以量子點及免疫螢光染色法探討維他命A酸誘導人類造骨細胞株hFOB1.19凋亡之路徑

維他命A酸是維他命A的代謝產物之一。維他命A酸在胚胎發育以及細胞分化上佔有重要的角色，對於治療急性骨髓性白血病有良好的成效。在先前的研究中，維他命A酸被用來調控不同類型之人類惡性腫瘤成長與分化，並影響多種正常組織的成長與分化。在本實驗中發現將hFOB1.19細胞株分別培養在無血清含有不同濃度維他命A酸之培養液，細胞會隨著濃度的增加細胞存活率下降的趨勢。分析細胞凋亡下游的凋亡相關蛋白質-PARP發現有活化斷裂的情形。在初步分析中，我們發現維他命A酸會誘導hFOB1.19產生細胞凋亡的現象，且隨著維他命A酸濃度的增加其相關蛋白質活化量越多。細胞凋亡是ㄧ種基本的生物學現象，它在生物體的進化、內環境的穩定以及多個系統的發育中起著重要的作用。細胞凋亡不僅是ㄧ種特殊的細胞死亡類型，而且具有重要的生物學意義及複雜的分子生物學機制。隨著分子生物學技術的發展對多種細胞凋亡的過程有了相當的認識，但是迄今為止凋亡過程確切機制尚不完全清楚。所以我們希望能夠釐清維他命A酸誘導hFOB1.19細胞凋亡的途徑及調控機制。在本研究中同時利用免疫螢光染色法觀察p-JNK在細胞內活化以及所在位置的情形。免疫分析方法在臨床、藥物學以及環境化學中扮演一個十分重要的角色。迄今，建立於抗體-抗原結合理論的偵測方法，使得多樣性的傳導機制已經被鑑定出來。這些方法有，化學冷光、螢光以及放射化學。在這些技術中，螢光分析法是最廣泛使用的方法之一。然而目前廣泛應用的免疫螢光分析法，傳統以有機染劑或是綠螢光蛋白來做標定，其原理利用加在二級抗體上的螢光有機染劑，藉由抗原-抗體原理結合在特異性位置上發出螢光，可以在螢光顯微鏡下被偵測到，但是ㄧ般螢光染劑缺乏光穩定性，且無法長時間觀測，又由於每種螢光染劑的激發波長與吸收波長相近易發生螢光區段相互幹擾的情形發生，且隨著時間的增加螢光強度會下降，在觀測上有諸多限制而導致判讀不易，無法有效被運用。本研究擬藉由奈米半導體材料-量子點，藉由獨特之發光特性及穩定性，使得發螢光的量子點是一種非常好用的藥劑，特別在研究3D受限制的系統中，亦可應用於生物醫學以及臨床化學研究中。本研究主要有兩個部分：(1)利用維他命A酸誘導人類骨母細胞株產生程式性死亡，藉由一般凋亡分析方法－西方點墨法和免疫螢光染色法，於型態學及生化學上觀察細胞進行程式性死亡時凋亡相關因數的表現。以釐清維他命A酸誘導人類骨母細胞株產生細胞凋亡之路徑及機制。(2)利用量子點接枝上抗體應用於免疫螢光染色分析法，分別使用一級抗體(anti-PAK2)和二級抗體(anti-mouse IgG)與量子點做鍵結，由抗體再專一性的結合在細胞內蛋白質大分子上，觀察蛋白質分子在細胞內的結合位置。研究結果中發現量子點可以取代一般有機染劑在觀察上的缺失，且對細胞不具有毒性比一般染劑更適合進行生物檢測上的分析。藉由此項研究進ㄧ步瞭解量子點可用於細胞內多種蛋白質的追蹤與觀察，為生物奈米研究有了基礎的貢獻。

關鍵字

維他命A酸；量子點；細胞凋亡

並列摘要

Retinoic acid (RA) is a derivative of vitamin A. Retinoic acid has been found to be involved in the development of vertebrates and in cell differentiation. It has been used in the treatment of acute promyelocytic leukemia (APL) with good success. In the present study, retinoic acid has been used to modulate cell growth and differentiation in many types of human malignancies. It also affects the differentiation and growth of many normal tissues. Cell death was observed when serum starved human osteoblast cell cultures (hFOB1.19 cell-line) was exposed to different concentrations of RA in the concentration range 0 to 200 uM. The survival of the cells was dose-dependent. The concentration of RA increased the survival of hFOB1.19 cells and decreased the cell growth. To observe the RA-induced cell death we used western blot to assay the downstream cleavage of the apoptotic protein-poly (ADPribose) polymerase (PARP). In preliminary analysis we found a dose-dependent increase in RA-induced apoptosis of hFOB1.19 cells. The hFOB1.19 cell-line also expressed the activated form of apoptotic protein-pJNK which was localizing and determined by Immuno-Fluorescence staining. Immunoassay plays a critical role in clinical, pharmaceutical and environmental chemistry. To date, based on the detection of antigen-antibody binding, a variety of transduction mechanisms have been identified. Examples of immunoassays include chemiluminescent, fluorescent, and radiochemical. Of these techniques, fluorescence spectroscopy is one of the most widely used methods. And the most commonly used visualization tool is fluorescent markets. Fluorescent quantum dots (QDs) are useful reagents for scientific research of three-dimensionally confined systems. Several attractive fluorescent characteristics of QDs suggest that they might be valuable for fluorescent labeling and readout methods in the area of biomedical and clinical chemistry research. However, conventional labels, such as organic fluorescent dyes or green fluorescent protein (GFP), lack the photostability to allow the tracking of cellular events that happen over a period from minutes to days. In addition, the emission spectra of QDs is rather narrow and generally 50-100 times more stable then that of most traditional organic dyes against photobleaching. This enables QDs to be used to average the signal for extended periods of time to lower the detection limit. In addition, the excitation band of QDs is rather wide; different colors are emitted from the same QD material of different size, which allows the use of a single excitation source for multi-channel detection. This study has two aims: (1) the pathways of hFOB1.19 cells induced apoptosis by RA were determined by traditional methods (western blot and immuno-fluorescence assay). (2) the possible application of QD-labeled antibody in immuno-fluorescence assay was determined. We used water-soluble CdSe/ZnS nanocrystals conjugated to anti-mouse IgG instead of the traditional organic dye-FITC to investigate apoptotic protein activation. We found the detection of antibody labeled with QDs bound selectively to the captured antigen. Our results indicate that QDs are suitable fluorescent materials for immunomolecule labeling and are useful substitutes for traditional organic dyes. The potential of this method to function as a simple and efficient immuno assay for biological analysis is discussed.

並列關鍵字

Quantum dot ； apoptosis ； retinoic acid

參考文獻

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以量子點及免疫螢光染色法探討維他命A酸誘導人類造骨細胞株hFOB1.19凋亡之路徑

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