【作者】 李晓军； Kikumi Hata； 秦浚川； Junichiro Mizuguchi；

【Author】 LI Xiao-jun, QIN Jun-chuan (State Key Laboratory of Pharmacentical Biotechnology, Nanjing University, Nanjing 210093, China)

【机构】 南京军区南京总医院全军医学检验中心； 东京医科大学免疫系； 南京大学医药生物技术国家重点实验室；

【摘要】 目的研究CD40配体(CD40L)对小鼠WEHI-231 B淋巴瘤细胞分化抑制因子3(Id3)表达的影响。方法用RT-PCR技术从WEHI-231细胞总RNA中扩增小鼠Id3(mId3)cDNA,将其亚克隆至p3FLAG-CMV-10载体;以p3FLAG-Id3-CMV-10为模板,用PCR扩增3FLAG-Id3融合基因,将其亚克隆至逆转录病毒载体pMX-CITE/IRES-EGFP中;通过脂质体转染技术将重组逆转录病毒载体(pMX-3FLAG-Id3-EGFP)转染Phoenix-ecotropic包装细胞系;将pMX-3FLAG-Id3-EGFP逆转录病毒转导WEHI-231细胞,转导48 h后的WEHI-231细胞与CD40L/NIH3T3和NIH3T3细胞共培养24-48 h。转导细胞经有限稀释法建立mld3基因稳定转染细胞系。mId3基因稳定转染细胞与CD40L/NIH3T3和NIH3T3细胞共培养24h后用Western blot法检测3FLAG-mId3融合蛋白的表达。结果pMX-3FLAG-Id3-EGFP转导WEHI-231细胞后,EGFP阳性细胞比例随时间呈下降趋势;单独用培养基培养或与NIH3T3细胞共培养的pMX-3FLAG-Id3-EGFP/WEHI-231细胞,24 h-48 h后,EGFP阳性细胞率呈逐渐下降趋势;而与CD40L/NIH3T3细胞共培养的pMX-3FLAG-Id3-EGFP/WEHI 231细胞,EGFP阳性细胞率基本保持不变。稳定表达3FLAG-Id3融合蛋白的WEHI-231细胞与CD40L/NIH3T3和NIH3T3细胞共培养24 h后,Western blot结果显示,CD40L刺激可明显抑制3FLAG-Id3-EGFP/WEHI-231中Id3的表达。结论CD40L可能通过对Id3的降解而缓解Id3诱导的细胞生长抑制作用。更多还原

【Abstract】 Objective To study mouse Id3 (mId3) protein expression in WEHI-231 B lymphoma cells after CD40 ligand (CD40L) treatment. Methods The mId3 cDNA was amplified from total RNA of WEHI-231 cells by RT-PCR and introduced into the p3FLAG-CMV-10 vector. The 3FLAG-W3 fusion gene was amplified from p3FLAG-Id3-CMV-10 vector and subcloned into retroviral vector pMX-CITE/IRES-EGFP. Recombinant retrovirus was produced by transient transfection of the Phoenix-ecotropic packaging cell line with pMX-3FLAG-Id3-EGFP vector using FuGene-6 transfection reagent. WEHI-231 cells were transduced with recombinant retrovirus. EGFP expression was monitored by flow cytometry. Two cell lines stably expressing 3FLAG-Id3 fusion protein were generated. Results The proportion of EGFP-positive cells in the population transduced with pMX-3FLAG-EGFP remained constant up to 2 days after transduction, whereas the proportion in the population of pMX-3FLAG-Id3-EGFP-transduced cells decreased with time. After coculturing with NIH3T3 cells or medium alone, the proportion of EGFP-positive cells in pMX-3FLAG-Id3-EGFP-transduced WEHI-231 cells decreased with time, whereas after coculturing with CD40L-NIH3T3 cells, the proportion of EGFP-positive cells in the transduced cells remained constant up to 2 days, suggesting that CD40L could rescue WEHI-231 from Id3 overexpression-induced growth arrest. After coculturing with CD40L/NIH3T3 or NIH3T3 cells, 3FLAG-Id3 fusion protein stably expressing WEHI-231 cells were harvested, lysed, and immunoblotted by using anti-FLAG antibody. The results showed that treatment via CD40 ligation induced significant downregulation of Id3 expression in pMX-3FLAG-Id3-EGFPtransduced WEHI 231 cells. Conclusions更多还原