摘要目的 观察感染Ad5-KAI1前后人胰腺癌细胞MiaPaCa-2自噬水平的变化,并初步探讨其机制.方法 应用无KAI1表达的人胰腺癌细胞MiaPaCa2,通过感染带有KAI1目的基因的复制缺陷型腺病毒Ad5-KAI1使细胞表达KAI1,以Ad5 -null感染作为阴性对照,亲本细胞为空白对照.用透射电镜观察细胞自噬小体,共聚焦显微镜观察自噬标志LC3颗粒.应用阻断剂PD98059和LY294002干预细胞,蛋白质印迹法检测自噬相关蛋白beclin 1、LC3-Ⅱ、LC3-Ⅰ及ERK-1/2、磷酸化ERK-1/2(p-ERK-1/2)、AKT、p-AKT的表达.结果 以100 MOI Ad5-KAI1感染细胞24h,表达KAI1蛋白的细胞达(84.97±8.56)％；LC3颗粒从4个左右增加到20个以上；细胞线粒体肿胀、变性,胞质内双层膜样结构增加；Beclinl表达增加(1.4±0.3)倍,LC3-Ⅱ/LC3-Ⅰ表达增加(8.00 ±2.78)倍.PI3K阻断剂LY294002预处理细胞后可以有效地抑制MiaPaCa-2细胞AKT的磷酸化(2.756降至1.516),但不能抑制LC3-Ⅱ/LC3-Ⅰ比值的增加(0.770增加到1.403).ERK阻断剂PD98059预处理细胞后不仅可以有效地抑制MiaPaCa-2细胞ERK的磷酸化(1.637降至0.403),而且可以抑制beclin 1蛋白表达的上调(2.377降至1.150)和LC3-Ⅱ/Lc3-Ⅰ比值的增加(2.225降至0.680).结论 KAI1明显促进MiaPaCa2细胞内自噬,它是通过ERK而不是AKT磷酸化途径促进自噬的.

abstractsObjective To study the change of autophagy of human pancreatic cancer cell MiaPaCa-2 before and after Ad5-KAI1 tranfection,and to investigate the possible mechanism.Methods The MiaPaCa-2 cells without KAI1 expression were infected with Ad5-KAI1 with KAI1 target gene,and Ad5-null was used as negative control,and parental cell was used as blank control.The formation of autophagosomes was observed by electromicroscopy.The green fluorescent protein-labeled light chain 3 (LC3) associations with autophagosome membranes was detected by confocal microscopy.PD98059,LY294002 were applied to pre-treat the cells.The expression levels of beclin 1,AKT,ERK,the phosphorylation of AKT and ERK protein and the ratio of LC3-Ⅱ to LC3- Ⅰ were detected by Western blotting.Results After 100 MOI Ad5-KAI1 infections for 24 h,the rate of cell expressing KAI1 protein reached (84.97 ±8.56)％,number of LC3 increased from 4 to 20; and swelling,degeneration of mitochondria was observed,and bilayer-like structure in cytoplasm was found.The expression of beclinl increased (1.4 ±0.3 ) folds,and the expression of LC3-Ⅱ/LC3- Ⅰ increased (8.00 ±2.78) folds.PI3K blockade LY294002 pretreatment significantly suppressed the phosphorylation of AKT of MiaPaCa-2 (2.756 vs 1.516),but it did not inhibit the increase of ratio of LC3-Ⅱ to LC3- Ⅰ (0.770 vs 1.403).ERK blockade PD98059 pretreatment not only significantly suppressed the phosphorylation of ERK of MiaPaCa-2 ( 1.637 vs 0.403 ),but also inhibit the up-regulation of beclin 1 protein expression ( 2.377 vs 1.150) and increase of ratio of LC3- Ⅱ to LC3- Ⅰ (2.225 vs 0.680).Conclusions KAI1 can significantly induce autophagy of human pancreatic cell line MiaPaCa-2 through phosphorylation of ERK rather than AKT.