Yasuda Noriko, Matzno Sumio, Iwano Chihiro, Nishikata Mayumi, Matsuyama Kenji
School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien, Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan.
J Pharm Biomed Anal. 2005 Sep 15;39(3-4):712-7. doi: 10.1016/j.jpba.2005.04.022.
The purpose of this study was to evaluate the apoptosis and necrosis induced by five kinds of statins in IM-9 human lymphoblasts with fluorescence-enhanced flow cytometry using avidin-biotin complex. IM-9 human lymphoblasts (2 x 10(4) cells/cm2) were seeded into tissue culture plates and incubated with five kinds of statins. Statin-treated cells were first incubated with biotin-annexin V, followed by addition of avidin-FITC and propidium iodide, and then subjected to flow cytometry. The fluorescence intensity was enhanced using an avidin-biotin complex system, resulting in successful separate determination of the statin-induced apoptosis and necrosis by flow cytometry, which enabled us to quantitatively evaluate the statin-induced cell damage. Flow cytometric analysis results in the intensity of statin-induced apoptosis in IM-9 cells as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The intensity of statin-induced necrosis in IM-9 cells was expressed as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The total damage of IM-9 cells induced by five kinds of statins were expressed as the sum of both percentages of apoptosis and necrosis as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. Our studies show that fluorescence enhancement with avidin-biotin complex is useful for the identification and quantitation of annexin-positive apoptosis cells and thus, the fluorescence-enhanced flow cytometry was shown to be applicable for screening of statins as new anti-leukemia agents.
本研究的目的是使用抗生物素蛋白-生物素复合物，通过荧光增强流式细胞术评估五种他汀类药物在IM-9人淋巴母细胞中诱导的凋亡和坏死。将IM-9人淋巴母细胞（2×10⁴个细胞/cm²）接种到组织培养板中，并与五种他汀类药物一起孵育。先用生物素化膜联蛋白V孵育经他汀类药物处理的细胞，然后加入抗生物素蛋白-异硫氰酸荧光素（avidin-FITC）和碘化丙啶，接着进行流式细胞术检测。使用抗生物素蛋白-生物素复合物系统增强荧光强度，从而通过流式细胞术成功分别测定他汀类药物诱导的凋亡和坏死，这使我们能够定量评估他汀类药物诱导的细胞损伤。流式细胞术分析得出IM-9细胞中他汀类药物诱导凋亡的强度如下：阿托伐他汀>西立伐他汀>氟伐他汀>辛伐他汀>普伐他汀。IM-9细胞中他汀类药物诱导坏死的强度如下：阿托伐他汀>西立伐他汀>氟伐他汀>辛伐他汀>普伐他汀。五种他汀类药物诱导的IM-9细胞总损伤表示为凋亡和坏死百分比之和，如下：阿托伐他汀>西立伐他汀>氟伐他汀>辛伐他汀>普伐他汀。我们的研究表明，抗生物素蛋白-生物素复合物增强荧光对于鉴定和定量膜联蛋白阳性凋亡细胞很有用，因此，荧光增强流式细胞术被证明适用于筛选作为新型抗白血病药物的他汀类药物。
