Verification of gene expression differences on a protein level - An IHC study
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Background: Inflammatory bowel disease, a collective term that includes ulcerative colitis and Crohns disease, is an inflammatory disorder that affects the gastrointestinal tract, with symptoms like severe diarrea, pain, fatigue and weight loss. Northern Europe has one of the highest incidence rates of IBD in the world. The pathophysiology is complex, which includes both genetic predisposition, alterations in gut microbiota and disruption of the intestinal. Aims: This thesis is based on research done by Atle van Beelen Granlund, a member of the IBD research group at St. Olav, where the mRNA expression levels of both UC and CD colonic mucosa was explored with whole genome expression sequencing. In this thesis, the purpose was to further investigate the protein expression for some of the genes investigated in Granlunds research; RARα, RARβ, RXRαβγ, HLA-‐II and CD86. Materials and methods: Colonic biopsies from five different patient groups; active UC, active CD, inactive UC, inactive CD and healthy individuals were stained with immunohistochemistry to visualize the protein expression of RARα, RARβ, RXRαβγ, HLA-‐II and CD86. Results: The IHC stain of RARα, RARβ and RXRαβγ showed no significant differences in intensity or coverage in the groups UCA, CDA, UC or CD compared to healthy individuals, except the intensity of the staining in the lamina propria for the RARα antibody. Negative tissue stained positive for the RARβ antibody, and Western Blot with RARβ antibody indicated a low sensitivity. The IHC stain of MHC-‐II showed a higher intensity and coverage in the epithelium in the UCA and CDA biopsies compared to healthy individuals. In the CD86 IHC stain, there was no difference in the intensity, but a qualitative difference was seen, with a stronger basolateral epithelial stain in the UCA and DCA groups compared to healthy individuals. Conclusion: This study failed to show a correlation between mRNA expression levels measured using whole genome gene expression analysis and the protein expression pattern as viewed using IHC of the receptors RARA, RARB and RXRABG. However, we suspect that the antibodies used in the project have a low specificity, leading to this result. We have shown an upregulated protein expression of MHC class II in the intestinal epithelial cells in patients with inflamed IBD compared to normal controls. The MHC class II 5 expression pattern supports the gene expression analysis, and suggests that there is an increase in MHC class II in the colon epithelium during IBD inflammation. The CD86 analysis failed to verify the difference in gene expression levels observed. This might be due to poor specificity of the antibody used. Thus, no conclusions can be drawn on basis of our results on CD86 expression.