| Gene therapy has been a novel method of malignant tumor treatment for more than ten years. But most clinical assays show that the single gene treatment did not obtain satisfied results. So many researchers become paid increasingly attention to the fusion gene therapy. Based on the study of our group, we here selected two therapy genes. One is a suicide gene, cytosine deaminase(CD) gene, whose product can transfer the pro-drug 5-FC to 5-FU that destroys tumor cells directly. The other gene we selected was endostatin (ES) gene. It's product, ES, plays an indirect role in checking tumor cells through inhibiting the formation of new blood vessels and then blocking the apply of oxygen and nutrients necessary for tumor cells by means of restraining the generation of the blood vessel endothelial cells in the tumor. Furthermore, ES has the ability to keep tumor cells from distant metastasis. We constructed CD and ES fusion gene and then inserted it into an adenovirus vector to make a recombinant virus capable of both direct and indirect roles in inhibition of tumor cells.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 carrying CDglyES fusion gene.pAdTrackCMV-CDglyES was transferred to BJ5183 bacteria combined with pAdEasy-1 by a high efficient and simple method of homologous recombination in bacteria to construct the recombinant adenoviral plasmid prAd-CDglyES. The recombinant clones were screened by kanamysin. And recombinant pAd-CDglyES was identified by PCR, restriction endonulease digestion, and sequencing of CDglyES gene.Recombinant pAd-CDglyES, which was transfected to 293 cells by liposome, packaged and amplified in the cells. The virus plaque was observed under thefluorescent microscope by the expression of the green fluorescent protein of the report gene GFP. The recombinant virus was purified by CsCl gradient centrifuge and was tittered by TCID50 method.To verify the double activities of CD and ES genes in CDglyES fusion gene, the purified recombinant virus was diluted to 1*1010 TCIDso/ L, which infected hepatic cancer cell strain SMMC-7721 and cervical cancer cell strain Hela for 24 hours respectively. Then the infected cells were treated with 5-FC. The growth inhibition effect of the vims on tumor cells were observed after another 48 hours and compared with that of rAd-CD and rAd-LacZ at the same period 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 statistically significance compared with that of rAd-CD (p>0.5). That confirm 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). This result also makes sure the genetic fusion does not affect the activity of ES gene.In conclusion, we made the elementary inhibition test of tumor cells in vitro with CDglyES fusion gene transferred by the recombinant adenovirus vector. The results reveal that rAd-CDglyES has the ability to inhibit rumor cells directly and indirectly in vitro and initially confirm that the idea of our study is reasonable. But there are...
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