| Based on the study of our group, we here selected two therapy genes. One is cytosine deaminase (CD) gene, whose product can transfer the pro-drug 5-FC to 5-FU that destroys tumor cells directly. The other is endostatin (ES) gene, it's product, endostatin, plays an indirect role in checking tumor cells through inhibiting the formation of new blood vessels and then blocking the apply of oxygen and nutrition necessary for tumor cells by means of restraining the generation of the blood vessel endothelial cells in tumor. Furthermore ES has the ability of keeping the tumor cells from distant metastasis. The purpose of this study was to evaluate whether the recombinant fusion gene has a significant antitumor effect and enhances the tumor killing effect of radiation.First, the CD gene containing specific endonulease digestion sites was amplified from rAd-CD carrying CD gene we constructed before by PCR. The amplified CD gene was inserted into the shuttle plasmid pAdTrack-CMV to construct pAdTrackCMV-CD, which was identified by endonulease digestion and PCR. The gene segment glyES was amplified from prokaryotic secretary expression plasmid pEZZ-18-ES constructed before with the sense and antisense primers. The glyES gene was then inserted into pAdTrackCMV-CD to construct adenovirus shuttle plasmid pAdTrackCMV-CDglyES, which carrying CDglyES fusion gene.pAdTrackCMV-CDglyES was transferred to BJ5183 bacteria combined with pAdEasy-1 by a simple and high efficient method of homologous recombination in bacteria to construct the recombinant adenoviral plasimd pAd-CDglyES. The recombinant clones were screened by kanamycin. The recombinant pAd-CDglyES was identified by PCR, restriction endonulease digestion, and sequencing of CDglyES gene. Recombinant pAd-CDglyES was transferred to 293 cells and amplified in the cells. The recombinant virus was purified by CsCl gradient centrifuge and was tittered by TCIDso method. To verify the double activities of CD and ES genes in CDglyES fusion gene, the purified recombinant virus was diluted to 1 1010TCID50/L, which infected hepatic cancer cell strain SMMC-7721 and cervical cancer cell strain Hela for 24 hours respectively. The infected cells were treated with 5-FC, the growth inhibition effect of the virus on tumor cells were observed after another 48 hours and compared with that of rAd-CD and rAd-LacZ at the sameperiod of time. The results show that 78.2 percent of SMMC-7721 cells were inhibited by rAd-CDglyES. The difference was statistically significant compared with rAd-LacZ (52.2%), p<0.01. 78.1 percent of Hela cells were inhibited by rAd-CDglyES and the difference was also statistically significant compared with rAd-LacZ (50.2), p<0.01 . But the variance was no statistical significance compared with that of rAd-CD (p>0.5). That confirmed that the genetic fusion of CD and ES has no influence on the activity of CD gene. The inhibition of umbilical vessel endothelial cells ECV-304 by the concentrated cellular culture supernatant of rAd-CDglyES (78.7%) was remarkably different compared with the control group of the concentrated cellular culture supernatant of rAd-CD (24.2%), p<0.01. That result also makes sure that the genetic fusion does not affect the activity of ES gene.As to the in vivo trial, we observed the inhibition ratio of CDglyES fusion gene on a human mammal carcinoma model named MA737. 66 jin bai II mice were divided into 7groups by random numbers and implanted MA737 cells. They were control group, radiation group, CDglyES group, CDglyES+radiation (I4Gy)group, CDglyES+5-FC group, CDglyES+5-FC+radiation (14Gy)group, CDglyES+5-FC+ radiation (7Gy) group. The fusion gene was intramuscular injected daily for five times, and 5-FC was intraperitoneal injected daily for five times, too. Radiation was given after the use of fusion gene and pro-drug. Several factors were observed to determine if the recombinant fusion gene is effect. Combined treatment group with recombinant fusion gene and IR produced tumor growth inhibition than any other group. By day 20, tumors receiv... |
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