| Objective Induction human macrophage elastase catalytic region (HMEcd) gene prokaryotic expression plasmid of E.coli expression of HMEcd fusion protein. After purification and renaturation obtaining human macrophage metalloelastase (HME), intervention in gastric cancer cells of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression in vivo.Methods Induced transformation of the prokaryotic expression vector pET-28a (+)-HMEcd E.coli BL21(DE3) to express HMEcd fusion protein. The expression product was identified by SDS-PAGE and Western blot. The fusion protein was purfied with His-Bind Collumn and refolded by dialysis and assaied after concentration. The enzymatic activity of recombinant protein was confirmed by gelatin zymography. Subcutaneously transplant tumor model of human gastric cancer was established in 36 BALB/c nu/nu nude mice. Then the nude mice were randomly divided into blank control group, negative control group, COX-2 inhibitor group and HME-intervention groups. COX-2 inhibitor group was treated with Celecoxib 10mg/kg, HME-intervention groups were treated with HME 0.2mg/kg, 0.4mg/kg, 0.8mg/kg each. Blank control group and negative control group were treated with same volume of sterile saline and no activity of HME protein solution. Animals in each group every two days intra-tumor injection one time, before the delivery measurement body weight and xenografts size at each nude mice. All tumor bearing mice were sacrified on 6th week after transplantation. Measurement of mice weight, tumors weight, tumors size and calculation of tumor inhibition rate. The expression of COX-2 and VEGF was detected by immunohistochemical SP method and Western blot assay. Microvessel density (MVD) were analyzed in CD34-stained vascular endothelial cell.Results HMEcd fusion protein was expressed in E.coli BL-21(DE31), which was demonstrated by SDS-PAGE analysis and Wsetern blot. After re-folding and renaturing, identification of fusion protein HMEcd gelatin decomposition activity by gelatin zymography. Contrasted with blank control group, the growth of xenografts were significantly inhibited by each dose groups of HME and COX-2 inhibitor group (P<0.05), but no obviously differences between two control groups (P>0.05). Among intervention groups the depressant effect was most powerful in HME 0.8 mg/kg dose group (P<0.05), but the effects had not significantly differences between COX-2 inhibitor group and HME 0.2 mg/kg dose group (P>0.05). Immunohistochemistry and Western blot results showed that the expression of COX-2 and VEGF in xenografts decreased in COX-2 inhibitor group and each dose groups of HME (P<0.05). COX-2 and VEGF reduced more obviously in HME 0.8 mg/kg group and 0.4 mg/kg group than COX-2 inhibitor group. Compared with blank control group, MVD decreased obviously in all intervention groups(P<0.05), especially in HME 0.8 mg/kg dose group.Conclusions Induced pET-28a(+)-HMEcd BL-21(DE3) to express fusion protein HMEcd successfully, after re-folding and renaturing, obtaining high-purity protein HME with enzyme activity. HME had significant antitumor effects when concentrations ranged from 0.2 mg/kg to 0.8 mg/kg. The antitumour effects was a dose-dependent manner. Among all intervention groups the antitumor effects were most powerful in HME 0.8 mg/kg dose group. HME 0.2 mg/kg had the same antitumor effects with Celecoxib 10mg/kg. The expression of COX-2, VEGF and tumor angiogenesis in xenografts were significantly inhibited by HME. The antitumor effect of HME in nude mice may attribute to the anti-angiognesis through inhibiting expression of COX-2 and VEGF, which effects were dose-dependent. |
