Genetic introduction of thyroid hormone transporter into the Bood-Brain-Barrier

通过血脑屏障系统实现运输系统的重建是非常重要的。最近，我们发现有机阴离子转运体OATP2和OATP3在脉络丛中表达，并运输甲状腺激素。本研究的目的是建立一个腺病毒介导的基因转移系统，将药物输送到脑内。将我们已分离到的LST-1和LST-2基因的编码区插入到腺病毒载体中，包装后与腺病毒基因组DNA共转染HEK293细胞，产生同源重组的细胞尖叫并形成空斑。用氯化铯超速离心法纯化重组腺病毒，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蛋白均有较强的表达。Western印迹分析也证实了条带的大小。表达LST-1和LST-2的哺乳动物细胞可运输各种有机阴离子作为底物，其摄取牛磺胆酸的表观Km值分别为24微米和32微米，与已报道的天然细胞的Km值相当。我们的研究表明，我们的系统是非常有效和有用的工具，通过细胞膜输送药物。

It is very important to claify the transport system via blood-brain barrier system. Recently, we have identified that organic anion transporter oatp2 and oatp3 are expressed in the choroid plexus and it transport thyroid hormones.The aim of this study is to extablished an adenovirus-mediated gene transfer system into cells to deliver drugs in to brain. 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 taurocholate uptake is 24 micro M and 32 micro M, respectively and both of which is a comparative value of the native cells reported. Our study revaled that our system is very effective and useful tool to deliver drugs through cell membrane.