The Study Of Mechanisms Of Fibrinolyric Protein (EFP) From Eupolyphage Sinesis Walker Inhibiting Tumor Angiogenisis

Eupolyphaga sinensis Walker, belonging to Eucorydia genera, Corydiidae famly, Blattaria order, has the effect of removing blood stasis efficacy, anti-inflammatory, promoting blood circulation and analgesic efficacy. It is used clinically, such as blood-stasis syndrome, hemostasis swelling pain.A novel of Eupolyphaga fibrinolytic protein (EFP)was separated and purified by salting-out, DEAE-52 cellulose column chromatography from fresh tradional Chinese medicine Eupolyphaga sinensis Walker in the paper. The molecular weight of EFP was determined by sodium dodecyl Sulfate polyacrylamide gel electrophoresis analysis, SDS-PAGE results indicate that the molecular weight of EFP is 79.2 KDa, The bradford method was used to detect protein content of 13.6%.Enthronlsulphuric acid method using a glucose standard was used to detect sugar content of 85.6%.The fibrin plate assay was used to test the fibrinolysin activity of EFP, fibrinolytic activity is 23U/mg.It is proved that Eupolyphaga fibrinolytic protein has the effect to inhibit the vasculogenesis of chick chorioallantoic membrane. Through in vivo experiments, to build tumor-bearing mouse model, Experimental drugs were divided into positive control group (cyclophosphamide), negative control group (saline), the high drug concentration group, the middle drug concentration group, the low concentration of the drug group.The microvessel density (MVD)of mouse S180 and H22 tumor tissue was detected by immunohistochemistry(IHC),the expression of VEGF in mouse serum was mensurated through enzyme linked immunosorbent assay(ELISA).EFP drug serum was produced by injection EFP to mice. Cells was cultured with 10% drug serum, using MTT colorimetric assay to observe the inhibition to the proliferation of cells and double antigen sandwich enzyme-linked immunosorbent assay (ELISA) to study the effect of EFP on the expression levels of VEGF and bFGF by determining the content in cell culture. Teomerase activity was detected by silver carbonate staining method.The results are that EFP group has lower MVD value than saline group,the same as VEGF value,EFP drug serum has obvious inhibition on HUVEC,A549 and MCF-7 cells proliferation and EFP drug serum can produce remarkable inhibition on the expression levels of VEGF and bFGF in HUVEC, A549 and MCF-7 cells.