论文标题:hTERT启动子调控腺病毒介导的HSV-TK/GCV基因系统治疗膀胱癌的实验研究
The Selective Killing Effect of Adenoviral Mediated HSV-TK/GCV Suicide Gene System Controlled by hTERT Promoter on Bladder Carcinoma Cells
论文作者 白志杰
论文导师 黎玮,论文学位 硕士,论文专业 外科学
论文单位 河北医科大学,点击次数 91,论文页数 65页File Size1797k
2005-03-01论文网 http://www.lw23.com/lunwen_134135747/ 膀胱癌;重组腺病毒;自杀基因;人端粒酶逆转录酶启动子;旁观者效应
bladder carcinoma; recombinant adenovirus; suicide gene; hTERT promoter; bystander effect
目的:膀胱肿瘤是泌尿系最常见的肿瘤。治疗膀胱肿瘤目前有多种方法,在过去的几年中基因治疗膀胱癌已经成为了重要的研究重点之一。分子化学治疗又称自杀基因治疗,他是将编码某些特定酶的基因导入肿瘤细胞,这些酶可将无活性或低毒的药物前体转化为具有较强细胞毒性的药物,从而杀死肿瘤细胞。单纯疱疹病毒胸苷激酶基因(herpes simplex virus thymidine kinase , HSV-TK)/ 丙氧鸟苷(ganciclovir,GCV)系统是分子化疗中研究最为深入的一种。HSV-TK 酶可将GCV 磷酸化为GCV—磷酸,继而在哺乳动物细胞TK 酶的催化下最终转变为GCV 三磷酸,抑制DNA 聚合酶的活性,掺入DNA 合成,产生细胞毒性。而且研究发现该方法还具有“旁现者效应”,即转染细胞可以杀死附近未转染细胞的特性,从而弥补转染效率低的缺陷,发挥更强大的治疗作用。在分子化学治疗中,要实现选择性地消除肿瘤细胞,必须将基因的表达限定于肿瘤细胞,即实现肿瘤基因治疗的靶向性。肿瘤基因治疗中为了获得肿瘤特异靶向性,可以使用特异性启动子在转录水平控制目的基因的表达水平。近年来人端粒酶逆转录酶(human telomerase reverse transcriptase hTERT)基因的启动子已被证实在90%以上的人类肿瘤组织中活性上调,现在成为肿瘤基因治疗中转录靶向的研究热点。病毒介导的酶解前体药疗法
Objective: Bladder carcinoma is the most common urological malignancy in china. Many methods have been used to the therapy for bladder carcinoma. Gene therapy is one of the most important progress in the past years. Molecular chemotherapy is a gene therapy approach designed to achieve selective eradication of cacinoma cells via a genetically expressed toxin. This method is often named as “suicide gene therapy”. One of the strategies utilizes a enzyme/prodrug system which can convert a prodrug into a toxic metabolite leading to cell death. The most frequently utilized system is the thimidine kinase (tk) gene from the herpes simplex virus(HSV),given in combination with ganciclovir (GCV).The HSV-tk product has high affinity for acyclovir and its analogues , including ganciclovir,mainly producing the phosphorylated product. Subsequent modification by the host cell to a triphosphorylated form and incorporation during replication halts growth of the developing DNA strands and inhibits DNA polymerase activity. Mammalian TK has a much lower affinity for the prodrug such that tumor cells once transduced are selectively killed in the presence of ganciclovir. The efficiency of this approach can be amplified by a “bystander effect”, killing the nontransduced neigbouring cells. Thus,suicide gene therapy theoretically is not necessarily targeting 100% of tumor cells for effective treatment. However,gene expression in nontarget cells is a central problem in cancer gene therapy. Efficient gene therapy regiments require transgene expression especially in tumors. In recent years the specific promoters are often used to control the therapeutic gene expression by regulation of transcription in order to achieve tumor specific target in cancer gene therapy. It has been already confirmed that the activity of human telomerase reverse transcriptase (hTERT) rise in approximately 90% human tumors whereas most normal cells do not express the activity and it becomes a hotspot in transcriptional target of cancer gene therapy. Virus-directed enzyme prodrug therapy (VDERT) has been popular in cancer gene therapy due to its potential foe tumor-specific cytotoxicity. Several biologic features of adenovirus have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. To investigate the efficacy of adenovirus -mediated gene therapy using herpes simplex thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment for human bladder carcinoma cell line (253J). Methods: we use a recombinant adenovirus vectorcontaining the enhanced green fluorescent protein (EGFP) gene. One construct with a cytomegalovirus (pCMV)-driven enhanced green fluorescent protein (EGFP) reporter gene (Ad-CMV-EGFP), the other construct with a human telomerase reverse transcriptase gene promoter (hTERTp )-driven enhanced green fluorescent protein (EGFP) reporter gene (Ad-hTERT-EGFP) transfected 253J and MRC-5 cells. The percent of transfection was determined by the detection of flurorescence microscopy. Human bladder carcinoma cell line 253J and MRC-5 was infected with Ad-hTERT-HSV/TK and Ad-CMV-HSV/TK, then various amount of GCV was added. Using the hTERT as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector(Ad-hTERT-HSV/TK) , the therapeutic efficacy of adenovirus mediated HSV-TK gene transduction,followed by ganciclovir(GCV)administration,was studied. As a control,a recombinant adenoviral vector(Ad-CMV-HSV/TK) which was driven by the CMV promoter,was constructed. The percentage survival of cells is presented as a percentage of the survival cell in the GCV-treated cells divided by that in the cells without GCV treatment(mean 士SD).The relative survival rate of cells in presence of prodrug GCV was tested using MTT method. Different mix culture cells , which contained different per
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