建構 CCL5 報導細胞平台以作為評估呼吸致敏物的非動物替代方法

Abstract

現代人在日常生活中暴露於各類化學物質的機會日益增加。其中，許多為低分子量的呼吸致敏物，可經由吸入途徑進入人體，誘發免疫反應，進而引起呼吸道致敏的現象。目前，化學物質所引起之呼吸致敏的診斷主要仰賴臨床個案報告，而相關的動物模式與細胞實驗研究仍相對有限。因此，極需更深入的探討與建立評估之方法。呼吸致敏物的鑑別與風險評估，為化學品毒理學中一項關鍵的安全性評估指標。如何在保障人類健康的同時，減少實驗動物使用，已成為全球毒理學領域極需面對的重要課題。因此，發展替代方法與預測模型，成為化學品安全評估制度的核心策略。有鑑於皮膚致敏與呼吸致敏有相似的免疫活化機制，因此，參考已成熟的皮膚致敏危害結局路徑架構，可能有助於研究呼吸致敏危害結局路徑的發展。在氣喘患者中，趨化因子CCL5於呼吸道上皮細胞中高度表達，顯示其可能在呼吸道發炎病理中扮演關鍵角色。CCL5可透過與趨化因子受器結合，誘導免疫細胞的活化與遷移，並引導其聚集至發炎部位，進一步放大免疫反應。在本研究中，我們透過設計以趨化因子CCL5為標的的螢光素酶質體，並將其轉染至人類非小細胞肺癌細胞株A549細胞株中，建立穩定表現的螢光素酶報導基因試驗系統。此系統旨在探討CCL5是否可作為一個靈敏且具代表性的分子終點，用於識別呼吸致敏物，提供一項可行且具科學基礎的評估工具，強化化學品毒理風險評估的精準度。 本研究評估了12種化學品，其包含7種呼吸致敏物: 氯鉑酸銨、頭孢美唑、戊二醛、海卓拉肼鹽酸鹽、六亞甲基二異氰酸酯、哌嗪、螺旋黴素；以及5種非呼吸致敏物: 甘油、異丙醇、乳酸、1,2-丙二醇、水楊酸。將12種化學品分別刺激A549、A549-NF-κB以及A549-NF-κB-NF-IL6-ISRE三種細胞株，並檢測細胞株在化學品刺激後的CCL5蛋白以及CCL5 mRNA的程度。藉由比較A549- NF-κB以及A549-NF-κB-NF-IL6-ISRE細胞模型對不同化學物質的反應特性，探討這兩種細胞株在呼吸道致敏物識別上的適用性與敏感性。 我們發現呼吸致敏劑顯著上調CCL5的蛋白和基因表達量，說明呼吸致敏劑可能參與引發呼吸高反應性等免疫相關病理反應。此外，A549-NF-κB-NF-IL6-ISRE細胞模型展現出良好的靈敏度，可評估化學品的致敏潛力，說明A549-NF-κB-NF-IL6-ISRE細胞模型作為替代試驗平台的應用潛力。

People are increasingly exposed to various chemical substances of low molecular weight respiratory sensitizers in their daily lives, which can enter the human body through inhalation, induce immune responses, and cause respiratory sensitization. Currently, the diagnosis of respiratory sensitization caused by chemicals mainly relies on clinical case reports, while related animal models and cell experimental studies are still relatively limited, therefore, more in-depth exploration and development of evaluation methods is needed. Identification and risk assessment of respiratory sensitizers is a key safety assessment indicator in chemical toxicology. How to reduce the use of experimental animals while protecting human health has become an important issue that the global toxicology field needs to face. Therefore, the development of alternative methods and predictive models have become the core strategies for the chemical safety assessment system. Because of the, skin sensitization and respiratory sensitization have similar immune activation mechanisms, therefore, we need to the recognized skin sensitization Adverse Outcome Pathway (AOP) framework to develop the respiratory sensitization adverse outcome pathway. In asthmatic patients, C-C Motif Chemokine Ligand 5 (CCL5) is highly expressed in airway epithelial cells, suggesting that it may play a key role in airway inflammatory pathology. CCL5 can bind to chemokine receptors to induce the activation and migration of immune cells, and guide them to recruit at the site of inflammation, further amplifying the immune response. In this study, we designed a luciferase plasmid targeting the chemokine CCL5 and transfected it into the human non-small cell lung cancer cell line A549 to establish a stable expression luciferase reporter gene assay system. This system aims to explore whether CCL5 can be used as a sensitive and representative molecular endpoint to identify respiratory sensitizers, providing a feasible and scientifically based assessment tool to enhance the accuracy of chemical toxicological risk assessment. This study evaluated twelve chemicals, including seven respiratory sensitizers: ammonium chloroplatinate (ACP), cefmetazole (CMZ ), glutaraldehyde (GA), hydralazine hydrochloride (HHC), hexamethylene diisocyanate (HMDI), piperazine (PPZ), and spiramycin (SPM); and five non-respiratory sensitizers: glycerol (GLY), isopropanol (ISO), lactic acid (LA), 1,2-propylene glycol (PG), and salicylic acid (SA). twelve chemicals were used to stimulate three cell lines, A549, A549-NF-κB, and A549-NF-κB-NF-IL6-ISRE, respectively, and the CCL5 protein level and CCL5 mRNA level of the cell lines after chemical stimulation were detected. By comparing the response characteristics of A549-NF-κB and A549-NF-κB-NF-IL6-ISRE cell models to different chemicals, the applicability and sensitivity of these two cell lines in the recognition of respiratory allergens were explored. We found that respiratory sensitizers significantly upregulated the protein and gene expression of CCL5, indicating that respiratory sensitizers may be involved in inducing immune-related pathological reactions such as respiratory hyperresponsiveness. In addition, the A549-NF-κB-NF-IL6-ISRE cell model showed good sensitivity and could evaluate the sensitizing potential of chemicals, indicating the potential application of the A549-NF-κB-NF-IL6-ISRE cell model as an alternative test platform.