细胞自噬在甲氨蝶呤耐药绒癌中的保护性作用  

The protective role of autophagy in methotrexate resistant human choriocarcinoma JEG-3 cell lines.

作　　者：沈芸[1,2] 赵静[2] 肖长纪[2] 向阳[2] 

机构地区：[1]国家人口计生委科学技术研究所,北京100081 [2]中国医学科学院北京协和医院妇产科,北京100730

出　　处：《现代妇产科进展》2013年第11期884-887,891,共5页Progress in Obstetrics and Gynecology

基　　金：国家自然科学基金资助(No:81272890)

摘　　要：目的:通过检测耐药绒癌细胞内自噬的变化,以及评估抑制自噬或敲除自噬相关基因后细胞凋亡情况,探讨细胞自噬在耐药绒癌中的作用。方法:以人绒毛膜上皮癌JEG-3细胞及本课题组前期构建的甲氨蝶呤(MTX)耐药绒癌JEG-3/MTXR细胞为研究对象。采用透射电镜、免疫荧光法观察MTX处理后细胞内自噬的变化;Western blot法检测自噬相关蛋白LC3、Beclin-1的表达变化。RNA干扰法沉默自噬相关基因LC3后,采用Western blot法和Annexin-Ⅴ/PI双染法流式细胞仪检测MTX作用后JEG-3/MTXR耐药绒癌细胞凋亡的变化。结果:透射电镜和荧光显微镜下均可观察到MTX处理后JEG-3、JEG-3/MTXR细胞内自噬小体的产生;IC30浓度MTX处理后,两种细胞内自噬相关蛋白LC3、Beclin-1表达水平上调。相同浓度MTX处理后,JEG-3细胞内LC3、Beclin-1表达下调。沉默自噬相关基因LC3后,沉默LC3联合MTX组的JEG-3/MTXR和JEG-3细胞内凋亡相关蛋白caspase-9表达上调,凋亡率显著增多。结论:MTX可诱导耐药绒癌产生保护性自噬。Objective：To explore the effect of autophagy on methotrexate resistant human chonocarcinoma JEG-3 eel1 lines. Methods：Human choriocarcinoma JEG-3 cell lines, and metho- trexate resistant ehorioearcinoma JEG-3 （MTXR/JEG-3） cell lines which was set up in our previous work, were used in our present study. Autophagy flux after methotrexate treatment was evaluated by the methods of transmission electron microscope, immunofluorescence staining assay. Autophagy related proteins LC3 and Beclin 1 were analyzed by Western blot after methotrexate exposure in both cell lines. Autophagy related gene LC3 was knockdown by LC3-siRNA in MTXR/JEG-3 cell lines, and cell apoptosis was evaluated by Western blot and flow cytometry analysis. Results：Autophagy was triggered after methotrexate treatment in the methotrexate-resistant JEG-3 cells by the methods of transmission electron microscope,immunofluorescence staining assay. Autophagy-related proteins LC3 and Beelin-1 were demonstrated to be upregulated after the same IC30 methotrexate concentration treatment in both cell lines ,while they were down-regulated after the same dosage of MTX treatment in JEG-3 cells. Moreover,methotrexate combined with autophagy related gene LC3 knockdown contributed to the up-regulation of apoptosis compared to methotrexate alone treatment group using FACS. Conclusion ： The protective role of autophagy might exist in the methotrexate-resistant choriocarcinoma cell lines.

关 键 词：绒癌 耐药 自噬 甲氨蝶呤 

分 类 号：R737.33[医药卫生—肿瘤]