The Initial Investigation Of The Mechanism Of N-terminal Domain Of Recombinant Human Pro-EMAPâ…¡

Tumor angiogenesis system has been a new target for cancer therapy。It is a an effective "hunger therapy" by destroying or inhibiting tumor angiogenesis in order to inhibit formation and development of tumor。Pro-EMAPⅡalso known as p43 protein, is thought to be a precursor of endothelial monocyte-activating polypeptideⅡ(EMAPⅡ). The recent report showed that, p43 protein showed a good inhibition of neovascularization both in vivo and in vitro, also could regulate the expression of several cytokines which trigger proinflammation in leukocytes and macrophages,so it is expected to be developed as a new drug of inhibition tumor angiogenesis.More studies have showned,p43 protein had more biological functions and regulated more cytokines expression than EMAPⅡ.The difference of biological function between p43 and EMAPⅡfrom structural,is the existence of N terminal domain,thus we believe that N-terminal domain of p43 plays an important role. But the structure and function of N terminal domain have not well understand.It is reported that N-terminal domain of p43 protein plays the role of immune enhancer, however,what its specific role to play, through what mechanism to promote the activity of p43 protein in anti-vascular increase is not reported.In order to further study of the mechanism of N terminal domain of p43 protein, we constructed both of the N terminal domain(p43N) and C terminal domain (p43C) of p43 protein prokaryotic expression vector, and to achieve expression in E. coli. The much more purity protein was achieved by SP ion exchange chromatography and Ni-agarose affinity chromatography. The purified protein was experimented on endothelial cells, at the same time,tube formation, cell migration assay and the double sandwich ELISA was used to identify their biological activity,and the differences of biological activity between p43 protein, p43N, p43C, and p43N + p43C with same molar concentrations were comparied.The results show that both of the p43N and p43C have biological activity of depressing angiogenesis and migration of endothelial cell in anti-angiogenic activity way,and both of them have raised IL8 and MCP-1 expression in the role of cytokines.In the same molar concentration condition, p43N shows weak activity of inhibiting angiogenesis but strong activity of inducing cytokine expression.The biological activity is improved when p43N was combined with p43C, Thus, we speculate that p43N and p43C has a certain synergy. These results also suggest that the activities for the endothelial cell migration and apoptosis are not separable by the different domains of p43, but its whole structure is involved for the full activity. At the same time,the activity of inhibiting angiogenesis may not be p43N's primary role, which may be primarily used as an immune enhancer, or cytokines inducing a variety of cytokines and inflammatory cytokine production, thus synergistic p43C promoting antiangiogenic activity of p43 protein increased. Differences in biological functions first reflected in the gene level, in order to study related gene expression differences of the human microvascular endothelial cells HMEC-1 by p43N and p43C protein treated, we used real-time RT-PCR technique to study the expression differences of IL8, IL1β, IRF7, IFI16, MX1, VCAM 1, STAT1 and TNFRSF9 of HMEC-1 cells which was stimulated by p43 (50μg/mL), p43N and p43C after 12h. The results showed that, mRNA of this eight gene was increased most obvious by p43N, and the effect of p43N in IL genes (IL8, IL1β), and interferons genes (IRF7, IFI16, MX1) was most significantly, so we may speculate that p43N activat related signal pathway by upregulating related immune gene expression and then play a biological function, but the specific mechanism needs further study.In summary, p43N and p43C proteins were constructed and expressed successfully, and the much more purity proteins were achieved through certain ways of purification. The activity of them was identified by tube formation,cell migration and double sandwich ELISA in vascular endothelial cell,at the same time, the activity of p43,p43N,p43C and p43N+ p43C with same molar concentration was compared real-time RT-PCR techniques was used to analyze the differences of gene expression of endothelial cells by p43N and p43C treated, which is the foundation of further studying the mechanism of p43N.