| Suicide gene therapy has been a new hot spot of cancer gene therapy research. Previous studies focused mainly on single suicide gene therapy, which have virtues of easy control and induce immunity as well as tumor target function. However, every knife has two edges, suicide gene therapy has some deficiencies too. Many experiments have demonstrated that different malignant tumors have various responses to different system of suicide gene/prodrug and the bystander effect has distinctness too. On the other side, the product of double suicide gene has the activities of two enzymes. Cytotoxicity would strongly increased by the complementary of double suicide gene expressing products. In this experiment, we probe to effects of fusion gene combined with radiotherapy to human colorectal cells and nude mice bearing human colorectal carcinoma. For this purpose, we construct a replication-defective adenovirus vector (Ad.CDglyTK). Adenovirus vector is the most effective vector at present, which can receive an insertion of large gene. We can get a high titer also (1012-1013pfu/ml) through this method. Combination the irradiation enhancement effect of CD/5-FC and TK/GCV we can kill the tumor cells more strongly. At the same time, it unites the radiotherapy/ chemotherapy with gene therapy into one. We try to find a novel way and to provide a foundation of clinical malignant tumor's therapy. Methods:1 The construction of cosmid vector inserting CDglyTK gene (pAxcAwt/CDglyTK) DNA fragment reclaimed from the plasmid of pWZLneoCDglyTK. Then the DNA was inserted into cosmid vector pAxcAw. The recombinant cosmid was amplified and was confirmed to be the correct gene . At last the correct clones containing target gene in right direction were selected.2 The construction of replication-defective recombinant adenovirus coding for CDglyTK gene (Ad.CDglyTK) Human embryonic kidney 293 cells cultured in a 6cm dish were transfected with DNA-TPC and pAxcAwt/CDglyTK by the calcium phosphate method. After 8-18 days, virus clones were isolated and propagated to for restriction analysis. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated in 293 cells. Whether there has a little wild type adenovirus was also examined at the same time. 3 Transfection of Ad.CDglyTK to carcinoma cells and its effects of enhancement of radiosensitization in vitro Three kinds of gastrointestinal cells(LoVo, HCT-8, SGC-7901) were isolated and cultured. Transfection efficiency of Ad.CDglyTK on these cells was examined at various titrations by immunofluorescence staining. And then Co60 radiotherapy of different dose in vitro were completed compared with prodrug treatment of 5-FC or GCV. Cell doubling time was tested after three kinds of cells were transfected by recombinant adenovirus. Fusion protein expression was evaluated by Western blot respectively. The cell survival curve was calculated by cell cloning forming test. Cell survival rate was examined by MTT assay. Bystander effect in vitro was observed. D0,Dq of the cell survival curve were measured and the SER(Sensitizing Enhancement Ratio) were compared in different groups. 4 To research into the therapeutic effect of double suicide gene combining radiotherapy to nude mice bearing human colorectal carcinoma First, the model of nude mice bearing carcinoma was established. Then the prodrug experiment was completed. In the period of prodrug treatment, Co60 therapy to nude mice in some groups was completed on the time. In the end, tumor of nude mice was examined. Cell apoptosis was checked through FCM method, DNA gel electrophoresis and transmission electron microscope . Results: 1 The segment of DNA obtained from pWZLneoCDglyTK plasmid was identical to that expected. After the target DNA was inserted into cosmid vector pAxcAw, the recombinant cosmid was amplified and confirmed to be the correct gene. 2 An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-defective adenovirus vectors coding for...
|
PDF Full Text Request