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  • 學位論文

第一型及第二型有機陽離子轉運蛋白在大腦微血管內皮細胞的分布位置以及其與MPTP所造成紋狀體毒性關係之研究

Cellular localization of the organic cation transporters, OCT1 and OCT2, in brain microvessel endothelial cells and its implication for MPTP-induced dopaminergic toxicity

指導教授 : 林君榮

摘要


攝入1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) 會增加其具毒性之代謝物1-methyl-4- phenylpyridinium (MPP+) 於大腦的量,並進而選擇性的對多巴胺神經產生毒性,使得腦中多巴胺濃度降低,造成類似巴金森氏症的症狀。Amantadine在臨床上可用於治療巴金森氏症,但其機制尚未完全清楚。已知amantadine為有機陽離子轉運蛋白 (organic cation transporters,OCTs) 的受質。本研究目的於探討amantadine對於MPTP在血腦屏障穿透與MPTP引發之多巴胺毒性的影響,同時,本研究也探討腦血管內皮細胞上OCTs的表現和分布位置,以及OCTs在腦血管內皮細胞攝取MPTP扮演中的角色。 由腦部微透析實驗發現amantadine明顯的降低了大鼠與小鼠之腦透析外液中MPTP與MPP+時間對濃度做圖的曲線下面積;同樣地,相較於只給予MPTP的組別,amantadine明顯增加了小鼠腦部紋狀體中多巴胺與多巴胺代謝物濃度。小動物正子掃描之實驗結果顯示MPTP在小鼠腦中所引起的多巴胺毒性可被amantadine改善。在本研究中使用共軛焦顯微鏡和西方墨點法兩種實驗方式探討有機陽離子轉運蛋白OCT1與OCT2在腦血管內皮細胞上分布的位置,發現OCT1與OCT2主要分布在細胞膜的luminal側,並且在OCT1/OCT2基因靜默後腦血管內皮細胞攝取MPTP減少。 總結以上的實驗結果,我們發現amantadine可減少MPTP通過血腦屏障以及MPTP在囓齒類中引起的多巴胺毒性,並且OCT1與OCT2對MPTP通過血腦屏障扮演重要的角色。

並列摘要


Exposure to the chemical 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) can lead to the presence of its toxic metabolite, 1-methyl-4-phenylpyridinium (MPP+), in the brain, which induces a selective destruction of dopaminergic nigrostriatal neurons and the occurance of Parkinson’s disease. Amantadine is used in the treatment of Parkinson’s disease. However, its mechanism is not fully understood. Amantadine is known to be as substrate of organic cation transporters (OCTs). The purpose of this study was to evaluate the effects of amantadine on blood-brain barrier (BBB) transport of MPTP, and on MPTP-induced dopaminergic toxicity. Also, the expression and cellular localization of OCTs were investigated. The role of OCTs on MPTP uptake was studied in brain microvessel endothelial cells (BMECs). The results of in vivo brain microdialysis showed that amantadine significantly reduced the area under the time-concentration curves of MPTP and MPP+ in brain extracellular fluid in rats and mice. Likewise, amantadine increased the level of dopamine and dopamine metabolites in brain striatum of mice, compared with mice recieving MPTP only. PET scan showed amantadine ameliorated MPTP-induced dopaminergic toxicity in the brain of mice. The confocal and Western blot analyses showed that OCT1 and OCT2 were mainly expressed on the luminal side of BMECs and adult rat brain endothelial cells (ARBECs). Cellular uptake of MPTP was decreased with OCT1/OCT2 silencing in BMECs and ARBECs. In conclusion, amantadine reduces the BBB transfer of MPTP and MPTP-induced dopaminergic toxicity in rodents, and OCT1 and OCT2 are important for MPTP transfer across the BBB.

參考文獻


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