本文關(guān)鍵詞：組織基質(zhì)金屬蛋白酶抑制劑-4真核表達(dá)載體的構(gòu)建和功能研究　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 組織基質(zhì)金屬蛋白酶抑制劑-4 HMEC-1 真核表達(dá) 血管形成

【摘要】： 人組織基質(zhì)金屬蛋白酶抑制劑-4(tissue inhibitor of matrix metalloproteinases type-4,TIMP-4)是近年發(fā)現(xiàn)的TIMPs家族新成員,1996年由Greene等首先從人心臟中克隆出來,之后小鼠的TIMP-4基因于1997年被Olson等人克隆出來。人TIMP-4基因位于第3號(hào)染色體,而小鼠的則位于第6號(hào)染色體。 自TIMP-4被克隆以來,其相關(guān)研究一直較少。近年來,逐漸發(fā)現(xiàn)了TIMP-4的一些功能,特別是涉及到腫瘤及血管新生方面對(duì)于MMP-2的抑制作用,由此吸引了更多學(xué)者的目光。但是對(duì)于其作用的具體機(jī)制,更多的是基于其它TIMP家族成員功能和一些臨床檢測(cè)結(jié)果的推測(cè),缺乏較為有力的直接證據(jù)。 目的本研究的目的驗(yàn)證人類微血管內(nèi)皮細(xì)胞株HMEC-1內(nèi)皮細(xì)胞特性,并構(gòu)建起TIMP-4的真核表達(dá)載體,獲得高表達(dá)TIMP-4的HMEC-1細(xì)胞株,在此基礎(chǔ)上探討TIMP-4體外對(duì)內(nèi)皮細(xì)胞遷移能力和血管形成能力的影響。 方法首先通過免疫組化、ELISA及PCR方法對(duì)HMEC-1細(xì)胞株內(nèi)皮特異性標(biāo)志物進(jìn)行驗(yàn)證。根據(jù)已公布的基因序列設(shè)計(jì)相應(yīng)引物,用RT-PCR方法從人原代微血管內(nèi)皮細(xì)胞(HDMEC)的cDNA中擴(kuò)增TIMP-4片段,采用限制性內(nèi)切酶HindⅢ和XholⅠ將目的片段插入pcDNA3.1/V5-His-TOPO真核表達(dá)載體中。經(jīng)過酶切、PCR和測(cè)序鑒定后通過脂質(zhì)體介導(dǎo)轉(zhuǎn)染至HMEC-1細(xì)胞中進(jìn)行表達(dá)。以Realtime-PCR和Western-Blotting分別檢測(cè)TIMP-4在HMEC-1中mRNA和蛋白水平的表達(dá);利用Transwell小室進(jìn)行細(xì)胞遷移實(shí)驗(yàn),并且在Matrigel上進(jìn)行體外血管形成實(shí)驗(yàn),觀察TIMP-4對(duì)血管新生的影響。 結(jié)論1.驗(yàn)證了HMEC-1具有原代微血管內(nèi)皮細(xì)胞所特有的分子標(biāo)志物和性質(zhì)。2.成功構(gòu)建了pcDNA3.1/V5-His-TOPO-TIMP-4真核表達(dá)質(zhì)粒,并獲得高表達(dá)TIMP-4的HMEC-1細(xì)胞株。3.證實(shí)了TIMP-4能于體外抑制血管形成。
[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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