窄頻中紅外光於鉤端螺旋體病免疫反應與黑色素癌之效應暨奈米力學在腸病毒檢測之研究

The Effect of Narrow Band Mid-Infrared on Immune Response of Leptospirosis and Melanoma & The Investigation of the Detection of Enterovirus Based on Nanomechanics

洪宗宏(Chung-Hung Hong)

指導教授 ： 李嗣涔

國立臺灣大學/電機資訊學院/電子工程學研究所/博士(2019年)

https://doi.org/10.6342/NTU201904397

摘要

隨著傳染性疾病與癌症導致之抗藥性威脅與日俱增，傳統的治療方法面臨著巨大的挑戰。因此，開發替代性之療法是當前無法避免的趨勢。此外，近年來研究發現紅外光可以調節細胞生理機能，例如影響基因表達、調節粒線體之機制及刺激免疫反應，進而可以調控細胞之生長與代謝。本論文之目的是設計一套可以結合可攜式窄頻中紅外光發射器之細胞培養箱，並進一步研究受到窄頻中紅外光照射後之鉤端螺旋體病的免疫反應與黑色素癌細胞之反應機制。另外，除了研究窄頻中紅外光之生物效應外，本論文另外開發一種透過改良原子力顯微鏡來直接檢測腸病毒71型之技術。
先前的研究發現中紅外光照射對於癌細胞有重大影響，然而窄頻中紅外光對於免疫反應與傳染性疾病之影響仍然未知。因此，本論文之第一個主題是研究窄頻中紅外光對於已受到鉤端螺旋體感染之宿主細胞的影響。本研究中通過鉤端螺旋體外膜蛋白(LipL32)與類鐸受體(TLR2)之間的交互作用，觀察到先天免疫反應的增強。此後，近端腎小管細胞啟動了一系列增強單核球趨化蛋白-1產生之反應。利用波導型窄頻中紅外光發射器產生的六微米波長光源在抗原受體複合物產生階段誘導羰基(C=O鍵)進行拉伸振動，在低劑量抗原(LipL32)刺激下與無受到照射之組別相比，窄頻中紅外照射後的實驗組之單核球趨化蛋白-1基因表達量增加了2.5倍。此外，透過酵素免疫分析法與共軛焦熒光顯微鏡也同時發現通過窄頻中紅外光照射後，低濃度與高濃度抗原(LipL32)刺激的組別之單核球趨化蛋白-1濃度均有顯著增加之現象。另外，本研究發現到一種特殊現象，即窄頻中紅外光透過增強抗原與受體之間的交互作用可以放大微弱的免疫反應之訊號。本研究顯示了明確之證據，即窄頻中紅外光照射可以調節傳染性疾病之早期免疫反應，並且在未來開法宿主定向治療中可發揮潛在作用。
本論文之第二個主題是研究檸檬醛與窄頻中紅外光刺激下對於黑色素癌細胞之影響。實驗結果表明，檸檬醛可以有效抑制黑色素癌細胞之生長。此外，窄頻中紅外光除了可以抑制黑色素癌細胞成長外，並且進一步被發現窄頻中紅外光可以通過抑制活性氧類之產生來調節腫瘤微環境，並經由細胞骨架重新排列而影響細胞遷移。
本論文之第三個主題是開發一種直接檢測腸病毒71型的方法。手足口病是一種對於兒童具有高度傳染力之常見疾病，而腸病毒71型是引起該疾病的主要病原體之一。因此本研究之目的是提出一種快速並且有效之技術且基於生物分子間作用力之機制並通過改良式的原子力顯微鏡技術來直接檢測腸病毒71型。首先，將腸病毒71型顆粒固定至雲母表面，並通過數種化學方法將腸病毒71型抗體固定在原子力顯微鏡探針上。然後使用化學修飾過原子力顯微鏡探針並通過接觸模式測量腸病毒71型顆粒與其抗體之結合力。最後藉由使用原子力成像計算軟體，腸病毒71型之尺寸（平均值±標準差）為31.36±3.87 奈米，該結果與以前文獻一致。此外，腸病毒71型抗原與抗體之間結合力（平均值±標準差）為336.9±64.7皮牛頓，腸病毒71抗體與非特異性樣本之間的力（平均值±標準差）為47.1±15.1皮牛頓且顯著性較小。很明顯結果表明透過測量力之大小並觀察腸病毒71型顆粒/抗體之解除結合事件之發生可以在樣品之間準確地判別出是否有腸病毒71型之感染。因此，結合原子力顯微鏡系統與化學修飾之探針具有成為直接檢測腸病毒71型的快速有效方法之潛力。

關鍵字

窄頻中紅外光發射器 ； 鉤端螺旋體 ； 先天性免疫反應 ； 類鐸受體 ； 單核球趨化蛋白-1 ； 黑色素癌細胞 ； 原子力顯微鏡 ； 腸病毒71型 ； 量子化學

並列摘要

As the threats of resistances of infections and cancer diseases increase gradually, traditional therapeutics confront the tremendous challenges. Therefore, the developments of alternative therapies are unavoidable trends currently. In addition, recent studies found that infrared light can regulate cell physiological functions such as affecting the gene expressions, modulating the mechanisms of mitochondria, and stimulating the immune response, and thereby the cell growth and metabolism can be regulated by infrared light. The purpose of this dissertation is to design the cell incubator equipped with portable narrow band mid-infrared emitters, and further investigate the mechanisms of the immune response of leptospirosis and melanoma exposed to narrow band mid-infrared. In addition to study the biological effects of narrow band mid-infrared, this dissertation was to develop a technique for direct detection of enterovirus 71 (EV71) by the modified atomic force microscopy additionally.
Previous studies revealed significant impact on cancer cell by mid-infrared exposure. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. Therefore, the first topic of this dissertation was to study the effect of narrow band mid-infrared on the host cell infected with leptospira. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 μm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (C=O bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.
The second topic of this dissertation was to investigate the effects of melanoma cells treated with citral or stimulated by narrow band mid-infrared exposure. The results revealed that citral could inhibit the growths of melanoma cells effectively. In addition, narrow band mid-infrared could inhibit the developments of melanoma cells, and it was further found that narrow band mid-infrared could modulate the tumor microenvironment by restraining the production of ROS and affect the cell migration as a result of the cytoskeleton rearrangement.
The third topic of this dissertation was to develop an approach for direct detection of EV 71. Hand, Foot and mouth disease (HFMD) is a common disease with high infectivity for children, and enterovirus 71 (EV71) is one of the main pathogens to cause the type of illness. Therefore, the aim of this study was to propose a rapid and effective technique for detecting EV71 directly based on the mechanism of biological intermolecular force by using the technique of modified atomic force microscopy (AFM). At first, EV71 particles were coated on the mica surface and made the EV71 antibodies (anti-EV71) fixed on the AFM tip by means of several chemical procedures. Then, AFM chemically modified tip was applied to measure the unbinding forces between EV71 and anti-EV71 by contact mode. Finally, by using AFM imaging calculating software, the EV71 particle size (mean±SD) was 31.36±3.87 nm (n = 200) and this result was concordance with previous literature. Besides, the force (mean±SD) between EV71 antigen and antibody complex was 336.9±64.7 pN. The force (mean±SD) between anti-EV71 and non-specific specimens was 47.1±15.1 pN and was significantly smaller. Apparently, the results show that EV71 infection could be identified precisely among the samples by measuring the force magnitude and observing the occurrence of EV71/anti-EV71 unbinding events. Therefore, the combination of AFM system and the chemically modified tip has the potential to be a rapid and effective method for detecting EV71 directly.

並列關鍵字

narrow band mid-infrared emitter ； Leptospira ； innate immune response ； toll-like receptor (TLR) ； monocyte chemotactic protein-1 (MCP-1) ； melanoma cell ； atomic force microscopy ； enterovirus 71 ； quantum chemistry

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