| Malignant tumors do heavily harm to human health and life. It has taken the lead over the disease of the heart and cerebral veins and become the first-leading disease in life. It's estimated that there will be more than 7,000,000 death every year worldwide, among which ocurred 2,000,000 new cases of tumor and 1,600,000 death yearly in our country.The routine methods such as surgery, radiotherapy and chemotherapy, are not competent enough for the treatment. Therefore the gene therapy is a very good substitution method to cure the cancer.The gene therapy is to transfer the human normal gene or the functional gene to the target cell by a certain way in order to correct the defective gene or to pruduce the treatment function, thus achieved a new biomedicine technology that can cure disease. The gene therapy must solve three key questions to obtain the breakthrough: the highly effective gene delivery system, the controllability of gene expression and the better functional gene.Adenoviral vectors are widely used as gene transfer vectors in cancer therapy because of their ability to transfect a wide range of host tissues ,to infect both proliferating and nonproliferating cells and the production of high titers of current good-manufacturing-practice quality adenoviruses is established. Futhermore, adenoviruses DNA is not integrated into the host genome, and the risk of mutagenesis is low.These advantages of adenoviral vectors, however, result in a lack of specificity in their infection pattern leading to transfection of normal host tissues with an unfortunate accompanying potential for toxicit.Tumor-specific replicative viruses overcome these disadvantag- es.Specifically replicative viruses have evolved to infect tumor cells,replicate,induce cell death and release of viral particles.And these progeny viruses released by virus-mediated lytic infected cells continue to infect surrounding tumor cells,finally spread in tumor tissues.We hypothesize that gene therapy utilizing replication-selective oncolytic adenoviruses as vectors,entension of virus-mediated gene delivery,will offer several potential advantages.The therapeutic gene inserted into the genome of the virus will specifically and highly express in the tumor cell with the viruses selectively proliferating in the tumor cells.Several mechanistic approaches for targeting have been reported,and one of them is engineer tumor/tissue-specific promoter into viruses to limit expression of gene necessary for replicatiom in cancer cell.Survivin is a novel member of the inhibitor of apoptosis (IAP) protein family, which plays an important role in the survival of cancer cells and the progression of malignancies. Survivin expression is found during embryonic and fetal development, but is undetectable in terminally differentiated adult tissue. Recently, various investigations have evaluated the activity of the survivin promoter in breast cancer, pancreatic cancer, esophageal carcinoma, primary glioblastoma, melanoma, and ovarian cancer. The survivin promoter shows noticeable activity in them and appears to be negligible in the majority of adult tissues.We hypothesized that the survivin promoter may be used for tumor-specific expression of gene necessary for viral replication in cancer cell.Cyclooxygenase (COX), a rate-limiting enzyme in the synthesis of prostanoids, is encoded by two genes, COX-1 and COX-2, which are differentially expressed and regulated. The expression of COX-1 is maximal in quiescent cells; COX-2 expression is induced by growth factors and cytokines. COX-2 is virtually undetectable in most tissues under physiological conditions. In contrast, it is expressed in several cancer tissues and may have an important role in carcinogenesis. The up-regulation of COX-2 has been well established in breast, colon, and lung cancers, bladder cancer as well as in melanoma. And the COX-2 promoter was demonstrated to have favorable utility in many cancers'gene therapy with a'tumor-on/ normal tissue-off'status such as in melanoma.In this study, we construct a series of recombinant plasmid ,all of which were inserted into the Ad-RGD vector. Ad-RGD-SUR-E1 is a replication-selective adenovirus in which the adenoviral El gene was driven by survivin promoter. Ad-RGD-COX-2-TK-IRES-NTR- SUR-E1 is a replication-selective adenovirus in which the suicide gene was driven by COX-2 promoter.And Ad-RGD-COX-2-TK-IRES-NTR is a non replication adenovirus. Ad-RGD-SUR-E1 was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Vital titer achieved 5.8×106 pfu/ml with TCID50 method. Viral replication experiments were performed to evaluate the selective replicationbility of Ad-RGD-SUR-E1. In tested tumor cell lines, Ad-RGD-SUR-E1 can replicate.and has the ability of selectively killing ZR-75-30 cells in vitro.We used virus proliferation assay, cell viability assay and western blot to evaluate the Ad-RGD-SUR-E1. Results demonstrated tha Ad-RGD-SUR-E1 EIA is expressed in survivin-positive ZR-75-30 cancer cells but not in survivin-negative normal CCD-11-lu cells. virus proliferation multiples in ZR-75-30 cancer cell is 100 folds after 72 hours,which is 10 folds in CCD-11-lu cells. Ad-RGD-SUR-E1 can cause about half of the cells to die within 7 days in ZR-75-30 cancer cell lines at MOI 5 PFU/cell. The results prove that Ad-RGD-SUR-E1 has highly proliferation selectivity on ZR-75-30 cancer cell lines.In this study, we construct three recombinant adenovirus plasmid .Experiments in vitro showed that Ad-RGD-SUR-E1 selectively replicated in and lyses survivin-positive tumor cells. The results of this study will give some important information for the following tumor therapy. |
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