Study On The Mechanism Of Angiostatin In Antiangiogenesis Of Tumor

Primary hepatocellular carcinoma is one of the most common neoplasms in 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 obtained. 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. However, the mechanism of antiangiogenic action of angiostatin is still unclear. In the present study, we constructed the recombinant human angiostatin (hANG) expression vector, observed the stable expression of recombinant hANG in hepatocellular carcinoma cells, examined itsinhibitory effects on tumor growth, and investigated the mechanism of its pro-apoptotic role of endothelial cells by in vitro and in vivo methods. The results of present study will provide experimental basis for angiostatin gene therapy for hepatocellular carcinoma. The experiment is divided into three parts:Part I: Construction of recombinant hANG expression vector and its stable expression in hepatocellular carcinoma cellsIn order to investigate the antiangiogenic mechanisms of angiostatin in vitro and in vivo, we first constructed the recombinant hANG expression vector. PfuTurbo DNA polymerase was used to amplify a DNA fragment (1100 bp in length) encoding Kringle 1-4 of human angiostatin from human embryonic tissue. This fragment was then cloned into pCR-Blunt II-TOPO vector. Colony PCR, restriction enzyme digestion and sequencing were used to verify the obtained hANG DNA fragment. The result showed that the obtained hANG fragment was identical to the sequence of reported human angiostatin. The hANG DNA fragment was further subcloned into the Kpn I and Xho I sites of pcDNA3.1 (+) expression vector. The positive clones were obtained after confirmed by restriction enzyme digestion, suggesting that the recombinant hANG expression vector was successfully constructed. To further detect if the constructed hANG DNA could be expressed in hepatocellular carcinoma cells, we transfected recombinant pcDNA3.1-hANG plasmid DNA into SMMC-7221 cells by lipofectin transfection method. RT-PCR result indicated that palsmid DNA was integrated into the genome of SMMC-7221 cells. Western blot was then used to detect its expressionproduct and a protein band at 38 KD was found, indicating that CMMC-7221 cells successfully expressed hANG. In addition, no difference in the growth speed was observed between untransfected SMMC-7221 cells, SMMC-7221 cells transfected with pcDNA3.1(+) vector and SMMC-7211 cells transfected with recombinant pcDNA3.1-hANG. This indicated that angiostatin had no inhibition on the growth of SMMC-7221 cells but it could be secreted from SMMC-7221 cells transfected with pcDNA3.1-hANG. From this part of experiment, we successfully constructed recombinant pcDN A3.1 -hANG expression vector and obtained SMMC-7221 cells that could stable express hANG, thus providing the basis for further study.Part II: Inhibitory effect of hANG on subcutaneous and liver transplanted tumors in nude miceIn order to verify if the recombinant hANG could play its inhibitory role on tumor growth, we designed the following in vivo experiments. Firstly, SMMC-7221 cells that could stable express hANG were injected subcutaneously and into liver of nude mice and the volume of tumor at different time points were measured. Mice injected with pcDNA3.1-hANG-transfected SMMC-7221 cells were experimental group, mice injected with normal SMMC-7221 cells or SMMC-7221 cells transfected with pcDNA3.1(+) vector were regared as blank control or vector control. T...