以乳鐵蛋白結合量子點進行造骨細胞株核內體標定之研究

Lactoferrin Conjugated with Quantum Dots to MC3T3-E1 Cell Endosome labelling

方詠涵(Yung-Han Fang)

指導教授 ： 張 恒 雄

中原大學/工學院/醫學工程研究所/碩士(2006年)

https://doi.org/10.6840/CYCU.2006.00508

摘要

本研究試圖將具有多種生物功能的乳鐵蛋白(lactoferrin，LF)與具有特殊發光性質的量子點(quantum dot)鍵結，利用乳鐵蛋白引發細胞產生內吞作用（endocytosis），將量子點與乳鐵蛋白複合體吞入並包存在細胞的核內體(endoosome)中，並藉由量子點的螢光標定，觀察核內體和乳鐵蛋白在細胞內的分佈情形。

本研究主要目的在找出量子點與乳鐵蛋白在何種條件下鍵結能有最佳的生物活性。在實驗部分，先將疏水性量子點以高分子改質為親水性後，再利用改變反應物濃度比例與調控活化劑數量的方式，調整乳鐵蛋白與量子點產生共價鍵結的反應環境。最後根據量子點光學
特性變化、電泳分析結果以及直接與造骨細胞株(MC3T3-E1 cell)共培養，來判斷何種鍵結條件能有最佳的標定效果。另外實驗過程中，利用不同官能基和鏈長之聚乙二醇( poly(ethylene glycol)，PEG )修飾親水性量子點表面，探討PEG 能否有效改善量子點非專一性鍵結
（nonspecific binding）的問題。

由實驗結果顯示，量子點與乳鐵蛋白鍵結實驗中，發現乳鐵蛋白與量子點濃度比為1：2 時，可純化出量子點表面只接特定個數乳鐵蛋白的複合體。而乳鐵蛋白與量子點的濃度比為1：25 時，若調控活化劑的濃度，可鍵結出大小和電荷較一致的複合體。而在與細胞共培
養實驗中發現，細胞質確實有粒狀的量子點分佈，由此現象推測，量子點是由內吞作用進入細胞中，標定細胞的核內體。另外，PEG 修飾量子點的實驗部分,發現PEG 修飾的量子點有降低親水性量子點非專一性鍵結的現象，可提高量子點在細胞培養液中的鹽穩定性。

關鍵字

量子點 ； 乳鐵蛋白 ； 聚乙二醇 ； 造骨細胞株 ； 核內體

並列摘要

In this study, the multifunctional Lactoferrin(LF) was conjugated with quantum dot (QD) that have greater and longer fluorescence.
Because LF can induce receptor-mediated endocytosis of cell, so the LF of QD and LF complexes (QD-LF) were used to induce cell endocytosis and bring QD-LF into intracellular endosome. Then the distribution of endosome and LF could be monitored by detecting the fluorescence of QD.
The purpose of this study was to find the best conjugated conditions of QD and LF without losing LF its own function. First of all, polymer
coating method was used to modify hydrophobic QD becoming hydrophilic QD. Then the reaction surrounding was modified by controlling reactant concentrations and linker numbers for forming
covalent bind of QD and LF. After that, the results of conjugation were determined based on optical property, electrophoresis analysis, and
incubated with MC3T3-E1 cell directly. In addition, this polymeric QD surface was modified by conjugating different functional group and
length of poly(ethylene glycol) (PEG), and was attempted to reduce nonspecific binding problem of QD.
The results of conjugated experiment of QD and LF showed that when the ratio of QD to LF is 1:2, the QD-LF that only have several LF on the surface of QD was obtained. And when the ratio of QD to LF is 1:25, the linker numbers of conjugated conditions were modulated. The products of conjugated QD with LF under this condition had unity size and electric charges. In the cell uptake experiment, there was distribution of granular QD in the cytoplasm. Because of this phenomenon, it was conjectured that QD was uptake by receptor-mediated endocytosis of cell. And QD really labeled the endosome. In addition, in the experiment of PEG conjugated with QD, the nonspecific binding was reduced and the salt stability of polymeric QD was increased by PEG-QD.

並列關鍵字

MC3T3-E1 cell ； endosome ； quantum dot ； lactoferrin ； poly(ethylene glycol) (PEG)

參考文獻

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