平成 23 年度 修士論文 三重大学大学院工学研究科 博士前期
Contents
1. D NA RNA AGPC RNA Guanidium thyocyanate GTC RNase DNA GTC R NA RNA2. HUBS MRKA NJI EE BR SACR EI AREA E 1
3. Fig 1 2A microvilli MV cilium C junctional complex JC lamellar body LB elastic lamina EL mast cell M fibroblast F Pericyte P endothelium E capillary CAP basement membrane BM Fig 1 2A 50 150um C EWIK 2 acti e OE LL BR x v7 2E ze ns c Ola ms EL OO JR JJ LE 2 3um 0 08um PWEDE EDAT Hig 1 2B uiepRudgye Fig 1 2B
4. 2 Ul Fig1 1 O O CIE C ex C Uus Fig 1 1 1 2 1 2 1
5. Change to frames persecond O add resize 640x480 Filters add grayscale OK 6 Cinepak Codec by Radius LOK C 1OK 2 49 9 5 RT PCR 9 5 L AGPC ma gt RNA DNA 2 1 DNA
6. 56C 30 lt suprement gt PS Penicilhn Streptomycin Sml 20C LG L Glutamine 5ml 4C bFGF FGF basic Ds Tu ZI EELA 20 59 0C CSTELAE 2 PBS 0 2g 0 2g 12 4142 2 90g 8g IL HRERS EE 3500ml 121 C 40 3 TRYPSIN EDTA SOLUTION 20 25 ml 4 ml 20 41 4 D 1L
7. Hig1 2D m OpAciFicATION BM THICKENING AND REDUPLICATION NEW COLLAGEN Fig 1 2D 1 2 4 1 2 3 2 co 0 5um min Figl 2E 0 12 24 36 48 60 72 84 96 hour Fig 1 2E
8. PCR 30 30 35 lt gt PCR PCR 61 i denature AA METI 94 Cx30 1 30 93C 94 ii annealing
9. 46 9 4 lt EHRE RR R Eae 350 OLYMPUS ERE Table Bohr i Tun 47 PP U gt I D
10. 1000cells cm 635 Appendix 1 10min 1h EERDE T B Pp Pu p 3 4 i bs P d lt gt Fig 3 1 ImageJ ver 1 40g ImageJ Hile lImport gt Image Sequence IOK wi Edit Image Process Analyze Plugins Window Help New amp je o mjs 2 Z e gt Open Cr Open Next Ctri Shift O Open Samples d to switch Open Recent d Import Image Sequence Close crew P9 Close All LUT Save Ctrl S Text Ima
11. L 18 85177 8 Fig 5 1 25 Rho G Rho Cdc42 Rac Rho ROCK Cdc42 N WASP Rac WAVE H bFGF bFGF 3 Cofilin ThymosinB4 Cofilin G F 1 1 F ThymosinB4 G
12. GAPDH a actin B actin 2 A B X N A B X i X A B a b A XX B XX alb n ii N A B m n A N B N m n A N B N Gm n iii X ii N X a m bn 9 6 69 9 6 0 S E s amp Geaemwes e ees
13. Fig5 6 1 1 pd 1 SH Mk PI3K SH bFGF T bFGF PIBK bFGF m To aues SH2 Fig 5 5 30 TJPA 76 Dok Fig 5 6 PIBK LY294002
14. 15 1 596 20 U9 G F Bd 5 1 3
15. POWER PAC 300 80V 80mA 70 9 5 5 PCR lt gt 280nm UV 2 DNA Ethidium Bromide PCR b 2 DNA 280nm UV Ethidium Br omide 590nm Ethidium Bromide 300nm 300nm 280nm UV Ethidium Bromide ITOT I EDT EL Ethidium Bromide DNA 2 DNA
16. x7 2 MMP IV MMP2 T MMP9 Hig3 2 ColIv Y YI XT FNH eE h Fig 5 2 Cofilin ThymosinB4 MMP2 26 Cofilin ThymosinB4 MMP2 mRNA bFGF 3 DbEGF Cofilin ThymosinB4 MMP2 bFGE bFGFE mRNA
17. gt RNA OD ODoso OD 4 OD gt s RNA 1 5 RNA 0 2 7 0 5ug ul OD gt o 0 085 gt Smart Spec M 3000 Spectrophotometer BIO RAD 170 2501 Cuvette BIO RAD 170 2505 MINI CENTRIFUGE COSTER MVSS 06618 Vortex 0 5m 72 699S PE na ATE 53 S 200 62011 REYNI PAZA 0 1N HCl aq DEPC lt RNA gt D AGPC 10ul 1 0 5ml
18. PARAFILM Pechiney Plastic Packaging Inc PM 996 NuSieve GTG Agarose BMA 50080 Ultra Pure Agarose Invitrogen 15510 019 50x TAE buffer INTERGEN S4300 10x TAE buffer Invitrogen 15558 042 Ethidium Bromide SIGMA E 1510 Blue Juice M Invitrogen 10816 015 123 bp DNA Ladder Invitrogen 15613 011 DEPC 66 lt Agarose Gel 3 wt vol OfE8 gt D Wide Mini Sub Cell amp GT System Gel caster 2 NuSieve amp GTG Agarose Ultra Pure Agarose Z TEEEL PARE 9 5 X es X PERUCICAZST 7e lv RVNL E S 50x TAE buffer ZPA UE ID CGUAT UL ITAN DEPC 50 lx TAE buffer from 30x 9 9 9 5 Agarose Gel 3 wt vol NuSieve GTG Agarose g 1 2 50x TAE buffer ml
19. 2 DNA HASTO DNA 2 DNA DNA DNA gt AGPC RT PCR better RNase Ethidium Bromide 65 E
20. 121C 40 WEIT oE 5 LY294002 No LY294002 Cayman chemical 70920 5mg dimethylsulfoxide DMSO Sigma D8418 0 5ul ref 2006 E 5 mg ml 80 10uM DMSO 0 22um LY294002 5mg DMSO 1ml 10uM 1ml 0 61 ul 42 9 2 CUADE du H pus Phosphate Buffered Saline PBS D 8537 SIGMA 75 cm MS 21230 35 Biologix Counting chamber 03 202 1 Erma CO KUBOTA 2010 RD3132 Vortex S 100 TAITEC BT 15 Yamato lt gt HMS human mesenchymal stem cell Cell No 0043 Cell name MSC R36 2 bFGF FGF basic PAARI KV teL
21. 3 34 7 1 X 10 cells amp C45257 35P 8 15 cells 1 335 6 31 0um 50mm 1 4 1 200mm 14mm 1 5 2 0m 1 2 3 2 P
22. er PIE PIP2 EE 0 xd 9 C P p ON D D w jp De uet Fig 5 7 PBK 32 Rho Rho Rho Rho ROCK2 ROKe ROCK1 ROKB 2 isoform ROCK1 ROKB PI Rho G E O GS Q RhoA GEF RhoA m Fig 5 8 Rho 33 bFGF PI3K Cdc42 Rho P ko PI3K Akt
23. UV UV djpeg 70 4 Positive Control lt gt Gel Pro amp Analyz er Version 3 1 for WindowsTW gt Gel Pro amp Analyzer Version 3 1 for WindowsTMW 2 nl lt gt 2 i PCR Size bp i max OD an
24. Hig 1 3D 225 200 175 150 125 100 75 TEESE control IL 6 SDF1 VEGF bFGF beta Fig 1 3D bFGF bFGF n T Ji T 4 y a H Ji ES eu 9 2 pcd Mihiy MSC bFGF bFGF bFGF
25. 1 3 bFGE 1 3 1 bFGF bFGF basic Fibroblast Growth Factor FGF 2 T7 18 22 22 5 24 34 kDa 5 isoform bFGF MAPK Fig 1 3A bFGF bFGF Proliferation Migration Differentiated Phenotype Fig 1 3A bFGF OH 1 3 2 bFGF bFGF l Hig1 3B 1N NaOH 2 H uni i us 0h 24h 72h 0h 24h 72h 24h 72h B bFGF C HGF A D VEGF I T L ii E fibronectin 0h 2
26. Wide Mini Su b Cell amp GT System 1x TAE buffer from 10x 600ml Ethidium Bromide 20ul D X 3 PARAFILMGZ 20cm Blue JuicerM 3ul X Blue JuicerMV 1 4 123 bp DNA Ladder 3uD iX Blue JuicerM 3u Ix TAE buffer fr om 30x 3l PCR product 10ul Blue JuicerM 3ul 6 Positive Control 10u Blue JuicemM 3ul 13 CD
27. RNA 330ml 1 5ml 7 D isopropanol 50001 Vortex I THR 80C Over Night lt AGPC gt D 12000G 4C 10 X D 75 EtOH DEPC 500uu Vortex 10000G 4 5 D 2 5cm PARAFILM 10 X PARAFILM 52
28. 17 4 1 1 0 1 FBS 10 FBS bFGF 0 1 FBS without bFGF ji 20ng ml bFGF 10 FBS without bFGF 20ng ml bFGF j 100ng ml bFGF Fig 4 1C bFGF 18 4 1 2 1h 0 1 FBS bFGF 20ng ml bFGF 100ng ml bFGF 10 FBS 20ng ml bFGF 100ng ml bFGF without bFGF 45 40 0 1 FBS without bFGF 23 0 1 FBS 20ng ml bFGF g 30 0 196FBS 100ng ml bFGF 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58
29. 23 1 1 Id Hu E d d ccotuieddtad 1 1 2 REDE EL I MMMMMMMMMHMMHMHMIMIHIHIIIIH 1 1 2 1 8 C ne een REA MM 1 1 2 2 88 ces dr CN SERES eee MMMMHIMIIRIHIISR 3 1 2 2 1 8 SAT MM 3 1 2 2 2 IMRIBe e HIIS 3 1 2 3 MEDE E A MM MMMMMMMMIHIMHIIHIIHHMHHHHHHPH 4 1 2 4 R8 BOO EE TR CABE e MIRI 4 1 2 5 5 3E A REALI HH 5 1 345 AP ERUME SERRE ARIKHT DEGE e n MM MMMMMMRIRRIRRHRHRRHRR8s 5 1 3 1 bFGP e M IIl 5 1 3 2 bFGF eee III G 1 3 3 bFGF 15 Z8 XA AREE e MS 7 2 lt Q LE SERERE m 9 3 NANO PRRPEPITM Gn G GGG g 9 a np uem MESE B erdiersedai eisque beesubei videa torbapes daas 9 3 3 8 52 2 RID DS EMT AR MH 13 3 4 13 3 5 PBK 7 JJ VBIB AE SABER 14 3 6 C
30. control 1 71
31. 4 7cells 0 03cm2 asd e n n noe a t 13 100 3 S PI3K bFGF PBK LY294002 Cayman Chemicals 3 3 e35 3000cells cm DMEM 1096FBS 4 3ng ml bFGF confluent DMEM 0 1 FBS growth arrest 2 20uIHI PBS 0 2 24 30 36h
32. Image DDMEM 0 1 FBS WDMEM 10 FBS DMEM 0 1 FBS 100ng ml bFGF DMEM 10 FBS 100ng ml bFGF DMEM 0 1 FBS 100ng ml bFGF LY294002 DMEM 10 FBS 100ng ml bFGF LY294002 14 3 6 Cofilin Thymosinp4 MMP2 mRNA Cofilin 2 ThymosinB4 MMP2 mRNA RT PCR 935 Vx VAC TERES EE 1000cells cm 0 1 2 3 mRNA 935 4 1sample RT PCR Appendix DMEM 10 FBS DMEBM 10 FBS 20ng ml bFGF PCR primer primerBid 7l PCR 5 GATGTC ATC ATA TTT GGC AGG TT 3 GAPDH CCT GCA CCA CCA ACT GCT TAG CCC 3 5 CAT GTC TGA CAA ACC C 3 Thymosinp4 f 5 TTCACC AAC ATG CAA GTT CT 3 cofilin Lu PPM 5 GCT TCT CTG CCA GGG IG 3 S ACA GCC TGG CAT GGG GCA GT 3 5 TTC TCC TCC ATC CAG TGG AG 3 Fig 3 3 PCR 15 94C 30s
33. bFGF 3 5 1k 3 1 30 passage number 2 3 HMS00S1 S000cells cm DMEM 10 FBS 3ng ml bFGF sub confluent 10 Trypsin PBS P 4 7 CD29 CD44 CD45 CD34 CD14 BECKMAN COULTER America Flow cytometory pg Sigma Ardrich Japan Appendix 3 2 bFGF
34. bFGF 28 bFGF EBS FBS bFGF 0 1 FBS bFGF 10 FBS bFGF bFGF FBS bFGF 101 Ing ml bFGF 10ng ml bFGF 10 FBS
35. Eq A 3 60 9 5 PCR 5 i Taq DNA polymerase i ii 774 0f518 iv v DNA i i PCR 25 30 ii v
36. Q 3 TOP STAGE HEATER 37 D CO gt AIR 200 ml min CO 10 ml min 5 CO IL D 37 5 CO XCAP XCAP O III D XCAP D 3 O CO Virtual Dub 48
37. Tm 1 2 2 iii extension 72C 720 1000bp 1 1000bp 1 lt Primer gt 1 18728 20724 DNA NN petu EC ii CG G C A C CG 50
38. 2001 19 4 430 432 18 Bernstein B W et al ADF cofilin a functional node in cell biology Trends Cell Biol 2010 20 4 187 195 19 Santis M D et al B thymosins and interstitial lung disease study of a scleroderma cohort with a one year follow up 2011 12 1 22 20 Mimura S et al Growth factor defined culture medium for human mesenchymal stem cells nt J Dev Biol 2011 55 2 181 187 21 Sotiropoulou P A et al Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells STEM CELLS 2006 24 462 471 221ATOPY COM http allatopy wakabagari com index html 23 Voet D 2 2000 241CTS http www cstj co jp 23 PI3K 2001 19 4 438 444 26 Nakashio A et al Topotecan inhibits VEGF and bFGF induced vascular endothelial cell migration via downregulation of the PIS3K Akt signaling pathway Int J Cancer 2002 98 36 41 27 Gao F et al L 5F an apolipoprotein A I mimetic inhibits tumor angiogenesis by suppressing VEGF basic FGF signaling pathways Integr Biol Camb 2011 3 4 479 489 28 Maffucci T et al A phosphoinositide 3 kinase phospholipase Cgammalpathway regulates fibroblast growth factor induced capillary tube formation PLoS ONE 2009 4 Is
39. DbFGF Cofilin Thymosinp4 MMP2 mRNA Rho Cdc42 Rac mRNA 36 7 1 Ria 2004 2 RA PD 2008 3 Michiel G H et al The Mesothehal Cells in CAPD Effluent and Their Relation to Peritonitis Incidence Peritoneal Dialysis International 1991 11 22 26 4 Yamamoto T et al Morphological studies of mesothelial cells in CAPD effluent and their clinical significance Am J Kidney Dis 1998 32 6 946 952 5 Gokal R et al The Text book of Peritoneal Dialysis Kluwer Academic Publishers 1994 6 Horiuchi T et al An image analysis algorithm for kinematics and morphology measurement of cells for the evaluation of tissue construction Kidney International 2003 64 2280 2290 7 2002 8 Brzoska M et al Epithelial differentiation of human adipose tissue derived adult st
40. DEPC ug ul OD gt x x RNA ug uug u x il 54 ul RNA ug ug ul DEPC ul ul ul RNA 0 04 ug pl X 60 AGPC 10u RNA 1ul 9ul 39 9 5 3 RT PCR ma PCR DNA mRNA PCR DNA mRNA DNA
41. PI3K 31 bFGF PI3K LE IS PT3K LY294002 bFGF Rho Rho bFGF bFGF PBK Rho DPBK PI3K PBK PIP PIP PIP PDK1 PBK PP Akt PA Akt
42. DNA mini 2 1 3 5 D DEPC 10ul Vortex MINI CENTRIFUGE 2 RNA tT 55 5 4C 9 RNA RT PCR 9 5 2 RNA lt gt OD ODoso RNA PCR RT PCR mRNA DNA DNA mRNA PCR
43. DbFGF bFGF PI3K Cofilin ThymosinB4 MMP2 mRNA bFGF bFGE bFGF bFGF 0 1 FBS 100ng ml bFGF HGF GDNF PI3K
44. 29 5 3 bFGF bFGF PI3K bFGF TGF B P 208 ETT TS eI ue 7 Fig 5 5 Sre 2 SH2 SH akii SH
45. A RNA DNA 50 o LAZE o RNase free o O O O O AGPC o mE pmi x E f Hl pias ar AED CR13B DNA mini HETO LAB EQUIPMENT ID 872146 MINI CENTRIFUGE COSTER MVSS 06618 Vortex UJ 4 4 258A 1 5ml 72 690S Janni pee Crash ice
46. RNA Bee M ISOLATIO N OF RNA RNA RNA DNA gt DNA mRNA mRNA RNA RNa se RNA RNase RNA RNase 121C RNase
47. Crash ice RT Master Mix gt 9 2 RT Master Mix RT Master Mix 1 2 9 3 RNase inhibitor Super ScriptTM 9 2 RT Master Mix NENNEN mm bb E me eb bb ss RC RC RC NR EN CHNNENEN EMEN EE eme p po po p ps Lom Depp ebbe zx 9 3 RNase inhibitor Super Script bep llb Sample Super Script 58 lt Reverse Transcription gt OD RT Master Mix RNA AGPC R eaction Tube with Cap 9ul 3 9ul RT Master Mix 9ul Vortex LAME MINI CENTRIFUGE 4 PROGRAM TEMP CONTROL SYSTEM RT pro gram 01 5D
48. jp n I Eq A 1 I DX Eq A 5 1 DNA 100 2 n E ddn Eq A 5 2 d dn E xI Eq A 5 2 E E Eq A 5 2 Eq A 5 3 I xE XE e Eq A 5 3 PCR E PCR 9 5
49. 2 DEPC 59u1 3 Vortex C THY MINI CENTRIFUGE HCl aq 0 1N Cuvette Ze O Cuvette DEPC 60u Smart SpecTM 3000 Spectrophotometer Read Blank DEPC 1 60ul Smart Spec 3000 3 Rea d Sample Cuvette 9 lt gt 2 i i
50. SH2 PI3K p85 PBK PIBK PIP PIP PIP PH pH Akt PLCyl Sos Ras GAP PI3K PI3K P 5 1 Rho G Rho
51. bFGF bFGF mRNA DMEM 10 FBS 20ng ml bFGF mRNA FBS 0 1 FBS mRNA 10 FBS 100ng ml bFGF bFGF
52. X Y mage E PEON PT OS x 409 40 y 33 56 z 2 52 value 91 1018 1073 83x859 05 um 1280x1 024 8 bit 91MB 4326 945 423 534 215 276 11 Oh X 0 0 h X Yn Xn Xo yn yo xy h X xo 0 X yy h Y yo 0 Y OExcell Oh X Y t um h S Gi xe yc ye 1 Ze xt X xui t 1 X yt Y yui 1 Y Ze t Li t 1 12 3 3
53. 3 2 K DNA 1 DNA DNA 207 30 DNA DNA 9 4 59 cDNA Primer antisense E TTTTTT 5 3 AAAAAA Primer antisense E TTTTTT TTTTT M Primer sense Primer 5 3 3 5 AAAAAA TTTTTT AA AAAA 3 5 B 3 zl ZI 5 3 mov denature E 3 5 3 A AAAAA n cycles 1 Y 2 9 4 PCR PCR gt PCR PCR PCR 1 DNA 2
54. PARAFILMQG Pechiney Plastic Packaging Inc PM 996 TERUMO NN 2432R RNA Bee M ISOLATION OF RNA Tel Test Inc CS 104B chloroform isoamylalcohol 24 1 SIGMA C 0549 isopropanol SIGMA I 9516 PBS SIGMA D 8537 75 BtOH DBPC EtOH DEPC EtOH Wako 321 00025 51 DEPC Diethylpyrocarbonate CALBIOCHEM 298711 Crash ice lt AGPC gt CD 35 PBS Iml well A PBS RNA Beerw 1ml well D 2 wel 10 1 5ml X lt gt 5 X wel O chloroform isoamylalcohol 24 1 200p 1 15 D R T 15 12000G 4 15 3
55. PROGRAM TEMP CONTROL SYSTEM 77 PCR program PCR product 20C 9 5 4 Gel Electrophoresis Jm IOLA a EA ar 2 DNA DNA DNA
56. p35 3000cells cm DMEM 10 FBS 3ng ml bFGF confluent DMEM 0 1 FBS growth arrest 2 20ul PBS 0 12 24 30 36h Image J Oh So h Si SySo X 100 l Confluent 2day 36h Growth arrest DDMEM 0 1 FBS DMEM 10 FBS DMEM 0 1 FBS 20ng ml bFGF D DMEM 1096FBS420ng ml bFGF DMEM 0 1 FBS 100ng ml bFGF 0DMEM 1096FBS 4 100ng ml bFGF Fig 3 2 3 4
57. 12 196 02835 Wako 198 01673 Wako TRYPSIN EBDTA SOLUTION 10x T4174 SIGMA 100 ml bFGF FGF basic DTT Dithiohtreitol 1261627 Invitrogen 500 ul PS Penicillin Streptomycin P0781 SIGMA LG L Glutamine G7513 SIGMA 1 DMEM gt DITS LG 5ml 15m 0 22um DMEM 40 FBS gt D 20 FBS 37 8 55 56 30 STERICUPTM 50ml 20 25ml lt gt 20C
58. 1 2 M T Cytokeratin 18
59. mRNA cDNA cDNA PCR mRNA PCR RT PCR 9 3 AAAAAA cDNA i mRNA fRNase H cDNA TTTTTT 5 _ sense Primer TTTTTT 2 K DNA PCR Ist cvele sense Primer A TTTTTT antisense Primer i PCR 3 2 A DNA 9 3 RT PCR 9 4 3 Reverse Transcription ma mRNA Primer Random Hexamer mRNA 1 DNA cDNA DNA RNA 23382375 2 PCR denature 1 56 DNA fcx zc EE LCv 5 mRNA S RNA RNase cDNA 9 3 CHER
60. 3 EE ALA GRO 3 24 2 AKA 39 9 Appendix 9 1 PETRI OO E lt EHEER mH BR 21B EXE 10 ml SS 10SZ TERUMO NN 2432R TEBRUMO MILLBEX GP SLGP033RS MILLIPORE STERICUPTM SCGVU0SRE MILLIPORE BT 15 Yamato 0 5ml A 050W Sml 60 9921 523S 15ml 1815 8 725172 117 VIOLAMO S0ml EE 725172 217 VIOLAMO 1L lt gt DMEMIDulbeccos Modified Eagle s Medium D6046 SIGMA 500 ml FBS Fetal Bovine Serum 500 ml PBS Phosphate Buffered Saline 169 04245 Wako 163 0354S Wako
61. Ale bu 9 0 1 FBS bFGF FBS 3 0 1 FBS bFGF 0 1 FBS bFGF bFGF bFGF bFGF bFGF
62. PCR Master Mix NINE 3 1 RT Master Mix 1 2 PCR Master Mix AmpliTaq GoldrV 9 4 PCR Master Mix 28 eme pe pm m ar p x e e ajajaj lt Polymerase Chain Reaction gt D PCR Master Mix AmpliTaq GoldTM D RT RT product 2ul Reaction Tube with Cap 3 PCR Master Mix AmpliTaq GoldrW Vortex CKE MINI CENTRIFUGE PCR Master Mix 48ul Vortex DAFL MINI CENTRIFUGE 1 64 GeneAmpG PCR system 2400 P CR MINERAL OIL SIGMA MS904 50ul 3 r
63. 5 2 bFGF S000cells cm 1 4 Fig5 3 27 0 1 FBS 1096FBS Fig 5 3 0 1 FBS bFGF 0 1 FBS bFGF 0 1 FBS bFGF 10 FBS 0 1 FBS bFGF
64. Cofilin MMP2 ThymosinB4 0day bFGF 2 0 9 n 2 3 0 8 II II II 0 7 0 6 0 5 Thymosinp4 B Cofilin BNMP2 0 4 0 3 mRNA vs GAPDH 0 1 mmm l m 0 0 EN E Oday 1day 2day 3day bFGF Fig 4 S Cofilin ThymosinB4 MMP2 mRNA 24 5 25 2x 5 1 bFGF bFGF 2 3
65. Ethidium Bromide 68 2 DNA x DNA EZET 2 DNA Ethidium Bro mide gt PCR DNA DNA PCR DNA GAP DH 3 house keeping gene
66. 2 1 2 2 1 2 2 1 Fig1 2C 4 1 30 Fig 1 2C 1 2 2 2 1 2 2 1
67. RNase inhibitor Super ScriptTM 1ul 2 2ul 4 0 PROGRAM TEMP CONTROL SYSTEM RT pro gram 02 RT product 20C 9 4 3 2 Polymerase Chain Reaction ma gt PCR 3 DNA 2 RDNA 1 denature DNA 2 DN A 2 annealing DNA 3 DNA DNA extension 2 2 DNA
68. Cdc42 Akt bFG l T f discs PIP gt PIP3 EE E Es G ed C Grb2 CTS Arp2 Am23 Amp2 3 Fig 5 9 bFGF PBK 34 Rho Akt GSK3 B mTOR PHBA bFGF bFGF PIP PIP3 B B ARR RR 2 2 lt 4 20 EN ORO ep A N GSK3 B mTOR cydinD1 im AE Fig 5 10 bFGF PIBK Akt PI3K 35 6
69. PCR o PROGRAM TEMP CONTROL SYSTEM P CR program o 4 2 o RT product cDNA PCR o RT Reaction Tube with Cap lt gt PROGRAM TEMP CONTROL SYSTEM ASTEC PC 708 MINI CENTRIFUGE COSTER MVSS 06618 Vortex Reaction Tube with Cap MicroAmp N801 0540 S uu ATE Crash ice dATP Amersham Biosciences 27 2050 01 dTTP Amersham Biosciences 272080 dCTP Amersham Biosciences 272060 dGTP Amersham Biosciences 272070 Primer sense antisense 4 fE AmpliTaq Gold M Applied Biosystems N808 0242 10x PCR buffer PERKIN ELMER E12387 DEPC 63 Crash ice PCR Master Mix gt 9 4
70. RNase 9 6 1 AGPC RT o PROGRAM TEMP CONTROL SYSTEM RT program 01 02 ZUNE o Reaction Tube with Cap o Reaction Tube with Cap o Reaction Tube with Cap lt fE H FERT Ar e RAIE gt PROGRAM TEMP CONTROL SYSTEM ASTEC PC 708 MINI CENTRIFUGE COSTER MVSS 06618 Vortex Reaction Tube with Cap Micro Amp N801 0540 c eres Crash ice Random Primers Invitrogen 48190 011 dATP Amersham Biosciences 27 2050 01 dTTP Amersham Biosciences 272080 dCTP Amersham Biosciences 272060 dGTP Amersham Biosciences 272070 10x PCR buffer PERKIN ELMER E12387 Recombinant RNasin Ribonuclease Inhibitor Promega N2511 super Script M RNase H Reverse Transcriptase Invitrogen 18053 017 DTI Invitrogen Y00147 DEPC 57
71. XCAP D XCAP PIXCL 1Open Close Multiple Devices D 2 Use Common amp Controls W Cameras Synchronized 1 Use Individual Format amp Control LOK 1 OPEN ppc im io 3 M O jig PRETA C ARXVUBEHI3 OUBQUERO Unit Capture minit Capture Video to Image File gmile Browse Jpeg Capture Tmagse to Save Time Interval Snap Video at Time Linear Seaquence Event Free D Record Virtual Dub D Virtual Dub D
72. bFGF 20ng ml 100ng ml bFGF bFGF Fig5 4 0 196 FBS 0 196 FBS 0 196 FBS 1096 FBS 20ng mlbFGF 100ng mlbFGF zu Sa m 10 FBS 10 FBS 20ng ml bFGF 100ng ml bFGF m ms 4 om hi Fig 5 4 bFGF 10 FBS bFGF confluent
73. h 40 109 6FBS without bFGF e 10 FBS 20ng ml bFGF 10 FBS 100ng ml bFGF 15 is 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 h 0 1 FBS 10 FBS 19 4 2 bFGF Hig4 2A without bFGF 20ng ml bFGF 100ng ml bFGF 7 m without bFGF 20ng ml bFGF 100ng ml bFGF Oh vmi 36h Fig 4 2A bFGF M kA Ri 3 26 SERRE OO D SEHE 8 M 0 RE BA ZEE EX 20 0 1 FBS 20ng ml bFGF bFGF 100ng ml bFGF mm Fig4 2B 10 FBS 20ng ml bFGF 100ng ml bFGF bFGF Hig4 2B n 3 4 0 1 FBS without bFGF
74. N N X gt Ethidium Bromide PCR o o o jpeg ERR t t ACD OARA UVYYT4W A TOYOBO FAS III Gel Pro amp Analyzer Version 3 1 for Windows Media Cybernetics 50N31000 11851 Jes pe euet ZUwyt Tz4AZ Windows UP ar A PCR gt D
75. 6 10 ml 3 ml 7 10 5x10 cells ml 37C 5 CO gt Aux NES UI 3 Imm 0 1mm x10 cells ml lt gt lt gt D 37 D oss 6 d 2 9 1 d ERIS rie c SOS Tte H 44 9 3 88 Jg D ORG EUR 2
76. 0 192 FBS 20ng ml bFGF 0 19eFBS 100ng ml bFGF 10 FBS without bFGF e 10 FBS 20ng ml bFGF 1095FBS 100ng ml bFGF m H gR S R 9 n t Wn iR 21 4 3 bFGF Hig 4 4 0 1 FBS bFGF 10 FBS 0 1 FBS bEFGF 2 BELTS ES Fig 4 3 22 4 4 PIBK PBK bEGF DO 196FBS 100ng ml bFGF LY294002 100ng ml bFGF
77. 3 AT iii Tm Tm Melting Temperature PCR DNA 2 de nature 1 55 80 2 Tm Tm A T 2C C G 4C Tm iv 2 3 62 2 PCR v PCR gt RNase 9 6 1 AGPC
78. 60 C Imin 72 1min 94 C 30s 60 C Imin 72 C 1 min 94 C 30s 60 C Imin 72 C 1 min 94 C 5min 94 C 30s 60C Imin 72 C 1min30s 94 C 30s 60C Imin 72 C 1min 94 C 30s 60 C Imin 72 Imin 94 C Imin 60 C Imin 72 C Imin 94 C 40s 60 C 10min 94C 40s 60 C 10min 94 C 40s 60 C 10min 4 4 1 bFGF 24h 48h 72h Fig4 1A B 0 1 EFBS 10 FBS bFGF 0 1 EBS Without bFGF 20ng ml bFGF Fig 4 1A bFGF WIN LAM ERRADO HERE M 00 SEE E TH 2E BRACELET 0 1 FBS 16 10 FBS Without bFGF Fig 4 1B bFGF 10 FBS bFGF
79. CIA DMEM Dulbecco s Modified Eagle s Medium D6046 SIGMA 500 ml FBS Fetal Bovine Serum 500ml SEINE lt gt 37 5 CO D 1 2 3 PBS 10 ml7Scm 4 PBS 1 Trypsin EDTA solution 1ml PBS 9 ml UOTE FRU ZAR ELT 43 2 HUZ zv 10 ml 75ScmS lt 30 10 2 3 FBS 2ml 75 cm lt 4 10 ml 15 ml 35 DECAT 5 50 G 800 rpm5 4C
80. Ultra Pure Agarose g o6 DEPC ml 1x TAE buffer from 50x buffer ACTRESS X 1 X buffer 1x TAE buffer from 30x D Ethidium Bromide 6 5ul 10 10x TAE buffer DBPC 1 0 1x TAE buffer from 10x 10 67 Gel Electrophoresis gt D
81. YE 80C br EE gt 2 ml 72 693 S Erma lt gt DMEMIDulbeccos Modified Eagle s Medium D6046 SIGMA 500 ml FBS Fetal Bovine Serum 500ml PBS e TRYPSIN EDTA SOLUTION 10x T4174 SIGMA 100 ml 637 0221 WAKO lt gt CD 1 PBS 5ml 25cm 10ml 75cm 2 10 trypsin 37C 3 s se FBS 2Scm Iml 75cm 2ml 3 15ml 50g 5 4 10 FBS
82. bFGF B 0 195 FBS without bFGF 0 196 FBS 100ng ml bFGF 0 196 FBS 1 00ng ml bFGF LY 10 20 h Fig 4 4A PI3K 0 1 FBS 100ng mlbFGF 10 FBS 100ng ml bFGF LY294002 100ng ml bFGF bFGF JA AH 1096 FBS without bFGF 1096 FBS 100ng ml bFGF 1096FBS 100ng ml bFGF LY 20 30 40 h Fig 4 4B PBK 10 FBS 100ng mlbFGF 23 4 5 Cofilin Thymosinp4 MMP2 mRNA RT PCR Cofilin Thymosinp4 MMP2 mRNA Cofilin Oday 1 2day 3day bFGF ThymosinB4 Cofilin 0day bFGF
83. 4h 72h 0h 24h 72h D g 8 g etc o e g 5 3 8 oz lt gs E Fig 1 3B 6 bFGF mRNA 13 ep Fig1 3C E 0 19c glucose control z um 4 05c glucose BE 0 1 glucose control mi 4 09c glucose bFGF mRNA B actin mRNA bFGF protein pg ml 6 hours 12 hours 24 hours 48 hours Time After Medium Change 6 hours 12 hours 24 hours 48 hours Time After Medium Change Fig l 3C bFGF 1 3 3 bFGF 1 3 1 bFGF bFGF bFGE
84. 5 ml 50g 5 NCTE I HEZITON 5 Cell Pellet 1ml 6 Sx10 1x10 cells ml 80C D 1 10 FBS 5m 37C 5 CO 45 2 80C IA 3 3 4 37 4 15ml X DL UC 10ml 5 50g 750rpm 5 4C UL 6 1ml
85. em cells Biochemical and Biophysical Research Communications 2005 330 142 150 9 Baer P C et al Conditioned medium from renal tubular epithelial cells initiates differentia onof human mesenchymal stem cells Cell Prolif 2009 42 29 37 10 Nugent M A et al Fibroblast growth factor 2 Int J Biochem Cell Biol 2000 32 115 120 11 Tsutsumi S et al Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF Biochem Biophys Res Commun 2001 288 413 419 12 Fujgikawa K et al Expression of mRNA for growth factors and extracellular matrix proteins after injury to cultured peritoneal cells does the healing process contribute to peritoneal ultrastructural alteration J Artif Organs 2003 6 253 259 13 Ogata S et al Glucose and Prednisolone Alter Basic Fibroblast Growth Factor Expression in Peritoneal Mesothelial Cells and Fibroblast J Am Soc Nephrol 2001 12 2787 2796 14 Schmidt A et al Basic Fibroblast Growth Factor Controls Migration in Human Mesenchymal Stem Cells STEM CELLS 2006 24 1750 1758 15 2006 16 2004 37 17 A Enc
86. ge Save As Text File Revert CHrLR Results URL 2 Analyze 2 5 Set Measurement Area Centroid Rectangular or rounded rectangular selections right click to switch Set Measurements ly Area Mean Gray Value Standard Deviation Modal Gray Value Min amp Max Gray Value Center of Mass Perimeter Bounding Rectangle FitEllipse Shape Descriptors Ferets Diameter Integrated Density Median Skewness Kurtosis Area Fraction Stack Position Me T 4 XZ Analyze 2 6 Set Scale Distance in pixels 1298 Known Distance 250 KUnit of Length Tum File Edit Image Process Analyze Plugins Window Help Soele s a jJ x 872 48 y 147 65 z 0 value 106 L06 uim 1280x1024 8 bit 91MB z Set Scale Distance in Pixels Known Distance Pixel Aspect Ratio Unit of Length Click to Remove Scale Global Scale 1 192 pixels um OK Cancel Polygon selections 3 2 H ZHP LT Cl M
87. ofilin Thymosinf4 MMP2 mRNA Ji SjBlXE re r 15 4 16 ji pde 5s A EU EC PET nEPEA DES PEES 16 4 1 1 42 B JOOWJL eee ehh III 18 4 1 2 42 JR Ee eee ee eh hh HH hh hh 19 4 2 5 28 x5 ERR OO EET RANA MIRI 20 4 3 bFGF ee 29 4 4 PBK 777 7 VIR SERA HMM 29 4 5 Cofilin ThymosinB4 MMP2 mRNA ee 24 5 I bFGF C A Eppe Eee 25 5 2 bFGF CA EEICT RJAD E He e ee eee BBBBBRRRRRRSeeeeee 27 5 3 bFGF EARI RDAs ee IIIHMHHHSHnH 30 6 o 1 00A 36 0 D ADDODOdIX 5995 56378 E TES QS Ei RI YS Qd e SE dee embeds Red epu E a E 40 9 2 AMADER RE hh HII HH 43 OC ME ee 45 9 4 EIRE RR 47 0 5 RT PCR s eee ehh 50 1 Till 1 1
88. sue 12 e8285 29 Abe M et al Evidence that PI3K Rac Rho and Rho kinase are involved in basic fibroblast growth factor stimulated fibroblast Collagen matrix contraction Jounal of Cellular Biochemistry 2007 102 1290 1299 30 T 277 VE TA y7 NF EtE2004 31 Springer 2002 32 Lee J G et al FGF 2 induced wound healing in corneal endothelial cells requires Cdc42 activation and Rho inactivation through the phosphatidylinositol 3 kinase pathway IOVS 2006 47 4 1376 1386 33 Vemula s et al ROCKI functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability Blood 2010 115 9 1785 1796 34 PTEN SR 38 8 GE bFGF
89. thidium Bromide Ei HE Ethidium Bromide 2 DNA DN A Ethidium Bromide buf fer O fi HH 18 OC 8 Over Nisht Fia iH t SACS gt Wide Mini Sub Cell GT System BIO RAD 170 4485 POWER PAC 300 BIO RAD 282 BR 11443 LIBROR AEL 200 100mt 100ml
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