The effect of NƐ - Carboxymethyl-lysine on the expression of the advanced glycation end-products receptor (AGER)
Cardiff Metropolitan University
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Advanced Glycation End-products (AGE's) are proteins that become glycated and oxidised (non enzymatically) with reducing sugars or lipids. Age's form naturally within the body but excess levels of glucose (e.g. in diabetes mellitus) and oxidative stress are associated with excess AGE formation. Once AGE's form the compound has been found by Schmidt, (1999) cited in Goldin, et al., (2006) to be almost irreversible. The receptor for AGE is found on the surface many different cell types throughout the human body such s endothelial cells and macrophages (Toth, et al., 2008) which links the receptor to a multitude of vascular disorders such as the chronic complications which arise in diabetic sufferers after prolonged exposure to AGE's. The binding of AGE's initiates an intra-cellular signal cascade which activates the NF-xB pathway and induces the up-regulation of RAGE on the cell surface. AGE's have been found to be increased in people with diabetes due to the hyperglycaemia associated with the disease. Abnormally high blood glucose levels found in both types of diabetes gradually affects small and large vessel walls, as AGE's have been found to accumulate at vessel walls and have thus been linked to the micro and to macro vascular complications which arise in diabetics (Goldin, et al., 2006). Carboxymethyl lysine (CML) is a modified AGE protein, which is possibly the most abundant AGE found in vivo (lkeda  cited in Southern, ). High levels of CML in serum has been found to be particularly higher in patients suffering with retinopathy and microalbuminaemia than diabetic sufferers without these conditions (Wautier,  Hirata"  cited in Southem et al., ). In-house grown THP-1 cell lines used for the base of RNA extraction were treated with 5.0mg/µl of CML for 24hrs. After cDNA extraction, conventional PCR assays were performed using 1.5% agarose gels, 0.5% ethidium bromide solution under a UV detection lamp and Q-PCR work was carried out using the Bio-Rad CFX96™ optical reaction module in conjunction with the C1000™ thermal cycler. In this investigation we succeeded in detecting RAGE expression via both conventional PCR and QPCR methods but failed to observe an up-regulation of RAGE expression on THP-1 cells and found that CML actually had a mild down-regulation effect on RAGE expression (P = 0.12), when compared to the novel housekeeping gene GAPDH. However numerous other current research papers suggest that AGE's such as CML do have an up-regulation on RAGE expression on monocytic cell lineages. Q-PCR analysis showed that the Bio-Rad iQ™ SYBR® green supermix was the most ideal supermix for use with the Bio-Rad CFX96™ optical reaction module in conjunction with the C1000™ thermal cycler (P = 3.572 x 105 when compared to Power SYBR® green).
MSc Biomedical Science
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