Differential effects of TNFalpha and IL1beta on FLS global gene expression profile

TNFalpha and IL1beta play a pathogenic role in rheumatoid arthritis. Both cytokines are known to activate cytokine and metalloproteinase secretion by synovial fibroblasts. In the present study, we wanted to investigate whether TNFalpha and IL1beta displayed differential effects on cultured Fibroblast-like Synovial Cells derived from RA patients. Global gene expression analyses indicated that both cytokines induced similar genes in these cells. Fibroblast-like cells (FLS) were purified from synovial biopsies from RA patients. Briefly, minced synovial fragments were digested in 1 mg/ml hyaluronidase solution (Sigma Aldrich, St Louis, MO) for 15 minutes at 37°C and 6 mg/ml collagenase type IV (Invitrogen, Paisley, UK) for 2 hours at 37°C. Next, cells were washed, resuspended in high-glucose Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 1% antibiotics-antimycotics (Invitrogen) and 1% MEM sodium pyruvate (Invitrogen), and seeded at 10,000 cells/cm2 in 6-well plates. When the cells reached confluence, adherent cells were detached using sterile 0.5% trypsin-EDTA (Invitrogen) and used as FLS between passages 3 and 9. Cells were seeded in 24-well plated at 25.000/well and incubated overnight with or without the following cytokines : TNF-α (R&D Systems, Minneapolis, MN) 10 ng/ml, IL-1β (R&D Systems) 10 ng/ml. After overnight incubation with the indicated cytokines, cells were harvested and total RNA was extracted using the Nucleospin® RNA II extraction kit (Macherey-Nagel, Düren, Germany), in order to be labeled according to a standard Affymetrix procedure and hybridized on HGU133 Plus 2.0 Human Genome slides.

肿瘤坏死因子-α（TNF-α）与白细胞介素-1β（IL-1β）在类风湿关节炎中发挥致病作用。已知两种细胞因子均可通过滑膜成纤维细胞激活细胞因子与金属蛋白酶的分泌。本研究旨在探究TNF-α与IL-1β对类风湿关节炎（RA）患者来源的培养成纤维样滑膜细胞（Fibroblast-like Synovial Cells, FLS）是否存在差异化调控效应。全局基因表达分析结果显示，两种细胞因子在该类细胞中诱导的基因表达谱较为相似。

本研究从RA患者的滑膜活检组织中纯化得到成纤维样细胞（FLS）。具体操作简述如下：将切碎的滑膜组织片段置于1 mg/ml透明质酸酶（Sigma Aldrich，美国密苏里州圣路易斯市）溶液中，于37℃消化15分钟，随后加入6 mg/ml IV型胶原酶（Invitrogen，英国佩斯利市），于37℃继续消化2小时。随后洗涤细胞，将其重悬于高糖杜氏改良伊格尔培养基（Dulbecco’s Modified Eagle Medium，Invitrogen）中，添加1%抗生素-抗真菌剂混合液（Invitrogen）及1% MEM丙酮酸钠（Invitrogen），以10,000细胞/cm²的密度接种于6孔培养板中。待细胞汇合后，使用无菌0.5%胰蛋白酶-乙二胺四乙酸（Invitrogen）消化贴壁细胞，取第3至9代细胞作为实验用FLS。

将细胞以25,000个/孔的密度接种于24孔培养板，过夜培养后，分别加入或不加入以下细胞因子：TNF-α（R&D Systems，美国明尼苏达州明尼阿波利斯市）10 ng/ml、IL-1β（R&D Systems）10 ng/ml。经上述细胞因子过夜处理后，收集细胞，使用Nucleospin® RNA II总RNA提取试剂盒（Macherey-Nagel，德国迪伦市）提取总RNA，随后按照标准Affymetrix实验流程进行标记，并在HGU133 Plus 2.0人类基因组芯片上进行杂交。