Abstract
Introduction Aberrations in the balance of chondrocyte metabolism play an integral role in the degeneration of articular cartilage and subsequent arthritis. Gene expression profiling is a powerful tool which allows identification of differences in levels of mRNA expression of large numbers of genes simultaneously. The objective of this study was to compare mRNA expression from osteoarthritic cartilage with that of normal cartilage and by use of the Affymetrix system, identify target genes for further investigation.
Methods Human cartilage samples were obtained from osteoarthritic knees and hips at the time of joint replacement surgery. Non-arthritic cartilage samples were obtained from notchplasty at time of cruciate ligament replacement surgery or from trauma surgery. Cartilage samples were either snap frozen in liquid nitrogen and RNA directly isolated from the frozen tissue or enzymatically digested and established in primary culture prior to RNA isolation. The RNA was reverse transcribed to cDNA, labelled with a fluorochrome and then hybridised to gene chips. This will allow us to: 1. Compare whether RNA expression in cell culture accurately reflects that in the tissue itself. 2. Determine whether there are differences between the gene profiles of knee and hip osteoarthritis. 3. Select candidate genes for further analysis.
Results At present primary cell culture lines have been successfully established and are ready for RNA isolation. Frozen cartilage samples have undergone RNA isolation. Currently techniques are underway to maximise RNA extraction and sufficiently purify it to process a gene chip. Once the gene chip is made a list of up or down-regulated genes will be available for analysis. Human articular cartilage lends itself to gene profiling using cDNA arrays as it contains only one cell type. Thus any changes in gene expression levels can be directly attributed to the chondrocyte.
Conclusions This technology opens the door to a new search for the ‘arthritis gene’.
In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source.
The abstracts were prepared by Mr Jerzy Sikorski. Correspondence should be addressed to him at the Australian Orthopaedic Association, Ground Floor, William Bland Centre, 229 Macquarie Street, Sydney NSW 2000, Australia.
