| Background: At present, suicide gene is one of widely prospect in all scheme of gene therapy of tumor. The most common suicide gene is coded for the herpes simplex type thymidine kinase(HSV-TK), which converts nontoxic nucleoside such as ganciclovir into toxic triphosphated compounds. Ganciclovir triphosphate is a purine analogue that competes with normal nucleotides and can inhibit DNA polymerase and thus lead to cell death.Bystander effects is an special phenomena of suicide gene therapy .Now, suicide gene transfers into tumor cells has been proposed for the treatment of various tumor, and several prototypes of gene therapy protocols have been investigated in clinical and precKnical studies for the treatment of cancer. To study the local/distant bystander effects of the HSV-TK gene system on bladder carcinoma, we have investigated the killing effects on T739 mouse bladder cancerl, and the mechanism of tumor cell death induced by HSV-TK/GCV system. The method and results are listed here as follows.The methods and the results of the experiment:1. The recombinant retroviral vector PL(HyTK)SN containing HyTK gene was transferred into packaging cell line PA317 with DNA-liposome complexes. Recombinant retroviral supernate was obtained and infected murine bladder cancer cell line T739 . After Hygromycin B selection, B-resistant colonies were obtained. RT-PCR showed that HyTK gene was transferred into the T739 cells and expressed successfully.2. In vivo, experiment on T739 syngeneic mice bladder cancer were performed in T739 syngeneic mice .100%,50%TK+ carcinoma were induced on flank in experiment groups.while TK-carcinoma were induced in control groups,then the GCV treatment wasgiven to the mice. After 7,14days, the growth of the tumor was subsequent observed slowly in experiment groups,and then animal survival time was longer than the control group. HSV-TK/GCV oncolysates can induce the kill effect to the carcinoma cell ,,and the local bystander effect is obviously.3. To investigate the distent bystander effect,in syngeneic murine T739 bladder cancer cell line transfer tumor model, HSV-TK gene was transferred by injection of retrovirus, TK+ carcinoma were induced on left flank while TK-carcinoma were induced in right flank,then the GCV treatment was given to the urime and the effect on the growth of the tumor was subsequent observed. After 7,14 days administration of GCV, no obvious difference was seen in right flank between experiment and control group, and the survival of the mice was not prolonged. It suggested that distant bystander effect to the distant carcinoma cell in other organ were not obviously.4. To evaluated the mechanism of local/distant bystander effects of the HSV-TK gene system on bladder carcinoma. We evaluated the GJIC of cells and CD4+, CD8+ Lymphocytic cell by filleting of the tumor constitution, and observed the expression of Cx26 protein and CD4+, CD8+ Lymphocytic cell by immunocytochemistry were obviously positive in tumor section in the local bystander effect experiment goups,but the expression of Cx26 protein and CD4+, CD8+ Lymphocytic cell by immunocytochemistry were not positive in tumor section in the local bystander effect experiment goups.5. We evaluated the expression of Cx26 by immunohistochemistry in normal mice bladder tissues and mice bladder carcinoma tissues . The expression of Cx26 was intensive in mucosa transitional epithelium cells in normal badder tissues, but faint or negative in the bladder carcinoma tissues. The expression of Cx26 was reduced in early highly differentiated bladder cancers, and was related to the malignant type.Conclusion:The growth of TK+carcinoma were inhibited obviously by HSV-TK/GCV system while animal survival time was longer than the control group,but the growth of TK+carcinoma in transfer tumor model were not inhibited obviously by HSV-TK/GCV system and animal survival time was not different between the control group, the local bystander effect to the carcinoma cell were obviously,while the distant bystander effect to the distant carcinoma cell in other organ were not obviously. The expression of Cx26 is evidently decreased in bladder cancers, which may induce the reduced function of GJIC and the effection of the bystander effect of HSV-TK/GCV system... |
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