以金奈米粒子聚集檢測去氧核醣核酸甲基化及以高靈敏度螢光顯微鏡觀測肝癌細胞中的微小核醣核酸

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题名:	以金奈米粒子聚集檢測去氧核醣核酸甲基化及以高靈敏度螢光顯微鏡觀測肝癌細胞中的微小核醣核酸
其它题名:	Colorimetric Determination of DNA Methylation by Gold Nanoparticles Aggregation, and Observation of MicroRNA From Hepatocellular Carcinoma Cells by Ultra-sensitive Fluorescence Microscopy
作者:	林宜蓁 Yi-Zhen Lin
贡献者:	張柏齡 Po-Ling Chang 化學系
关键词:	微小核醣核酸;高靈敏度螢光顯微鏡;去氧核醣核酸甲基化;金奈米粒子 Ultra-sensitive Fluorescence Microscopy;DNA Methylation;Gold Nanoparticles;MicroRNA
日期:	2014
上传时间:	2015-03-06T07:36:39Z (UTC)
摘要:	我們發展一個簡單且新穎檢測去氧核醣核酸甲基化（DNA methylation）的奈米感測器，將含四種含氮鹼基之寡核?酸使用此奈米感測器表現顏色，由實驗結果可區分成A/T及C/G二組，與Watson?Crick鹼基對相符。學生先將genomic DNA進行重亞硫酸鹽（sodium bisulfite）反應，再使用聚合?連鎖反應（polymerase chain reaction, PCR）進行DNA擴增。將鼻咽癌細胞使用抗癌藥物5-aza-2’-deoxycytidine處理0至5天，其甲基化程度經由此奈米感測器表現顏色後可由肉眼觀察辨別。12種癌細胞於DNA擴增長度只有342個鹼基對之情況下，甲基化程度亦可在此奈米感測器顯示出異質性。實驗結果證明此奈米感測器有助於表觀遺傳學（epigenetics）之研究。論文第二部分為以高靈敏度螢光顯微鏡觀測固定之培養肝癌細胞中的微小核醣核酸（microRNA）。學生建構之HILO螢光顯微系統相較於TIRF螢光顯微系統能觀測到較多層之螢光影像，且可靈敏地觀測到單一螢光分子。使用Molecular beacon進行microRNA之雜合作用，miR10b之表達程度可由此系統直接觀測。由影像圖計算Huh7及Hep3B之單顆細胞平均螢光訊號，分別為1147 ± 129及838 ± 66，即表示Huh7肝癌細胞相較於Hep3B肝癌細胞之miR-10b含量高。Huh7及Hep3B之間的差異與定量聚合?連鎖反應之實驗結果相符。 A simple, novel colorimetric nanosensor for DNA methylation based on the strength of hydrophobic interaction between DNA and gold nanoparticles was proposed. The nanosensing of oligonucleotides with four nitrogen bases was first demonstrated by dividing the bases into two groups (A/T and C/G) using the representative colors that correspond to Watson?Crick base pairing. By treatment of the genomic DNA with sodium bisulfite followed by PCR amplification, the methylation level of nasopharyngeal carcinoma cells treated with 5-aza-2’-deoxycytidine for up to 5 days could be discriminated by naked eye observation. Furthermore, 12 cancer cell lines that demonstrate heterogeneity with respect to DNA methylation could also be distinguished using the nanosensor, even for amplicons as long as 342 bp. These results demonstrate that the proposed colorimetric nanosensor could potentially be useful in epigenetic studies. The second part of this thesis is focus on the development of highly inclined and laminated optical sheet (HILO) microscopy for miRNA observation from fixed cultured cells. The HILO microscopy provided better depth of fluorescence image than total internal reflection fluorescence microscope and remains single-molecule sensitivity. Using the molecular beacon of miR10b* for miRNA hybridization, the expression level of miR10b* could be observed directly by HILO microscopy. The average fluorescence signal of Huh7 and Hep3B were 1147 ± 129 and 838 ± 66 per cell respectively. This difference between Huh7 and Hep3B corresponds with the data that obtained from quantitative polymerase chain reaction.
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