| Radionuclide therapy can affect tumor cells by ionization radiation, lead to necrobiosis, radiation injury of normal tissue close to tumor and radiation resistance of some tumors, and influence the curative effect and application of radiotherapy. By transferring objective gene into target cell, tumor gene therapy may enable the target cell to achieve the specific function of killing or inhibiting the tumor. Gene-radionuclide therapy can couple the control sequence of irradiation inductivity gene with anti-tumor gene, and then transfect the tumor cells. At the same time of local internal irradiation, gene-radionuclide therapy can enhance the anti-tumor gene expression, raise local the radiation sensitivity and lower radiation injury of the normal tissue close to tumor, generate the anti-tumor function of radiation and gene expression products. Therefore, gene-radionuclide therapy has become the central issue in the tumor therapy field recently. On the basis of irradiation inductivity of Egr-1 gene promoter and the function of killing malignant tumor cells by 125I-UdR and immunological regulation of anti-tumor by IFNγ. The recombinant plasmid pcDNAEgr-IFNγwas constructed in this study. The radiation inducible expression of recombinant plasmid pcDNAEgr-IFNγby ionizing radiation of 125 I-UdR as well as anti-tumor effects and its mechanism of pcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy were studied. 1 Construction of recombinant plasmid pcDNAEgr-IFNγThe recombinant plasmid pcDNAEgr-IFNγwas constructed successfully with gene recombinant technique. It was proved to be right after identification by restriction enzyme.2 Radiation inducible expression of recombinant plasmid pcDNAEgr- IFNγby ionizing radiation of 125I-UdR2.1 Doses of 125I-UdR absorbed by nucleusAfter 48 h transfection, 125I-UdR in 0-50kBp/ml was added to culture solution. And 24 h later, the cells were harvested and splitted. Then, the nucleus were extracted and the intensity of radioactivity were detected. The results showed that the dose of 125I-UdR absorbed by B16 cell nucleus increased with the increasing of concentration of 125I-UdR in the culture solution.2.2 Expression of IFNγin B16 cells transfected by pcDNAEgr-IFNγThe harvested cell lysate was detected by Western blotting. The results showed that through the internal irradiation of 125I-UdR in 1-50kBp/ml, The IFNγprotein expressed in the cell lysates of B16 cells transfected by recombinant plasmid pcDNAEgr-IFNγ, and the expression level increased with concentration of 125I-UdR, while there was no expression of IFNγwithout the internal irradiation of 125I-UdR.Dose-effect relationship between IFNγand 125I-UdR in the harvested cell supernatant was detected by ELISA. The results showed that through the internal irradiation of 125I-UdR, the expression of IFNγin the supernatant of B16 cells transfected by recombinant plasmid pcDNAEgr-IFNγincreased with the concentration of 125I-UdR in the cell culture fluid. The IFNγexpression levels in B16 cells treated with 5, 10, 20, 50 kBq/ml doses of 125I-UdR were obviously higher than that of B16 cells treated with 0 kBq/ml (P<0.05-0.001). The IFNγexpression level in 50 kBq/ml dose of 125I-UdR was the highest and was 3.66 times of that of 0 kBq/ml.2.3 Expression of IFNγin B16 cells transfected by pcDNAEgr-IFNγat different time after 50 kBq/ml dose of 125I-UdR irradiationAfter 48 h transfection, 125I-UdR in 50kBp/ml was added to culture solution. The expression of IFNγin the harvested cell supernatant was detected seperately by ELISA after culture for 2 h, 6 h, 12 h, 24 h and 48 h .The results showed that the IFNγexpression level at 6 h was obviously higher than that of 0 h(P<0.001). The IFNγexpression was in a time-dependant manner at 2-48 h, and the peaked at 48 h, the highest level was 6.51 times of that of 0 h.3 Anti-tumor effect of pcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy and its mechanism3.1 Anti-tumor effect of pcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy3.1.1 Effect of gene-radionuclide therapy on tumor growth rate of miceThe right hind limbs of mice were implanted with H22 cells. Then 100ul recombinant plasmid pcDNAEgr-IFNγmixed with liposome was injected into tumor when tumor diameter was about 5mm. 48 h later, 370 kBq 125I-UdR was injected into tumor. 6-15d after gene-radionuclide therapy, the tumor growth rates of 125I-UdR group, pcDNAEgr-1+125I-UdR group and pcDNAEgr-IFNγ+ 125I-UdR group were obviously lower than those of control group and pcDNAEgr-IFNγgroup (P<0.05-0.01). There was no significant difference between control group and pcDNAEgr-IFNγgroup (P>0.05). The tumor growth rate of pcDNAEgr-IFNγ+ 125I-UdR group was obviously lower than those of 125I-UdR group and pcDNAEgr-1+125I-UdR group (P<0.05-0.01). There was no significant difference between 125I-UdR group and pcDNAEgr-1+125I-UdR group (P>0.05).3.1.2 Effect of gene-radionuclide therapy on tumor weight of miceAfter 15 d gene-radionuclide therapy, the tumor weights of mice in 125I-UdR group, pcDNAEgr-1+125I-UdR group and pcDNAEgr-IFNγ+125I-UdR group were obviously lower than those of control and pcDNAEgr-IFNγgroup (P<0.05-0.01). There was no significant difference between control group and pcDNAEgr-IFNγgroup (P>0.05). The tumor weight of pcDNAEgr-IFNγ+ 125I-UdR group was obviously lower than those of 125I-UdR group and pcDNAEgr-1+125I-UdR group (P<0.05). Also, there was no significant difference between 125I-UdR group and pcDNAEgr-1+125I-UdR group (P>0.05). The tumor-restraining rates of pcDNAEgr-IFN group, 125I-UdR group, pcDNAEgr-1+125I-UdR group and pcDNAEgr-IFNγ+125I-UdR group were 1.6%, 9.8%, 11.4% and 21.1%, respectively.3.1.3 Effect of gene-radionuclide therapy on survival time of miceThe results showed that the mean survival time of the mice in 125I-UdR group, pcDNAEgr-1+125I-UdR and pcDNAEgr-IFNγ+125I-UdR group was obviously longer than that of the mice in control and pcDNAEgr-IFNγgroup (P<0.05-0.01). There was no significant difference between control group and pcDNAEgr-IFNγgroup (P>0.05). The mean survival time of the mice in pcDNAEgr-IFNγ+ 125I-UdR group was the longest in all groups. The mean survival time of the mice in pcDNAEgr-IFNγ+125I-UdR group was longer for 11d than that of control group. The mean survival time of pcDNAEgr- IFNγ+125I-UdR group was obviously longer than that of 125I-UdR group and pcDNAEgr-1+125I-UdR group (P<0.05). There was no significant difference between 125I-UdR group and pcDNAEgr-1+125I-UdR group (P>0.05).3.2 The mechanism of pcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy3.2.1 Expression of IFNγin cytoplasm of H22 cells implanted into mice after gene-radionuclide therapyAfter 3 d gene-radionuclide therapy, the concentration of IFNγin cytoplasm of H22 cells was detected by ELISA. The results showed that there was no IFNγprotein in cytoplasm of H22 cells in control group, pcDNAEgr-IFNγgroup, 125I-UdR group and pcDNAEgr-1+125I-UdR group. However, IFNγprotein was found in cytoplasm of H22 cells in pcDNAEgr-IFNγ+125I-UdR group. The concentration of IFNγreached (413.33±35.47) pg/mg in cytoplasm of H22 cells in pcDNAEgr-IFNγ+125I-UdR group.3.2.2 Immunological mechanism of anti-tumor effects of gene-radionuclide therapyAfter 3 d gene-radionuclide therapy, cytotoxic activities of splenic CTL of the mice in different groups were examined. The results showed that cytotoxic activity of splenic CTL of the mice in pcDNAEgr-IFNγ+125I-UdR group was significantly higher than those in control group, pcDNAEgr-IFNγgroup, 125I-UdR group and pcDNAEgr-1+125I-UdR group(P<0.01). There was no significant difference among the mice in control group, pcDNAEgr-IFNγgroup, 125I-UdR group and pcDNAEgr-1+125I-UdR group (P>0.05). 3.2.3 Pathological changes of tumor tissue After 15 d gene-radionuclide therapy, mice were killed and tumor was examined with histological method. Histological sections were stained with hematoxylin/eosin (HE). The results showed that there were rich blood vessels and wide hemorrhage in tumor of control group. There were rich blood vessels, wide hemorrhage and less lymphocytes in tumor of pcDNAEgr-IFNγgroup. There were less blood vessels, part of necrosis and less lymphocytes in tumor of 125I-UdR group and pcDNAEgr-1+125I-UdR group. There were less blood vessels, part of necrosis and rich lymphocytes in tumor of pcDNAEgr- IFNγ+125I-UdR group.All things considered, in this study, the recombinant plasmid pcDNAEgr- IFNγwas constructed successfully, which had the property of enhanceing expression of IFNγinduced by ionizing radiation of 125I-UdR, and IFNγlevels were dose-dependant and time-dependant. PcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy could obviously restrain hepatoma growth. The anti-tumor effects in vivo of pcDNAEgr-IFNγgene therapy combined with 125I-UdR radionuclide therapy were better than that of pcDNAEgr-IFNγand 125I-UdR therapy. The mechanism might be the combination of radiation- inducible IFNγexpression, the direct and indirect anti-tumor effects of IFNγand the direct killing effects of 125I-UdR. This study combined radionuclide therapy with immunogene therapy and set up a novel strategy for cancer gene-radionuclide therapy. These findings would provide the theoretical and experimental basis for applying gene-radionuclide therapy in clinical treatment for cancer.
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