Study Of Human Macrophage Metalloelastase Inhibiting Angiogenesis In Hepatocellular Carcinoma

The growth, invasion and metastasis of malignant solid tumor are depending on angiogenesis. Multiple experiments show that inhibiting angiogenesis can prevent growth and metastasis of malignant tumor. So the inhibition of angiogenesis is considered to be one of the most promising strategies that might lead to the development of novel antineoplastic therapies. Recently as the mechanism of angiogenesis regulation was elucidated, more and more anti-angiogenesis drug is found and applied. In these drugs, angiostain is now considered to be the most efficient angiogenesis inhibitor, which was isolated from serum of tumor-bearing mice by O'reilly in 1994. Research shows that angiostatin can inhibit neovasculation by preventing endothelia cells' proliferation and migration. Angiostatin can significantly inhibit growth of primary tumor and prevent metastasis. Moreover angiostatin can make primary tumor shrink to small dormant microtumor because tumor cell is deficient of oxygen and nutrients. The effects of angiostatin have exceeded other anti-angiogenesis drugs, for example angiogenesis inhibitor TNP-470. Recently gene therapy technology has applied to anti-angiogenesis in antineoplastic therapies, which avoidi-6-mmany shortcomings in purely drug therapy, for example, repeatedly administrated, high costs etc. Gene therapy now is considered to be a primary means in tumor anti-angiogenesis therapy. However, research shows that angiostatin is an internal proteolytic fragment of plasminogen containing Kringle structure, which produced by proteolysis and reduction. No cells in body can direct synthesize and discrete angiostatin. So the cell transected with angiostatin gene unnecessarily can produce functional angiostatin, moreover it will decrease the anti-angiogenesis effect of angiostatin. More research about mechanism of angiostatin's production shows that macrophage which infiltrating in tumor can discrete metalloelastase(MMP-12). This enzyme can direct produce functional angiostatin by cleaving plasminogen. So if we can transfect macrophage metalloelastase gene into related cells, we should get indirect anti-angiogenesis effect by producing functional angiostatin. Now this hypothesis has been proved by experiment.Primary hepatocellular carcinoma is one of the most common neoplasms in China. Hepatocellular carcinoma has double blood supply system, which is one of the tumors with large blood vessels. To explore an new target gene in anti-angiogenesis gene therapy and investigate effect and mechanism of macrophage metalloelastase in anti-angiogenesis gene therapy, in the experiment we isolated total RNA from macrophage which was purified from ascites. Then we got human macrophage metalloelastase(HME) gene clone by using RT-PCR. Product of PCR was inserted into the eukaryotic expression vector pcDNA3.1(-)A after digestion and ligation by using restriction endonucleases and ligase, so we constructed an newrecombinant eukaryotic expression vector pcDNA3.1(-)A/HME. Then we transfected the recombinant vector into SMMC-7721 cells by using liposome. We got the positive cell clone that can express recombinant protein by using G418. The positive cell clone was proved by immunohistochemical staining. To observe the changes of microvessel density (MVD) and study the mechanism of retarding tumor of HME with immunohistochemical staining. The results are as follows:1. Enzymatic identification and sequencing results stated that the HME gene clones had been transfected into eukaryotic expression vector of pcDNA3.1(+),and we obtained the restructing plasmid.2. pcDNA3.1(-)A/HME plasmid had been transfected into human hepatocellular cancer line SMMC-7721 with lipofectin transfection. The transfected cell clones were selected by G418 after four weeks and the RT-PCR result and immunohistochemical staining showed that pcDNA3.1(-)A/HME had been transfected into SMMC-7721 successfully.3. Cultivated three groups of human hepatocellular carcinoma (SMMC-7721 ,SMMC-7721...