放线菌酮快速诱导小鼠肝细胞凋亡的实验研究

目的　研究放线菌酮 (CHX)诱导小鼠肝细胞凋亡的作用机制。方法　利用琼脂糖凝胶电泳、流式细胞术、电镜、活细胞荧光染色、HE染色等方法观察CHX诱导肝细胞凋亡的情况。结果　 ( 1)2 0mg kg以上剂量的CHX作用 1h即可出现典型的肝细胞凋亡 ,溴化乙啶和吖啶橙荧光染色为桔黄色 ,HE染色可见核固缩、浓染以及凋亡小体形成 ,凋亡细胞周围无炎性细胞浸润。电镜观察可见肝细胞体积变小 ,染色质固缩。 ( 2 )电泳结果显示 :不同剂量的CHX处理的小鼠其肝细胞NDA呈典型的“Ladder”电泳条带。 ( 3 )流式细胞术分析 :在CHX 2 0mg kg ,作用 1h以后即可见到肝细胞凋亡峰 (AP峰 )。随CHX剂量增加和作用时间的延长 ,凋亡率也逐渐增高 ,凋亡细胞的NDA含量减少。 ( 4)C myc基因表达量随CHX剂量增加和作用时间的延长而增加 ,但bcl 2基因表达呈下降趋势。结论　CHX可以快速诱导小鼠肝细胞凋亡 ,在凋亡过程中C myc基因表达量增加 ,bcl 2基因表达量降低 ,细胞凋亡率与CHX剂量和作用时间有一定的线性关系

Experimental study on rapid induction of hepatocyte apoptosis in mouse by cycloheximide

Objective To study the role of cycloheximide inducting hepatocyte apoptosis in mouse. Methods The hepatocyte apoptosis was studied with agarose gel electrophoresis,flow cytometry,transmitting electron microscopy,fluorescence labeling of alive cells and staining with H and E. Results (1)After injecting CHX with dose of >2.0 mg/kg for>1 hour,the typical apoptosis was appeared.Alive hepatocytes were differintiated from apoptotic cells after stained with ethidium bromide (EB) and acidine orange (AO), and observed with fluorescence microscopy.The pathological characteristics of apoptotic hypatocytes were that the nucleus of hepatocyte became small and dark blue,and apoptotic bodies appeared after stained with H and E.The volume of apoptotic cells became small and nucleus irregular,in the ultrastructure.(2)The results of electrophoresis showed that clear ladder" DNA extracted from liver tissue of mouse were observed.(3)The results of flow cytometry showed that after giving CHX with 2.0 mg/kg for 1 hour,AP peak was appeared.The rate of apoptosis was increased and DNA content of apoptotic cells was decreased along with increased doses of CHX in prolonged time.(4)The expressivity of C myc gene increased and bcl 2 decrease according to increaseing doses of CHX and prolonging time for which CHX was injected. Conclution CHX could rapidly induce hepatocyte apoptosis in mouse,the expressivity of C myc gene increased and bcl 2 gene reduced during apoptosis period and there was a defintie lnear relationship between rate of the apoptosis and the dose of CHX and time of CHX effecting.