Medizinische Klinik

, Volume 94, Issue 4, pp 228–232 | Cite as

Analyse des Genexpressionsmusters von synovialen Fibroblasten von Patienten mit rheumatoider Arthritis mittels RAP-PCR Differential Display

  • Ulf Müller-Ladner
  • Martin Judex
  • Hans-Peter Jüsten
  • Dieter Wessinghage
  • John Welsh
  • Michael McClelland
  • Steffen Gay
  • Jürgen Schölmerich
  • Frank Kullmann
Article

Zusammenfassung

□ Fragestellung

Im rheumatischen Gelenk finden sich transformiert erscheinende Fibroblasten an der Invasionszone in den Knorpel und Knochen. Über die Faktoren, die zu diesem aggressiven Phänotyp führen, ist bisher wenig bekannt. Ziel der Untersuchungen war daher, mittels RAP-PCR (RNA arbitrarily primed PCR) zwischen rheumatoiden Fibroblasten und Osteoarthritis-Fibroblasten differentiell exprimierte Gene zu erfassen.

□ Methodik

Die Gesamt-RNS von synovialen Fibroblasten wurde im ersten Schritt der RAP-PCR mittels arbiträren Zehn- bis Zwölf-Basen-Primern in cDNS überschrieben. Die Amplifikation des zweiten Schrittes erfolgte dann ebenfalls mittels Zehn- bis Zwölf-Basen-Primern unter niedrigstringenten Bedingungen für insgesamt 35 Zyklen, gefolgt von elektrophoretischer Auftrennung, Klonierung und Sequenzanalyse der differentiell exprimierten RAP-PCR-Produkte.

□ Ergebnisse

Im Durchschnitt konnten je Primerpaar ca. 70 verschiedene RNS amplifiziert werden, wobei das Expressionsmuster der RA- und OA-Fibroblasten eine hohe Homologie aufwies. Insgesamt konnten aus den mit 26 verschiedenen Primerkombinationen durchgeführten RAP-PCR Differential Displays zwölf sehr stark differentiell exprimierte RNS nachgewiesen werden. Die Sequenzanalysen der hierbei in den rheumatoiden Fibroblasten hochregulierten und bisher identifizierten Gensegmenten zeigten sehr große Homologien zu verschiedenen in den Zellmetabolismus involvierten Gene.

□ Schlußfolgerung

Die Untersuchungen zeigen, daß in rheumatoiden synovialen Fibroblasten mittels RAP-PCR Differential Display spezifisch exprimierte Gene identifiziert werden können, die möglicherweise eine wichtige Rolle in der Pathophysiologie dieser Erkrankung spielen.

Schlüsselwörter

Rheumatoide Arthritis Synoviale Fibroblasten Differential Display Osteoarthritis 

Analysis of gene expression patterns in rheumatoid synovial fibroblasts using RAP-PCR for differential display

Abstract

□ Objective

Destruction of articular cartilage and bone by invading synovial fibroblasts is a typical histopathologic feature in rheumatoid arthritis (RA). However, little is known about specific up- or downregulation of genes leading to this aggressive phenotype. Thus, our aim was to identify genes, which are differentially expressed in RA synovial fibroblasts as compared to synovial fibroblasts derived from patients with osteoarthritis (OA) using RAP-PCR for differential display.

□ Methods

After extraction of total RNA, the first step of RAP-PCR was performed using various different arbitrary 10–12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stringency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplified fingerprint products.

□ Results

On average, approximately 70 different RNAs were obtained per primer, of which most were expressed both by RA and OA synovial fibroblasts. Using 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong amplification of distinct PCR products suitable for sequencing could be observed. Sequence analysis identified these PCR products as highly homologous to various genes involved in regulation of cell cycle and metabolism.

□ Conclusion

The data indicate that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts potentially involved in the specific pathophysiology of RA.

Key Words

Rheumatoid arthritis Synovial fibroblasts Differential display Osteoarthritis 

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Literatur

  1. 1.
    Aicher WK, Heer AH, Trabandt A, et al. Overexpression of zinc-finger transcription factor Z-225/egr-1 in synoviocytes from rheumatoid arthritis patients. J Immunol 1994;152:5940–8.PubMedGoogle Scholar
  2. 2.
    Buckland R, Malvoisin E, Beauverger P, Wild F. A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion. J Gen Virol 1992;73:1703–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Chomcynski P, Sacchi N. Single-step method of RNA isolation by acid guadinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9.Google Scholar
  4. 4.
    Fassbender HG. Histomorphologic basis of articular cartilage destruction in rheumatoid arthritis. Coll Relat Res 1983;3:141–55.PubMedGoogle Scholar
  5. 5.
    Gay S, Gay RE, Koopman WJ. Molecular and cellular mechanisms of joint destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction? Ann Rheum Dis 1993;52:S39–47.CrossRefGoogle Scholar
  6. 6.
    Jung B, Vogt T, Mathieu-Daudé F, et al. Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment. Carcinogenesis 1998;19:1901–6.PubMedCrossRefGoogle Scholar
  7. 7.
    Kalden JR, Gay S. Retroviruses and autoimmune rheumatic diseases. Clin Exp Immunol 1994;98:1–5.PubMedCrossRefGoogle Scholar
  8. 8.
    Kriegsmann J, Keyszer GM, Geiler T, Bräuer R, Gay RE, Gay S. Expression of vascular cell adhesion molecule-1 mRNA and protein in rheumatoid arthritis synovium demonstrated by in situ hybridization and immunohistochemistry. Lab Invest 1995;72:209–13.PubMedGoogle Scholar
  9. 9.
    Landschulz WH, Johnson PF, Adashi E, Graves BJ, McKnight SL. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev 1988;2:786–800.PubMedCrossRefGoogle Scholar
  10. 10.
    Liang PL, Averboukh L, Keyomarsi K, Sager R, Pardee AB. Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells. Cancer Res 1992;52:6966–8.PubMedGoogle Scholar
  11. 11.
    Liang PL, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992;257:967–71.PubMedCrossRefGoogle Scholar
  12. 12.
    Liang PL, Pardee AB. Differential display. Materials and methods. Totowa: Humana Press, 1997.CrossRefGoogle Scholar
  13. 13.
    McClelland M, Mathieu-Daudé F, Welsh J. RNA finger-printing and differential display using arbitrarily primed PCR. Trends Genet 1995;11:242–5.PubMedCrossRefGoogle Scholar
  14. 14.
    Müller-Ladner U. T cell independent pathways in rheumatoid arthritis. Curr Opin Rheumatol 1995;7:222–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Müller-Ladner U, Kriegsmann J, Gay RE, Gay S. Oncogenes in rheumatoid arthritis. Rheum Dis Clin N Am 1995;21:675–90.Google Scholar
  16. 16.
    Rattner JB, Rees J, Arnett FC, Reveille JD, Goldstein R, Fritzler MJ. The centromere kinesin-like protein CENP-E. An autoantigen in systemic sclerosis. Arthritis Rheum 1996;39:1355–61.PubMedCrossRefGoogle Scholar
  17. 17.
    Shinoura N, Shamraj OI, Hugenholz H, et al. Identification and partial sequence of a cDNA that is differentially expressed in human brain tumors. Cancer Lett 1995; 89:215–20.PubMedCrossRefGoogle Scholar
  18. 18.
    Sullivan BA, Schwartz S. Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum Mol Genet 1995;4:2189–97.PubMedCrossRefGoogle Scholar
  19. 19.
    Trabandt A, Aicher WK, Gay RE, Sukhatme VP, Fassbender HG, Gay S. Spontaneous expression of immediately-early response genes c-fos and egr-1 in collagenase-producing rheumatoid synovial fibroblasts. Rheumatol Int 1992;12:53–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Trabandt A, Gay RE, Gay S. Oncogene activation in rheumatoid synovium. APMIS 1992;100:861–75.PubMedCrossRefGoogle Scholar
  21. 21.
    Tucker S, Srinivas R, Copmans R. Molecular domains involved in oligomerization of the Friend leukemia virus envelope protein. Virology 1991;16:2431–44.Google Scholar
  22. 22.
    Vogt TM, Welsh J, Stolz W, et al. RNA fingerprinting displays UVB-specific disruption of transcriptional control in human melanocytes. Cancer Res 1997;57: 3554–61.PubMedGoogle Scholar
  23. 23.
    Welsh J, Chada K, Dalal SS, Cheng R, Ralph D, McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res 1992;20:4965–70.PubMedCrossRefGoogle Scholar
  24. 24.
    Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 1990; 18:7213–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Wilkinson LS, Edward JC, Poston RN, Haskard DO. Expression of vascular cell adhesion molecule-1 in normal and inflamed synovium. Lab Invest 1993;68:82–8.PubMedGoogle Scholar
  26. 26.
    Yeatman TJ, Mao W. Identification of a differentially expressed message associated with colon cancer liver metastasis using an improved method of differential display. Nucleic Acids Res 1995;19:4007–8.CrossRefGoogle Scholar
  27. 27.
    Zvaifler NJ, Firestein GS. Pannus and pannocytes. Alternative models of joint destruction. Arthritis Rheum 1994;37:783–9.PubMedCrossRefGoogle Scholar

Copyright information

© Urban & Vogel 1999

Authors and Affiliations

  • Ulf Müller-Ladner
    • 1
  • Martin Judex
    • 1
  • Hans-Peter Jüsten
    • 2
  • Dieter Wessinghage
    • 2
  • John Welsh
    • 3
  • Michael McClelland
    • 3
  • Steffen Gay
    • 4
  • Jürgen Schölmerich
    • 1
  • Frank Kullmann
    • 1
  1. 1.Klinik und Poliklinik für Innere Medizin I der UniversitätRegensburg
  2. 2.Klinik und Poliklinik für Orthopädie der Universität RegensburgRegensburgDeutschland
  3. 3.Sidney Kimmel Cancer CenterSan DiegoUSA
  4. 4.Zentrum für Experimentelle RheumatologieDepartment für Rheumatologie des Universitätsspitals ZürichSchweiz

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