The Research On Combination Of Adenovirus-mediated Rb94 Gene Wirh γ-radiation In Inhibiting The Growth Of Human Malignant Tumor Cells

The retinoblastoma gene (Rb) is the earliest isolated humansuppressor gene,Rb94 gene is part of Rb gene, RB94 protein lacks theNH₂-terminal 112 amino acid residues of RB protein. Studies indicatethat the function of the Rb94 gene is better than the function of thefulllength Rb gene in the inhibiting tumor effect. The purpose of thisstudy is to construct an Adenovirus-mediated expression vector of Rb94gene (Ad-Rb94) using the pENTR^TM1A vector and pAd/CMV/V5-DEST^TMexpression vector, and to check the inhibiting effect for tumor cellsgrowth.Gene therapy is the Hotpoint of the tumor biological treatment atpresent, and also the trend of the tumor therapy in the future. However,there has not been breakthrough on gene therapy for a while, and manyproblems are still unresolved. Thus, it alone is not an applicablemeans of cancer treatment. Radiotherapy has been the major method ofthe cancer treatment. The effect of inhibiting tumor is enhancedeach other between gene therapy and radiotherapy, so we research thecombining function of Ad-Rb94 andγ-radiation to provide theexperimental data for clinical therapy of the cancer gene therapycombined with radiotherapy.We extracted the total RNA from the human embryo, reversetranscription into cDNA by specific primer in this experiment, andused it as the template DNA to amply the target gene fragment by DNAhigh fidelity polymerase PCR technology. The pENTR^TM1A vector wasdigested with EcoRV,Xmnâ…¡The amplified Rb94 gene fragment was ligatedinto the digested pENTR^TM1A vector,and then transformed into E.coli,and then isolated as plasmid DNA. The extracted plasmid DNA wasrecombined with pAd/CMV/V₅-DEST^TM expression vector by LR recombinant technology to get the adenovirus expression vector containing the Rb94gene. The recombinant adenovirus vector was restricted with Pacâ… ,thentransfected 293A cells by liposomes to amplify the recombinantadenovirus express vector. Finally we got the recombinant adenovirusparticles. Esophageal cancer cells and breast cancer cells, which weretransfected the tumor cells using recombinant adenovirus particlescontaining Rb94 gene, were choiced to research the tumor cell's growth,cells cycle and cells apoptosis situation, for checking the anti-tumorfunction of the Rb94 gene.After transfected 24 hours,the tumor cellswere irradiated with 2Gyγ-ray.We can observe the inhibiting tumoreffect of the combinating function of Rb94 gene withγ-radiation.Results showed that expected Rb94 gene was comfirmed by agrose gel.The direction of the inserted target gene fragment in the PENTR^TM1Avector was right by restricting with MSâ…¡and Nheâ… .Construction ofthe recombinant adenovirus express vector was correct by PCR. Thegrowth of esophageal cancer cells transfected with recombinant Ad-Rb94express vector containing Rb94 gene were effectively inhibited, thegrowth of breast cancer cells was inhibited slightly usingrecombinant Ad-Rb94 express vector.And the combining effect of Rb94gene andγ-radiation was better than any one of them in inhibitingtumor.The experiment results were consistent with our expectation.Anti-tumor effect was observed in some types of tumor cells which weretransfected with the recombinant adenovirus particles. So thisrecombinant adenovirus particles can be used for the future researchof the function of the Rb94 gene.There was an enhanced inhibitingtumor effect on combined Ad-Rb94 withγ-radiation. The mechanism maybe: radiation improves the efficiency of gene transfection and (or)increases the expression of protein; on the other hand, thetransfection of the Rb94 gene improve the sensitivity of the radiation. Therefore, combinating gene therapy with radiotherapy could providea new treatment method for clinical tumor therapy.