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研究生:蔡秀雯
研究生(外文):Hsiu-Wen Tsai
論文名稱:利用反轉錄病毒攜帶受缺氧調控之endostatin基因治療肺腺癌
論文名稱(外文):Retrovirus mediated hypoxia-inducible endostatin gene expression for the treatment of lung adenocarcinoma
指導教授:吳昭良
指導教授(外文):Chao-Liang Wu(吳昭良)
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:63
中文關鍵詞:反轉錄病毒肺癌基因治療缺氧調控
外文關鍵詞:lung cancerretrovirusHIF-1endostatin
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  肺腺癌是目前最常發生的惡性腫瘤之一,其早期症狀不明顯且不易察覺,因此提供有效的治療是非常的重要。肺癌的治療主要以手術、化學治療及放射線治療為主,但是治療能力有限,在本實驗中我們運用基因治療以期達到更佳的治療效果。腫瘤在生長的階段需要新生的血管提供其氧氣及養分,目前有許多研究致力於阻斷腫瘤生長中血管的新生作用以達到治療癌症的目的。在癌症的基因治療上,為了達到安全及治療的目的,載體對腫瘤細胞的專一性非常重要。腫瘤生長時內部會呈現缺氧狀態進而引發受缺氧調控的因子(HIF-1)的累積,HIF-1會和HRE 作用以調控下游基因表現。Endostatin 會抑制血管的生長、移動並促進血管的凋亡,目前已廣泛運用在癌症的治療。在此研究中,我們利用腫瘤缺氧的特性建構一由反轉路病毒攜帶受缺氧調控的endostatin基因,Retro/6xHRE-ES,治療肺癌。 在細胞試驗中,我們證明6xHRE確實會受到缺氧的調控。Retro/6xHRE-ES在缺氧調控下產生的endostatin 蛋白具有抑制血管內皮細胞移動的能力。在肺癌的動物實驗上,我們可以發現 Retro/6xHRE-ES能有效的抑制腫瘤的生長及腹水的產生。Retro/6xHRE-ES 合併化學治療(cisplatin)可以達到更好的治療的效果。我們更進一步建立小鼠的惡性腹水的模式證明Retro/6xHRE-Luc能專一的感染腫瘤細胞以提高其安全性。由結果顯示本論文成功的利用腫瘤缺氧的特性建構一由反轉錄病毒攜帶受缺氧調控的endostatin基因,更有效及安全地治療肺癌。
  Lung adenocarcinoma is one of the most human malignant tumors. As this disease has a poor prognosis, an effective therapeutic modality is urgently needed. Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of antiangiogenic factors is one of the promising therapeutic approaches for cancer. For cancer-specific gene therapy, it is important to distinguish the physiological differences between normal tissues and tumors. Hypoxia is generally detected in central regions of solid tumors and regulates a variety of transcription factors, including hypoxia-inducible factor-1 (HIF-1). Therefore, we have exploited hypoxia response element (HRE) to control antiangiogenic gene expression for tumor-targeted gene therapy. Endostatin, a 20-kDa carboxyl-terminal fragment of collagen XVIII, inhibits endothelial cell migration in vitro and tumor growth in vivo. We constructed retroviral vectors encoding endostatin where expression of the transgene was regulated by 6 copies of HRE ligated to CMV minimal promoter (Retro/6xHRE-ES) or by constitutive CMV promoter (Retro /CMV-ES) for treatment of lung cancer. In wound healing and Boyden chamber assays, the conditioned medium from Retro/HRE-ES-transduced NIH3T3 cells inhibited the migration of human microvascular endothelial HMEC-1 cells in vitro. Intratumoral injection of Retro/HRE-ES resulted in suppression of tumor growth in C57BL/6 mice bearing subcutaneous Lewis lung (LL2) carcinomas. Combination of retrovirus-mediated antiangiogenesis gene therapy and chemotherapy exhibited higher antitumor efficacy compared with either treatment alone. In the ascites LL2 tumor model, mice treated with tumor-targeted Retro/HRE-ES had lower tumor numbers and reduced ascites volume. Taken together, these results suggest that hypoxia-inducible antiangiogenic gene therapy mediated by retrovirus gene transfer may be used for the treatment of lung cancer or other solid tumors.
Content
Chinese abstract…………………………………………………………………I
Abstract……………………………………………………………………………III
Content………………………………………………………………………………V
Figure content……………………………………………………………………VIII
Abbreviation………………………………………………………………………X
Acknowledgement……………………………………………………………………XI
Introduction
A. Retrovirus……………………………………………………………………1
A-1 Structure of retrovirus…………………………………………………1
A-2 Retroviral vector for gene therapy………………………………2
B. Antiangiogenesis in tumor therapy – Endostatin…………………3
C. Improvement of retroviral vector targeting………………………4
D. Lung cancer…………………………………………………………………6
E. Treatment strategies………………………………………………………6
Materials and Methods
A. Materials
Plasmids…………………………………………………………………………7
Oligonucleotides……………………………………………………………7
Cell lines……………………………………………………………………8
Experiential animal…………………………………………………………8
B. Methods
Cell………………………………………………………………………………9
Transfection……………………………………………………………………9
Production of retroviruses………………………………………………9
Determination of viral titers…………………………………………10
Transcription activity of promoter……………………………………10
RNA isolation and RT-PCR…………………………………………………11
Wound healing assay………………………………………………………12
Boyden chamber assay………………………………………………………13
Experimental Animals………………………………………………………13
Statistical analysis………………………………………………………16

Results
A. Determination of the infectivity of retrovirus in ascites LL2 tumor
model………………………………………………………………………………17

B. Detection of the endostatin mRNA by RT-PCR Retro/CMV-ES
prolonged survival in mice bearing ascites LL2 tumors……………17

C. Survival rate of Retro/CMV-ES and Retro/CMV-Luc treated mice on
intraperitoneally inoulated LL2 tumors ……………………………… 17

D. Antitumor effects of Retro/CMV-ES in the LL2 subcutaneous animal
model…………………………………………………………………………………18

E. Detection of the HIF-1 mRNA by RT-PCR in NIH3T3 and LL2 cells.18

F. Activation of 6xHRE-CMVmini promoter in response to hypoxia
F.1 CoCl2 ……………………………………………………………………19
F.2 HIF-1a………………………………………………………………………19

G. Functional assay – cell migration assay
G.1 Wound healing assay……………………………………………………20
G.2 Boyden chamber assay………………………………………………………20

H. Antitumor effects of Retro/6xHRE-ES in the subcutaneous LL2
animal model……………………………………………………………………20

I. Reduction of angiogenesis after Retro/6xHRE-ES gene therapy……21

J. Effects of Retro/6xHRE-ES gene therapy combined with chemo- therapy in vivo
J.1 Subcutaneous LL2 tumor model…………………………………………21
J.2 Ascites LL2 tumor model…………………………………………………22

K. Detection of total VEGF protein level in ascites……………………22

L. Retro/6xHRE-Luc could targeted more specificity in mice bearing LL
tumor cells………………………………………………………………………22

Dicussion..........................................................23
References…………………………………………………………………………27
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