CAJ | 학술논문

目的：通过饥饿诱导人神经胶质瘤细胞发生自噬，利用 siRNA 技术部分沉默细胞内氯通道蛋白4（CLIC4），探讨 CLIC4在细胞自噬中的作用。方法根据 CLIC4的基因序列构建 CLIC4 siRNA 质粒，建立稳定转染CLIC4 siRNA 的 U251细胞株，分析 CLIC4蛋白表达；MTT 法检测转染 pSH1Si-CLIC4对细胞生存率的影响；利用Western Blot 检测转染 CLIC4 siRNA 在 U251细胞凋亡相关蛋白 Bax、Bcl-2表达；检测饥饿条件下 U251细胞 LC3蛋白、Bax、Bcl-2的表达；检测抑制 CLIC4表达对于饥饿条件下细胞白 caspase-3和胞浆 cytc 以及 LC3的表达。结果Western Blot 显示，与空质粒对照组相比，转染 CLIC4 siRNA 的 U251细胞 CLIC4表达显著下降；与对照组相比，转染空质粒细胞组以及瞬时转染 pSH1Si-CLIC4载体细胞组 U251细胞生存率均无明显差异；单纯 CLIC4 siRNA 对于U251细胞 Bax、Bcl-2表达无影响；与对照组相比，饥饿8 h 能够诱导 U251细胞自噬，LC3水平显著增加，而细胞 Bax、Bcl-2表达无明显变化；转染 CLIC4 siRNA 后饥饿8 h，caspase-3、cytc 以及 LC3表达增加。结论单纯 CLIC4 siRNA对于 U251细胞自噬和凋亡均无显著影响，饥饿条件下 U251细胞发生自噬的同时并没有明显细胞凋亡过程，抑制CLIC4表达促进了饥饿条件下的 U251细胞自噬，同时能够启动线粒体相关途径的细胞凋亡。
목적：통과기아유도인신경효질류세포발생자서，이용 siRNA 기술부분침묵세포내록통도단백4（CLIC4），탐토 CLIC4재세포자서중적작용。방법근거 CLIC4적기인서렬구건 CLIC4 siRNA 질립，건립은정전염CLIC4 siRNA 적 U251세포주，분석 CLIC4단백표체；MTT 법검측전염 pSH1Si-CLIC4대세포생존솔적영향；이용Western Blot 검측전염 CLIC4 siRNA 재 U251세포조망상관단백 Bax、Bcl-2표체；검측기아조건하 U251세포 LC3단백、Bax、Bcl-2적표체；검측억제 CLIC4표체대우기아조건하세포백 caspase-3화포장 cytc 이급 LC3적표체。결과Western Blot 현시，여공질립대조조상비，전염 CLIC4 siRNA 적 U251세포 CLIC4표체현저하강；여대조조상비，전염공질립세포조이급순시전염 pSH1Si-CLIC4재체세포조 U251세포생존솔균무명현차이；단순 CLIC4 siRNA 대우U251세포 Bax、Bcl-2표체무영향；여대조조상비，기아8 h 능구유도 U251세포자서，LC3수평현저증가，이세포 Bax、Bcl-2표체무명현변화；전염 CLIC4 siRNA 후기아8 h，caspase-3、cytc 이급 LC3표체증가。결론단순 CLIC4 siRNA대우 U251세포자서화조망균무현저영향，기아조건하 U251세포발생자서적동시병몰유명현세포조망과정，억제CLIC4표체촉진료기아조건하적 U251세포자서，동시능구계동선립체상관도경적세포조망。
Objective To investigate the mechanism and effects of CLIC4 protein in glioma cells autophagy induced by starvation through siRNA silencing CLIC4.Methods Based on the gene sequence,CLIC4 siRNA plasmid and stable transfected U251 cells were constructed.The level of CLIC4 expression was analyzed.To detect cell vitality,MTT method was applied.To investigate the effect of CLIC4 siRNA on the apoptosis induced by starvation,the levels of Bax, Bcl-2 were detected.To investigate the effect of CLIC4 siRNA on the apoptosis and autophagy induced by starvation, the levels of cleaved caspase-3,cytosolic cyt c and LC3 were detected.Results Compared with the control group,the results indicated that there was obvious decrease of CLIC4 expression in U251 cells with CLIC4 siRNA transfected. MTT results indicated that there was no obvious effect of cell vitality with CLIC4 siRNA transfected There were no ob-vious changes in protein Bax and Bcl-2 with CLIC4 siRNA transfected U251 cells.Compared with control group,the level of LC3 had a significant increase under starvation in U251 cells at 8 hours.Meanwhile,protein Bax,Bcl-2 had no significant change under starvation.Western Blot indicated cleaved caspase-3,cytosolic cyt c and LC3 expression in-creased in CLIC4 siRNA transfected U251 cells under starvation.Conclusion The results showed that CLIC4 siRNA had no effect on cell autophagy and apoptosis.Under starvation,autophagy was detected in U251 cells but no apoptosis. CLIC4 siRNA enhanced the autophagy under starvation,on the same time,triggered caspase-dependent mitochondrial apoptosis.