Expression And Purification Of Human Macrophage Metalloelastase And Effects On The Expression Of VEGF,COX-2 In Gastric Cancer Cells

Objective Human macrophage metalloelastase(HME),also referred as matrix metalloproteinase 12,played an important role in the generation of angiostatin,an internal fragment of plasminogen,which shows inhibition of tumor.Vascular endothelial growth factor(VEGF) was believed as a most important factor promoting the generation of the new vessels in tumor.Cyclooxygenase-2(COX-2),a rate-limiting enzyme of catalyzing arachidonic acid into prostaglandin,was associated with lymph node involvements,infiltration depth of gastric carcinoma when it was overexpressed.Meanwhile,COX-2 cooperated with VEGF in the advancement of gastric carcinoma.This research induced E eoli.BL-21(DE3) to express HME,then purificated and refolded the fusion protein,subsequently,to investigate the effect of HME on the VEGF and COX-2 expressed by gastric cancer cells,we interfere in gastric cancer cells with HME.Methods To induce E coli.BL21(DE3) which was transformed into the recombinant pET-28a(+)-HMEcd by IPTC4 The production of expression was analyzed by SDS-PAGE and detected by Western-blot.The fusion protein was purified with His Bind Collumn and refolded by dialysis and assaied after concentration.The enzymatic activity of recombinant protein was confirmed by gelatin zymography.Results SDS-PAGE analysis and Western-blot demonstrated HMEcd protein was expressed in E coli.BL-21(DE3),gelatin zymography showed the enzymatic activity. The VEGF expression level after intervening for 36 h was reduced significantly compared with control group,P＜0.05,after intervening for 12 h,the level of VEGF of control group increased significantly compared to 0 h,P＜0.05.HME at 1 ug/ml,2 ug/ml,4 ug/ml,5 ug/ml had no significant differences between 0 h and 12 h,P＞0.05; HME did not cause conspicuous change in the expression level of VEGF with combined use of plasminogen,P＞0.05;HME could not decrease the expression of COX-2 compared to control group,P＞0.05.Conclusions We induced pET-28a(+)-HMEcd/ BL-21(DE3) to express HMEcd protein and established the purification and then refolded HMEed protein in vitro successfully.HME at 1 ug/ml-5 ug/ml inhibited the expression level of VEGF significantly after intervening for 36 h.the mechanism did not achieve through decomposing plasminogen and producing angiostatin.HME at 1 ug/ml-5 ug/ml could not have effect on gastric cancer cells...