摘要
目的 观察阻抑核心岩藻糖基化修饰对前B细胞信号传导的调节作用.方法 利用逆转录病毒包装技术建立核心岩藻糖转移酶Fut8基因沉默前B细胞株（70Z/3-Fut8-RNAi细胞）.用实时定量聚合酶链反应（Real-time PCR）及Western blot检测Fur8 mRNA、蛋白表达和细胞内信号分子的酪氨酸磷酸化水平.结果 成功构建重组pSINsi-hU6-Fut8 siRNA质粒,逆转录病毒包装后病毒滴度可达2.1×10^5 CFU/ ml.Real-time PCR及Western blot检测结果表明70Z/3-Fut8-RNAi细胞的Fut8基因和蛋白表达明显下降,蛋白表达水平下调70％ - 80％,mRNA水平下调70％ -80％,以及pre-BCR介导的细胞信号传导,即CD79a及BTK磷酸化显著下降.结论 成功构建70Z/3-Fut8-RNAi细胞株,Fut8修饰的核心岩藻糖基化可能在早期B细胞的细胞信号传导过程中发挥重要的调节作用.
Objective To investigate the function of core fucosyltransferase Fut8 in the intracellular signal transduction in mice pre-B cells.Methods Using recombinant DNA techniques,complementary 82 bp oligonucleotides designed with hairpin loop for Fut8 siRNA were annealed,and then inserted into the retrovirus expression vector pSINsi-hU6 with BamH Ⅰ and Cla Ⅰ sites.The recombinant retroviral vector pSINsi-hU6-Fut8 siRNA was packaged with 293T cells and the generated recombinant retrovirus infected mice pre B cell line （70Z/3 cells）.The expressions of Fut8 mRNA,protein and enzyme activity were detected by Real-time polymerase chain reaction （Real-time PCR）,Western blotting and high performance liquid chromatography （HPLC）.The phosphorylation of cell signal molecules were detected by Western blotting.Results The restriction enzyme analysis with BamH Ⅰand Cla Ⅰshowed that the recombinant retroviral vector pSINsi-hU6-Fut8 small interference RNA siRNAwas constructed correctly.The titer of generated recombinant retrovirus was up to 2.1 × 10^5 CFU/ml.The Fut8 mRNA,protein and enzyme activity were significantly down-regulated in Fut8-KD-70Z/3 cells.The pre-BCR-mediated tyrosine-phosphorylation of CD79a and activation of BTK were suppressed in Fut8-KD-70Z/3 cells.Conclusion The Fut8-KD-70Z/3 cells transfected with the pSINsi-hU6-Fut8 siRNA plasmid were established successfully.Core fucosylation mediates the intracellular signaling via pre-BCR in pre-B cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第11期2402-2404,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30972675、31270864）
大连市科技局计划启动项目（2010J21DW011）
