5-氟尿嘧啶对人类绒癌JAR细胞增殖及凋亡的影响

Effect of 5-fluorouracil on proliferation and apoptosis of human choriocarcinoma JAR cells

目的:研究5-氟尿嘧啶(5-fluorouracil,5-FU)对人类绒毛膜癌JAR细胞增殖及凋亡的影响。方法:MTT法检测5-FU对JAR细胞的增殖抑制作用;流式细胞术检测5-FU对JAR细胞DNA含量的改变;倒置显微镜及荧光显微镜下观察JAR细胞凋亡过程中细胞核染色质的形态学改变。结果:MTT抑制实验得出5-FU对JAR细胞的增殖具有一定程度的抑制作用,且抑制作用呈时间、剂量依赖性;5-FU0μg/ml、20μg/ml、40μg/ml、60μg/ml、80μg/ml处理JAR细胞24h,通过倒置光学显微镜可观察到JAR细胞的形态变化,可见随着5-FU浓度的增加,JAR细胞逐渐缩小、变圆、分散,细胞核减少;用流式细胞仪检测5-FU对JAR细胞DNA含量的改变发现:12h均未诱导出凋亡,直到24h5-FU处理组才诱导出凋亡,48h由于JAR细胞耐酸性低,出现自溶现象,干扰实验结果,因此只选择24h为实验时间点。0μg/ml(对照组)、20μg/ml、40μg/ml、60μg/ml、80μg/ml5-FU处理JAR细胞24h后,流式细胞术检测发现5-FU处理组可诱导绒癌细胞凋亡。5-FU在0μg/ml^40μg/ml范围内可以呈剂量依赖性促进JAR细胞凋亡,在40μg/ml^80μg/ml范围内,凋亡反而降低。40μg/ml、60μg/ml5-FU处理组凋亡率与对照组相比差异有统计学意义。在0μg/ml^80μg/ml范围内5-FU处理组细胞坏死增加,呈剂量依赖性。坏死率高于不加5-FU的对照组,差异具有统计学意义;Ho-echst33342/PI荧光染色结果显示,典型的细胞凋亡形态学改变,可见典型的凋亡小体,表现为核染色质聚集、核固缩、核碎裂。20μg/ml、40μg/ml、60μg/ml、80μg/ml各处理组的凋亡率均显著高于对照组(P<0.01),80μg/ml处理组的坏死率也显著高于对照组。结论:5-FU对JAR细胞的增殖具有一定程度的抑制作用,5-FU对绒毛膜癌JAR细胞有凋亡诱导作用。

Objective：To investigate the effect of 5-fluorouracil on proliferation and apoptosis of human choriocarcinoma JAR cells.Methods：MTT assay was used to determine the proliferative and inhibitive effects of 5-fluorouracil on JAR cells;flow cytometry was applied to detect the changes of DNA content of JAR cells;invert microscope and fluorescence microscope were used to observe the morphological changes of cell nuclei and chromatin in the course of cell apoptosis.Results：5-fluorouracil had a certain inhibitive effect on proliferation of JAR cells,showing time-dependent manner and dose-dependent manner;after treated JAR cells with 5-fluorouracil of different doses（0 μg/ml,20 μg/ml,40 μg/ml,60 μg/ml and 80 μg/ml）for 24 hours,the morphological changes of JAR cells could be seen under invert microscope,JAR cells became small,round,dispersion,and the amount of cell nuclei decreased with the increase of 5-fluorouracil dose;after 12 hours,apoptosis was not induced,until 24 hours,apoptosis was induced in 5-fluorouracil group,autolysis phenomenon occurred because of low acid resistance of JAR cells,which affected the results;thus,24 hours were selected as the time point;treating JAR cells with 5-fluorouracil of different doses（0 μg/ml,20 μg/ml,40 μg/ml,60 μg/ml and 80 μg/ml）for 24 hours,the apoptosis of choriocarcinoma cells was induced in 5-fluorouracil group,which was found by flow cytometry;within the range of 0 μg/ml～40 μg/ml,5-fluorouracil promoted apoptosis of JAR cells,showing a dose-dependent manner;within the range of 40 μg/ml～80 μg/ml,the apoptosis rate decreased;there was significant difference in apoptosis rate of JAR cells between 40 μg/ml group and 60 μg/ml group;within the range of 0 μg/ml～80 μg/ml,the cell necrosis rate increased,showing a dose-dependent manner,the cell necrosis rate was higher than that in non-5-fluorouracil group;Hoechst33342/PI fluorescence staining showed typical morphological changes of apoptosis,apoptotic bodies were seen,the manifestations included nuclear chromatin aggregation,karyopyknosis and nuclear fragmentation;the apoptosis rates of 20 μg/ml group,40 μg/ml group,60 μg/ml group and 80 μg/ml group were significantly higher than that of control group（P0.01）;the necrosis rate of 80 μg/ml group was significantly higher than that of control group.Conclusion：5-fluorouracil can inhibit the proliferation of JAR cells to a certain extent,5-fluorouracil can induce the apoptosis of JAR cells.