Construction And Study Of Tissue Inhibitor Of Matrix Metalloproteinases Type-4 Eukaryotic Expression Vector

Tissue inhibitor of matrix metalloproteinases type-4 (TIMP-4) is a new member of TIMPs family. It was first cloned from human heart at 1996 by Greene, and mouse TIMP-4 was cloned by Olson. The gene for TIMP-4 was mapped to the human 3 chromosome and in the mouse map to the 6 chromosome, respectively.After cloned in 1996,there are few reports about TIMP-4. The fuction about inhibiting MMP-2 during developing of tumor and angiogenesis was attented by some people.But we need more evidences for the mechanism of TIMP-4. In the investigation, we got the gift of HMEC-1. The cell line would be detected for making sure its resource.Objective To construct human TIMP-4 eukaryotic expression vector, and to study the effect of TIMP-4 on angiogenesis in vitro.Methods Detected the HMEC-1 by immuno-labelling, ELISA and RT-PCR.The human TIMP-4 cDNA which was in line with the reported human TIMP-4 gene was amplified from human dermal microvascular endothelial cell(HDMEC) by RT-PCR. The interested fragment was inserted into eukaryotic expression vectors pcDNA3.1/V5-His-TOPO by restriction enzymen. The recombinant expression plasmid was transfected into HMEC-1 cells and realtime-PCR and Western blotting were used to analyse TIMP-4 expression both on mRNA and protein levels. And transfected HMEC-1 cells were reconstructed on Matrigel to observe the inhibition effect of TIMP-4 on angiogenesis in vitro.Conclusion 1.HEMC-1 was identified with immuno-labelling and ELISA. 2.The pcDNA3.1/V5-His-TOPO-TIMP-4 eukaryotic expression vector and high expression TIMP-4- HMEC-1 were contructed. 3.TIMP-4 can inhibited the process of angiogenesis in vitro.