| Objective To construct double-regulated conditionally replicating adenovirus SG504-SEA. SEA gene construct carrying the selective adenovirus. In vitro experimental observation of SEA gene expression, the anti-tumor function of its stimulated lymphocytes, and the auxo-action function of SEA on cytokines.Methods The PSA promoter was amplified from prostate cancer tissues by PCR method and the 522bp PSA promoter gene sequence was cloned into pSG504 plasmids after restriction enzyme cutting. The plasmids pSG504 was co-transfected with PPE3-ccdb-SEA in 293 cells to generate recombinant adenoviruses SG504-SEA. The recombinant adenoviruses were verified by PCR, purified by cesium chloride density purification and propagated in 293 cells. Virus titer was measured by TCID50 method and its titre was 2.0×1010pfu/ml. The mRNA expression of SEA in prostate cancer DU145 cells is detected for each period of time through RT-PCR. We extract the total RNA lines after 12, 24, and 48 hours respectively and detected the SEA expression at nucleic acid levels. The tumor cells DU145 is infected by recombinant virus with the titer of 5MOI and is taken out after 12, 24, and 48 hours respectively. The SEA expression is located in prostate cancer cells through immunofluorescence localization. Western blot is used in determination of SEA protein expression. Through microscope, the co-culture of lymphocytes and prostate cancer cells are observed dynamically. ELISA is used in detection of TNF and IL-2 secretion.Results It is proved that the successful cloning of PSA promoter and SEA gene into the oncolytic adenovirus vector could realize the expression of the SEA gene, by PCR amplification, restriction enzyme cutting identification. And the virus titer was 2.0×1010pfu/ml. The mRNA expression of SG504-SEA in tumor cells is detected for each period of time through RT-PCR. Strips are seen clearly at agarose electrophoresis 252bp. Prostate cancer DU145 cells are transfected for 12, 24, and 48 hours in the continued expression of SEA mRNA. Obvious fluorescence expression can be seen by locating the immunofluorescence on the cell surfaces. Thus it can be determined that the SEA gene is expressed in prostate tumor cell membrane, that the brightness of cells fluorescence expression differs for each period of time, and that as time increases, the fluorescence expression is enhanced gradually. After SDS-PAGE electrophoresis, with Coomassie brilliant blue staining, strips could be seen clearly around 27kDa. But no strips are seen for the untransfected cells. The Western-blot results confirm the successful expression of SEA protein. The lymphocytes and tumor cells are co-cultured for 12, 24, and 48 hours respectively. The tumor cells in the experimental group are less than that in the contrast group. The above shows that during the co-culturing process of DU145 cells and lymphocytes, compared to the untransfected cells, the tumor cells with transfected SEA gene are more vulnerable to be damaged by lymphocytes and have a certain chemotactic effect on the lymphocytes. Through ELISA detection, the TNF and IL-2 secretion in the experimental groups are higher than those in the contrast groups.Conclusion We have successfully construct double-regulated conditionally repli -cating adenovirus SG504-SEA, which lays the foundation for further research on application of SEA in targeted gene therapy for prostate cancer and the underlying immunological mechanisms. It is proven that the in vitro stimulated lymphocytes has a killing effect on prostate cancer cells and has a promoting effect on the secretion of cytokines surrounding the tumor cells. |
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