近年來，奈米科技蓬勃發展，奈米材料被廣泛應用於日常生活中，由於銀具有抗菌殺菌效果，將銀奈米化生成之銀奈米粒子(silver nanoparticle, AgNPs)，表面積大幅增加，提升抗菌效果，廣泛應用於日常用品中，增加人體暴露AgNPs機會。已有研究指出AgNPs可透過皮膚接觸、攝食及吸入方式進入人體，透過血液循環系統至各器官，並且可經由腦微血管通過血腦屏障進入腦組織及中樞神經系統中，產生活性氧物質(reactive oxygen species)，造成神經細胞發炎反應和神經細胞凋亡，進而可能誘發神經退化性疾病，但目前對於AgNPs如何破壞血腦屏障進入神經系統中仍不清楚。因此，本研究目的為探討AgNPs暴露是否增加體外血腦屏障通透度及減少緊密連接(tight junctions)分泌之情形，以及觀察AgNPs是否通過血腦屏障進入腦神經細胞中，影響發炎相關基因表現。
本研究利用小鼠腦微血管內皮bEnd.3細胞(mouse brain endothelial cells)、星狀膠ALT細胞(mouse brain astrocyte cells)及神經元N2a細胞(mouse neuroblastoma)之三層細胞共培養系統，模擬體外血腦屏障暴露AgNPs，觀察其對於三層細胞共培養系統之緊密連接及發炎影響。結果顯示單層bEnd.3細胞暴露AgNPs (1、2及4 g/mL) 24小時，會增加發炎和氧化還原相關基因(C-X-C motif chemokine 13 (CXCL13)、macrophage receptor with collagenous structure (MARCO)、glutathione synthetase (GSS)、tumor necrosis factor α (TNFα)、interlukin-6 (IL-6)及interlukin-1β (IL-1β))表現，降低緊密連接之claudin-5 (Cldn-5)基因和蛋白表現及其上游基因low density lipoprotein receptor-related protein 5 (Lrp5)表現，對於緊密連接之zona occludens-1 (ZO-1)及occludin (Ocln)基因表現則無顯著影響。在bEnd.3細胞與ALT細胞共培養系統中，隨著AgNPs暴露濃度上升，bEnd.3細胞電阻值越低，導致bEnd.3細胞通透度上升，使AgNPs通過血腦屏障。三層細胞共培養系統暴露AgNPs (2、3及4 g/mL) 24小時後，bEnd.3細胞中發炎相關基因interleukin-2 (IL-2)、interleukin-17A (IL-17A)、interferon gamma (IFN-γ)、IL-6、IL-1β和monocyte chemoattractant protein-1 (MCP-1)表現量下降；ALT細胞中IL-2、IL-17A、IFN-γ和MCP-1基因表現量上升，IL-1β和IL-6基因表現量下降；N2a細胞中僅MCP-1基因表現量上升。此外，三層細胞共培養系統暴露AgNPs，在上層培養液中增加MCP-1和IL-6蛋白分泌量，降低IL-1β蛋白分泌量，在下層培養液中增加MCP-1蛋白分泌量。
本研究得知在單層bEnd.3細胞暴露AgNPs增加發炎相關基因表現及降低緊密連接Cldn-5 基因表現。在三層細胞共培養系統中，AgNPs暴露降低bEnd.3細胞緊密連接蛋白claudin-5和ZO-1分泌，增加細胞通透度，使AgNPs通過血腦屏障，促使ALT細胞發炎相關基因表現及MCP-1蛋白分泌，但N2a細胞之發炎相關基因表現大多未受影響，顯示bEnd.3細胞和ALT細胞能抵禦AgNPs對於N2a細胞之影響。由上述結果可知體外血腦屏障模型暴露AgNPs會影響緊密連接及發炎相關蛋白及基因表現，因此AgNPs對於人體影響應受關注。

Due to the vigorous development of nanotechnology, nanomaterials are used in a variety of fields including commercial products and biomedical applications. Silver nanoparticles (AgNPs) are applied to daily utilities due to antimicrobial activity, which increase the chance of AgNPs exposure in human. AgNPs can get into human body via dermal contact, ingestion and inhalation, and distribute to various organs via blood circulation. AgNPs can cross through blood brain barrier (BBB) to potentially induce neurotoxicity for neurodegenerative diseases. However, there is not clearly how AgNPs disrupt BBB. Thus, this study investigated the potential effects of 3-5 nm AgNPs on inflammation and tight junctions individually in mouse brain endothelial bEnd3 cells and in vitro model of mouse BBB.
In vitro model of mouse BBB was constrcted by bEnd3 cells, mouse brain ALT astrocytes and mouse neuroblastoma N2a cells to investigate the potential effects of 3-5 nm AgNPs on gene expression of inflammation and tight junctions. The results showed AgNPs exposure increased the gene expression relevant to inflammation (CXC motif chemokine 13 (CXCL13), macrophage receptor with collagenous structure (MARCO), glutathione synthetase (GSS), tumor necrosis factor α (TNFα), interlukin-6 (IL-6) and interlukin-1β (IL-1β)) and decreased the gene expression of claudin-5 (Cldn-5) and Lrp5 in bEnd.3 cells. The zona occludens-1 (ZO-1) and occludin (Ocln) gene expression had no significant effect in bEnd.3 cells after AgNPs exposure. In bEnd.3 cells and ALT cells co-culture system, as AgNPs exposure concentration increased, the lower the TEER value in bEnd.3 cells which lead to AgNPs cross through BBB. Additionally, immunofluorescence images showed reduction of claudin-5 and ZO-1 protein for BBB disruption in bEnd.3 cells of co-culture system. After the triple cell co-culture system exposed to AgNPs (2, 3 and 4 g/ml) for 24 hour, the gene expression relevant to inflammation (interleukin-2 (IL-2)、interleukin-17A (IL-17A)、interferon gamma (IFN-γ)、IL-6、IL-1β and monocyte chemoattractant protein-1 (MCP-1) decreased in bEnd.3 cells；The gene expression of IL-2, IL-17A, IFN-γ and MCP-1 increased in ALT cells；Only the MCP-1 gene expression increased in N2a cells. Besides, 4 g/mL AgNPs exposure increased MCP-1 and IL-6 cytokine secreation and decreased IL-1β cytokine secreation in the upper medium of in triple cell co-culture system.
This study found that AgNPs exposure increased the gene expression relevant to inflammation and decreased the gene expression of Cldn-5 in bEnd.3 cells. In triple cell co-culture system, AgNPs exposure reduced claudin-5 and ZO-1 protein expression leading to permeability of bEnd.3 cells rise. AgNPs can cross through BBB leading to inflammation in ALT cells. Besides, AgNPs exposure obviously altered the gene expression of tight junctions and inflammation in vitro model of mouse BBB. Therefore the effect of AgNPs to human body should be concern.

摘要 2
Abstract 4
目錄................................................................................................................................6
表目錄 9
圖目錄 11
第一章 前言 13
第一節 研究動機 13
第二節 研究目的 14
第二章 文獻回顧 16
第一節 奈米銀AgNPs 16
第二節 AgNPs暴露路徑 16
第三節 AgNPs的毒性 17
第四節 AgNPs導致毒性機制 18
第五節 腦內皮細胞與血腦屏障 19
第六節 緊密連接 20
第七節 神經血管單元 21
第八節 血腦屏障與神經退化性疾病 22
第九節 發炎與緊密連接相關基因 23
第三章 材料與方法 25
第一節 實驗材料...............................................................................................25
第二節 細胞培養 26
第三節 三層細胞共培養模型建立 26
第四節 RNA萃取 26
第五節 反轉錄和定量聚合酶鏈鎖反應定量奈米銀暴露細胞基因表現 27
第六節 內皮細胞增生率之測定 29
第七節 免疫螢光染色 29
第八節 跨內皮細胞膜之電阻值測定 30
第四章 結果 31
第一節 AgNPs暴露對於bEnd.3細胞生存率之影響 31
第二節 AgNPs暴露對於bEnd.3細胞之發炎相關基因表現之影響 32
第三節 AgNPs暴露對於bEnd.3細胞緊密連接之claudin-5及其上游基因表現之影響.............................................................................................34
第四節 AgNPs暴露對於bEnd.3細胞緊密連接之ZO-1及其上游基因表現之影響 35
第五節 AgNPs暴露對於bEnd.3細胞緊密連接之occludin及其上游基因表現之影響 36
第六節 AgNPs暴露對於共培養系統之bEnd.3細胞緊密連接之claudin-5和ZO-1蛋白表現之影響 37
第七節 共培養bEnd.3細胞與ALT細胞之跨內皮細胞膜電阻值測定 38
第八節 AgNPs暴露對於bEnd.3細胞與ALT細胞共培養系統之跨內皮細胞膜電阻值變化.....................................................................................39
第九節 AgNPs進入三層細胞共培養系統之N2a細胞 40
第十節 AgNPs暴露於三層細胞共培養系統之bEnd.3細胞緊密連接之claudin-5及其上游基因表現之影響 41
第十一節 AgNPs暴露對於三層細胞共培養系統之bEnd.3細胞緊密連接之ZO-1及其上游基因表現之影響 42
第十二節 AgNPs暴露對於三層細胞共培養系統之bEnd.3細胞緊密連接之occludin及其上游基因表現之影響 43
第十三節 AgNPs暴露對於三層細胞共培養系統之發炎相關基因表現之影響 44
第十四節 AgNPs暴露於三層細胞共培養系統cytokine分泌至細胞培養液之
影響..........................................................................................................46
第五章 討論 47
第一節 AgNPs暴露影響bEnd.3細胞發炎相關基因表現 48
第二節 AgNPs影響緊密連接蛋白表現 49
第三節 AgNPs影響三層細胞共培養系統緊密連接與發炎相關基因表現及
蛋白分泌 50
第六章 結論 53
第七章 參考文獻 54

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