Genetic introduction of thyroid hormone transporter in Bood-Brain-Barrier

我们建立了腺病毒介导的细胞内基因转移系统，将我们分离到的LST-1和LST-2的编码区分别导入腺病毒载体，包装后与腺病毒基因组DNA一起转入HEK293细胞，产生同源重组的细胞尖叫并形成空斑。制备大规模感染培养基后，用氯化铯超速离心法对重组腺病毒进行纯化，测定纯化后的LST-1和LST-2腺病毒的感染滴度，IC50分别为1.26和1.48×10~(-1)和1.48×10~(-1)pfu。将重组病毒应用于培养的细胞株，在作用后24小时内LST-1和LST-2蛋白有较强的表达。Western印迹分析也证实了条带的大小。表达LST-1和LST-2的哺乳动物细胞转运各种有机阴离子作为底物，对牛磺胆酸摄取的表观Km值分别为24微米和32微米，两者都是报道的天然细胞的比较值。腺病毒介导的基因转移系统的建立将为脑、肝、肾导向的药物输送和治疗工具的筛选提供各种应用系统。

We have established adenovirus-mediated gene transfer system into cells.The coding region of LST-1 and LST-2, both of which have isolated by us have introduced into adenovirus vector, packaged, and transfected into HEK293 cell with adenovirus genomic DNA.The cells that produces homologous-recombinant shrieked and made plaque formation. After producing large scale infected medium, the recombinant adenovirus was purified by CsCl ultracentrifuge.The infection titer of the purified LST-1 and LST-2 adenoviruses were measured and the IC 50 of each virus was 1.26 and 1.48 X 10^<10> pfu, respectively.When recombinant virus was applied to cultured cell line, the LST-1 and LST-2 protein was strongly expressed within 24 hr after application. The size of the band was also confirmed by Western blot analysis. The LST-1 and LST-2 expressing mammalian cell transports various organic anions as substrates and the apparent Km value of tauro-cholate uptake is 24 micro M and 32 micro M, respectively and both of which is a comparative value of the native cells reported.The establishment of the adenovirus-mediated gene transfer system wiil lead to the various applied sysem for screening of the brain-, liver and kidney oriented drug delivery and therapeutic tools.