【作者】 陈晓宁；

【导师】 顾建新；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 β1，4-半乳糖基转移酶V(GalT V)是β-，4-半乳糖基转移酶(β1，4-GalT)家族成员之一。在以往的研究中发现，GalT V的表达水平与胶质瘤的恶性程度呈正相关。在星型胶质细胞瘤SHG44细胞中，过表达GalT V可以加快细胞生长速率，增加细胞在软琼脂上的集落形成数，促进了SHG44细胞的恶性程度。同时，大量的研究已表明，由细胞表面黏附分子——整合素(integrin)介导的细胞黏附、迁移能力的变化与肿瘤细胞的恶性表型密切相关。整合素integrin由α和β亚基组成。β1亚基是一个N—糖基化蛋白，其糖链的修饰是依次在内质网和高尔基体中进行的，在糖链的加工过程中，形成了分布于胞内的未成熟型(105 kD)和定位于细胞膜的成熟型(125 kD)integrinβ1亚基。我们发现，与对照相比，在转染反义GalT V的SHG44细胞中(GalT V-AS／SHG44)，细胞表面integrinβ1明显增多，同时伴随有105 kD型式的integrinβ1减少，125 kD型式的integrinβ1增多；通过糖苷内切酶Endo H消化实验，我们进一步确证了105 kD型式的integirnβ1是糖链修饰不完全的未成熟型；在GalT V-AS／SHG44细胞中，integrinβ1除了与高尔基体共定位外，还呈簇状分布于胞内接近细胞膜的位置。这一结果表明，下调GalT V的表达，可以干涉integrinβ1的糖基化修饰过程，影响了integrinβ1的成熟及细胞膜转运。与之相应，进一步研究表明，随着细胞表面integrinβ1的减少，integrinβ1和α5的相互作用和integrin下游信号分子粘着斑激酶FAK的磷酸化水平都降低，进而导致GalT V-AS／SHG44细胞在Fn上的黏附能力增强、迁移能力下降。为了研究GalT V在脑星型胶质瘤中的特异性转录调控机制，我们构建了一系列GalT V启动子区的缺失突变体。我们发现，-200～+120启动子区域对维持GalT V在SHG44细胞中的转录至关重要，并且这一启动子区域在SHG44细胞中的转录活性相对较高。对这一区域的分析表明，其中有转录因子E1AF的结合序列。进一步研究发现，E1AF在SHG44细胞中高表达，并能够促进GalT V-200～+120启动子区域的转录活性。综上所述，我们认为脑星型胶质瘤SHG44细胞通过特异性表达转录因子E1AF，促进了GalT V的转录，进而影响了integrinβ1的成熟与细胞膜定位，改变了细胞对Fn的黏附、迁移能力。吡咯喹呤醌(pyrrolo quinoline quinone，PQQ)是一种分子量为330的小分子物质，最早发现于微生物中。PQQ是细菌中多种重要酶类的辅基，并能影响呼吸链功能与体内自由基水平。现已证实，高等真核生物体内也有PQQ存在。在研究中发现PQQ缺乏的小鼠生长缓慢，生殖能力差，并容易产生关节炎症，因此也有报道认为PQQ是体内的必需元素之一。本实验从微生物中分离纯化得到PQQ，并发现其对铅中毒和酒精性脂肪肝的具有治疗作用。更多还原

【Abstract】 Astrocytoma is one of the most common malignant tumors in human central nervous system. Until now, the pathogenesis of astrocytoma has not been elucidated clearly. The researches on the changes and significance of β1,4-GalT V (GalT V) in malignancy of human astrocytoma will be helpful to make a prognosis of astrocytoma.In previous study, we have shown that GalT V functions as a positive growth regulator in glioma. Here we reported that down-regulation the expression of GalT V in SHG44 cells by transfection with antisense cDNA specifically up-regulated the expression of cell surface integrin β1 without the change of its mRNA, and with integrin β1 125 kD mature form increased and 105 KD precursor form decreased. It is well known that the N-glycans of integrins modulate the location and functions of integrins. The SHG44 cells transfected with antisense cDNA of GalT V demonstrated decreased Golgi localization of integrin β1, strengthened the interaction between integrin α5 and β1 subunit, and enhanced the adhesion ability to fibronectin and the level of focal adhesion kinase (FAK) phosphorylation. Our results suggested that the down-regulation expression of GalT V could promote the expression of cell surface integrin β1 and subsequently inhibit glioma malignant phenotype.Previous study showed that increased GalT V transcript which appeared in the process of astrocytoma progress with the highest level in grade IV. We found that the sequence of GalT V promoter between -200 and +120 were very important for GalT V transcription in SHG44 cells. More interestingly, this sequense showed specifically higher activity in SHG44 cells than in other cells. We found dose dependence up-regulation of the -200 ～ +120 GalT V promoter-Luc constructs activity by transfactor E1AF, which specifically high expression in SHG44 cells.Pyrrolo-quinoline quinine (PQQ), which has a molecular weight of 330, is purified from methanol-utilizing bacteria. Its crystal structure and chemical synthesis have been discovered. In recent years PQQ is also found in eucaryotes. Subsequent studies identified that PQQ is cofactor of many important enzymes in the bacteria and could affect function of electron transport chain and level of free radical in humanbody. Animal experiment indicated decelerated growth, decreased fertility and defects in immune function have occurred with PQQ-deficient mice. So PQQ is thought to role as an essential vitamin and helpful nutrient in vivo. Our experiment suggested that PQQ could treat lead poisoning and alcoholic fatty liver obviously.更多还原