Role of nuclear factor kappa B (NFkB) in the vascular complications of diabetes.

摘要

Diabetes mellitus (DM) is the most common metabolic disease worldwide. This condition accounts for the majority of renal failure and blindness in adult’s aged between 20 to 74 years of age. After 20 years, some of type 1 diabetic patients and around 60% of type 2 diabetic patients will be diagnosed with retinopathy. In 2011, a survey was presented and supplied data on the number of people who are suffering from diabetes. In Scotland, they found that 247,278 people were diagnosed with diabetes. In addition, about 36.6% of patients’ were diagnosed with type 1 diabetes (T1DM) and 31.7% of those with type 2 diabetes (T2DM). In this research, the role of inflammatory mediators in the progression of the vascular complications of diabetes was investigated. Specifically, the role of nuclear factor kappa B1 (NFĸB1). There were 2 approaches used in this study: i) a bioinformatics approach to determine what single nucleotide polymorphisms (SNP) may occur within the NFĸB1 gene in T2DM and ii) a molecular biology approach to investigate the role of the SNP of interest in a human monocyte cell line using polymerase chain reaction (PCR) and restriction enzymes. Methods: Genetic polymorphisms were investigated using bioinformatics tools through computational analysis and databases, such as, Ensembl and the National Center for Biotechnology Information (NCBI), NEB cutter, Primer Quest and NetPhos. The SNP of interest was identified due to its role in diabetes. Primers were designed to span the region for the SNP of interest, amplified using PCR and resolved in an agarose gel. The SNP of interest was restricted with three restriction enzymes (HpyCH4iii, Alu1 and PvuII) followed by sequencing of the product. The U937 monocyte cell line was used to investigate the expression of the NFĸB gene. Cells were incubated in RPMI 1640 with 2 mM L-glutamine, 100 U/mL Penicillin, Streptomycin and 10% heat inactivated foetal bovine serum (FBS) with H2O2 for up to 180 minutes with 5.5mM, 20mM and 40mM glucose +/- H2O2. RNA extraction was followed by cDNA amplification, which was subsequently used in reverse transcriptase polymerase chain reaction (RT-PCR) and for a period of time to address the biological significance of NFĸB activation in these conditions. Results: A DNA fragment corresponding to the 206bp fragment was identified that spanned the area containing the SNP. Successful restriction with HpyCH4iii has not been possible, therefore, DNA sequencing was carried out. Sequencing identified the A allele indicating the presence of the homozygous wild type form of this region. Also, determining the role of NFĸB activation, HIF1-α and β2-microglobulin in U937 cells mRNA via culturing U937 under high glucose (HG) condition in different concentrations (5.5mM, 20mM and 40mM). The NFĸB and β2-microglobulin PCR product was not detected during incubations with various glucose concentrations. Conclusion: The DNA sequence of the U937 cell line corresponds to the wild type form of the SNP under investigation. This model has enabled the investigation of the effect of glucose concentration on the expression of the NFĸB and HIF1-α genes and this has provided useful insight that can be used to develop this work further.