氨基酸饥饿诱导人胶质瘤细胞表达COX-2的机制研究

发布时间：2018-03-03 11:15

本文选题：胶质瘤　切入点：氨基酸饥饿　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：胶质瘤是一种神经系统常见的恶性肿瘤,其以发病率低、致死率高为主要特点。目前,治疗胶质瘤的标准方法是手术切除辅助以化疗和放疗。但是,该方法不能彻底治愈胶质瘤,经过治疗后患者预后差,复发率高,5年生存率不足10%。胶质瘤细胞在体内迅速增殖需要消耗大量营养物质,超出了周围环境的供给能力,导致微环境中营养物质供应不足。但是,在营养物质供应不充分的条件下,胶质瘤细胞是如何保持着顽强的生存能力成为该领域研究的热点问题。环氧化酶-2(Cyclooxygenase-2,COX-2)作为前列腺素合成的限速酶,在维持细胞的正常生命活动方面发挥着关键作用。近些年的研究发现,COX-2在肿瘤细胞内的表达能够提高肿瘤细胞的增殖能力、迁移能力和血管化进程,同时抑制肿瘤细胞的凋亡。COX-2在细胞内的表达受活性氧、炎症和代谢压力等因素的调节。那么,营养物质供应不足是否会对胶质瘤内COX-2的表达产生影响,如果产生影响,具体的机制是什么?对于这一问题,在国内外还未见到相关报道。因此,本研究通过细胞的氨基酸饥饿模型模拟体内营养物质供应不足的生存环境,探索营养物质供应不足对胶质瘤细胞内COX-2表达的影响,并对相关的机制进行进一步的探索。本研究通过甲硫氨酸/半胱氨酸(Methionine/Cysteine,Met/Cys)缺失的培养基和谷氨酸盐/精氨酸/赖氨酸(Glutamine/Arginine/Lysine,Gln/Arg/Lys)缺失的培养基培养U87细胞和SF767细胞,收集不同时间点的细胞样品,通过荧光定量PCR和免疫印迹检测法,从m RNA和蛋白质水平对COX-2的表达进行检测。然后,我们利用p38-MAPK、MEK和JNK的特异性抑制剂SB202190(SB)、U0126(U)和SP600125(SP)阻断这些信号通路的信号传递功能,通过荧光定量PCR和免疫印迹检测法,从m RNA和蛋白质水平对COX-2的表达进行检测。为了更加准确的阐明p38-MAPK信号通路对COX-2表达的影响,我们通过脂质体转染法将p38-MAPK特异性的si RNA导入到胶质瘤细胞内,通过荧光定量PCR和免疫印迹检测法,从m RNA和蛋白质水平对p38-MAPK的基因敲除效果和COX-2的表达进行检测。为了进一步探索p38-MAPK下游基因SP1对COX-2表达的影响,我们通过脂质体转染法将COX-2-luciferase报告基因导入到胶质瘤细胞内,再利用SP1特异性的si RNA和抑制剂mithramycin探索SP1对COX-2表达的影响,通过检测荧光的强度确定COX-2表达量的高低。最后,我们利用COX-2特异性的si RNA和抑制剂celecoxib抑制COX-2的功能,研究COX-2对VEGF表达和自噬发生的影响。通过荧光定量PCR确定COX-2对VEGF表达的影响;通过免疫印迹的方法检测LC3-II的表达水平,进而评价细胞自噬的强度。实验结果显示:Met/Cys缺失的培养基和Gln/Arg/Lys缺失的培养基均可以诱导COX-2在U87和SF767细胞中的表达,在诱导2h时,细胞内开始表达COX-2,4-6h趋于稳定。但是,相比之下,Met/Cys缺失能够更快速的诱导COX-2的表达。因此,在后续的实验研究中,我们选择通过Met/Cys缺失建立细胞饥饿模型。经过p38-MAPK、MEK和JNK的特异性抑制剂SB202190(SB)、U0126(U)、SP600125(SP)以及si RNA处理后,我们发现p38-MAPK抑制剂SB对氨基酸饥饿诱导的COX-2表达的抑制效果最明显,而JNK特异性抑制剂SP能够显著抑制U87细胞中COX-2的表达,却对SF767细胞中COX-2的表达几乎没有影响,U0126的作用效果刚好与SP相反。同样,我们发现氨基酸饥饿能够同时在U87细胞和SF767细胞诱导p38-MAPK的磷酸化,而JNK的磷酸化仅发生在U87细胞中。进一步实验发现p38-MAPK是通过SP1调节COX-2的表达,SP1基因的敲除和抑制剂的应用能够显著降低氨基酸饥饿诱导的COX-2的表达。对COX-2下游的作用途径进行检测分析发现,COX-2的表达能够促进VEGF的表达及诱导自噬的发生。综上所述,本研究以氨基酸饥饿模型模拟胶质瘤组织内营养物质供应不足的生存环境,探索在该种条件下诱发COX-2表达的相关机制。研究结果阐明氨基酸饥饿诱导的COX-2表达主要受上游p38-MAPK/SP1信号通路的调控;COX-2能够提高胶质瘤细胞内VEGF的表达,为肿瘤组织内的血管化奠定基础;COX-2通过激发胶质瘤细胞的自噬作用,为细胞的生存提供所需能量。
[Abstract]:Glioma is the most common malignant tumor of a nervous system, with its low incidence, high death rate as the main characteristics. At present, the standard method for the treatment of glioma is surgical resection assisted by chemotherapy and radiotherapy. However, this method cannot be completely cured after treatment in patients with glioma, pre post difference, high recurrence rate. The 5 year survival rate of less than 10%. glioma cells need to consume a large amount of nutrients in the body rapidly proliferating, beyond the supply capacity of the surrounding environment, resulting in insufficient supply of nutrients to the micro environment. However, the supply of nutrients is not sufficient under the condition of curing glioma is how to maintain a strong survival ability has become a hot issue in the the field of research. Cyclooxygenase -2 (Cyclooxygenase-2, COX-2) is the rate limiting enzyme of prostaglandin synthesis, play a key role in maintaining the normal life activities of cells. Recent studies have found that in COX-2 The expression of tumor cells can enhance tumor cell proliferation, migration and vascularization, expression and inhibit the apoptosis of.COX-2 tumor cells in cells by regulating inflammation and metabolism of active oxygen, pressure and other factors. So, whether the nutrient supply will have an impact on the expression of COX-2 in glioma, if have an impact, what is the specific mechanism? For this problem, at home and abroad has not been reported. Therefore, this research through the model simulation of amino acid starvation cell nutrient in a living environment in short supply, insufficient supply of nutrients to explore effect on the expression of COX-2 in glioma cells, and further explore the related mechanism. This study by methionine / cysteine (Methionine/Cysteine, Met/Cys) medium and glutamate / lack of arginine / lysine (Glutamine/Argi Nine/Lysine, Gln/Arg/Lys) medium lack of cultured U87 cells and SF767 cells were collected at different time points of cell samples was detected by fluorescence quantitative PCR and Western blot, were detected from the m level of RNA and protein expression of COX-2. Then, we use p38-MAPK, a specific inhibitor of SB202190 MEK and JNK (SB). U0126 (U) and SP600125 (SP) signal pathway blocking the transfer function was detected by fluorescence quantitative PCR and Western blot, were detected from the m level of RNA and protein expression of COX-2. In order to accurately elucidate the p38-MAPK signaling pathway on the expression of COX-2, we transfected into Si RNA p38-MAPK specific to glioma cells was detected by fluorescent quantitative PCR and Western blotting, from m RNA and protein levels of p38-MAPK gene knockdown the expression and effect of COX-2 were detected. Further explore the effect of p38-MAPK on the expression of COX-2 downstream gene SP1, we transfected COX-2-luciferase gene into glioma cells, then using SP1 specific Si RNA and explore the effect of SP1 inhibitor mithramycin on the expression of COX-2, COX-2 expression level was determined by measuring the fluorescence intensity. Finally, we use the COX-2 specific Si and RNA Inhibitor Celecoxib can inhibit the function of COX-2, the effects of COX-2 on the expression of VEGF and autophagy. Effect of COX-2 on the expression of VEGF was determined by fluorescence quantitative PCR; expression by Western blot analysis to detect LC3-II, and evaluate the autophagy intensity. Experimental results show that the medium of Met/Cys the lack of culture medium and deficiency in Gln/Arg/Lys could induce the expression of COX-2 in U87 and SF767 cells, induced by 2H, the cells began to express COX-2,4-6h Stable. But, in contrast, more rapid loss of Met/Cys can induce the expression of COX-2. Therefore, in the following experiments, we chose to establish a cell model by Met/Cys. The lack of hunger by p38-MAPK, a specific inhibitor of SB202190 MEK and JNK (SB), U0126 (U), SP600125 (SP) and Si after RNA treatment, we found that the inhibitory effect of p38-MAPK inhibitor SB on the expression of amino acid starvation induced COX-2, JNK specific inhibitor SP can inhibit the expression of COX-2 in U87 cells, but the expression of COX-2 in SF767 cells does not influence the effect of U0126 is just the opposite of SP. Also, we found that the amino acid at the same time to starvation induced p38-MAPK in U87 cells and SF767 phosphorylation, JNK phosphorylation occurs only in U87 cells. Further experiments found that p38-MAPK regulates COX-2 expression through SP1, SP1 gene The application of knockout and inhibitors can significantly reduce the expression of amino acid starvation induced COX-2. The effect of COX-2 downstream of the detection analysis showed that the expression of COX-2 can promote and induce autophagy of VEGF expression. In summary, based on the amino acid starvation model to simulate glioma tissue nutrient supply environment. To explore the related mechanisms of COX-2 expression induced by this kind of conditions. The results illustrate the amino acid starvation induced COX-2 expression is regulated by upstream of the p38-MAPK/SP1 signaling pathway; COX-2 can enhance the expression of glioma cells VEGF, lay the foundation for tumor angiogenesis; COX-2 through autophagy stimulate glioma cells, provide as the energy required for cell survival.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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