【作者】 张乐；

【导师】 关方霞；

【作者基本信息】 郑州大学 ， 细胞生物学， 2019， 硕士

【摘要】 食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)是一种由消化道上皮异常增生所形成的恶性病变,在我国食管鳞癌患者约占全世界发病总数的70%,是世界食管癌高发区,严重威胁着我国人民的身体健康和生活质量。伴随着我国环境问题和老龄化的加快,ESCC的病发率和病死率呈上升趋势。近年来,我们对ESCC的研究在基础和临床方面取得了些许进展,但其发生和发展的分子机制目前尚未明了。线粒体作为细胞能量代谢和凋亡相关重要的细胞器,在肿瘤细胞的发生发展过程中起着重要作用。第二线粒体源的caspase激活物(Second mitochodria-adrived activator of caspase,Smac)是一种由线粒体释放到胞浆的促凋亡调节因子。在凋亡刺激作用下,从线粒体释放到胞浆中,通过与凋亡抑制蛋白(Inhibitor of apoptosis protein,IAP)结合从而解除对Caspase的抑制作用,引发细胞凋亡过程。研究发现线粒体的功能变化影响着肿瘤细胞的能量代谢方式,进而可以改变细胞的增殖、凋亡、迁移等生命活动过程。Warburg效应是肿瘤细胞特有的能量代谢方式,这一过程为肿瘤细胞的转移提供了充足的物质、能量以及微环境的条件。但是肿瘤细胞的代谢方式并不是一成不变的,它会随着基因、微环境、药物等因素的改变而改变,因此本研究旨在发现Smac蛋白是否能够通过改变ESCC细胞的能量代谢方式进而影响肿瘤细胞的生长存活状态。目的本研究通过改变食管鳞癌细胞中Smac蛋白的表达,在体内外水平上检测Smac蛋白对ESCC细胞增殖、凋亡、迁移以及线粒体代谢功能的影响,以Warburg效应为切入点,从能量代谢水平揭示Smac蛋白对食管鳞癌的影响作用。方法1.Western blot检测ESCC组织以及细胞中Smac蛋白的表达,并通过慢病毒LV5-Smac和LV-Smac-RNAi感染ESCC细胞构建Smac过表达和干扰细胞株。2.通过CCK-8检测氧化损伤条件下Smac过表达和干扰后ESCC细胞的增殖和存活变化。3.线粒体膜电位和Annexin V-FITC/PI双染法检测Smac蛋白表达改变后对ESCC细胞的凋亡的影响。4.Transwell小室实验检测Smac表达改变对ESCC细胞迁移的影响。5.分光光度计法和化学荧光探针法分布检测Smac过表达和干扰后ESCC细胞中乳酸的释放、葡萄糖的吸收以及细胞内活性氧(ROS)的表达变化。6.Western blot检测细胞中第二线粒体凋亡通路的蛋白、糖酵解过程相关蛋白、N-cadherin、E-cadherin、Vimentin、P-AMPK、HIF-1α蛋白的表达变化。7.建立ESCC细胞移植瘤模型,测量肿瘤块的体积以及裸鼠体重变化,处死后取肿瘤块称重并TUNEL法检测移植瘤组织细胞凋亡情况。8.免疫荧光法检测移植瘤组织中N-cadherin、E-cadherin以及Vimentin蛋白和糖酵解相关蛋白LDH和HK2的表达水平。9.化学荧光探针法检测四组移植瘤组织中ROS水平的变化。结果第一部分Smac过表达和siRNA干扰后对食管鳞癌细胞表型的影响1.Smac蛋白在ESCC组织和细胞中的表达量下调,成功构建Smac过表达和siRNA干扰的Eca109和EC1细胞株。2.在氧化应激作用下,Smac过表达的Eca109和EC1细胞存活率显著低于对照组,而Smac-siRNA干扰的Eca109和EC1细胞存活率则高于对照组;线粒体膜电位以及流式细胞术检测结果显示,在氧化刺激条件下Smac过表达可促进Eca109和EC1细胞的凋亡,而Smac-siRNA干扰后则抑制Eca109和EC1细胞的凋亡;同时,Western blot结果显示Smac过表达抑制了凋亡抑制蛋白Bcl-2和X-IAP的表达,凋亡执行蛋白Caspase-3的前体表达量也显著下降。3.与对照组相比,Smac过表达的Eca109和EC1细胞迁移数量显著减少,Smac-siRNA干扰的Eca109和EC1细胞迁移数量则明显增加;Smac过表达使Eca109和EC1细胞中钙粘附蛋白E-cadherin、波形蛋白Vimentin表达量增多;且Smac过表达抑制了Eca109和EC1细胞中乳酸释放和葡萄糖的吸收,而Smac-siRNA干扰的Eca109和EC1细胞中乳酸释放和葡萄糖的吸收则明显升高。4.Smac过表达可降低Eca109和EC1细胞中ROS水平,而Smac-siRNA干扰则提高了Eca109和EC1细胞中ROS的水平;Western blot结果显示Smac过表达抑制了Eca109和EC1细胞中HIF-1α和P-AMPK的表达量,Smac-siRNA干扰则促进Eca109和EC1细胞中HIF-1α和P-AMPK的表达。第二部分Smac通过Warburg效应促进Eca109移植瘤组织中细胞的迁移1.Smac过表达和siRNA干扰对Eca109移植瘤的体积大小和生长速度影响不显著。2.免疫荧光结果显示Smac siRNA干扰对Eca109移植瘤组织中促进迁移过程的蛋白E-cadherin、Vimentin表达量增多;糖酵解过程关键酶LDH和HK2的表达量增多;Smac siRNA干扰促进Eca109移植瘤组织中ROS的生成。结论1.ESCC组织和细胞中的Smac蛋白表达量明显低于正常组织细胞。2.Smac过表达增强了ESCC细胞对氧化应激的敏感性,ESCC细胞中Smac蛋白的低表达一方面降低细胞对氧化刺激的敏感性,另一方面通过细胞能量代谢的变化促进细胞迁移过程。3.Smac过表达和siRNA干扰对Eca109移植瘤的体积大小和生长速度影响不大,但是可能通过Warburg效应进而影响肿瘤细胞的迁移能力。更多还原

【Abstract】 Esophageal cancer is a malignant lesion formed by abnormal proliferation of the digestive tract epithelium.China is one of the countries with the highest incidence of ESCC in the world.With the aging of the population,the total morbidity and mortality of ESCC keeps rising.Although some progress has been made in the basic and clinical research of ESCC in recent years,the genetic basis and molecular mechanism of its occurrence have not been revealed,so researches on its pathogenesis can be used as the molecular basis for future targeted drug therapy.As an important organelle involved in cell energy metabolism and apoptosis,mitochondria play an important role in the development of tumor cells.The Smac protein is a pro-apoptotic regulator released from the mitochondria to the cytosol.Under the stimulation of apoptosis,it is released from the mitochondria into the cytoplasm,and the inhibition of Caspase is released by binding to the Inhibitor of apoptosis protein to induce apoptosis.Studies have shown that changes in energy metabolism of tumor cells affect the function of mitochondria,which in turn changes the life processes of cell proliferation,apoptosis and migration.The Warburg effect is a unique form of energy metabolism in tumor cells,which provides sufficient material,energy,and microenvironment conditions for tumor cell metastasis.However,themetabolic mode of tumor cells is not static.It will change with the changes of genes,microenvironment,drugs and other factors.Therefore,this study aims to find out whether the Smac gene can change the energy of ESCC cells.Metabolic pathways in turn affect the growth and survival of tumor cells.ObjectiveThis study will examine the effects Smac protein on cell proliferation,apoptosis,migration,and mitochondrial metabolism by overexpressing and si RNA interfering with the Smac gene in esophageal squamous cell carcinoma cells.Combining cell and animal experiments,the effects of Smac on esophageal squamous cell carcinoma were revealed via energy metabolism.Methods1.Western blot was used to detect the expression of Smac protein in ESCC tissues and cells.Smac overexpressed and si RNA interfered ESCC cell lines were constructed mediated by lentivirus.2.CCK-8 was used to detect the proliferation and survival of Smac-overexpressed and-interfered ESCC cells under oxidative damage condition.3.Mitochondrial membrane potential and Annexin V-FITC/PI double staining were employed to detect the apoptosis of Smac-overexpressed and-interfered ESCC cells.4.Transwell chamber was used to detect cell migration when Smac was overexpressed and interfered.5.The lactic acid release,glucose uptake and expression of reactive oxygen species in the cells were detected by spectrophotometer and chemical fluorescence probe method respectively.6.The changes of the second mitochondrial apoptosis pathway,the protein related to glycolysis process,N-cadherin,E-cadherin,Vimentin,P-AMPK and HIF-1α protein were detected by Western blot.7.The ESCC xenograft model was established.After tumor-bearing,the volume of the tumor mass and the weight of the nude mice were measured and recorded every 3 days.After 3 weeks,the tumor mass was weighed and the apoptosis level of four groups of transplanted tumor cells was detected by TUNEL method.8.The expression levels of N-cadherin,E-cadherin and Vimentin protein and glycolytic process related proteins LDH and HK2 in transplanted tumor tissues were detected by immunofluorescence assay.9.Chemical fluorescence probe method was used to detect the changes of ROS levels in the four groups of transplanted tumor tissues.ResultsPart Ⅰ: Effect of Smac expression changes on phenotype of esophageal squamous cell carcinoma1.The expression of Smac protein was down-regulated in ESCC tissues and cells,Eca109 and EC1 cell lines with Smac overexpression and si RNA interference were successfully constructed.2.The Smac overexpressing and si RNA interfering Eca109 and EC1 cells under oxidative stress,Smac overexpressing cell line survival rate was significantly smaller than the control group,Smac si RNA interference cell line survival rate was higher than the control group.Both mitochondrial membrane potential and flow cytometry showed that Smac overexpression promoted apoptosis of Eca109 and EC1 cells under oxidative stimulation,while Smac si RNA interfered with apoptosis of Eca109 and EC1 cells.At the same time,Western blot analysis showed that Smac overexpression inhibited the expression of apoptosis inhibitory proteins Bcl-2 and X-IAP,and the expression of apoptosis-executing protein Pro-Caspase 3 was also significantly decreased.3.The results of Transwell assay showed that the number of migration of Eca109 and EC1 cells overexpressed by Smac was decreased compared with the control group,and the number of migration of Eca109 and EC1 cells by Smac was increased.Western blot analysis showed that Smac overexpression increased theexpression of E-cadherin and Vimentin proteins in Eca109 and EC1 cells.Smac overexpressed Eca109 and EC1 cells had less lactate release than control cells,and glucose uptake was lower than normal Eca109 and EC1 cells.The amount of lactic acid released from Eca109 and EC1 cells si RNA interfered by Smac was significantly higher,and the absorption of glucose was also higher than that of the control group.4.Smac overexpression reduced ROS levels in Eca109 and EC1 cells,while Smac si RNA interference increased ROS levels in Eca109 and EC1 cells.Western blot analysis showed that Smac overexpression down-regulated the expression of HIF-1α and P-AMPK in Eca109 and EC1 cells,while Smac si RNA interference promoted the expression of HIF-1α and P-AMPK in Eca109 and EC1 cells.Part Ⅱ: Smac promotes cell migration in Eca109 xenografts through the Warburg effect1.Smac overexpression and si RNA interference had no significant effect on the size and growth rate of Eca109 xenografts.2.The results of immunofluorescence showed that Smac si RNA interference increased the expression of E-cadherin and Vimentin,which promoted the migration process in Eca109 xenografts,and increased the expression of key enzymes in the glycolysis process.Smac si RNA interference promotes ROS production in Eca109 xenografts.Conclusion1.The expression level of Smac protein in ESCC tissues and cells was significantly lower than that in normal tissue cells.2.Smac overexpression enhances the sensitivity of ESCC cells to oxidative stress.The low expression of Smac protein in ESCC cells reduces the sensitivity of cells to oxidative stimulation on the one hand,and promotes cell migration through changes in cellular energy metabolism.3.Smac overexpression and si RNA interference have little effect on the size and growth rate of Eca109 xenografts,but may affect the migration ability of tumor cells through the Warburg effect.更多还原