【摘要】 目的:探讨简便易行的在光镜下通过形态学观察检测细胞凋亡的方法,并对其进行比较。方法:首先用6%的乙醇作用6h诱导人肝细胞癌细胞系HCC-9204细胞凋亡,然后进行未固定细胞的台盼蓝染色、吖啶橙/溴化乙啶(AO/EB)双染色,细胞固定后的Hoechst33258荧光染色、May-Grunwald-Giemsa(MGG)染色和常规HE染色等形态学观察,并进行末端脱氧核苷酸转移酶(TdT)介导的dUTP缺口末端标记(TUNEL)法检测。结果:用低浓度乙醇诱导细胞凋亡后,大部分肝癌细胞为台盼蓝拒染,TUNEL阳性着色。除了台盼蓝染色法以外,AO/EB、Hoechst33258、MGG和HE等染色法,以及TUNEL检测法均能够不同程度地显示典型的细胞变圆,细胞核深染,染色质边集、呈环状、团块或新月体状,以及细胞浆浓缩和“出泡”现象等细胞凋亡的形态学特征。AO/EB双染色法还能够显示呈致密浓染的黄绿色染色或出现黄绿色碎片的早期凋亡细胞,呈致密浓染的鲜红色染色或出现鲜红色碎片的晚期凋亡细胞,以及呈鲜红色膨大状的坏死细胞。结论:上述六种染色方法均可作为检测细胞凋亡的手段。除了台盼蓝染色以外,其余5种染色方法均可用于观察凋亡细胞的形态学变化。其中,从AO/EB双染色法还可以区分早期凋亡。晚期凋亡和坏死细胞。TUNEL法不仅能够通过检测凋亡细胞的DNA片段化来判别细胞凋亡,还可以从形态学上观察凋亡细胞。更多还原

【Abstract】 Objective To investigate some simple and convenient morphological methods to detect apoptosis under optical microscopic observation, and perform contrast study in them.Methods At first, human hepatocelluar carcinoma cell line HCC-9204 cells were affected by 6% ethanol for 6 h to induce apoptosis. The non-fixed cells were stained with trypan blue and by the doubly-staining method of acridine orange/ethidium bromide (AO/EB),then the cells were fixed and stained with fluorescent dye Hoechst 33 258,May-Grunwald-Giemsa (MGG) and normal H.E stainings, and at last Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining was performed.Results After affected by low-concentration ethanol to induce apoptosis,most hepatoma cells were negative in trypan blue staining and TUNEL-positive.Except for by trypan blue staining, typically morph ologically apoptotic characters such as pyknosis, circled,dense-stained nucleus and cytoplasm,nucleus granuling,ringing or crescenting,and cytoplasm bubbling in different extent could be seen in the cells stained by AO/EB,Hoechst 33 258,MGG,H.E and TUNEL Besides,early apoptotic cells which were densely green yellow-stained or displaying green yellow fragments,late apoptotic cells which were densely cardinal red-stained or displaying cardinal red fragments, and cardinal red swollen necrotic cells could be found in the AO/EB doubly stained cells.Conclusions All the above 6 staining methods can be used to detect apoptosis.The morphological characters of apoptotic cells can be observed after stained by all the other 5 methods except for trypan blue staining. Besides detecting apoptosis, early apoptotic cells,late apoptotic cells and necrotic cells can be distinguished by AO/EB doubly-staining Not only apoptosis can be detected according to DNA fragmentation, but also the morphological characters of apoptotic cells can be observed in TUNEL staining.更多还原