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

将甲状腺激素转运蛋白基因引入血脑屏障

基本信息

批准号：
11557001
负责人：
YAWO Hiromu
金额：
$ 8.64万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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.

阐明血脑屏障系统的转运系统非常重要。最近，我们发现有机阴离子转运蛋白oatp2和oatp3在脉络丛中表达，并转运甲状腺激素。本研究的目的是建立一个腺病毒介导的细胞基因转移系统，将药物输送到大脑。将我们分离得到的LST-1和LST-2的编码区导入腺病毒载体中，包装后用腺病毒基因组DNA转染HEK293细胞，产生同源重组的细胞尖叫并形成空斑。产生大量感染培养基后，通过CsCl超速离心机纯化重组腺病毒。测量纯化的LST-1和LST-2腺病毒的感染滴度，每种病毒的IC 50 分别为1.26和1.48 X 10^10 pfu。当重组病毒应用于培养细胞系时，LST-1和LST-2蛋白在应用后24小时内强烈表达。条带的大小也通过蛋白质印迹分析得到证实。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，牛磺胆酸盐摄取的表观Km值分别为24μM和32μM，并且这两者都是报道的天然细胞的比较值。我们的研究表明，我们的系统是通过细胞膜递送药物的非常有效和有用的工具。