摘要
目的前期构建的真核表达载体pSUPER-CD3ζRNAi和pSUPER-CD59RNAi在Jurkat细胞中稳定表达,研究CD59与CD3分子在T细胞活化信号转导中的相互作用,进一步探讨CD59向T细胞内传递信号的相关机制。方法酶切鉴定构建的pSUPER-CD3ζRNAi和pSUPER-CD59RNAi;脂质体法将两种载体分别转染Jur-kat细胞,经G418筛选建立稳定转染细胞系;RT-PCR技术检测转染后CD59和CD3ζ在mRNA水平的表达抑制效果;采用噻唑蓝比色法检测转染前后各组Jurkat细胞的增殖效应,Western Blot技术检测各组细胞ZAP-70酪氨酸磷酸化的水平,激光共聚焦扫描显微镜检测各组细胞内Ca2+变化,ELISA法检测细胞上清液中白细胞介素2(IL-2)的变化。结果重组载体转染后RT-PCR结果表明,转染pSUPER-CD3ζRNAi和pSUPER-CD59RNAi质粒的细胞组CD3ζ分子和CD59分子的表达分别受到抑制。转染干扰质粒的细胞组细胞增殖能力、ZAP-70酪氨酸磷酸化水平、Ca2+浓度及IL-2水平均明显低于未转染组和空质粒组,差异有显著性(F=96.13～328.60,q=6.27～32.664,P<0.01)。结论 CD59和CD3在T细胞活化信号转导中具有协同作用。
Objective To study the synergistic effect of CD59 and CD3 in T cell activation signal transduction, and fur- ther explore the mechanism concerning CD59 transmitting signals to T cell. Methods The eukaryotie expression vector contai- ning CD3ξRNAi and CD59RNAi was identified by enzyme digestion and transfeeted into Jurkat cells by liposome, while steady cells cloned could get through G418 screening. CD59 and CDSξ mRNA level were detected by RT-PCR. The cell proliferation activity was measured by MTT colorimetry after anti-CD3 mAb and anti-CD59 mAb. The phosphorylation levels of ZAP-70 protein tyro- sine were investigated by immunoblot analysis, the kinetic changes of [Ca2＋ ] in T cell were determined by laser scanning eonfocal microscope imaging, while interleukin 2 （IL-2） in cell supernatants fluid was detected by ELISA, Results CD59 and CDSIξ ex- pression was successfully inhibited in transfeeted with siRNA plasmid group cells. The cell proliferation, phosphorylation levels of ZAP-70, degree of [Ca2＋ ] and IL-2 in transfeeted siRNA plasmid group cells were decreased compared with non-transfeeted and Jurkat cells after anti-CD3 mAb and anti-CD59 mAb, the differences were statistically significant （F： 96.13-328.60,q= 6.27- 32. 664,P〈0.01）. Conclusion There is a synergistic effect of CD59 and CD3 in T cell activation signal transduction.
出处
《青岛大学医学院学报》
CAS
2012年第4期287-290,共4页
Acta Academiae Medicinae Qingdao Universitatis
基金
国家自然科学基金资助项目(30972677)
