IMMORTALIZED HUMAN SKIN ENDOTHELIAL CELLS

内皮细胞在多种生理过程中起着至关重要的作用。 和病理过程，这些过程主要发生在 微血管系统。已经清楚地证明，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一直很严重 受分离和繁殖这些病毒的固有困难的限制 细胞。尽管分离和培养微血管是可能的 来自人类皮肤的内皮细胞，只有相对较少的细胞 可以获得非常有限的寿命。这限制了 可以解决的科学问题。关于人的研究 微血管内皮细胞将得到极大的促进 永生化人微血管内皮细胞的建立 保持表型和功能特性的品系(S) 人类微血管内皮细胞的特征。这样一间牢房 LINE将在研究一些 生理和病理过程，从基因调控和 内皮细胞肿瘤生物学的细胞间通讯。它还会 消除因混合使用 来自多个供体的微血管内皮细胞。很遗憾，我不会 人类微血管内皮细胞系存在，它们保留了 内皮细胞表型。在本计划中，我们计划1)创建 转化为纯净的微血管内皮细胞永生化 人真皮微血管内皮细胞的体外培养 猴病毒40大T抗原。我们还将生成转换后的HDMEC 使用SV40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 在不允许的温度下永生表型；2)表征 永生化细胞系为其保留必需的HDMEC 表型和功能特征，包括细胞黏附 分子表达与调控；3)建立稳定的转染体 使用这些永生化的HDMEC和部分转录的 人细胞间黏附分子1(ICAM-1)的调节区 基因来表征细胞环境在调节细胞生长发育中的作用 ICAM-1的表达；4)信号和分子特征 皮肤特异性白细胞归巢蛋白E的调节机制 选择素。这些研究将产生重要的新型试剂和新的 对微血管内皮细胞的作用和黏附的认识 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.