国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (8): 641-646.doi: 10.3760/cma.j.issn.1673422X.2017.09.001

• 论著 • 上一篇    下一篇

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

邱梅清,王涛,佟仲生,贾勇圣   

  1. 277100山东省枣庄市立医院肿瘤科
  • 出版日期:2017-09-08 发布日期:2017-10-31
  • 通讯作者: 王涛，Email: wangtao1968@126.com E-mail:wangtao1968@126.com

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

Qiu Meiqing, Wang Tao, Tong Zhongsheng, Jia Yongsheng   

  1. Department of Oncology, Zaozhuang Municipal Hospital of Shandong Province, Zaozhuang 277100, China
  • Online:2017-09-08 Published:2017-10-31
  • Contact: Wang Tao E-mail:wangtao1968@126.com

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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（PDT）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Western blotting分别检测人乳腺癌T47D细胞中pEGFP组和pEGFPMfn2组Mfn2 mRNA和Mfn2蛋白的表达；pEGFP组、pEGFPMfn2组转染T47D细胞48 h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pEGFP+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2 mRNA的表达量为1.01±0.12，转染pEGFPMfn2后T47D细胞中Mfn2 mRNA的表达量为1 067.00±41.72，差异有统计学意义（t=67.541,P＜0.001）。Western blotting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5.00 μmol/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%±1.21%、46.50%±1.72%（t=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基因可能增强T47D细胞对PDT治疗的敏感性，其机制可能与改变线粒体形态结构，诱导线粒体途径凋亡有关。

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

Abstract: ObjectiveTo investigate the effects and possible mechanisms of Mfn2 gene overexpression on photodynamic therapy (PDT) sensitivity of T47D cells in human breast cancer. MethodspEGFP 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 (MTT) 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 ultrastructure of mitochondria in pEGFP group, pEGFPMfn2 group, pEGFP+PDT group, and pEGFPMfn2+PDT group. ResultsRT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=34.403, P＜0.001）. Flow cytometry assay showed that the cell apoptosis rates in pEGFPMfn2+PDT group was markedly higher compared with the pEGFP+PDT group ［(81.21±2.13)% vs. (68.82±2.64)%, P=0.024］, with statistical significance. Also, the mitochondrial membrane potential was obviously lower in pEGFPMfn2+PDT group  compared with the pEGFP+PDT group ［(1.37±0.12)% vs. (23.33±1.86)%,P＜0.001］, with statistical significance. Laser scanning confocal fluorescence microscope showed that cells in pEGFP group showed network structure, both pEGFPMfn2 group and pEGFP+PDT group could cause mitochondrial fusion, and the pEGFPMfn2+PDT group could induce mitochondrial disintegration and lose its normal morphology completely. ConclusionMfn2 may enhance the PDT sensitivity of T47D cells in human breast cancer, which is possibly related with the normal morphology alteration of mitochondria and the inducement of mitochondrial apoptosis.

Key words: Breast neoplasms, Genes, mitochondrial, Photochemotherapy, Molecular mechanisms of pharmacological action