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研究生:賴韋蓁
研究生(外文):Wei-Chen Lai
論文名稱:合併雙標靶療法BikDD和內皮抑制素-胞嘧啶脫氨酶毒殺肝癌細胞
論文名稱(外文):Combination of Targeted BikDD and Endostatin-Cytosine Deaminase for Killing Liver Cancer Cells
指導教授:李龍緣
指導教授(外文):Long-Yuan Li
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:癌症生物學研究所碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:52
中文關鍵詞:肝癌標靶基因治療甲型胎兒蛋白促凋亡基因血管內皮抑制素-嘧啶脫氨酶合併治療
外文關鍵詞:Hepatocellular carcinomaeAFPBikDDEndostatin-cytosine deaminaseCombination therapy
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肝癌高居世界所有癌症致死率的第三名。而且,目前使用的治療方式並不適用於所有的肝癌病患。所以,有迫切的需要發展新的肝癌治療方法。我們之前已經發展出針對肝癌進行標靶基因治療的eAFP-VISA-BikDD載體,它包含了具有肝癌特異性的甲型胎兒蛋白啟動子與增強子組合(eAFP)和突變的模擬持續磷酸化狀態的促凋亡基因Bik,稱為 BikDD。在肝癌原位腫瘤的小鼠模型中,我們利用肝癌標靶基因進行系統性的治療,發現BikDD有效的抑制腫瘤生長和延長小鼠的存活率。另外,血管內皮抑制素(Endostatin)是一種內生性的血管生成抑制劑,它具有阻斷新生血管內皮細胞的功能,進而達到抑制腫瘤的效果。之前的研究指出,血管內皮抑制素-嘧啶脫氨酶(EndoCD)融合蛋白,擁有腫瘤標靶的特性,而且可以把前驅藥物5-FC轉換成化療藥物5-FU,進而殺死腫瘤細胞。在本實驗中,我們將進一步探討合併使用BikDD和EndoCD對於肝癌的治療效果。結果顯示,跟單獨治療比起來,合併治療能殺死更多的肝癌細胞。因此我們進一步的評估在肝癌原位腫瘤的小鼠模型中,合併使用BikDD和EndoCD的治療效果。由我們的實驗結果得知,跟對照組或5-FU組相比,單獨使用eAFP-VISA-BikDD,EndoCD/5-FC,或是合併治療,都可以有效的抑制腫瘤生長,並且延長老鼠的存活率。然而,跟單一治療比起來,合併治療更可以增加抑制腫瘤生長的效果,和延長老鼠存活率,這表示跟單一治療比起來,合併治療擁有更好的抑制腫瘤效果。而且,跟對照組或5-FU比較起來,單一治療和合併治療可以減少肝癌中的血管密度,並導致細胞凋亡的現象,但是這些現象並沒有出現在正常的肝臟細胞中。此外,跟5-FU比較起來,單一治療和合併治療也不會導致心臟毒性。這些實驗數據證實了跟單一治療比較起來,合併使用eAFP-VISA-BikDD和EndoCD/5-FC,擁有更好的治療效果和安全性,未來,值得發展成新的肝癌療法。
Hepatocellular carcinoma (HCC) has been ranked as the third most common cause of cancer death in the world. In addition, current remedies for HCC are not applicable to all HCC patients. So, there is an urgent necessity to develop the new therapeutic strategies for HCC. Previously, we developed a liver cancer targeted gene therapy vector eAFP-VISA-BikDD consisting of the liver cancer-specific α-fetoprotein promoter combined with AFP enhancers and a mutant mimicking constitutively phosphorylated form of pro-apoptotic gene Bik (named BikDD). Systemic treatment of liver cancer-targeted BikDD gene therapy DNA effectively inhibited liver cancer growth and prolonged survival in orthotopic mouse models of HCC. Additionally, endostatin is an endogenous angiogenesis inhibitor that targets neovascular endothelial cells and suppresses tumor growth. Previous study showed that endostatin fused with cytosine deaminase (EndoCD) exhibited tumor-targeting property and could convert 5-FC into the highly toxic chemotherapeutic compound 5-FU, thereby killing tumor cells. Here, we further investigated the effects of eAFP-VISA-BikDD combined with EndoCD on liver cancer cells in vitro and in vivo. Compared with the single treatment, the combination of EndoCD with BikDD showed enhanced killing effect on HCC in vitro. We further assessed the therapeutic efficacy of combined EndoCD/5-FC and eAFP-VISA-BikDD in orthotopic mouse models of HCC. Our data showed that eAFP-VISA-BikDD alone, EndoCD/5-FC alone or combination therapy effectively inhibited tumor growth and prolonged mice survival compared with control and 5-FU treatment. However, enhanced repression of tumor growth and prolonged survival were observed in mice with combination therapy compared with mice receiving single treatment, indicating combination therapy exerted better anti-tumor effect than single treatment in vivo. Moreover, both single therapy and combination therapy reduced the vascular density and induced cell apoptosis in liver tumor but not in normal liver cells compared with control and 5-FU therapy. In addition, in contrast to 5-FU therapy, combination therapy and single therapy did not caused cardiotoxicity. Together, these results show that combination of eAFP-VISA-BikDD with EndoCD/5-FC exerts better therapeutic efficacy and safety than single treatment and is worth developing for HCC therapy in the future.
中文摘要 IV
Abstract V
1. INTRODUCTION 1
1.1 Hepatocellular carcinoma 1
1.2 Etiology of hepatocellular carcinoma 1
1.3 Current treatments of hepatocellular carcinoma 1
1.4 eAFP-VISA-based expression vector 3
1.5 Proapoptotic protein-BikDD 4
1.6 Endostatin-cytosine deaminase (EndoCD) 5
2. SPECIFIC AIM 7
3. MATERIALS & METHODS 8
3.1 Cell lines & Cell culture 8
3.2 Animal model 8
3.3 Reagents 8
3.4 Chemical solution 8
3.5 Plasmid preparation 10
3.6 Transient transfection 12
3.7 Western blot analysis 12
3.8 MTT assay 13
3.9 CMV-EndoCD/5-FC therapeutic effect in vivo 13
3.10 Combination therapy therapeutic effect in vivo 14
3.11 Immunofluorescence staining 14
3.12 Statistics analysis 15
4. RESULTS 16
4.1 Expression of eAFP-VISA-BikDD and Endostatin-Cytosine Deaminase (EndoCD) in HCC cell lines 16
4.2 In vitro cell killing effects of endostatin-cytosine deaminase / 5-fluorocytosine (EndoCD/5-FC) and 5-fluorouracil (5-FU) 16
4.3 Comparison of the in vitro cell killing effects of different amounts of EndoCD on HCC cell lines 17
4.4 EndoCD/5-FC and eAFP-VISA-BikDD combination enhance cytotoxic effect on HCC cell lines 17
4.5 EndoCD/5-FC in combination with eAFP-VISA-BikDD gene therapy exerts synergistic or additive cytotoxicity in HCC cell lines 18
4.6 EndoCD suppresses tumor growth in orthotopic xenograft mouse models 19
4.7 EndoCD/5-FC therapy induces tumor cell apoptosis 20
4.8 Combination of eAFP-VISA-BikDD and EndoCD/5-FC therapy enhances repression of tumor growth in orthotopic xenograft mouse model 20
4.9 Combination of eAFP-VISA-BikDD and EndoCD/5-FC therapy induces cell apoptosis in liver tumor but not in normal liver cell 21
4.10 Combination of eAFP-VISA-BikDD and EndoCD/5-FC therapy and single gene therapy exert no cardiotoxicity 22
5. DISCUSSION 23
FIGURES 25
REFERENCES 42
APPENDIXES 47
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