CAJ | 학술논문

目的：研究自噬抑制药物氯喹（chloroquine ，CQ）诱导肝癌细胞死亡的作用机制，探索自噬相关药物可能的临床应用价值。方法：选取3种肝癌细胞系，给予不同浓度CQ干预24 h ，采用M T T法检测细胞的增殖情况，采用碘化丙啶（PI）染色法检测细胞死亡，采用 Hochest33342染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色、Western blotting （caspase‐9）法检测凋亡。采用GFP‐LC3转染、Western blotting （LC3）及细胞电镜技术检测细胞自噬功能。结果：M T T检测显示，CQ作用24 h能够显著抑制肝癌细胞增殖。CQ （≥20μmol／L ）干预后，PI染色阳性细胞明显增多，Hochest33342核染色显示核浓聚、核碎裂，TUNEL染色阳性细胞明显增多，caspase‐9活化；而CQ（5、10μmol／L）干预后PI染色阳性细胞数目无明显变化。CQ处理细胞后，GFP‐LC3阳性细胞明显增多，LC3‐Ⅱ蛋白表达明显增高。同时用caspase抑制剂Z‐VAD‐FMK和3‐MA或沉默Atg5的表达预处理，可显著抑制40μmol／L CQ诱导的MHCC97‐L细胞的死亡。结论：CQ可以抑制肝癌细胞自噬降解，导致自噬小泡及自噬标志性蛋白积聚。大剂量CQ直接诱导肝癌细胞死亡，其死亡形式既具有凋亡的特征又具有自噬样死亡的特征。
목적：연구자서억제약물록규（chloroquine ，CQ）유도간암세포사망적작용궤제，탐색자서상관약물가능적림상응용개치。방법：선취3충간암세포계，급여불동농도CQ간예24 h ，채용M T T법검측세포적증식정황，채용전화병정（PI）염색법검측세포사망，채용 Hochest33342염색、말단탈양핵감산전이매개도적dUTP결구말단표기（TUNEL）염색、Western blotting （caspase‐9）법검측조망。채용GFP‐LC3전염、Western blotting （LC3）급세포전경기술검측세포자서공능。결과：M T T검측현시，CQ작용24 h능구현저억제간암세포증식。CQ （≥20μmol／L ）간예후，PI염색양성세포명현증다，Hochest33342핵염색현시핵농취、핵쇄렬，TUNEL염색양성세포명현증다，caspase‐9활화；이CQ（5、10μmol／L）간예후PI염색양성세포수목무명현변화。CQ처리세포후，GFP‐LC3양성세포명현증다，LC3‐Ⅱ단백표체명현증고。동시용caspase억제제Z‐VAD‐FMK화3‐MA혹침묵Atg5적표체예처리，가현저억제40μmol／L CQ유도적MHCC97‐L세포적사망。결론：CQ가이억제간암세포자서강해，도치자서소포급자서표지성단백적취。대제량CQ직접유도간암세포사망，기사망형식기구유조망적특정우구유자서양사망적특정。
Objective:To investigate the role of chloroquine (CQ) during inducing the death of hepatic carcinoma cells and to explore the clinical application of autophagy‐related agents .Methods:Three kinds of hepatic carcinoma cell lines were chosen and treated with CQ at different concentrations for 24 hours .Then MTT assay was applied to determine the proliferation ,and prodium iodide (PI) stalning assay was used to detect the cell death .The specific apoptosis was examined by Hochest 33342 stalning , TUNEL stalning and Western blotting (caspase‐9 ) . The autophagic function was explored by GFP‐LC3 redistribution ,Western blotting (LC3) ,and electron microscopy .Results:MTT assay revealed that the proliferation of hepatic carcinoma cells was significantly inhibited by using CQ for 24 hours .The PI‐positive cells increased significantly after the treatment with CQ (≥ 20 μmol/L) .Hochest33342 stalning assay showed karyopyknosis and karyorrhexis .Meanwhile , TUNEL positive cells significantly increased and caspase‐9 was activated .However ,there was no significant change in the number of PI‐positive cells after the CQ treatment at concentrations of 5 μmol/L and 10 μmol/L .CQ treatment resulted in significant increase of GFP‐LC3‐positive cells and expression of LC3‐II .When the caspase inhibitor Z‐VAD‐FMK and 3‐MA or Atg5 siRNA were applied at the same time , 40 μmol/L CQ‐induced M HCC97‐L cell death was significantly inhibited . Conclusions:CQ can inhibit the autophagic degradation of hepatic carcinoma cells , and resulted in the accumulation of autophagosomes and autophagic proteins .High‐dose CQ directly induces the death of hepatic carcinoma cells ,and the death has both characteristics of apoptosis and autophagic death .