ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.47 No.1 (2010) pp.27-31


2QNQZCOGT ߩࠕࡐ࠻࡯ࠪࠬ⺃ዉ⢻ߣ &&5 ߣߒߡߩ᦭↪ᕈ↰᧛ ୶ሶ ⩲⼱ 㤗Ꮧ㧖ਭን ኼ㧖㧖

Poloxamer 407 induces p53 related apoptosis in human leukemic cell line
Michiko TAMURA㧖, Maki SUGAYA㧖, Hisashi HISATOMI㧖
ABSTRACT㧦Poloxamer block copolymers have been studied in multiple applications as drug deliverysystems. These A-B-A amphiphilic block copolymers can up-regulate the expression of selected genes incells and alter genetic responses to antineoplastic agents in cancer. One example is poloxamer 407, also
known as pluronic F127, which may be a promising carrier in drug delivery systems (DDS). To clarify
the possible mechanistic role of pluronic F127 in leukemia cell lines, we tested whether pluronic

F127-inducible factors would cause apoptosis. The influence of pluronic F127 in cell lines was examinedusing a comprehensive analysis. Based on these studies, it was proposed that treatment of NALM-6 cellswith 3% pluronic F127 resulted in an arrested G2/M phase of the cell cycle, followed by caspase

activation and accumulation of apoptotic cells. When used as a carrier in a DDS, pluronic F127 may

provide a synergistic effect on the drug of interest. Although the mechanisms of the function of pluronic

F127 are not fully understood, our study suggests that pluronic F127 may act as a useful carrier in DDS
for purposes of leukemia therapy.

Keywords㧦pluronic, drug delivery system, p53, apoptosis(Received March 23, 2010)㧝 ߪߓ߼ߦߚ⮎೷ࠍ቟ቯߒߡᖚㇱ߳ャㅍน⢻ߥ⮎‛ࠠࡖ࡝ࠕ࡯ߩ㐿⊒߇ᔅⷐߢ޽ࠆ‫⃻ޕ‬࿷, ⮎‛ࠠࡖ࡝ࠕ࡯ߣߒߡ᳓ṁᕈ㜞 ⮎ߪᴦ≮ㇱ૏ߩ⚦⢩ߦ೔㆐ߒ, ⚦⢩ߦೝỗࠍਈ߃ࠆߎಽሶߢ޽ࠆࡐ࡝ࠛ࠴࡟ࡦࠣ࡝ࠦ࡯࡞ (PEG) ߇⥃ᐥᔕ↪

ߣߢ⮎ലࠍ⊒ើߔࠆ‫ߒ߆ߒޕ‬, ታ㓙ߦ૞↪ㇱ૏߳೔㆐ߔߐࠇߡ޿ࠆ‫ޕ‬PEGߪ⚦⢩߿࠲ࡦࡄࠢ⾰ߥߤߩ↢૕ᚑಽߣࠆ㊂ߪ߶ࠎߩ৻ㇱߢ޽ࠅ, ߶ߣࠎߤߪઍ⻢࡮ឃᴭߐࠇߚߩ⋧੕૞↪߇ዊߐߊ, ࠲ࡦࡄࠢ⾰߳ߩୃ㘼ߦࠃࠅ᛫ේᕈࠅ, ૞↪ㇱ૏એᄖ߳૞↪ߔࠆߎߣߢ೽૞↪ߩේ࿃ߣ߽ߥߩᷫዋ߅ࠃ߮ⴊᶧᓴⅣᕈߩჇᄢߥߤߩലᨐ߇޽ࠆ‫ࠇߎޕ‬ࠆ‫߼ߚߩߘޕ‬, ೽૞↪߇⺃⊒ߐࠇߥ޿ࠃ߁̌ᔅⷐ㊂ߩ⮎ࠄߩലᨐࠃࠅ, ⮎‛߇ᮡ⊛⚦⢩ߦ೔㆐ߔࠆ㊂߇Ⴧᄢߒ,ࠍ, ᔅⷐߥ႐ᚲߦ, ᔅⷐߥߣ߈ߦଏ⛎ߔࠆ̍࠼࡜࠶ࠣ࠺࡝ᮡ⊛߳ߩ᥸㔺ᤨ㑆߇ᑧ㐳ߔࠆߚ߼⮎೷ലᨐ߇ะ਄ߔࠆ‫ޕ‬ࡃ࡝࡯ࠪࠬ࠹ࡓ (DDS㧦Drug Delivery System) ߇ℂᗐታ㓙ߦ, PEGߢୃ㘼ߐࠇߚ࠲ࡦࡄࠢ⾰߇PEGୃ㘼࠲ࡦࡄ⊛ߥᛩਈᣇᴺߣߒߡឭ᩺ߐࠇߡ޿ࠆ‫ޕ‬DDSߪ, ೽૞↪ߩᛥ೙ߛߌߢߥߊ, ⮎೷ߩᜬ⛯⊛ߥ᡼಴, ⮎೷ߩ૕ౝඨᷫ

ࠢ⾰ක⮎ຠߣߒߡ⊒ᄁߐࠇߡ޿ࠆ‫ߡߒߣ଀৻ߩߘޕ‬, ࠕ࠺ࡁࠪࡦ࠺ࠕࡒ࠽࡯࠯ߣ PEG ࠍ⚿วߐߖߚ㊀∝఺∉ਇᦼߩᑧ㐳, ⒳‫ߩޘ‬ㇱ૏ߢߩ⮎೷ߩๆ෼ଦㅴ߅ࠃ߮⮎೷ߩోⶄว∔ᖚᴦ≮⮎Adagen®߇᜼ߍࠄࠇࠆ2)‫ޕ‬Adagen®ࠍᮡ⊛⚵❱߳ߩ․⇣⊛ߥㅍ㆐ߥߤߩലᨐ߇ᦼᓙߐࠇߡ޿᦯↪ߔࠆߎߣߦࠃࠅ, ࠕ࠺ࡁࠪࡦ࠺ࠕࡒ࠽࡯࠯ߩ૕ౝỚ‫ޕ‬DDSߩ⎇ⓥߦߪౝኈ‛ߩ⮎೷ߛߌߢߥߊ, ኽ౉ߒᐲ߇਄᣹ߒ, ૕ౝ⛽ᜬᦼ㑆߇ᢙㅳ㑆߆ࠄᦨ㐳㧢߆᦬߹ߢ1,2)

ࠆะ਄ߒߚ‫ߒ߆ߒޕ‬, PEGߪ⇹᳓ᕈߩൻቇ᭴ㅧࠍ᦭ߒߚ⮎㧖㧦ℂᎿቇ⎇ⓥ⑼ℂᎿቇኾ᡹ඳ჻೨ᦼ⺖⒟‛ࠍ቟ቯߒߡᮡ⊛ㇱ૏߹ߢャㅍߢ߈ߥ޿ᮡ⊛⚦⢩߳㧖㧦‛⾰↢๮ℂᎿቇ⑼ഥᢎ

ߩㆬᛯᕈࠍ㜞߼ࠆߎߣ߇ߢ߈ߥ޿ߥߤߩ໧㗴ὐ߇᜼ߍ㧖㧦‛⾰↢๮ℂᎿቇ⑼ᢎ᝼ JKUCVQOK"UVUGKMGKCELRࠄࠇࠆ‫ޕ‬㧙㧙 PEG ߦઍࠊࠆวᚑ㜞ಽሶߣߒߡਔⷫᇦᕈ㜞ಽሶߢ޽

iodide (PI, SIGMA)ࠍ 30 µL ᷝടߒFlow cytometry

Pluronic ߣ๭߫ࠇࠆߩ߇৻⥸⊛ߢ, ethylene oxide (EO)⢩๟ᦼࠍ⸃ᨆߒߚ

ࠆPoloxamer߇᜼ߍࠄࠇࠆ ‫ޕ‬Poloxamerߪ໡ຠฬߢ޽ࠆ

(FCM, Cell Lab Quanta SC, Beckman Coulter)ࠍ↪޿⚦3)®

ߣ propylene oxide (PO) ߆ ࠄ ᭴ ᚑ ߐ ࠇ ,9,10)‫ޕ‬

EOX-POY-EOX (A-B-A)ߩ᭴ㅧࠍ␜ߔࡉࡠ࠶ࠢ౒㊀ว૕ߢ޽ࠆ‫ⷫޕ‬ #PPGZKPΧࠍ↪޿ߚࠕࡐ࠻࡯ࠪࠬ⸃ᨆ෸߮᳓ᕈߢ޽ࠆ EOX ߣ⇹᳓ᕈߢ޽ࠆ POY ߦࠃࠅⷫ᳓ⷫᴤᱜᏱ࡝ࡦࡄ⃿ࠍ↪޿ߚ⚦⢩๟ᦼ᷹ቯࡃ࡜ࡦࠬߦᓇ㗀ࠍਈ߃ࡉࡠ࠶ࠢ౒㊀ว૕ߣߒߡߩ‛ᕈ #PPGZKPΧࠍ↪޿ߚࠕࡐ࠻࡯ࠪࠬ⸃ᨆ

ᄌൻߦࠃࠅಽሶ㊂ߩ೙ᓮ߇น⢻ߢ޽ࠆ4, 5)‫ޕ‬PEGߣᲧセ  ߣห᭽ߦછᗧߩỚᐲߢࡅ࠻ၭ㙃⚦⢩ߦฦ⒳ൻว‛ߒߡ⇹᳓ᕈߩൻቇ᭴ㅧࠍ᦭ߒߚ⮎‛ߩኽ౉߇น⢻ߢ޽ࠅ,૶↪↪ㅜߦㆡߒߚಽሶ㊂ߩㆬᛯ߽น⢻ߢ޽ࠆ4-6)‫ߚ߹ޕ‬᷷ᐲߦᔕ╵ߒߡᒻ⁁߇ᄌൻߔࠆߚ߼,ㆮવሶᴦ≮ߦ↪޿
ࠆGeneࠠࡖ࡝ࠕ࡯߿ੱᎿ⊹⤏૞⵾↪ᡰᜬ૕ߥߤ߳ߩᵴ↪߇ᦼᓙߐࠇߡ޿ࠆ

7, 8)

‫ޕ‬

એ೨, ᚒ‫ߪޘ‬Pluronic F68 (PF68) ߦࠃࠆᘟᕈ㛽㜑ᕈ®

⊕ⴊ∛⚦⢩ᩣK562⚦⢩․⇣⊛ߥࠕࡐ࠻࡯ࠪࠬ⺃ዉ⢻ࠍ9)ࠍᛩਈߒ48 h ၭ㙃ࠍⴕߞߚ‫ޕ‬ၭ㙃ᓟ㆙ᔃಽ㔌ࠍ↪޿ߡၭ࿾ࠍ㒰෰ߒ⚂ 3×105 ୘ߩ⚦⢩ߦ 100 µL ߩ Annexin

Χ Binding Buffer (Beckman coulter)ࠍ↪޿ߡ⚦⢩ Ộᶧ ࠍ ૞ ⵾ ߒ ߚ ‫ ߩ ߘ ޕ‬ᓟ 1 µL ߩ Annexin Χ -FITC

solution (Beckman coulter)ߣ 0.75 mM PI ࠍ 5 µL ᷝടߒ, ᳖ᶎਛ, ᥧᚲਅߢ 15 min 㕒⟎ߒߚ‫ޕ‬㕒⟎ᓟ 400 µLߩ Annexin Χ Binding Buffer ࠍᷝടߒ, FCM ࠍ↪޿⊒⷗ߒߚ ‫ߪߣߎߩߎޕ‬ᘟᕈ㛽㜑ᕈ⊕ⴊ∛ࠍ࠲࡯ࠥ࠶࠻

ߡࠕࡐ࠻࡯ࠪࠬ⸃ᨆࠍⴕߞߚ 9)‫ޕ‬

ߣߒߚDDS ߩ⮎‛ࠠࡖ࡝ࠕ࡯ߣߒߡF68ߩ᦭↪ᕈࠍ 7 ࡝ࡦࡄ⃿ߩᐜ⧯ൻ⚂㧞୚ߩPluronic F127 (PF127) ߦ⌕⋡ߒ, ઁ∔ᖚߦ߅ࠍ↪޿ߡஜᏱੱߩ਄⣨⢀㕒⣂ࠃࠅ⚂ mL ណⴊߒߚ‫ޕ‬ណ␜ໂߔࠆ߽ߩߢ޽ࠆ‫⎇ᧄޕ‬ⓥߢߪ, PF68ߦᲧߴಽሶ㊂߇
ߌࠆ DDS ߣߒߡߩน⢻ᕈࠍ⏕⹺ߔࠆߚ߼ߦ, PF127ᛩਈߦࠃࠆ↢⚦⢩ᢙჇട₸, ⚦⢩๟ᦼ߅ࠃ߮ㆮવሶ⊒⃻ߩᄌൻࠍ⺞ᩏߒߚߩߢႎ๔ߔࠆ‫ޕ‬

 EDTA࡮2Na (10.5 mg)౉ࠅࡊ࡜ࠬ࠴࠶ࠢ⵾⌀ⓨណⴊ▤ⴊߒߚஜᏱੱᧃ᪳ⴊࠍ 1000 ×g, 25 min ㆙ᔃಽ㔌ࠍⴕ޿᦭ᩭጀࠍ࿁෼ߒߚ‫ޕ‬࿁෼ߒߚ᦭ᩭጀߦ╬㊂ߩ PBS ࠍട߃ߡᏗ㉼ߒߚ‫ޕ‬3 mL ߩᏗ㉼ߒߚ᦭ᩭጀࠍ 3 mL ߩ࡝ࡦࡎ࠮ࡄ࡯࡞Τ ఺ ∉ ↢ ‛ ⎇ ⓥ ᚲ ߦ ㊀ ጀ ߒ 400 ×g, 30

min ㆙ᔃಽ㔌ࠍⴕߞߚ‫ޕ‬ኒᐲ൨㈩ߦࠃࠅಽ㔌ߐࠇߚ࡝ࡦ㧞㧚᧚ᢱߣᚻ㗅ࡄ⃿ጀࠍ࿁෼ߒ112 ×g, 5 min ㆙ᔃಽ㔌ࠍⴕߞߚ‫ޕ‬㆙ᔃ ⚦⢩ၭ㙃 ࡅ࠻ B ⚦⢩⊕ⴊ∛↱᧪⚦⢩ᩣ NALM-6 ⚦⢩ࡅ࠻ᄢ⣺≸↱᧪⚦⢩ᩣ DLD-1 ⚦⢩߅ࠃ߮ࡅ࠻⢄≸↱᧪⚦⢩ᩣ

Huh-7 ⚦⢩ߪ᧲ർᄢቇട㦂කቇ⎇ⓥቶක↪⚦⢩⾗Ḯ࠮ࡦ࠲࡯߆ࠄ౉ᚻߒߚ‫ߚ߹ޕ‬, ࡅ࠻⢗≸↱᧪⚦⢩ᩣ MKN-45⚦⢩ߪࡅࡘ࡯ࡑࡦࠨࠗࠛࡦࠬ⎇ⓥ⾗Ḯࡃࡦࠢ߆ࠄ౉ᚻߒߚ‫ޕ‬Huh-7 ⚦⢩ߢߪ 10% Fetal Bovine Serum (FBS),100 U / mL Benzylpanicillin Potassium, 25 µg / mLSpreptmycin ࠍᷝടߒߚ D-MEM ၭ࿾ (Invitrogen), ߘߩઁߩ⚦⢩ߢߪ 10% FBS, 100 U / mL Benzylpanicillin

Potassium,25µg / mL Spreptmycin ࠍᷝടߒߚ RPMI1640
ၭ࿾ (Invitrogen)ࠍ↪޿, 37͠, 5% CO2 ᧦ઙਅߢၭ㙃ࠍⴕߞߚ‫ޕ‬ )ORZF\WRPHWU\ ࠍ↪޿ߚ⚦⢩๟ᦼ᷹ቯ ࡅ࠻ၭ㙃⚦⢩ߦ 6% PEG, 6% PF68, 3% PF127 ࠍߘࠇߙࠇᛩਈߒ, 48 h ၭ㙃ᓟ, ၭ㙃ߒߚ⚦⢩߆ࠄ㆙ᔃಽ㔌ࠍ↪޿ߡၭ࿾ࠍ㒰෰ߒ, ⚂ 310 ୘ߩ⚦⢩ߦ 300 µL ߩૐ㧡ᒛಣℂᶧ (0.05% Sodium citrate, 0.1% NP40)ࠍ↪޿ߡ⚦⢩ Ộᶧࠍ૞⵾ߒߚ‫ߩߘޕ‬ᓟ0.75 mM Propidiumಽ㔌ᓟ਄ᷡࠍ㒰෰ߒPBS ߢ 2 ࿁ᵞᵺߒߚ‫ޕ‬ᵞᵺᓟන

⃿ࠍ㒰෰ߔࠆߚ߼࡝ࡦࡄ⃿ࠍ RPMI ၭ࿾ࠍ↪޿ߡ⚵❱ၭ㙃↪࠺ࠖ࠶ࠪࡘߢ 1 h, 37͠5㧑 CO2 ᧦ઙਅߢၭ㙃ߒߚ‫ޕ‬࿁෼ߒߚ࡝ࡦࡄ⃿ࠍ PBS ߢᵞᵺߒ100ng / mL

anti-CD3 mbs (Beckman coulter) ߅ࠃ߮ 40 µg / mL
PHA/PBS ࠍᷝടߒߚ RPMI ၭ࿾ (Invitrogen)ࠍ↪޿ߡ
24 h, 37͠, 5㧑 CO2 ᧦ઙਅߢၭ㙃ࠍⴕ޿T ࡝ࡦࡄ⃿ࠍᐜ⧯ൻߐߖߚ‫ޕ‬ ᱜᏱ࡝ࡦࡄ⃿ࠍ↪޿ߚ⚦⢩๟ᦼ᷹ቯ ណⴊߒߚஜᏱੱᧃ᪳ⴊ߆ࠄណขߒߚ T ࡝ࡦࡄ⃿ࠍᐜ⧯ൻߐߖߚᓟ ߣห᭽ߦછᗧߩỚᐲߢࡅ࠻ၭ㙃⚦⢩ߦฦ⒳ൻว‛ࠍᛩਈߒ48 h ၭ㙃ࠍⴕߞߚ‫ޕ‬ၭ㙃ᓟ㆙ᔃಽ㔌ࠍ↪޿ߡၭ࿾ࠍ㒰෰ߒ⚂ 3105 ୘ߩ⚦⢩ߦ 300µL ߩૐᒛಣℂᶧࠍ↪޿ߡ⚦⢩ Ộᶧࠍ૞⵾ߒߚ‫ߩߘޕ‬ᓟ 0.75 mM PI ࠍ 30 µL ᷝടߒ, FCM ࠍ↪޿ߚ⚦⢩๟ᦼࠍ⸃ᨆߒߚ‫ޕ‬㧙㧙ߩ⚿ᨐࠃࠅ, PF127 ߩ⚦⢩๟ᦼห⺞⢻ߪ⊕ⴊ∛↱᧪⚦⢩ 573&5 ᴺߦࠃࠆ P51$ ⊒⃻㊂ߩ⸃ᨆᩣߦ․⇣⊛ߦ૞↪ߒߡ޿ࠆߎߣ߇⏕⹺ߐࠇDNA วᚑ 7RWDO51$ ᛽಴ ฦ⒳ൻว‛ᛩਈࠍⴕߞߚ⚦⢩߆ࠄ Quick Gene RNA߅ࠃ߮⚦⢩ಽⵚߩ㒖ኂࠍ⺃⊒ߒߡ޿ࠆߣផ᷹ߐࠇߚ‫ޕ‬

cultured cell HC kit S ን჻ࡈࠖ࡞ࡓ ࠍ↪޿ߡQuickNALM-6 ⚦⢩Gene mini80 ን჻ࡈࠖ࡞ࡓ ߦࠃࠅ total RNA ࠍ᛽಴ߒߚ‫ޕ‬ F'1$ วᚑ 22.5 µL ߩ Reaction Mixture (50 mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM MgCl2, 4 mM DTT, 0.5 mM

DLD-1 ⚦⢩dNTP, 15 ng / µL Random Primer, 1.6 U / µL M-MLVRTase (Invitrogen), 0.96 U/ µL ribonuclease Inhibitor(Takara)ߦ total RNA 2.5 µL (100 ng / µL)ࠍᷝടߒ

40͠1 h cDNA วᚑࠍⴕߞߚ‫ޕ‬ 3ULPHU ⸳ቯNCBI

(NationalCenterforBiotechnology

Huh-7 ⚦⢩Informetion) ࠍ ෳ ᾖ ߒ  TP53 mRNA ߦ ኻ ᔕ ߔ ࠆForward primer ࠍ 5’- TTT CAG ACC TAT GGA AAC

TAC TTC-3’, Reverse primer ࠍ 5’-TGG CAT TCT
GGG AGC TTC ATC T-3’ߣ⸳ቯߒߚ‫ޕ‬ 3&5 3RO\PHUDVH&KDLQ5HDFWLRQ MKN45 ⚦⢩ 10 µL ߩ PCR Mixture (10×Paq5000TM Reaction

Buffer (Stratagene), 0.25 mM
Paq5000

TMdNTP,0.8 U

DNA Polymerase (Stratagene), 100 pM

Forward Primer, 100 pM Reverse Primer)ߦวᚑߒߚ

cDNA ࠍ 1 µL ᷝടߒ95͠ / 30 sec, 58͠/ 30 sec,72͠ / 30 sec ࠍ 1 ࠨࠗࠢ࡞ߣߒߚ PCR ෻ᔕࠍ 35 ࠨࠗࠢ࡞ߢⴕߞߚ

Biosystems)‫ޕ‬(2720 Thermal cycler, Applied࿑㧝ฦ⒳ൻว‛ᛩਈߦࠃࠆ⚦⢩๟ᦼߩ⚻ᤨ⊛ᄌൻ❑ゲ⚦⢩ᢙᮮゲ:DNA ㊂G0 /G1 ᦼ:㑆ᦼS ᦼDNA
วᚑᦼG2 /M ᦼ⚦⢩ಽⵚᦼNALM-6 ⚦⢩ߦ߅޿ߡPF127 ᛩਈߦࠃࠆ G2/M ห⺞߇⏕⹺ߐࠇߚ‫ޕ‬ 㔚᳇ᵒേ ෻ᔕ⚳ੌᓟPCR Ⴧ᏷↥‛ࠍ 2㧑ࠕࠟࡠ࡯ࠬࠥ࡞ߦଏ

ߒ145 V, 35 min ߩ㔚᳇ᵒേࠍⴕߞߚ‫ޕ‬ᵒേᓟࠛ࠴ $QQH[LQ ൙ࠍ↪޿ߚࠕࡐ࠻࡯ࠪࠬ⸃ᨆ߅ࠃ߮ᱜᏱ࡝ࡦࡄ⃿ࠍ↪޿ߚ⚦⢩๟ᦼ᷹ቯ

ࠫ࠙ࡓࡉࡠࡑࠗ࠼ᨴ⦡ߒߚ PCR Ⴧ᏷↥‛ࠍ UV ࠻࡜ࡦ NALM-6 ⚦⢩ߦ߅޿ߡ PF127 ᛩਈߦࠃࠆࠕࡐ࠻࡯ࠪࠬࠗ࡞ࡒࡀ࡯࠲࡯ߦࠃߞߡ⏕⹺ߒߚ‫ޕ‬ࠬ⺃ዉ߇⏕⹺ߐࠇߚ ࿑㧞 ‫ߚ߹ޕ‬ᱜᏱ࡝ࡦࡄ⃿ߦ߅޿ߡฦ⒳ൻว‛ᛩਈߦࠃࠆ⚦⢩๟ᦼ߳ߩᓇ㗀ߪ⏕⹺ߐࠇߥ㧟⚿ ᨐ߆ߞߚ ࿑㧟 ‫ޕ‬ ߎࠇࠄߩ⚿ᨐࠃࠅPF127 ߪᱜᏱ࡝ࡦࡄ⃿ߦߪ⚦⢩๟ )ORZF\WRPHWU\ ߦࠃࠆ⚦⢩๟ᦼ᷹ቯ

 NALM-6 ⚦⢩ DLD-1 ⚦⢩ Huh-7 ⚦⢩߅ࠃ߮

MKN45 ⚦⢩ߦ߅ߌࠆ 6㧑PEG, 6㧑 PF68, 3㧑PF127ᛩਈߦࠃࠆ⚦⢩๟ᦼ߳ߩᓇ㗀ࠍ FCM ߢ᷹ቯߒߚ⚿ᨐࠍᦼ߳ߩᓇ㗀ߪߥߊ⊕ⴊ∛↱᧪⚦⢩ᩣߩߺߦ․⇣⊛ߥ⚦⢩๟ᦼห⺞⢻߅ࠃ߮ࠕࡐ࠻࡯ࠪࠬ⺃ዉ⢻ࠍ␜ߔߎߣ߇ផ᷹ߐࠇߚ‫ޕ‬࿑㧝ߦ␜ߒߚ‫ޕ‬FCM ᷹ቯߦࠃࠅᓧࠄࠇߚࡅࠬ࠻ࠣ࡜ 㪩㪫㪄㪧㪚㪩 ᴺ䈮䉋䉎 㫄㪩㪥㪘 ⊒⃻㊂ᄌൻ䈱⸃ᨆᦼ߳ߩ⚦⢩๟ᦼห⺞߇⏕⹺ߐࠇߚ‫ߚ߹ޕ‬, DLD-1 ⚦⢩,MKN45 ⚦⢩ߦ߅޿ߡฦ⒳ൻว‛ࠍᛩਈߒߚ⚦⢩߆ࠄᛩਈߦࠃࠆ⚦⢩๟ᦼߦᄌൻߪ⏕⹺ߐࠇߥ߆ߞߚ‫ࠄࠇߎޕ‬࿑㧠ߦ␜ߒߚ‫ޕ‬NALM-6 ⚦⢩ߦ߅޿ߡPF127 ᛩਈߦࡓ߆ࠄ, NALM-6 ⚦⢩ߦ߅޿ߡ PF127 ᛩਈߦࠃࠆ G2/MHuh-7 ⚦⢩߅ࠃ߮ MKN-45 ⚦⢩ߦ߅޿ߡ, ฦ⒳ൻว‛ NALM-6 ⚦⢩ DLD-1 ⚦⢩ Huh-7 ⚦⢩߅ࠃ߮᛽಴ߒߚ mRNA ࠍ↪޿ߚ RT-PCR ᴺߦࠃࠆ⸃ᨆ⚿ᨐࠍ㧙㧙ࠃࠅ TP53 ㆮવሶߦ⊒⃻㊂ߩᷫዋ߇⏕⹺ߐࠇߚ‫߅ߥޕ‬㧘࠻࡯ࠪࠬ⺃ዉߦߪ TP53 ㆮવሶ߇㑐ਈߒߡ޿ࠆߎߣ߇ផ

ߘߩઁߩ⚦⢩ߢߪ㧘TP53 ㆮવሶߦ⊒⃻㊂ߩᄌൻ᷹ߐࠇߚ‫ޕ‬㧠⠨ ኤ ᧄ⎇ⓥࠃࠅPF127 ߦ߅ߌࠆ⊕ⴊ∛↱᧪⚦⢩ᩣߦ․⇣⊛ߥ⚦⢩Ⴧᱺᛥ೙⢻⚦⢩๟ᦼห⺞⢻߅ࠃ߮ࠕࡐ࠻࡯ࠪࠬ⺃ዉ⢻ࠍᜬߟߎߣ߇⏕⹺ߐࠇߚ‫ࠠ‛⮎ߪߣߎߩߎޕ‬ࡖ࡝ࠕ࡯߇߇ࠎ⚦⢩ߦ⮎೷ࠍ੺ߐߕߦᓇ㗀ࠍਈ߃ߚὐߢ

DDS ߩၮᧄ᭎ᔨ߆ࠄㅺ⣕ߒߡ޿ࠆ‫࡝ࡖࠠ‛⮎ߒ߆ߒޕ‬ࠕ࡯ߘߩ߽ߩߩ⮎೷ߣߒߡߩലᨐ߇ౝ൮ߒߚ⮎೷ߩലᨐߣᜟ᛫ߒߥߌࠇ߫⮎‛ࠠࡖ࡝ࠕ࡯ߦ⮎ല߇޽ߞߡ߽૗ࠄ໧㗴ߪߥ޿‫ޕ‬Pluronic®ߩ᛫⣲≌ലᨐߪ߻ߒࠈౝ൮ߔࠆ᛫߇ࠎ೷ߣߩ⋧ਸ਼ലᨐ߇ᦼᓙߢ߈ࠆ‫ ߦ․ޕ‬G2/M ᦼ߳ߩ⚦⢩๟ᦼߣή㑐ଥߥ᛫߇ࠎ೷ߣߩ૬↪߇ᦼᓙߐࠇࠆ‫ޕ‬ Pluronic® ߦࠃࠆ⊕ⴊ∛⚦⢩ᩣߦኻߔࠆ⚦⢩ห⺞⢻߇࿑㧞ࠕࡐ࠻࡯ࠪࠬ⸃ᨆ 0#./ ⚦⢩ ❑ ゲ :PI ߩ Ⱟ శ ᒝ ᐲ  ᮮ ゲ :Annexin Χ ߩ Ⱟ శ ᒝ ᐲ Viable:↢⚦⢩Early apoptosis:ᣧᦼࠕࡐ࠻࡯ࠪࠬ⚦⢩Apoptosis:ᓟᦼࠕࡐ࠻࡯ࠪࠬ⚦⢩Dead:ᱫ⚦⢩ PF127

૗ߦ⿠࿃ߔࠆ߆ߪ, ੹ߩߣߎࠈਇ᣿ߢ޽ࠆ‫ޕ‬ஜᏱੱᧃ᪳ⴊ↱᧪ߩᱜᏱ࡝ࡦࡄ⃿߿ๆ⌕ᕈߩ࿕ᒻ≸↱᧪⚦⢩ߥߤ߳ߩ⚦⢩ห⺞⢻߇ోߊ⏕⹺ߐࠇߥ޿ߎߣ߆ࠄ, ၭ㙃᭴ᚑᚑᛩਈߦࠃࠅNALM-6 ⚦⢩ᩣߦ․⇣⊛ߥࠕࡐ࠻࡯ࠪࠬಽ, ଀߃߫ၭ㙃ᶧ߿ⴊᷡߥߤ߳ߩ⚿วߦࠃࠅ, ᚑಽ⚵ᚑ߇ᄌൻߔࠆߚ߼ߣ߽⠨߃ࠄࠇࠆ‫ޕ‬Pluronic ߩ⒳㘃ߦࠃߞ®

⺃ዉ߇⏕⹺ߐࠇߚ‫ޕ‬ߡၭ㙃᭴ᚑᚑಽ߳ߩ⚿วᕈ߇⇣ߥࠅ, ⚵ᚑߩᄌൻ߇ේ࿃
ߩ⇣ߥࠆ⚦⢩ห⺞ࠍ⺃ዉߒߡ޿ࠆߣߔࠇ߫, ℂ⸃ߒ߿ߔ޿‫ߒ߆ߒޕ‬, ၭ㙃᭴ᚑᚑಽߩᄌൻ߇, ⊕ⴊ∛↱᧪⚦⢩ߦߩߺᓇ㗀ߒߡ޿ࠆߣߪ⠨߃ߦߊ޿‫ᧄޕ‬ႎ๔ߢߪ⊕ⴊ∛⚦⢩࿑㧟ᱜᏱ࡝ࡦࡄ⃿߳ߩᛩਈߦࠃࠆ⚦⢩๟ᦼᄌൻ❑ゲ㧦⚦⢩ᢙ, ᮮゲ:DNA ㊂, G0 /G1 ᦼ:㑆ᦼ, S ᦼ:DN วᚑ

ᦼ, G2 /M ᦼ:⚦⢩ಽⵚᦼ. ᱜᏱ࡝ࡦࡄ⃿ߢߪൻว‛ᛩਈߦࠃࠆ⚦⢩๟ᦼߩᄌൻߪ⏕⹺ߐࠇߥ߆ߞߚ‫ޕ‬ᩣߣߒߡ NALM-6 ⚦⢩ߩߺߩ⚿ᨐࠍႎ๔ߒߚ߇, ታ㓙ߦߪઁߩ⊕ⴊ∛⚦⢩ᩣߣߒߡ, K562 ⚦⢩, HL-60 ⚦⢩,U937 ⚦⢩, Molt-4 ⚦⢩, Molt-17 ⚦⢩ߢ߽ห᭽ߩታ㛎ࠍⴕ޿, ⚦⢩ห⺞ߦᄌൻ߇ߥ޿ߎߣࠍ⏕⹺ߒߡ޿ࠆ(ᧂ⊒⴫࠺࡯࠲)‫ߡߞ߇ߚߒޕ‬, ᚒ‫߇ޘ‬એ೨ߦ⏕⹺ߒߚ K562 ⚦⢩ߦኻߔࠆ PF68 9), ᧄႎ๔ߦࠃࠆ NALM-6 ⚦⢩ߦኻߔࠆ PF127 ߩ⚵ߺวࠊߖએᄖߪ, ଀߃߫ K562 ⚦⢩ߦኻߒ⚦⢩ฬ

control ࡮ PF68 ࡮ PF127

ߡߩ PF127 ߢߪ⚦⢩ห⺞⢻ߪ⏕⹺ߐࠇߥ޿‫ޕ‬K562 ⚦⢩NALM-6

ᚑಽߩᄌൻߪ, NALM-6 ⚦⢩ߦᲧߴ K562 ⚦⢩ߩ⚦⢩Ⴧ

DLD-1
Huh-7

MKN45

ߪห⺞ߐߖࠆ߇ NALM-6 ⚦⢩ߪห⺞ߐߖߥ޿ၭ㙃᭴ᚑᱺ⢻߇ᒝ޿ߚ߼, ⠨߃ߦߊ޿‫⚦ޕ‬⢩ห⺞⢻ߩㆬᛯᕈߦ㑐ߒߡߪ, ේ࿃ߩⶄ㔀ߐ߇ኈᤃߦផ᷹น⢻ߢ޽ࠅ, ᄙߊߩ࿑㧠ฦ⚦⢩ߦ߅ߌࠆฦ⒳ൻว‛ߦࠃࠆ 62O40# ⊒⃻㊂Ყセ⚿ᨐNALM-6 ⚦⢩ߦ߅޿ߡPF127 ᛩਈߦࠃࠅ TP53 mRNAߩ⊒⃻ૐਅ߇⏕⹺ߐࠇߚ‫ޕ‬ߦ㗼⪺ߦ㗼⪺ߥᏅ⇣ߪ⹺߼ࠄࠇߥ߆ߞߚ‫ࠄࠇߎߡߞࠃޕ‬ߩ⚿ᨐ߆ࠄPF127 ߩ⊕ⴊ∛↱᧪⚦⢩ᩣߦ․⇣⊛ߥࠕࡐᬌ⸽ࠍⷐߔࠆߣផ᷹ߐࠇࠆ‫ޕ‬B ⚦ ⢩ ߩ ᕆ ᕈ ࡝ ࡦ ࡄ ᕈ ⊕ ⴊ ∛ (ALL) ߦ ↱ ᧪ ߔ ࠆNALM-6 ⚦⢩ߦኻߔࠆ PF127 ᛩਈߪp53 ࠲ࡦࡄࠢ⾰ࠍ੺ߒߚࠨࡐ࠻࡯ࠪࠬࠍ⺃ዉߔࠆߎߣ߇᣿ࠄ߆ߦߥࠅ⋥ធߩℂ↱ߪਇ᣿ߥ߽ߩߩPF127 ߩ ALL ߦ߅ߌࠆDDS ߣߒߡߩ᦭↪ᕈࠍ␜ໂߔࠆߎߣߣߥߞߚ‫ޕ‬એ਄ߦࠃࠅ Pluronic ߪ⊕ⴊ∛↱᧪⚦⢩ᩣߦ․⇣⊛®

ߥࠕࡐ࠻࡯ࠪࠬ⺃ዉ⢻ࠍ᦭ߒߚ‛⾰ߢ޽ࠅ⊕ⴊ∛ࠍ࠲㧙㧙࡯ࠥ࠶࠻ߣߒߚ DDS ߩ⮎‛ࠠࡖ࡝ࠕ࡯ߣߒߡ᛫߇ࠎ೷10) Rabinovitch PS. DNA content histogram and cellcycle analysis. Meth Cell Biol. 1994; 41:263-96

ߣ૬↪ߔࠆߎߣߢ, ኽ౉ߒߚ᛫߇ࠎ೷ߣߩ⋧ਸ਼ലᨐ߇ᦼᓙߐࠇࠆ‫ޕ‬ ෳ⠨ᢥ₂1) ↰┵ᐽᒾ, ࠼࡜࠶ࠣ࠺࡝ࡃ࡝࡯ࠪࠬ࠹ࡓ DDS ᛛⴚߩᣂߚߥዷ㐿ߣߘߩᵴ↪ᴺ, ᩣᑼળ␠ ࡔ࠺ࠖࠞ࡞࠼࠘, 20032)  Ellis KM, Mazzoni L, Fozard JR. Role ofendogenous adenosine in the acute and late

response to allergen challenge in actively sensitized

Brown Norway rats. Br J Pharmacol. 2003; 139:1212-1218

3) Kabanov AV, Bratrakova EV, Alakhov VY. Pluronic

block copolymers as novel polymer therapeutics

for drug and gene delivery. J Conrol Release.

2002;82:189-212

4)  Newman MJ, Actor JK, Balusubramanian M,Jagannath C. Use of nonionic block copolymers invaccines and therapeutics. Crit Rev Ther Drug
Carrier Syst. 1998;15:89-142

5) Slepnev VI, Kuznetsova LE, Gubin AN, Bratrakova

EV, Alakhov VYu, Kabanov AV. Micelles ofpoly(oxyethlene)-poly(oxypropylene)

blockcopolymer (pluronic) as a tool for low-molecularcompound delivery into a cell: phoasphorylation of
intracellular proteins with micelle incorporated[gamma 32P]ATP. Biochem Int. 1992;26:587-595

6) Zhang L, Parsons DL, Navarre C, Kompalla UB,Development and in-vitro evaluation of sustained
release poloxamer 407 (P407) gel formulations ofceftiofur. J Control Release. 2002;85:73-81

7)  Kwon GS, Naito M, Yokoyama M, Okano T,Sakurai Y, Kataoka K. Physical entrapment ofadriamycin in AB block copolymer micelles.
Pharm Res. 1995;12:192-195

8) Kwon GS, Okano T. Soluble self-assembled block

copolymersfor1999;16:597-600

drugdelivery.

PharmRes.

9) Aoki N, Tamura M, Ohyashiki JH, Sugaya M,Hisatomi H. Poloxamer 188 enhances apoptosis
-pathway in human leukemia cell line. Mol MedRep. 2010; in press.

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