| Insulin resistance, also referred to as insulin insensitivity, is one of the most essential pathogenesis of type 2 diabetes mellitus. Recently, it is reported that insulin resistance is in correlation with microalbuminuria, the pathological mechanism of which is the histological changes in diabetic nephropathy (DN) caused by hyperglycemia: glomerular basement membrane (GBM) thickening and extracellular matrix (ECM) accumulation. Matrix metalloproteinases (MMPs) comprise a superfamily of at least 20 members of endopeptidase involving in ECM degradation. MMP-1 is the human interstitial collagenase, the most appropriate substrate of which is type III collagen. In patients who suffer from DN, type III collagen deposits in glomerular mesangium, which is the marker of the onset of glomerularsclerosis. So, we postulate that insulin resistance might affect the regulation of MMP-1 expression via its contribution to hyperglycemia. Up to date, there haven't been any closely related articles on the correlation between insulin resistance and MMP-1. To answer this, we used a one-step sandwich enzyme-linked immunoassay (ELISA) to exam the subjects' plasma MMP-1 concentrations. For estimation of severity of insulin resistance (IR), we used a homeostasis model assessment ( HOMA ) recommended equation for IR value ( HOMA-IR ). We expect to establish a simple and convenient method of detecting MMP-1 with less hurt, good accuracy and precision while elucidating the implication of the correlation between IR and MMP-1, and its possible clinical importance. ProtocolsSubjectsThis study included 60 type 2 diabetics (29 men, 31 women) aged 59.48 ?11.41 (x眘) who were recruited from No.l Affiliated Hospital, medicine School, Zhejiang University. The inclusion criteria are the revised diagnosis and classification of diabetes mellitus recommended by ADA in 1997, confirmed by WHO in 1999. The exclusion criteria are those who are with: recent trauma or operation; malignant tumors; clinical rheumatic diseases; severe cardiac, hepatic, pulmonary or renal diseases; non-diabetic related renal diseases; secondary hypertension; other endocrinopathies; receiving insulin and other kinds of hormone-therapy; severely beta-cell dysfunction; acute diabetic complications and those who are cigarette and/or alcohol abusers.30 normal controls were also included, aged 58.93 ?8.44(x s). They were recruited among those who were attending routine physical examination sponsored by No.1 Affiliated Hospital, College of Medicine, Zhejiang University,all having no type 2 diabetes first-degree relatives. MMP-1 DetectionWe stringently followed the human plasma EL1SA kit (purchased from Chemicon Inc. USA) instructions. Briefly, pipette 100 uL of each standard curve solution or specimen (1 : 10 diluted) into a well of antibody coated microplate. Remove the solution and wash 4 times with washing solution. Pipette lOOuL enzyme-labeled antibody solution into each specimen or standard containing well. Cover micro plate with lid, and incubate at 20癈 for exactly 60 minutes without shaking. Remove the solution and wash 4 times with washing solution. Add 100uiL of Color Reagent to each specimen or standard containing well. Cover microplate with lid, and incubate at 20癈 for exactly 30 minutes without shaking. Stop enzyme reaction by adding lOOuL of 1mol/L sulfuric acid to each well. Using well containing only Buffer Solution as a blank, read the absorbance at 450 nm for each well. For specimen, read in duplicate. Plot the net absorbance value for each proMMP-1 concentration, subtracting the value for the Buffer Solution alone (Ong/mL) from the value for individual dilutions. Multiply the ng/mL value by additional dilution of specimen. For standard, read the absorbance directly. Employing the standard concentrations as dependent, standard absorbance as independent, set a regression equation. Using the net absorbance for specimen as independent, figure out the genuine values standing for proMMP-1 concentrations. Determination of HOMA-IRFasting plasma gl... |
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