The Growth Inhibition Of Adenovirus-mediated Rb94 Gene Therapy Combined With Radiotherapy On Human Malignant Tumor Cells

ObjectiveGene therapy is the hot point of the tumor biological treatment at present,and also the trend of the tumor therapy in the future.Radiotherapy has been the major method of the cancer treatment,and the effect of inhibiting tumor is enhanced each other between gene therapy and radiotherapy.The mechanism may be:radiation improves the efficiency of gene transfection and(or) increases the expression of protein,on the other hand the transfection of the gene improves the sensitivity of the radiation.The retinoblastoma gene(Rb) is the earliest isolated human suppressor gene,Rb94 gene is the part of Rb gene.Rb94 protein lacks 112 amino acid residues at NH₂-terminal of Rb protein.Studies indicate that the function of the Rb94 gene is better than that of the full length Rb gene in the inhibiting tumor effect.The aim of this study is to check the inhibiting effect of tumor cells growth by transfecting human hepatoma cell lines HepG2 and human cervix cancer Hela cells with adenovirus-mediated expression vector of Rb94 gene(Ad-Rb94).We study the combined effect of Ad-Rb94 with X-radiation to provide the experimental data for clinical therapy of the cancer gene therapy combined with radiotherapy.MethodsThe recombinant adenovirus vector was restricted with PacI,then transfected 293A cells by liposomes to amplify the recombinant adenovirus express vector, finally we got a lot of the recombinant adenovirus Rb94 particles.Results showed that Rb94 gene was confirmed in Ad-Rb94 vector by PCR.We choosed human hepatoma cell lines HepG2 and human cervix cancer Hela cells.The experiment groups were designed as blank control,Ad-lacZ,Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy.The cell growth inhibition was assessed by MTT,and cell cycle and apoptosis were detected by flow cytometry.ResultsFirst Results showed that expected Rb94 gene was confirmed in Ad-Rb94 vector by PCR.Second The titer of adenovirus was 1.6×108 pfu/ml,which reached the standard of experiment.Third There was no statistical significance compared with Ad-lacZ group and blank control.Fourth The growth curve of HepG2 cells:The growth of HepG2 cells that was treated with Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy was slower than that of both Ad-lacZ and blank control significantly(p＜0.05), especially at 96h after transfection.The growth inhibition rate of HepG2 cells of combined Ad-Rb94 with radiotherapy group at 96h after transfection was 55.63%±8.97%,which was significantly higher than that of Ad-Rb94 group(31.61%±3.25%,p＜0.05) and radiotherapy group(20.06%±4.33%,p＜0.05).Fifth The growth curve of Hela cells:The growth of Hela cells that was treated with Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy was slower than that of both Ad-lacZ and blank control significantly(p＜0.05),especially at 96h after transfection.The growth inhibition rate of Hela cells of HepG2 cells of combined Ad-Rb94 with radiotherapy group at 96h after transfection was 51.84%±3.84%,which was significantly higher than that of Ad-Rb94 group(37.40%±5.65%,p＜0.05) and radiotherapy group(35.42%±2.47%,p＜0.05).Sixth The cell cycle and apoptosis of HepG2 cells:The number of cells of G₂/M phases that was treated Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy increased,while the number of cells of G₀/G₁ and S phases decreased; cells of apoptosis increased.The apoptotic rate of HepG2 cells of combined Ad-Rb94 with radiotherapy group was 27.1%,which was significantly higher than of Ad-Rb94 group(18.2%) and radiotherapy group(11.3%).ConclusionsFirst We got high titer adenovirus particles containing Rb94 gene by amplifying recombinant adenovirus express vector of Rb94 gene,and results showed that expected Rb94 gene was confirmed by PCR.It indicated that amplification was successful. Second Recominant adenovirus mediated retinoblastoma 94 gene can inhibit the growth of human hepatoma cells and human cervix cancer cells.Compared with Ad-lacZ control group,the inhibition of Ad-Rb94 arised from Rb94 gene,rather than adenovirus.Third There was a time-dependent inhibition of cell proliferation in Ad-Rb94,radioterapy and combined treatment groups on HepG2 and Hela.Ad-Rb94 gene therapy combined with radiotherapy showed synergism of the inhibition. Ad-Rb94,radiotherapy and combined administration of Ad-Rb94 and radiotherapy arrested HepG2 cells in the G₂/M phase,and increased levels of apoptosis.Ad-Rb94 gene therapy combined with radiotherapy showed synergism of the inhibition.And synergism of combined group could provide a new treatment for clinical tumor therapy.