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目的探讨甲氨蝶呤(MTX)耐药绒癌的发生机制。方法 2012年5月至2013年1月于北京协和医院以人绒毛膜上皮癌JEG-3细胞及本课题组前期构建的MTX耐药绒癌JEG-3/MTXR细胞为研究对象,采用免疫印迹、RTPCR法检测MTX处理后细胞内P62的变化。流式细胞仪检测MTX诱导后JEG-3/MTXR细胞内活性氧(ROS)变化,采用ROS清除剂Mn Tm Py P检测MTX作用后JEG-3/MTXR细胞内P62蛋白水平的变化。RNA干扰法沉默P62基因免疫印迹法和流式细胞仪检测MTX诱导后JEG-3/MTXR耐药细胞内PARP蛋白的表达和凋亡率的变化。结果 MTX可诱导耐药绒癌P62蛋白和m RNA表达水平呈时间依赖性上调。MTX可诱导耐药绒癌ROS表达水平升高,Mn Tm Py P降低P62蛋白的表达。P62-si RNA可促进JEG-3/MTXR细胞内MTX诱导的凋亡反应蛋白PARP的切割和激活,流式细胞仪检测RNA干扰联合药物组细胞凋亡率(14.4±1.02)%,高于单纯药物应用组(9.1±1.34)%,差异有统计学意义(P<0.05)。结论 ROS介导P62蛋白激活参与MTX耐药绒癌发生机制,为耐药绒癌临床靶向治疗提供新思路。
Objective To investigate the mechanism of methotrexate (MTX) -mediated choriocarcinoma. METHODS: Human choriocarcinoma JEG-3 cells and the MTX-resistant choriocarcinoma JEG-3 / MTXR cells previously constructed in our group were studied at Peking Union Medical College Hospital from May 2012 to January 2013. Western blot, The RTPCR method was used to detect the intracellular P62 changes after MTX treatment. The changes of reactive oxygen species (ROS) in JEG-3 / MTXR cells induced by MTX were detected by flow cytometry. The levels of P62 protein in JEG-3 / MTXR cells were detected by MTT assay using Mn Tm Py P as ROS scavenger. The expression of PARP protein and apoptosis rate in JEG-3 / MTXR-resistant cells induced by MTX were detected by Silencing P62 gene by RNA interference and by flow cytometry. Results MTX induced the expression of P62 protein and m RNA in choriocarcinoma in a time-dependent manner. MTX can induce ROS expression in drug-resistant choriocarcinoma, Mn Tm Py P decreases P62 protein expression. P62-si RNA could promote the cleavage and activation of MTX-induced apoptotic reactive protein PARP in JEG-3 / MTXR cells, and the apoptotic rate was 14.4 ± 1.02% by flow cytometry (FCM) Drug application group (9.1 ± 1.34)%, the difference was statistically significant (P <0.05). Conclusion ROS-mediated activation of P62 protein is involved in the pathogenesis of MTX-resistant choriocarcinoma and may provide new ideas for the clinical targeted therapy of drug-resistant choriocarcinoma.