6A8α-甘露糖苷酶表达抑制使Jurkat细胞间的黏附性增强  

Inhibition of 6A8α-mannosidase expression causes an enhancement of cell-cell adhesion among Jurkat cells

作　　者：曲莉[1] 向志光[1] 史耕先[1] 刘音[1] 崔巍[1] 杨海鹏[2] 朱立平[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所免疫学系 [2]北京大学首钢医院检验科

出　　处：《中华微生物学和免疫学杂志》2004年第6期456-461,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家 973重大基础研究项目 ( 2 0 0 1CB5 10 0 0 4) ;国家自然科学基金项目 ( 3 0 170 868)

摘　　要：目的　探讨 6A8α 甘露糖苷酶表达抑制对人T淋巴母细胞Jurkat生物学行为的影响。方法　采用反义核酸技术抑制细胞中 6A8α 甘露糖苷酶的表达。用PCR检测转导基因在基因组中的整合。用单抗 6A8作探针 ,Western印迹和免疫荧光染色流式细胞术检测细胞中 6A8α 甘露糖苷酶的表达状况。用ConA结合试验检测细胞蛋白质的糖基化改变。普通光学显微镜观察细胞的形态。用DNA芯片技术分析AS细胞和M细胞mRNA表达的差异。用RT PCR和免疫荧光染色流式细胞术验证基因芯片结果的可靠性。结果　制备了 6A8α 甘露糖苷酶表达抑制的Jurkat细胞 (AS)。与对照细胞 (野生型细胞W和转导空载体的细胞M)相比 ,AS细胞在培养中黏附成大团块。DNA芯片分析见与M细胞相比 ,AS细胞中有 19个基因的表达上调 ,2 0个基因的表达下调。上调的基因中有一些与细胞间黏附相关 ,如CD5 4、CD11a、CD2 4、CD82、整合素αX、整合素α7、整合素αE、IL 1R、IL 2Rγ和TNFSF9。RT PCR肯定了DNA芯片分析的可靠性。免疫荧光染色流式细胞术进一步肯定了CD5 4和CD11a在AS细胞表达的上调。结论　 6A8α 甘露糖苷酶表达抑制使Jurkat细胞间黏附增强 ,CD5 4和CD11a等黏附分子表达的增高可能与此相关。Objective To study the effect of inhibition of 6A8α-mannosidase expression on the biological behaviors of human T lymphoblastoid cell Jurkat. Methods Antisense technique was used to suppress 6A8α-mannosidase expression in cells. Integration of the transduced gene was detected by genomic PCR. Reduction of 6A8α-mannosidase expression was examined by Western blot and immunofluorescence staining using McAb 6A8 as a probe. Con A binding assay was carried out to detect the modification of protein glycosylation. The morphology of cells in culture was examined upon a microscope. Differential display of genes between AS and M cell was analyzed by means of DNA microarray. The differential display was confirmed by RT-PCR and immunofluorescence staining. Results Jurkat cell with reduction of 6A8α-mannosidase expression (AS) was developed. In comparison to the controls, the wild type cell (W) and the cell transduced with the mock (M), AS cells formed larger aggregates in culture. DNA microarray analysis on a DNA chip involving the known immune-related genes revealed 19 up-regulated and 20 down-regulated genes in AS cell in comparison to M cells. Several genes related to cell-cell adhesion, such as CD54, CD11a, CD24, CD82, integrin αX, integrin α7, integrin αE, IL-1R1, IL-2Rγ and TNFSF9 were up-regulated. RT-PCR confirmed the datasets of the DNA microarray analysis. Flowcytometry was used to further confirm the up-regulation of CD54 and CD11a. Conclusion Inhibition of 6A8α-mannosidase gene expression caused enhancement of cell-cell adhesion among Jurkat cells, which might be related to the enhanced expression of CD54, CD11a and other adhesion molecules.

关 键 词：6A8α-甘露糖甘酶 表达抑制 JURKAT细胞 黏附性 生物学 糖基化 

分 类 号：R392[医药卫生—免疫学]