survivin反义寡核苷酸诱导人肝癌细胞凋亡的相关信号转导途径

目的 利用人肝癌细胞株SMMC-7721细胞,探讨survivin基因诱导肝癌细胞凋亡的相关信号转导途径。方法 采用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞。采用Western印迹及原位杂交检测细胞内survivin蛋白及mRNA的表达。采用流式细胞仪及TUNEL染色检测细胞凋亡的变化。采用western印迹,免疫沉淀,逆转录-聚合酶链反应（RT-PCR）及激酶活性检测方法测定细胞内丝裂原活化蛋白激酶p38(p38MAPK)及半胱氨蛋白水解酶3表达及活性的变化,观察p38MAPK与半胱氨蛋白水解酶3活性变化之间的关系。结果 脂质体介导转染survivin反义寡核苷酸后,SMMC-7721肝癌细胞内survivin蛋白及mRNA表达可分别被下调84.6%及69.7%。凋亡细胞比率由转染前的0.70%增加至31.51%。转染后细胞内p38MAPK及半胱氨蛋白水解酶3表达无明显变化。但其活性显著升高,其中p38MAPK活性升高发生于半胱氨蛋白水解酶3活性升高之前。结论 转染survivin反义寡核苷酸可以有效诱导肝癌细胞发生凋亡,其诱导肝癌细胞凋亡的信号通过顺序激活p38MAPK-半胱氨蛋白水解酶3信号途径传导。

The signal transduction pathway related to hepatocellular carcinoma apoptosis induced by survivin antisense oligonucleotide

Objective The expression of survivin gene in hepatocellular carcinoma cell line SMMC-7721 was blocked by means of antisense oligonucleotide (ASODN) transfection. To investigate the signal transduction pathway of apoptosis induced by survivin ASODN. Methods survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent. The expression of survivin protein and mRNA was detected by western-blot and in situ hybridization method, respectively. Flow cytometer and TUNEL method were used to detect apoptosis. The changes in the expression and activity of p38MAPK and caspase-3 were assessed by western-blot, immuno-precipitation, RT-PCR, and kinase activity assess to study the relationship between the changes of p38MAPK and caspase-3 activity. Results The expression of survivin protein and mRNA decreased 84.6% and 69.7% respectively while apoptosis increased from 0.70% to 31.15% after transfected with survivin ASODN. P38MAPK activity increased significantly followed by increasing of caspase-3 activity after survivin ASODN transfection. Both p38MAPK and caspase-3 activity were inhibited after treated with inhibitor of p38MAPK, SB202190. Conclusion Transfection of survivin ASODN could induce hepatocellular carcinoma cells apoptosis effectively through activated p38MAPK-caspase-3 signal pathway sequentially.