c-fos反义寡核苷酸激活caspase 3诱导人肝癌HepG2细胞凋亡的研究

Effect of C-fos antisense oligodeoxynucleotide-induced activation of Caspase 3 on inducing apoptosis of human hepatocellular carcinoma HepG2 cells

目的探讨c-fos反义探针诱导人肝癌HepG2细胞凋亡以及caspase3在其中的作用。方法人肝癌HepG2细胞分为3组,①对照组:加生理盐水10μl;②c-fos正义寡核苷酸(SO)处理组:加入SO10μl(5μg/μl);③c-fos反义寡核苷酸(ASO)处理组:加入ASO10μl(5μg/μl)。各组细胞再培养1h后进行后续实验。采用Hoechst 33258染色检测细胞凋亡情况,并分别运用实时荧光定量PCR和Western blotting检测caspase 3在mRNA和蛋白水平的表达变化。结果Hoechst 33258细胞染色显示,对照组和SO处理组的细胞核为弥散均匀的圆形或椭圆形荧光,而ASO处理组的细胞核或细胞质内可见致密浓染的颗粒、新月体或环状荧光,核固缩,部分切片可见核碎裂。PCR和Western blotting检测显示,与对照组和SO处理相比,ASO处理组caspase 3 mRNA和蛋白的表达均明显上调。结论c-fos反义探针可以诱导人肝癌HepG2细胞凋亡,且caspase 3参与了此过程。

Objective Proliferation and apoptosis play a major role in the development of tumor cells, and the intranuclear transcriptional factor c-fos is significantly up-regulated in the primary hepatocellular carcinoma and involved in early carcinogenesis. The purpose of the present study is to investigate the apoptotie effect of c-fos antisense oligodeoxynucleotide （ASO） on human hepatocellular carcinoma HepG2 cells and the participation of Caspase3 in this process. Methods Cell culture, Hoechst 33258 staining, real-time PCR and Western blotting were used in present study. Cultured HepG2 ceils were divided into 3 groups： 1） control group., cultured with 10μl saline; 2） sense oligodeoxynucleotide （SO, used as a negative control） treated group： co-cultured with 10μl SO （5μg/μl） ; 3） ASO treated group： co- cultured with ASO 10μl （5μg/μl）. The subsequent experiments were performed lh after cultivation for each group. Hoechst 33258 staining was performed to detect the apoptosis by observing the staining of nuclear chromatin. Real-time PCR and Western blotting were used respeetively to detect the expression of Caspase 3 at mRNA and protein levels after different treatments. Results Hoechst 33258 staining revealed that the nuclei of HepG2 cells showed diffuse and adqulis fluorescence in control and SO-treated groups, while dense and clark fluorescence was observed in ASO-treated group, which indicated that c-fos ASO bad significantly induced apoptosis of HepG2 cells. The expression of Caspase 3 in ASO group was enhanced both at mRNA and protein levels compared to that in control groups. Conclusion C-fos ASO significantly induces apoptosis in human hepatoeellular carcinoma HepG2 cells, as shown by Hoechst 33258 staining and higher expression of Caspase 3 mRNA and protein. Moreover, Caspase 3 activation is involved and probably plays an important role in c-fos ASO- induced apoptosis of HepG2 cells.