QDs-SA和Cy3荧光标记阿尔茨海默病细胞模型β淀粉样蛋白的比较研究  

Comparative Study on the Fluorescence Imaging of AβProtein of Alzheimer's Disease with QDs-SA Quantum Dot and Cy3

作　　者：唐薇婷[1] 冯莉[1] 肖波[1] 李蜀渝[1] 龙莉莉[1] 杨晓苏[1] 龙小艳[1] 

机构地区：[1]中南大学湘雅医院神经内科,410008

出　　处：《中国临床神经科学》2010年第3期225-230,共6页Chinese Journal of Clinical Neurosciences

基　　金：国家自然科学基金资助项目(30770736)

摘　　要：目的:观察利用量子点(QDs)标记的链霉亲和素复合物(QDs-SA)和传统荧光染料Cy3分别标记的阿尔茨海默病(AD)转基因细胞模型β淀粉样蛋白(Aβ)在荧光强度和抗光漂白性能的差异。方法:将pcDNA3.1/APP质粒转染至HEK293细胞建立稳定表达株,Western blot法检测转染细胞APP蛋白的表达,细胞免疫荧光检测Aβ蛋白生成。建立AD转基因细胞模型,分别应用QDs-SA和Cy3细胞免疫荧光染色技术靶向标记转基因细胞模型的Aβ蛋白,激光共聚焦显微镜下对荧光强度和抗光漂白特性进行检测。结果:Western blot法在稳定转染pcDNA3.1/APP质粒的细胞中检测到APP目的蛋白谱带,而未转染的空载体转染组细胞未见APP蛋白表达。共聚焦荧光显微镜下观察到:①APP基因转染后能够生成大量Aβ蛋白;②QDs-SA特异标记的Aβ蛋白主要分布在细胞膜和胞质,显现出均匀分布的高密度橙红色强荧光;③Cy3特异标记Aβ蛋白荧光强度较QDs-SA标记弱,且细胞膜染色不均匀,部分细胞膜区域有染料少量团聚;④QDs-SA相较于传统Cy3荧光染料的平均荧光强度值更大(P<0.05),488 nm激发光连续激发12 min,QDs-SA荧光标记的Aβ蛋白仍发射较强的橙红色荧光,荧光强度下降了29.25%,而传统荧光染料Cy3荧光强度下降幅度达76.82%。结论:QDs-SA能有效识别AD转基因细胞模型中Aβ蛋白,且在光稳定性和荧光强度等方面均优于传统的Cy3荧光染料标记的免疫荧光成像。Aim： To compare the fluorescent intensity and duration of quantum dots streptavidin conjugate （QDs-SA） with Cy3 as molecular probes of β-amyloid protein（Aβ） protein. Methods： The pcDNA3.1/APP plasmid was transfected into HEK293 cell line. APP and Aβ proteins were identified by Western blot and immunofluorescent cytochemical method, respectively. With the help of laser scanning confocal microscope, the flurescenct probe based on QDs-SA had been used to detect Aβ protein in HEK293 cells stably transfected by pcDNA3.1/APP plasmid, and compared with conventional Cy3-conjugated immunofluorescent staining. Results： The APP protein band was only detected in HEK293 cells stably transfected with pcDNA3.1/APP plasmid by Western blot, but no expression in control groups. Immunofluorescent studies showed that the transfected cells expressed a great quantity of Aβ proteins. By using the confocal technique, Aβ proteins labeled with QDs-SA were expressed in the membrane and cytoplasm of transfected cells and were possessed bright salmon pink florescence, the fluorescence of Cy3 labeled Aβ was weaker than that of QDs-SA labeled Aβ. The mean fluorescence intensity of QDs-SA labeled Aβ was greater than that of Cy3 labeled Aβ under confocal fluorescence microscopy（P〈0.05）. The fluorescence intensity of Aβ protein labeled with QDs-SA decreased nearly by 29.25%, but that labeled with Cy3 decreased by 76.82%. Conclusion： The QDs-SA fluorescence probes can effectively recognize Aβ protein and exhibit good sensitivity and exceptional photostability, suggesting that QDs-SA fluorescence probes could be a potential method in Aβ detection and offer a novel way to the diagnosis of Alzheimer＇ s disease.

关 键 词：阿尔茨海默病 量子点 Β淀粉样蛋白 分子成像 激光共聚焦显微镜 

分 类 号：R741[医药卫生—神经病学与精神病学]