臺灣博碩士論文加值系統

癌症的產生主要是因為細胞在生長及死亡之間無法取得平衡。不適當的表現或過度活化細胞內的proto-oncogenes或是腫瘤抑制基因缺失都會使細胞的生長失去控制。前列腺癌是每年男性最常被診斷出來的癌症。過去研究發現PKCε overexpression與前列腺癌的復發有很大的相關性，使癌細胞的生長更不受到控制。PKC它是PKC中唯一可做為致癌基因的isozyme。已知PKC可透過抑制細胞死亡及促進細胞增生來執行它致癌基因的角色。另一方面實驗室過去的研究也指出在前列腺癌細胞中Disabled-2(DAB2)表現量的上升具有生長抑制的功能，DAB2主要是藉由影響生長抑制來達到抑制腫瘤的活性及維持正常細胞的平衡。增加DAB2的表現會抑制許多癌細胞的生長，因此過去的研究建議DAB2是一個腫瘤抑制基因。我們主要的目的是探討在前列腺癌細胞中DAB2是否藉由影響PKC的表現進而去執行腫瘤抑制的功能。在前列腺癌細胞株及K562細胞株中發現DAB2的存在與否與PKCε表現有拮抗的關係。且我們利用細胞轉染的方式將DAB2 過度表現或抑制後，發現PKCε的表現受其影響。藉由western blot的方式，發現表現DAB2的細胞都會降低PKCε的表現量。鑑於此實驗結果推測DAB2可能會影響細胞內PKCε的表現。而進一步使用不同的藥物處理細胞，利用MTT assay的方式來了解DAB2的存在與否，是否會影響細胞對藥物的抵抗能力進而達到其生長抑制的功能。研究發現當DAB2表現量增加時會降低PKCε的表現量，進而降低細胞的生長速率及對MSA(Methyseleninic acid)的抗藥性。因此我們推測在前列腺細胞株中DAB2藉由調控PKCε的表現量進而執行它tumor suppressor的角色

The imbalance between cell proliferation and cell death leads to cancer formation. Inappropriate expression or activation of proto-oncogene or lost of tumor suppressor gene disrupts cell growth. Prostaste cancer (PrCa) is a leading cause of cancer-related death in men. According to previous studies, overexpressed of PKCε promotes survival of human prostate cancer cells through interact with Bax. PKCε acts as an oncoprotein to inhibit cell death and promote cell growth. Several studies found that Disabled-2 (DAB2) inhibits the growth of cancer cells, suggest that DAB2 acts as an tumor suppressor gene. In this study, we used prostate cancer cell line model to study the role of DAB2 in prostate cancer, and whether it acts as a tumor suppressor through influence PKCε expression. We manipulated DAB2 and PKCε expression in DU145 cells by transient transfection. By western blotting, overexpresssion of DAB2 cell have less PKCε protein expression. Our results showed that DAB2 overexpression contributes to PKCε depletion and decreases cell growth of DU145 cells.We further observed the effect of drug-sensitivity while overexpression or knockdown of DAB2 or PKCε. Our data shown that DAB2 might increase drug susceptibility through inhibiting PKC expression in DU145 cell. Taken together, these results suggest that DAB2 overexpression decreased PKCε protein expression, accompanied increase in cell growth rate and methylseleninic acid resistance. These results implicate that the level of DAB2 expression may affect the outcome of cancer chemotherapy

指導教授推薦書……………………………………………………………………...
口試委員會審定書…………………………………………………………………...
授權書………………………………………………………………………………... iii
致謝…………………………………………………………………………………... iv
中文摘要……………………………………………………………………………... v
英文摘要……………………………………………………………………………... vi
縮寫表………………………………………………………………..…………….… vii
第一章、 Background and Significance………………………………………….….. 1
1. PKC在cancer progression中所扮演的角色............................................. 1
2. PKC所參與的signal transduction pathway……………………………... 4
3. DAB2在cancer中所扮演的角色............................................................... 5
4. DAB2所參與的signal transduction pathway…………………………….. 6
5. 使用抗癌藥物誘導前列腺癌細胞走向凋亡…………………………….. 7
6. Ibuprofen…………………………………………………………..……... 7
7. Methyseleninic acid………………………………………………………. 8
8. Specific Aim………………………………………………………………. 10
第二章、 Materials and Methods…………………………………………………..... 13
1. 實驗材料……………………………………………………………...…... 13
2. 實驗方法…………………………………………………………….……. 14
第三章、 Result………………………………………………………........................ 23
第四章、 Discussion………………………………………….……………................ 32
第五章、 References………………………………………………………………… 38
圖表………………………………………………………………………………….. 43
指導教授推薦書……………………………………………………………………...
口試委員會審定書…………………………………………………………………...
授權書………………………………………………………………………………... iii
致謝…………………………………………………………………………………... iv
中文摘要……………………………………………………………………………... v
英文摘要……………………………………………………………………………... vi
縮寫表………………………………………………………………..…………….… vii
第一章、 Background and Significance………………………………………….….. 1
1. PKC在cancer progression中所扮演的角色............................................. 1
2. PKC所參與的signal transduction pathway……………………………... 4
3. DAB2在cancer中所扮演的角色............................................................... 5
4. DAB2所參與的signal transduction pathway…………………………….. 6
5. 使用抗癌藥物誘導前列腺癌細胞走向凋亡…………………………….. 7
6. Ibuprofen…………………………………………………………..……... 7
7. Methyseleninic acid………………………………………………………. 8
8. Specific Aim………………………………………………………………. 10
第二章、 Materials and Methods…………………………………………………..... 13
1. 實驗材料……………………………………………………………...…... 13
2. 實驗方法…………………………………………………………….……. 14
第三章、 Result………………………………………………………........................ 23
第四章、 Discussion………………………………………….……………................ 32
第五章、 References………………………………………………………………… 38
圖表………………………………………………………………………………….. 43
圖1、比較DU145、PC3、C4-2細胞中PKC protein及mRNA的量.................. 43
圖2、比較重新表現T7-DAB2、si2112的DU145細胞中PKC的量…………… 44
圖3、比較K562、D-1-4、v7、si8細胞中PKCprotein及mRNA的量………… 45
圖4、比較重新表現T7-DAB2、si2112的K562細胞中PKC的量…………….. 46
圖5、比較使用Ibuprofen處理DU145後觀察細胞死亡的情況….………......…. 47
圖6、比較DAB2 knockdown後在DU145是否會影響Ibuprofen誘導細胞死亡. 48
圖7、比較在DU145 PKC overexpression是否會影響Ibuprofen誘導細胞死亡. 49
圖8、利用MTT assay分析不同濃度的MSA 處理DU145觀察細胞存活率....... 50
圖9、利用flow cytometer分析不同濃度的MSA處理DU145後細胞死亡情況.. 51
圖10、利用flow cytometry分析在DU145細胞內PKC overexpressio後，處理
MSA是否會影響細胞死亡率………………………………………….........
52
圖11、利用MTT assay分析比較在DU145細胞內PKC、DAB2 overexpression或knock -down後，處理MSA是否會影響細胞存活率…………………….
54
圖12、圖十二.利用growth rate分析比較在DU145細胞內PKC、DAB2
overexpression或knock -down後，是否會影響細胞生長速率…………..
. 56

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