The Construction of Recombinant Adenovirus rAdCDglyTK and the Study of Inhibiting Effect of It in Vitro and in Vivo

Abstract: Malignant tumor therapy with suicide genes as well as prodrugs has become one of studyfocuses of professionals in fields both of basic and clinical medicine. In our study, therecombinant adenoviral plasmid rpAdCDglyTK containing E. coli cytosine deaminase (EC-CD)gene and herpes simplex virus thymidine kinase (HSV-TK) gene, constructed earlier by ourresearch group, were packaged and multiplied in 293 cells to produce rAdCDglyTK. Theexperiment study of tumor inhibition effect of the recombinant adenovirus containing confusionsuicide genes rAdCDglyTK compared with single suicide gene in vivo and in vitro accumulatedrich experimental basis for the clinical application of rAdCDglyTK.Firstly, our study transfected rpAdCDglyTK, constructed earlier successfully by ourresearch group, into 293 cells to get it packaged. The successful package was verified byobserving the expression of green fluorescence protein encoded by the reporter gene GFP andobtaining specific segment of CDglyTK by multiplying the supernatant solution of infected cellsby PCR. Subsequently, the virus was further multiplied by passage and then purified by CsCIdensity grads centrifuge to get high concentration of recombinant adenovirus containing fusionsuicide genes rAdCDglyTK, which was titrated by TCID_(50). The results of the growth inhibitiontest of Hela cells verified that both CD gene and TK gene in the recombinant adenoviruscontaining fusion suicide gene rAdCDglyTK presented functional activity individually. There was no obviously difference from the activities of the recombinant adenoviruses containingsingle suicide gene rAdCD and rAdTK (P>0.05).To discuss the tumor inhibition effect of the recombinant adenovirus containing fusionsuicide gene rAdCDglyTK, the recombinant adenovirus systems rAdCDglyTK/5-FC+ACV,rAdCD/5-FC, rAdTK/ACV, and rAdlacZ/5-FC+ACV were activated onto human cervical cancercell Hela, human liver cancer cell SMMC-7721, human lung cancer cell A549, humancystadenoma of ovary cell SK-OV-3 and humanglioma cell U251. 48 hours after culture, thegrowth inhibition rates of tumor cells were tested by MTT. The results showed that the inhibitionrates of recombinant adenovirus rAdCDglyTK our study constructed on those above five tumorcelis (54-79%) were significant difference from those of rAdCD/5-FC (30-46%), rAdTK/ACV(31-45%), and rAdlacZ/5-FC+ACV(4-11%) (P<0.05). There was also obvious difference fromthe negative control of tumor cells (P>0.05).Meanwhile,our study discussed the bystander effect of the recombinant adenoviruscontaining fusion suicide genes rAdCDglyTK. Firstly, rAdCDglyTK, rAdCD, rAdTK andrAdlacZ systems infected Hela, SMMC-7721, A549, SK-OV-3 and U251, respectively. The cellswere harvested 12 hours after culture at 37℃5%CO_2. Then, those five infected cells weremixed with the uninfected cells of same strains with different scales and cultured under the samecondition for 48 hours. The results showed that the rAdCDglyTK system presented higher ratesof tumor inhibition than rAdCD, rAdTK, or rAdlacZ system in each scale. Moreover, thebystander effect was more evident in low scale(10%, 25%) (P<0.05). With the increase of thescale (75%), there was no obvious difference between rAdCDglyTK and rAdCD and rAdTK(P>0.05).To discuss the tumor inhibition effect of the recombinant adenovirus rAdCDglyTK in vivo, our study injected the lung cancer strain Lewis into the left gastrocnemius of inbreeding linemice C57BL and subsequently administered rAdCDglyTK, rAdCD, rAdTK, and rAdlacZ withrelating prodrugs according to the plan. By observing tumor size, tumor weight, median survivalperiod, and pathological changes, the results showed that both the growth rate of tumor andweight of tumor of the experimental group rAdCDglyTK/5-FC+ACV were markedly lower thanthose of control groups rAdCD/5-FC、rAdTK/ACV、rAdlacZ/5-FC+ACV (P<0.05). Thepathological slice of mice tumor tissue showed that the tumor tissue of mice of the experimentalgroup presented spotted necrosis while the tumor tissue of mice of the control ones showed largenecrosis, which also proved that rAdCDglyTK contained higher tumor inhibition effect thanrAdCD or rAdTK in vivo.Our study discussed that the rAdCDglyTK/5-FC+ACV system presented the perfect tumorinhibition effect by the experimental research of the tumor inhibition effect of the recombinantadenovirus rAdCDglyTK in vivo and in vitro. The results set up a strong experimental basis forgene therapy of tumor with suicide gene…
Key words: malignant tumor; suicide gene; cytosine deaminase; thymidine kinase; fusion gene; recombinant adenovirus

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