TIMP-4真核表达载体的构建及其作用机制的研究

Construction and Study of Tissue Inhibitor of Matrix Metalloproteinases Type-4 Eukaryotic Expression Vector

目的构建人组织基质金属蛋白酶抑制剂-4(tissue inhibitor of matrix metalloproteinases type-4,TIMP-4)的真核表达载体,并探讨TIMP-4体外对血管新生的影响。方法根据已公布的基因序列设计相应引物,用RT-PCR方法从人微血管内皮细胞株HMEC-1的cDNA中扩增TIMP-4片段,采用限制性内切酶HindⅢ和XholⅠ将目的片段插入PcDNA3.1/V5-His-TOPO真核表达载体中。经过酶切和PCR鉴定后通过脂质体介导转染至HMEC-1细胞中进行表达。以Realtime PCR和West-ern Blotting分别检测TIMP-4在HMEC-1中mRNA和蛋白水平的表达;利用Matrigle进行血管形成试验,观察TIMP-4对血管新生的影响。结论构建PcDNA3.1/V5-His-TOPO-TIMP-4真核表达质粒,并获得高表达TIMP-4的HMEC-1细胞株,证实了TIMP-4体外抑制血管形成的作用。

Objective To construct human tissue inhibitor of matrix metalloproteinases type-4（TIMP-4） eukaryotic expression vector, and to study the effect of TIMP-4 on angiogenesis in vitro.Methods The human TIMP-4 eDNA which was in line with the reported human TIMP-4 gene was amplified from human microvascular endothelial cell Iine---HMEC-1 by RToPCR.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 on mRNA and protein levels.And transfected HMEC-1 cells were cultivated on Matrigel to observe the effect of TIMP-4 on angiogenesis in vitro.Conclusion The PcDNA3.1/V5-His-TOPO-TIMP-4 eukaryotic expression vector and transfected HMEC- 1 with high expression of TIMP-4 were constructed.TIMP-4 can inhibited the process of angiogenesis in vitro.