六种染色后光镜观察法检测肝癌细胞凋亡
染色,,],细胞凋亡；检测；形态学；肝细胞癌,1材料和方法,2结果,3讨论,参考文献：
[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6 h诱导人肝细胞癌细胞系HCC9204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Hoechst 33 258荧光染色、MayGrunwaldGiemsa(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、Hoechst33 258、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。[关键词] 细胞凋亡；检测；形态学；肝细胞癌
Detection of Apoptosis in Hepatoma Cells under Optical Microscopic Observation after Stained by Six Methods
YANG Lianjun1,SI Xiaohui1,WANG Wenliang2,et al
(1.Bethune International Peace Hospital of PLA, Shijiazhuang,Hebei 050082，China;2.Fourth Military Medical University,Xi'an,Shaanxi 710032,China)
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 HCC9204 cells were affected by 6% ethanol for 6 h to induce apoptosis. The nonfixed cells were stained with trypan blue and by the doublystaining method of acridine orange/ethidium bromide (AO/EB),then the cells were fixed and stained with fluorescent dye Hoechst 33 258,MayGrunwaldGiemsa (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 lowconcentration ethanol to induce apoptosis,most hepatoma cells were negative in trypan blue staining and TUNELpositive.Except for by trypan blue staining ......
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