| Objective:To investigate the inhibition effect of recombinant adenovirous which contain the cytosine deaminase (CD)gene alone or the CDglyES fusion gene when combined with radiotherapy on Lewis lung cancer of mice ,and the radiosensitization of recombinant adenovirus containing different genes. We hope to find a new useful program in the treatment of cancer. Methods:1 First, we get the gene CD and the gene segment gly ES from the recombinant adenovirus rAdCD and the secretory plasmid pEZZ-18-ES which have been constructed by our department by PCR. Then we constructed the recombinant adenovirus plasmid containing CDglyES fusion gene and got the recombinant adenovirus by amplified them in the 293 cells. And also verified the activities of CD and ES genies respectively in CDglyES fusion gene.2 We make a animal test model by inoculating gastrocnemius of the CSTBlinbreeding mice with Lewis lung cancer cells.3 C57BL inbreeding mices were divided into 8 groups randomly, 9 mices evry group, and from group 1 to 8, each group received PBS .radiotherapy alone, rAdCD/5-Fc, rAdCD/5-Fc+radiotherapy, rAdCDglyES, rAdCDglyES+radiotherapy, rAdCDglyES/5-Fc and rAdCDglyES/5-Fc+ radiotherapy respectively.4 We record the survival days, growing delay, inhibition and the pathology state (such as necrosis, apoptosis and the number of vascular )of the tumor .Investigate the mechanisms of synergism and radiosensitization when the recombinant adenovirus combined with the radiotherapy for the treatment of cancer.Results:1 The identification of recombinant adenovirus containing CDglyES fusion geneAfter being digested by double restriction endunulease, amplified by PCR and sequenced , we identified that the fusion gene CDglyES was inserted into the the recombinant adenovirus.in vitro, we infected the hepatic cancer cell strain SMMC-7721 and cervical cancer cell strain Hela with the recombinant adenovirus. When treated with 5-Fc, about 80 percent of cancer cells were inhibited by rAdCDglyES .The difference was statistically significant compared with rAd-LacZ (52.2%) (P<0.01). Then we concluded that the fusion gene CDglyES has the activity of gene CD. The inhibition of umbilical vessel endothelia cells ECV-304 by the concentrated cellular culture supernatant of rAdCDglyES (78.7%)was remarkably different compared with the control group of the concentrated cellular culture supernatant of rAdCD(24.2%)( P<0.01).This result also makes sure the genetic fusion does not affect the activity of ES gene. 2 in vivo(1) the group received rAdCD/5-Fc+radiotherapy and the group received radiotherapy aloneThe results of this study showed us: the weight of the tumor of the group which received rAdCD/5-Fc system combined with radiotherapy was lighter than that of the group which received radiotherapy alone, and the later was lighter than that of the control group. Pathology: there are many apoptosis in both the group received rAdCD/5-Fc+radiotherapy and the group received radiotherapy alone, and the former was much more than the later ,but there is no apoptosis in the control group. On the contrary, there are much more necrosis in the control group and nearly no in the other groups. The time for the tumor growing to 2000mg of the former was 1.64 when compared with the later. The median survival time of the former was longer than the later too. There is a statistically significant difference (P=0.019) between them. Then we concluded that the rAdCD/5-Fc system have a sensitiziation when combined with the radiotherapy.(2) the group received rAdCDglyES/5-Fc+radiotherapy and the group received rAdCDglyES/5-FcThe weight of tumor of the former is lighter than that of the later, there are many apoptosis and few necorsis in the former, it was contrary in the later .On the other hand , the median survival times of the former is nearly 2 times of the later( P<0.01). The times for the tumor growing to 2000mg and SOOOmg of the former is also2 times of that of the later. There is a synergic effection ofrAdCDglyES/5Fc in... |
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