【作者】 龚铖；

【导师】 刘志苏；

【作者基本信息】 武汉大学 ， 外科学， 2013， 博士

【摘要】 研究背景肝细胞肝癌是严重危害人类生命的恶性肿瘤,揭示其分子发病机制极为重要。激活的T细胞核因子5(nuclear factor of activated T-cells5, NFAT5)也被称为TonEBP (Tonicity-responsive enhancer binding protein),是经典的Rel同源转录因子,因为作为渗透压调节者的角色在高渗或低渗状态下调节细胞平衡而被人们熟知。凋亡的特征是细胞收缩,核固缩,DNA断裂及凋亡小体生成。这些特征使凋亡区别于其他类型的细胞死亡如坏死。而一些细胞凋亡的标志,如磷脂酰丝氨酸外化,改变的线粒体功能或具有细胞类型或凋亡信号依赖的激活caspases,凋亡细胞体积减小(apoptotic cell volume decrease, AVD)是早期而普遍的标志事件。而高渗下正常的肝细胞皱缩能减小细胞体积,诱导细胞凋亡。由于没有任何关于癌组织的渗透压的文献数据,是否恶性肿瘤组织渗透压与周围组织或血液明显不同仍是未知的。明确肝癌发生过程中渗透压应激对肝癌细胞凋亡和增殖的影响,能为肝癌的分子靶向治疗提供新的靶位点。而将来后续的深入研究将提供更好地理解对NFAT5的刺激是如何调控相关基因表达的,而这与健康和疾病息息相关。研究目的观察高渗应激对肝细胞癌Huh7细胞增殖和凋亡的影响,并探讨其调控机制。研究方法1.免疫组化法检测64例肝细胞癌组织中渗透压调控的转录因子NFAT5的表达。2. RT-PCR检测肝细胞癌组织NFAT5mRNA的水平。3.氯化钠模拟肿瘤高渗微环境,蛋白免疫印迹法检测NFAT5蛋白的表达。4.流式细胞仪检测氯化钠模拟肿瘤高渗微环境下肝癌细胞凋亡5.噻唑蓝(MTT)比色法检测高渗微环境下肝癌细胞体外增殖活力。研究结果1.肝癌组织NFAT5的阳性表达率为9.38%,明显低于癌旁组织68.75%(P<0.01)。2.癌旁组织NFAT5基因mRNA水平明显高于癌组织(P<0.01)。3.高渗24h (100mmol/LNaCl)处理能使Huh7细胞NFAT5蛋白的表达显著升高(P<0.01)。4.高渗24h (100mmol/LNaCl)处理肝癌细胞凋亡率(26.0±3.2)%明显高于空白对照组(0.8±0.2)%(P<0.01)。5.高渗24h (100mmol/LNaCl)能明显抑制Huh7细胞增殖,细胞生长抑制率(31.35±2.09)%(P<0.01)。结论作为哺乳动物唯一的渗透压调节相关因子NFAT5,肝细胞癌组织与癌旁组织在NFAT5高表达率上存在明显差异,高渗应激能上调肝癌细胞NFAT5的表达,诱导肝癌细胞凋亡,并抑制细胞增殖。更多还原

【Abstract】 BackgroundIt is critical to understand the underlying molecular mechanisms of the development of hepatocellular carcinoma (HCC) which is a malignant seriously threatening people’s life. Toni city-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cells5,NFAT5) is a Rel homologytranscription factor classically known for its osmosensitive role in regulating cellular homeostasis during states of hypo-and hypertonic stress.Apoptosis is characterized by cell shrinkage,nuclear condensation, DNA fragmentation and apoptoticbody formation. These features distinguish apoptosis from other types of cell death, such as necrosis.Whereas some signs of apoptosis, such as externalization of phosphatidylserine, altered mitochondrial function or activation of caspases are cell type-and death signal-dependent, apoptotic cell volume decrease (AVD) is an early and ubiquitous event. Hyperosmotic hepatocyte shrinkage could decrease cell volume and induce cell apoptosis. Since there is no documentation of carcinoma tissue osmolality,it is unknown if cancerous tumors possess an osmolality vastly different from that of the surrounding tissues or blood. Since there is no documentation of carcinoma tissue osmolality,it is unknown if cancerous tumors possess an osmolality vastly different from that of the surrounding tissues or blood.In conclusion, the aim of this research is to find the influence of osmotic stress in tumor microenvironment on hepatoma cell apoptosis and proliferation to provide a new molecular targeted therapy in HCC. Continuing research will provide a better understanding as to how these and other alternative TonEBP stimuli regulate gene expression in both health and disease.ObjectiveTo investigate the effect of hyperosmotic stress on the proliferation and apoptosis of hepatocellular carcinoma Huh7cells,and preliminarily explore the mechanism. Methods1. The expression of mammalian osmotic regulator Nuclear factor of activated T-cells5(NFAT5) was detected in64cases of hepatocellular carcinoma by immunohistochemistry staining method.2. The NFAT5mRNA level was analyzed by RT-PCR.3. NaCl was used as a chemical hyperosmotic-inducible reagent to mimic tumor hyperosmotic microenvironment. The expression of NFAT5protein was analyzed by Western blotting Successfully construct the NFAT2overexpressed lentiviral vector and package the lentivirus.4. Cell apoptosis was examined by flow cytometry.5. Cell growth was measured by Methyl-Thiazol-Tetrazolium (MTT) assay.Results1. NFAT5positive expression rate was low(9.38%) in hepatocellular carcinoma tissue but was high in the corresponding peritumoral tissue(68.75%)(P<0.01).2. NFAT5mRNA level was low in hepatocellular carcinoma tissue but was high in the corresponding peritumoral tissue(P<0.01).3. Under hyperosmotic stress(100mmol/LNaCl) for24h,the Huh7cell protein expression levels of NFAT5were up-regulated(p<0.01).4. The apoptosis rate in experimental group (26.0±3.2%) was significantly higher than that in blank control group (0.8±0.2%)(P<0.01).5. Hyperosmotic stress(100mmol/LNaCl) also inhibit growth of Huh7cells, cell growth inhibitory rate was31.35±2.09%(P<0.01).更多还原