C_(2)-神经酰胺在A549/PC9细胞凋亡中的作用及影响

The role and influence of ceramide in A549/PC9 cell apoptosis

目的探讨C_(2)神经酰胺(鞘磷脂类物质之一)对非小细胞肺癌(A549和PC9)细胞凋亡的影响。方法培养非小细胞肺癌细胞系(A549和PC9),提取总蛋白进行Western blot,检测两种细胞中凋亡蛋白Caspase-3及cleaved Caspase-3蛋白的表达情况;使用CCK-8比色法筛选药物浓度;Hoechst 33258凋亡染色检查细胞凋亡情况;流式细胞化学术检测细胞凋亡比率,RT-PCR检测凋亡蛋白Caspase-3的表达。结果神经酰胺在50μmol/L浓度处理后细胞活力均在70%左右;与对照组相比,神经酰胺组凋亡蛋白表达升高(P<0.05);流式细胞化学术及凋亡染色检测显示神经酰胺处理组凋亡率与对照组相比增加(P<0.05);在基因水平检测mRNA显示凋亡蛋白Caspase-3表达增加(P<0.05)。结论C_(2)-神经酰胺可以促使非小细胞肺癌细胞凋亡,从而为临床肺癌的化疗提供新的治疗靶点。

Objective To explore the effect of C_(2)-ceramide,one of the sphingomyelin substances,on apoptosis of non-small cell lung cancer cells(A549 and PC9).Methods Non-small cell lung cancer cell lines(A549 and PC9)were cultured,total proteins were extracted and Western blot was performed to detect the expression of apoptotic protein Caspase-3 and cleaved Caspase-3 in the two cells.CCK-8 colorimetric method was used to screen drug concentration.Hoechst 33258 apoptosis staining was used to detect apoptosis.The apoptosis rate was detected by flow cytometry,and the expression of apoptotic protein Caspase-3 was detected by RT-PCR.Results The cell viability was about 70%when ceramide was treated at 50μmol/L.Compared with the control group,the expression of apoptotic proteins increased in the ceramide group(P<0.05).Flow cytometry and apoptosis staining showed that the rate of apoptosis increased in the ceramide treatment group compared with the control group(P<0.05).mRNA detection at gene level showed increased the expression of apoptotic protein Caspase-3(P<0.05).Conclusion C_(2)-ceramide can promote the apoptosis of non-small cell lung cancer cells,thus providing a new therapeutic target for clinical lung cancer chemotherapy.