目的探讨龙葵碱诱导HepG2细胞凋亡与线粒体损伤的关系。方法倒置显微镜观察细胞形态,Annexin V/PI双染激光共聚焦扫描显微术检测细胞凋亡,流式细胞仪检测细胞凋亡率。透射电镜观察细胞线粒体超微结构,CDFH-DA染色激光共聚焦扫描显微术检测活性氧(ROS)相对量,比色法检测还原型谷胱甘肽(GSH)量。结果龙葵碱组HepG2细胞贴壁细胞数量减少,部分出现死亡。Annexin V/PI双染激光共聚焦扫描显微镜下观察发现龙葵碱组细胞Annexin V-FITC高染,细胞膜呈绿色荧光;PI低染,细胞核呈红色荧光,呈现明显的早期凋亡特征。流式细胞术分析0.4、2、10μmol/L龙葵碱作用HepG2细胞24 h后,早期凋亡率分别为4.0%、8.5%、20.1%。透射电镜观察发现龙葵碱作用HepG2细胞24 h,细胞线粒体超微结构出现肿胀,嵴排列紊乱、嵴消失,重度空泡样变性等变化;细胞内ROS水平升高,GSH的水平降低。结论龙葵碱通过降低HepG2细胞内GSH量,使ROS不能被及时清除而损伤线粒体结构,导致HepG2细胞凋亡。

Objective To study the relationship between apoptosis and mitochondria damage.MethodsThe cell morphology was observed under inverted microscope.Staining with Annexin V/PI,the cell apoptosis was observed by laser confocal scan microscope and the apoptosis rate was analyzed by flow cytomety.Transmission electron microscope (TEM) was used to observe mitochondria) structure.ROS was determined by confocal. Colorimetry assay was adopted to determine the reduced glutathione hormone (GSH).Results The cell number in solanine groups was fewer than that in control group,and some died.Staining with Annexin V/PI,Annexin V-FITC was high fluorescent in solanine groups，which is obviously apoptosis features.The early apoptosis rate is 4.0%，8.5%,and 20.1%inducing by 0.4,2,and 10mol/L solanine for 24 h,respectively. Swelling mitochondria,absence of mitochondria crests and vesicles were found in mitochondria of HepG2 cell treated by solanine for 24h. The ROS levels increased and GSH level decreased in solanine groups.ConclusionSolanine could induce the apoptosis and the effect may be attributed to decrease GSH,which interrupts the immediate remove of ROS so as to damage the mitochondria.

教育部博士点基金项目(200802400001);黑龙江省博士后基金资助项目;国家自然科学基金资助项目(30400591);哈尔滨市青年基金资助项目(2004AFQXJ035)