第七章 : 圖表
第一節、 表
Table 1 利用 PhytoViewerBR 2.2 分析 SKOV-3 細胞株投予松杉靈芝
萃取物之 Affymetrix 人類微陣列基因表現資料進行各組間
PSI (Phytomics similarity index) matrix 之分析比較
(a)
MeGt vs. EtGt – union-1
Me2 Me3 Me4 Et6 Et7
Me2 Me3 Me4 Et6 Et7
1
1
1
1
1 MeGt vs. EtGt
– union-1
Me2 Me3 Me4 Et6 Et7
Me2 Me3 Me4 Et6 Et7
1
1
1
1
1
(b)
Me2 Me3 Me4
1
1
1 Me2
Me3 Me4 Union-1
Me2 Me3 Me4
1
1
1 Me2
Me3
Me4
Union-1
(c)
MeGt vs. EtGt - intersect
Me2 Me3 Me4 Et6 Et7
Me2 Me3 Me4 Et6 Et7
1
1
1
1
1 MeGt vs. EtGt
- intersect MeGt vs. EtGt
- intersect
Me2 Me3 Me4 Et6 Et7
Me2 Me3 Me4 Et6 Et7
1
1
1
1
1
(d)
Me2 Me3 Me4
Me2 Me3 Me4
1
1
1 Intersect
Me2 Me3 Me4
Me2 Me3 Me4
1
1
1 Intersect
Tab. 1 PSI (Phytomics Similarity Index)
SKOV-3 細胞株投予松杉靈芝萃取物 24 小時，抽取其 total RNA，利用 Affymetrix 人類微陣列基因表現技術分析其 mRNA 量的改變；經過
PhytoViewerBR 2.2 聯集分析(a, b)，得到所有出現於各組 data 中的 genes，及交集 分析(c, d)，得到各組 data 中都有出現的 genes。各組間相似度以 PSI value(0~1; 0, complete difference; 1, complete similarity)來表示。由 PSI matrix 可看出投予松杉 靈芝甲醇萃取物的組別(Me2~Me4)與投予松杉靈芝乙醇萃取物的組別(Et6、
Et7)，其 gene expression 不同但相似(a, c)。若只比較投予松杉靈芝甲醇萃取物的 組別(Me2~Me4)，則 PSI value 皆大於 0.95，表示此三組間極相似。
Table 2 Altered protein spots review
Zinc finger protein 578, ZNF5780.34 LIM and SH3 protein 1, LASP1, Lasp-3-7101 1 PCTAIRE protein kinase 1, isoform CRA_b, PCTK1, PCTGAIRE 4-F heterogeneous nuclear
ribonucleoprotein U-like 2(HNRPU2), HNRPUL2 VIME, Vimentin, VIM
4.72 ENPL, Endoplasmin precursor, Heat
shock protein 90 kDa beta member 1, (94 kDa glucose-regulated protein) (GRP94) (Tumor rejection antigen 1), HSP90B1,GP96 4F2, 4F2 cell-surface antigen heavy
chain (4F2hc), solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform f, SLC3A2, 4F2HC, 4T2HC 5-1802 GRP78, 78 kDa glucose-regulated
protein precursor, HSPA5, BIP, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
3.11 VIME, Vimentin, VIM
5-1301
HDGF, Hepatoma-derived growth factor (HDGF) (High-mobility group protein 1-like 2) (HMG-1L2)
2.44 GNL3, Guanine nucleotide-binding
protein-like 3 (Nucleolar GTP-binding protein 3) (Nucleostemin) 5-1103 LDHB, L-lactate dehydrogenase B
chain (EC 1.1.1.27) (LDH-B) (LDH heart subunit) (LDH-H)
4.79
＊TBB2, Tubulin beta-2 chain, TUBB 5-5003 LASP1, LIM and SH3 domain protein 1 (LASP-1) (MLN 50) HINT1, Histidine triad
nucleotide-binding protein 1(Adenosine 5'-monophosphoramidase) PSA2, Proteasome subunit alpha type 2 (EC 3.4.25.1) (Proteasome component C3), PSMA2
0.5 HNRH1, Heterogeneous nuclear
ribonucleoprotein H (hnRNP H), HNRPH1 FABPE, Fatty acid-binding protein,
epidermal (E-FABP) (Psoriasis-associated fatty acid-binding pr), FABP5, EFABP CAPG, Macrophage capping protein (Actin-regulatory protein CAP-G) 5-6001 CATZ, Cathepsin Z precursor
(Cathepsin X) (Cathepsin P), CTSZ 5-1109 Zinc finger protein 578, ZNF578
0.34 LIM and SH3 protein 1, LASP1, Lasp-3-7101 1 PCTAIRE protein kinase 1, isoform CRA_b, PCTK1, PCTGAIRE 4-F heterogeneous nuclear
ribonucleoprotein U-like 2(HNRPU2), HNRPUL2 VIME, Vimentin, VIM
4.72 ENPL, Endoplasmin precursor, Heat
shock protein 90 kDa beta member 1, (94 kDa glucose-regulated protein) (GRP94) (Tumor rejection antigen 1), HSP90B1,GP96 4F2, 4F2 cell-surface antigen heavy
chain (4F2hc), solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform f, SLC3A2, 4F2HC, 4T2HC 5-1802 GRP78, 78 kDa glucose-regulated
protein precursor, HSPA5, BIP, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
3.11 VIME, Vimentin, VIM
5-1301
HDGF, Hepatoma-derived growth factor (HDGF) (High-mobility group protein 1-like 2) (HMG-1L2)
2.44 GNL3, Guanine nucleotide-binding
protein-like 3 (Nucleolar GTP-binding protein 3) (Nucleostemin) 5-1103 LDHB, L-lactate dehydrogenase B
chain (EC 1.1.1.27) (LDH-B) (LDH heart subunit) (LDH-H)
4.79
＊TBB2, Tubulin beta-2 chain, TUBB 5-5003 LASP1, LIM and SH3 domain protein 1 (LASP-1) (MLN 50) HINT1, Histidine triad
nucleotide-binding protein 1(Adenosine 5'-monophosphoramidase) PSA2, Proteasome subunit alpha type 2 (EC 3.4.25.1) (Proteasome component C3), PSMA2
0.5 HNRH1, Heterogeneous nuclear
ribonucleoprotein H (hnRNP H), HNRPH1 FABPE, Fatty acid-binding protein,
epidermal (E-FABP) (Psoriasis-associated fatty acid-binding pr), FABP5, EFABP CAPG, Macrophage capping protein (Actin-regulatory protein CAP-G) 5-6001 CATZ, Cathepsin Z precursor
(Cathepsin X) (Cathepsin P), CTSZ 5-1109
5.01 70.48
11/47 gi|11960 65
8027 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa), isoform CRA_b, HSPA5, MIF2, FLJ26106, GRP78, BIP
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9), KRT9 protein disulfide isomerase family A, member 3, isoform CRA_a, phospholipase C-alpha, ER-60 protease, PDIA3, ERp60, GRP57 heterogeneous nuclear
ribonucleoprotein K, HNRPK, CSBP, TUNP
＊keratin 2, KRT2 620
＊keratin 9, KRT9, K9, EPPK 899
2.92 Chain A, Heat-Shock 70kd Protein 42kd Atpase N-Terminal Domain, HSPA5 protein disulfide isomerase family A, member 3, isoform CRA_a, phospholipase C-alpha, ER-60 protease, PDIA3, ERp60, GRP57 KHSRP protein, KHSRP, KSRP,
MGC99676, FBP2, FUBP2, KH-type splicing regulatory protein (FUSE binding protein 2)
Tetratricopeptide repeat protein 19
(TPR repeat protein 19), TTC19
＊keratin 2, KRT2 9-4311
＊keratin 9(type I keratin 9), KRT9 899 9-8703 FUBP1 protein, FUBP1, FBP, FUBP 2 8-5612 domain-associated protein, isoform CRA_a, TRRAP, TR-AP
73.07 23/55
gi|11958 176 9503 KH-type splicing regulatory protein (FUSE binding protein 2), isoform CRA_c KHSRP protein, KHSRP, KSRP,
MGC99676, FBP2, FUBP2, KH-type splicing regulatory protein (FUSE binding protein 2)
Tara, HRIHFB2122, KIAA1662,
TRIOBP dynein, axonemal, heavy polypeptide 3, DNAH3 keratin 6 irs4, K6IRS4, keratin 74, KRT74 dynein, axonemal, heavy polypeptide 3, DNAH3
keratin 3, KRT3 8552
0.15
＊keratin 2, KRT2 8-2401 vimentin variant
3.11 actin, alpha 1, skeletal muscle,
ACTA1
＊actin, gamma 1 propeptide, ACTG1
＊actin, beta, ACTB 5-2212
＊tubulin, beta 2B, isoform CRA_c, TUBB tubulin, beta 4, TUBB4
0.07
＊tubulin, beta 2C, isoform CRA_b 8775 5-5213
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9) (Keratin-9), KRT9 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa), isoform CRA_b, HSPA5, MIF2, FLJ26106, GRP78, BIP
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9), KRT9 protein disulfide isomerase family A, member 3, isoform CRA_a, phospholipase C-alpha, ER-60 protease, PDIA3, ERp60, GRP57 heterogeneous nuclear
ribonucleoprotein K, HNRPK, CSBP, TUNP
＊keratin 2, KRT2 620
＊keratin 9, KRT9, K9, EPPK 899
2.92 Chain A, Heat-Shock 70kd Protein 42kd Atpase N-Terminal Domain, HSPA5 protein disulfide isomerase family A, member 3, isoform CRA_a, phospholipase C-alpha, ER-60 protease, PDIA3, ERp60, GRP57 KHSRP protein, KHSRP, KSRP,
MGC99676, FBP2, FUBP2, KH-type splicing regulatory protein (FUSE binding protein 2)
Tetratricopeptide repeat protein 19
(TPR repeat protein 19), TTC19
＊keratin 2, KRT2 9-4311
＊keratin 9(type I keratin 9), KRT9 899 9-8703 FUBP1 protein, FUBP1, FBP, FUBP 2 8-5612 domain-associated protein, isoform CRA_a, TRRAP, TR-AP
73.07 23/55
gi|11958 176 9503 KH-type splicing regulatory protein (FUSE binding protein 2), isoform CRA_c KHSRP protein, KHSRP, KSRP,
MGC99676, FBP2, FUBP2, KH-type splicing regulatory protein (FUSE binding protein 2)
Tara, HRIHFB2122, KIAA1662,
TRIOBP dynein, axonemal, heavy polypeptide 3, DNAH3 keratin 6 irs4, K6IRS4, keratin 74, KRT74 dynein, axonemal, heavy polypeptide 3, DNAH3
keratin 3, KRT3 8552
0.15
＊keratin 2, KRT2 8-2401 vimentin variant
3.11 actin, alpha 1, skeletal muscle,
ACTA1
＊actin, gamma 1 propeptide, ACTG1
＊actin, beta, ACTB 5-2212
＊tubulin, beta 2B, isoform CRA_c, TUBB tubulin, beta 4, TUBB4
0.07
＊tubulin, beta 2C, isoform CRA_b 8775 5-5213
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9) (Keratin-9), KRT9
5-7002 1 4
2.29 cathepsin D preproprotein, CTSD
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9) (Keratin-9), KRT9
glutathione transferase, GSTP1, GST3 cytokeratin 8, KRT8
Chain W, Crystal Structure Of The Mammalian 20s Proteasome At 2.75 A Resolution
Q5VVZ6 155 Q32Q68 capping protein (actin filament)
muscle Z-line, beta, isoform CRA_a, F-actin capping protein beta subunit, CAPZB 78 kDa glucose-regulated protein
precursor, BiP protein (GRP 78) (Heat shock 70 kDa protein 5), HSPA5
＊actin, gamma 1 propeptide, ACTG1
＊ACTB protein, ACTB 3
＊beta actin, ACTB
0.44
＊actin, gamma 1 propeptide, ACTG1 Enoyl-CoA hydratase, mitochondrial precursor (Short chain enoyl-CoA hydratase), ECHS1
heat shock protein 27, HSPB1, Hsp25, HSP27, HSP28, heat shock 27kDa protein 1 calponin 3, acidic, Cnn3
＊KRT9 protein, KRT9
＊tubulin beta polypeptide, TUBB enolase 1, ENO1, NNE, PPH, MPB1
0.18
enolase 1 variant
nebulin, isoform CRA_f, NEB 09
0.26
＊Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (K2e)(Keratin 2), KRT2 chromosome 10 open reading frame 30, C10orf30, MGC35247 heterogeneous nuclear
ribonucleoprotein K, isoform CRA_f
0.24 heterogeneous nuclear
ribonucleoprotein K isoform a variant, HNRPK, CSBP, TUNP
＊actin, gamma 1 propeptide, ACTG1
＊ACTB protein, ACTB prolyl 4-hydroxylase, beta subunit
precursor, P4HB, Prolyl 4-hydroxylase subunit beta, Cellular thyroid hormone-binding protein, PDI, PO4HB Chain , Human Protein Disulfide
Isomerase, Nmr, 40 Structures
＊Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (K2e) (CK 2e), KRT2 elongation factor 2, EEF2
enolase 1, ENO1, NNE, PPH, MPB1 cathepsin D preproprotein, CTSD
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9) (Keratin-9), KRT9
glutathione transferase, GSTP1, GST3 cytokeratin 8, KRT8
Chain W, Crystal Structure Of The Mammalian 20s Proteasome At 2.75 A Resolution
Q5VVZ6 155 Q32Q68 capping protein (actin filament)
muscle Z-line, beta, isoform CRA_a, F-actin capping protein beta subunit, CAPZB 78 kDa glucose-regulated protein
precursor, BiP protein (GRP 78) (Heat shock 70 kDa protein 5), HSPA5
＊actin, gamma 1 propeptide, ACTG1
＊ACTB protein, ACTB 3
＊beta actin, ACTB
0.44
＊actin, gamma 1 propeptide, ACTG1 Enoyl-CoA hydratase, mitochondrial precursor (Short chain enoyl-CoA hydratase), ECHS1
heat shock protein 27, HSPB1, Hsp25, HSP27, HSP28, heat shock 27kDa protein 1 calponin 3, acidic, Cnn3
＊KRT9 protein, KRT9
＊tubulin beta polypeptide, TUBB enolase 1, ENO1, NNE, PPH, MPB1
0.18
enolase 1 variant
nebulin, isoform CRA_f, NEB 09
0.26
＊Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (K2e)(Keratin 2), KRT2 chromosome 10 open reading frame 30, C10orf30, MGC35247 heterogeneous nuclear
ribonucleoprotein K, isoform CRA_f
0.24 heterogeneous nuclear
ribonucleoprotein K isoform a variant, HNRPK, CSBP, TUNP
＊actin, gamma 1 propeptide, ACTG1
＊ACTB protein, ACTB prolyl 4-hydroxylase, beta subunit
precursor, P4HB, Prolyl 4-hydroxylase subunit beta, Cellular thyroid hormone-binding protein, PDI, PO4HB Chain , Human Protein Disulfide
Isomerase, Nmr, 40 Structures
＊Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (K2e) (CK 2e), KRT2 elongation factor 2, EEF2
enolase 1, ENO1, NNE, PPH, MPB1
2.78 cytokeratin 8, KRT8 3
pyruvate kinase 3 isoform 1 variant, PKM2
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9), KRT9 pyruvate kinase, pyruvate kinase 3 isoform 1,Pyruvate kinase isozymes M1/M2, PKM2, PK3, cytosolic thyroid hormone-binding protein (EC 2.7.1.40)
EDAR-associated death domain, isoform CRA_b, EDARADD peroxiredoxin 2 isoform a, thiol-specific
antioxidant protein, PRDX2, TSA, TDPX1, Natural killer cell-enhancing factor B cytokeratin 8, KRT8 3
pyruvate kinase 3 isoform 1 variant, PKM2
＊Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9), KRT9 pyruvate kinase, pyruvate kinase 3 isoform 1,Pyruvate kinase isozymes M1/M2, PKM2, PK3, cytosolic thyroid hormone-binding protein (EC 2.7.1.40)
EDAR-associated death domain, isoform CRA_b, EDARADD peroxiredoxin 2 isoform a, thiol-specific
antioxidant protein, PRDX2, TSA, TDPX1, Natural killer cell-enhancing factor B
17-6004 5 6
<備註>
Probability Based MOWSE Score : Protein score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 65 are significant (p<0.05). For a score that is exactly on the default significance
threshold, (p<0.05), the expectation value is also 0.05. Increase the score by 10 and the expectation value drops to 0.005. The lower the expectation value, the more significant the score.
＊有部份蛋白質點經 MALDI-TOF 分析，乃含有相同蛋白質，但其二維膠體染色分 析數據卻同時有兩倍以上增加及減少的相反情形出現
Table 3 比對 SKOV-3 細胞投予靈芝甲醇萃取物後 2D-MALDI/TOF
(蛋白質表現) 及 Affymetrix (基因表現) 分析結果
10.4 Mammalian 20s Proteasome
2.4 Mammalian 20s Proteasome
2.4
欄：表現量減少(control 組的二分之ㄧ以下)的蛋白質(共 23 個)，以及右欄：2D-MS 分析中表現量有增加亦有減少的蛋白質(共 6 個)。左欄中標紅色的蛋白質(HSPA5，SLC3A2，HSP90B1，P4HB)在 Affymetrix microarray 結果中亦見增加。
中欄中標綠色的蛋白質(CAPG，LASP1，TUBB4，HNRPK，Cnn3)則是減少。左 中二欄皆有出現的淺藍綠標記表示在Affymetrix microarray 數據分析中，三個批 次的結果並未完全相同，有可能是此基因表現有改變但不夠明顯。較奇特的是左 欄中標藍色的蛋白質(ECHS1，CAPZB，PKM2)，其 microarray 數據顯示其基因 表現下降，與蛋白質的上升表現相反。
Table 4 2D-MS 分析所得蛋白質之功能分類 Proteosome /
Proteosomal
Transcription factors/mRNA processing protein/Tranlation transport /
trafficking Metabolism
Chaperone
tubulin, beta 4↓
PKM2↑
tubulin, beta 2C KRT2
Vimentin↑
CAPG↓
tubulin, beta 2B KRT3↓
Cytoskeleton / Cytoskeleton-binding protein
PCTK1↑ Proteosome /
Proteosomal
Transcription factors/mRNA processing protein/Tranlation transport /
trafficking Metabolism
Chaperone
tubulin, beta 4↓
PKM2↑
tubulin, beta 2C KRT2
Vimentin↑
CAPG↓
tubulin, beta 2B KRT3↓
Cytoskeleton / Cytoskeleton-binding protein
Table 4
分析所得的 51 個蛋白質，根據其功能可大致分類為：chaperone，代 謝相關蛋白，細胞骨架蛋白，細胞骨架結合蛋白，與胞內物質運輸相關之蛋白質，異質性細胞核醣核蛋白(heterogeneous nuclear ribonucleoprotein, hnRNP)，轉錄因 子，調節mRNA splicing 的蛋白質，轉錄活化的輔因子，轉譯因子，生長因子，
溶酶體及蛋白酶體相關蛋白，具解毒功能的蛋白質，具抗氧化功能的蛋白質，甲 狀腺素結合蛋白，一般性酵素蛋白，以及其他蛋白質。表中箭號代表此蛋白質表 現量(跟據其相對蛋白點的 2D 影像分析)為增加或減少，無箭號標示的蛋白質為 在2D 影像分析中表現量有增加亦有減少的蛋白質(共 6 個)。