Slaninová Iva, Slanina Jirí, Táborská Eva
Faculty of Medicine, Department of Biology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
Cytometry A. 2007 Sep;71(9):700-8. doi: 10.1002/cyto.a.20423.
Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells.
Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry.
All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping.
It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content.
季铵型苯并[c]菲啶生物碱（QBAs）是从紫堇科、罂粟科、毛茛科和芸香科植物中分离出的天然化合物。它们除了具有广泛的生物活性外，还因其荧光特性而备受关注。我们观察到QBAs中的马卡品（MA）、血根红碱（SR）、白屈菜红碱（CHR）、血根黄碱（SL）、白屈菜黄碱（CHL）、血根碱（SA）和白屈菜红碱（CHE）与活细胞相互作用后呈现出有趣的荧光特性。
将生物碱的水溶液储备液（10 - 100微克/毫升）加入完整细胞中，短暂孵育后观察细胞。实验使用了人类细胞系HL60（人早幼粒细胞白血病细胞）、HeLa（人宫颈腺癌细胞）和LEP（人肺成纤维细胞）以及仔猪血液。血细胞用MA与异硫氰酸荧光素（FITC）偶联的抗CD45表面标志物抗体进行染色。通过荧光显微镜和流式细胞术对细胞进行分析。
所有测试的生物碱都能立即进入活细胞，MA、CHR和SA与DNA结合。MA表现出最佳的DNA染色特性。对MA、CHR和SA染色细胞的荧光显微镜观察描述了细胞核结构，并清晰地显示了活细胞中的染色体和凋亡片段。此外，MA能够以足以进行细胞周期分析的分辨率快速反映活细胞的细胞DNA含量。QBAs可使用发射波长在575 - 755纳米范围内的普通氩离子激光器（488纳米）激发（即荧光探测器FL2 - 5）。MA的光谱特性允许同时进行表面免疫表型分析。
结果表明，MA、CHR和SA可对活细胞中的核酸进行染色。它们可作为超活荧光DNA探针，用于荧光显微镜和流式细胞术，包括对外周血和骨髓的多参数分析。MA以化学计量方式结合DNA，并可提供有关DNA含量的信息。
