【作者】 张一鸣；

【导师】 德伟；

【作者基本信息】 南京医科大学 ， 生化与分子生物学， 2004， 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，前者是生物大分子核酸的组成成分；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。近年来的研究表明转醛醇酶在多发性硬化、恶性肿瘤的发生、肝硬化、器官发育、一些细胞因子引起的细胞凋亡甚至胚胎发生中都起着非常重要作用。转醛醇酶的表达和活性表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。 Lachaise等以及本研究室已有的研究表明，在无脊椎动物螃蟹的蜕皮甾酮和小鼠睾丸间质细胞的睾酮生成过程中，转醛醇酶的表达和活性与之呈密切正相关。并且蜕皮甾酮合成的负调控因素—蜕皮抑制激素也能同时抑制着转醛醇酶的表达和活性。刺激间质细胞分泌睾酮的HCG同时也升高转醛醇酶的活性。在这些过程中转醛醇酶都极有可能是通过调节NADPH的生成来控制这些类固醇类分子的合成的。 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，我们试图探讨在以NADPH为辅酶的羟化酶活跃作用的瘢痕疙瘩中转醛醇酶与成纤维细胞增生之间的关系。 同时，在Banki等的研究中也表明，转醛醇酶与恶性肿瘤的发生之间有相关性，我们也将在胃癌发生中研究转醛醇酶的作用。 南京医科人学硕!‘学位论文 本实验共分为两个部分进行:第一部分研究人疲痕疙瘩和正常组织中转醛醇酶在核酸和蛋白两个水平的表达变化;第二部分研究在胃部肿瘤组织中转醛醇酶的活性变化，并试图解释在肿瘤发生的分子机制中转醛醇酶的作用。第一部分:人疲痕疙瘩中转醛醉醉活性的特异性表达研究 汉，J定疲痕组织(15cases)和正常皮肤组织(scases)中转醛醇酶的活性，同时以RI’- PCR方法检测转醛醇酶mRNA水平差异，运用SDS-聚丙烯酞胺凝胶电泳、免疫印迹方法测定转醛醇酶蛋白表达水平差异，并且培养疲痕疙瘩和正常皮肤组织的成纤维细胞。结果显示:疲痕疙瘩与正常皮肤组织相比，转醛醇酶虽然在转录及蛋白两个表达水平皆没有显著性差别，但瘫痕疙瘩中酶活性明显比正常皮肤组织中低，差异显著;细胞培养的结果表明分别来自疲痕疙瘩和正常皮肤组织的成纤维细胞在动态生长、增殖上的差异无统计学意义。此结果显示在痕痕疙瘩中成纤维细胞的数量没有增加，但可能每个成纤维细胞合成胶原的能力增加，这一点导致了疲痕疙瘩的形成，而胶原的合成与NADPH提供还原能力是紧密相连的，转醛醇酶可能正是通过调节磷酸戊糖途径中NADPH的生成而在痕痕疙瘩的发生、发展中起到重要作用。这将为研究疲痕的发生提供一个新的切入点，有可能通过调节该酶的活性达到抑制瘫痕疙瘩中胶原的合成，从而控制疲痕的生成。 南京医科人学硕}学位论文第二部分:转醛醉醉在胃癌中特异性表达的研究 坝，l定胃癌(4Cases)、癌旁组织(4eases)和正常胃组织(3cases)中转醛醇酶的活性，利用双向电泳比较胃癌和正常两种组织中蛋白表达的变化，运用Westem blot比较转醛醇酶在蛋白表达水平的差异。结果显示:在胃癌中转醛醇酶活性与癌旁组织及正常皮肤组织相比明显升高，差异显著;癌旁组织中活性比正常组织低，也有显著性差异。而在蛋白水平皆没有显著性差别。正常胃私膜上皮细胞存在着增殖与凋亡的动态平衡，而肿瘤细胞中存在不同程度的细胞凋亡也是一个公认的事实，转醛醇酶在细胞凋亡上的作用也日渐受到关注。本实验结果中在癌旁组织转醛醇酶活性低于正常组织显示，在肿瘤的发生起始阶段有一个凋亡被抑制的过程，随着病程的发展，凋亡被激活，这时候细胞的凋亡增加将反馈性的刺激胃补膜细胞不断增殖，结果引起凋亡/增殖比例失调，最终促进肿瘤的发生。如果这一点被证实的话，转醛醇酶是否可以作为一个病变的mark，这非常令人感兴趣。这是本次研究一个新的发现，在国内外杂志上皆未见有报道。 总结前面两部分实验的结果，可以认为转醛醇酶作为糖代谢途径的一个关健酶对维持细胞的正常生理功能上是必需的，但除此之外，在组织细胞的病变过程中也起着非常重要的作用，甚至有可能成为病变的一个mark。对糖代谢酶的其他功能的研究将有可能成为后基因组计划中的一个新研究方向。更多还原

【Abstract】 Study on Specific Activity of Transaldolase in theHumann Keloid and Gastric CarcinomaTransaldolase (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 Lachaise’s lab and our lab had demonstrated that the expression and activity of Transaldolase was positively related to the production of ecdysterone in the invertebrate-crab and testosterone in the mouse Leydig cells. Molt inhibiting hormone, the negative regulator of ecdysterone, can also inhibit the expression and activation of Transaldolase; HCG, the stimulator of Leydig cells secreting testosterone, can increase the activity of Transaldolase and had no change in mRNA and protein levels. In these similar biosynthesis it is possible thatTransaldolase influence the synthesis of these steroids through regulating NADPH.Due to the high conservation of Transaldolase gene and the homology of Transaldolase protein structure in defferent creature. We are trying to study the relationship between the activity of Tranaldolase and fibroblasts development in the keloid formation in which the activity of hydroxylase is high which uses the NADPH as the Co-Enzyme.Meanwhile, study of Banki group showed that Transaldolase functions was related to the malignant tumor development. So the second portion of our research will focus on the function of Transaldolase in the development of gastric carcinoma.The project will be executed as two parts: the first part was to study the expression level of Transaldolase in keloid tissue and normal tissue at mRNA and protein level. The second part would study the activity of Transaldolase in the gastric carcinoma tissue, and set out to indicate the function of Transaldolase in the molecular mechanism of tumor development.1 The specific expression research of Transaldolase in the human keloidWe measure the activity of Transaldolase in the keloid tissue and normal tissue by the method described by Lachaise, and using PT-PCR and SDS-PAGE, Western blotting Transaldolase expression in mRNAlevel and protein level were determined. At the same time we culture flbroblasts from keloid and normal tissue. Our results showed that the activity of Tansaldolase was down regulated in the keloid tissue, but the expression levels of both messenger and protein are no significant difference. The results from cell culture show that there is no statistical difference in dynamic growth and proliferation of keloid and the normal tissue. Compared with normal tissue, the quantity of flbroblasts from keloid isn’t change. A significantly increase of the collagen expression per flbroblasts might lead to the formation of keloid. Collagen composition is well related to NADPH, thus Transaldolase may be a vital role in the keloid genesis and development by regulating the quantity of NADPH in the ppp. It would be a new path on study in keloid genesis. It is likely to inhibit the growth of flbroblasts by controlling the activity of Transaldolase in the keloid tissue. 2 The specific expression of Transaldolase in the gastric carcinomaWe did 2-Dimentional electriphoresis and Western blot to check the protein level of Transaldolase in the gastric carcinoma tissue. We found the activity of Transaldolase was much higher in the carcinoma tissue than it in the normal tissue or tissue around the tumor. There were significant differences in Transaldolase activity between sur更多还原