【作者】 张勇；

【导师】 蔡文清；

【作者基本信息】 河北医科大学 ， 外科学， 2005， 博士

【摘要】 目的:在肿瘤治疗中,许多药物缺乏特异性靶向作用是现今许多治疗方案所共同面临的一个主要问题,因此研发出新的肿瘤靶向机制的治疗方法显得非常必要。肿瘤细胞特异性重组腺病毒,它具有选择性地在肿瘤细胞中表达目的基因、在通过“旁观者效应”在肿瘤细胞间扩散杀伤的优点,且同现有的治疗手段无交叉耐药作用,同时由于其肿瘤靶向性,在杀伤肿瘤细胞的同时又不影响正常细胞,因此,在肿瘤治疗中极具前景。到目前为止,已有十余种不同类型的选择性重组腺病毒进入到临床试验中。为达到肿瘤靶向性的目的,对腺病毒最常采用如下两种分子改造策略:其一选择性缺失病毒,它缺乏在正常细胞中复制所必需而在肿瘤细胞中复制非必需的基因,如腺病毒的E1A基因及E1B基因缺失,使其只能在肿瘤细胞中复制。这种改造后的病毒在动物实验中疗效显著,并已进入临床试验,经典的如美国ONYX药物公司研制的E1B-55KD缺失的ONYX-O15,其联合常规化疗(5-Fu、顺铂)治疗30例复发的头颈部肿瘤患者,临床有效率可达63%,但作为单剂治疗疗效仅在20%左右,可能同E1B基因同时也参与晚期mRNA转运出核的过程这一特性有关。另一改造策略是利用组织或肿瘤特异性启动子,如AFP、MUC1,PSA及pS2等,来调控腺病毒表达目的基因,这种改造后的病毒在动物实验中非常成功,前列腺肿瘤特异性重组腺病毒CN-706及CV-787已进入临床试验,然而经此途径改造的病毒其增殖被局限于仅能表达相应肿瘤特异性抗原的肿瘤类型。 肿瘤细胞的无限增殖是恶性实体瘤的主要特性。肿瘤的无限增殖能力主要靠端粒酶的激活来不断地维持端粒的长度而达到的。端粒酶在大多恶性肿瘤细胞中处于激活状态而在正常细胞中却没有活性或者活性很低,是目前所报道最为广谱的肿瘤生物分子标记。人端粒酶逆转录酶(human telomerase transcriptase, hTERT)是维持端粒酶活性所必需的催化亚单位,它的表达调控在转录水平上。利用hTERT启动子来调控病毒携带的目的基更多还原

【Abstract】 Objective: Prostate carcinoma accounts for more than 90% of all prostate cancers. It ranks the third in frequency among all geneurological malignancies. As it is known, surgery such as prostate resection is the choice of treatment for prostate cancers. Yet the most of patients with prostate carcinoma have little opportunity to undergo surgery. General treatment or combined treatment has drawn great attention due to the poor prognosis of prostate carcinoma. Palliative treatments including chemoembolization, radiation therapy and systemic chemotherapy have been beneficial complementarities for prostate cancer treatment. A major problem with many cancer treatments is that they are lack of tumor specificity. Therefore, development of effective alternative approaches with novel tumor-targeting mechanism is needed. Replication-selective virotherapy holds great promise for the treatment of cancer. Appealing features include tumor-selective targeting, viral self-spreading in cancer cells, and no cross-resistance to current treatments. Several types of conditionally replicative and replicative defecated viruses have already been tested in clinical trials, including conditionally replicative adenovirus (CRAD), herpes simplex virus, vaccinia virus, reovirus and Newcastle disease virus.Conditionally replicative defecated adenovirus has attracted considerable interest among these replicating viruses at the beginning. Two molecular strategies have been exploited to target the CRAD selectively to tumor cells. The first strategy involves the deletion of adenovirus genes that are necessary for virus replication in normal cells but not in tumor cells. These include the adenovirus E1A and E1B genes, which are responsible for the inactivation of tumor suppressor Rb and p53 genes that are often mutated in cancer cells. Thisstrategy has been effective in animal models, and led to clinical trials combining the application of the ElB/55 Kda-deleted Onyx-015 adenovirus with chemotherapy. The second strategy involves the use of tumor- or tissue-specific promoters, such as AFP, MUC1, PSA, kallikrein-1 and pS2, to drive adenoviral genes that are essential for replication. This strategy has also been successful in animal models, and the prostate-specific CRADs CN-706 and CV-787 have been tested in clinical trials. This approach, however, is limited to specific tumor types that express the corresponding tumor-specific antigens.The selective reactivation of telomerase in tumor cells offers an attractive therapeutic target for developing new broad-spectrum antitumor agents. Telomerase are essential elements at chromosome termini that preserve chromosomal integrity by preventing DNA degradation, end-to-end fusion, rearrangements, and chromosome loss. Each cell replication is associated with the loss of 30-150 bp of telomeric DNA that can be compensated by telomerase, an RNA-dependent DNA polymerase. Most human somatic cells exhibit neither hTERT expression nor telomerase activity, whereby the number of cell divisions is limited because of the reduction of telomeres to a critical length. In contrast to quiescent somatic cells, in highly proliferative cells, such as germ-line, hematopoetic stem, or transformed cancer cells, diverse molecular mechanisms are necessary to maintain telomere length. Although some tumors activated a yet unknown alternative mechanism of telomere extension, the majority (>90%) of human cancer cells acquire immortality by expression of the hTERT. It has been shown that hTERT expression is regulated at the transcriptional level, thereby providing a promising tool for tumor-specific gene expression.Therefore, we hypothesized that the hTERT promoter could be used to restrict adenoviral to tumors and thus achieve tumor-selective killing tumor . On the basis of this rationale, we generated a hTERT promoter-regulated replication-defecated adenovirus Ad-hTERT-HSV-tk in which adenovirus El A gene expression was driven by the hTERT promoter. This vector displayedefficient tumor-selective killing ability both in vitro and in vivo.Methods and Results: 1. Construction of hTERT promote-regulated replication-defecated adenovirus Ad-hTERT-HSV-tk. By using overlap PCR, endogenous adenovirus El A promoter and ElB promoter was deleted and a series of new restriction endonucleases sites including Age I-Bst BI-Not I-Spe I-Sal I-Xho I-Swa I and Spe I was introduced upstream of El A and ElB gene respectively. Human telomerase reverse transcriptase promoter with three E-boxes downstream were synthesized, verified by sequence confirmation and cloned into the upstream of El A gene to generate a new plasmidpCA13-CMV-EGFP^ PCA13-hTERT-EGFP> pCA13-CMV-HSV/tk^ and pCA13-hTERT-HSV-tk. The plasmid PCA13 was co-transfected with pBHGE3 into 293 cells to produce pCA13-CMV-EGFP > pCA13-hTERT-EGF, pCA13-CMV-HSV/tk and pCA13-hTERT-HSV-tk the new recombinant adenovirus by homologous recombination. These recombination was confirmed by PCR analysis using specific primers covering the adenovirus El A promoter or ElB promoter region, propagated in 293 cells and purified by cesium chloride gradient centrifugation. Viral titer achieved 1.9X 1010pfu/ml with TCID50 method. 2. Selective infection and oncolysis of Ad-hTERT-HSV-tk/GCV in vitro. Viral infection experiments were performed to evaluate the selective infection ability of Ad-hTERT-HSV-tk. In tested tumor cell lines, infection of Ad-hTERT-EGFP is as well as that of Ad-CMV-EGFP, but Ad-hTERT-EGFP hardly replicate, greatly attenuated compared with the Ad-CMV-EGFP adenoviruses in MRC-5 cell line. When containing reporter gene green fluorescent protein gene, Ad-hTERT-EGFP was observed to replicate efficiently in prostate cancer cells but poorly in normal cells under fluorescence microscope. To assay the cytolytic effects of Ad-hTERT-HSV-tk/GCV on tumor and normal cells, we performed a crystal violet assay. All LNCaP cells were killed at an MOI of 10,GCV at the concentration of 100 u g/ml and PC-3 cells were not killed at the same condition. MR-5 cells, the normal fibroblast cells, were not killed even at an MOI as high as 100 indicating that Ad-hTERT-HSV-tk/GCV更多还原