The Experiment Study Of Gene Transfer Of Endostatin Combined With Cisplatin On Lung Adenocarcinoma A549

The growth,invasion, and metastasis of solid tumor are depended on the neovascularization, which is controlled by both angiogenic and antiangiogenic factors. Endostatin,which was firstly isolated from culture medium of mouse hemangio-endothelioma by Michael S. O'Reilly and his colleagues in 1997, is one of the strongest anti-angiogenetic factors. Studies demonstrated that endostatin can inhibit new vessels forming as a result of preventing the proliferation and migration of endothelial cells. Tumors will remain a state of dormancy without enough supply of blood from vessels. Although antiangiogenetic factors can hold back tumor growth and metastasis, it couldn't kill all tumor cells. Therefore,the investigation of the therapeutic effect of antiangiogenesis combining with the conventional treatment on malignant tumor has been becoming the focus of malignant tumor study. So far, the effects of endostatin combining with chemotherapy on lung Adenocarcinoma has been reported in a few papers. Thus, we devise this study to observe the effects of the expressed product of endostatin gene on the proliferation of vascular endothelial cell and implanted lung adenocarcinoma using gene techniques. Further study was taken to evaluate the efficacy of endostatin combining with cisplatin for the treatment of lung adenocarcinoma. These results would supply new strategics and convincing evidence for the treatment of lung cancer.Part I Retrovirus-mediated Enodstatin Gene Transfer and Effects of Its Expressed Product on the Proliferation of Vascular Endothelial cellPurpose: To establish a method of retrovirus-mediated H-enodstatin gene transfer and to estimate the effects of its expressed product on the proliferation of vascularendothelial cell.Methods and Materials: H-endostatin (human endostatin) gene wastransferred into lung adenocarcinoma A549 cell by the vector of retrovirus to obtain a A549/Endo cell . PCR was used to confirm the transfer and expression of endostatin gene. Its expressed product of A549/Endo cells, H-endostatin , was detected by ELISA.. MTT was applied to estimate the suppressive effects of H-endostain on the proliferation of endothelial cell (EA.hy926) in vitro.Flow cytometry (FCM) studies and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay were performed to observe whether the endostatin-treated cells underwent apoptosis after roughly 24 hours. The expression of Bax ,Bcl-2 and Bel-XL were detected in the endostatin-treated cells by Immunohistochemic staining and Insitu hybridization (ISH) respectively. At the same time, the activity of the intracellular protease caspase 3 was also examined .Results: PCR proved that the h-endostatin gene was successfully inserted intoA549 genomic DNA. Endostatin gene can express mRNA that was confirmed by RT-PCR. ELISA showed that the H-endostain in the supernatant of culture medium of A549/Endo cells (1.10"/ml) was about 348?20pg/ml at 48 hour after the cells was cultivated. MTT confirmed that the H-endostatin in the concentrated supernatant of A549/Endo cells could effectively inhibit the proliferation of endothelial cell EA.hy926 . Moreover, the FCM and TUNEL confirmed that the H-endostain could induce the apoptosis of EA.hy926 cell .After 24 h the expression of the Bcl-2 and Bel-XL ,anti-apoptotic proteins, in the endostatin-treated EA.hy926 cells was reduced and the caspase 3 activity was elevated at the same time . But the Bax-mRNA's expression showed no difference between the control and treated samples.Conclusions: The endostatin gene can be highly transfered into A549 cells byretroviruses . The endostatin gene's expression product can significantly inhibit the vascular endothelial cell proliferation and induce the cell apoptosis. Reducing the expression of the vascular endothelial cell's Bcl-2 and Bel-XL may be one of the mechanism of cell apoptosisvinduced by endostatin.