组织基质金属蛋白酶抑制剂-4真核表达载体的构建和功能研究

发布时间：2018-01-11 13:31

  本文关键词：组织基质金属蛋白酶抑制剂-4真核表达载体的构建和功能研究　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 组织基质金属蛋白酶抑制剂-4 HMEC-1 真核表达 血管形成

【摘要】： 人组织基质金属蛋白酶抑制剂-4(tissue inhibitor of matrix metalloproteinases type-4,TIMP-4)是近年发现的TIMPs家族新成员,1996年由Greene等首先从人心脏中克隆出来,之后小鼠的TIMP-4基因于1997年被Olson等人克隆出来。人TIMP-4基因位于第3号染色体,而小鼠的则位于第6号染色体。 自TIMP-4被克隆以来,其相关研究一直较少。近年来,逐渐发现了TIMP-4的一些功能,特别是涉及到肿瘤及血管新生方面对于MMP-2的抑制作用,由此吸引了更多学者的目光。但是对于其作用的具体机制,更多的是基于其它TIMP家族成员功能和一些临床检测结果的推测,缺乏较为有力的直接证据。 目的本研究的目的验证人类微血管内皮细胞株HMEC-1内皮细胞特性,并构建起TIMP-4的真核表达载体,获得高表达TIMP-4的HMEC-1细胞株,在此基础上探讨TIMP-4体外对内皮细胞迁移能力和血管形成能力的影响。 方法首先通过免疫组化、ELISA及PCR方法对HMEC-1细胞株内皮特异性标志物进行验证。根据已公布的基因序列设计相应引物,用RT-PCR方法从人原代微血管内皮细胞(HDMEC)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、PCR和测序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Western-Blotting分别检测TIMP-4在HMEC-1中mRNA和蛋白水平的表达;利用Transwell小室进行细胞迁移实验,并且在Matrigel上进行体外血管形成实验,观察TIMP-4对血管新生的影响。 结论1.验证了HMEC-1具有原代微血管内皮细胞所特有的分子标志物和性质。2.成功构建了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。3.证实了TIMP-4能于体外抑制血管形成。
[Abstract]:Human tissue inhibitor of matrix metalloproteinases type-4. TIMP-4 is a new member of TIMPs family discovered in recent years. In 1996, it was first cloned from human heart by Greene et al. In 1997, the TIMP-4 gene of mice was cloned by Olson et al. The human TIMP-4 gene is located on chromosome 3, while the mouse gene is located on chromosome 6. Since the cloning of TIMP-4, its related research has been relatively few. In recent years, some functions of TIMP-4 have been gradually discovered. In particular, the inhibition of tumor and angiogenesis on MMP-2 has attracted more and more scholars' attention, but the specific mechanism of its action. It is more based on the function of other TIMP family members and some clinical test results, which is lack of strong direct evidence. Objective to verify the characteristics of human microvascular endothelial cell line (HMEC-1) and construct the eukaryotic expression vector of TIMP-4. HMEC-1 cell lines with high expression of TIMP-4 were obtained, and the effects of TIMP-4 on endothelial cell migration and angiogenesis in vitro were investigated. Methods Endothelial specific markers of HMEC-1 cell line were verified by immunohistochemistry Elisa and PCR method, and corresponding primers were designed according to published gene sequence. The TIMP-4 fragment was amplified by RT-PCR from the cDNA of human primary microvascular endothelial cells (HDMECs). The target fragment was inserted into the eukaryotic expression vector of pcDNA3.1/V5-His-TOPO by restriction endonuclease Hind 鈪，

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