Tran N D, Wong V L, Schreiber S S, Bready J V, Fisher M
Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Stroke. 1996 Dec;27(12):2304-10; discussion 2310-1. doi: 10.1161/01.str.27.12.2304.
Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA.
Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture.
Both in situ hybridization and PCR studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however, astrocyte-endothelial cocultures had markedly decreased thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures.
These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.
内皮细胞部分通过血栓调节蛋白（一种有效的抗凝血蛋白）的表达来调节止血。本研究的目的是分析脑毛细血管内皮细胞血栓调节蛋白mRNA的表达。
牛脑毛细血管内皮细胞在血脑屏障模型中培养，其中内皮细胞形成毛细血管样结构。采用原位杂交和聚合酶链反应（PCR）检测血栓调节蛋白的表达。然后将内皮细胞与星形胶质细胞共培养。我们检测了共培养物和单培养物中的γ-谷氨酰转肽酶（GGTP），这是血脑屏障的一个标志物。然后，我们使用定量竞争PCR比较培养1天和7天后内皮细胞单培养物和星形胶质细胞-内皮细胞共培养物中血栓调节蛋白的表达。
原位杂交和PCR研究均表明内皮细胞表达血栓调节蛋白mRNA。在星形胶质细胞-内皮细胞共培养1周期间，（1）星形胶质细胞与毛细血管样结构逐渐结合，（2）GGTP表达；内皮细胞单培养物不表达GGTP。共培养物与单培养物在培养1天后血栓调节蛋白mRNA表达无显著差异。然而，1周后，与单培养物相比，星形胶质细胞-内皮细胞共培养物中的血栓调节蛋白mRNA明显减少（9±2对189±62 pg/mL；P<.025）。当血脑屏障表型的成分可被证明时，即当星形胶质细胞与毛细血管样结构的结合最大且共培养物中表达GGTP时，血栓调节蛋白mRNA就会减少。
这些发现表明星形胶质细胞在体外对血栓调节蛋白mRNA表达有调节作用，并提示血脑屏障在血栓调节蛋白的调节中起重要作用。
