TIGAR调节TRX1核转运影响人脑胶质瘤细胞放射敏感性的作用机制研究

发布时间：2018-05-13 11:03

本文选题：TIGAR + TRX1核转运　；参考：《苏州大学》2014年博士论文

【摘要】：目的：干扰TP53诱导的糖酵解及凋亡调节蛋白（TIGAR）表达可以增加人脑胶质瘤细胞的放射敏感性，但其机制尚不明了。硫氧还原蛋白-1（TRX1）是一个通过核转运参与电离辐射后DNA损伤修复过程的重要的还原蛋白。由于TRX1依赖NADPH保持还原状态从而发挥功能，而在氧化应激条件下，增加TIGAR表达有助于产生更多的NADPH从而帮助细胞对抗氧化。本课题试图通过改变TIGAR的表达水平，研究TIGAR对TRX1核转运的影响及其在调节胶质瘤细胞放射敏感性中的作用，从而阐述TIGAR调节胶质瘤细胞放射敏感性的机制；通过比较TIGAR在胶质瘤细胞与正常胶质细胞中功能的差异，探讨TIGAR作为胶质瘤放疗增敏靶点的可能性；最后，通过建立人脑胶质瘤裸鼠原位种植模型，验证TIGAR调节细胞放射敏感性的机制。达到阐明干扰TIGAR表达增加人脑胶质瘤放射敏感性机制的目的。方法：采用Western blot技术检测电离辐射对细胞TIGAR表达水平及p53蛋白水平的影响；通过实时荧光定量PCR技术检测电离辐射后细胞TIGAR、TP53转录水平的改变；通过转染TIGAR siRNA干扰TIGAR表达，构建pcDNA3.1-TIGAR瞬时过表达TIGAR；采用流式细胞技术、NADPH检测试剂盒检测电离辐射前后细胞的氧化还原水平；通过转染野生型TRX1过表达质粒（pcDNA3.1-WT-TRX1）及突变型TRX1过表达质粒（pcDNA3.1-MT-TRX1）、G418筛选得到野生型及突变型TRX1稳定过表达细胞株；采用克隆形成试验检测电离辐射后细胞的克隆存活率；通过免疫荧光技术评价电离辐射后不同时间点（0.5-12h）胶质瘤细胞DNA损伤的修复水平；通过Western blot技术检测干扰TIGAR表达对细胞TRX1核转运的影响；采用氧化还原Western blot技术检测电离辐射前后TRX1蛋白的氧化还原水平。最后通过原位种植胶质瘤细胞建立人脑胶质瘤裸鼠原位种植模型；通过原位注射TIGAR shRNA慢病毒实现基因治疗，并采用核磁共振成像（MRI）技术评价基因治疗联合放疗的治疗效果。结果：（1）电离辐射后，p53野生型人脑胶质瘤A172细胞内TIGAR表达水平在照后1h开始增加，照后8h回落至基础水平；p53蛋白水平于照后0.5h开始增加，先于TIGAR表达增加。而在受照的p53突变型（M237I）T98G细胞中，，TIGAR与p53蛋白水平均无显著增加。克隆形成试验显示，干扰TIGAR表达显著降低受照A172、T98G细胞克隆存活率。（2）干扰TIGAR表达显著增加受照胶质瘤细胞内的活性氧（ROS）水平并造成受照细胞内NADPH水平耗竭，在单纯照射组细胞内NADPH的含量仅较照射前下降了40%，而TIGAR干扰联合照射组细胞内NADPH的含量较照射前下降了约75%。相似的，干扰TIGAR表达也引起了受照胶质瘤细胞内GSH/GSSG比例的进一步下降，而TIGAR过表达则可显著增加受照胶质瘤细胞内的NADPH含量与GSH/GSSG的比例。Western blot结果显示，电离辐射后细胞内TRX1可发生核转运，核转运的峰值时间为照后2h。而干扰TIGAR表达可显著抑制受照胶质瘤A172、T98G细胞内的TRX1核转运。（3）干扰TIGAR表达无法抑制野生型TRX1过表达A172细胞发生电离辐射诱导的TRX1核转运。氧化还原Western blot结果表明A172细胞受照后，胞浆内TRX1于照后0.5h内被氧化，于照后8h回复到还原状态，而干扰TIGAR表达可显著延缓细胞浆内TRX1的还原进程，结果显示胞浆内TRX1于照后8h仍处于氧化状态。而在野生型TRX1过表达细胞内，TIGAR干扰无法延缓TRX1的还原进程，电离辐射后细胞核内TRX1的氧化还原进程与细胞浆内TRX1类似，也可被干扰TIGAR表达延缓。克隆形成试验的结果显示，野生型TRX1过表达可显著抑制TIGAR干扰引起的放射增敏效应，而突变型TRX1过表达联合TIGAR干扰无法在单纯干扰TIGAR表达的基础上进一步减少受照胶质瘤细胞的克隆存活率。更为关键的是，增加TIGAR表达对TRX1突变型细胞的放射敏感性无显著影响，表明TRX1核转运在TIGAR调节胶质瘤细胞放射敏感性中发挥了重要作用。（4）免疫荧光实验的结果显示，受照A172γ-H2AX的点状荧光在照后4h内消失，而在TIGAR干扰组A172细胞内γ-H2AX点状荧光直到照后12h依然存在，表明细胞内DNA损伤修复进程被显著延迟。与Western blot的结果相似，免疫荧光实验的结果显示受照A172细胞内TRX1核转运的峰值时间为照后2h，而TIGAR干扰组细胞内TRX1核转运则完全消失。在野生型TRX1过表达细胞中，干扰TIGAR表达导致的DNA损伤修复延迟与TRX1核转运抑制均得到了显著恢复。（5）与胶质瘤细胞不同，改变TIGAR表达无法改变正常脑胶质细胞的放射敏感性。并且尽管电离辐射后，星形胶质细胞内可以观察到TRX1核转运现象，但干扰TIGAR表达无法抑制电离辐射引起的星形胶质细胞TRX1核转运，也无法延缓受照星形胶质细胞内TRX1的还原进程。氧化还原Western blot的结果还显示，受照星形胶质细胞内TRX1的氧化程度显著低于相同照射条件下脑胶质瘤细胞内TRX1的氧化还原程度。（6）通过TIGARshRNA慢病毒基因治疗，证实了干扰TIGAR表达可增加荷瘤小鼠颅内胶质瘤的放疗敏感性。结论：本课题的研究首次提出在人脑胶质瘤细胞内干扰TIGAR表达显著抑制电离辐射引起的TRX1核转运，此现象很可能与干扰TIGAR表达的放射增敏机制有关。干扰TIGAR表达抑制TRX1核转运的原因可能与破坏细胞内氧化还原稳态、导致受照细胞内TRX1过氧化及还原进程延缓有关。干扰TIGAR表达无法抑制野生型TRX1过表达胶质瘤细胞内TRX1发生核转运。干扰TIGAR表达对正常脑胶质细胞的放射敏感性无显著影响，并且干扰TIGAR表达可能增加胶质瘤的放疗敏感性。
[Abstract]:Objective : To interfere with the expression of TP53 - induced glycolytic and apoptosis - regulated proteins in human glioma cells , but the mechanism remains unknown .
To compare the difference of the function between the glioma cells and the normal glial cells , the possibility of the ADAR as the target for radiotherapy of glioma was discussed .
Finally , by establishing the in - situ implantation model of human glioma nude mice , the mechanism for regulating the radiosensitivity of human glioma cells was demonstrated .

Methods : Western blot was used to detect the effect of ionizing radiation on the expression level and p53 protein level .
The transcription level was changed by real - time fluorescence quantitative PCR ( RT - PCR ) .
pcDNA3 . 1 - 4 AR was constructed transiently by siRNA interfering with grade 4 AR expression .
Flow cytometry was used to detect the redox level of cells before and after ionizing radiation .
By transfection of the wild type TRX1 overexpression plasmid ( pcDNA3.1 - WT - TRX1 ) and the mutant TRX1 overexpression plasmid ( pcDNA3.1 - MT - TRX1 ) , the wild - type and mutant TRX1 stably expressing cell lines were obtained by G418 selection ;
cloning and forming test to detect the clone survival rate of the cells after ionizing radiation ;
The repair level of DNA damage in glioma cells at different time points ( 0.5 -12h ) after ionizing radiation was evaluated by immunofluorescence technique .
Western blot was used to detect the effect of the expression on TRX1 nuclear translocation .
The oxidation - reduction level of TRX1 protein before and after ionizing radiation was detected by oxidation - reduction Western blot .
Gene therapy was carried out by in situ injection of the slow virus , and the therapeutic effect of gene therapy combined with radiotherapy was evaluated by nuclear magnetic resonance imaging ( MRI ) .

Results : ( 1 ) In A172 cells of wild type human glioma after ionizing radiation , the expression level in A172 cells began to increase at 1 h after irradiation , and then returned to the basal level at 8h after irradiation .
The results showed that TRX1 could not inhibit TRX1 nuclear transport in glioma cells . The results showed that TRX1 could not inhibit TRX1 in glioma cells . ( 5 ) The expression of TRX1 could not change the radiosensitivity of normal brain glioma cells . However , after ionizing radiation , TRX1 nuclear transport could not be inhibited in astrocytes .

Conclusion : It is suggested that TRX1 nuclear transport induced by ionizing radiation can be significantly inhibited in human glioma cells . This phenomenon is probably related to the radiosensitivity mechanism which interferes with the expression of TRX1 in human glioma cells .

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41;R730.55

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