摘要
目的 观察KAI1基因诱导的自噬在人胰腺癌MiaPaCa-2细胞中调节凋亡的分子途径.方法 实验分为用细胞外调节蛋白激酶（ ERK）的磷酸化阻断剂PD98059预处理组、Caspase-3的活化阻断剂VAD-FMK预处理组和未用PD98059、VAD-FMK预处理组3大组；每大组再分3小组进行不同处理,感染腺病毒空载体AD5-null对照组、感染人KAI1基因的重组腺病毒载体AD5-KAI1组和用自噬阻断剂3 MA预处理阻断自噬后再感染AD5-KAI1组.通过膜朕蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染法检测细胞凋亡,流式细胞术评价Caspase-3活化水平,Western印迹检测ERK磷酸化水平和PARP蛋白的裂解情况.结果 感染AD5-KAI1后癌细胞表达KAI1蛋白的同时绿色荧光蛋白（GFP） LC3绿色颗粒增加,Caspase-3活化、PARP-1裂解、ERK磷酸化和凋亡均明显增多.自噬阻断剂3-MA预处理后,凋亡率由（63.0±7.9）％升至（88.0±4.5）％,Caspase-3活化由（34.0±2.8）％升至（44.2±4.0）％,同时PARP-1裂解更多.Caspase-3阻断剂VAI-FMK预处理可完全抑制3-MA预处理导致的促凋亡作用.ERK磷酸化抑制剂PD98059不能抑制3 MA预处理导致的促凋亡作用.结论 KAI1诱导的自噬通过抑制细胞中Caspase-3活化和PARP裂解而不是通过抑制ERK磷酸化来拮抗KAI1诱导的凋亡.
Objective To explore the pathway of KAI1 induced autophagy regulating apoptosis in human pancreatic cancer cell line MiaPaCa-2.Methods There were three groups in the experiment,which were extracellular regulated protein kinases （ERK） phosphorylation inhibitor PD98059 pretreated group,Caspase-3 activation inhibitor VAD-FMK pretreated group and no PD98059 or VAD-FMK pretreated groups.And each group was divided into three sub groups with different treatment,which were adenovirus AD5-null vector infected control group,the human KAI1 gene recombinant adenovirus vector AD5 KAI1 infected group and autophagy inhibitor 3-MA pretreated and AD5-KAI1 infected group.The cell apoptosis was observed by AnnexinV-FITC/PI double staining.Caspase-3 activation level was evaluated by flow cytometry.ERK phosphorylation and poly（ADPribose） polymerase （PARP） cleavage were determined by Western blot.Results After the cancer cells infected with AD5 KAI1,KAI1 protein was expressed and GFP-LC3 green particles increased.Caspase-3 activation,PARP cleavage,ERK phosphorylation and apoptosis increased obviously.After autophagy inhibitor 3-MA pretreated,the percentage of apoptosis increased from （63.0 ± 7.9）% to （88.0±4.5） % and Caspase-3 activation increased from （34.0±2.8） % to （44.2±4.0） % and PARP cleavage more.The apoptosis induced by 3-MA could be totally inhibited by Caspase-3 activation inhibitor VAD-FMK pretreated but could not be inhibited by ERK phosphorylation inhibitor PD98059.Conclusion KAI1- induced autophagy inhibits apoptosis through the downregulation of Caspase-3activation and PARP cleavage instead of ERK phosphorylation.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2011年第12期817-821,共5页
Chinese Journal of Digestion
基金
国家自然科学基金（81071982,30470798）
