Abstracts (complete list) - Wissenschaft Online

Nadja Hilger, Frank Emmrich, Ulrich Sack
Gene expression in microdissected invasive fibroblast isolated
from arthritic joints from patients with RA
The etiology of Rheumatoid Arthritis remains incompletely understood. Inflammatory
fibroblasts (FB) are necessary for joint destruction and the propagation of inflammation
while cytokines and their receptors play an important role in the pathogenesis, namely
in the intercourse of FB with inflammatory and lymphoid cells and in the destruction of
cartilage and bone.
Studies of invasive FB isolated from homogenized synovial membrane failed to provide
detailed insight into the characteristics of erosive FB at the site of cartilage and bone
destruction. By modifying the latest microdissection technologies, we have analysed
small cell groups from destructive regions in biopsies of patients and compared them
with FB from non-arthritic regions. In this way, differential expression of cytokines and
further candidate genes can be investigated. So we investigated to what extent an
investigation of mRNA from few cells is possible and whether RNA amplification should
be attached.
Cells were isolated by laser capture microdissection. High laser intensity and specialized
slides were found to be pre-requisites for successful isolation of small cellular units.
After optimisation of the different methods, RNA of sequential cell numbers starting with
1000 cells was isolated. One part was reverse transcribed and the other fraction was
amplified. In order to examine differences, expression of different genes was examined
and compared in both preparations.
We were able to dissect small cell groups out of erosive sites and non-inflammed parts
of synovial tissues from RA patients. We showed that RNA isolation from very small cell
numbers with following expression analysis is possible. The house keeping gene and a
FB-specific genes could be detected in all samples without amplification. The
quantification of cytokine expression was limited.
We could show that through our approach an optimized investigation of RNA from few
cells derived from RA synovial tissue is possible. By identifying function-specific
markers, erosive FB will be better accessible in future and can be investigated more
easily.