大肠癌细胞凋亡模型的建立及凋亡细胞内质网膜系统的变化

Colorectal Carcinoma Cell Apoptosis and Morphological Changes of Endoplasmic Reticulum Induced by Retinoic Acid

目的 :建立全反式维甲酸 (ATRA)诱导人大肠癌细胞凋亡的体外模型 ,观察凋亡细胞内质网膜系统的变化。方法 :应用光镜、透射及扫描电镜、DNA凝胶电泳、流式细胞术及末端脱氧核苷酰转移酶原位标记 (TU NEL法 ) ,观察维甲酸诱导的人大肠癌 CCL2 2 9细胞凋亡特征。结果 :维甲酸诱导 CCL2 2 9细胞凋亡 ,产生典型的形态学改变 ,在琼脂糖凝胶电泳上呈现特征性的 DNA ladder,DNA直方图上显示亚二倍体峰 ,具有时间和剂量依赖性。CCL2 2 9细胞在被维甲酸诱导成熟分化后发生凋亡。凋亡过程中内质网膜系统的结构与分布变化为 :内质网逐渐减少 ,向核周聚集 ,部分呈扩张状态 ,形成大小不等的小泡状或短管状结构。结论

Objective:We wanted to explain the mode and mechanisms of cell death in human colorectal carcinoma cells induced by treatment with all trans retinoic acid (ATRA). Methods: In vitro, a model of apoptosis was developed. Apoptosis induced by retinoic acid (RA) in cell line CCL229 was studied by fluorescence microscopy, electron microscopy, agarose gel electrophoresis, flow cytometry, and TdT mediated dUTP biotin in situ nick end labeling (TUNEL).Results: ATRA was found to induce apoptosis and extensive DNA fragmentation in the CCL229 cell line as early as 12 hours after treatment. Degradation of the nuclear DNA of apoptotic cells was demonstrated by visualization of “DNA ladders” on gel electrophoresis. DNA flow cyto fluorimetric analysis showed the appearance of a discrete population of cells containing hypodiploid DNA. A concentrations between 10 -5 mol/L and 10 -8 mol/L, RA treated 229 cells were induced to apoptosis in a dose and time dependent manner. During RA induced apoptosis, there was a gradually decrease and dilatation of ER in the cytoplasm, and dilated vesicular or tubular cisternae of endoplasmic reticulum (ER) were found either in the cytoplasm or in the apoptotic bodies.Conclusion:ATRA induced apoptosis in colorectal carcinoma cells at pharmacologically relevant doses. The changes of structure and distribution of ER induced by RA were probably related to the changes of protein synthesis in ER.