Study On Vasostatin Protein Preparation And Anti-tumor Mechanism

Anti-angiogenesis is an important part of comprehensive anti-tumor treatment.Vasostatin was found by Sandra E.pike in 1998,it is a N-terminal fragment of calreticulin protein comprised with 180 amino acids.Vasostatin exists extensively in liver and pancreas,it has the inhibitory effect on endothelial cell proliferation,angiogenesis and tumor growth,but its mechamism is not clear.Firstly,active vasostatin protein were prepared by affinity chromatography and the purity was over 95%.Secondly,a series of qulity assays had been done for the protein,such as molecular mass,purity,isoelectric point,concentration.These physical and chemical assays all comply with the requirement of Chinese pharmacopia. Thirdly, HHEC anti-migration assay and HM2 anti- proliferation method were established to detect vasostatin's bioactivity in cell level. The results showed that vasostatin play an important role in inhibiting endothelial cell's migration and proliferation. Fourthly, tumor-inhibition assay was established to assess the protein's bioactivity using H22 tumor mouse model,and the inhibition rate were about 61.7% on the dose of 160μg/mouse/day(8mg/kg/d).Pathology examination indicated that the blood vessel desity of treatment groups was much lower than that of negative control groups,and there were no toxic to other organs.The results indicated that vasostatin could inhibit tumor growth by inhibiting angiogenesis. The same test was done using SMMC7721 tumor nude mouse model,the inhibition rate was about 46.2% on the dose of 1.5mg/mouse/day (75mg/kg/d),which was significantly different from negative control group.After animial experiments,the third generation yeast two-hybrid technique was used to further investigate vasostatin protein's anti-tumor mechanism.Through screening human endothelial cell cDNA library,three positive clones were obtained.They were lamininα5 and the fragment of integrin,respectively,and belonged to the family of laminin and integrin,which were closely related to cell migration.We could find a clue for vasostatin protein's activity mechamism from these results.When vasostatin protein was combined with laminin and integrin,the...