| Background Radiotherapy is one of the key countermeasures required for comprehensive therapy of tumor. However, in the local treatment of certain tumor, radiotherapy may inevitably impair normal tissues around the tumor. In clinical, radiation dosage or duration was controlled to mitigate the radiation injury to normal tissue, which leads to decreased therapeutic effects of radiotherapy. Some methods such as multileaf collimator, precise fixation technique and radioprotectors have been used to eliminate the radiation injury, following with unsatisfactory results. For example, radioprotector failed to improve the efficacy of radiotherapy for the lack of tumor-targeting property and the protection against tumor tissues. Radiosensitizer enhanced the tumor radiosensitivity, but also the normal tissues, resulting in severer radiation injury. Recently, gene therapy has become a spotlight in tumor therapy, by activating suppressor gene, suppressing oncogene, importing cytotoxic gene,improving tumor immunogenicity and changing the tumor microenvironment. Superoxide dismutase(SOD) 2 locates in mitochondria and serves as an effective radioprotector for its potential in clearing cellular oxygen radicals and thus alleviating oxidative damage. It has been demonstrated that SOD2 could block tumor proliferation, metastasis, and improve the radiosensitivity of tumor cells. Unfortunately, the lack of effective methods for gene transfection and regulation of gene expression targeting radiation field limits the clinical application of gene therapy using SOD2. The promoter region of Egr-1 gene contains six CAr G boxes, and this region is radiation-inducible regulatory sequence that responses directly to radiation. Construction of chimerical promoter containing CAr G box can optimize the driven efficiency of promoter, thus decrease the background expression. Additionally, the construction of radiation-inducible chimerical promoter that contains SOD2 gene in the downstream may precisely regulate the SOD2 gene expression through controlling radiation dosage and duration, and ultimately realize the space-time restrictive expression of SOD2 gene therapy.Objective To solve the clinical problem presented as increased tumor radiosensitivity and enhanced normal tissue damage, we attempted to construct the SOD2 overexpressed lentivirus vector that driven by radiation-inducible chimerical promoter. By utilizing this kind of lentivirus vector, we can control the radiation dosage and duration, and realize the targeting and regulatable gene therapy, as well as increase the sensitivity of tumor radiotherapy and decrease the radiation-induced normal tissue damage. Because of the increased efficacy on tumor radiotherapy and improved life quality in tumor patients, gene therapy targeting SOD2 shows great potential in tumor radiotherapy.Methods 1. The preparation of lentivirus that contains reporter gene and therapeutic gene First of all, the chimerical promoter sequence containing CAr G box and CMV basic promoter was synthesized. Then, based on the structure of lentivirus vector p LVX-Ac GFP1-N1, through three successive clonal methods, the therapeutic gene lentivirus vectorp LVX-C9BC-SOD2-T2A-Ac GFP1 and reporter gene lentivirus vector p LVX-C9BC-Ac GFP1-N1 was respectively synthesized. These two lentivirus vectors were driven by radiation-induced promoter. Whether these lentivirus vectors were correctly constructed was confirmed by enzyme cleavage and sequencing. After the amplification of vector transformed stb13 bacterial strain, and removal of endotoxin, the plasmids were abstracted. 293 FT cell and three-plasmid lentivirus packing system was used to package lentivirus and the virus titer was determined at last.2. Radiation-inducible property and quantitative regulation character of chimerical promoter HT-29 and CCD41 Co N cell strains were respectively transfected with reporter gene and therapeutic gene lentivirus, and the stably transfected cell strain was harvested by drug screening. In order to verify the promoting activity and characteristics of the radiation-inducible promoter, these stable cell strains were exposed to different doses of irradiation and the expression levels of reporter gene and therapeutic gene were determined at various time points after irradiation. 3. The SOD2 expression and phenotypic change in irradiation-treated stable transfected cell lines The expression levels of SOD2 in irradiation treated HT-29 and CCD41 Co N cell lines were measured. The cell proliferation, apoptosis in different groups were also observed and compared. 4. In vivo study on the treatment of xenografted tumor in naked mice through irradiation induced SOD2 expression The xenografted tumor model in naked BALB/c mice was established by using HT-29 cell. The lentivirus containing therapeutic gene were locally injected into the tumor at 72 h before irradiation treatment. At different time points, the gene expression levels, tumor growth condition, histopathology and in-situ apoptosis were entirely observed.1. Reporter gene lentivirus(p LVX-C9BC-Ac GFP1-N1) and therapeutic gene lentivirus(p LVX-C9BC-SOD2-T2A-Ac GFP1) were successfully obtained and the correction of the sequence was confirmed by gene sequencing. The lentivirus titer was above 5×108 TU/ml, which meets the experimental requirements. Stably transfected cell lines were screened out by puromycin, and stored after amplification. 2. In vitro experiments were performed by using stably transfected HT-29 and CCD41 Co N cell lines, and the results demonstrated that radiation-inducible chimerical promoter can effectively initiate the expression of downstream gene under 2 Gy γ-irradiation. The expression levels of reporter gene Ac GFP1 and therapeutic gene SOD2 increased a lot 24 h after irradiation, and picked at 48 h. 3. In vitro experiments also demonstrated that the expression level of SOD2 significantly increased in 6 Gy γ-irradiation treated and therapeutic gene stably transfected HT-29 cell lines. Meanwhile, significant suppressed cell proliferation or growth and increased cell apoptosis ratio were observed, compared with control group. In stably transfected CCD841 Co N cell lines, the SOD2 level markedly increased, minor suppression in cell proliferation or growth and relative lower cell apoptosis ratio were observed, compared with control group. 4. In vivo experiments, the xenografted tumor model in naked BALB/c mice was successfully established by using HT-29 cell, following by Results injection of lentivirus 72 h before receiving local 6 Gy γ-irradiation. A marked increase in SOD2 expression level in tumor cells, delayed tumor growth, elevated apoptosis ratio of tumor cells and decreased apoptosis ratio of skin cells were observed after the mice were treated with lentivirus and local irradiation, compared with tumor-bearing naked mice without any therapy.Conclusion Chimerical promoter consisting of CAr G box and preliminary CMV promoter possesses obvious radiation-inducible property. This promoter can initiate the gene expression in the downstream and regulate the gene expression within certain range based on the radiation dosage. The in vitro and in vivo experiments demonstrated that combined application of SOD2 overexpressed gene therapy and radiotherapy can inhibit the proliferation and growth of tumor cells and improve tumor radiosensitivity. What more important is that the combined therapy can protect normal tissues from radiation damage by decreasing the apoptosis of normal cells in radiation field. |
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