CXCR7及其配体SDF-1介导细胞迁移的初步研究

发布时间：2018-04-13 22:29

本文选题：原核表达 + 多克隆抗血清　；参考：《第四军医大学》2007年硕士论文

【摘要】： 细胞迁移指的是细胞在接收到迁移信号或感受到某些物质的浓度梯度后而产生的移动。趋化因子作为一种信号分子通过与细胞膜表面上的受体结合,启动细胞内信号,激活或抑制肌动蛋白结合蛋白的活性,最终改变细胞骨架的状态,引起细胞的移动。基质细胞衍生因子-1(Stromal cell derived factor-1,SDF-1)就是一种重要的趋化因子,广泛地表达于各种细胞和组织中,包括免疫细胞、脑、心脏、肾、肝、肺和脾,在免疫系统、循环系统及中枢神经系统的发育中起着至关重要的作用。同时与HIV感染、炎症反应、造血细胞的调控作用、肿瘤细胞的迁移等密切相关。历来认为,SDF-1是通过CXCR4这个唯一的受体来调控这些不同的过程。但近来发现了SDF-1的一个新受体,称为CXCR7。CXCR7表达于许多肿瘤细胞系,活化的内皮细胞以及胎肝细胞,大多数正常细胞表面不表达,尽管这些正常细胞表面不表达CXCR7分子,但其细胞内却表达了CXCR7 mRNA。目前的研究表明在体外CXCR7具有促进细胞存活的作用,并对小鼠肿瘤生长有促进作用,但关于SDF-1/CXCR7对细胞迁移的作用并不明确,我们进行了如下两个方面的研究: 1、SDF-1α的原核表达。用已有的pMD-18T-SDF-1α质粒与pGEX-4T-1表达载体构建融合表达载体pGEX-4T-1-SDF-1α,并在大肠杆菌BL21中进行诱导表达。确定目的基因的表达情况后,进一步大量表达,利用Glutathione Sepharose 4B柱,对获得的融合蛋白进行纯化。2、CXCR7抗血清的制备、鉴定以及对MCF-7细胞迁移影响。合成两条肽段H458、H459,和KLH交联后免疫新西兰兔,制备CXCR7的多克隆抗血清,饱和硫酸胺法纯化,酶联免疫反应(ELISA)检测抗血清滴度, Western-blot和间接免疫荧光检测多克隆抗体对CXCR7受体的识别能力。采用Millicell小室来研究SDF-1/CXCR7对细胞迁移的影响,分别对抗体封闭组、趋化因子组和对照组的迁移细胞进行计数。结果:双酶切鉴定证明我们成功地构建了重组融合表达载体pGEX-4T-1-SDF-1α,IPTG诱导融合蛋白GST-SDF-1α表达,主要以包涵体的形式存在。对包涵体充分洗涤后,利用Glutathione Sepharose 4B柱,获得了纯化的GST-SDF-1α融合蛋白。用与KLH交联的肽段H458、H459免疫新西兰兔,获得多克隆抗血清,间接ELISA检测抗血清效价约为1:8000。Western-blot检测多克隆抗血清可以与Hela和MCF-7细胞裂解液特异结合,形成单一的结合条带。间接免疫荧光显示,阳性物质分布于Hela和MCF-7细胞表面。细胞迁移实验中抗体封闭组和趋化因子组在细胞迁移数量上有明显的差异。结论:成功获得了纯化的GST-SDF-1α融合蛋白和CXCR7多克隆抗体,CXCR7多克隆抗体可抑制SDF-1/CXCR7介导的细胞迁移。为进一步阐明SDF-1/CXCR7在细胞迁移中的机制奠定基础。
[Abstract]:Cell migration is the movement of a cell after it receives a migration signal or feels the concentration gradient of certain substances.Chemokines act as signaling molecules by binding to receptors on the surface of cell membranes to initiate intracellular signals, activate or inhibit the activity of actin binding proteins, and ultimately change the state of the cytoskeleton and cause cell movement.Stromal cell derived factor-1 (SDF-1) is an important chemokine that is widely expressed in a variety of cells and tissues, including immune cells, brain, heart, kidney, liver, lung and spleen, in the immune system.The development of circulatory system and central nervous system plays an important role.It is closely related to HIV infection, inflammation, hematopoietic cell regulation and tumor cell migration.It has been thought that SDF-1 regulates these different processes through CXCR4, the sole receptor.But recently, a new receptor for SDF-1, called CXCR7.CXCR7, was found in many tumor cell lines, activated endothelial cells and fetal liver cells, most of which are not expressed on the surface of normal cells, although these normal cells do not express CXCR7 molecules.However, CXCR7 mRNA was expressed in the cells.Current studies have shown that CXCR7 can promote cell survival in vitro and promote tumor growth in mice. However, the effect of SDF-1/CXCR7 on cell migration is not clear, we have carried out the following two aspects of research:1 prokaryotic expression of SDF-1 伪.The fusion expression vector pGEX-4T-1-SDF-1 伪 was constructed by using the existing pMD-18T-SDF-1 伪 plasmid and pGEX-4T-1 expression vector, and was induced to express in Escherichia coli BL21.After the expression of the target gene was confirmed, the fusion protein was purified by Glutathione Sepharose 4B column, and the antiserum of CXCR7 was prepared, identified and the effect on the migration of MCF-7 cells was studied.The polyclonal antiserum of CXCR7 was prepared by immunizing New Zealand rabbits with two peptides, H458H 459 and KLH crosslinked. The polyclonal antiserum was purified by saturated sulfate-amine method.Elisa was used to detect the titer of antiserum, Western-blot and indirect immunofluorescence to detect the ability of CXCR7 receptor recognition by polyclonal antibodies.Millicell chamber was used to study the effect of SDF-1/CXCR7 on cell migration. The migration cells in antibody blocking group, chemokine group and control group were counted respectively.Results: we successfully constructed the recombinant fusion expression vector pGEX-4T-1-SDF-1 伪 -IPTG to induce the expression of GST-SDF-1 伪, mainly in the form of inclusion body.After washing the inclusion bodies, the purified GST-SDF-1 伪 fusion protein was obtained by using Glutathione Sepharose 4B column.The polyclonal antiserum was obtained by immunizing New Zealand rabbits with the peptide H458H459 linked with KLH. The titer of indirect ELISA detection of polyclonal antiserum was that 1:8000.Western-blot detection of polyclonal antiserum could specifically bind to Hela and MCF-7 cell lysate and form a single binding band.Indirect immunofluorescence showed that the positive substances were distributed on the surface of Hela and MCF-7 cells.In cell migration assay, there were significant differences in the number of cell migration between antibody blocking group and chemokine group.Conclusion: purified GST-SDF-1 伪 fusion protein and CXCR7 polyclonal antibody, CXCR7 polyclonal antibody, can inhibit cell migration mediated by SDF-1/CXCR7.It lays a foundation for further elucidating the mechanism of SDF-1/CXCR7 in cell migration.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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