The Study Of Inhibitory And Chemosensitivity Of Adenovirus-mediated PTEN For Gastric Cancer And Its Mechanisms

Object: To object the effect of Ad-PTEN gene in SGC-7901tumor cells. To study the inhibition and potential molecular mechanisms of adenovirus-mediated expression of Ad-PTEN gene in SGC-7901 gastric cancer cells .Methods: The PTEN gene was transfected into human gastric cancer cell SGC-7901 with a replication-incompetent adenovirus vector. Using RT-PCR detected the mRNA expression of PTEN. In vitro divided into 5 groups: (1)PBS group, (2) Ad-GFP group, (3)Ad-PTEN group (experimental group), (4) DDP group (positive control), (5)Ad-PTEN DDP group (combination group). Using light microscope and fluorescence microscope observe the appearances of SGC-7901 cells infected by Ad-PTEN . Using MTT essay and flow cytometry test the cell growth and the apoptotic effect. Using flow cytometry (FCM) detect the synergies of Ad-PTEN on SGC-7901 gastric cancer cells. Using RT-PCR analysis apoptosis-related gene expression such as bax, bcl-2, p53, Survivin. In vivo groups are similar with in vitro ,using cancer cell line human SGC-7901 gastric cancer plant in nude mice to establish gastric cancer models, measure tumor volume, calculate tumor weight inhibition rate, the tumor Cell morphology HE staining, using immunohistochemical method detect the apoptosis-related proteins including bcl-2,bax,Survivin,Fas,Caspase-3,and tumor angiogenesis-related factors CD34, Cox-2, VEGF .Results :Ad-PTEN gene can prolifera in QBI-293A cells, the virus titer was (1-2)×10~9 pfu / ml. Infected with Ad-PTEN gene in gastric cancer cells SGC-7901, RT-PCR results showed that PTEN gene can transcript in gastric cancer cells SGC-7901.In vitro Ad-PTEN can inhibit human gastric cancer SGC-7901 cell growth and induce apoptosis.In vivo can also inhibit the growth of gastric cancer plant in nude mice, comparing with PBS group, Ad-GFP group was significantly inhibited (P <0.05).Ad-PTEN significantly increased bax, Caspase3 and reduced bcl-2, survivin and other apoptosis-related protein; significantly reduced tumor angiogenesis factors Cox-2, CD34, VEGF expression.Comparing with PBS group,Ad-GFP group had significant differences, P<0.05, having statistical significance.Ad-PTEN + DDP combination group in vivo inhibit growth of SGC-7901 gastric cancer cells and tumor growth in nude mice and induce apoptosis, respectively comparing with Ad-PTEN group and the DDP, P <0.05, statistically significant, which indicating PTEN has the effect of chemotherapy sensitivity.Conclusion:1.We successfully constructe recombinant adenovirus-mediated PTEN gene (Ad-PTEN); after many rounds of amplification and purification, we can get high purity recombinant adenovirus, which can prolifer in QBI-293A cells, and transcrip in gastric cancer cells SGC-7901.2. In vitro, Ad-PTEN can inhibit the growth of SGC-7901 gastric cancer cell and induce apoptosis, in vivo can inhibit the growth of gastric cancer plant in nude mice .3. Ad-PTEN can enhance chemosensitivity to DDP effect in suppressing cell growth and inducing cell apoptosis inSGC-7901 gastric cancer cells and gastric cancer transplanted tumor growth of nude mouse.4. The regulation of gastric cancer of Ad-PTEN may be related to cell cycle arrest and apoptosis related factors expression ,such as Fas, Bax and Caspase-3 and decreased expression of Bcl-2 , Cox -2and survivin, and reduced tumor angiogenesis-related factors expression of VEGF and CD34.Increased DDP chemotherapy may be related to increase the sensitivity factors of Fas, caspase-3 expression and the bax/bcl-2 ratio and reduced the expression of survivin and VEGF. The study of inhibitory effect of Ad-PTEN and the molecular mechanisms of chemotherapy sensitivity provide the experimental basis.