配體修飾之量子點應用於細胞表面分子動態變化之研究

Study of Membrane Molecules Dynamics Using Ligand-Modified Quantum Dots in Optical Imaging

蔡幸珊(Hsin-Shan Tsai)

指導教授 ： 張富雄

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02652

摘要

近年來隨著奈米科技以及光電顯微影像技術的蓬勃發展，於是科學家們整合奈米技術與光學影像兩大研究領域，將奈米粒子與顯微影像系統結合應用於生物醫學影像研究中。常見之奈米粒子如量子點，因具有獨特光學特性故常被應用於生物影像學上，作為螢光探針。
由於量子點比起傳統之螢光染劑具有極高的光穩定性，不易因為光源長時間激發而有光褪色的現象產生。因此，將量子點與超靈敏性光學技術結合不僅可提供較佳之訊雜比外同時更允許長時間觀察。藉由兩者結合之優勢，我們可以利用光學之方式去探討研究活細胞上之單一分子的動態變化、追蹤單一分子之移動軌跡或是分子間之交互作用，確切了解特定單一分子於時間以及空間上之變化，更進一步去了解細胞分子層次的改變。
因此本研究即是透過凝集素：刀豆凝集素與小麥凝集素；以及抗細胞黏附因子抗體：anti-integrin αv與anti-integrin β1分別辨識老鼠神經纖維瘤細胞Neuro2A與人類神經纖維瘤細胞SH-SY5Y上之特定分子，進一步追蹤表面特定分子之動態變化。於實驗中，我們利用中性脂質( DPPC/Cholesterol/DSPE-PEG/DOPE-biotin )包覆量子點，不僅可以降低量子點本身對於細胞之毒性同時因為DSPE-PEG之存在可以減少量子點非專一性的黏附於細胞膜上。由於量子點本身具有螢光訊號，而經過脂質修飾後之量子點因為界面差異故於三倍頻顯微影像系統之下亦具有強烈訊號存在。因此，在本研究中，我們透過螢光顯微鏡以及三倍頻顯微影像系統可以觀察到活細胞上被標定之特定分子的動態變化以及移動軌跡。
未來，脂質包覆量子點結合光學影像系統於活細胞影像之應用，可深入探討單一受體與藥物間之反應或分子與分子間之交互作用，並紀錄其動態變化。

關鍵字

量子點 ； 單一分子追蹤 ； 刀豆凝集素 ； 小麥凝集素 ； 抗細胞黏附因子抗體α1,β1 ； 三倍頻顯微影像系統

並列摘要

According to the progress in the nanotechnology and opto-electronics imaging, the scientists integrate nanoparticles with microscope imaging system and apply to molecular imaging in the biomedicine. Quantum dot is a familiar nanoparticle which has a lot of unique optical properties. Therefore, quantum dot has emerged as a new fluorescent probe for biomolecular imaging.
Quantum dot has better photostability and less photobleach in long term laser excitation than conventional dyes. Base on this, we can combine quantum dots with ultrasensitive optical technology to provide the high signal-to-noise ratio and allow for long term observation. In order to understand the dynamics of temporal and spatial at molecular scale, we can track the motions of the single molecule, record the trajectory, and even investigate the intermolecular interaction by optical technology.
In this research, we use lectins (ConA, WGA) and antibody (anti-integrin αv, β1) to recognize the surface makers on the mouse or human neuroblastoma cells (Neuro2A and SH-SY5Y) to track the dynamics of the specific molecule. In the experiments, we use natural lipids (DPPC/Cholesterol/DSPE-PEG/DOPE-biotin) coated quantum dots which is not only reduce the cytotoxity but also diminish the non-specific binding on the cell membrane. Due to quantum dot has fluorescence signal and lipid-coated quantum dots has third harmonic generation signal result from the difference of the interface. Therefore, we can observe the dynamic changes of the targeted molecule in live cells by fluorescent microscopy and third harmonic generation imaging system.
In the future, applications of lipid-coated quantum dots combine optical imaging system in live cell imaging can investigate the responses between single receptor and drugs or the intermolecular interactions, and record the dynamic changes at the same time.

並列關鍵字

Quantum dots ； Single molecule tracking ； ConA ； WGA ； Anti-integrin αv, β1 ； Third harmonic generation imaging system

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