thermoreversible gelation hydrogel; tgp ˘...

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臯腸腼腧膀腳膅膍膔臈臎 Vol. 36, pp. 371381, 2008 腒腕腁腍腑腏 腚腋腎腖腇腂腆腊腄腉 Thermoreversible gelation hydrogel; TGP腀腍腘腔腛腜 腅腈腃腊腂腆腀腙腓腗腌腐腊腅 腄腎 腇腋腈 腃腃 腊腍 腉腆 腌腋 : 20 9 9 腒腎腗腑 臉臑膴腛腰TGPHIF-1a膾膤膁臊臷腳腧 HBO腛臊臷膙膩 I腀 腆腂腇腅 膍腖腜膞腚膝腎腤膠臶臕膳腗腍腕膞臂 臸臦腛臉臑膴腛腰腗膘臚臬臍膳膖臬臊臷 腗膚臡膓膸膞臄腚腡腤膚臡膓腛膱膦腦臗腗 腍腕膼腓腕腂腤腋腛膺腖腜膞臂臸臦腛腰腗膘臚臬臍膳膞臄臺膪膷膎腛膱膦腚腔腂腕臠膐腎腤膫臓腖腜膌膥臬腵腨腲腾腪腽 ther- moreversible gelation hydorogel TGP腂腕臉 臑膴腛腰腦膼腓腕腂腤臉臑膴腛腰腱腏臏腛臸臦膹臻腦臒 膜臲臧腠腲腀膹臻腦腎腤腬腶腩腾腨腲腦 腍腕in vivo 腛臤in vitro 腖膛臇腖腇腤腋腗 腖腁腤腓腢臕膳腝腛臫膵腙腘腅腢腡腣臮 臏腍腑膹臻腦臐腔臼臂膬臜multicellu- lar tumor spheroid MCTS腁腤腂腜膜臔臂腗臢 膯臂heterospheroid 腛臆臰腜臀膶腛臁臮 腠膞膱膦腚腇腙膕腦腃腟腛腗膻腄腢腥 II腀 腖腏腐腔腋腈腊腍腉腌腅腃腁腄 腛腰腮膠臅腚腂腕腂腤膌膥臬腵腨腲腾 腪腽 TGP膘膈臬膾臌膋膿thermo- responsive polymer TRP腛膆腔腖腁腤 poly N- isopropylacrylamode῍ῌPNIPAAm腗臨自臬膾膋膿腖腁腤 polyethyren glycol PEG腛膨臝膿腅腢膹臭腌腥腕腂腤腋腛膋膿腔腆腗腍腕腮腽腀腪腽膄膉Sol-gel transitting temperature, SgTT腻腰膀腠膗腗膃腙腣 SgTT 腡腣膾腂膉腖腪腽臤 SgTT 膂膊腛膉腚腙腤腗腮腽臤膇腚腙 腔腆腆腁腤 12膇腖膡臖腍腙腂膵腣膌膥臬腛膋腦臒腎腨腛腰腚腋腛臬臔腦腱腮腎腤腗腂腈 腔腅腛腱腏腆膧腊腢腥腤腞腏膉腚腎腤 腒腉腖臂腛膑臛腆膌腖腁腣臡膓腛腁腤腱 腼腷腫膀腙腘腛臟膋膽臷腦臋腃腟腯腆腙腂腋腗腢腚腋腛腵腨腲腾腪腽腛腖膞臂腛腰腎腤 臉臑膴腚臺臥腍腕臼臂膬臜multicellu- lar tumor spheroid MCTS腦膮臭腎腤腋腗腆腖腇 腤腋腗腍腅腟spiner rotator 腔腥腙臹腐腏腡腈CO2 腛腰腖腛臱腄腛腰MCTS 腦臆臰腎腤腋腗腆膌腏腒腖腁腤腋腛膄膉SgTTPNIPAAm 腦臝膿臮臭 腎腤臃腚N-isopropylacrylamide 腗腻膀腯腹膨臝 膿腌腐腤臵自臬腺腴腸腀腖腁腤 n-butylmetha- crylamide BMA腛膨臝膿膋腌腐腤腋 腗腚腡腣腎腤膉腚至腎腤腋腗腆膌腖腁 腛腰腂腤臣膿腚腜 22C SgTT 腆臋腂臞臘腆腡腈腵腨腲腾腪腽腟膢膜23 臯腸腼腧膀腳膅膍膒膍膔臟膋膟膒膍371 89

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Page 1: Thermoreversible gelation hydrogel; TGP ˘ ˇˆ˙˝˛igakukai.marianna-u.ac.jp/idaishi/www/364/04-36-11Satoshi Tukikaw… · Vol. 36, pp. 371 381, 2008 Thermoreversible gelation hydrogel;

� � ����������Vol. 36, pp. 371�381, 2008

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���� ��� �Thermoreversible gelationhydrogel; TGP� ������������ �������

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��� :�� 20� 9� 9��

��������� TGP HIF-1a ����� �HBO� �������

I� ��

��� !"#$%&'()*+, � !- �./������*0�12) � 31��*456 � 7!8"9%456��:;<=*+,>?,@%�A�B� !- �./��*0�12) 7!8CDEF�:;"G@,HI$%��JK� �LM1NOPQRS �ther-moreversible gelation hydorogel TGP� <�@,�����<>?,@%��������� T�*�U�./VW<X+ - YZ[\��VW<�$%]^_QOP<�+, in vivo�`�< in vitro�ab�c%A*�d%� e� ��()f�ghijkl 9mT�"�U+nVW<oGp- qr� �multicellu-lar tumor spheroid MCTS� d%@ Ys- *t�u- � heterospheroid �vw xy�zT�\!:;"�ci�{<�|}�*~�l�%�

II� � ������������

������&�"�@,@%�LM1NOPQRS �TGP� ��0�1������ �thermo-

responsive polymer TRP� ��G�d% poly �N-isopropylacrylamode� �PNIPAAm�*��1�������d% polyethyren glycol �PEG������klV���,@%�A����� !*+, �S�RS"���

�Sol-gel transitting temperature, SgTT� <�+ ����\�#*�im SgTT9m�@���RS`� SgTT �����"i%*�S`��$�"i% !�d%1�2�� $����+i@hmLM1�%�<X$� - ��"A�1s<��$%* @�Gk������l�%� �  ��<&�"$%¡¢�- �£¤�L'�dm - 56�d%¥�¦§�ij�¨�©�<ª|()�i@A* �l" A�NOPQRS�*�!- <��$%* ���+"C«+,p- qr� �multicellu-lar tumor spheroid MCTS� <¬�$%A*��c%A* +k} spiner rotator �� ,i­-<ª�®  .�� CO2 ��¯/��°-���MCTS <vw$%A*�L'i���d%�A�"��� �SgTT� PNIPAAm<��T�$%±" N-isopropylacrylamide *��²³����®%´�1µ¶���d% n-butylmetha-crylamide �BMA� ����0�1<%��®%A*"9m �2$%��"·3$%A*�L'�d%� ��"�@%¸�" 22�C� SgTT ·3���ª@¹º�9� NOPQRS}4»Y�5 2�3��������� ¼��¨�¯��6¼��

371

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Page 2: Thermoreversible gelation hydrogel; TGP ˘ ˇˆ˙˝˛igakukai.marianna-u.ac.jp/idaishi/www/364/04-36-11Satoshi Tukikaw… · Vol. 36, pp. 371 381, 2008 Thermoreversible gelation hydrogel;

������������� � ���BMA ���� 5mol� ����� �� BMA ����������� SgTT ��� � 7mol�!� 18�C�� 3�� SgTT ���� "#!�$�%&' � (�� �)�*+,-./012��34�56���� (7� 8� SgTT �9��37�C �:�;<!=%&' �>>� ?�%&' �=:�@A!��$��B � (�� %&����CD�EF!���GH�I �J"� TGP �� 10KL� 10� FCS �MNO:�@ 9 ml !�B � 10��w�w�PQ� ���� �� TGP �@�:�RS���� %&T��A!UV�:�� WVXYZ �multicel-lular tumor spheroid; MCTS� ���[\ � �MCTS �]^:��_ V`�a� U�b�c��bdefg�� � hiU�jk&lNm\�� 4��

III� ���� TGP ����

n'opUVDLD-1� TGP<!:�� �V�qrse��t MCTS ���� � ��[\O MCTS �:�uv=��C � wx�ry��o �z>� 100{�ml �V�Q� 20�FCS |�:�@!:� O}�� ~0K����_7�� :� 1 �!�� �wx 192.23�64.50

mm� 2�!� 404.37�153.42 mm� 3�!� 496.57�178.65 mm� 4 �!� 541.61�51.98 mm �a���!=�$���!� o�[��C� �Fig.1�� MCTS �Z����e�� � S�T���� � Gompertz curve ��\ ����� �Fig. 2��MCTS ��C�Q������ O��V�

Q��M� Fetal Calf Serum �FCS��PQ�_���3��> �

IV� MCTS ��

�1� � ���MCTS ��bde���� � v^�V^��^��� � A��������m\ � �3�� ���I V� ����� � BrdU ¡¢�£7� wx�i[� MCTS !�� ¡¢�¤V�MCTS <��=¥¦� � _ao�� MCTS!�� BrdU �¤� S D�V�� 2�3^���^�V^<�§¨ �¥¦ �� ���[\ �� A�������� S D�V�©m\��� P27 �¡¢� � 8�ª«¬/�©m\�<��­®7�¯��¤V��M � V°D�±² �� V�W>�m\ � A����:�uv�_���o� �W��� ³´µ�i[� MCTS !=��[\� apoptosis �¶� �� lNm\ 5��

Fig. 1. MCTS volumes and culture period

MCTS volumes are growing in size according to the culture period in

TGP.

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Page 3: Thermoreversible gelation hydrogel; TGP ˘ ˇˆ˙˝˛igakukai.marianna-u.ac.jp/idaishi/www/364/04-36-11Satoshi Tukikaw… · Vol. 36, pp. 371 381, 2008 Thermoreversible gelation hydrogel;

TGP����� 4��MCTS��� ������������ � ���������� � !��" 40��70��#$%� &'�(���)���"� !��*" 90�+,�-.& /01& 23� MCTS �"45�%���67/89��1�& /:;<'��

�2� MCTS ����������=>?�0@"� S A����BC�� D� G0�G1A���"E?�F=G�-.& /H3'�1�6�� >IJ�K"LMNOP�Q$�RS� LMN�T<'%��"LMNUV�WX.Y@� Z[G\]23^[G\]_`a�� ��bA"cd�� G0A���/89.�7�� Cuisnier et al.8�"�e,f>g�C1�� hGijLMNOP�Bk�� G0�G1 arrest ���/lm���nY� 20�89.�& �op��1�� &'3���"qrstuv=>?�(��F=G�-.�MCTS� trypsin������������ ��bA�;w.� G0�G1A; 80.4�� S A; 11.6��G2�M A; 8� 1xyz�#$%�

{|� 8}A��)���"� G0 �G1 A;59.2�� SA; 21.1�� G2�MA; 16.7�� �#$%& 23� MCTS �" G0�G1A����n*/0@� S A����n*/L1 ~�3'��Khaitan 39�"� BMG-1 �������C1

��)���R��8}A plateau �� MCTS���bA�;w�� �)���8}A�" G0�G1: 48.3�� S: 40.0�� G2�M: 12�� plateau �"�72�, 3�, 25� 1xyz�#$%� {|� �� 4��� MCTS �"� 58, 5, 23��#$% op��1�� MCTS ���.������bA"�)��� plateau �#������bA ����1�& /�2��&'"� MCTS /=>?�F=G�-.���{��#� ~�3'��

�3� MCTS �� �����j����>�">IJ�"LMN����������& /���#S� &������/=>?�(��F=G�-. ~�3'� E?�G����{� ����<'�1�� &�E?�

Fig. 2. PO2 of MCTS

The outer rim of MCTS shows 120 mmHg, the inner side shows lower PO2� thecentral zone shows about 19�20 mmHg.

 ¡¢£¤¥ LMNUV 373

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���������� �� ���������������� ��������������� !"#��$�� MCTS %� &'()�%� *+��,-

.�/�� ��01%23456*+��758"� ���9�:;�<6!�=>�?��@A ��BC�D4��EFG��HI� ��

JK���LM�NO"���������� ���� !�P�Q����R�� S5S��6� TGP����TU��#!V� �@� CO2��WE MCTS �X����YZE?�� = �[\%���S�#�MCTS ]����9^_%� 1980`a56bc!� de 200 mm � MCTS � PO2�^_����Fig. 3�f��g����9�hi��6!�� ��cj� 3456*+��7k"���9%:;S"lm� ���9�n� o�� ATP� pH� pqrstLM�uvw�g�hi�356]x:;�����6!�10�� �!6%� MCTS ]�P�yz�a{�b8"#�|�E?���%�}�~^E�������#"%��56 145 mm ���!������� �anoxia region� ������#c!"#�11�� ���*+����9�������

�� normoxia, hypoxia, anoxia �P���i�S"��� ���% 50�250 mm ������S"#�4������56� ����#� MCTS %� de

300 mm�500 mm ����� �u�������� ��JK�<Ig MCTS � TGP EX����%� DP 100 ��ml �P�ME� 20� FCS�����E 3��������)��� DLD-1� MCTS ��������E���

V� MCTS � HIF-1a� VEGF

�&']�P� ��BC���� HIF-1a��� !� ��P� ��BCx������ !�� ���� �1� �������� ��������� � �2� apoptosis ��¡� �3� ¢z�a{56yz�£a{x� shift� �4� P¤¥�¦§��8"��B¨������ ����%� ��LM� 21� �Po2; 120�150 mmHg� 56 7� �Po2;40�50 mmHg� �:;���� $©�� up-

regulation !�� 2����% 0.5����LM�Po2; 3 mmHg� �ª�«����¬ !"#�12�� ���­c� HIF-1a� VEGF ®¯°±²���� DLD-1�D4��� MCTS ��#" ³���� HIF-1a %D4��E%´mµ¶ !�#�

Fig. 3.

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����� MCTS ������� �������� ���� 100 mm ��� MCTS ��� 100mm ��� MCTS ����� ������ �!"#$�� #%�� 3&'��(%$�� ���)*+ MCTS ��� HIF-1a �,- �!"#$� �Fig. 4��.�./0%� MCTS ����1234�MCTS 56�789:;�<=�� 789�>#$�?@�1A���B./�CD#$B� War-tenberg %13�� MCTS ����1)//E� HIF-1a F P-glycoprotein �1A�� MCTS GH,-EIJ6�,-1A�K��./LMN���B�OPQ�89RSLTU#VBWX�YZ�� [\]89^X �Hyperbaric oxygen therapy HBO��_B� HBO �Y8UI`Fabc��d^+4�e�%$��B�� Od^f�ge�hijklm�_B/no%$B�MCTS L HBO �>���pq� HIF-1 a�

VEGF �TUL NBO �Normobaric oxygen ther-apy� /�r2B/� HBO s�� VEGF �tuv�wx2B��yz#$�� .$����� HIF-1a�HBO>�{ 12u'�{��� _�-)*+TU�|%$+0}�� Wang GL %14��,$~� 789�>#$�?@����89����B/�

HIF-1a���0 5�0% 15��wx� basal level�����B./�MN#$��B� VEGF �wx�PQ�89U�,B HIF-1a�wx����/no%$B �Fig. 5��

VI� ���������

TGP ��5��� O?@����v�1�2B./0%OPQf��O������fge���OPQ��������'�?@� �����?@���L��2B./�"�+��� TGP 5�����?@�3/ 41��+�./�¡%0�+}��B� O?@/���?@L¢q#V��������� ?@£ 1 : 1�����¤%$�� 1 : 4����¥�+B/� O?@�pq���� 10�¦§¨?@¥�7������[�©ª#$B��/+}����O?@/���?@L TGP 5�¢q��E� WST-8 �«¬§�O?@�|�1�§/3­Y®2B� .�./0%� WST-8 �¨?@¥L¯°2Bpq� ���?@����x+�/no%$��YW� ±"PQ0%(%$�OPQ²L TGP ��5�2B/� ³´£� 2&'{�EOPQL�µ���B./�¶·#$��

Fig. 4. The result of western blotting of HIF-1a, VEGF, AKT, PI3K in MCTS.

HIF-1a shows strongly in larger sized and longer culture period of MCTS.

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93

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�������� �� TGP � ���������������� ��������� !"15��

VII� ��������� ��

#$�%&'����()��*��+����� ,-.�/0�������123� 5FU47.6���45�67� �892�:�3 90��;<12�=!"� 5FU ����>?3@����ABC� DE�FGHIJ��K L�M7�N"16����12�OPQ� RS()�� stage IV T

�U�C���������V;� WXYZ��/0�K[�\;C]^_�=�� `��� abcd�e�+�fVgN� h-�.��U�C sec-ond line, third line chemotherapy �V<ij���9 ���kl� �`NC;�17�� P"��m�noF18�� ()��/0��pYZ� �stageIII� ef+��C;�19��

VIII� HBO ������� MCTS

���<�� ����������� �9 ���kl�������� !Cq"� ��� ��� rVh-()��U�C� st�YZ��FOLFOX, FOLFIRI� �uV�CfDE�340�50��=�� d�v�f��w\��x����y�z{������8�|� }y�=�� d�~����������C������������������������� �S I������`��� 12����92���������I�`N�� HBO ����d�~��3�� ������A��C����Y�� �;������w`N�����M`NC;�20�� `��� ��d��3��������3� �¡¢�=�� �N�£ �¡¢��Y����� ����¤¥*�F���*���9��¦§`NC;���¨������©�$����©�� ª«���� ¬­®¯°¬±²©� ���³´�������µ� `NCq"�� �9 ¶·3��`NC

Fig. 5. Comparison of HIF-1 and VEGF change between NBO and HBO.

HIF-1 has no change under HBO condition, but VEGF was decreasing

after HBO day by day.

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������������ ��������������� HBO�������� !"#$�%&!��21��HBO '(�)*+,-�./01�23������ Wells &�)4� 19775678!��22��9:&23�24�� HBO ;�<*= �CBDCA� >?@�"AB� NBO ��C�DE�FG:�HI�.HI�J"!KL>67�� HBO MN;�OPQR- CBDCA STUV�)WXQR:CBDCA HI�YZ[�.\]^�<�_`a>

bc���de���KL>f�"�HBO gh;<*=��>i1jkl*�mhn

KL�deop3q?K!>rsX�tunEv� wx*yz{DLD-1� MCTS >h��� HBO �)<*=|}`a�~��� �����ABLD������"������� CDDP, 5FU, TAXOL �!��=� HBO �)<*=�|}`a���&!��Fig. 6�� Dose-response curve � NBO �L HBO��� �L�������%&!"� �����OP 3 �=� IC50 \� �Table 1�� NBO �LHBO ��!�!� CDDP: 36.513�23.269 mg�ml�24.901� 1.358 mg �ml� 5 FU: 392.613� 25.758�437.490�47.562 mg�ml� TAXOL: 11.532�9.716 mg

�ml� 18.571�17.469 mg�ml L��� ���X�������&!�q4"���MCTS��� CDDP, 5FU, TAXOL�~��� Fig. 7�)3� dose-response curve �%&!���!��=�O��� HBO ����yz �¡;��&!� 5FU L TAXOL �O�� HBO �)���<*=|}`a�%&!"� ¢HI�£10���� �¡;�¤&!� ¥� 5FU �O��

Fig. 6. Dose response curve of CDDP, 5FU and TAXOL under monolayer condition.

There is no enhancement e#ect by HBO.

Table 1. The IC 50 of three drugs under monolayer

condition.

There is no enhancement e#ect by HBO.

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������������ ������ IC50���������� �Table 2�� CDDP: 22.944�11.296 mg�ml� 15.946�4.586 mg �ml� 5FU: 1000�mg �ml�8.939�7.651 mg�ml�p�0.00465�� TAXOL: 58.901�10.915 mg�ml� 45.608�8.217 mg�ml�0.0208� ���� HBO �� CDDP 31.7�� 5FU 97.3� ���TAXOL 18.7� IC50� !"#� HBO $%�&'() ����

Raa �25�*� +,- DMBA ./�012�HBO ./�34�56��7��8� 9:;<= apoptosis� ./�&>�?@��7��AB#21�� C�$� HBO $ 5FU �D0#�EF�GH$%�� �I�EF�()�&J() ��7��K#��L�� Takiguchi�26�M 5FU�HBO�D0#2

./�NO() &J��7��AB#21��HBO �PQ GH$R��&'()* hypoxic

fraction 10� �STUVW�MXYC21�22��HBO HZ"$EF�GH�[\7�*� ]^ 10m �_`"�EF�GH�[�21�$a#1�7�7�LLbcd0��$*� e��fg ��� #�#� �� ./h�ijkl�mnCo�7�$%�2� EF��()�&JCo�7� �pqr$*I��$����st� 7�!ijuv$wx#2./�NO�y�z��{| �C21��HIF-1a�}~���?@��T�*� E./(

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Fig. 7. Dose response curve of CDDP, 5FU and TAXOL in TGP.

HBO increases the e#ect of anticancer drugs.

Table 2. The IC50 of three drugs under HBO condition.

5FU and TAXOL showed enhanced e#ects by

HBO. Particulary, 5 FU showed remarkable

enhanced e#ect by HBO.

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