長夜暗羅樹（P. longifolia var. pendula）是一種常青樹，生長於印地安和斯里蘭卡。而在南台灣則也有大比率的種植用來美化風景。由長夜暗羅樹的樹皮萃取出一種新的化合物16-hydroxycleroda-3,13-dien-15, 16 -olide（CD）。高雄醫學大學初步實驗對此化合物研究顯示具有抑制細胞生長的特性。我們利用MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay測CD對大腸直腸癌細胞株的抗細胞生長活性得到可抑制50%細胞的藥物濃度IC50 （HT29=10μg/ml；HCT116=5μg/ml）。而加入CD後隨著劑量的增加會造成caspase 3和PARP活化且細胞凋亡的現象也會增加。我們利用流式細胞儀可發現CD誘導sub-G1的堆積且會釋放出ROS。而前處理抗氧化劑NAC（N-Acetyl-L-cystine）則可使原本CD所引發的細胞毒殺作用被擷抗。而後也發現CD能使細胞的粒線體膜電位改變。另一方面，我們利用二維電泳法分析出差異性蛋白質並找出藥物反應的蛋白質有HSP10、Profilin-1、Peroxindoxin-1。我們更利用了RT-PCR以及西方墨點法去證實這三個蛋白在RNA和蛋白質層次其表現量均受到CD藥物處理後有改變。這些蛋白質的改變對大腸直腸癌研究中所扮演的角色具有進一步研究的價值。

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. Today, P. longifolia var. pendula is in large-scale cultivation in south Taiwan as a landscape plant. A new compound, clerodane diterpenoid 16-hydroxycleroda-3,13-dien-15, 16 -olide（CD）, was isolated from the bark of Polyalthia longifolia var. pendula. The compound was shown with cell growth inhibitory property at first screen in KMU（Kaohsiung Medical University）. In our study, antiproliferative activity of CD on human colorectal adenocarcinoma cells（HT29 and HCT116）were tested and the inhibitory concertration of 50﹪cell viability（IC50）is 5 μg/ml（HCT116） and 10 μg/ml（HT29）determined by MTT(3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The caspase 3 and PARP cleavage experiment results indicated that CD induced apoptosis is dose dependent manner. We found that CD induced sub-G1 accumulation and reactive oxygen species(ROS) release by flow cytometry analysis. Pretreatment of N-Acetyl-L-cystine(NAC), an antioxidant agent, can reverse the anti-proliferation effect by CD. We found that CD induced loss of mitochondria membrane potentiol（ΔΨm）. We also analysis differentially expressed proteins by 2-D electrophoresis and find drug response proteins, e.g. HSP10, Profilin-1, Peroxindoxin-1. RT-PCR and western blot was performed to confirm the protein expression changes. It is interesting to reveal the role of these proteins in the colorectal cancinogenesis and anti-tumor drug response.

中文摘要 I
英文摘要 II
英文縮寫表 III
致謝 IV

壹、前言 1
1.1長夜暗羅樹（Polyalthia longifolia var. pendula）背景知識 1
1.2對Polyalthia longifolia var. pendula有效成份 2
1.3對Polyalthia longifolia var. pendula提煉出的化合物作用的研究 3
1.4大腸直腸癌 4
1.4.1 大腸直腸癌位居癌症第三位 4
1.4.2 大腸直腸癌的症狀 4
1.4.3 大腸直腸癌的病因學 5
1.4.4 大腸直腸癌的病理學與預後 5
1.4.5 大腸直腸癌的化學治療 6

貳、實驗流程（Experiment flowchart） 7

叁、材料與方法（Materials and Methods） 11
3.1 Clerodane 雙萜類化合物 11
3.2大腸直腸癌細胞培養 11
3.3 MTT 細胞抗癌活性測試 11
3.4 流式細胞儀分析（Flow cytometric analysis） 12
3.4.1測量DNA的含量（cell cycle and sub-G1 accumulation） 12
3.4.2 Annexin V-FITC和PI雙染 12
3.4.3 測量細胞內活性氧ROS（Reactive Oxygen Species） 12
3.4.4 測量粒線體膜電位（mitochondria membrane potential（ΔΨm） 13
3.5 蛋白質萃取與濃度分析 13
3.6 西方墨點電泳法（Western Blotting） 13
3.7二維電泳法（Two-dimensional electrophoresis）(2-DE) 14
3.7.1 聚電焦電泳（Isoelectric focusing electrophoresis） 15
3.7.2 Immobiline DryStrip Equilibration 16
3.7.3聚丙烯硫胺膠體電泳Sodiumdodecylsulfate Polyacrylamide Gel 16
Electrophoresis（SDS-PAGE）
3.7.4 銀離子染色（Silver stain） 17
3.7.5 Coomassie Brilliant Blue G-250 17
3.7.6 In gel digestion 17
3.7.7 MALDI-TOF質譜操作 17
3.7.8 PSD（Post Source Decay）（Tandem TOF/TOF MS）操作 18
3.7.9 使用ProteinProspector之MS-Fit來辨認為之的蛋白質 18
3.8 RT-PCR quantization 19
3.8.1 大腸癌細胞RNA萃取 19
3.8.2 PCR聚合酶連鎖反應 19
3.8.3 PCR引子之設計 20
3.9 Real time-PCR 20
3.10統計分析（Statistical analysis） 21

肆、實驗結果（Result） 22
4.1 16-hydroxycleroda-3,13-dien-15,16-olide的細胞毒性 22
4.2 16-hydroxycleroda-3,13-dien-15,16-olide誘導細胞凋亡 22
4.3 16-hydroxycleroda-3,13-dien-15,16-olide誘導ROS產生 23
4.4 Caspase-3和PARP參與16-hydroxycleroda-3,13-dien-15,16-olide機制 23
4.5 16-hydroxycleroda-3,13-dien-15,16-olide 誘導失去粒線體膜電位 24
4.6.二維電泳膠片之分析結果 24
4.6.1 大腸直腸癌細胞處理CD之二維電泳分析 24
4.6.2 軟體比對分析及統計出差異性與非差異性之二維電泳膠片上spot 24
4.6.3 MALDI-TOF質譜分析與蛋白質資料庫比對 25
4.6.4 差異性蛋白質身份鑑定 25
4.6.5 差異性蛋白質西方墨點法結果分析 26
（A）HSP10蛋白質表現之結果 26
（B）Profilin-1蛋白質表現之結果 26
（C）Peroxiredoxin-1蛋白質表現之結果 26

伍、實驗討論（Discussion） 27
5.1 CD的細胞毒性 27
5.2 CD誘導sub-G1 accumulation以及lose of mitochondrial membrane 27
potential（ΔΨm）
5.3 CD處理大腸直腸癌細胞之二維電泳討論 28
5.4差異性蛋白質介紹與討論 29
5.4.1 Profilin-1 30
5.4.2 HSP10 32
5.4.3 Peroxiredoxin-1 35
陸、結論（Conclusion） 36

柒、參考文獻（Reference） 37

捌、Figures and Tables Countents 40
Figure 1. Structure of 16-hydroxycleroda-3,13-dien-15,16-olide 1
Figure 2. Five structure of 16-hydrocleroda-13-ene-15,16-olide-3-one 2
Figure 3. The role of Hsp10/60 in apoptosis 35
Figure 4. The cytotoxic effect of CD in HT29 and HCT116. 40
Figure 5. CD induced HT29 and HCT116 apoptosis. 42
Figure 6. CD induced sub-G1 accumulation. 43
Figure 7. CD induced ROS generation. 45
Figure 8. Antioxidant repressed ROS generation and increased cell proliferation. 47
Figure 9. CD induced lose of mitochondrial membrane potential（ΔΨm）. 49
Figure 10. CD induced apoptosis by Annexin V-FITC and PI staining. 50
Figure 11. 2-DE gels of colorectal cancer cell line HCT116. 51
Figure 12. Down regulated proteins in 2-DE gels. 52
Figure 13. Mass analysis result of HSP10 protein. 56
Figure 14. Mass analysis result of Peroxiredoxin-1 protein. 57
Figure 15. Mass analysis result of Profilin-1 protein. 58
Figure 16. Western blotting of HSP10, Profilin-1 and Peroxiredoxin-1. 59
Figure 17. Simplified diagram of activation of JNK pathways and their possible 60
cross talk at the level of downstream targets
Table 1. Primer of RT-PCR from NCBI homepage. 20
Table 2. Identification of 9 down regulated proteins after CD treatment on 53
HCT116 colon cancer cell line.
Table 3. Heat shock protein family. 33
Table 4. The IC50 of CD for HT29 and HCT116 cytotoxicity. 41
Table 5. Function of differentially expressed proteins. 54
Table 6. Comparison result of protein sequence of HSP10 protein. 56
Table 7. Comparison result of protein sequence of Peroxiredoxin-1 protein. 57
Table 8. Comparison result of protein sequence of Profilin-1 protein. 58
Table 9. The information of colonrectal cancer cell line HCT116. 61

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