TIGAR调节自噬活性影响人脑胶质瘤放射敏感性的机制研究

发布时间：2018-02-24 16:34

本文关键词： TIGAR 自噬氧化应激放射敏感性　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:干扰TP53诱导的糖酵解及凋亡调节蛋白(TIGAR)表达可以增加肿瘤细胞自噬活性,课题组前期工作表明干扰TIGAR表达可增加人脑胶质瘤细胞的放射敏感性,但其与自噬的相关性仍不明了。本课题通过改变TIGAR表达水平,研究受照胶质瘤细胞自噬水平对其存活作用,从而进一步阐述TIGAR调节胶质瘤细胞放射敏感性的机制。建立脑胶质瘤原位接种模型,通过慢病毒转染,在动物体内研究干扰TIGAR表达对胶质瘤放疗敏感性影响。最后,检测TIGAR蛋白在恶性胶质瘤及瘤旁组织中表达,分析其在恶性胶质瘤中表达的临床意义。方法:采用Western blot技术检测8Gy 6MV-X线照射后A172及T98G胶质瘤细胞内TIGAR表达及磷酸化p53蛋白水平的变化;运用实时荧光定量PCR技术检测电离辐射后A172细胞TIGAR转录水平的改变;分别通过转染TIGAR siRNA干扰TIGAR表达,pcDNA3.1-TIGAR过表达TIGAR,通过克隆形成试验检测不同TIGAR表达水平细胞受照后的克隆形成能力;采用NADPH检测试剂盒检测不同TIGAR表达水平细胞照射前后细胞内的氧化还原水平;采用Western blot技术检测改变TIGAR表达对细胞受照后自噬水平的影响;通过抑制受照细胞的自噬水平,观察电离辐射诱导自噬在细胞存活中作用,在此基础上探讨电离辐射诱导的自噬发生的机制;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,采用TIGAR shRNA慢病毒实现基因治疗,并利用核磁共振成像(MRI)技术评价基因治疗联合放疗的治疗效果,病理免疫组化观察放疗后肿瘤组织内TIGAR蛋白表达以及治疗后病理反应;最后采用免疫组化技术检测恶性胶质瘤中TIGAR表达水平,分析其与恶性胶质瘤的病理分级相关性,以及与患者放疗疗效、生存时间等临床资料的相关性。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1 h开始增加,照后8 h回落至基础水平;p53蛋白于照后0.5 h开始发生磷酸化,照后1 h达到峰值,而后缓慢下降。TIGAR mRNA水平受照后0.5 h开始增加,于受照后2 h达到峰值,而后开始回落,至受照后4-8 h回落至照前水平;而在受照后各时间点,TP53 mRNA水平均无显著变化(*p0.05)。在受照的p53突变型(M237I)T98G细胞中,TIGAR水平无显著变化。克隆形成试验显示,干扰TIGAR表达显著降低A172、T98G细胞受照后克隆形成率,相反TIGAR过表达则显著增加了受照胶质瘤细胞的克隆形成能力。(2)干扰TIGAR表达导致受照细胞内NADPH水平耗竭,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比例较单纯照射组明显下降。(3)干扰TIGAR联合电离辐射导致胶质瘤细胞损伤性自噬活性明显增加,其与细胞ROS通路相关。(4)通过TIGAR shRNA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。(5)TIGAR蛋白主要在恶性胶质瘤组织中表达,而在瘤旁组织中无明显表达,TIGAR表达与患者病理分级具有相关性,TIGAR表达可影响恶性胶质瘤患者放疗近期疗效。结论:本课题的研究创新性地提出干扰TIGAR表达可引起受照人脑胶质瘤细胞发生损伤性自噬,其机制可能与受照胶质瘤细胞氧化还原水平失衡相关。干扰TIGAR表达可在整体水平增加胶质瘤的放疗敏感性。TIGAR表达可能参与了胶质瘤发生发展,对病理诊断以及预测放疗近期疗效有一定价值。
[Abstract]:Objective: to regulate protein interference TP53 induced glycolysis and apoptosis (TIGAR) can increase the expression of autophagy activity of tumor cells, the prophase showed that interfering TIGAR expression can increase the radiosensitivity of human glioma cells, but its correlation with autophagy remains unknown. This topic by changing the expression level of TIGAR, according to research by the role of autophagy in glioma cells survive on the mechanism to explain TIGAR regulating radiosensitivity of glioma cells. The establishment of brain glioma orthotopic model, through lentivirus transfection, in vivo animal studies the interference effect of TIGAR expression on the radiosensitivity of human glioma. Finally, detect the expression of TIGAR protein in malignant glioma and its'contiguous in the organization, the clinical significance of the expression in malignant glioma. Methods: using Western blot technology to detect 8Gy 6MV- X-ray irradiated A172 and T98G glioma cells TIGA The expression of R and phosphorylated p53 protein level in A172 cells; change the transcription level of TIGAR by real-time fluorescence quantitative PCR detection of ionizing radiation; were expressed by transfection of TIGAR siRNA interference TIGAR pcDNA3.1-TIGAR, overexpression of TIGAR by clonogenic assay and TIGAR expression level of different clone cells after irradiation formation was detected by NADPH; kit for detecting TIGAR expression level of cells before and after irradiation with different intracellular redox levels; using Western blot technology to detect the change of TIGAR expression on the effects of autophagy induced by irradiation; through inhibition of autophagy by cells, induces autophagy in cell survival observed in ionizing radiation, on the basis of mechanism induced by ionizing radiation the occurrence of autophagy; through orthotopic glioma cells established in situ glioma xenograft model, using TIGAR shRNA Lentiviral gene therapy, and the use of magnetic resonance imaging (MRI) technique to evaluate gene therapy combined with radiotherapy treatment, immunohistochemical observation after radiotherapy TIGAR protein expression in tumor tissue and pathological response after treatment; the immunohistochemical detection of malignant glioma and to analyze the expression levels of TIGAR in malignant glioma the correlation between tumor pathological grading, and radiotherapy effect, the correlation of clinical data and survival time. Results: (1) after irradiation, the expression level of H began to increase after 1 in the TIGAR p53 wild type human glioma A172 cells after exposure to 8 h down to the basic level of p53 protein in 0.5 after irradiation; the beginning of H phosphorylation at 1 h reached the peak, and then slowly decreased.TIGAR mRNA levels after exposure to 0.5 h began to increase after exposure to 2 h reached the peak, and then began to fall, after exposure to 4-8 h down to the water before irradiation Ping; after exposure in each time point, the TP53 mRNA level had no significant changes (*p0.05). In the irradiated p53 mutant (M237I) in T98G cells, no significant changes in TIGAR levels. Colony formation test showed that the interference of TIGAR expression significantly decreased A172, T98G cells after exposure to clone formation rate, on the contrary over expression of TIGAR was significantly increased by glioma cells clone forming ability. (2) the expression of TIGAR by NADPH interference leads to depletion of intracellular levels, the content of the irradiated group of intracellular NADPH is only before irradiation decreased by 40%, while the content of TIGAR interference combined with radiation group NADPH cells than before irradiation decreased about 75%. similar, interfering TIGAR expression also caused by glioma cells in GSH/GSSG proportion compared with irradiation group decreased significantly. (3) TIGAR interference combined with ionizing radiation leads to injury of glial tumor cell autophagy activity was significantly increased, and the cells of ROS pathway. (4) by TIGAR shRNA lentiviral gene therapy, confirmed that the interference of TIGAR expression can increase the radiosensitivity of intracranial glioma bearing mice. (5) TIGAR protein mainly expressed in malignant glioma tissues, and in tumor adjacent tissue and no obvious expression, the relationship between the expression of TIGAR in patients with pathological grading, TIGAR expression the short-term effects of radiotherapy in patients with malignant glioma. Conclusion: the innovation of this research put forward the interference of TIGAR expression induced by irradiation of glioma cell injury of autophagy, and its mechanism may be exposed to oxidation of glioma cells is imbalance. Primary interference TIGAR expression can increase the radiosensitivity of.TIGAR glioma expression participate in the development of glioma in the overall level, have a certain value for diagnosis and prediction of the short-term efficacy of radiotherapy.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41;R730.55

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