Heterogeneity of endothelial cells - The specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization
Heterogeneity of endothelial cells - The specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization
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DOI：
10.1016/s0002-9440(10)65523-x
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发表时间：
1999-12-01
影响因子：
6
通讯作者：
Amalric, F
中科院分区：
文献类型：
--
作者：
Girard, JP;Baekkevold, ES;Amalric, F
高内皮微静脉（HEVs）是在淋巴器官和慢性炎症组织中发现的特化毛细血管后微静脉，其支持高水平的淋巴细胞从血液外渗。戊型肝炎病毒内皮细胞（HEVECs）的分子特征已受到其纯化和体外维持的困难。为了克服这些局限性，我们开发了一种策略，结合使用新鲜纯化的HEVEC（类似于98%阳性的HEV特异性标记MECA-79）和最近描述的基于聚合酶链反应（PCR）的cDNA消减克隆程序称为抑制消减杂交（SSH）。这导致了22个cDNA的克隆，这些cDNA优先在HEVEC中表达，它们编码混杂的趋化因子受体DARC、线粒体组分和基质细胞蛋白。后者包括hevin、血小板反应蛋白-1和mac 25/IGFBP-rP 1，mac 25/IGFBP-rP 1是一种分泌的生长因子结合蛋白，先前发现其特异性地在肿瘤血管中积累。生化和组织化学分析证实了mac 25和DARC作为HEVEC的新标记物的鉴定。超微结构免疫定位显示mac 25和MECA-79抗原与内皮细胞连接附近的微绒毛过程有明显的关联，表明mac 25在控制淋巴细胞迁移中的作用。这项研究表明，基于PCR的SSH是有用的克隆差异表达基因在非常小的样本。
High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs (similar to 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH), Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.