内质网应激和自噬在顺铂诱导肺癌细胞凋亡中作用的研究

发布时间：2018-08-02 12:48

【摘要】：肺癌是一种常见的,严重威胁人群健康和生命的肺部恶性肿瘤,非小细胞肺癌约占肺癌细胞的80%。近50年来许多国家都报道肺癌的发病率和死亡率都明显增高,肺癌的病因目前还不是完全清楚,但越来越多的文献表明肺癌的常见病因包括吸烟、电离辐射、大气污染和和既往肺部慢性感染等。顺铂(cis-diamminedichloroplatinum II,CDDP)是临床上治疗肺癌最常用的化疗药物。顺铂作用位点在癌细胞DNA的嘌呤和嘧啶碱基,它通过干预DNA损伤修复机制和抑制DNA复制诱导癌细胞凋亡。目前,顺铂在临床上广泛应用于肿瘤治疗过程中,并取得了良好的效果。但顺铂在使用过程中机体容易对其产生耐受性,同时顺铂所具有的耳毒性、神经毒性和肾毒性等毒副作用也限制了它的临床应用。因此,明确顺铂诱导细胞凋亡的机制尤为重要,越来越多的文献表明顺铂在诱导癌细胞凋亡过程中伴随着内质网应激和自噬的发生。内质网(Endoplasmic Reticulum,ER)是真核细胞内的一种膜结合细胞器,它的主要功能是对膜蛋白和分泌蛋白进行正确折叠和翻译后修饰。此外,内质网在脂类生物合成,能量代谢,细胞内Ca~（2+）稳态和氧化还原平衡方面也发挥重要的作用。内质网中蛋白质的折叠功能对细胞外和细胞内的刺激都非常敏感,包括缺血再灌注,炎症,糖基化和Ca~（2+）平衡失调等。内质网管腔中错误折叠或未折叠蛋白的聚集会诱导内质网应激(Endoplasmic Reticulum stress,ER stress),进而会激活未折叠蛋白反应(unfolded protein response,UPR)。UPR可以增加内质网对蛋白质的折叠和修饰的能力,减弱m RNA的翻译,通过内质网相关蛋白降解(ER-associated degradation,ERAD)和自噬降解未折叠和错误折叠蛋白。在内质网应激的早期阶段,内质网内的蛋白质合成会减少,调控蛋白质翻译和正确折叠的相关基因会被激活,这都有利于维持细胞的正常生理学功能,从而促进了细胞存活。然而,当ER stress持续时间过久或UPR功能受到损伤时,内质网内就会聚集大量的错误折叠或未折叠蛋白。这时,促凋亡信号就会被激活。目前,有研究表明心肌细胞、胰腺细胞的内质网功能失调可能是心脑组织缺血阻塞、糖尿病的主要发病机制。因此,ER stress既可以作为维持细胞存活的生存手段,也可以诱导细胞凋亡的重要机制。在大部分真核细胞内,自噬是一种广泛存在的生命现象,是一种依赖于溶酶体途径对细胞内代谢产物和细胞器进行降解并具有高度保守性的过程。自噬具有降解和回收代谢物以及细胞器的功能,在维持细胞内环境稳态方面起到至关重要的作用。自噬在肿瘤细胞中的作用具有双重性:一方面,自噬可以通过清除有缺陷的蛋白质或受损的细胞器,最终通过维持细胞内环境的稳态促进癌细胞存活;另一方面,过度活跃的自噬通过诱导细胞器的损伤和细胞内环境稳态失调,激活自噬性程序性细胞死亡。越来越多的研究表明自噬在肿瘤中所发挥的具体作用依赖于肿瘤发展的特定阶段和特殊肿瘤类型。因此,探讨自噬在顺铂诱导肺癌细胞凋亡中的作用具有很重要的意义。本文以人肺癌A549和H460细胞为研究对象,探讨了内质网应激-自噬反应在顺铂诱导肺癌A549和H460细胞凋亡中的作用及机制,为临床治疗肺癌提供新的思路。方法:(1)分别用不同浓度的顺铂(5μM、10μM、20μM、40μM和80μM)处理肺癌A549和H460细胞24 h。MTT法检测不同浓度的顺铂对肺癌A549和H460细胞生存率的影响,LDH试剂盒检测A549和H460细胞LDH漏出率。(2)分别用不同浓度的顺铂(10μM、20μM和40μM)处理肺癌A549和H460细胞24 h。通过流式细胞仪和荧光分光计分别检测细胞凋亡和线粒体膜电位的变化,Western blot方法检测cleaved caspase3、cleaved PARP、胞浆Cyt c、Grp78、PERK、IRE1的变化。(3)顺铂联合内质网应激抑制剂4-PBA和TUDC。MTT检测联合内质网应激抑制剂对A549和H460细胞生存率的变化,Western blot检测cleaved caspase3和Cyt c的变化。(4)观察自噬在顺铂诱导A549和H460细胞凋亡过程中的变化。Western blot检测LC3、Beclin 1蛋白的变化。(5)顺铂联合自噬抑制剂3-MA和CQ。MTT检测联合自噬抑制剂对A549和H460细胞生存率的变化,Western blot检测cleaved caspase3和Cyt c的变化。结果:(1)顺铂以剂量依赖性的方式抑制肺癌A549和H460细胞生长,同样地,顺铂也能以剂量依赖性的方式诱导了LDH释放率升高。(2)顺铂诱导了A549和H460细胞凋亡并降低其线粒体膜电位,顺铂诱导cleaved caspase3和cleaved PARP的活化,胞浆Cyt c、Grp78、PERK和IRE1表达上调。(3)用4-PBA和TUDC抑制内质网应激,可以增加顺铂对细胞的生长抑制作用,增加顺铂诱导的凋亡,增加顺铂诱导的cleaved caspase3和Cyt c的表达。(4)顺铂诱导了A549和H460细胞内自噬相关蛋白LC3II/LC3I、Beclin 1表达上调。(5)用3-MA和CQ抑制自噬,可以增加顺铂对细胞的生长抑制作用,增加顺铂诱导的凋亡,以及增加顺铂诱导的cleaved caspase3和Cyt c的表达。结论:我们的实验数据表明顺铂能诱导人非小细胞肺癌A549和H460细胞凋亡。一方面,通过线粒体途径介导的细胞凋亡;一方面,通过内质网应激相关凋亡途径。减轻内质网应激可以显著地增加顺铂诱导的细胞凋亡。顺铂在诱导细胞凋亡的同时也激活了自噬。自噬在顺铂诱导的细胞凋亡过程中发挥着促存活的作用,抑制自噬增加了肺癌细胞对顺铂的敏感性。本研究探讨了内质网应激-自噬反应在顺铂诱导肺癌A549和H460细胞凋亡中的作用及机制,进一步揭示顺铂的作用机制,为临床治疗肺癌提供了可行的理论依据。
[Abstract]:Lung cancer is a common type of malignant tumor that seriously threatens the health and life of the population. Non small cell lung cancer accounts for approximately 50 years of lung cancer cells. In recent 50 years, many countries have reported that the incidence and mortality of lung cancer are obviously increased. The cause of lung cancer is not completely clear, but more and more literature shows the common cause of lung cancer. Including smoking, ionizing radiation, air pollution and chronic pulmonary chronic infection. Cis-diamminedichloroplatinum II (CDDP) is the most commonly used chemotherapeutic drug for the treatment of lung cancer. The cisplatin action site is in the purine and pyrimidine bases of the cancer cell DNA. It can induce the repair mechanism of DNA damage and inhibit DNA replication to induce the apoptosis of cancer cells. Before, cisplatin is widely used in the clinical treatment of cancer and has achieved good results. However, cisplatin is easily tolerated during the use of cisplatin. At the same time, the toxic side effects of cisplatin, such as ototoxicity, neurotoxicity and nephrotoxicity, also restrict its clinical application. Therefore, the mechanism of cisplatin induced apoptosis is clearly defined. In particular, more and more literature shows that cisplatin is accompanied by endoplasmic reticulum stress and autophagy during the induction of apoptosis in cancer cells. Endoplasmic Reticulum (ER) is a membrane binding organelle in eukaryotic cells. Its main function is to fold and post translational modifications to membrane proteins and secretory proteins. The mass network also plays an important role in lipid biosynthesis, energy metabolism, intracellular Ca~ (2+) homeostasis and redox balance. The folding function of protein in the endoplasmic reticulum is very sensitive to both extracellular and intracellular stimuli, including ischemia reperfusion, inflammation, glycosylation and Ca~ (2+) imbalance. The aggregation of unfolded proteins induces endoplasmic reticulum stress (Endoplasmic Reticulum stress, ER stress), and then activates the unfolded protein reaction (unfolded protein response, UPR).UPR that can increase the ability of the endoplasmic reticulum to fold and modify proteins, weaken the translation of M RNA, and degrade through endoplasmic reticulum related proteins. On, ERAD) and autophagy degrade unfolded and erroneous folding proteins. In the early stages of endoplasmic reticulum stress, protein synthesis in the endoplasmic reticulum will be reduced, and genes related to protein translation and correct folding are activated, which all contribute to the maintenance of normal physiological functions of the cells and thus promote cell survival. However, when ER stress continues, it continues. In the endoplasmic reticulum, a large number of misfolded or unfolded proteins are aggregated in the endoplasmic reticulum when the time is too long or the UPR function is damaged. At this time, the apoptosis signal can be activated. At present, there is a study showing that the dysfunction of the endoplasmic reticulum in the pancreatic cells may be the main pathogenesis of ischemia and diabetes. Therefore, ER stress is the main mechanism of the disease. In most eukaryotic cells, autophagy is a widespread life phenomenon in most eukaryotic cells. It is a process that relies on lysosome pathway to degrade intracellular metabolites and organelles and is highly conserved. Autophagy degrades and returns. The function of metabolites and organelles plays a vital role in maintaining the homeostasis of the cell. Autophagy plays a dual role in tumor cells: on the one hand, autophagy can promote the survival of cancer cells by maintaining the homeostasis of the intracellular environment by eliminating defective proteins or damaged organelles; On the other hand, hyperactive autophagy activates autophagic programmed cell death by inducing cellular organelle damage and homeostasis of intracellular homeostasis. More and more studies have shown that the specific role of autophagy in tumors depends on the specific stage and specific tumor type of tumor development. Therefore, the study of autophagy induces lung cancer cells in cisplatin. The role of apoptosis is of great significance. In this paper, the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin in human lung cancer A549 and H460 cells were studied. Methods: (1) different concentrations of cisplatin (5 M, 10 u M, 20 mu M, 40 mu) were used respectively. The effects of different concentrations of cisplatin on the survival rate of A549 and H460 cells in lung cancer were detected by M and 80 M H460 cells. The LDH kit was used to detect the LDH leakage of A549 and H460 cells. (2) different concentrations of cisplatin (10 micron, 20 mu and 40 micron) were used to treat lung cancer and 24 cells through flow cytometer and fluorescent spectrometer. The changes in apoptosis and mitochondrial membrane potential were detected respectively. Western blot method was used to detect cleaved Caspase3, cleaved PARP, cytoplasmic Cyt C, Grp78, PERK, IRE1. (3) cisplatin combined with endoplasmic reticulum stress inhibitors and the changes in the survival rate of endoplasmic reticulum stress inhibitors Changes in ED Caspase3 and Cyt C. (4) observe the changes of autophagy during the apoptosis of A549 and H460 cells induced by cisplatin and.Western blot to detect the changes of LC3, Beclin 1 protein. (5) the changes of cisplatin combined with autophagy inhibitor, 3-MA and CQ.MTT detection combined autophagic inhibitors Results: (1) cisplatin inhibited the growth of lung cancer A549 and H460 cells in a dose-dependent manner. Similarly, cisplatin also induced the increase of LDH release rate in a dose-dependent manner. (2) cisplatin induced apoptosis of A549 and H460 cells and reduced the mitochondrial membrane potential. Cisplatin induced the activation of cleaved Caspase3 and cleaved PARP, cytoplasmic Cy. T C, Grp78, PERK and IRE1 are up-regulated. (3) inhibition of endoplasmic reticulum stress by 4-PBA and TUDC can increase the inhibition of cisplatin on cell growth, increase cisplatin induced apoptosis, increase the expression of cisplatin induced cleaved Caspase3 and Cyt C. (4) cisplatin induced A549 and intracellular autophagy related protein, up regulation of 1. (5) Inhibition of autophagy with 3-MA and CQ can increase cisplatin's inhibitory effect on cell growth, increase cisplatin induced apoptosis, and increase cisplatin induced cleaved Caspase3 and Cyt C expression. Conclusion: our experimental data suggest that cisplatin can induce apoptosis of A549 and H460 in human non-small cell lung cancer. On the one hand, it is mediated by mitochondrial pathway. Apoptosis, on the one hand, through endoplasmic reticulum stress related apoptosis pathway. Reduction of endoplasmic reticulum stress can significantly increase cisplatin induced apoptosis. Cisplatin also activates autophagy while inducing apoptosis. Autophagy plays a role in promoting survival during cisplatin induced apoptosis, and inhibition of autophagy increases lung cancer cells. This study explored the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin, and further revealed the mechanism of cisplatin, which provides a feasible theoretical basis for the clinical treatment of lung cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

【参考文献】