KISEP Original Article 생물정신의학 Vol. 12, No. 1, May 2005 Ginsenoside Rg1 및 Rb1 을처리한신경세포주 (SH-SY5Y 세포 ) 의 유전자발현양상 이준노 1) 양병환 2) 최승학 2) 김석현 2) 채영규 3) 정경화 3) 이준석 4) 최강주 5) 김영숙 5) Gene Expression Profiling of SH-SY5Y Human Neuroblastoma Cells Treated with Ginsenoside Rg1 and Rb1 Joon-Noh Lee, M.D., Ph.D., 1) Byung-Hwan Yang, M.D., Ph.D., 2) Seung-Hak Choi, M.D., 2) Seok-Hyun Kim, M.D., Ph.D., 2) Young-Gyu Chai, Ph.D., 3) Kyoung-Hwa Jung, M.S., 3) Jun-Seok Lee, M.D., Ph.D., 4) Kang-Ju Choi, Ph.D., 5) Young-Suk Kim, Ph.D. 5) ABSTRACT O bjectives - Methods Results Seoul National Hospital, Seoul, Korea Department of Neuropsychiatry, College of Medicine & The Mental Health Research Institute, Hanyang University, Seoul, Korea Division of Molecular and Life Science, College of Science, Hanyang University, Ansan, Korea Department of Psychiatry, College of Medicine, Kwandong University, Myongji Hospital, Goyang, Korea KT & G Central Research Institute, Daejeon, Korea - 42 -
- Conclusion- - KEY WORDS 서론 - - - - 43 -
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- - - - - 1. 세포배양 실험재료및방법 2. MTT assay - - - 45 -
3. Total RNA 분리 4. cdna microarray - - - - - - - 46 -
실험결과 1. MTT assay 결과 1) 세포증식결과확인 ( 현미경 ) Control Rb1 Rg1 0hr 400l 800l 400M 800M 400M 800M 12 hr 24 hr 36 hr 48hr Fig 1. The microscopic pictures200 of SH-SY5Y cells treated with ginsenoside Rb1 or Rg1 for 12, 24, 36 and 48 hours. Treatment of SH-SY5Y cells with 80M Rb1 for 36hours showed maximal proliferation compared with other concentrations or control. - 47 -
2) MTT assay 250 Total ginsenoside 2. cdna Microarray 결과 - 200 8g/10ml 16g/10ml 32g/10ml 200 Rb1 150 150 % 100 % 100 50 50 0 12 24 36 48 Timehr 0 12 24 36 48 Timehr Fig 2. MTT assay of SH-SY5Y cells treated with total ginsenosides. Cells were treated in triplicate with each 8, 16, 32 g of total ginsenosides for 12, 24, 36 and 48hours and MTT assays were performed. Y axis shows the percentages of each treatment group relative to the untreated matching control. 140 Rg1 20M 40M 80M Fig 4. MTT assay of SH-SY5Y cells treated with Rb1. Cells were treated in triplicate with each 20, 40, 80M Rb1 for 12, 24, 36 and 48 hours and MTT assays were performed. Y axis shows the percentages of each treatment group relative to the untreated matching control. 120 100 6 % 80 60 4 2 40 20 M 0 2 0 12 24 36 48 Timehr 4 6 20M 40M 80M Fig 3. MTT assay of SH-SY5Y cells treated with Rg1. Cells were treated in triplicate with each 20, 40, 80 M Rg1 for 12, 24, 36 and 48 hours and MTT assays were performed. Y axis shows the percentages of each treatment group relative to the untreated matching control. 6 8 10 12 14 A Fig 5. The MA plot of cdna microarray of SH-SY5Y cells following treated with Rg1 for 36 hours. 96 genes were up-regulatedcy5/cy33 and seven genes were down-regulatedcy5/cy30.33. Mrelative expression ratio=log2cy5/cy3 ASignal intensity=[log2cy5xcy3]/2. - 48 -
Table 1-1. Up-regulated genes and average expressed ratio of SH-SY5Y cells treated with ginsenoside Rg1 for 36 hours Function Protein biosynthesis Regulation of translation Regulation of transcription Signal transduction Gene symbol RPS21 RPS15A RPS15 UCHL1 RPL41 RPL18A UBA52 RPS12 RPS28 RPS27A RPS3A RPL23 RPL35 RPS10 RPL9 RPL14 RPL31 MRPL3 RPL38 RPL3 RPL23A RPS29 RPL10A RPS14 RPL13 RPL35 RPS3 RPS23 RPL12 RPS20 RPL6 RPL17 RPL11 RPL7A RPL37 RPS4X RPS7 EIF3S5 EEF2 EEF1B2 RPLP0 HMGB1 HMGB2 MLL2 ZNF45 RAN YWHAE YWHAQ RGS5 PLA2G1B CALM2 Expression ratiomlog2cy3/cy5 Gene name ribosomal protein S21 ribosomal protein S15a ribosomal protein S15 ubiquitin carboxyl-terminal esterase L1 ribosomal protein L41 ribosomal protein L18a ubiquitin A-52 residue ribosomal protein fusion product 1 ribosomal protein S12 ribosomal protein S28 ribosomal protein S27a ribosomal protein S3A ribosomal protein L23 ribosomal protein L35 ribosomal protein S10 ribosomal protein L9 ribosomal protein L14 ribosomal protein L31 mitochondrial ribosomal protein L3 ribosomal protein L38 ribosomal protein L3 ribosomal protein L23a ribosomal protein S29 ribosomal protein L10a ribosomal protein S14 ribosomal protein L13 ribosomal protein L35 ribosomal protein S3 ribosomal protein S23 ribosomal protein L12 ribosomal protein S20 ribosomal protein L6 ribosomal protein L17 ribosomal protein L11 ribosomal protein L7a ribosomal protein L37 ribosomal protein S4, X-linked ribosomal protein S7 eukaryotic translation initiation factor 3, subunit 5 epsilon, 47kDa eukaryotic translation elongation factor 2 eukaryotic translation elongation factor 1 beta 2 ribosomal protein, large, P0 high-mobility group box 1 high-mobility group box 2 myeloid/lymphoid or mixed-lineage leukemia 2 zinc finger protein 45 RAN, member RAS oncogene family tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide regulator of G-protein signalling 5 phospholipase A2, group IB calmodulin 2-49 - *Exp ratio 1.9 1.9 2.0 2.0 2.0 2.2 2.2 2.2 2.2 2.3 2.3 2.3 2.4 2.4 2.5 2.5 2.6 2.6 2.6 2.6 2.7 2.7 2.2 2.3 1.9 2.0 Gene bank access number AI026740 AA292861 J02984 AI928978 AI125571 F28484 AA522790 AA314429 AI929726 AA583926 M77234 AI147195 AA305945 AI066801 AI625598 D87735 AA039258 X06323 AI554584 NM_000967 AA857067 AA715449 NM_007104 AI928982 AI382216 AI815757 AA593872 D14530 L06505 AL037652 AI888138 X53777 L05092 AI625430 AI879226 M58458 AA315981 U94855 Z11692 X60489 AI92 X12597 X62534 AF010403 L758479696 NM_006325 U54778 AF070556 AI082260 M21054 D45887
1) Rg1 Table 1-2. Up-regulated genes and average expressed ratio of SH-SY5Y cells following treatment of ginsenoside Rg1 for 36 hours Function Gene symbol Gene name Cell proliferation and cell growth AF1Q SUI1 DDX5 FTH1 ALL1-fused gene from chromosome 1q putative translation initiation factor DEAD/HAsp-Glu-Ala-Asp/His box polypep 5 ferritin, heavy polypeptide 1 Neurogenesis GPI glucose phosphate isomerase and SCG10 Superior Cervical ganglion 10Stathmin-like 2 differentiation MLP MARCKS-like protein Regulation of CDK2AP1 CDK2-associated protein 1 cell cycle CCND1 cyclin D1 Energy transport ATP5L ATP synthase, H transporting, mitochondrial F0 complex, subunit g ATP5J ATP synthase, H transporting, mitochondrial F0 complex, subunit F6 UQCRH ubiquinol-cytochrom c reductase hinge protein Miscellaneous OK/SW-cl.56 beta 5-tubulin SNRPD2 small nuclear ribonucleoprotein D2 polypeptide 16.5kDa ACTG1 actin, gamma 1 KATNB1 katanin p80wd repeat containingsubunit B1 QP-C low molecular mass ubiquinone-binding protein9.5kd AAMP angio-associated, migratory cell protein XPO7 exportin 7 FLJ10468 hypothetical protein FLJ10468 NPM1 nucleophosmin HSPA8 heat shock 70kDa protein 8 HSPCB heat shock 90kDa protein 1, beta H3F3B H3 histone, family 3BH3.3B XAB2 XPA-binding protein 2 ABCF2 ATP-binding cassette, sub-family FGCN20, member 2 GNB2L1 guanine nucleotide binding proteing protein, beta polypeptide 2-like 1 PPIA peptidylprolyl isomerase A ART3 ADP-ribosyltransferase 3 H3F3A H3 histone, family 3A C20orf4 chromosome 20 open reading frame 4 GNAS GNAS complex locus TPT1 tumor protein, translationally-controlled 1 MDS006 x 006 protein DKFZp45-1G182 hypothetical protein DKFZp451G182 Expression ratiomlog2cy3/cy5 *Exp ratio 1.9 2.2 1.9 1.9 2.0 2.2 2.4 2.5 2.7 2.7 Genebank access number AL038143 AI832315 X15729 AA102267 K03515 D45352 AI341990 AB006077 X59798 W94335 AA452026 NM_006004 AF070561 AI126579 X04098 AF052432 AL036415 M95627 AB018288 AA134589 AA173870 AL044172 M16660 NM_005324 AI613115 AL050291 AL047767 AA304657 AI201027 AA313375 AI300103 X04409 AI979107 AI016298 AA931319-50 -
- - Table 2. Up-regulated genes and average expressed ratio of SH-SY5Y cells following treatment of ginsenoside Rb1 for 36 hours Function Gene symbol Gene name *Exp Genebank ratio access number Protein Biosynthesis RPL23A RPL3 RPS27A RPS29 RPS3A MRPL3 RPL10A RPL13 RPL35 RPL38 RPS3 RPL9 RPS10 RPL31 RPL37 RPL7A RPL17 RPS20 RPS23 RPL12 RPL11 RPL6 RPS4X RPS7 ribosomal protein L23a ribosomal protein L3 ribosomal protein S27a ribosomal protein S29 ribosomal protein S3A ribosom mitochondrial ribosomal protein L3 ribosomal protein L10a ribosomal protein L13 ribosomal protein L35 ribosomal protein L38 ribosomal protein S3 ribosomal protein L9 ribosomal protein S10 ribosomal protein L31 ribosomal protein L37 ribosomal protein L7a ribosomal protein L17 ribosomal protein S20 ribosomal protein S23 ribosomal protein L12 ribosomal protein L11 ribosomal protein L6 ribosomal protein S4, X-linked ribosomal protein S7 1.1 1.1 1.1 1.1 1.1 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.3 1.3 1.3 1.3 1.4 1.4 1.4 1.5 1.5 1.5 AA857067 NM_000967 AA583926 AA715449 M77234 X06323 NM_007104 AI382216 AI815757 AI554584 AA593872 AI625598 AI066801 AA039258 AI879226 AI625430 X53777 AL037652 D14530 L06505 L05092 AI888138 M58458 AA315981 Regulation of translation EEF1B2 RPLP0 eukaryotic translation elongation factor 1 beta 2 ribosomal protein, large, P0 1.1 1.4 X60489 AI929696 Regulation of transcription XAB2 ZNF45 XPA-binding protein 2 zinc finger protein 45 1.1 1.1 AI613115 L75847 Cell proliferation TSPAN-1 tetraspan 1 1.1 AF065388 Miscellaneous ART3 C20orf4 H3F3A PPIA TPT1 GNAS DKFZp451 MDS006 ADP-ribosyltransferase 3 chromosome 20 open reading frame 4 H3 histone, family 3A peptidylprolyl isomerase A(cyclophilin A) tumor protein, translationally-controlled 1 GNAS complex locus hypothetical protein DKFZp451G182 x 006 protein Expression ratiomlog2cy3/cy5 1.2 1.3 1.3 1.4 1.4 1.5 AI201027 AI300103 AA313375 AA304657 AI979107 X04409 AA931319 AI016298-51 -
M 6 4 2 0 2 4 6 2) Rb1 6 8 10 12 14 16 A Fig 6. The MA plot of cdna microarray of SH-SY5Y cells with Rb1 for 36 hours. 40 genes were up-regulated Cy5/Cy32 and no single gene was down-regulatedcy5/cy30.5. Mrelative expression ratio=log2cy5/cy3. ASignal intensity=log2cy5xcy3/2. - - 고 - - 찰 1. MTT Assay 결과에대한고찰 - 52 -
- 53 -
2. cdna Microarray 결과에대한고찰 - - 1) 전사, 번역및단백질생합성의조절 (Regulation of transcription, translation and protein biosynthesis) - - - 54 -
- - - 2) 신호변환조절 (Regulation of signal transduction) - - - 55 -
- - - 3) 세포증식, 성장및세포주기조절 (Regulation of cell cycle, proliferation, and growth) - - - - 4) 신경발생과분화조절 (Regulation of Neurogenesis and Differentiation) - - - 56 -
- - 결 - 론 - 57 -
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