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THU0003 Altered gene expression in rheumatoid arthritis synovial fibroblasts after few culture passages
  1. U Müller-Ladner1,
  2. E Neumann1,
  3. M Judex1,
  4. F Kullmann1,
  5. J Grifka2,
  6. K Masuda3,
  7. S Gay3,
  8. J Schölmerich1
  1. 1Internal Medicine I
  2. 2Department Orthopedics, University Hospital, Regensburg, Germany
  3. 3Ctr Exp Rheum, Department Rheum, Zurich, Switzerland

Abstract

Objectives Inflammation, synovial hyperplasia and articular destruction are typical features of rheumatoid arthritis (RA) pathophysiology modulated by activated synovial fibroblasts. To evaluate distinct metabolic pathways and gene expression, for the majority of experimental settings cultured RA synovial fibroblasts derived from synovial tissue samples are used. To analyse, whether RA synovial fibroblasts can be cultured for an extended period of time without changes in their gene expression pattern, a combination of RNA arbitrarily primed PCR (RAP-PCR) and cDNA array was used.

Methods RA synovial fibroblasts of 2 patients with RA were isolated and cultured under standard conditions for up to 9 passages. RNA was extracted from these cells after each of the passages 2–9. RAP-PCR was performed and the PCR reactions were hybridised to cDNA expression array membranes (Atlas™, Clontech). For gene expression analysis, cDNA expression array membranes with 190 tumour suppressor genes and oncogenes and 9 control genes were used. The gene expression pattern of RA samples after each passage was compared to the pattern of the previous and following passages.

Results RA synovial fibroblasts showed a stable cDNA expression pattern for passages 2 to 4. After passages 5 to 6, expression pattern started to change (approximately 5% of the expressed genes). After passages 7 to 8, expression pattern of more than 5% of the 190 analysed tumour suppressor genes and oncogenes were altered. Of these, oncogene (e.g. bcl-1) and tumour suppressor-associated gene (e.g. EB-1 and CDK tyrosine kinase) expression had changed. Of interest, cell proliferation and growth decreased after passages 6 to 7 but distinct oncogenes and tumour-suppressor genes (e.g. jun-B and bcl-1) were still found to be upregulated.

Conclusion The results of this study reveal that gene expression of cultured RA synovial fibroblasts starts to alter after few passages indicating that future experimental settings need to address this potential problem for interpretation of gene expression. In addition, the data support the hypothesis that lack of downregulation of oncogenes and tumour suppressor genes might be responsible for the long-term aggressive growth of RA synovial fibroblasts observed in RA joints.

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