総括研究成果報告書

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1 BILD 1

2 diethylstilbestrol (DES) estrogen DNA embryonic stem (ES) ES ES A3-1 ES ES Genetic Microsystems DNA 2

3 3 DNA DNA DNA BILD

4 ES ES ES RNA RNA cdna DNA DNA estrogen MCF-7 RNA DNA ES mrna Differential Subtracting Cloning ES 4

5 ES ES ES DMEM 48 1% dextran DCC 4 ES total RNA mrna ES ethynylestradiol (E2) testosterone propionate (TP) bisphenol A (BisA) DNA mouse chip set I (TAKARA) Atlas TM Glass MOUSE 1.0 Microarray (Clontech) mrna Cy5 Cy3 DNA DNA GMS TM 418 Array Scanner (TAKARA) Imagene TM Ver.3 (TAKARA) ES Mouse chip set I Atlas TM Glass MOUSE 1.0 Microarray 2 5

6 E2 ES 3 DNA 0.5 nm E2 50 nm E2 Cy5 Cy3 2 E2 10 E2 ( Cy3 Cy5) TP BisA TP mouse chip set I Bis-1 Atlas TM Glass MOUSE 1.0 Microarray TP BisA glutamate receptor 5 precursor (GLUR5); ionotropic glutamate receptor kainate1 (GRIK1) transcription factor 4 (TCF4); immunoglobulin transcription factor 2 (mitf2; ITF2); SL3-3 enhancer factor 2 (SEF2) ets-related transcription factor; E74-like factor 1 (ELF1) 40S ribosomal protein SA (RPSA); lamimin receptor 1 (LAMR1); 34/67-kDa laminin receptor ES ES DNA E2 BisA TP DDE DDT ES cdna subtraction PCR ES 2.5 x 10 5 /ml 1 1 x 10 6 /ml 4 x 10 5 LIF E2 5 x M BisA 4 x 10-6 M TP 2 x 10-9 M DDE 2 x 10-9 M IC50 6

7 E2 BisA 4 TP DDE 3 LIF 1 3 RNA Clontech TAKARA DNA RNA ES LIF LIF 1 3 mrna cdna stbtration PCR ES E2 BisA E2 41 BisA 27 4 ES TP DDE ES E2 BisA TAKARA DNA 1 3 Clontech DNA E2 3 BisA 1 ES TP DDE 1 TAKARA DNA 3 Clontech DNA TP 1 3 DDE 1 3 ES 1 3 TAKARA DNA E2 1 3 TP DDE Clontech DNA 7

8 DDE 816 cdna 200 1,000 DNA ES MCF nm E2 E2 100% DCC ( %) MCF-7 DCC 48 E2 5 MCF-7 total RNA mrna human DNA chip for endocrine disruption study version 1.0 (TAKARA, catalog #: X103) mrna (TAKARA mrna 2 mg Clontech mrna 5 mg) Cy5 Cy3 ImageneTM Ver.3 (TAKARA) E2 MCF-7 DNA Cy5 Cy3 human DNA chip for endocrine disruption study version Cy3 Cy5 E2 MCF-7 trefoil factor 1 Cy5 Cy3 15 8

9 E2 ( Cy3 Cy5) E2 DNA Cy5 dutp Cy3 Cy5 RNA cdna ( ) Clontech cdna ( ) TAKARA Label it (TAKARA) Clontech 2 human DNA chip for endocrine disruption study version 1.0 Cy5 Cy3 (Label A) (Label B) 2 E2 E2 trefoil factor 1 House keeping genes actin ( ; 24 spots ) ( ) Label A ( ) ( ) Label B ( ) ( ) DNA 1:1 E2 Cy3 Cy5 E E2 17 E2 v-fos FBJ murine osteosarcoma viral oncogene homolog Cy Cy topoisomerase (DNA) I Cy Cy Cy3 Cy

10 ES dimethylnitrosoamin (DMN) MCF-7 estrogen E2 BisA (CD-1) SD 19 CD-1 E2 BisA E2 BisA SD DMN 10mg kg 3 24 DMN 3 24 RNA mrna RNA DNA MCF-7 E2 BisA RNA mrna RNA Intelli Gene human DNA chip for endocrine disruption study DNA 19 CD-1 E2 BisA 24 E2 CD µg/kg µg/kg BisA CD-1 10

11 1.2 8 SD DMN (0h) 3 24 RNA mrna RNA DNA DMN MCF-7 BisA Intelli Gene human DNA chip for endocrine disruption study ES DNA DNA DNA DNA MCF-7 2x10 6 cells/plate 6 total RNA DES 8 5 total RNA Total RNA 15 g DNA Affymetrix 418 Array Scanner Cy3 Cy5 ImaGene Spot Signal Mean + 2 SD > Background Signal Mean Cy3 Cy5 Log(Cy3/Cy5) Mean 0 11

12 Human EST DNA EST 428 DNA ImaGene DNA GeneSight K-means Spot Signal Mean SD > Background Signal Mean total RNA 15 g total RNA 15 g total RNA 15 g total RNA 15 g MCF-7 E2 BisA 1/2 E2 BisA DES trefoil factor 1 EST EST 428 DNA E2 DES BisA Genistein 1/2 E2 DES BisA Genistein DES Genistein K-means E2 DNA DNA total RNA total RNA total RNA

13 DNA estrogen MCF-7 DNA ES estrogen E2 BisA estrogen androgen PT androgen DDE DNA DNA 13

14 ES DNA ES DNA 14

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