人ICOS基因转染细胞对抗体产生的影响

Effects of Human ICOS cDNA Transfected Cells on the Production of T-Lympho-cyte Dependent Antibodies

利用RT-PCR方法从扁桃体中激活的T细胞,mRNA中扩增出ICOS cDNA,继而将ICOS基因插入到逆转录病毒载体pGEZ-Term中,用脂质体法将重组逆转录病毒载体与两个辅助病毒载体共同转染包装细胞293T,用含有完整病毒颗粒的293T细胞的培养上清转染L929细胞,经Zeocin筛选获得稳定表达ICOS蛋白的L929细胞株;经RT-PCR、流式细胞仪表型检测证实L929基因转染细胞能稳定表达人ICOS蛋白。利用ICOS和CD40L基因转染细胞联合IL-10以及PWM驱动的B细胞体外培养系统,分析了ICOS在B细胞生长及抗体产生中的作用,实验结果表明,ICOS在PWM体外培养体系中,能有效地促进B细胞生长以及协同IL-10增加IgG的分泌。

Human ICOS (inducible costimulator ) is a new member of the CD28/B7 family and differs from CD28 molecules in molecular structures, expression pattern and functions. It was cloned from activated T cells of human tonsils by RT-PCR and then inserted into retrovirus vector pGEZ-Term. By means of lipofectin, the recombination was transferred into packing cell line 293T with two adjuvant virus vectors, and the L929 cells were transfected with culture supernatant from 293T cells containing the intact virus particles. A L929 cell line with stable expression of ICOS protein was obtained by Zeocin selection, and this was further confirmed by RT-PCR and FACS testing. The effects of the ICOS protein on the proliferation of B cells and the production of antibodies were analyzed by means of the in vitro cultivation system of B cells driven by ICOS and CD40L transfected cells in combination with IL-10 and PWM. The results showed thai ICOS could improve the growth of B cells efficiently in the in vitro PWM cultivation system and could increase the secretion of IgG in the presence of IL-10.