Abstract
Introduction: The intervertebral disc consists of three regions; the nucleus pulposus and the inner and outer annulus contain cells with individual phenotypes. However, no molecular markers are known to discriminate between the various cell types. Molecular markers would help identify the cell types in development, ageing, localisation and pathological states. Here we reveal major differences between the cell types using SDS-PAGE and mass spectrometry, which will help to develop molecular markers.
Methods: Intervertebral discs were isolated from bovine tails and separated into three distinct regions; the nucleus, inner and outer annulus. Cells were separated by enzyme digestion overnight and then washed extensively. The cells were then separated into membrane and supernatant fractions followed by SDS-PAGE analysis; various fractions revealed bands of interest that were then cut from the gel, digested with trypsin and analysed by mass spectrometry.
Results: Analysis of the membrane and supernatant fractions of the cells by SDS-PAGE revealed unique protein patterns between the regions and fractions. A broad band from the membrane of nucleus cells was analysed by mass spectrometry and revealed strong matches for myosin and clathrin. A control band from the outer annulus also revealed a strong match for myosin. A strong band from the membrane fraction of the outer annulus revealed a mass spectrometry match to actin. Analysis of the corresponding supernatant fraction revealed a strong match to actin, whereas a band of similar molecular weight from the inner annulus revealed another myosin chain.
Conclusion: The differences revealed in the protein profile of cells from the three regions of disc and the identification of prominent proteins demonstrates that such differences can be used to identify molecular markers. Specific molecular markers for cells from different disc regions will aid in both understanding disc pathology and in development of new biological treatments.
The abstracts were prepared by Editorial Secretary, Dr Charles Pither. Correspondence should be addressed to SBPR at the Royal College of Surgeons, 35–43 Lincoln’s Inn Fields, London WC2A 3PN
