࢝ࢽࢬ࣒ࡢゎᯒࡸࠊ✀ࠎࡢ⏕⌮άᛶศᏊࡸᢠ⒴ࡢస⏝ᶵᗎࢆ᳨ウࡍࡿࡇࡀྍ⬟࡞ࡿࠋ
ⴭ⪅ࡢᡤᒓࡍࡿ◊✲ᐊ࡛ࡣࠊཱྀ⭍⒴ࡢ⏕᳨⤌⧊ࡸᡭ⾡ษ㝖⤌⧊ࡼࡾࠊ↓⾑Ύᇵ㣴ἲࢆ⏝࠸࡚⒴
⣽⬊ᰴࡢᶞ❧◊✲ࢆ⾜࠸ࠊࡇࢀࡽ⣽⬊ᰴࡀⓎ⌧࣭⏘⏕ࡍࡿ⣽⬊ቑṪᅉᏊࠊཷᐜయཬࡧ exosome
࡞ࡢゎᯒࢆ㏻ࡋ࡚ࠊཱྀ⭍⒴⣽⬊ࡢ≉ᛶࢆ⣽⬊ෆศἪᏛⓗ᫂ࡽࡋ࡚ࡁࡓࠋ
5
ᮏ◊✲࡛ࡣࠊཱྀ⭍ᡥᖹୖ⓶⒴ཬࡧཱྀ⭍㡿ᇦࡢⓎ⏕ࡀ⛥࡛ࠊ⥅௦⥔ᣢྍ⬟࡞⣽⬊ᰴࡢᶞ❧ࡢሗ
࿌ࡢ࡞࠸ཱྀ⭍⭢ᡥᖹୖ⓶⒴ࠊࡉࡽ↓⾑Ύ᮲௳ୗ࡛ࡢᇵ㣴ᡤぢࡸච⤌⧊Ꮫⓗ᳨⣴ࡢ⤖ᯝࢆ
⌮㒊ሗᥦ౪ࡍࡿࡇ࡛☜ᐃデ᩿⮳ࡗࡓࠊྠࡌࡃཱྀ⭍㡿ᇦࡢⓎ⏕ࡀᴟࡵ࡚⛥࡛࠶ࡿཱྀ⭍
ཎⓎ⚄⤒ෆศἪ⒴ࡢ⣽⬊ᰴࡢᶞ❧◊✲ࢆࠊ↓⾑Ύᇵ㣴⣔ࢆ⏝࠸࡚⾜ࡗࡓࠋࡉࡽࡇࢀࡽ⣽⬊ࡢቑ
ṪᅉᏊせồᛶࡸ㑇ఏᏊⓎ⌧࡞ࡢ≉ᛶࢆࠊ⣽⬊࣭ศᏊ⏕≀Ꮫⓗ᫂ࡽࡍࡿࡇࢆ┠ᣦࡋࡓࠋ
6
➨ 2 ❶ ཱྀ⭍⒴⣽⬊ᰴࡢᶞ❧
➨ 1 ⠇ ᐇ㦂ᮦᩱཬࡧ᪉ἲ
(1) ᇵ㣴ᮦᩱཬࡧᇵ㣴᪉ἲ
ⴭ⪅ࡢᡤᒓࡍࡿ◊✲ᐊ࣭デ⒪⛉࡛⒪ࢆ⾜ࡗࡓࠊཱྀ⭍ᡥᖹୖ⓶⒴ࠊཱྀ⭍⭢ᡥᖹୖ⓶⒴ࠊཱྀ⭍ཎ
Ⓨ⚄⤒ෆศἪ⒴ᝈ⪅ࡢ⭘⒆⤌⧊ࢆ᥇ྲྀᚋࠊ㏿ࡸึ௦ᇵ㣴ࢆ⾜ࡗࡓ (ᗈᓥᏛࣄࢺࢤࣀ࣒⌮
ᑂᰝጤဨᢎㄆィ⏬◊✲➨ࣄ 58 ྕཬࡧ 191 ྕ)ࠋ
ᇶ♏ᰤ㣴ᇵᆅࡋ࡚ࠊDulbecco’s Modified Eagle’s Medium (Dulbecco and Freeman,
1959)(Sigma-Aldrich, St. ...