摘要
目的用前期构建好的CD59pSUPER-RNAi质粒转染Jurkat细胞,探讨CD59特异性沉默前后CD59对CD3介导Jurkat细胞活化信号转导的相关作用。方法采用Lipofectamine2000转染Jurkat细胞,经G418筛选建立稳定转染细胞系,利用RT-PCR检测转染后CD59在基因水平的表达抑制效果。实验分为未转染的Jurkat细胞组(A组),转染空质粒pSUPER组(B组)和转染CD59pSUPER-RNAi质粒的Jurkat细胞组(C组),用噻唑蓝(MTT)比色法,Western blot技术和激光共聚焦扫描显微镜分别检测交联CD59单抗与固相化CD3单抗联合作用对3组Jurkat细胞的增殖效应,ZAP-70酪氨酸磷酸化的水平及细胞浆内钙离子的变化。结果重组载体转染后,经G418筛选得到稳定转染细胞系,RT-PCR结果表明转染CD59pSUPER-RNAi质粒的Jurkat细胞组CD59分子的表达受到抑制。转染干扰质粒Jurkat细胞组CD59与CD3联合作用后细胞增殖能力,ZAP-70酪氨酸磷酸化水平及钙离子浓度均明显低于未转染组和转染空质粒组(P<0.05);但未转染组和转染空质粒组之间无差异。结论 CD59对CD3介导T细胞活化信号转导有增强效应。
In order to study the synergistic effects of glycosylphosphatidyl inositol（GPI）-anchored protein CD59 on CD3-mediated T cell signal transduction,human Jurkat cells were divided into 3 groups： Jurkat cells,blank plasmid-transfected Jurkat cells,and siRNA plasmid-transfected Jurkat cells.CD59 mRNA level was detected by RT-PCR.The cell proliferation activity of the three groups was measured by MTT colorimetry after immobilization with anti-CD3mAb and cross linkage with anti-CD59 mAb.The phosphorylation levels of ZAP-70 protein tyrosine were investigated by immunoblot analysis,and the kinetic changes of in T cell endochylema were determined by Laser scanning confocal microscope imaging.The results showed CD59 expression was successfully inhibited in siRNA plasmid-transfected group cells.The cell proliferation,phosphorylation levels of ZAP-70 and degree of in siRNA plasmid-transfected group cells were decreased compared with Jurkat cells and blank plasmid-transfected Jurkat cells（P 0.05）,but there was no difference between Jurkat cells and blank plasmid-transfected Jurkat cells.Our conclusion is CD59 can enhance the effects of CD3-mediated T cell signal transduction.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2011年第11期959-962,978,共5页
Immunological Journal
基金
国家自然科学基金资助项目(3170893)
