Effect Of Photodynamic Therapy Combined With VP3Gene Therapy For Nasopharyngeal Carcinoma

Photodynamic therapy(PDT) is a light physical and chemical reaction with photosensitizer accumulation in the tumor cells under certain wavelength light. PDT a non operation treatment to kill tumor cells through generating singlet oxygen and free radicals and other active substances to destroy the tumor microvessel and enhance immunity. However, PDT is restricted by tumor depth and oxygen limitations which limits application in tumor therapy. In recent years, as gene therapy has become one of the most promising treatment of tumor, PDT combined gene therapy may be a new strategy for tumor treatment. This article will discuss the effect of suicide gene VP3on nasopharyngeal carcinoma cells and killing effect of PDT combined with VP3gene in the treatment of nasopharyngeal carcinoma in vitro and in vivo.Chapter I The effect of VP3gene on the growth, proliferation and apoptosis of nasopharyngeal carcinoma CNE-2cellsObject To study the effect of VP3gene on the growth, proliferation and apoptosis of nasopharyngeal carcinoma cell line CNE-2and testify effect induced by VP3on apoptosis of nasopharyngeal carcinoma cell in vitro experiment. Method Using liposome transfection of PVP3and mock vector to transfect nasopharyngeal carcinoma CNE-2cells. Plasmid transfection efficiency were observed by fluorescence microscope, RT-PCR, Western blot was used to detect expression of VP3,CCK-8method was used to detect cell survival rate, cell apoptosis was detected by Annexin V.Result1. VP3mRNA is no obvious expression in the wild type and mock vector transfected cells, but expression in PVP3transfected cells, which relative expression level is about1.27±0.24. VP3protein (apoptin) is no obvious expression in the wild type and mock vector transfected cells, but expression in PVP3transfected cells, which relative expression level is about0.22±0.06.2. The survival rates of mock vector transfected cells are97.6±1.4%,94.2±1.7%and92.3±2.1%in24h,48h and72h respectively after transfection. The survival rates of PVP3plasmid transfected cells are79.7±3.6%,61.5±2.3%and52.4±3.8%in24h,48h and72h respectively after transfection. The survival rates between PVP3transfected cells and mock vector transfected celss has significant difference (P<0.001).3. The apoptosis rates of mock vector transfected cells are4.5±1.2%、5.1±0.8%and5.7±1.4%in24h,48h and72h respectively after transfection. The apoptosis rates of PVP3plasmid transfected cells are1.6±2.4%,17.8±1.4%and27.3±3.3%in24h,48h and72h respectively after transfection. The apoptosis rates between PVP3 transfected cells and mock vector transfected celss has significant difference (P<0.001).Conclusion Successfully established stable VP3gene expression nasopharyngeal carcinoma cell line CNE-2, and proved that VP3could obviously inhibit the growth and proliferation and induce the apoptosis of CNE-2cells in vitro experiments, which provides the basis for follow-up experiments. Chapter Ⅱ To study the effect of photodynamic therapy combined with VP3gene therapy on nasopharyngeal carcinoma CNE-2cells in vitro experimentsObject To study the effect of photodynamic therapy combined with VP3gene therapy on CNE-2cells.Method Stable expression of VP3gene CNE-2cells and control cells were treated with PDT with different energy density. CCK-8method was used to detect cell survival, Annexin V and Tunnel method was used to detect apoptosis.Result1. The survival rate of PVP3vector transfected cells was significantly lower than that in mock vector transfected cells after PDT treatment with different energy density. The survival rates were0.533±0.036,0.312±0.027and0.171±0.032in PVP3vector transfected cells at energy density of1,3,6.25J/cm2respectively. The survival rates were0.816±0.058,0.570±0.044and0.308±0.047in control cells at energy density of1,3,6.25J/cm2respectively. The survival rates between two groups have significant difference (P<0.001).2. The apoptosis rate was significantly higher in PVP3vector transfected cells than that of mock vector transfected cells by Annexin V assay. The apoptotic rate of PVP3vector transfected cells were0.363±0.025,0.672±0.043and0.741±0.039at PDT energy density of1,3,6.25J/cm2respectively; The apoptotic rate of the control cells were0.266±0.027,0.568±0.055and0.671±0.046at PDT energy density of1,3,6.25J/cm2respectively. The two groups had significant difference (P<0.001).Tunnel assay showed that cell expansion and nuclear fragmentation in most of cells in experimental group cells while membrane integrity and cell vacuolization and nuclear condensation in a few of control cells.Conclusion PDT combined with VP3gene therapy can significantly inhibit the growth and proliferation and increase the apoptosis of human nasopharyngeal carcinoma CNE-2cells compared with PDT. Chapter III To study the effect of photodynamic therapy combined with VP3gene therapy on CNE-2cells xenografts in nude miceObject To compare PDT combined with VP3with PDT or VP3gene therapy in vivo experiment.Method Construction of human nasopharyngeal carcinoma xenograft model in nude mice. Tumor size and weight were measured and recorded after PDT treatment. HE staining was used to compare pathology in different groups; immunohistochemistry was used to detect expression of apoptin.Result1. Compared four groups (mock vector, PVP3plasmid, CNE2/Mock+PDT and CNE2/VP3+PDT) of tumor volume by ANOVA analysis of variance in0,3,7,14days after PDT treatment. There is significant difference in tumor volume of four groups. The tumor volume of CNE2/VP3+PDT group is the smallest than others(P<0.001); the tumor volume of CNE2/VP3+PDT is smaller than that in CNE2/Mock+PDT (P<0.001).2. There are significant differences in four groups of tumor weight. The tumor weight of CNE2/VP3+PDT group is the lightest than others (P<0.001); the tumor weight CNE2/VP3+PDT is lighter than that in group CNE2/Mock+PDT (P<0.001).3. HE staining showed that normal morphology is observed in CNE2/Mock group with no obvious necrosis tissue; a spot of necrosis region is observed in CNE2/VP3group; massive tumor cell degeneration and nuclear pyknosis with partial necrosis region is observed in CNE2/Mock+PDT group; large area of necrosis with only a small amount of residual cancer cells island is observed in CNE2/VP3+PDT group.4. Immunohistochemistry showed strong expression of apoptin in CNE2/VP3group and CNE2/VP3+PDT group with mainly located in the nucleus, while no expression of apoptin in CNE2/Mock group and CNE2/Mock+PDT group.Conclusion Gene therapy or photodynamic therapy can inhibit the growth of nasopharyngeal carcinoma xenografts in nude mice. PDT combined with VP3has better killing effect than PDT or VP3gene therapy alone in nasopharyngeal carcinoma xenografts, which provide experimental basis for clinical treatment of NPC.