摘要
目的:探讨分裂原激活的蛋白激酶家族(MAPK)信号转导通路与脂质体生存素(survivin)反义寡核苷酸转染诱导人胃癌细胞系HS-746T凋亡的关系. 方法:设计合成survivin反义寡核苷酸(ASODN)转染胃癌HS-746T细胞,分空白对照组,脂质体和正义链转染组,100,200,400 nmoL/L反义链转染组及P38MAPK、ERK1/2抑制剂组.用流质细胞仪检测转染后2,4,8, 12,24 and 48 h各组细胞增生和凋亡指数.逆转录聚合酶链反应(RT-PCR)、免疫细胞化学染色法、Western blot 蛋白免疫印迹法、免疫沉淀法及激酶活性测定法检测各对照组及ASODN组转染后suevivin mRNA和P38MAPK, ERK1/2,survivin蛋白表达和活性的变化. 结果:不同时间空白组、脂质体和正义链转染组ERK1/2, P38MAPK表达均无明显差异,各浓度survivin ASODN转染组凋亡细胞增多,凋亡指数高于其他对照组.P38MAPK抑制剂组凋亡细胞减少,ERK1/2抑制剂组增多.Survivin mRNA和蛋白表达随着转染浓度的增加而降低.磷酸化和非磷酸化细胞外信号调节激酶(extracellular signal-regulated kinase1/2,ERK1/2)的表达随药物浓度增加而减弱.P38MAPK 的表达在各组均相同,但ASODN转染组活性增高. 结论:Survivin基因反义寡核苷酸在体外可能通过MAPK信号转导通路激活凋亡相关的信号途径P38MAPK,阻断与细胞增生相关的信号途径ERK1/2来诱导胃癌细胞凋亡, 抑制人胃癌细胞的增生.
AIM: To study the relation of mitogen-activated protein kinase (MAPK) signal transduction and apoptosis of human gastric carcinoma cells HS-746T induced by liposomes of survivin antisense oligonucleotide (ASODN). METHODS: Survivin ASODN was designed and synthesised to transfect human gastric carcinoma cells HS-746T. The cultured cells were divided into 6 groups: vacuity control group, liposome and sense oligonucleotide (SODN) group, 100, 200 and 400 nmoL/L ASODN group and P38MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor groups. Apoptotic index (AI) and proliferative index (PI) were examined by flow cytometry after transfection 2, 4, 8, 12, 24 and 48 h. RT-PCR, immunocytochemical stain, Western blot, immuno-precipitation and kinase activity assay were used to detect protein expression and activity of P38MAPK, ERK1/2, survivin and survivin mRNA after transfection. RESULTS: Expression of ERK1/2 and P38MAPK has not significantly different among vacuity control group, liposomes group and SODN group. The apoptotic cells increased in anisoconcentration survivin ASODN groups and AI was higher than that of other control group. Apoptotic cells decreased in P38MAPK inhibitor group while increased in ERK1/2. The protein and mRNA expression of survivin de- creased when transfection concentration was increased. The phosphorylated and nonphosphorylated ERK1/2 showed a dose-and time-dependent decrease whereas protein level of p38MAPK remained unchanged, but activity increased. CONCLUSION: Survivin ASODN can induce apoptosis of human gastric carcinoma cells in vitro though MAPK signal transduction including activating apoptosis-related signal P38MAPK and suppressing proliferation-related signal ERK1/2.
出处
《世界华人消化杂志》
CAS
2004年第5期1034-1039,共6页
World Chinese Journal of Digestology
