6A8α-甘露糖苷酶表达对Jurkat细胞粘附性的影响

发布时间：2018-06-27 10:26

本文选题：6A8α-甘露糖苷酶 + Jurkat细胞　；参考：《中国协和医科大学》2006年博士论文

【摘要】： 蛋白质糖基化在细胞的功能行为中起重要的作用。本实验室以往克隆到了1个新的人α-甘露糖苷酶cDNA(6A8)，并观察到了6A8α-甘露糖苷酶表达抑制影响包括Jurkat细胞在内的多种细胞的生物学行为。在预实验中观察到6A8α-甘露糖苷酶表达抑制的Jurkat细胞(AS)在培养中聚集成大的团块，这提示AS细胞间的粘附性增强。为研究其机制，首先用含有粘附分子和其它免疫分子基因在内的440个基因的DNA芯片对AS和M细胞(转导空载体的对照细胞)间的基因表达差异作了分析，见AS细胞有19个基因的表达上调，有20个基因的表达下调。表达上调的基因中包括CD11a(整合素α-L)、CD54(ICAM-1)、CD82、CD24、CD11c(整合素α-X)、整合素α-7、CD103(整合素α-E)、TNFSF9、IL-1R和IL-2Rγ等与细胞间粘附相关的基因。RT-PCR和免疫荧光染色支持芯片的结果。这些基因中以CD54和CD11a最为重要。本论文工作中对它们作了重点研究。与对照细胞(野生型细胞W与M细胞)相比，AS细胞与包被有CD54分子的培养板的粘附增强。封闭性CD11a单抗能阻断细胞与包被有CD54分子的培养板的粘附及细胞间的粘附。这证明了CD54和CD11a表达增强参与AS细胞间粘附增强。AS细胞LFA-1分子的亲和力也增高。另外，AS细胞间的粘附增强也与细胞骨架排列改变相关。异种细胞间的粘附在免疫应答等的生命活动中也起重要的作用。在对AS细胞与对照细胞和Raji细胞间粘附的比较中，观察到前者和Raji细胞间的粘附增强。在超抗原SEB存在下，Jurkat细胞与Raji细胞间可形成免疫突触，并有信号传导发生。6A8α-甘露糖苷酶的功能是修剪N-糖链中的甘露糖，Con A结合实验能检测细胞糖蛋白N-糖链中的甘露糖被α-甘露糖苷酶修剪的程度。与对照细胞相比，Con A与AS细胞的结合明显增强。关于6A8α-甘露糖苷酶表达抑制所致的蛋白质糖基化改变影响Jurkat细胞粘附性的确切机制须作深入研究。
[Abstract]:Glycosylation of proteins plays an important role in the functional behavior of cells. A new human 伪 -mannosidase cDNA (6A8) was cloned in our laboratory, and the inhibition of the expression of 6A8 伪 -mannosidase was observed to affect the biological behavior of many cells, including Jurkat cells. In the pre-experiment, Jurkat cells (as), whose expression of 6A8 伪 -mannosidase was inhibited, were observed to aggregate into large lumps in culture, which indicated that the adhesion between as cells was enhanced. In order to study its mechanism, the difference of gene expression between as and M cells (control cells of empty vector) was analyzed by DNA microarray containing 440 genes including adhesion molecules and other immunomolecular genes. There were 19 genes up-regulated and 20 genes down-regulated in as cells. The up-regulated genes included CD11a (integrin 伪 -L), CD54 (ICAM-1), CD82, CD24, CD11c (integrin 伪 -X), integrin 伪 -7CD103 (integrin 伪 -E), TNFSF9IL-1R, IL-2R 纬 and other genes associated with intercellular adhesion. Of these genes, CD54 and CD11a are the most important. In this paper, we focus on them. Compared with the control cells (wild-type cells W and M cells), the adhesion of as cells to the culture plates coated with CD54 molecules was enhanced. The closed CD11a McAb could block the adhesion of cells to the culture plate coated with CD54 molecule and the adhesion between cells. It is suggested that the increased expression of CD54 and CD11a may play an important role in the affinity of LFA-1 molecules in as cells. In addition, the enhancement of adhesion between as cells was also related to the change of cytoskeleton arrangement. Adhesion between xenogeneic cells also plays an important role in immune response and other life activities. The adhesion between as cells and Raji cells was observed in comparison with control cells and Raji cells. Immune synapses can be formed between Jurkat cells and Raji cells in the presence of superantigen SEB. The function of signal transduction. 6A8 伪 -mannosidase is to prune mannose Con A in N- sugar chain to detect the degree of mannose pruning by 伪-mannosidase in cell glycoprotein N- sugar chain. The binding of Con A to as cells was significantly increased compared with the control cells. The exact mechanism of the effect of 6A8 伪 -mannosidase expression inhibition on the adhesion of Jurkat cells needs further study.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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