mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines;: a new in vitro BBB model for drug targeting
mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines;: a new in vitro BBB model for drug targeting
复制标题
DOI：
10.3109/10611860008997912
复制
发表时间：
2000-01-01
影响因子：
4.5
通讯作者：
Terasaki, T
中科院分区：
文献类型：
--
作者：
Hosoya, K;Takashima, T;Terasaki, T
以新近开发的携带温度敏感猿猴病毒40 （ts SV 50）大t抗原基因（Tg大鼠）的转基因大鼠为材料，建立了脑毛细血管内皮细胞系（TR-BBB），并用于表征血脑屏障（BBB）内皮标志物、转运活性以及转运蛋白和紧密连接链蛋白的mRNA表达。这些细胞系表达有活性的大t抗原，在33℃下生长良好，生长时间约为12-31小时，但在39℃下不生长。tr - bbb表达典型的内皮标志物血管性血血病因子，并表现出乙酰化低密度脂蛋白摄取活性。虽然tr -血脑屏障的γ -谷氨酰转肽酶活性与正常大鼠脑毛细血管部分的13%相似，但它定位于脑顶侧，这表明它反映了体内血脑屏障的功能极性。TR-BBB13中表达claudin -5、occludin、连接黏附分子等紧密连接链蛋白mRNA，所有tr - bbb中均表达分子量为170 kDa的药物外排转运蛋白p -糖蛋白mRNA， RT-PCR检测到tr - bbb中mdr 1a、mdr 1b、mdr 2 mRNA。mrp1 mRNA在所有tr - bbb中均有表达。Western blot分析显示，内流转运蛋白GLUT-1在55kda表达。3- o -甲基-d -葡萄糖（3-OMG）摄取活性呈浓度依赖性，Michaelis-Menten常数为9.86 +/- 1.20 mM。tr - bbb中表达由lat1和4F2hc组成的大中性氨基酸转运体mRNA。综上所述，条件永生化大鼠脑毛细血管内皮细胞系（TR-BBB）具有内皮生成物，表达紧密连接链蛋白mRNA和内流酸外排转运蛋白mRNA，并产生具有3-OMG转运活性的GLUT-1。
Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 50) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degreesC with a doubling-time of about 12-31 hr, but did not grow at 39 degreesC. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma -glutamyltranspeptidase activity in TR-BBBs was similar to 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13, Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx acid efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.