Clerodane diterpenoid ( CD)，是從名叫Polyalthia longifolia var. pendula.的植物樹皮中所萃取出的天然化合物。在過去實驗室的研究中，CD證明能誘導細胞走向apoptosis的路徑。我們在給予藥物CD的狀況下利用二維電泳從大腸直腸癌的细胞中分析出給藥前後表現有差異性表達的蛋白質，像是10 kDa heat shock protein，Profilin-1，Peroxindoxin-1。而我們進一步用western和RT-PCR證實從蛋白質和mRNA level上的表現。我們認為這些蛋白質對於大結腸直腸癌的癌化和大腸直腸癌對於抗癌藥物反應是值得探討的。報告指出在大部分的癌症中Hsp10幾乎都是高度表現的。但是我們在給予CD的狀況下，Hsp10的表現量在大腸直腸癌是可以被抑制的。這項研究的主要目標是研究Hsp10的表現量對大腸直腸癌細胞SW480所造成的影響，並且研究在給予藥物後對Hsp10表現對其影響。SW480細胞株中高度表現Hsp10的表現量。之後利用MTT assay，流式細胞儀來分析研究Hsp10的作用以及高度表現Hsp10狀況下對於CD的細胞毒性所產生的影響。

A new compound, clerodane diterpenoid, CD, was isolated from the bark of Polyalthia longifolia var. pendula. . In previously study, CD was proved to induce cell apoptosis. We analysis differentially expressed proteins of colorectal cancer cells under drug CD treatment by 2-D electrophoresis and find drug response proteins, e.g. Hsp10, Profilin-1, Peroxindoxin-1. The expression change of protein had been further confirmed by RT-PCR and western blotting. It is interesting to reveal the role of these proteins in the colorectal cancinogenesis and anti-tumor drug response. In most kind of cancers, Hsp10 is often overexpress as indicated in many reports. Under CD treatment, Hsp10 is down-regulation in our experiment. The major aim of this study is to investigate the effect of Hsp10 on colorectal cancer cell lines SW480, and also investigate the relation of Hsp10 to drug treatment. Here,we overexpress Hsp10 in SW480. MTT assay, and flow cytometric analysis are utilized to investigate the effect of Hsp10 and CD cytotoxicity when overrxpress Hsp10 in SW480.

Contents
中文摘要 V
英文摘要 VI英文縮寫表 VII
致謝 VIII

壹、序言
1、大腸直腸癌 1
1.1大腸直腸位置與解剖圖 1
1.2大腸直腸癌好發區域 2
1.3病理特性與分期 2
1.4大腸直腸癌之病因學 4
1.5化學治療 6
1.6大腸直腸癌的病理學及其預後 7
2、10kDa heat shock protein (Hsp10) 7
3、Clerodane diterpenoid 雙萜類化合物

貳、實驗目的 12

叁、實驗流程 (Experiment flowchart) 12

肆、材料與方法 (Materials and Methods) 16
1. Clerodane diterpenoid 雙萜類化合物 16
2. 細胞培養 16
2.1實驗材料 16
2.2實驗方法 17
2.2.1 細胞培養液的配置 17
2.2.2 解凍細胞 18
2.2.3 繼代培養 18
2.2.4冷凍細胞 19
2.2.5 細胞計數 19
2.2.6大腸直腸癌細胞HT29、HCT116、SW480、SW620的培養與處理 20

3. DNA Cloning
3.1大腸癌細胞RNA萃取 21
3.2將mRNA反轉錄成cDNA 21
3.3 PCR引子之設計 22
3.4 PCR聚合酶連鎖反應 22
3.5 質體建構 (Plasmid construct) 24
4. 質體轉型 (Transformation) 26
5. 質體純化 (Plasmid purification)小量純化質體 27
6. 細胞轉殖 (Transfect transiently or stablely) 28
7. MTT抗癌活性測試 28
8. 流式細胞分析 (Flow cytometric analysis) 29
8.1測量DNA的含量 (cell cycle and sub-G1 accumulation) 29
8.2Annexin V-FITC 和 PI 雙染 30
8.3測量細胞內的活性氧ROS (Reactive Oxygen Species） 30
8.4測量粒線體膜電位 (mitochondria membrane potential(ΔΨm) 30
9. 蛋白質萃取和濃度分析 31
9.1 聚丙烯硫胺膠體電泳Sodiumdodecylsulfate Polyacrylamide Gel 31
10. 西方墨點電泳法 (Western Blotting) 32
11. 細胞遷移分析 (cell migration assay) 34
12. 統計分析 (Statistical analysis) 34

伍、實驗結果 (Result) 35
5.1 Hsp10在大腸直腸癌細胞株的內生性表現量 35
5.2 質體pAcGFP-Hsp10的建構和Hsp10的轉殖基因 35
5.3 高度表現Hsp10使SW480細胞株增生快速 36
5.4 Hsp10對於細胞的增生和16-hydroxycleroda-3,3-dien-15,16-olide的細胞毒性
36
5.5 Hsp10對於細胞增生和Cisplatin的細胞毒性 37
5.6 高度表現Hsp10使Cispaltin所誘導cell cycle arrest G2/M下降 37
5.7 高度表現Hsp10抑制ROS的生成 38
5.8 高度表現Hsp10增加粒線體膜電位的損失 38
5.9 高度表現Hsp10增加細胞的apoptosis 39
5.10高度表現Hsp10對細胞遷移的影響 39

陸、實驗討論 (Discussion) 40
6.1 CD的細胞毒性 40
6.2 10 kDa heat shock protein, (Hsp10)(10 kDa chaperonin)(CPN10) 41
6.3 高度表現Hsp10對細胞所造成的影響 43
6.4高度表現Hsp10對細胞遷移所造成的影響 44
6.5高度表現Hsp10對細胞所造成的影響 44
6.6 summary pathway 46

柒、Figures and Tables Countents
Figure1. Possible pathways and roles for Hsp10 and EPF trafficking. 46
Figure2. Endogenous expression Hsp10 in colorectal cancer cell line. 48
Figure3. Construction pAcGFP-N3-Hsp10 and pcDNA-Hsp10 from pAcGFP-N3 and pcDNA3.1 His A plasmid. 49
Figure 4. Transfect GFP-N3-Hsp10 to SW480 and western detect Hsp10 fusion protein
expression 50
Figure5. The proliferation effect of overexpression Hsp10 in SW480-Hsp10. 52
Figure 6. The cytotoxic effect of CD in SW480-Mock and SW480-Hsp10. 53
Figure7. The cytotoxic effect of Cisplatin in SW480-Mock and SW480-Hsp10. 54
Figure8. The chemoresistent in SW480-Mock and SW480-Hsp10. 55
Figure9. Cisplatin induced cell cycle G2/M arrest. 56 Figure10. Cisplatin induce ROS generation. 57
Figure 11. Cisplatin induced lose of mitochondrial membrane potential (ΔΨm) 58
Figure12. Cisplatin induced apoptosis 59
Figure 13. The wound healing assays in SW480-Mock and SW480-Hsp10 60
Table 1.The doubling time assay in SW480-Mock and SW480-Hsp10cc. . 51
Table 2. The IC50 of Cisplatin for SW480-Mock and SW480-Hsp10 cytotoxicity. 54

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