| Lung cancer is a gene disease. With the progress of molecular biology and molecular oncology, gene therapy for lung cancer becomes a prospective therapy in the field of medical studies. Because of the complexity of tumors and heterogeneity of human,gene therapy is often needed to combined with other therapies to obtain good effect. Wide-type p53 gene transfection combined with photodynamic therapy was first selected to treat lung cancer in our study.,which focused on effects of each therapy and interaction of these two therapies in A549 growth,apoptosis and tumor vascularization. This study was made up of two parts:in vitro and in vivo study.It was divided into six groups: A549 cells group(A) ,A549/pCDNA3.1-neo cells group(B),A549/ pCDNA3.1-wtp53 cells group(C),A549 cells + PDT group(D),A549/pCDNA3.1-neo cells + PDT group(E) and A549/ pCDNA3.1-wtp53 cells + PDT group(F).Part â… in vitro studyObjective:To study what roles wtp53 gene transfection combined with PDT played in cells cycle and apoptosis of human lung adenocarcinoma cell strain A549 and vascularization of tumors. Methods: Plasmids pCDNA3.1-wtp53 and pCDNA3.1-neo were transfected into our study object –human lung adenocarcinoma cell strain called A549 cells by cationic liposomes Lipofectamin 2000.After transfection,the sqRT-PCR and Western blot were used to analyse the integration and expression of exogenous genes. The cells entered the experiment after 24h of PDT(HPD concentration 10ug/ml,laser dosage 10J/cm2).The six group cells above-mentioned had been cultured for 7 days to draw the cells growth curve. Cell cycle and apoptosis rate were estimated by flow cytometry,furthermore, agarose gel electrophoresis and TUNEL were selected to observe the fragments of DNA and AI.SqRT-PCR and ELISA were used to determinate the expression of VEGF mRNA and VEGF protein. Results: The cells growth curve showed that group A and group B cells were all grew in log manner, group C,group D,group E cells had much longer latent period. The growth of group F cells were inhibited obviously , the cells quantity only increased 4.5×104 after 7 days.The outcomes of the cells cycle showed that C,D,E and F groups took on G0/G1 cycle arrest ,the proportion of G0/G1 cells of group F were the highest. Wtp53 gene transfection and PDT could cause apoptosis in various degrees. The apoptosis rate of group F achieved highest. Agarose gel electrophoresis of DNA revealed the appearance of DNA ladder,a biochemical hallmark of apoptosis. The DNA ladder of group F was the clearest. Apoptosis indexes of cells determined by TUNEL revealed that A and B groups had no apoptosis,the other four groups occurrented apoptosis in different degrees. The AI of group F were the highest. The expression of VEGF was much lower after wtp53 gene transfection by sqRT-PCR and ELISA(vs group A P<0.01), it was not increased significantly when combined with PDT(vs groups not combined with PDT P>0.05).Conclusions: Wtp53 gene transfection and PDT play a synergistic role in G0/G1 cycle arrest and apoptosis of A549 cells. After wtp53 gene transfection,A549 cells were more sensitive to PDT. Futhermore, wtp53 gene transfection can down-regulate the VEGF expression to inhibit vascularization of tumors,it can also inhibit the increasing of VEGF after PDT. |
