过表达C-Src激酶结合蛋白诱导Jurkat淋巴细胞表型改变和凋亡  

Phenotypic changes and apoptosis of Jurkat cells induced by over-expression of C-Src kinase-binding protein

作　　者：高美华[1] 丛蓓蓓[1] 王冰[1] 王丽娜[1] 邵志伟[2] 

机构地区：[1]青岛大学医学院免疫教研室,山东青岛266071 [2]青岛大学医学院神经外科,山东青岛266071

出　　处：《细胞与分子免疫学杂志》2015年第6期749-752,757,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81273206);青岛市公共领域科技支撑计划项目[12-1-3-5-C-(2)-n Sh]

摘　　要：目的研究上调C-Src激酶结合蛋白(CBP)表达对Jurkat细胞超微结构及相关生物学功能的影响。方法构建CBP-EGFP融合蛋白过表达病毒载体,转染建立稳定的高表达CBP-EGFP融合蛋白的Jurkat细胞株。光学显微镜下观察细胞的基础生长状态,共聚焦显微镜观察病毒转染效率及CBP蛋白在细胞上的定位,电镜观察细胞内细胞器的复杂程度。流式细胞术检测细胞的基本参数及凋亡情况,实时定量PCR(qRT-PCR)检测CBP基因及信号转导通路中C-Src激酶(CSK)、原癌基因蛋白Vav(Vav oncoprotein)mRNA水平。结果光镜下发现转染CBP-EGFP病毒的Jurkat细胞皱缩细胞增多,大小均一性较差。共聚焦显微镜显示细胞转染效率达到100%,同时发现CBP定位于细胞膜上。电镜可见CBP组细胞内细胞器较Jurkat细胞复杂,有线粒体,并出现大量的溶酶体样结构。与阴性组相比,转染了CBP-EGFP病毒后,死亡细胞的数量大大增加,而qRT-PCR结果显示CBP组CSK表达上调,Vav则表达下调。结论在Jurkat细胞中,CBP蛋白高表达可以使细胞的均一性下降,凋亡细胞增多。Objective To explore the effects of up-regulated expression of C-Src kinase-binding protein （CBP） on biological functions and cell ultrastructure in Jurkat cells. Methods We constructed the CBP-EGFP viral vector and established a stably transfected Jurkat cell line with it. Cell growing status was observed under a light microscope. The transfection efficiency and the location of CBP were examined with confocal microscopy. The complexity of cell organelles was observed by electron microscopy. Apoptotic cells were detected by flow cytometry. The expressions of C-Src kinase （CSK） and Vav oncoprotein in CBP signaling pathway were measured by real-time quantitative PCR （qRT-PCR）. Resalts We found more pyknotic cells and less homogeneity in the transfected Jurkat cells under light microscope. Confocal microscopy revealed that cell transfection efficiency was up to 100% and CBP was located on the cell membrane. Moreover, there were more complex organelles in CBP-EGFP transfected Jurkat cells with a lot of mitochondrions and the lysosome-like clumps accumulation. Detection of apoptosis showed that CBP-EGFP transfected Jurkat cells had more necrotic cells compared with the negative group. Finally, qRT-PCR indicated the expression of CSK increased and Vav decreased in the CBP-EGFP transfected group. Conclusion Up-regulated expression of CBP in Jurkat cells could reduce cell homogeneity and promote cell apoptosis.

关 键 词：C-Src激酶结合蛋白 细胞器 信号转导 

分 类 号：R392.11[医药卫生—免疫学] Q255[医药卫生—基础医学]