【作者】 张锐；

【导师】 德伟；

【作者基本信息】 南京医科大学 ， 生化与分子生物学， 2005， 硕士

【摘要】 转醛醇酶是磷酸戊糖途径非氧化阶段的关键酶,其主要作用在于调控磷酸戊糖途径产生5-磷酸核糖和尼克酰胺腺嘌呤二核苷酸磷酸,前者是细胞增殖过程中合成核苷酸不可缺少的原料;后者是生物体内重要的还原当量,不仅维持谷胱甘肽处于还原状态,还是体内多种代谢反应的供氢体,广泛参与胞内信号转导、抗氧应激等。因此,TAL既是细胞基础代谢所必需的,也与细胞功能转变、代谢状态变化密切相关,近年来的研究表明转醛醇酶在多发性硬化、恶性肿瘤、肝硬化、器官发育、胚胎发生、多种细胞因子引起的细胞凋亡中都起着非常重要作用,其表达和活性呈现出明显的组织特异性、发育阶段相关性和生理病理状态差异性。 Peter等的初步研究发现大鼠肝癌组织中TAL活性显著增高,在再生肝中没有同样变化,提示TAL活性似乎对细胞癌变是特异性的。Banki等的研究也表明,转醛醇酶与着色性干皮病这一癌前病变的发生之间有相关性 鉴于在不同物种中转醛醇酶基因的高度保守性及转醛醇酶蛋白结构的相似性,我们试图在人类研究模式来探讨转醛醇酶在肝癌组织中的作用。 本实验共分为两个部分进行:第一部分研究人肝癌组织中转醛醇酶的活性变化及在核酸和蛋白两个水平的表达情况;第二部分利用蛋更多还原

【Abstract】 Transaldolase (TAL) is an essential enzyme of the non-oxidative part of the pentose phosphate pathway(ppp) with produces reductive potential in the form of NADPH as well as ribose 5-phosphate for incorporation into nucleotides . NAPDH is not only a reductor to keep glutathione reducted , but also a H~+ supplier for many metabolic pathways. Recent studies have shown that Transaldolase plays an important role in the development of Multiple Sclerosis, malignant tumor, Liver Cirrhosis organ development and embryogenesis, and also functions in some factors induced apoptosis. The expression and activation of Tranaldolase have tissue-specificity, correlating with the different develop phase and the difference between physiological and pathological condition.Previous study of Peter’s lab had demonstrated that In the liver tumors, transaldolase activity was increased 1.5- to 3.4-fold over the activities observed in normal control rat liver,while it showed no change in the regenerating liver.Meanwhile, study of Banki group showed that Transaldolase functions was related to the malignant tumor development.Due to the high conservation of Transaldolase gene and the homology of Transaldolase protein structure in defferent creature. We are trying to study the potential role of Transaldolase on human hepatocarcinogenesisThe study was divided into two parts: the first part was to study the activity of Transaldolase in the hepatocellular carcinoma tissues and the expression level of Transaldolase in hepatocellular carcinoma tissue and normal tissue at mRNA and protein level. The second part was to probe whether Transaldolase has any isoforms.and research on the relationship between Transaldolase activity and post-translational modification. We set out to indicate the function of Transaldolase in the molecular mechanism of tumor development.1 The high activity of Transaldolase in the human hepatocellular carcinomaWe measured the activity of Transaldolase in normol liver 、 pericancerous land hepatocellular carcinoma tissues by the method described by Lachaise, and using PT-PCR and SDS-PAGE, Western blotting Transaldolase expression in mRNA level and protein level were determined. Our results showed that the activity of Transaldolase was much higher in the carcinoma tissue than it in the normal tissue or tissue around the tumor, but the expression levels of both messenger and protein are no significant difference. The change of activity may be associated with post-translational modification. This is a discovery which not reported in human carcinoma.Previous studies had demonstrated that nucleotide composition is well related to ribose 5-phosphate, thus Transaldolase may be a vital rolein the occurrence of hepacellular carcinoma by regulating the quantity of ribose 5-phosphate in the ppp. It would be a new path on study in hepacarcitogenesis. It is likely to inhibit the growth of cacinoma by controlling the activity of Transaldolase . 2 The expression of Transaldolase in the hepacellular carcinomaWe did 2-Dimentional electriphoresis Western blotting to check the protein level of Transaldolase in the hepatocellular carcinoma tissue. We separated more than 10 isoforms of Transaldolase. The a7 spot was hardly expressed in normol liver , while in hepacellular carcinoma it was highly expressed. The rapid growth of hepacellular carcinoma accompanies high frequency of cell metabolism, which may cause the over expression of Transaldolase gene. The a7 spot was highly expressed in hepacellular carcinoma, so it may be related to hepacarcitogenesis . The a2 spot was markly decreased in hepatoma, which indicated the change of activity may be associated with post-translational modification.These results show that Transaldolase is a key enzyme which maintain the well-balanced cellular function. In addition, Transaldolase may play a crucial role in pathological changes and may be a marker. It is a question for discussion to research for other function of the well-informed enzyme in carbohydrate metabolism.更多还原