印楝素对SL-1的细胞凋亡诱导作用

Induction of apoptosis by azadirachtin,a botanical insecticidal component,in Spodoptera litura cultured cell line SL-1

本文以喜树碱（camptothecin）作对比,以二甲基亚砜（DMSO）作对照,系统研究了印楝素（azadirachtin）对斜纹夜蛾Spodoptera litura离体细胞系（SL-1）的凋亡诱导作用。印楝素0.75μg/mL处理后SL-1细胞后12-72h,倒置显微镜观察可见大量细胞皱缩,体积变小,胞膜气泡化,与邻周细胞脱落,胞浆浓缩,胞膜突起,细胞器密集,核染色质浓缩并凝聚在核膜周边,出现大量凋亡小体;AO染色荧光显微镜观察可见细胞核内致密明亮黄绿色荧光和亮绿色或橘黄色的凋亡小体;透射电镜观察可见细胞皱缩、微绒毛消失、染色质浓缩和边缘化、核膜皱缩界限模糊、部分线粒体嵴结构消失和数量明显增加的吞噬泡等细胞凋亡典型形态学特征;TUNEL实验可见大量被标记为小圆形或环形黄绿色或绿色荧光的阳性凋亡细胞。流式细胞仪测定表明,印楝素0.75μg/mL处理后48h凋亡率最高达11.45%,比对照提高381.3倍;而喜树碱以1.72μg/mL处理亦对SL-1具有相似的诱导作用,处理后36h凋亡率最高达到17.42%。扫描电镜观察表明,印楝素和喜树碱处理后,SL-1细胞表面没有形成明显的“孔”、“洞”、“门”之类的结构破坏。推测印楝素与喜树碱对SL-1的凋亡信号转导方式和途径不同,导致细胞凋亡时序性不同。

The induction of apoptosis of azadirachtin in Spodoptera litura cultured cell line SL-1 at different time after treatment was determined and compared with comptothecin, with DMSO as the control. The results showed that in 12 - 72 h after treatment with azadirachtin at a concentration of 0.75 μg/mL, the typical morphological characteristics of apoptosis appeared. Observed with the inverted phase contrast microscope （IPCM）, the drastic morphological changes were found including cell shrinkage, cells diminishing, membrane blebbing, gaps increasing, organelle condensation, nuclei excursion, and a lot of apoptotic bodies. Stained with 5 μg/mL acridine orange （AO）, SL-1 cells were observed with yellow bright spots in the nuclei, chromatin condensation, and a few of green bright or saffron yellow apoptotic bodies by visual examinations in fluorescence microscopy （FM）. The uhrastructure of SL-1 cells treated with 0.75 μg/mL azadirachtin displayed apoptotic changes observed with transmission electron microscopy （TEM）, including cell shrinkage, microvilli decreasing, chromatin condensation and fragmentation, gaps of nuclear envelope, small vacuoles and so on. A number of apoptotic cells were marked with small rotund or ring fluorescence in TUNEL test. The maximum apoptotic rate reached 11.45 % 48 h after SL-1 cells treated with azadirachtin at a concentration of 0.75 μg/mL by flow cytometric, which was 381.3 times as high as that of the control. Camptothecin, a known natural component with extraordinary induction of cancer cells apoptosis, possessed similar induction of apoptosis to SL-1 cells treated with 1.72 μg/mL in a series of examinations, including IPCM, FM, TEM and TUNEL assay, and got 17.42% of the maximum apoptotic rate 36 h after treatment. The observations by scan electron microscopy showed, however, that SL-1 cells kept whole orbicular membrane surface after treatment with both azadiraehtin and camptothecin and showed no significant difference from the control cells. From these results, it was inferred that azadiraehtin and eamptotheein may possess different apoptotie signal transduetion pathways and/or approaches, causing /he different apoptotic time orders.