人Blys转染细胞对B细胞功能的影响

The construction of human B lymphocyte stimulator-transfected cells and effects of its signal on B lymphocytes

利用RT-PCR方法从经IFN-γ激发的正常人外周血单核细胞总RNA中扩增出全长BlyscDNA,继而将BlyscDNA插入真核表达载体逆转录病毒载体pSIV1中。以脂质体转染法将重组逆转录病毒载体与两个辅病毒载体共转染包装细胞293T,将含完整病毒颗粒的上清转染小鼠成纤维细胞L929,经G418筛选获得稳定表达Blys分子的L929细胞;RT-PCR和流式细胞术检测证实Blys分子在L929的稳定和高水平表达。继而以该转染细胞与PWM驱动的人扁桃体B细胞共培养,体外实验表明Blys介导的共刺激信号能促进该培养体系B淋巴细胞增殖,与抗μ抗体信号协同能刺激B细胞分泌IgG。

Human B lymphocyte stimulator (Blys) is a new member of TNF ligand family, which is an important costimulatory molecule for the physiobiology of B lymphocyte cells. By using RT-PCR, the full length gene coding for human Blys was cloned from IFN-γ stimulated-peripheral monocyte of healthy volunteer, and then inserted into retrovirus vector pSIV1. Through the means of lipofectamine, the recombinatecl vector was transferred to packing cell line 293T with two adjuvant virus vectors, and then the L929 cells was infected with the culture super-nant containing the intact virus particles. Fourthermore the L929 cell line with stable expression of Blys protein was obtained by G418 seletion, which was further confirmed by RT-PCR and FACS analysis. Our results evidenced that Blys protein expressed by Blys/L929 could promote the proliferation of the normal tonsil B lymphocyte, and enhance the IgG production by synergy with anti-μ antibody in the vitro culture system driven by PWM.