| 摘要: | 癌症是全球重大的公共衛生問題之一。根據 GLOBOCAN 2020 的數據，在全球所有年齡層中大腸直腸癌（CRC）佔的病例和死亡人數排名第三，而其生存率低很大程度是因為缺乏早期診斷和精準醫療。外泌體是由細胞分泌到體液中的細胞外囊泡，其大小介於 30 至 200nm 且內部含有許多的功能性生物分子，如蛋白質、脂質及核酸等，並具有在細胞間傳遞訊息的功能。exosomal miR-21 和癌胚胎抗原 （CEA）雖然已被證明可調節大腸直腸癌和多種疾病的發病機制，但早期診斷和個人化治療的非侵入性方法對分離和分析這些循環生物標誌物是一種挑戰。
本研究利用免疫親和法獲得純外泌體或分離潛在細胞外囊泡亞群的能力，並結合紙基裝置的優勢，提出了一種紙基免疫親和裝置。該裝置是通過將 Whatman 紙表面改質上對外泌體的雙層磷脂質表面 CD63 蛋白含有高專一性的 anti-CD63 抗體開發而成的。為了檢測該裝置是否能被有效應用，這邊分別使用紙基酵素結合免疫吸附分析法（P-ELISA）和掃描電子顯微鏡（SEM）評估捕獲的外泌體的數量和形態。在確認此裝置能獲取外泌體後，接著設計更進階的實驗，針對的外泌體裂解時所含有的核酸和蛋白質進行分析。實驗上我們使用高溫超純水來裂解外泌體，並使釋放出的核酸被二氧化矽吸附，然後通過 RT-qPCR 檢測 exosomal miR-21。此外，在實驗上也將外泌體裂解後的裂解液直接進行 ELISA，用於外泌體 CEA 的檢測和定量。首先，我們使用來自 HCT116（人類結腸癌細胞系）細胞培養基的外泌體標準品來確認實驗中對外泌體分離、萃取和定量上整個過程的可行性。接著，使用生物樣品，如血漿、慢性傷口組織液等，來證明所設計的方法對於臨床上的適用性。最後，還研究此方法具有的特性，例如專一性、與商業試劑盒相比的 miRNA 萃取效率以及抗體修飾的紙基對時間的穩定性。
由結果證明了本研究可從不同樣品中分離和純化外泌體，甚至具有對裝置上外泌體定量的能力。而後，也成功進行紙基對 exosomal miR-21 的萃取和外泌體 CEA 定量之實驗，並在研究結果中顯示，使用高溫超純水可成功應用於外泌體 miRNA 萃取，但在針對外泌體 CEA 檢測研究目前還在進行程序上的優化。此外，傷口組織液中外泌體 miRNA 的實驗結果也顯示了慢性傷口癒合與 miRNA-21 含量之間的關聯。此設計的程序不僅穫取了更多的 exosomal miR-21，而且與商業試劑盒相比所需樣品量更少。此方法除了適用於不同的樣本檢測，如血漿、慢性傷口組織液和細胞培養基等，也對外泌體內部成分分析存在優勢。;Cancer is a great worldwide public health concern with steadily rising incidence, especially in developing countries. According to GLOBOCAN 2020 data, colorectal cancer (CRC) ranks third in estimated number of incident cases and deaths worldwide, both sexes, all ages. The poor survival rate is largely because of lacking early diagnosis and precision medicine. Exosome is extracellular vesicle with diameter from 30 to 200 nm, secreted by all cells to body fluids, transporting functional biomolecules (nucleic acids, proteins, lipids, etc.) of normal physiology and acquired abnormalities. Namely, exosomal miRNA-21 and Carcinoembryonic antigen (CEA) have been revealed to regulate the pathogenesis of colorectal cancer and numerous diseases. However, non-invasive methods for early diagnoses and personalized treatments are hindered by challenges of isolating and profiling these circulating biomarkers.
Leveraging the capacity to obtain pure exosomes of immunoaffinity method and advantages of paper platform – an ideal candidate for point-of-care testing (POCT), this study presents a paper-based immunoaffinity device developed by fabricating Whatman paper surface with anti-CD63 antibodies to specifically target CD63 protein on exosome membrane. Number and morphology of captured exosomes were evaluated using paper-based enzyme-linked immunosorbent assays (P-ELISA) and scanning electron microscopy (SEM), respectively. Following initial successes, we designed further procedures for exosomal nucleic acids and proteins to be analyzed. First, we used high temperature double-distilled water, a highly purified and easily accessible laboratory water for biochemistry, to lyse the exosome and released nucleic acids were absorbed by silica particles before extracted miRNA-21 was evaluated by RT-qPCR. Otherwise, lysed solution of exosomes was directly performed ELISA for detection and quantification of exosomal CEA. At first, we used the standard exosomes from cell culture media of HCT116 (a human colorectal carcinoma cell line) to clarify the feasibility of the whole process from exosome isolation to extraction and quantification of exosomal nucleic acids and proteins. Afterward, biological samples (plasma, chronic wound fluids) were applied to demonstrate the clinical applicability of the designed methods. Finally, some properties of these approaches such as specificity, miRNA extraction efficiency compared with commercial kit as well as the stability of modified paper were also investigated.
The first success achieved was to demonstrate the feasibility of isolating and purifying exosomes from diverse samples and the ability to distinguish the number of captured exosomes. Thereafter, paper-based designed procedures for exosomal miRNA-21 extraction and exosomal CEA quantification were successfully experimented. Interestingly, the empirical results indicate that using high temperature double-distilled water as a lysis buffer was successfully applied for exosomal miRNA extraction, but not as effective as commercial lysis buffer in exosomal CEA study. Moreover, experimental results on wound fluids exposed an association between prolongation of healing and exosomal miRNA-21 reduction. Our designed procedure not only harvested more copious amount of exosomal miRNA but also consumed less sample volume than by commercial kit. These approaches feasibly adopt for different samples (plasma, chronic wound fluids, cell culture media) and manifest advantages for deeper inquiry on exosome-derived constituents. |
