第四章 研究結果
5.5 結論
後基因體的生物科技研究主題，生醫整合是一項很重要的議題。如何利用基因體時代所 發展出的關鍵技術，並應用於適合的研究對象。此二方面必需同時配合，才能真正發展 生物科技[陳培哲，2001]。透過肝癌(研究對象)臨床診斷資料與蛋白質體研究及生物資 訊(關鍵技術)資料整合，才能奠定國內後基因體時代的生物科技。
蛋白體學的研究，不僅增進對人類疾病的瞭解，也為提供疾病的更正確的診斷和早期治 療的研究。經由蛋白體學和生物資訊學研究，針對本土的重大疾病為研究對象，用來改 善國內健康並提昇國內的相關產業的發展。透過臨床資料與蛋白質體資料整合，奠定台 灣肝癌的研究基礎，進而開展肝癌之預後、診斷、和治療的新方法。維持台灣在肝病研 究的競爭優勢，並善加利用這些研究成果，以生物科技為基礎來發展穩固的經濟[國家 基因體計畫 website]。
第六章 文獻來源
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Website Information
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http://tw.expasy.org/tools/peptide-mass.html Accessed 2004.
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附錄一 肝癌有關的蛋白質點
Protein name(s) Accession No. References DNA replication /gene
regulation
High mobility group protein HMG1
P09429 N. Kawahara et al., Cancer Res, 56 (1996) 5330. Enhanced coexpression of thioredoxin and high mobility group pro tein 1 genes in human
hepatocellular carcinoma and the possible association with decreased sensitivity to cisplatin.
Purine nucleoside phosphorylase
P00491 L. Mohr et al., Hepatology, 31 (2000) 606. Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude mice by adenoviral transfer of the
Escherichia coli purine nucleoside
phosphorylase gene.a-Enolase) (EC 4.2.1.11)
(2-phospho-D-glycerate hydrolyase) (NON-neural enolase) (NNE)
(phosphopyruvate hydratase)
P06733 N. Durany et al., Br J Cancer, 75 (1997) 969.
Phosphoglycerate mutase,
2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymes in lung,
Creatine kinase, B-chain P12277 J. Joseph et al., Br J Cancer, 76 (1997) 600.
(EC 2.7.3.2) (B-CK) Creatine kinase activity and isoenzymes in lung, colon and liver carcinomas.
Nucleoside diphosphate kinase A (tumour metastatic
process-associated protein) of nm23-H1 in human hepatocellular carcinoma
(metastasis inhibition factor nm23) (nm23-H1)
P15531 Y. Fujimoto et al., J Gastroenterol, 33 (1998) 368. Reduced expression and rare genomic alteration Y. Fujimoto et al., J Gastroenterol, 33 (1998) 368.
Reduced expression and rare genomic alteration of nm23-H1 in human hepatocellular carcinoma.
Phosphoglycerate mutase, brain form (EC 5.4.2.1) (PGAM-B) (EC 5.4.2.4) (EC 3.1.3.13)
(BPG-dependent PGAM)
P18669 N. Durany et al., Br J Cancer, 75 (1997) 969 Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymes in lung, colon and liver carcinomas.
Glutathione synthetase (EC 6.3.2.3) (glutathione synthase) (GSH synthetase) (GSH-S)
P48637 Z. Huang et al., FASEB J, 15 (2001) 19. Mechanism and significance of increased glutathione level in human hepatocellular carcinoma and liver regeneration.
3-Hydroxyacyl-CoA dehydrogenase type II (EC 1.1.1.35) (Type II HADH) (endoplasmic reticulum-associated amyloid b-peptide binding protein) (short-chain type dehydrogenase/ reductase XH98G2)
Q99714 K. Suto et al., J Cancer Res Clin Oncol, 125 (1999) 83. Decreased expression of the peroxisomal bifunctional enzyme and carbonyl reductase in human hepatocellular carcinomas
Maspin precursor (protease inhibitor 5)
P36952 L. Yu et al., Electrophoresis, 21 (2000) 3058. Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography–ion trap mass spectrometry.
N. Maass et al., J Pathol, 195 (2001) 321. Decline in the expression of the serine roteinase inhibitor maspin is associated with tumour progression in ductal carcinomas of the breast.
Annexin V (lipocortin V) (endonexin II)
(calphobindin I) (CBP-I) (placental anticoagulant protein I) (PAP-I) (PP4)
P08758 Z.J. Gong et al., Hepatology, 29 (1999) 576. Transfection of a rat hepatoma cell line with a construct expressing human liver annexin V confers susceptibility to hepatitis B virus infection.
Glutathione S-transferase P (EC 2.5.1.18) (GST class-PI) (GSTP1-1)
P09211 J.C. Tchou et al., Int J Oncol, 16 (2000) 663. GSTP1 CpG island DNA
hypermethylation in hepatocellular carcinomas.
T. Zhou et al., Cancer Res, 57 (1997) 2749. Glutathione S-transferase expression in hepatitis B
virus-associated human hepatocellular.carcinogenesis.
Proteasome (prosome, 4506217 H. Higashitsuji et al., Nat Med, 6
non-ATPase, 10 retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.
S.A. Shah et al., Surg Oncol, 10 (2001) 43. Ubiquitin proteasome pathway:
implications and advances in cancer therapy.
Annexin I (lipocortin I) (calpactin II)
(chromobindin 9) (P35) (phospholipase A2 inhibitory protein)
P04083 T. Masaki et al., Hepatology, 24 (1996) 72. Enhanced expression of the protein kinase substrate annexin in human hepatocellular carcinoma.
14-3-3 Protein z /d
(protein kinase C inhibitor protein-1) (KCIP-1) (factor activating exoenzyme S) (FAS)
P29312 N. Iwata et al., Oncogene, 19 (2000) 5298. Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma.
14-3-3 Protein e (mitochondrial import stimulation factorL subunit) (protein kinase C inhibitor protein-1) (KCIP-1) (14-3-3E)
P42655 N. Iwata et al., Oncogene, 19 (2000) 5298. Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma.
Galectin-3 T.
(galactose-specific lectin 3) (MAC-2 antigen) (IgE-binding protein) (35 kDa lectin) (carbohydrate binding protein 35) (CBP
P17931 D.K. Hsu et al., Int J Cancer, 81 (1999) 519. Galectin-3 expression is induced in cirrhotic liver.
35) (laminin-binding protein) (lectin L-29) (L-31)
(galactoside-binding protein) (GALBP)
(laminin-binding protein) (lectin L-29) (L-31)
附錄二 肝功能檢查一覽表 (AST，ALT)
○
膽 鹼 酯 酶 (ChE)
○
BSP(ICG) 試 驗
○ ○
凝血酶原 時 間
○
膽固醇
B 型 肝 炎 (HBsAg)
○
特殊組合檢查如:
酒精性中毒： alcohol，MCV，folate，TG，GGT，AST，bilirubin 肝癌：α-fetoprotein，CEA，ALP，LDH，AST，ALT，GGT，HBsAg
附錄三 蛋白質體實驗條件
二維電泳分析：
肝臟組織在液態氮中，以硏砵及礎磨成細粉，其中的蛋白質以250 µl rehydration buffer (7M urea，2M thiourea， 4% CHAPS， 5 mM dithiothreitol， 2% IPG buffer，及微量的 bromophenol blue)溶解。將此含有待分析蛋白樣品的 250 µl rehydration buffer，加到 13 公分長的IPG 電泳梯度條(pH 3-10, Amersham Pharmacia)，在室溫下、30V、12 小時進 行rehydration 反應。等焦聚電泳在 20℃，5.5 小時完成。將完成的等焦聚電泳條帶，以 平衡液(6M urea， 50mM Tris-HCl buffer，pH8.8，30% glycerol，2% SDS 及微量的 bromophenol blue)，伴隨輕微的攪拌，浸潤電泳條 30 分鐘。將平衡好的電泳條平躺在 10% SDS 聚丙稀安胺電泳膠体(20 x 20 cm)上層，以 25 mA 定電流條件下跑第二維電泳，
直到追蹤用染劑跑到底。蛋白質點的呈色以銀染法或Colloid Coomassive blue 染之。蛋 白質點的的偵測及定量以Imagemaster (Amersham Pharmacia)軟體分析。
Tryptic in-gel digestion：
依Kaji et al 法，簡述如下。將對出有意義的蛋白質點經 CBB 染色自二維電泳膠片切 出，25nM ammonium carbonate 含 50% autonitile 溶液沖洗三次每次 15 分鐘。再以 autonitile 溶液將含蛋白質點的膠點重新浸潤 5 分鐘。直到膠塊呈半透明白色，最後以離 心抽氣法將膠體完全乾燥。將乾燥的膠以 50ul 的消化液(0.1 ug/l Trypsin 溶在 25mM ammonium carbonate 中 )重新濕潤，並在 37℃烘箱中反應一晚。隔日離心並取上清液。
其餘膠塊以50 ul 50% / 5% TFA 萃取數次，合併萃取液及上清液，在真空抽氣機中乾燥。
MALDI-TOF 質譜分析：
TM (Brucker Daltonics, Bremen, Germany)分析。質譜儀配有一氮素雷射，以 reflectron 模 式進行分析。蛋白質的比對以EMBL (http://matrixscience.com)提供的網路資料庫搜尋軟 體Mascort，至 NCBI 資料庫進行比對。先導離子閘門只選擇性讓單一 peptide 及 metastable 的片段到達 reflectron 以進行 PSD 序列分析.搜尋的標準以+1 Da 質譜不確定性及+10%
Mr 值(自二維電泳所推測)，輸出格式以比對相似度最高排起。
附錄四 上傳資料檔案格式
病患診斷資料csv 格式(每一病患為獨立一檔案)
欄位 中文說明 範例
patient_id 病患代碼 000215
Gender 性別(M、F) M
birth year 出生年(西元年) 1932 occurence year 病發年(西元年) 1989
blood_type 血型 O
Diagnosis 主訴 HCC
Site 病變部位(S1 ~ S8) S1,S3
Stage 期別 3
Alb Alb 檢測值
Got Got 檢測值 21.0
Gpt Gpt 檢測值 24.0
Alp Alp 檢測值
Ldh Ldh 檢測值
Tbil Tbil 檢測值
Dbil Dbil 檢測值 0.4
r_report r_report 摘要 A case of R/O HCC p_report p_report 摘要 HCC, grade 3
蛋白質定性分析預測資料格式：
欄位 範例(多筆資料)
SPOT_id Protein name MW
pI
Access No Diagnosis
0101,α2-HS-glywpwtein,36.28,5.2,P22589 0202,hnRNP,37.464,8.97,P12968
0203,Lirer GAPDH,37.46,8.97,P2569
0601,Glutamate dehydrogenase,61.7,7.66,P00367 0701,Peroxiredoxin 2,22.05,5.66,P32119
蛋白質點資料格式：X 軸
欄位 範例(多筆資料)
SPOT_id x-axis
0101,23.00, 0102,12.91,
蛋白質點資料格式：Y 軸
欄位 範例(多筆資料)
SPOT_id y-axis
0101,123.00, 0102,132.91,
附錄五 問卷