摘要
目的研究15-脱氧前列腺素 J_2(15-d-PGJ_2)体外诱导人肝癌细胞系 BEL-7402细胞失巢凋亡的特性,并探讨其机制。方法在纤连蛋白(Fn)或多聚2-羟乙基甲基丙烯酸脂(poly-HEMA)包被的培养板中,应用光学显微镜观察不同因素处理后的细胞生长状态和形态学变化;应用 DNA 片段分析和流式细胞术观察细胞的凋亡变化;应用 Western 印迹技术观察细胞焦点黏着斑激酶(FAK)和磷酸化 FAK(p-FAK)蛋白质的表达;应用小 RNA 干扰技术来沉默 FAK 基因。结果在 Fn 包被的培养板中贴壁生长的 BEL-7402细胞经15-d-PGJ_2处理24 h 或48 h 后,细胞变圆,脱离细胞外基质呈悬浮状态改变,而且这种改变呈时间和剂量依赖性关系;其中以浓度为20 μmol/L 的15-d-PGJ_2最为显著,24 h 和48 h 时间点细胞的黏附比率分别为(66.0±3.6)%和(35.0±5.0)%,与肝癌细胞对照相比差异有统计学意义(P<0.05)。经流式细胞术和 DNA 片段分析后发现肝癌细胞发生了失巢凋亡;Western 印迹显示 p-FAK 蛋白下降,而 FAK 蛋白质表达水平未发生改变。结论 15-d-PGJ_2在体外能够诱导 BEL-7402细胞失巢凋亡,这一过程与细胞 FAK 磷酸化水平降低有关。
Objective To investigate the effect of 15-deoxy-△^12＇ 14-prostaglandin J2 （ 15-d-PGJ2 ） on the anoikis of hepatocellular carcinoma （HC） cells and mechanisms thereof. Methods Fibronectin or polyhydroxyethylmethacrylate （poly-HEMA） were coated onto tissue culture plates, cell growth status and morphological changes were detected by optical microscope. DNA fragmentation analysis and Flow cytometry were used to measure cell apoptotic activity. Western blotting analysis was performed to detect the levels of focal adhesion kinase （FAK） and phosphorated FAK （p-FAK）. Furthermore, small interfering RNA （siRNA） was used to suppress FAK expression. Results The adhesion rate of the BEL-7402 cells treated with 15-d-PGJ2 began to decrease 12 h after the treatment, time- and dose-dependently compared with the HC cell control group （all P 〈 0.05 ） ; when the concentration of 15-d-PGJ2 was 20 μmol/L, the adherent cells ratio at 24 h and 48 h later were （66.0 ±3.6）% and （35.0 ±5.0）% respectively. Anoikis of BEL- T402 cells was observed by flow cytometry and DNA fragmentation analysis. Western blotting showed that the p-FAK level of the BEL-7402 cells treated with 15-d-PGJ2 for 24 h decreased dose-dependently, however, the total FAK protein did not change. Conclusion 15-d-PGJ2 induces anoikis and decreases the phosphorylated FAK expression of the hepatocellular carcinoma cells.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第42期3001-3005,共5页
National Medical Journal of China
基金
国家自然科学基金(30672053)
