出版年:

研究生:

劉俐伶

研究生(英文姓名):

Li-ling Liu

論文名稱:

利用反核酸序列及自殺基因治療小鼠膀胱癌

英文論文名稱:

gene therapy fo murine bladder cancer using antisense and suicide gene strategies

指導教授:

蕭璦莉

指導教授(英文姓名):

Ai-li Shiau

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46861050

學年度:

87

語文別:

中文

論文頁數:

73

關鍵詞:

反核酸序列 ; 自殺基因 ; 轉型生長因子 ; 大腸桿菌胞密啶去胺脢 ; 基因治療

英文關鍵詞:

antisense ; sucicde gene ; transforming growth factor ;
cytosine deaminase ; 5-FC ; gene therapy

被引用次數:

0

[ 摘要 ]

由於基因工程的進步，使得利用基因來治療癌症以成為一種具發展性的治療方式。在基因治療的策略中，利用反核酸序列(antisense)來抑制基因的表現，此方式不但可修復某些基因不正常的表現之外，在目前的發展趨勢中，更是一個新興的方式。另外利用自殺基因來治療癌症也已廣泛地被研究。因此在本研究中，我們試圖評估在小鼠MBT-2膀胱癌模式下，運用反轉錄病毒載體同時攜帶轉型生長因子-b1(Transforming growth factor-b1; TGF-b1)的反核酸序列及大腸桿菌胞嘧啶去胺 (E. coli cytosine deaminase; CD)，治療膀胱癌之可行性。另外也嘗試以TGF-b的反核酸序列所轉型的MBT-2細胞當作腫瘤疫苗，評估其治療之可行性。
TGF-b1在免疫調節方面，主要是扮演著免疫抑制的角色；它可抑制T細胞的增加、殺手細胞的活化等。另外，它與癌症的相關性，從一些研究也發現，腫瘤細胞本身會釋放免疫抑制因子，一方面促使腫瘤細胞的增生；另一方面則可逃避免疫系統的攻擊。從一些報告指出，在膀胱癌的病例中，TGF-b1的表現有偏高的趨勢。因此，我們試圖利用反核酸序列的方式，去阻斷TGF-b1的過度表現。在實驗中，我們利用一個帶有TGF-b的反核酸序列的反轉錄病毒載體pRUFCD- TGF-b(-)，將之轉染至包裝細胞株j CRE中，經過cytosine 及5-fluorouracil篩選出轉染成功的細胞株；再以聚合每連鎖反應進一步確認CD基因的表現。收集此細胞的上清液並與MBT-2細胞混合，一起注射至C3H/HeN小鼠的皮下，發現其腫瘤發生率及腫塊大小皆比對照組低。且同時應用自殺基因，CD基因及TGF-b反核酸序列去治療，所得到的治療效果遠比單一治療來的好。另外，以帶有TGF-b反核酸序列的反轉錄病毒所感染成功的MBT-2細胞株，發現其TGF-b的表現量降低，而且細胞增殖的速度也相對的降低。將此轉型過的MBT-2細胞株應用在動物實驗，則發現腫瘤切除過的小鼠，以經過 g-射線照射過的轉型細胞株(irradiated MBT-2/pRUFCD- TGF-b(-))免疫，發現小鼠的存活率比對照組高。而且從試管實驗中發現，也可提升部分的免疫反應，如毒殺T細胞反應。因此總結以上結果，TGF-b反核酸序列及綜合性的基因治療在膀胱癌的治療上應為可行的策略。

[ 英文摘要 ]

Advances in recombinant DNA technology have brought gene transfer for the treatment of cancer to a rapid expanding field. For cancer application, inhibition of gene expression by antisense provids rationale for identification of disease targets and offers a promise as a novel therapeutic intervention. Tumor-associated antigens have been demonstrated to stimulate antitumor immune responses and thus are good tumor vaccine candidates. The promise of the cytosine deaminase (CD) suicide gene therapy has also been documented as a potential antitumor treatment. However, transforming growth factor b (TGF-b) secreted by tumors exerts several potent immunosuppressive effects, including the inhibition of cytotoxic T-lymphocyte activation, which may contribute to the inefficacy of some tumor vaccines. The aim of this study is to test TGF-b antisense and CD gene therapies delivered by retroviral vectors for the treatment of murine bladder tumor. The retroviral vector pRUFCD-TGF-b(-) containing E. coli CD gene and antisense TGF-b1 oligonucleotide was transfected into a retroviral packaging cell line j CRE. The cell clones transduced with pRUFCD-TGF-b(-) were isolated by selection with cytosine and 5-fluorouracil. The PCR analysis shows that pRUFCD-TGF-b(-) was intergated into the chromosomal DNA of transduced cells. C3H/HeN mice injected with MBT-2 cells admixed with the supernatant of jCRE transduced with pRUFCD-TGF-b(-) showed lower tumor incidence. In addition, such mice treated with 5-fluorocytosine have even lower tumor incidence, smaller tumor mass, and higher survival rate. On the other hand, we tested whether bladder tumor vaccines modified to express antisense TGF-b(-) is more superior to the wild-type tumor vaccines. The data show that the modified MBT-2/pRUFCD-TGF-b(-) clones, either g-irradiated or not, expressed a lower level of TGF-b than MBT-2 cells. In animal studies, tumor-bearing mice undergone tumor resection and rechallenged with g-irradiated MBT-2/pRUFCD-TGF-b(-) cells showed lower tumor incidence, and higher survival rate as well as higher antitumor responses, such as CTL response in vitro. Taken together, the delivery of antisense TGF-b and CD transgenes by retroviral vectors as well as the use of tumor vaccines modified to express antisense TGF-b may be effective approaches in the treatment of bladder cancer.