Mfn-2基因对人乳腺癌T47D细胞光动力疗法敏感性的影响及其作用机制  

Effects and mechanism of overexpression of Mfn-2 gene on photodynamic therapy sensitivity of T47D cells in human breast cancer

作　　者：邱梅清[1] 王涛[1] 佟仲生 贾勇圣 

机构地区：[1]山东省枣庄市立医院肿瘤科,277100

出　　处：《国际肿瘤学杂志》2017年第9期641-646,共6页Journal of International Oncology

摘　　要：目的探讨过表达Mfn-2基因对人乳腺癌T47D细胞光动力疗法（POT）敏感性的影响及其可能机制。方法转染pEGFP和pEGFP-Mfn-2至T47D细胞，实时定量PCR（RT-PCR）和Westernblotting分别检测人乳腺癌T47D细胞中pEGFP组和pEGFP-Mfn-2组Mfn-2mRNA和Mfn-2蛋白的表达；pEGFP组、pEGFP—Mfn-2组转染Td7D细胞48h后，四甲基偶氮唑蓝（MTT）法检测细胞对PDT的敏感性；PDT照射转染了T47D细胞的pEGFP及pEGFP-Mfn-2，得到pEGrP＋PDT组和pEGFP-Mfn-2＋PDT组。流式细胞术检测pEGFP＋PDT组、pEGFP-Mfn-2＋PDT组T47D细胞凋亡及线粒体膜电位变化情况；激光共聚焦显微镜观察pEGFP组、pEGFP—Mfn-2组、pEGFP＋PDT组、pEGFP-Mfn-2＋PDT组线粒体超微形态结构。结果RT-PCR结果显示，转染pEGFP后T47D细胞中Mfn-2mRNA的表达量为1．01±0．12，转染pEGFP—Mfn-2后T47D细胞中Mfn-2mRNA的表达量为1067．004-41．72，差异有统计学意义（t=67．541，P〈0．001）。Westernblotting检测结果显示，与转染pEGFP相比，转染pEGFP-Mfn-2可明显提高T47D细胞中Mfn-2蛋白的表达。MTT分析结果显示，与转染pEGFP相比，pEGFP-Mfn-2转染可明显提高T47D细胞对PDT的敏感性，差异有统计学意义，如亚甲蓝为5．00μmoL／ml时，pEGFP＋PDT组和pEGFP—Mfn-2＋PDT组乳腺癌T47D细胞生存率分别为59．96％土1．21％、46．50％±1．72％（1=34．403，P〈0．001）。流式细胞术检测结果显示，与pEGFP＋PDT组相比，pEGFP—Mfn-2＋PDT组细胞凋亡率明显升高，差异有统计学意义[（81．21±2．13）％：（68．82±2．64）％，P=0．024]。与pEGFP＋PDT相比，pEGFP-Mfn-2＋PDT组线粒体膜电位明显降低，差异有统计学意义[（1．37±0．12）％：（23．33±1．86）％，P〈0．001]。激光共聚焦显微镜显示，pEGFP组细胞呈网状，pEGFP．Mfn-2组和pEGFP＋PDT组可导致线粒体融合，而pEGFP—Mfn-2＋PDT组可导致线粒体崩解，完全失去其形态结构。结论Mfn-2基因可�Objective To investigate the effects and possible mechanisms of Mfn-2 gene overexpression on photodynamic therapy （PDT） sensitivity of Td7D cells in human breast cancer. Methods pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells, and then the mRNA and protein expression of Mfn-2 gene in T47D cells were detected by real-time PCR （RT-PCR） and Western blotting in pEGFP group and EGFP-Mfn-2 group, respectively. After pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells for 48 hours, methyl thiazolil tetracolium （M3＋） assay was used to measure the PDT sensitivity of T47D cells in human breast cancer in pEGFP group and pEGFP-Mfn-2 group, pEGFP ＋ PDT group and pEGFP-Mfn-2 ＋ PDT group were obtained by PDT irradiating pEGFP and pEGFP-Mfn-2 which were transfec- ted into human breast cancer T47D cells. Cell apoptosis and mitochondrial membrane potential of T47D cells were assayed by flow cytometry in pEGFP ＋ PDT group and pEGFP-Mfn-2 ＋ PDT group. Laser scanning confocal fluorescence microscope was applied to observe the morphological uhrastructure of mitochondria in pEGFP group, pEGFP-Mfn-2 group, pEGFP ＋ PDT group, and pEGFP-Mfn-2 ＋ PDT group. Results RT-PCR showed that after transfecting T47D cells with pEGFP, the expression of Mfn-2 mRNA was 1.01 ＋0. 12. After transfecting T47D cells with pEGFP-Mfn-2, the expression of Mfn-2 mRNA was 1 067.00 ＋ 41.72. There was statistical significance （t =67.541 ,P 〈 0. 001 ）. Western blotting revealed that compared with the pEGFP group, the pEGFP-Mfn-2 transfection group had higher expression of Mfn-2 gene in T47D cells. MTT assay showed that pEGFP-Mfn-2 transfection significantly enhanced the PDT sensitivity of T47D cells in human breast cancer compared with the pEGFP ＋ PDT group. When the concentration of methylene blue was 5.00 μmol/ml, the survival rate of the pEGFP ＋ PDT group and pEGFP-Mfn-2 ＋ PDT group were （59.96%± 1.21% ） vs. （46.50%± 1.72% ）, with significant difference （t = 3

关 键 词：乳腺肿瘤 基因 线粒体 光化学疗法 药理作用分子作用机制 

分 类 号：R737.9[医药卫生—肿瘤]