Detection of differentially expressed genes in particle disease using array-filter analysis
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Ch P Rader1, B Baumann, O Rolf
1Department of Orthopadics, University of Wuerzburg.
Unlabelled:
The precise cellular mechanism of osteolysis in particle disease is still unknown. The aim of the study was to screen for new gene products in macrophages during particle contact.
Method:
In an established macrophage model THP1-cells (human monocytic cells) were differentiated under the influence of vitamin D3 and GM-CSF into macrophage-like cells (MLC). MLCs were incubated each with different concentrations of polyethylene particles, Lipopolysaccharids (LPS) and controls. Isolated RNA was transcribed into complementary radioactive 32P labeled cDNA. This probe was hybridised on an human cDNA expression array and analysed by autoradiography. To obtain a more reliable method quantifying mRNA, the reverse transcriptase polymerase chain reaction (RT-PCR) was used.
Results:
The arrays showed an upregulation of the following genes by particles: TNF-Rezeptor 2, IL-1 Receptor Antagonist, Bone Morphogenic Protein 4 and HM 145. This was proven three times using RT-PCR and statistically significant in comparison to the controls. LPS induced the same upregulation except for HM145 whereas particles caused downregulation of this mRNA expression.
Conclusion:
Our results prove that the model of differentiated THP-1 cells treated with PE particles is a suitable system to analyse differential gene expression patterns, since the induction of the major positive control genes TNF alpha and IL1 beta were detected by this approach. BMP 4 is known as signal protein which mediates ectopic bone formation and can also be interpreted as a contra regulatory gene. HM 145 belongs to the leukocyte chemotactic peptide receptor family. HM 145 seems to be one of the first genes that is enhanced along the septical pathway but less expressed by contact with particles. Analysis of HM 145 expression might help to diagnose septic versus aseptic loosening of prosthesis.
Insights
This study identifies key genes, including Bone Morphogenic Protein 4 and HM 145, involved in macrophage responses to polyethylene particles, offering insights into particle disease mechanisms and potential diagnostic markers for prosthesis loosening.
Area of Science:
- Biomedical Science
- Molecular Biology
- Immunology
Background:
- Osteolysis in particle disease lacks a clear cellular mechanism.
- Identifying genes expressed by macrophages during particle contact is crucial.
Purpose of the Study:
- To screen for novel gene products in macrophages responding to polyethylene particles.
- To investigate gene expression patterns in a macrophage model.
Main Methods:
- Differentiated human monocytic THP1-cells into macrophage-like cells (MLCs).
- Incubated MLCs with polyethylene particles and lipopolysaccharide (LPS).
- Utilized cDNA expression arrays and reverse transcriptase polymerase chain reaction (RT-PCR) for gene expression analysis.
Main Results:
- Particles upregulated TNF Receptor 2, IL-1 Receptor Antagonist, Bone Morphogenic Protein 4 (BMP 4), and HM 145.
- LPS induced similar upregulation, except for HM 145, which was downregulated by particles.
- RT-PCR confirmed these findings with statistical significance.
Conclusions:
- Differentiated THP-1 cells treated with PE particles provide a suitable model for differential gene expression analysis.
- BMP 4 may act as a contra-regulatory gene in ectopic bone formation.
- HM 145 expression patterns could aid in diagnosing septic versus aseptic prosthesis loosening.
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