VASCULAR ENDOTHELIUM FORM AND FUNCTION: EFFECTS OF GLIA

这项建议的目标有两个；(1)开发一种体外培养的 两种不同细胞类型之间相互作用的共培养系统 可以研究，以及(2)在体内评估神经胶质细胞在 血脑屏障(BBB)的分化与维护 脑毛细血管内皮细胞的特性。 体内研究将利用小鼠冷冻损伤模型来研究这些 再生脑微血管中的突起。具体目标是 彻底调查时间序列和关系 内皮紧密连接形成，早期出现神经胶质足 血管基底膜上的突起，胶质细胞的完成 再生血管周围的鞘，恢复屏障功能 示踪剂、内皮细胞因子VIII和血栓调节蛋白(TM)的命运 再生和诱导(或激活)过程中的抗原或 γ-谷氨酰转肽酶(GGTP)位于新生内皮细胞内。这些 研究将采用GGTP的酶组织化学，免疫细胞化学 GGTP、TM和第VIII因子、超微结构研究和萝卜 巴里尔功能的过氧化物酶示踪分析。 体外研究的目的是确定通过什么机制 内皮细胞在胶质细胞存在的情况下获得GGTP 共培养，以测试神经胶质细胞诱导其他分化的能力 内皮细胞特征，并描绘胶质细胞的程度 细胞可诱导和/或激活颅外血管内皮细胞GGTP 网站。这些研究将使用放射免疫分析、放射自显影、酶 GGTP的组织化学和生物测定，TM的免疫化学和生物测定， 以及各种不同的共同文化方案。 这种双重方法的好处是体内研究将告诉我们 在胶质细胞/内皮细胞-细胞相互作用中应该寻找什么，以及在 体外研究将提供一个场所来测试或验证体内的 观察。 这些目标的实现将大大增加我们的 对多种微血管再生过程的理解 血管改变的临床和病理情况 诚信会导致不同程度的脑损伤。(梗塞， 创伤、缺氧、肿瘤、感染等)。同样重要的是 体外共培养方案的开发和测试，使 对分化的内皮细胞功能的研究，这些功能经常在 纯细胞培养。

The objective of this proposal is twofold; (1) to develop an in vitro co-culture system in which the interaction between two different cell types can be studied, and (2) to evaluate in vivo the role of the glial cell in the differentiation and maintenance of the Blood-Brain Barrier (BBB) properties in capillary endothelium of the brain. The in vivo studies will utilize a mouse freeze-injury model to study these processes in the regenerating cerebral microvessels. The specific aims are to thoroughly investigate the temporal sequence and relationships of endothelial tight junction formation, early appearance of glial foot processes on the vascular baseement membrane, the completion of the glial sheath around the regenerating vessel, restoration of barrier function to tracers, the fate of endothelial cell Factor VIII and Thrombomodulin (TM) antigens during regeneration, and induction (or activation) or gamma-glutamyl transpeptidase (GGTP) ion the new endothelium. These studies will employ enzyme histochemistry for GGTP, immunocytochemistry for GGTP, TM and Factor VIII, ultrastructural studies and horse radish peroxidase tracer analysis for barrir function. The Aims of the in vitro studies are to determine the mechanisms by which endothelial cells acquire GGTP in the presence of glial cells in co-culture, to test the glial cell's ability to induce other differentiated endothelial characteristics, and to delineate the extent to which glial cells can induce and/or activate GGTP in endothelium from extracranial sites. These studies will use radioimmunoassay, autoradiography, enzyme histochemistry and bioassay for GGTP, immunochemistry and bioassay for TM, as well as varied schemes of co-culture. The advantage this dual approach is that the in vivo studies will tell us what to look for in the glial/endothelial cell-cell interaction, and the in vitro studies will provide a place to test or validate the in vivo observations. The accomplishment of these aims will add significantly to our understanding of the microvascular regeneration process following a variety of clinical and pathological conditions where alterations in vascular integrity have resulted in varying degrees of brain damage. (Infarcts, trauma, anoxia, neoplasms, infection, etc.). Of equal importance will be the development and testing of in vitro co-culture schemes that allow the study of differentiated endothelial cell functions that are often lost in pure cell culture.