摘要
目的：用缺口末端标记技术（TUNEL）对肝硬变、肝细胞肝癌及培养肝癌细胞系的细胞调亡进行形态学观察．方法：采用德国宝灵曼公司生产的原位末端标记试剂盒对福尔马林固定、石蜡包埋的肝组织和培养肝癌细胞进行TUNEL染色，激光共聚焦显微镜观察肝细胞凋亡的形态特征．结果：TUNEL阳性信号为黄绿色或黄色强荧光，位于胞核，呈环行、小圆形或颗粒状，提示凋亡细胞有典型的新月体状染色质边集、核浓缩以及有大量微核体的形成．在阿霉素诱导的HCC-9204细胞系还可见调亡细胞的出泡现象．TUNEL阳性细胞大多HE染色表现为调亡的细胞．结论：TUNEL技术加激光共聚焦显微镜观察凋亡细胞是目前原位观察调亡的理想方法，特别适用于调亡细胞的早期检测．
AIM: The purpose of this study was to examinethe occurrence of apoptotic cell death in HCC - 92O4 cell lineand 2l formalin fixed, paraffin-embedded biopsy specimensfrom liver cirrhosis (6 cases ) and hepatocellular carcinoma(l5 cases ) by TUNE1, assay under laser confocal microscope.METHoDS: Apoptosis of HCC-92O4 cell line was induced byadriamycin (2O pmol/I,). Modified TUNEL assay was con-ducted using in situ cell death detection kit. The morphoIogical changes were observed under MRC lO24l aser confocalmicroscope mounted on the microscope with a 4OXoil immer-sion lens. Control experiments were also per formed, includ-ing DNase-digestion before TUNEL as a positive control andTdT and FITC-dUTP deletion as a negative control. RoutineHE staining was conducted on serial sections. RESULTS:Con focaI microscopic evaluation of FlTC-labeled TUNELpositiye celIs revealed nuclei with morphologic characteristicsof apoptosis, consisting of typical crescent-shaped chromatinmargination, nuclear fragmentation and formation of numer-ous micronuclei, especially in apoptosis of HCC-92O4 cell lineinduced by adriamycin. Nearly all sections had positive signalsof TUNEL staining, which were observed on the nuclearsites. ln noncancerous tissues, most of TUNEL-positive cellswere scattered. In HCC tissues, TUNEL-positive cells weremorphologically scattered or clustered. CoNCLUSIoN:TUNEI, assay is at present a useful method to detect earlymorphological change of apoptotic cells in situ under laserconfocal microscope.
出处
《第四军医大学学报》
1999年第2期102-105,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金!39470771
关键词
细胞凋亡
TUNEL
肝细胞癌
肝硬变
肝肿瘤
apoptosis
laser confocal microscope
TUNEL
hepatocellular carcinoma
liver cirrhosis
