| Cancer is a disease of genetic and epigenetic disorder. Abnormalities in genes that control cell proliferation lead to the unrestrained growth and other malignant phenotypes. Initiation and development of cancer is caused by both environmental and hereditary factors and the process of transformation from a normal cell to cancer cell remains unknown. Besides surgery, radiotherapy and chemotherapy, a new strategy, gene therapy, is proposed as a new approach to control or cure cancer. However, up to now, there were more than 600 approved gene therapy clinical trials world wide, but unfortunately, most of them are not as satisfied as expected. Therefore, further serious efforts should be made on the cancer-targeting vectors and therapeutic genes.In 1971, J. Folkman proposed a concept of the role of blood vessels in tumor growth in the form of a hypothesis that tumor growth is angiogenesis dependent. The new blood vasculatures embedded in a tumor bring nutrition to the tumor and provide as well a gateway for tumor cells to metastasize to distant sites. Angiogenesis has been currently concerned as an essential process of the development and progression of cancer. Angiopoietin-1(Ang1) is an angiogenic factor that signals through the endothelial cell Tie2 receptor tyrosine kinase. Unlike VEGF, Ang1 does not directly promote the proliferation of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates the involvement of Angiopoietin-1 in endothelial developmental processes. Angiopoietin-2, a member of Ang family, which can also bind to the same receptor Tie2, was shown to be a naturally occurring antagonist for Ang1. It is reported that Tie2 is specifically expressed in endothelial cells, but by immunohistochemistry, we found that cancercells themselves from many kind of malignancies such as breast cancer, lung cancer etc., also have high expression of Tie2 receptor.Now we attempt to develop a gene delivery system targeting Tie2 of the endothelial cell of the tumor blood vessels, which could be potentially used to inhibit the angiogenesis and cancer growth.Part I GA1 gene delivery system transducing (-gal gene mediated by Tie2 receptor in vitro and in vivoSince the binding domain of angiopoietin to its receptor Tie2 remains unknown, first of all, we have to narrow down the sequence of the binding area based on the available data. By analyzing the sequence homology between Ang1 and Ang2, and the hydrophobility data, we picked up a candidate oligopeptide sequence subjected to further biological studies.With heterobifunctional crosslinking agent SPDP, we linked GA1 and HA20 to poly-L-lysine. In normal physiological pH, polylysine has positive ion on its surface, while DNA carries negative charges, thereby forming a electrostatic complex. With immunohistochemistry screening, we found lung adenocarcinoma cell line SPC-A1 expresses Tie2 receptor at a high level. With this cell line, we transduce GA1-PL/pCMV-(-gal/HA20-PL in vitro. After 2 days, the activity of (-gal could be detected in cells transduced by GA1 gene delivery system but not in cells treated with normal saline and naked DNA of plasmid CMV-(-gal.After animals received injection of GA1-PL/CMV-(-gal/HA20-PL around the subcutaneously transplanted tumor, the expression of reporter gene in tumor and other tissues was examined. No (-gal activity was observed in heart, liver, lung, kidney, stomach and small intestine except spleen. The expression of (-gal at various times was also examined, at the first day after injection, blue staining could be found and it reached its peak at the second day. 1 week after injection, the activity still could be found.We also injected the GA1 gene delivery complexes subcutaneously to various tumor-bearing nude mice, including SPC-A1, SMMC-7721, A375, SGC-7901, SK-OV-3, cervical xenograft and two heptocarcinoma xenograft. Results showed thatGA1 gene delivery system could transduce reporter gene to some of the endothe...
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