Expression Of Human Angiostatin(K1-3) Gene In Bombyx Mori Cells And Larvae Of Sikworms And Its Activity Assays In Vitro & In Vivo

Human angiostatin, a 38 kD protein which is composed of 1-4 kringles of plasminogen, was separated and purified from serum and urine of lewis lung carcinoma by O'Relly, et al. Angiostatin can inhibit the angiogenesis of tumor, the tumor growth and metastasis through preventing tumor's blood and nutrition from supplying. Now the inhibition of the angiogenesis has become a new target to cure the tumor. Some researches showed that inhibitory effect of human angiostatin (Kl-3) on angiogenesis was stronger than that of human angiostatin (Kl-4), and kringle4 of plasminogen had no direct action on inhibition of angiogenesis. In the present thesis, it is mainly to express the human angiostatin (Kl-3) in Bombyx mori cells (BmN) and larvae of silkworms with baculovims expression vector system (BEVS), to investigate the effect of human angiostatin (kl-3) on cell apoptosis in endothelial cells, and on vascular growth in chick embryo, and to elucidate cooperation between angiostatin and endostatin.Angiostatin (Kl-3 ) gene was inserted into Bombyx mori baculovims transfer vector pBacPAKS and cotransfected with lineared DNA of Bm-BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus BacPAK-angiostatin was expressed, as identified by PCR and DNA dot blotting. The BmN cells and fifth instars were infected by the recombinant virus BacPAK-angiostatin. The bioactivity of the protein product was determined by human umbilical vein endothelial cells (ECV304) growth assay in vitro and inhibition of vascular growth assay in CAM in vivo. Angiostatin showed significantly inhibitory effect on endothelial cells. The expression activity reached the highest point at the 72nd hour in BmN cell (22 u/2x!06cells) and at 144th hourin larvae (159 u/ml), respectively. ELISA assay showed that expression product had angiostatin's immunoreactivity. Western blotting assay also showed that product expressed in cultured cells was a 36 kD band, while product expressed in larvae of silkworms was two proteins, and the molecular weights were 37 kD and 42 kD, respectively. These three proteins represented that angiostatin was glycosylated hi different positions.In addition, the mechanism and bioactivity of angiostatin in vivo and synergy between angiostatin and endostatin had also been studied in this thesis. Endothelial cells were continuously cultured for 36 h after treating with angiostatin, endostatin and both of them, respectively. Through transmission electron-microscope, we found all of them could induce apoptosis in endothelial cells. The effect of synergy between angiostatin and endostatin was stronger than either of angiostain or endostatin. CAM assay also showed synergy between angiostatin and endostatin could inhibit the angiogenesis and its effect was stronger than either of angiostain or endostatin.In conclusion, using BEYS to express angiostatin, we could obtain lots of active angiostatin protein, could also cut down production cost grately and lay a foundation for clinical application.