论文部分内容阅读
目的:研究糖基磷脂酰肌醇(GPI)锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法:应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER-siCD55,pSUPER-siCD59。实验分为未转染的Jurkat细胞组(Ⅰ组)、转染pSUPER空质粒的Jurkat细胞组(Ⅱ组)、转染pSUPER-siCD59重组质粒的Jurkat细胞组(Ⅲ组)及转染pSUPER-siCD55重组质粒的Jurkat细胞组(Ⅳ组)。RT-PCR检测转染细胞中CD55和CD59基因的表达噻唑蓝(MTT)比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化、结果:稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组(P<0.05),Ⅰ组和Ⅱ组之间无差异结论:CD59和CD55在T细胞活化信号转导通路中存在协同效应。
Objective: To study the synergistic effect of glycosylphosphatidylinositol (GPI) anchoring proteins CD59 and CD55 in lipid raft-mediated T cell signal transduction pathway. Methods: Recombinant vectors pSUPER-siCD55 and pSUPER-siCD59 were constructed by using siRNA technique to specifically target CD55 and CD59 genes. The experiment was divided into untransfected Jurkat cells (group Ⅰ), Jurkat cells transfected with pSUPER empty plasmid (group Ⅱ), Jurkat cells transfected with pSUPER-siCD59 recombinant plasmid (group Ⅲ) and pSUPER-siCD55 Recombinant plasmid Jurkat cell group (group Ⅳ). The expression of CD55 and CD59 in transfected cells was detected by RT-PCR. MTT colorimetric assay and confocal laser scanning microscopy were used to detect the proliferative effects of CD55 and CD59 on Jurkat cells and the changes of intracellular Ca2 + RESULTS: After stable transfection, the expression of CD59 in group Ⅲ and CD55 in group Ⅳ were successfully inhibited. The proliferation and calcium concentration of CD55 and CD59 cells in group Ⅰ and group Ⅱ were significantly higher than those in group Ⅲ and group Ⅳ (P <0.05). There was no difference between group Ⅰ and group Ⅱ: the expression of CD59 and CD55 in T cell There is a synergistic effect in activating signaling pathways.