摘要
目的观察无血清条件下bcl2对神经酰胺诱导人肝癌细胞HepG2凋亡的影响。方法通过脂质体介导的基因转染,建立稳定转染表达反义bcl2mRNA的cDNA片段的真核表达质粒的人肝癌细胞HepG2(HepG2antibcl2)并鉴定,采用AOEB荧光染色、流式细胞术DNA倍体分析和DNA琼脂糖凝胶电泳检测细胞凋亡,分光光度法测定Caspase3活性。结果终浓度50μmol/LC2神经酰胺作用24h,可诱导无血清培养的HepG2细胞、HepG2vector细胞及HepG2antibcl2细胞发生凋亡。AOEB染色可见典型的细胞凋亡表现。流式细胞仪检测显示C2神经酰胺作用12,18,24h,HepG2antibcl2细胞凋亡率较同时间HepG2细胞及HepG2vector细胞凋亡率显著增加(P<0.05)。终浓度50μmol/LC2神经酰胺作用12h,HepG2antibcl2细胞DNA凝胶电泳呈现梯状条带,而HepG2细胞及HepG2vector细胞作用24h出现DNA梯状条带。HepG2细胞和HepG2vector细胞Caspase3活性随C2神经酰胺(50μmol/L)作用时间延长而增强,HepG2antibcl2细胞Caspase3活性始终维持较高水平。结论低表达bcl2可通过增加Caspase3活性从而促进C2神经酰胺诱导的HepG2细胞凋亡。
Objective To observe the effect of low expression bcl-2 on the ceramide-induced apoptotic in hepatoma cells HepG2. Methods The recombinant plasmids pcDNA3.1/Hygro（-）bcl-2 with antisense bcl-2 eDNA sequence and vector pcDNA3.1 were transfected into HepG2 cells by liposome respectively. HepG2, HepG2-vector and HepG2-anti-bcl-2 cells were incubated without serum for 48 h and then treated with 50 μmol/L C2-ceramide in serum-free DMEM medium for 24 h. RT-PCR and Western blotting detected the expression of bcl-2. The apoptotic cells were detected by AO-EB double fluorescent staining, flow cytometry and DNA agarose gel electrophoresis. The activities of caspase-3 were examined by colorimetric. Results After 12 h treatment with 50 μmol/L C2-ceramide, HepG2-anti-bcl-2 cells displayed an apoptotic phenotype confirmed by a high（peak〉20%） proportion by flow cytometer and DNA ladder by DNA electrophoresis. C2-ceramide induced caspase-3 activation in HepG2, HepG-rector, and HepG2-anti-bcl-2 cells. However the activities of caspase-3 in HepG2-anti-bcl-2 cells increased significantly and keep in high level. Conclusion Down-regulated of bcl-2 increased activities of caspase-3 and promotes C2-ceramide-induce apoptosis in hepatoma HepG2-anti-bcl-2 cell.
出处
《福建医科大学学报》
2006年第2期121-124,共4页
Journal of Fujian Medical University
基金
福建省教育厅科研基金资助项目(K20044)
