利用蛋白質體學探究體外血腦障壁模組暴露奈米銀影響腦神經細胞退化之研究

A Proteomic Study of Neurodegenerative Effect of Exposure to Silver Nanoparticles in an in Vitro Blood-Brain Barrier Model

林合真(Ho-Chen Lin)

指導教授 ： 莊淳宇

國立清華大學/原子科學院/生醫工程與環境科學系/博士(2019年)

摘要

奈米銀(silver nanoparticles, AgNPs)具有抗菌特性，由於廣泛應用於食物容器、牙膏及紡織物中，增加人類暴露AgNPs的機會。已有小鼠暴露實驗指出AgNPs可通過血腦障壁(blood-brain barrier, BBB)進入中樞神經系統，累積於腦部誘發活性氧化物質(reactive oxygen species, ROS)產生，導致神經細胞損傷。先前本研究室實驗亦發現單層培養小鼠神經元細胞暴露AgNPs後，會誘導類澱粉蛋白(amyloid beta, Aβ)生成，腦部Aβ沉積與臨床上阿茲海默症(Alzheimer’s disease, AD)病程發展有關。BBB是由腦血管內皮細胞的緊密連接蛋白(tight junction protein)建構而成之腦部屏障，圍繞在血管內皮細胞旁之神經膠質細胞能將通過BBB之物質經過篩選後供給神經元細胞使用。由於AgNPs暴露通過腦部細胞BBB後，對於影響神經膠質細胞和神經元細胞代謝功能，進而可能驅使AD發展在蛋白質體方面之探究仍不明確。因此，本研究以小鼠腦內皮細胞(mouse brain endothelial cells, bEnd.3)、小鼠腦星狀細胞(mouse brain astrocytes, ALT)及小鼠腦神經母細胞(mouse neuroblastoma neuro-2a cells, N2a)建構三層細胞之共培養系統，來模擬腦部細胞暴露AgNPs通過BBB之情形，並利用蛋白質體學(proteomics)探討暴露AgNPs是否改變神經元細胞內蛋白質生成，進一步瞭解受到影響之蛋白質是否與神經退化性疾病有關。此外，本研究亦探討星狀細胞及神經元細胞中Aβ生成以及血管內皮細胞清除Aβ能力，以觀察AgNPs是否影響代謝Aβ的能力。研究結果顯示AgNPs會破壞bEnd.3細胞緊密連接蛋白claudin-5及zona occludens-1 (ZO-1)之完整性和促使BBB通透性增加，能累積於ALT細胞及N2a細胞之細胞質中。蛋白質體學分析結果發現，N2a細胞暴露AgNPs後有298個蛋白質表現改變(differentially expressed proteins)，這些蛋白質與脂肪酸(fatty acid)代謝有關聯性，其中棕櫚酸(palmitic acid)的增加可能會促使AD特徵蛋白Aβ生成。此外，AgNPs暴露會增加Aβ前驅蛋白(beta amyloid precursor protein, APP)生成，誘發Aβ生成相關之分泌酵素(secretase)，如早老素1 (presenilin-1, PSEN1)、早老素2 (PSEN2)及β-分泌酵素(beta-site APP cleaving enzyme, BACE)剪切APP，造成Aβ40及Aβ42累積。另一方面，AgNPs暴露降低bEnd.3細胞中Aβ清除受器(receptor)的基因表現，包含p-醣蛋白 (p-glycoprotein, p-gp)及低密度脂蛋白受體相關蛋白-1 (low density lipoprotein receptor-related protein 1, LRP-1)，導致類澱粉蛋白清除(Aβ clearance)能力降低。增加的Aβ堆積於N2a細胞表面，促進細胞分泌單核細胞趨化蛋白-1 (monocyte chemoattractant protein-1, MCP-1)及細胞激素介白素-6 (interleukin 6, IL-6)，產生發炎反應及誘發N2a細胞自然凋亡(apoptosis)。因此，本研究推測AgNPs暴露會破壞腦內皮細胞緊密連接蛋白，增加BBB通透性，增加神經元細胞中棕櫚酸含量，促進Aβ生成並降低清除Aβ能力，導致Aβ累積及發炎反應，促使神經元細胞凋亡，此可能會誘發阿茲海默症病症之發展。

關鍵字

奈米銀 ； 血腦障壁 ； 緊密連接蛋白 ； 類澱粉蛋白 ； 阿茲海默症 ； 蛋白質體學

並列摘要

Silver nanoparticles (AgNPs) have the antibacterial feature, and widely used in daily supplies such as food container, toothpaste and textiles. Therefore, human may suffer the increased risk of exposure to AgNPs. Previous studies in mice exposure to AgNPs presented that AgNPs can enter the central nervous system through the blood-brain barrier (BBB), and accumulate in the brain to induce reactive oxygen species (ROS) and cause neural cells damage. Our previous study also has found that exposure to AgNPs increases amyloid beta (Aβ) generation in mouse mono-cultured neuronal cells. In clinical, the deposition of Aβ in the brain is a hallmark of the progression of Alzheimer’s disease (AD). The BBB is a brain barrier constructed by tight junction proteins of brain vascular endothelial cells. The astrocytes surrounding the vascular endothelial cells can select the substances passing through BBB to supply to the neuronal cells. Since the exposure of AgNPs can pass through BBB, whether AgNPs affect the metabolism of astrocytes and neuronal cells and subsequently lead to AD progression remains unclear in proteomic exploration. Thus, this study constructed a triple-cell coculture model using mouse brain endothelial (bEnd.3) cells, mouse brain astrocytes (ALT), and mouse neuroblastoma neuro-2a (N2a) cells to simulate the exposure of brain cells to AgNPs through BBB for investigating whether exposure to AgNPs alters protein production in neuronal cells, and further understanding whether these affected proteins were associated with neurodegenerative disease. Moreover, this study also explored the generation of Aβ in astrocytes and neuronal cells, and the clearance activity of Aβ in endothelial cells to observe whether AgNPs alter Aβ metabolism in the brain. The results of this study showed that AgNPs disrupted the integrity of tight junction proteins, claudin-5 and zona occludens-1 (ZO-1) to increase BBB permeability in bEnd.3 cells, and accumulated in the cytoplasm of ALT cells and N2a cells. The proteomic profiling of N2a cells after AgNPs exposure identified 298 differentially expressed proteins related to fatty acid metabolism. The AgNPs induced palmitic acid production might promote Aβ generation. In addition, exposure to AgNPs increased the protein expression of amyloid precursor protein (APP) and Aβ generation-related secretases, presenilin-1 (PSEN1), presenilin-2 (PSEN2), and beta-site APP cleaving enzyme (BACE), for APP cleavage to stimulate Aβ40 and Aβ42 accumulation. Additionally, AgNPs decreased the gene expression of Aβ clearance-related receptors, p-glycoprotein (p-gp) and low density lipoprotein receptor-related protein-1 (LRP-1), in bEnd.3 cells to attenuate the activity of Aβ clearance. The increased Aβ further aggregated on the surface of N2a cells to enhance the secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6), to induce inflammatory response and cause the programed cell death. Thus, this study suggested that exposure to AgNPs can disrupt tight junction proteins to increase BBB permeability of brain endothelial cells, and increase the production of palmitic acid and attenuate the Aβ clearance activity in neuronal cells to induce inflammation and apoptosis subsequently for AD progression.

並列關鍵字

silver nanoparticles ； blood-brain barrier ； tight junction protein ； amyloid ； Alzheimer’s disease ； proteomics

參考文獻

Y. Lee, P. Kim, J. Yoon, B. Lee, K. Choi, K. H. Kil, and K. Park, Serum kinetics, distribution and excretion of silver in rabbits following 28 days after a single intravenous injection of silver nanoparticles. Nanotoxicology, 2013. 7(6): p. 1120-1130.

F. Piccinno, F. Gottschalk, S. Seeger, and B. Nowack, Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. Journal of Nanoparticle Research, 2012. 14(9).

Silver Nanoparticles Market Size By Application (Healthcare & Lifesciences, Textiles, Electronics & IT, Food & Beverage), Industry Analysis Report, Regional Outlook (U.S., Canada, Germany, UK, France, Italy, Spain, Poland, Russia, Netherlands, China, India, Japan, South Korea, Australia, Indonesia, Malaysia, Brazil, Argentina, Mexico, Saudi Arabia, UAE, South Africa), Growth Potential, Price Trends, Competitive Market Share & Forecast, 2016 – 2024. Global Market Insights, 2017.

Q. L. Li, S. Mahendra, D. Y. Lyon, L. Brunet, M. V. Liga, D. Li, and P. J. J. Alvarez, Antimicrobial nanomaterials for water disinfection and microbial control: Potential applications and implications. Water Research, 2008. 42(18): p. 4591-4602.

X. Jiang and A. Yu, Low dimensional silver nanostructures: synthesis, growth mechanism, properties and applications. Journal of Nanoscience and Nanotechnology, 2010. 10(12): p. 7829-7875.