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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 investigate the effects of up-regulation of C-Src kinase-binding protein (CBP) expression on the ultrastructure and related biological functions of Jurkat cells. Methods CBV-EGFP fusion protein overexpression vector was constructed and transfected into Jurkat cell line which was stable and highly expressed CBP-EGFP fusion protein. Under the light microscope, the basic growth state of the cells was observed. The transfection efficiency of the virus and the localization of the CBP protein on the cells were observed with a confocal microscope. The complexity of intracellular organelles was observed under an electron microscope. The basic parameters and apoptosis of cells were detected by flow cytometry. The levels of C-Src kinase (CSK) and Vav oncoprotein mRNA in CBP and signal transduction pathways were detected by real-time quantitative PCR (qRT-PCR) . Results Under the light microscope, Jurkat cells transfected with CBP-EGFP virus showed an increase in shrinkage cells with poor uniformity of size. Confocal microscopy showed that the transfection efficiency reached 100%, and CBP was found to be located on the cell membrane. Electron microscopy showed that intracellular organelles in CBP group were more complex than Jurkat cells, with mitochondria and a large number of lysosome-like structures. Compared with the negative group, the number of dead cells was greatly increased after transfection with CBP-EGFP virus, while qRT-PCR results showed that the expression of CSK was up-regulated and Vav was down-regulated in CBP group. Conclusion In Jurkat cells, high expression of CBP protein can decrease the cell homogeneity and increase the number of apoptotic cells.