| Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide, and has been ranked the 2nd cancer killer in China. Surgery has been recognized as the most effective treatment option for HCC. Nonetheless, only a very small fraction of patients with HCC qualifies for surgical resection. Far fewer patients undergo orthotopic liver transplantation (OLT), although OLT can produce excellent outcomes in early HCC. The vast majority of liver caner patients remain untreated, since other therapies are mostly palliative, and novel therapies are urgently needed. Generally, tumor growth is critically dependent on blood supply. Folkman showed that suppression of tumor angiogenesis leads to tumor starvation and tumor regression. Therefore, the tumor vascular system has become an important target for cancer therapy. In contrast to malignant cells that are able to become resistant to conventional chemotherapy or radiotherapy, a body of data indicates that endothelial cells do not develop resistance to antiangiogenic agents. An increasing number of antiangiogenic agents have been discovered. Among these, endostatin was identified by O'Reilly et al.Endostatin is a Mr 22000 protein that specifically and strongly inhibits tumor angioginesis. It was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line and represents a COOH-terminal fragment of collagen Xâ…§. The mechanism of action of endostatin is unclear. It inhibits endothelial cell proliferation in vitro. Different investigators have proposed interference with vascularendothelial cell gowth factor and fibroblast growth factor-2 (basic fibroblast growth factor) pathways, induction of endothelial cell apoptosis, and inhibition of matrix metallopreteases. Inhibition of endothelial cell migration by modulating c-myc levels has also be proposed. Animal studies demonstrated that recombinant endostatin strongly inhibits the growth of a variety of murine and xenotransplanted human tumors including HCC.The production of bioactive recombinant endostatin has proven challenging because of its unstably physical property. In addition, antiangiogenic therapy with endostatin in cancer requires prolonged administration and high doses of the recombinant protein. It will result in heavy economic burden and inconvenience to recipients by repeated administration. Therefore, transfer of foreign endostatin gene into host cells represents an alternative method to treat tumor by generating high efficient endostatin in areas around tumor. A few groups have demonstrated that antiangiogenic gene therapy with viral vectors is a potentially useful approach for inhibiting tumor growth in mouse model. Adeno-associated virus (AAV) has several characteristics which make it extremely attractive as a gene transfer vector: (1) no known pathogenicity; (2) high efficiency and the ability to remain latent; (3) a minimal number of antigens ensuring minimal immunogenicity; (4) the ability to transduce post-mitotic cells; (5) possible advantages of site-specific integration; and (6) a broad host and cell range. It was reported that recombinant AAV vectors accumulated predominantly in liver cells, when directly injected intravenously in mice, suggesting that AAV may possess in vivo organ-tropism for liver.In the present study, we constructed the recombinant adeno-associated virus vector containing human endostatin gene (rAAV2-hEN), and investigated the inhibitory effects of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma in vitro and in vivo. The experiment was carried out at three steps.Part one: cloning of human endostatin gene and construction of recombinant Adeno-associated virusThe primers containing specific enzyme-cutting sites were designed according to human endostatin (hEN) gene sequence of the Genebank. Total RNA was extracted from human liver tissue, and the hEN cDNA was amplified by reverse transcriptional polymerase chain reaction (RT-PCR) and cloned into the pUC19 vector. The constructed plasmids pUC19-hEN was identi...
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