艰难梭菌毒素A对SMMC-7721细胞增殖和凋亡的影响

Effects of toxin A from Clostridium difficile on SMMC-7721 cells proliferation and apoptosis

目的:研究艰难梭菌毒素A对人肝癌细胞株(SMMC-7721)增殖和凋亡的影响。方法:克隆分析及四甲基偶氮唑盐(MTT)法用于细胞增殖的分析;透射电镜及单细胞凝胶电泳用于凋亡细胞形态学变化及DNA片段的观察;流式细胞术检测凋亡细胞数及Bcl-2和P53蛋白的表达。结果:0.018～4.690mg/L的毒素A明显抑制SMMC-7721细胞的克隆形成,并以时间和浓度依赖方式抑制SMMC-7721细胞的增殖;SMMC-7721细胞与4.690mg/L毒素A共同培养48h,透射电镜观察到典型的凋亡形态学变化,单细胞凝胶电泳显示细胞DNA的损伤;流式细胞仪分析结果显示:0.073～4.690mg/L毒素A诱导6.8%～41.8%细胞凋亡;0.293～4.690mg/L毒素A降低Bcl-2、增高p53蛋白的表达。结论:艰难梭菌毒素A对SMMC-7721细胞有明显的增殖抑制活性,此作用通过诱导细胞凋亡产生。

Aim： To study the effects of Clostridium difficile toxin A on the cytotoxity and apoptosis of human heparoma cell line SMMC-7721. Methods： Clone inhibiting experiment and MTT calorimetric assay were used to assay SMMC-7721 proliferation. Morphological changes relevant to the apoptosis and the DNA damage were ana- lyzed by transmission electron microscope and single cell gel assay. The number of apoptosis cells and the expression of Bcl-2 and p53 protein were detected by the flow cytometry. Results： 0. 018- 4. 690 mg/L toxin A significantly decreased the colony information of SMMC-7721 cells, and greatly inhibited SMMC-7721 proliferation in a time- and concentration-dependent manner. Morphological changes related to apoptosis were evident under transmission electron microscope and DNA damage was detected by single cell gel assay when SMMC-7721 cultured with 4. 690 mg/L toxin A for 48 h. Toxin A 0. 073-4. 690 mg/L induced apoptosis in SMMC-7721 cells from 6.8% to 41.8%. In addition, Toxin A 0, 293-4. 690 mg/L significant decreased Bcl-2 protein expression and increased p53 protein expression in SMMC-7721. Conclusion： These results showed that clostridium difficile toxin A had a significantly cytotoxicity in human SMMC-7721 which was attributed to toxin A induced apoptosis.