IMMORTALIZED HUMAN SKIN ENDOTHELIAL CELLS

内皮细胞在多种生理学过程中是至关重要的。 和病理过程，主要发生在水平的 微脉管系统 已经清楚地表明，微血管 内皮细胞明显不同于大血管内皮细胞， 但对微血管内皮细胞的广泛研究 由于在分离和传播这些病毒时存在固有的困难， 细胞 虽然分离和培养微血管是可能的， 来自人类皮肤的内皮细胞，只有相对少量的细胞 寿命非常有限。 这限制了 可以解决的科学问题。 研究人类 微血管内皮细胞将大大促进 永生化人微血管内皮细胞的开发 保留表型和功能特性的品系 人微血管内皮细胞的特征。 这种细胞 线将有广泛的效用，在研究一些 生理和病理过程，从基因调控， 细胞间通讯到内皮细胞肿瘤生物学。 它还将 消除使用混合物所产生的困难， 来自多个供体的微血管内皮细胞。 可惜没有 存在人微血管内皮细胞系， 内皮细胞表型 在本提案中，我们计划1）创建 永生化微血管内皮细胞 培养人真皮微血管内皮细胞（HDMEC）， 猴病毒40大T抗原。 我们还将生成转换后的HDMEC 使用SV 40大T的温度敏感突变体， 像其他永生化细胞一样增殖，但恢复为非永生化细胞。 在非允许温度下的永生化表型; 2）表征 永生化细胞系保留了必需的HDMEC 表型和功能特征，包括细胞粘附 分子表达调控; 3）建立稳定的转染子 使用这些永生化的HDMEC和转录因子的部分， 人细胞间粘附分子1调节区 基因来表征细胞环境在调节 ICAM-1表达;和4）表征信号和分子 调节皮肤特异性白细胞归巢蛋白E- 选择素 这些研究将产生重要的新型试剂和新的 深入了解微血管内皮细胞和粘附的作用 皮肤炎症中的分子。

Endothelial cells are critically important in a variety of physiologic and pathologic processes which occur primarily at the level of the microvasculature. It has been clearly demonstrated that microvascular endothelial cells differ distinctly from large vessel endothelial cells, but extensive study of microvascular endothelial cells has been severely limited by inherent difficulties in isolation and propagation of these cells. Although it is possible to isolate and culture microvascular endothelial cells from human skin, only relatively small numbers of cells with a very limited lifespan can be obtained. This limits the scope of the scientific questions that can be addressed. The study of human microvascular endothelial cells would be greatly facilitated by the development of an immortalized human microvascular endothelial cell line(s) which retains the phenotype and functional properties characteristic of human microvascular endothelial cells. Such a cell line would have widespread utility in the study of a number of physiologic and pathologic processes ranging from gene regulation and cell-cell communication to endothelial cell tumor biology. It would also eliminate the difficulties engendered by the use of mixtures of microvascular endothelial cells from multiple donors. Unfortunately, no human microvascular endothelial cell lines exist that retain an endothelial cell phenotype. In this proposal, we plan to 1) create immortalized microvascular endothelial cells by transforming pure cultures of human dermal microvascular endothelial cells (HDMEC) using simian virus 40 large T antigen. We will also generate transformed HDMEC using temperature sensitive mutants of SV40 large T which will proliferate like other immortalized cells, but revert to a non- immortalized phenotype at the non-permissive temperature; 2) characterize the immortalized cell lines for their retention of essential HDMEC phenotypic and functional characteristics including cell adhesion molecule expression and regulation; 3) establish stable transfectants using these immortalized HDMEC and portions of the transcriptional regulatory region of the human intercellular adhesion molecule 1 (ICAM-1) gene to characterize the role of cellular context in the regulation of ICAM-1 expression; and 4) characterize the signals and molecular mechanisms regulating the skin specific leukocyte homing protein E- selectin. These studies will produce important novel reagents and new insights into the role of microvascular endothelial cells and adherence molecules in cutaneous inflammation.