采用形态学实验研究双脱甲氧基姜黄素对人血管内皮细胞的诱导凋亡作用

A morphologic investigation on apoptosis of endothelia cells induced by bisdemethoxycurcumin

目的 探讨双脱甲氧基姜黄素 (又称姜黄素 3 )对人肺微血管内皮细胞的诱导凋亡作用。方法 (1 )采用Ｇｉｅｍｓａ染色法观察浓度分别为 4μｇ/ｍｌ、8μｇ/ｍｌ和 1 2 μｇ/ｍｌ的双脱甲氧基姜黄素诱导内皮细胞凋亡的效果 ; (2 )采用透射电镜观察浓度分别为4μｇ/ｍｌ、8μｇ/ｍｌ的双脱甲氧基姜黄素诱导内皮细胞凋亡的效果 ; (3 )采用丫啶橙荧光染色观察内皮细胞在 8μｇ/ｍｌ双脱甲氧基姜黄素诱发凋亡后发生的形态结构改变。结果 (1 )Ｇｉｅｍｓａ染色实验表明 ,双脱甲氧基姜黄素作用 2 4ｈ后 , 4μｇ/ｍｌ组细胞凋亡率为1 5. 8% ,与阴性对照组 (1 3 . 2 % )比较无明显差异 (Ｐ >0 . 0 5) , 8μｇ/ｍｌ组和 1 2 μｇ/ｍｌ组细胞凋亡率分别为 2 0 . 6%和 3 8% ,与阴性组比较差异显著 (Ｐ < 0 . 0 1和Ｐ < 0 . 0 0 1 ) ,凋亡细胞多表现为胞体缩小、变圆 ,核浓缩 ,呈紫黑色 ; (2 )透射电镜分析表明 , 4μｇｍｌ组的内皮细胞形态结构与阴性对照组基本相同 ,无明显异常改变 , 8μｇ/ｍｌ组有大量凋亡小体形成 ,并观察到较多的典型凋亡细胞 ;(3 )丫啶橙荧光染色可较细致地显示细胞凋亡时的多种结构、形态改变 ,能够发现较早期的凋亡细胞 ,因而其敏感度可能更高。结论 (1 )

Objective To investigate the effect of bisdemethoxycurcumin applied in different concentrations to induce apoptosis of human pulmonary microvessel endothelial cell line. Methods (1) After bisdemethoxycurcumin treatment in different concentrations for 24 hours, the apoptosis rates of human pulmonary microvessel endothelial cell line in vitro were investigated with Giemsa staining. (2) Transmission electron microscope (TEM) was used to investigate effect of bisdemethoxycurcumin treatment in different concentrations on inducing apoptosis of human pulmonary microvessel endothelial cell line in vitro. (3) The morphologic changes of human pulmonary microvessel endothelial cell line in vitro were observed by means of fluorescence staining with acridine orange. Results (1) Giemsa staining indicated that the apoptosis rates of endothelial cell were 15.8%, 20.6% and 38% respectively after bisdemethoxycurcumin treatment in concentration of 4μg/ml, 8μg/ml and 12μg/ml for 24 hours. (2) TEM indicated that apparent morphologic change of human pulmonary microvessel endothelial cell line in vitro wasn't observed after bisdemethoxycurcumin treatment in concentration of 4μg/ml when compared with the control, while lots of apoptotic bodies and typical apoptotic cells were induced after bisdemethoxycurcumin treatment in concentration of 8μg/ml. (3) Acridine orange staining indicated that apoptosis cell showed many phenotypes of nucleus ,preferable to indicate the morphologic changes of apoptotic cell. Conclusion (1) The minimal effective concentration of bisdemethoxycurcumin used to induce apoptosis of blood capillary endothelial cell from human lung is 4μg/ml; (2) fluorescence staining with acridine orange is not only fit for calculating of the cell apoptosis rate as well as Giemsa staining, but preferable to investigate morphologic changes of cell apoptosis.