Li Ning-Li, Nie Hong, Yu Qi-Wen, Zhang Ji-Ying, Ma An-Lun, Shen Bai-Hua, Wang Li, Bai Jun, Chen Xue-Hua, Zhou Tong, Zhang Dong-Qing
Department of Immunology, Shanghai Second Medical University, Shanghai Institute of Immunology, Shanghai 200025, China.
World J Gastroenterol. 2004 Nov 1;10(21):3151-6. doi: 10.3748/wjg.v10.i21.3151.
To investigate the role of soluble Fas ligand in autoimmune diseases.
RT-PCR was performed to amplify sFasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. DNA fragments were cloned into PCR vector. After sequenced, sFasL gene fragments were inserted into pQE-31 vector and expressed in E. Coli M15 respectively. Proteins were purified through affinity chromatography column with ligand of 6XHis tag and identified by SDS-PAGE and Western blot. Mice were immunized with sFasL protein and specific anti-serum was harvested 6 wk after immunization. Monoclonal anti-human FasL antibody was made from the immunized mice. Serum level of sFasL in different patients was detected using anti-FasL antibodies from the immunized mice.
The protein expressed was 24 ku by SDS-PAGE electrophrosis. The protein was specially bound to anti-human FasL antibody by Western blot analysis. The sFasL protein could induce Jurket cell apoptosis in vitro. The concentration of serum sFasL in patients with autoimmune diseases was higher than that in normal individuals. sFasL could reduce arthritis in collagen induced arthritis (CIA) mice model by subcutaneous injection.
sFasL may be involved in either induction of apoptosis or autoimmune diseases. Furthermore, sFasL may have potential application in treatment of autoimmune diseases.
探讨可溶性Fas配体在自身免疫性疾病中的作用。
采用逆转录-聚合酶链反应(RT-PCR)从活化的人外周血淋巴细胞提取的总RNA中扩增可溶性Fas配体(sFasL)cDNA。将DNA片段克隆到PCR载体中。测序后,将sFasL基因片段分别插入pQE-31载体并在大肠杆菌M15中表达。通过6XHis标签配体亲和层析柱纯化蛋白,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法进行鉴定。用sFasL蛋白免疫小鼠,免疫6周后收集特异性抗血清。从免疫小鼠制备单克隆抗人FasL抗体。用免疫小鼠产生的抗FasL抗体检测不同患者血清中sFasL水平。
SDS-PAGE电泳显示表达的蛋白分子量为24 ku。蛋白质印迹分析表明该蛋白能与抗人FasL抗体特异性结合。sFasL蛋白在体外可诱导Jurket细胞凋亡。自身免疫性疾病患者血清sFasL浓度高于正常个体。皮下注射sFasL可减轻胶原诱导性关节炎(CIA)小鼠模型的关节炎症状。
sFasL可能参与细胞凋亡的诱导或自身免疫性疾病的发生。此外,sFasL在自身免疫性疾病治疗中可能具有潜在应用价值。