發(fā)布時(shí)間：2018-06-27 10:26

  本文選題：6A8α-甘露糖苷酶 + Jurkat細(xì)胞�。� 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》2006年博士論文

【摘要】： 蛋白質(zhì)糖基化在細(xì)胞的功能行為中起重要的作用。本實(shí)驗(yàn)室以往克隆到了1個(gè)新的人α-甘露糖苷酶cDNA(6A8)，并觀察到了6A8α-甘露糖苷酶表達(dá)抑制影響包括Jurkat細(xì)胞在內(nèi)的多種細(xì)胞的生物學(xué)行為。在預(yù)實(shí)驗(yàn)中觀察到6A8α-甘露糖苷酶表達(dá)抑制的Jurkat細(xì)胞(AS)在培養(yǎng)中聚集成大的團(tuán)塊，這提示AS細(xì)胞間的粘附性增強(qiáng)。為研究其機(jī)制，首先用含有粘附分子和其它免疫分子基因在內(nèi)的440個(gè)基因的DNA芯片對(duì)AS和M細(xì)胞(轉(zhuǎn)導(dǎo)空載體的對(duì)照細(xì)胞)間的基因表達(dá)差異作了分析，見(jiàn)AS細(xì)胞有19個(gè)基因的表達(dá)上調(diào)，有20個(gè)基因的表達(dá)下調(diào)。表達(dá)上調(diào)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等與細(xì)胞間粘附相關(guān)的基因。RT-PCR和免疫熒光染色支持芯片的結(jié)果。這些基因中以CD54和CD11a最為重要。本論文工作中對(duì)它們作了重點(diǎn)研究。與對(duì)照細(xì)胞(野生型細(xì)胞W與M細(xì)胞)相比，AS細(xì)胞與包被有CD54分子的培養(yǎng)板的粘附增強(qiáng)。封閉性CD11a單抗能阻斷細(xì)胞與包被有CD54分子的培養(yǎng)板的粘附及細(xì)胞間的粘附。這證明了CD54和CD11a表達(dá)增強(qiáng)參與AS細(xì)胞間粘附增強(qiáng)。AS細(xì)胞LFA-1分子的親和力也增高。另外，AS細(xì)胞間的粘附增強(qiáng)也與細(xì)胞骨架排列改變相關(guān)。異種細(xì)胞間的粘附在免疫應(yīng)答等的生命活動(dòng)中也起重要的作用。在對(duì)AS細(xì)胞與對(duì)照細(xì)胞和Raji細(xì)胞間粘附的比較中，觀察到前者和Raji細(xì)胞間的粘附增強(qiáng)。在超抗原SEB存在下，Jurkat細(xì)胞與Raji細(xì)胞間可形成免疫突觸，并有信號(hào)傳導(dǎo)發(fā)生。6A8α-甘露糖苷酶的功能是修剪N-糖鏈中的甘露糖，Con A結(jié)合實(shí)驗(yàn)?zāi)軝z測(cè)細(xì)胞糖蛋白N-糖鏈中的甘露糖被α-甘露糖苷酶修剪的程度。與對(duì)照細(xì)胞相比，Con A與AS細(xì)胞的結(jié)合明顯增強(qiáng)。關(guān)于6A8α-甘露糖苷酶表達(dá)抑制所致的蛋白質(zhì)糖基化改變影響Jurkat細(xì)胞粘附性的確切機(jī)制須作深入研究。
[Abstract]:Glycosylation of proteins plays an important role in the functional behavior of cells. A new human 偽 -mannosidase cDNA (6A8) was cloned in our laboratory, and the inhibition of the expression of 6A8 偽 -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 偽 -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 偽 -L), CD54 (ICAM-1), CD82, CD24, CD11c (integrin 偽 -X), integrin 偽 -7CD103 (integrin 偽 -E), TNFSF9IL-1R, IL-2R 緯 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 偽 -mannosidase is to prune mannose Con A in N- sugar chain to detect the degree of mannose pruning by 偽-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 偽 -mannosidase expression inhibition on the adhesion of Jurkat cells needs further study.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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相關(guān)期刊論文 前2條

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本文編號(hào)：2073614