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

甲状腺激素转运蛋白在血脑屏障中的基因导入

• 批准号: 11557188
• 负责人: ABE Takaaki
• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557188/
• 关键词: organic anion transporter; Thyroid hormone transporter; adenovirus; blood-brain-barrier; choroid plexus; gene transfer; トランスポーター; 有機アニオン; プロスタグランジン; 胆汁酸; 海馬

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.

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。

项目成果

Abe T et al.: "Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1."J Biol Chem. 274. 17159-17163 (1999)

Abe T 等人：“编码人肝脏特异性有机阴离子转运蛋白 LST-1 的新基因家族的鉴定。”J Biol Chem。

Abe T., Kakyo M., Tokui T., Nakagomi R., Nishio T., Nakai D., Nomura H., Unno M., Suzuki M., Naitoh T., Matsuno S.and Yawo H.: "Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1."J.Biol.Chem.. 274. 17159-1

Abe T.、Kakyo M.、Tokui T.、Nakagomi R.、Nishio T.、Nakai D.、Nomura H.、Unno M.、Suzuki M.、Naitoh T.、Matsuno S.和 Yawo H.：“识别

Kakyo M., Sakagami H., Nishio T., Nakai D., Nakagomi R., Tokui T., Naitoh T., Matsuno S., Kondo H., Abe T., and Yawo H.: "Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide2 (oatp2)."FEBS Lett.. 4

Kakyo M.、Sakagami H.、Nishio T.、Nakai D.、Nakagomi R.、Tokui T.、Naitoh T.、Matsuno S.、Kondo H.、Abe T. 和 Yawo H.：“免疫组织化学分布和功能

Tokui T. et al.: "Pravastatin, an HMG-CoA reductase inhibitor, is transported by rat liver organic anion transporting polypeptide, oatp2."Pharmaceutical Res.. 16. 912-916 (1999)

Tokui T. 等人：“普伐他汀是一种 HMG-CoA 还原酶抑制剂，由大鼠肝脏有机阴离子转运多肽 oatp2 转运。”Pharmaceutical Res.. 16. 912-916 (1999)

Abe T., Kakyo M., Sakagami H., Tokui T., Nishio T., Tanemoto M., Nomura H., Hebert S.C., Matsuno S., Kondo H., and Yawo H.: "Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transport thyroid hormo

Abe T.、Kakyo M.、Sakagami H.、Tokui T.、Nishio T.、Tanemoto M.、Nomura H.、Hebert S.C.、Matsuno S.、Kondo H. 和 Yawo H.：“分子特征和组织分布