Development of in vitro blood-brain barrier culture system from temperature sensitive SV40T antigen gene transgenic animal

本研究的目的是建立体外血脑屏障（BBB）系统。用SV 40 T抗原转基因小鼠和大鼠建立了条件永生化脑毛细血管内皮细胞系。两种细胞系均呈梭形纤维形状，并表达SV 40 T抗原基因产物。33 ℃培养的细胞中SV 40 T抗原的量显著高于37 ℃和39 ℃培养的细胞。33度的生长率也大于37度和39度。BBB选择性标记，即，对建立的细胞系进行乙酰化LDL受体、碱性磷酸酶、γ-谷氨酰转肽酶的检测。RT-PCR分析显示，这些细胞株表达GLUT-1和mdr 1a的mRNA。Western blot分析也显示GLUT-1基因产物和P-糖蛋白的表达。小鼠细胞系对3-O-甲基-D-葡萄糖（3-OMG）的初始摄取速率为2.6-5.3μl（min mg蛋白）。米氏常数为6.6 mM，与体内报告值非常相似，而转运活性比BBB的体内摄取速率小约10倍。建立的细胞系的紧密连接不足以进行跨细胞转运研究，而通过RT-PCR分析检测到紧密连接蛋白如claudin-5、occludin、连接粘附分子的mRNA。结论：本实验成功建立了小鼠和大鼠脑毛细血管内皮细胞条件永生化细胞系。这些细胞系表现出至少一部分体内特性。紧密连接蛋白基因产物和转运蛋白基因产物的诱导将是今后研究的方向。

The purpose of the present study is to develop the in vitro blood-brain barrier (BBB) system. A conditionally immortalized brain capillary endothelial cell line was established from the temperature sensitive SV40T antigen gene transgenic mouse and rats. Both cell lines had spindle fiber shape and expressed SV40T antigen gene product. The amount of SV40T antigen in the cell cultured at 33 degree was significantly greater than those of 37 and 39 degree. Growth rate at 33 degree was also greater than those of 37 and 39 degree. BBB selective marker, i.e., acetylated LDL receptor, alkalinephosphatase, gamma-glutamyltranspeptidase, was detected for the cell lines established. RT-PCR analysis revealed that these cell line express the mRNA of GLUT-1 and mdr1a. Western blot analysis also indicated the expression of GLUT-1 gene product and P-glycoprotein. Initial uptake rate of 3-O-methyl-D-glucose (3-OMG) by the mouse cell line was 2.6-5.3μl(min mg protein). The Michaelis constant was 6.6 mM which is very similar to the in vivo reported value, while the transport activity was approximately ten fold smaller than that of in vivo uptake rate at the BBB. Tight junction of the established cell lines was not strong enough for the transcellular transport study, whereas mRNA of tight junction proteins such as claudin-5, occludin, junctional adhesion molecule was detected by the RT-PCR analysis. In conclusion, we could establish conditionally immortalized cell line of brain capillary endothelial cell in mouse and rats. These cell lines exhibited at least a part of in vivo characteristics. The induction of tight junction protein gene products and transporter gene product would be a subject for the future projects.