Experimental Therapeutic And Relative Clinical Studies Of Gene Transfer Of Endostatin Combined With Ionizing Radiation On Lung Adenocarcinoma

The growth and metastasis of malignant tumor are depended on the neovascularization, which is controlled by both angiogenic and anti-angiogenic factors. For example, endostatin is an anti-angiogenic factor, which was isolated from culture medium of mouse hemangio-endothelioma by Michael S. O'Reilly and his colleagues in 1997. Initial studies demonstrated that endostatin potently inhibited proliferation of vascular endothelial cells and subsequent tumor growth and metastasis. However, anti-angiogenic factors can only inhibit tumor growth, but have not been proven effective to fight cancer on their own. Therefore, they may serve as adjunct therapeutic reagents and cannot replace the conventional treatment for tumor. The investigation the therapeutic effect of antiangiogenic inhibitors combined with conventional radiation therapy on malignant tumor has very important idea on anti-cancer treatment. So far, the effects of endostatin combining with ionizing radiation (IR) on malignant tumor has only been reported in a few papers published from abroad, and has not yet been examined in our country.Therefore, this study was initiated to observe these effects of endostatin alone on the vascular endothelial cell proliferation and implanted lung adenocarcinoma using gene engineering techniques. Further studies were taken to observe the effects of treatment on lung adenocarcinoma by endostatin conbining with ionizing radiation (IR). Finally, this study was focused on investigating the effect of IR on the level of serum angiogenic factor in patients with lung adenocarcinoma. These results would provide further insights into the issue whether combined retrovirus-mediated endostatin gene therapy with IR could improve the response to a human pulmonary adenocarcinoma. The final goal of this study is to supply new strategics and convincing evidence for treatment of malignant tumor. Part I Effects of Retrovirus-mediated Gene Transfer and Its Expressed Product on the Proliferation of VascularEndothelial cellPurpose: To establish the system of retrovirus-mediated enodstatin gene transfer and to investigate the effects of its expressed product on the proliferation of vascular endothelial cellMethods and Materials: Retroviral-encoding Human endostatin gene vector LEndoSN was constracted by standard molecular cloning techniques. Combination of the liposome mediation with "Ping-Pang transfer" was used to achieve high concentration of endostatin-engineeredvirus, which can then infect the lung adenocarcinoma A549 cells at high titers. PCR was used to confirm the transfer of endostatin gene and expression of transgenic messages. ELISA was performed to detect the expression of endostatin protein. Cell counting and 3H-TdR incorporation methods were applied to detect the inhibition of endothelial cell proliferation in vitro. Flow cytometry (FCM) studies and DNA ladder analysis were performed to observe the apoptosis of endothelial cells as the effects of endostatin.Results: PCR confirmed that endostatin gene was inserted into the human lung adenocarcinoma A549 genomic DNA. Endostatin gene can express mRNAs was confirmed by RT-PCR and the protein in Endostatin was examined by ELISA. Cell counting and 3H-TaR incorporation methods showed the suppressive effects of endostatin on the endothelial cell proliferation. The apoptosis of the endothelial cells induced by endostatin were confirmed by the FCM and DNA ladder analysis.Conclusions: Retroviruses can highly mediate the transfer of endostatin gene into the adenocarcinoma cells. The gene-encoded protein significantly inhibits vascular endothelial cell proliferation and induces the cell apoptosis.