氧化砷诱导食管癌细胞凋亡线粒体形态改变

Morphological changes of mitochondria in apoptosis of esophageal carcinoma cells induced by As 2O 3

目的 通过研究食管癌细胞株SHEEC1细胞凋亡早期线粒体的形态改变和bcl 2、bax的表达 ,以了解三氧化二砷 (As2 O3 )作用于食管癌细胞的机制。方法 食管癌细胞株SHEEC1用 1、2、3μmol/LAs2 O3 作用 2、4、6、1 2、2 4h。用Annexin V荧光探针 ,流式细胞仪和DNA组方图检测早期凋亡细胞 ;光镜和电镜检测凋亡细胞的形态学改变 ;用罗达明 (Rhodamin 1 2 3)荧光探针检查活细胞线粒体的改变 ;用免疫组织化学SP方法检查bcl 2和bax的表达。结果 SHEEC1细胞经As2 O3 作用 2 4h出现细胞凋亡的典型形态改变。 4h可用Annexin V查出早期凋亡细胞。线粒体的超微结构改变如下 :As2 O3 作用 2～ 4h内线粒体增生 ,伴有电子密度高物质出现在基质 ,是早期细胞损伤的现象。作用 6h线粒体进行性肿胀 ,1 2h出现气球样外观 ,甚至外膜破裂。不同剂量皆引起损伤脱落细胞bcl 2表达下调和bax上调。结论 由As2 O3 诱导的SHEEC1细胞凋亡 ,线粒体形态改变是早期改变 ,细胞凋亡和bax表达上调及bcl

Objective In order to make clear how As 2O 3 targets the cells of esophageal carcinoma, the morphology of mitochondria and expression of bcl 2/bax were selected to study the SHEEC1 cell line during early apoptosis. Methods The esophageal carcinoma cell line SHEEC1 was treated by As 2O 3 with concentrations of 1, 2 and 3 μmol/L for the periods of 2, 4, 6, 12 and 24 h. Annexin V Fluorescein and DNA histogram were used to detect the early apoptotic cells by flow cytometry. The morphologic changes of the apoptotic cells were examined by means of light and electron microscopy. Changes in mitochondria of living cells were detected with Rhodamin 123 fluorescent probe by fluorescent microscopy. The expression of bcl 2 and bax in floating cells were determined by immunohistochemical procedures. Results Typical apoptotic morphological changes were found in SHEEC1 at 24 h after As 2O 3 treatment. Cells in the early stage of apoptosis were detected by Annexin V labeled at 4 h. The ultrastructural alterations of mitochondria were found as following: within 4 h after administrating As 2O 3, mitochondrial hyperplasia with small electron dense materials in its matrix was the earliest manifestation of cell injury. At 6 h progressive mitochondrial swelling lead to balloon like appearance and its outer membrane disrupted at 12 h. In all groups, down regulated expression of bcl 2 and over expression of bax were always found in damaged cells. Conclusion In the SHEEC1 cell line morphological changes of mitochondria are the early events in apoptosis induced by As 2O 3 and apoptosis is closely related to over expression of bax and down regulating expression of bcl 2. It is possible that As 2O 3 is a mitochondriotoxic anticancer agent for esophageal carcinoma.