Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene

摘要

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TM-BBBs expressed the typical endothelial marker, von Willberand factor and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were −10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-Mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59±1.16 mmol/1. Moreover, P-glycoprotein (P-gp) with a molecular weight of −170 kDa was expressed in all TM-BBBs. Both mdr 1a and mdr 1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 μmol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.