Investigation For The Expression Of Angiostatin CDNA In Hepatocellular Cancer Cell Line SMMC-7721 And Its Effects On Primary Carcinoma Of Nude Mouse

Angiogenesis, the formation of new capillary blood vessels from pre-existing ones, is a fundamental process required for a variety of physiological and pathological processes. Under physiological conditions, such as wound healing, menstrual cycle in ovaries and endomentrium, angiogenesis is in active situation. Angiognesis occurs over a limited time period and proliferating endothelial cells are rapidly returning to their normal state of quiescence. However, many disease states can arise and are maintained by the persistence of angiogenesis and the loss of normal regulatory mechanisms. These include chronic inflammation, lepra alphos, tumor proliferation and metastasis.The triggering and regulation of angiogenesis is the result of various factors effect, among which angiogenesis stimulators and inhibitors play a direct role on endothelial cells and matrix cells. Angiogenesis is dependent on the balance between angiogenesis stimulators and inhibitors. A tumor is unable to grow above 2mm3 and then towards into the dormancy period without development of a new blood vessel. When angiogenesis stimulators is stronger than angiogenesis inhibitors, the switch of angiogenesis is on and increases angiogenesis, which result in the proliferation and metastasis of tumor. In recent years, a new strategy based on tumor dependence of blood supply, antiangiogenesis for tumor therapy, has been a hot point, which possesses characteristic advantages than normal therapies such as chemotherapy and radiotherapy. With the development of genetic technology, the combination of antiangiogenesis with gene therapy may be is an attractive choice in clinic.Primary hepatocellular carcinoma is one of the most common neoplasmsin China. Most previous experimental researches were against tumor cells which had characters of unable transmissibility, high heterotype and high mutation rate, so satisfied curative effects could not be attained. Hepatocellular carcinoma has double blood supply system, which is one of the tumors with large blood vessels. The treatment of antiangiogenesis will become a new method to the primary hepatocellular carcinoma's therapy.Angiostatin is one of the most powerful angiogenesis inhibitors which specifically affect endothelial cells, blocking their proliferation and migration, sequentially retarding tumor growth and metastasis and keeping the tumor in the dormancy period. It is a fairly useful target gene in antiangiogenesis gene therapy. In order to investigate the function of angiostatin on hepatocellular carcinoma cells and its possible usage for treatment of hepatocellular carcinoma, we contracted angiostatin cDNA into an eukaryotic expression plasmid pcDNA3.1-mAST and transfected it into SMMC-7721 with lipofectin transfection. After selection by G418, the stable expression on SMMC-7721 of angiostatin cDNA was detected with Western blot and the changes of tumorigenic capacity was also be studied. To observe the changes of microvessel density (MVD) and study the mechanism of retarding tumor of angiostatin with immunohistochemical staining.The results are as follows:1. Enzymatic identification and sequencing results stated that the angiostatin gene clones had been transfected into eukaryotic expression vector of pcDNA3.1(+), i.e. obtained the restructing plasmid.2. pcDNA3.1-mAST plasmid had been transfected into human hepatocellular cancer line SMMC-7721 with lipofectin transfection. The transfected cell clones were selected by G418 after two weeks and the RT-PCR result showed that pcDNA3.1-mAST had been transfected into SMMC-7721 successfully.3. Western-blot detected that the stable expression on SMMC-7721 of angiostatin gene was obtained.4. Cultivated three groups of human hepatocellular carcinoma (SMMC-7721 ,SMMC-7721 /pcDNA3.1 ,SMMC-7721 /pcDNA3.1 -mAST) without G418, the growth curves showed that there were no significant difference between the growth speed of transfected cells and non-transfected cells which stated that angiostatin had no inhibitory effect on the growth of SMMC-7721.