TIGAR基因调控Bcr-Abl阳性白血病细胞耐药的机制研究

Imatinib resistance has become a major obstacle that has to be overcome in the treatment of chronic myelogenous leukemia (CML). An increased TIGAR gene transcription level has been found in the imatinib resistant CML patients by us in the previous study. TIGAR gene is closely related to glucose metabolism, oxidative stress and apoptosis. However, it was quite rarely studied in pathogenesis and treatment of leukemia, even no data can be found in CML so far. Reactive oxygen species (ROS) was found significantly increased after imatinib treatment in the Bcr-Abl positive cell lines in vitro. So we proposed that the Bcr-Abl positive imatinib resistant cells protect them from apoptosis induced by oxidative stress via up-regulating TIGAR gene. To clarify the mechanism of imatinib resistance controlled by TIGAR via oxidative stress regulation, we will use the RQ-RT-PCR, Western blot, vector transfection and RNA interference techniques in cell lines K562, and K562-R which is induced to imatinib resistance and primary leukemic cells from the imatinib resistant patients. The purpose of this study is to confirm the restoration of sensitivity to imatinib by TIGAR gene silence in the imatinib resistant cells.

耐药已成为影响伊马替尼（imatinib）治疗慢性粒细胞性白血病（CML）疗效的主要因素。我们前期研究发现imatinib耐药的CML患者TIGAR基因转录水平明显增高。TIGAR基因与糖代谢、氧化应激和细胞凋亡密切相关，但在白血病中研究甚少，在CML中未见报道。我们的体外试验表明imatinib处理后Bcr-Abl阳性细胞株K562中活性氧明显增加，TIAGR高表达。据此我们推测耐imatinib Bcr-Abl阳性细胞通过上调TIGAR适应imatinib所致的氧化应激，阻止细胞凋亡。为验证这一假说，我们将对K562、耐imatinib细胞株（K562-R）及耐imatinib CML患者原代细胞，采用RT-PCR、Western blot、病毒载体转染、RNA干扰等手段，明确TIGAR对氧化应激的调控及其在CML耐药中的机制，证实沉默TIGAR基因能增加对imatinib的敏感性。

耐药已成为影响伊马替尼（Imatinib）治疗慢性髓细胞性白血病（CML）疗效的主要因素。前期研究发现Imatinib耐药的CML患者TIGAR基因转录水平明显增高。TIGAR基因与糖代谢、氧化应激和细胞凋亡密切相关，本研究组在体外试验发现Imatinib处理后Bcr-Abl阳性细胞株K562中活性氧明显增加，TIAGR表达水平降低。通过构建Imatinib及多药耐药细胞株K562R为工具，进行了体外、体内实验后得出以下结论：1. K562/TIGAR-shRNA2 + 2-DG细胞的胞浆中有明显的细胞色素C条带，而K562 + 2-DG组中细胞色素C只存在于线粒体中，胞浆中无条带，证实沉默TIGAR基因后细胞凋亡增加是通过促进线粒体释放细胞色素C机制引起的。2. Nutlin-3α激活p53导致TIGAR表达水平下调，反之采用PFT-α抑制P53将导致TIGAR表达水平上调，证实了TIGAR基因主要受P53基因调控；3. TIGAR与MDR1基因的表达密切相关，TIGAR敲减后将导致MDR1/P-pg水平明显下调。4. K562、K562-R两株细胞中以ROS诱导剂（H2O2）和ROS清除剂N-乙酰半胱氨酸（NAC）刺激ROS产生或清除ROS后均可见TIGAR表达水平降低或升高。Imatinib处理细胞株K562后ROS明显升高，TIGAR水平下降，而在耐药细胞株K562-R中ROS未见升高，TIGAR水平也有所下降，提示TIGAR水平不完全受ROS影响；以lenti-virus转染的方式使TIGAR的主动表达增强可导致ROS水平降低，GSH水平增高。5. 通过转染提高TIGAR水平后能够看到K562细胞株出现对Imatinib的耐药。6. K562-R/PCDNA-TIGAR 在裸鼠体内成瘤的体积和重量均高于K562-R和K562-R/TIGAR-shRNA成瘤。综合以上实验，本实验组证实了耐Imatinib Bcr-Abl阳性细胞通过上调TIGAR适应Imatinib所致的氧化应激，阻止了细胞凋亡。沉默TIGAR基因能使Bcr-Abl阳性耐Imatinib细胞恢复敏感性。