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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 characteristics of 15-deoxy-prostaglandin J_2 (15-d-PGJ_2) induced apoptosis in vitro and to explore its possible mechanism. Methods The growth and morphological changes of cells treated with different factors were observed under optical microscope in fibronectin (Fn) or poly-HEMA-coated culture plates. DNA fragmentation and flow cytometry were used to observe the changes of cell apoptosis. Western blotting was used to observe the expression of FAK and p-FAK protein. The small interfering RNA (FAK) gene was used to silence the FAK gene . Results BEL-7402 cells adherent to Fn-coated plates were round after treatment with 15-d-PGJ 2 for 24 h or 48 h. Cells were detached from the extracellular matrix in suspension and changed in time (66.0 ± 3.6)% and (35.0 ± 5.0)%, respectively, at a concentration of 20 μmol / L for 24 h and 48 h, respectively, in a dose-dependent manner Compared with the control group, the difference was statistically significant (P <0.05). Apoptosis was observed in HCC cells by flow cytometry and DNA fragment analysis. Western blotting showed that p-FAK protein was decreased but FAK protein expression level did not change. Conclusion 15-d-PGJ 2 can induce apoptosis of BEL-7402 cells in vitro, which is related to the decrease of FAK phosphorylation.