细胞自噬在天冬酰胺酶杀伤喉癌细胞中的作用及机制的研究

发布时间：2018-06-03 23:41

本文选题：喉癌 + 天冬酰胺酶　；参考：《第二军医大学》2017年硕士论文

【摘要】：目的:头颈部肿瘤是常见威胁人类健康的恶性肿瘤,而喉癌是最主要的头颈部恶性肿瘤之一,并且喉癌的发病率仍在逐年增加。尽管对喉癌的治疗方法不断改善,其五年生存率未明显提高。因此,寻找治疗喉癌的新疗法迫在眉睫。耗竭肿瘤生长所必需的氨基酸,达到“饿死”肿瘤细胞效果的氨基酸耗竭酶成为抗肿瘤研究的热点。对于大多数肿瘤细胞自身不能合成天冬酰胺,必须依赖外源的天冬酰胺才能生存。天冬酰胺酶(asparaginase)可通过降解天冬酰胺,从而抑制肿瘤细胞中蛋白质的合成,导致肿瘤细胞的死亡。目前天冬酰胺酶临床已被用于白血病、淋巴瘤和黑色素瘤的治疗。然而,针对于喉癌的治疗却尚未报道。细胞自噬是通过依赖于溶酶体降解蛋白质和细胞器,对降解产物循环利用的过程。正常情况下,自噬保持低水平状态。在内外界压力下自噬可快速上调,以维持细胞稳态。近年来的研究表明,自噬在肿瘤治疗方面起“双刃剑”作用,治疗肿瘤时细胞自噬既能提高肿瘤细胞的生存能力,也能加速细胞死亡。对于天冬酰胺酶耗竭天冬酰胺是否在喉癌细胞中诱导自噬,及自噬在其起的作用未有报道。方法:1、MTT法检测天冬酰胺酶对喉癌细胞Tu212和Tu686的细胞毒性,以及联合自噬抑制剂氯喹(CQ)对细胞的毒性影响;检测抗氧化剂N-乙酰-L-半胱氨酸(NAC)在天冬酰胺酶对喉癌细胞生长作用的影响;2、流式细胞术检测细胞凋亡。天冬酰胺酶单独、联合自噬抑制剂CQ对Tu212和Tu686细胞凋亡的影响;抗氧化剂NAC对天冬酰胺酶诱导喉癌细胞凋亡的影响;3、透射电子显微镜(TEM)观察天冬酰胺酶诱导Tu212和Tu686细胞中自噬体的产生;4、Western blot法检测在Tu212和Tu686细胞中天冬酰胺合成酶(ASNS)的表达情况;检测天冬酰胺酶单独作用、联合CQ作用时,喉癌细胞中自噬特异蛋白LC3-II的表达情况;检测凋亡相关蛋白Caspase 3、PARP表达量的变化;5、激光共聚焦显微镜(Confocal microscopy)检测自噬及自噬流的产生情况。结果:喉癌细胞Tu212和Tu686中天冬酰胺合成酶表达缺失;天冬酰胺酶通过耗竭天冬酰胺对喉癌细胞Tu212和Tu686细胞显著杀伤。天冬酰胺酶能诱导喉癌细胞产生显著细胞凋亡,并且诱导凋亡相关蛋白Caspase 3、PARP剪切,Caspase抑制剂z-VAD-fmk明显减弱天冬酰胺酶诱导的Tu212和Tu686细胞凋亡,因此表明天冬酰胺酶诱导细胞凋亡是依赖Caspase途径。天冬酰胺酶能诱导喉癌细胞中自噬体的出现,诱导LC-II表达,以及自噬流的产生。天冬酰胺酶引起的自噬伴随信号通路Akt/mTOR的抑制和信号通路Erk1/2的激活,表明Akt/mTOR信号通路和Erk1/2信号通路参与了天冬酰胺酶诱导喉癌细胞自噬中的分子调控机制。值得注意的是,联合自噬抑制剂CQ作用于喉癌细胞时,显著增强天冬酰胺酶对喉癌细胞Tu212和Tu686的生长抑制以及细胞凋亡。这表明,当天冬酰胺酶作用于喉癌细胞时,细胞自噬在此过程中起保护性作用。抗氧剂NAC可以明显抑制天冬酰胺诱导的细胞自噬,并且天冬酰胺引起的细胞毒性也显著性减弱,表明ROS在天冬酰胺酶诱导的自噬及细胞毒性中发挥重要作用。综上所述:天冬酰胺酶对喉癌细胞Tu212和Tu686有显著的细胞毒性。天冬酰胺酶能诱导喉癌细胞发生自噬。抑制细胞自噬明显增强天冬酰胺酶对喉癌细胞杀伤作用。因此,为喉癌治疗提供新的思路。
[Abstract]:Objective: head and neck tumors are one of the most common malignant tumors that threaten human health, and laryngeal cancer is one of the most important head and neck cancers, and the incidence of larynx cancer is still increasing year by year. Although the treatment methods for larynx cancer are constantly improving, the five-year survival rate is not obviously improved. Therefore, the new therapy for the treatment of larynx cancer is imminent. The amino acid depleting enzymes necessary for the growth of the cancer cells are the hot spots in cancer research. For the inability of most tumor cells to synthesize asparagine itself, it is necessary to rely on exogenous asparagine to survive. Asparaginase can inhibit the tumor by degrading asparagine. The synthesis of proteins in the cell and the death of tumor cells. Currently, asparaginase has been used in the treatment of leukemia, lymphoma and melanoma. However, treatment for laryngoma has not been reported. Autophagy is a process of recycling the degradation products by relying on lysosomes to degrade proteins and organelles. Autophagy maintains a low level of autophagy. Autophagy can be rapidly up-regulated under internal and external pressure to maintain cell homeostasis. Recent studies have shown that autophagy plays a "double-edged sword" role in cancer treatment. Autophagy can improve the viability of tumor cells and accelerate cell death in the treatment of tumors. Whether or not amides induce autophagy in larynx cells, and the role of autophagy in it has not been reported. Methods: 1, MTT assay was used to detect the cytotoxicity of asparaginase to Tu212 and Tu686 in laryngeal cancer cells, and the toxicity of chloroquine (CQ) combined with autophagic inhibitor (CQ), and the detection of antioxidant N- acetyl -L- cysteine (NAC) in asparaginase to larynx cancer The effect of cell growth; 2, flow cytometry to detect apoptosis. The effect of asparaginase alone, combined with autophagy inhibitor CQ on apoptosis of Tu212 and Tu686 cells; the effect of antioxidant NAC on asparaginase induced apoptosis in larynx cells; 3, transmission electron microscopy (TEM) observed the autophagy induced by asparaginase in Tu212 and Tu686 cells. 4, Western blot method was used to detect the expression of asparagine synthetase (ASNS) in Tu212 and Tu686 cells; the expression of autophagic specific protein LC3-II in larynx cells was detected by the action of asparaginase alone and combined with CQ; changes in the expression of apoptosis related protein Caspase 3, PARP expression; 5, laser confocal microscopy (Conf) Ocal microscopy) detects the production of autophagy and autophagic flow. Results: the expression of asparagine synthetase in Tu212 and Tu686 of larynx cancer cells was missing; asparaginase was killed by asparagine by exhaustion of asparagine. Asparaginase induced significant apoptosis in larynx cancer cells and induced apoptosis related eggs. White Caspase 3, PARP shear, and Caspase inhibitor z-VAD-fmk obviously weaken the apoptosis of Tu212 and Tu686 cells induced by asparaginase, so the apoptosis induced by asparaginase is dependent on the Caspase pathway. Asparaginase can induce the appearance of autophagic in the larynx cells, induce the expression of LC-II, and the production of autophagic flow. The inhibition of autophagy accompanied by the signaling pathway Akt/mTOR and the activation of the signal pathway Erk1/2 indicate that the Akt/mTOR signaling pathway and the Erk1/2 signaling pathway participate in the molecular regulation mechanism of the autophagy induced by asparaginase in larynx cells. It is worth noting that the combination of autophagy inhibitor CQ on larynx cells significantly increases the asparaginase to the larynx. The growth inhibition and apoptosis of Tu212 and Tu686 cells in cancer cells showed that the autophagy played a protective role in the process of the action of asparaginase on Laryngocarcinoma Cells that day. Antioxidant NAC could significantly inhibit the autophagy induced by asparagine, and the cytotoxicity of asparagine caused by asparagine was also significantly weakened, indicating that ROS was in asparagine. Aminase induced autophagy and cytotoxicity play an important role. To sum up, asparaginase has significant cytotoxicity to Tu212 and Tu686 in larynx cells. Asparaginase can induce autophagy in laryngeal cancer cells. Inhibition of autophagy significantly enhances the killing effect of asparaginase on laryngeal cancer cells. Therefore, a new thought is provided for the treatment of larynx cancer. Road.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

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