Transfecting Human Canstatin Gene Secretory Eukaryotic Expression Vector Into CHO-K1 Cells Stably And Identifing Biological Activity Of The Supernatant

[Objectives] Stably transfecting the Secretory eukaryotic expression vector of human canstatin gene into CHO-K1 cells to get the cell strain that can express the canstatin proteins stably. Centrifuging and collecting the supernatant to process the human umbilical vein endothelial cells(HUVEC-12) in order to study more biologic action of canstatin.[Methods] Stably transfect the Secretory eukaryotic expression vector of human canstatin gene into CHO-K1 cells to get the cell strain that can express the canstatin proteins stably. Collect the cells and Detect the expression of canstatin gene in CHO-K1/pSecTag2B/canstatin cells by RT-PCR assay. Collect the supernatant and Detect the expression of canstatin protein by Western-blotting assay. Identify the anti-angiogenesis biological activity of the supernatant protein by chick embryochorioallantoic membrane (CAM) assay. Then study more biologic actions of canstatin to the HUVEC-12 by Curve of growth assay and Flow cytometry.[Results] Secretory eukaryotic expression vector of recombined human canstatin gene was successfully transfected CHO-K1 cells stably by liposome method. The result of RT-PCR showed that the canstatin containing plasmid was successfully transfected into CHO-K1 cells. The result of Western-blotting showed that the cell strain can express the canstatin proteins stably. In vivo, the supernatant collected from the culture medium of CHO-K1/pSecTag2B/canstatin cells could significantly inhibit angiogenesis in CAM. In experiment group, the new microvessels grew slowly. However, in control groups, the new microvessels grew actively and the figures of them were distinct. The supernatant collected from the culture medium of CHO-K1/pSecTag2B/canstatin cells also showed that it can inhibit the growth of the...