Key regulatory molecules of cartilage destruction in rheumatoid arthritis:: an in vitro study
Key regulatory molecules of cartilage destruction in rheumatoid arthritis:: an in vitro study
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DOI：
10.1186/ar2358
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发表时间：
2008-01-01
影响因子：
4.9
通讯作者：
Sittinger, Michael
中科院分区：
文献类型：
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作者：
Andreas, Kristin;Luebke, Carsten;Sittinger, Michael
背景类风湿性关节炎（RA）是一种慢性炎症性全身性自身免疫性疾病，会导致进行性软骨破坏。 RA 相关软骨破坏的治疗进展需要深入了解软骨退化的分子机制。到目前为止，有关软骨细胞分子 RA 相关功能障碍的综合数据还很有限。因此，本研究的目的是建立一个标准化的体外模型，以分析人软骨细胞表达的与 RA 相关的软骨破坏的关键调节分子。方法将人软骨细胞在藻酸盐珠中三维培养 14 天，随后用 SV40 T 抗原永生化人滑膜的上清液刺激 48 小时。 成纤维细胞 (SF) 分别来自正常供体 (NDSF) 和 RA 患者 (RASF)。为了鉴定 SF 释放的 RA 相关因子，使用基于抗体的蛋白质膜阵列分析 RASF 和 NDSF 的上清液。受刺激的软骨样培养物用于随后通过寡核苷酸微阵列进行基因表达谱分析。使用 Affymetrix GeneChip 操作软件和稳健多阵列分析 (RMA) 来鉴定差异表达基因。通过实时 RT-PCR 验证所选基因。结果 基于抗体的滑膜成纤维细胞上清液蛋白膜阵列鉴定出 RASF 释放的 RA 相关可溶性介质（IL-6、CCL2、CXCL1-3、CXCL8）。 RASF 刺激的软骨细胞的全基因组微阵列分析揭示了与软骨破坏相关的独特表达谱，涉及炎症标记基因（腺苷 A2A 受体、环氧合酶-2）、NF-κ B 信号通路（Toll 样受体 2、精胺合酶、受体相互作用丝氨酸-苏氨酸激酶 2）、 细胞因子/趋化因子和受体（CXCL1-3、CXCL8、CCL20、CXCR4、IL-1β、IL-6）、软骨降解（基质金属蛋白酶 (MMP)-10、MMP-12）和抑制基质合成（软骨寡聚基质蛋白、硫酸软骨素蛋白聚糖 2）。 结论 受刺激的人软骨细胞的差异转录组分析揭示了软骨细胞功能的分解代谢-合成代谢稳态受到干扰，并揭示了软骨破坏的相关药理学靶基因。这项研究提供了对 RA 相关软骨破坏期间人类软骨细胞诱导的分子调节过程的全面见解。所建立的模型可作为RA相关软骨破坏的人类体外疾病模型，并可能有助于阐明抗风湿药物对人类软骨细胞基因表达的分子效应。
Background Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.Methods Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. of selected genes was verified by real-time RT-PCR.Results Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1-3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-kappa B signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1 beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).Conclusion Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of antirheumatic drugs on human chondrocyte gene expression.