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目的探讨鞘氨醇激酶2(SphK2)在5-氟尿嘧啶(5-FU)诱导的人结肠癌HCT116细胞凋亡中的作用。方法采用Hoechst33342染色检测5-FU作用48h后HCT116细胞凋亡形态学的变化;流式细胞术检测5-FU作用48h后HCT116细胞的凋亡率,并分析SphK2的干扰或过表达对5-FU诱导的HCT116细胞凋亡率的影响;用蛋白质印迹法观察5-FU诱导HCT116细胞SphK2活性的改变及干扰或过表达SphK2对5-FU诱导凋亡中凋亡标记蛋白的影响。结果 5-FU能诱导HCT116细胞凋亡,细胞出现了明显的核固缩、染色质凝集等凋亡形态变化。细胞SphK2被干扰后,与未被干扰的细胞相比,5-FU诱导后的细胞凋亡率增高(P<0.01),凋亡标记蛋白表达水平升高;而过表达SphK2的细胞在5-FU作用下其凋亡率较对照组(空质粒处理)下降(P<0.01),凋亡标记蛋白表达水平亦下降;5-FU可诱导磷酸化SphK2水平增高。结论 SphK2对5-FU诱导的HCT116细胞凋亡具有负调控作用。
Objective To investigate the role of SphK2 in the apoptosis of human colon cancer HCT116 cells induced by 5-fluorouracil (5-FU). Methods Hoechst33342 staining was used to detect the morphological changes of HCT116 cells induced by 5-FU for 48 hours. Flow cytometry was used to detect the apoptosis rate of HCT116 cells after treated with 5-FU for 48 hours. The effects of SphK2 on 5-FU Induced apoptosis of HCT116 cells. Western blotting was used to observe the effect of 5-FU on SphK2 activity in HCT116 cells and the effect of SphK2 on apoptosis induced by 5-FU. Results 5-FU could induce the apoptosis of HCT116 cells, and the apoptotic cells such as nuclear condensation and chromatin condensation were observed. When SphK2 was interfered, the apoptosis rate of 5-FU induced by 5-FU increased (P <0.01) and the expression of apoptosis marker protein increased compared with the untreated cells. However, FU significantly decreased the apoptotic rate (P <0.01) and the expression of apoptotic marker protein decreased compared with the control group (P <0.01). 5-FU induced an increase in the level of phosphorylated SphK2. Conclusion SphK2 can negatively regulate the apoptosis of HCT116 cells induced by 5-FU.