Experimental Study On Sensitization Of Radiotherapy And Chemotherapy Of E1A Gene To Cervical Carcinoma And Correlated Mechanism Of Action

Objective To investigate transfer activity of PEI-Fe₃O₄NP gene carrier and the growth inhibition and radiochemosensitivity of E1A gene on cervical carcinoma cell , and the correlated mechanism of which as well, to provide the experimental evidence for the feasibility of cervical carcinoma gene therapeutics .Methods â‘  E1A gene in adenovirus was transfected into human cervical carcinoma cell ,growth curve was drown , the doubling time and the number of soft agar colony was calculated by cell count method .The effect of E1A gene on the multiplication of the cell in vitro was observed.â‘¢The cell was treated by cisplatin ,5-fluorouracil ,paclitaxel respectively , then the changes of sensitivity were evaluated .â‘¢ Cell line Hela untransfected , transfected by idler plasmid and transfected by E1A gene respectively were irradiated by 6MV X ray with dose of 0,2,4,6,8,10 Gy .The effect of E1A gene on radiosensitiveness of cervical carcinoma cell was observed by drawing cell survival curve .â‘£ The growth inhibition of E1A gene on the transplantation tumor in nude mice was observed by the experiment of carcinogenesis in nude mice . Chemosensitivity of E1A gene on the transplantation tumor in nude mice was observed by the experiment imitating the therapeutics of nude mice by E1A gene. ⑤ Contents of DNA in human cervical carcinoma cell were tested by flow cytometry before and after transfection and cell cycle was analyzed by computer. The changes on cell cycle was also observed .â‘¥The expression level of HER-2/neu before and after transfection was tested by Immunocytochemical stain to observe the effect of E1A gene on HER-2/neu expression. Tumor cell Apoptosis and the expression level of Survivin gene and Caspase-3 gene before and after transfection were tested by immunohistochemistry stain to observe the effect of E1A gene on tumor cell Apoptosis and the expression of Survivin gene and Caspase-3 gene.Results â‘ Human cervical carcinoma cell transfected with E1A gene grew slowly with longer doubing time which was 1.53 times ,1.58 times as long as that of Hela-vect and parent cell Hela respectively .Thegenerating rate of the cell colony were 61.48%,58.2% and 13.62 % respectively in cell line Hela untransfected , transfected by idler plasmid and transfected by ElA gene ,indicating that in ElA gene group was lower than that in the Hela and Hela-vest groups. The suppressed colony generation rate were 77.84% and 76.65% respectively compared with Hela and Hela-vect group.(2)Radiosensitivity of Hela-EIA increased 5 times as long as that in the cell line Hela untransfected , transfected by idler plasmid and that in the two .Latter was closer. ?Human cervical carcinoma cell transfected with ElA gene was remarkably more sensitive to cisplatin and paclitaxel ,but not sensitive to 5-fluorouracil and the sensitivity was increased by 10 times and 15 times compared with that of Hela cells and Hela-vect cells. ?The experiment of carcinogenesis in nude mice showed that Hela-EIA cells elongated the time of tumor formation and more effectively suppressed the growth of the tumor compared with Hela and Hela-vect, and the suppressed rates of tumor growth was 83.42 and 84.74 .The experiment imitating the therapeutics of nude mice by ElA gene showed that the tumor deflation in paclitaxel group was deflated most obviously one week after administration and then came to a platform period .Until 21 days after administration the volume of the tumor was 198.54 mm3 and the tumor of three mice deflated by more than 50% (PR30%).The tumor deflation in paclitaxel+ElA gene group was most obvious 3 days after administration ,and continued to decline later . To 21st day, the average volume of the tumor was 11.46 mm ,that of four mice deflated by more than 50% (PR40%) and that of five mice disappeared entirely(CR50%),where CR+PR=90%. The suppressed rates of tumor growth of paclitaxel+ElA gene was 94.23% which was obviously higher than that of paclitaxel .?Results form flow cytometry showed that ElA gene could change the distribution of human cervical carcinoma cell cycle with the occurrence of S stage suppression and G2/M stage blockage .? Results from Immunocytochemical stain showed ElA gene transferred by PEI-Fe3O4NP could stably expressed in Hela cell and decreased expression level of HER-2/neu gene. ElA gene could suppress the expression of the HER-2/neu gene . Result fromimmunohistochemistry stain showed E1A gene could activate Caspase-3 gene, suppress the expression of Survivin gene and promote apoptosis.Conclusions E1A gene can effectively suppress the multiplication of human cervical carcinoma cell in vitro and the growth of the transplantation tumor in nude mice ,moreover remarkably enhance the radiosensitivity and chems-sensitivity to chemotherapeutics drug such as cisplatin and paclitaxel .The probable mechanism of action are as follows :d)ElA gene can change the distribution of human cervical carcinoma cell cycle with the occurrence of S stage suppression and G2/ M stage blockage .(2) E1A gene can suppress the expression of the HER-2/neu gene . (3)E1A gene can activate Caspase-3 gene , ? E1A gene can suppress the expression of Survivin gene .