引起巴金森氏症之相關因子的血腦屏障穿透之研究

Abstract

許多環境因子，如重金屬(鐵離子、銅離子以及錳離子)、殺蟲劑(paraquat、organochlorine和carbamate衍生物) 以及其他神經毒素(MPTP和rotenone)和巴金森氏症的發生有關;另外方面也發現保護因子如nicotine、selegiline以及caffeine，可以降低巴金森氏症的發生風險。 本實驗中我們以大腦微血管內皮細胞(SV-ARBEC)培養於transwell或者plate上藉此模擬中樞血腦屏障(Blood and Brain Barrier)利用此培養模式探討部分巴金森氏症的環境因子如何穿透血腦屏障到達中樞神經。我們發現以巴金森氏症神經毒素MPP＋在此可透過有機陽離子轉運蛋白organic cation transport 1 (OCT1)(63%)以及多巴胺轉運蛋白dopamine transporter (DAT)(36%)有效進入大腦微血管內皮細胞中，Km和Vmax 分別為11.81±0.95μM、7.55±0.59 pmole/ mg protein /10min，而且MPP＋能有效穿透大腦微血管內皮細胞到transwell另一側。 研究其他環境因子於MPP＋進入細胞的結果發現nicotine(0.5mM)、selegiline(0.5mM)、paraquat(0.5mM)、tetraethylammonium(0.5mM)、choline(0.5mM)、dopamine(0.5mM)以及rotenone (50µM)，可以有效抑制MPP＋進入細胞，分別降低為37.78%、21.48%、84.6%、62.82%、84.52%、60.9%、74.5%。而nicotine (0.5mM)存在之下使MPP＋之Km從11.81±0.95μM增加為46.73±1.49μM，Vmax則沒有改變，得知nicotine對於MPP＋抑制作用為競爭型抑制，此外nicotine也可有效進入大腦微血管細胞內Km、Vmax 分別為29.13±0.13μM、276.12±5.61 pmole/ mg protein /20s，以OCT抑制劑decynium22 (10μM)以及DAT抑制劑GBR12909 (5μM) 研究對於nicotine進入大腦微血管內皮細胞的影響，得知nicotine部分是經由OCT1 (16%)進入細胞但不經由DAT。另外 paraquat (3mM)存在之下使MPP＋之Vmax從7.55±0.59 pmole/ mg protein /10min降低為3.43±0.87 pmole/ mg protein /10min，Km則沒有改變，得知paraquat對於MPP＋抑制作用為非競爭型抑制。 在本實驗中發現nicotine可和透過競爭性抑制作用和MPP＋競爭OCT1進入細胞內，此結論可提供解釋抽煙為何能降低巴金森氏症的發生率，也提供一個相關的研究基礎理論在於防治巴金森氏症上。另外其他環境因子selegiline、TEA、choline、dopamine以及rotenone也可有效抑制MPP＋進入細胞，但抑制機轉尚未明確，這將是未來研究方向之一。

The etiology of Parkinson’s disease (PD) is unknown. Epidemiological studies suggest the risk factors of PD associated with environmental factors, such as heavy metals (iron, copper and manganese), pesticides (paraquat, organochlorine and carbamate derivatives) and neurotoxins (MPP+ and rotenone). On the other hand, nicotine, selegiline, and caffeine had been reported as protective factors of PD. The aim of our study was to investigate how environmental factors penetrate blood-brain-barrier (BBB) and enter the central nerve system contributing the pathogenesis of PD. We used neonatal rat brain endothelial cell (SV-ARBEC) culture on transwell or plate as our experimental model. The results showed that MPP+ can be taken up by SV-ARBEC cell and transported to the other side of the transwell by organic cation transporter (OCT) and dopamine transporter (DAT). Other environmental factors, such as nicotine (0.5mM), selegiline (0.5mM), paraquat (0.5mM), tetraethylammonium (0.5mM), choline (0.5mM), dopamine (0.5mM) and rotenone (50µM) can inhibit the uptake of MPP+ by the SV-ARBEC cells. The inhibitory ratio was 37.78%, 21.48%, 84.6%, 62.82%, 84.52%, 60.9%, and 74.5%, respectively. Nicotine (0.5mM) increased the Km of MPP+ from 11.81±0.95μM to 46.73±1.49μM without changing Vmax. It showed a competitive pattern for the inhibition of nicotine. OCT inhibitor (decynium22, 10µM), but not for DAT inhibitor (GBR 12909, 5µM), inhibited the accumulation of nicotine into SV-ARBEC cell (16%) suggestting that nicotine can enter the SV-ARBEC cell via OCT but not DAT . Paraquat (3mM) decreases the Vmax of MPP+ from 7.55±0.59 to 3.43±0.87 pmole/mg protein/10min without changing Km, indicating the non-competitive inhibitory mechanism. Our study suggested that nicotine effectively reduces the entrance of MPP+ into SV-ARBEC cell by a competitive manner. These results may explain the possibility that smoking probably decrease the risk of PD.