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    題名: 單一活細胞內溫度分布對轉錄因子NF-κB動力學的即時偵測
    Real-time probing the intracellular temperature distribution on NF-κB dynamics in single living cells
    作者: 蔡建遠
    Tsai, Chien-Yuan
    關鍵詞: 微尺度溫探針;Rhodamine B;NF-κB
    日期: 2018
    上傳時間: 2018-12-25 12:55:49 (UTC+8)
    摘要: 背景：
    NF-κB是調節DNA轉錄的蛋白質複合物，可以透過多種的刺激來使NF-κB活化。癌症和自身免疫疾病等相關的疾病與NF-κB的調節失衡有關。相關文獻表明，不同的溫度會影響NF-κB的轉錄速率。然而很少有文獻研究單細胞中溫度分布與NF-κB之間的關係。在這篇研究中，運用微尺度溫度探針探討單細胞內的溫度分佈與NF-κB轉錄速率的關係。在溫度探針研究裡許多的研究把溫度探針運用在工程上，但很少運用在生物研究上，這是因為溫度探針尺寸越小，所要克服的溫度量測的空間解析度和準確度就顯得很重要。為了可以瞭解細胞內部的溫度和細胞內部機能的相關性，因此微尺度溫度探針在生物上的相關研究是必要的。
    目的：
    本論文運用Ti-6Al-4V粉末吸收近紅外光並轉換成熱的特性製造一個溫度場，再根據Rhodamine B的螢光強度跟溫度變化的相關性製造微尺度溫度探針，並且利用受體媒介的方式把溫度探針送入細胞內。最後利用觀察溫度探針螢光強度的變化，並分析螢光強度來推斷出細胞內溫度的分布，以便探討溫度分布與NF-κB轉錄速率的相關性。
    材料與方法：
    本研究製作溫度探針的方式是藉由Streptavidin – Biotin鍵結方式把Rhodamine B 鍵結在乳膠微珠上，此溫度探針是利用Rhodamine B 的螢光強度會隨著不同溫度變化的特性來量測溫度，並利用溫控系統校正溫度探針的螢光強度。
    在微環境溫度量測裡，運用Ti-6Al-4V粉末與近紅外光雷射製造溫度場並利用溫度探針量測微環境的溫度分布，最後藉由受體媒介的方式把溫度探針送進細胞並探討細胞溫度分布與NF-κB轉錄速率的相關性。對於螢光強度的計算，本研究運用MATLAB程式分析螢光影像並轉換成溫度分布圖。
    結果：
    本研究使用熱電偶校正溫控平台的設定溫度和微流道內的溫差。利用此關係來校正溫度探針，實驗結果證實溫度探針的螢光強度隨著溫度的升高而降低。在微環境的溫度量測結果顯示溫度探針根據不同的位置會有不同的ΔI的變化，此結果證實在溫度探針可以反應出微環境裡溫度的分布。另外，利用A549細胞膜上的表皮生長因子受體( EGFR )，使得溫度探針可以藉由受體媒介方式送入細胞，從實驗結果觀察到有EGF的鍵結增加溫度探針進入A549細胞的機率。
    結論：
    本論文已完成溫度探針的製作，實驗觀察到Rhodamine B的螢光強度隨著溫度的升高而降低，不管是在螢光再現性量測、微環境溫度量測都反應出了此特性，這跟先前的文獻相符合。而溫度探針也藉由鍵結EGF的方式增加進入細胞的機率，並且具有專一性結合的功能，在未來的實驗裡會利用此方式把溫度探針送入細胞內，並且觀察細胞內的溫度分布和觀察NF-κB轉錄速度跟溫度的關係。
    Background：
    The NF-κB is a protein complex that regulates DNA transcription. Incorrect regulation of NF-kB has been linked to cancer, inflammatory and autoimmune diseases. Recent studies indicate that different temperatures can affect the transcription rate of NF-κB. However, few researches have studied the relationship between temperature distribution and NF-κB in single living cell. In recent year, many researchers have begun to study on micro-scale temperature probes. They use micro-scale temperature probes in microfluidic or microelectronic systems. But the temperature probes are rarely used in biological research. If the temperature probe is smaller, the spatial resolution of the temperature measurement to overcomes that is important. In order to understand the correlation between the temperature inside the cell and the internal function of the cell. Therefore, biologically relevant research on microscale temperature probes is necessary.
    This study is a study of micro-scale temperature probes. According to the fluorescence intensity of fluorescent molecules will change with the temperature. This feature is used to fabricate micro-scale temperature probes. The temperature probes are passed into the cell by endocytosis. The intracellular temperature is then monitored by analyzing the fluorescence intensity.
    Aims：
    Using microscale temperature to detection of intracellular temperature distribution and exploring the related mechanisms of NF-κB in single living cell
    Material and Method：
    In this study, the Rhodamine B was coated on Polystyrene particles by Streptavidin – Biotin’s bonding. This kind of bonding is stronger than hydrogen bonding and isn’t easy to be destroyed. To avoid the micro-scale temperature probe bonding is destroyed when the temperature or pH value changes. Finally, the temperature probe is sent into the cell by receptor-mediated endocytosis.
    We measured the temperature is the use of Rhodamine B fluorescence intensity will change with the temperature. If the temperature increase, the fluorescence intensity will be weakened. The experiment combines with the lab’s microscope integrated system, temperature control system and sample replacement system for temperature measurement experiments. Finally, using the MATLAB program to analyze the signal that the fluorescence intensity. Showing a temperature versus fluorescence intensity correction curve or cell temperature profile.
    Results：
    Experiment apply thermocouple to calibrate the temperature difference between the set temperature of sample holder and temperature inside the flow chamber. The experimental results show that the temperature of the microchannel will indeed be lost from the objective lens. Then use this relation to calibrate micro-scale temperature probes. The results show that the fluorescence intensity of the temperature probes decreased as the temperature increased. In addition, temperature probes can be endocytosed in cells by receptor-mediated endocytosis.
    Conclusion：
    We have completed the temperature probes production and observe that the fluorescence intensity of rhodamine B decreases with increasing temperature. Therefore, rhodamine B labeled polystyrene beads can measure the temperature distribution in the microchannel.
    Microscale temperature probe;Rhodamine B;NF-κB
    描述: 碩士
    楊自森
    Tzu-Sen Yang
    資料類型: thesis
    顯示於類別:[生醫材料暨組織工程研究所] 博碩士論文

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