| Diabetic Nephropathy (Diabetic Nephropathy, DN), is the more serious complications of diabetes, is the main reason for end-stage renal disease. Diabetic nephropathy with glomerular basement membrane thickening and extracellular matrix accumulation is the main pathology. Glomerular lesions of mesangial cells is one of the important target cells, their proliferation, differentiation, disease progression in the evolution of glomerular play a leading role. In diabetic nephropathy, mesangial cells are significantly influenced by high glucose and high insulin. Mesangial lesions of diabetic nephropathy changes is the most prominent pathological one. This change in the DN other basic pathological changes, such as glomerular hypertrophy, extracellular matrix (Extracellular matix, ECM) and glomerular sclerosis produced an increase in the incidence of the development process has important pathological significance, but the exact cause of these changes occurred mechanism is not fully understood. Although studies have found that high glucose can lead to mesangial cell hypertrophy and increased ECM synthesis, but not enough to use it to fully clarify the pathogenesis. Type 2 diabetes, we found that liver and muscle activity in the abnormal increase of GSK-3β, GSK-3βis in a wide variety cells, involved in cell differentiation, proliferation and apoptosis, and other important physiological processes in diabetes weaken the signal transduction of insulin, so that the use of glucose, the process of glycogen synthesis was inhibited, and thus give rise insulin resistance. The role of hyperinsulinemia may be localized in the kidney by increasing the activity of GSK-3βto promote extracellular matrix accumulation, leading to or aggravated the occurrence and development of DN.Objective:This experimental in vitro mesangial cell, mesangial cell fusion to be 80% -90%, the synchronization after the use of Lipofectamine 2000 transfection reagent (Invitrogen) to GSK-3βsiRNA short clips to the Department of transfected membrane cells, fluorescence microscope, 24 to 48 hours after the cells were collected by Real-time PCR detection of transfected mesangial cell fibronectin expression level of GSK-3βin the changes under the action of high insulin. Clarify to clarify when GSK-3βin insulin signaling pathway DN role in the hyperinsulinemia in the activity of GSK-3βaccumulation of extracellular matrix, provide a theoretical basis for the prevention and control DN, and R & D to prevent the occurrence and delay the DN DN the progress of new drugs to provide a theoretical basis and experimental evidence.Methods:The experimental mesangial cells cultured in vitro, trypsin digestion and passage, and into 24-well plates and cultured until the mesangial cell fusion to 80% -90%, the synchronization with DMEM for 24 hours. Divided into 9 groups: a, control group b, high insulin alone group i, the negative control group transfected with c, saline + siRNA (6331) group d, saline + siRNA (6057) group e, saline + siRNA ( 6058) group. f, high insulin + siRNA (6331) group g, high insulin + siRNA (6057) group h, high insulin + siRNA (6058) group. With 50μL DMEM diluted siRNA, mix gently blowing and drawing 3-5. Mix gently reverse transfection reagent, diluted with 50μL DMEM 1.0μL LipofectamineTM2000, mix gently blowing and drawing 3-5, room temperature for 5 min. SiRNA transfection reagent and mixed diluent, mix gently blowing and drawing 3-5, room temperature for 20 min. Transfection complex added to the 24-hole cell plate, 100μL / hole, before and after mixing gently rocking the cell plate. Cell plate in 37℃, 5% CO2 incubator for 24-48 hrs. Fluorescence microscope, after 24 to 48 hours the cells were collected after centrifugation to the supernatant, the extracted RNA (total RNA extraction of all the steps in accordance with QIAGEN's RNeasy @ Mini Kit kit instructions), Real-time PCR detected by the group transfer mesangial cell fibronectin expression level of GSK-3βin the changes under the action of high insulin.Results:The fluorescent labeled siRNA was transfected into mesangial cells 24H, the use of fluorescence microscopy, the visible part of up to 40-60% transfection efficiency, cell density slightly larger area of up to 80% transfection efficiency. RNA was extracted after the cells were collected after the RealtimePCR detected fibronectin in the expression levels of each group (Figure). Test results appear in the role of high insulin expression of fibronectin was significantly higher than control group, after the silence of GSK-3β, fibronectin expression was significantly increased, and the high insulin group, higher levels of fibronectin expression.Conclusion:1 successfully transfected mesangial cells is the key to this experiment, observed under a fluorescence microscope higher transfection efficiency;2.RealtimePCR test results show that high insulin secretion increased glomerular extracellular matrix can promote, GSK -3βas a negative regulator of its regulatory role to a certain extent, in the GSK-3βsiRNA short segment transfected mesangial cells, the result of high insulin secretion of extracellular matrix significantly reduced the inhibitory effect of aggregation.3. Hyperinsulinemia in the activity of GSK-3βon the impact of extracellular matrix accumulation, DN provides a theoretical basis for the prevention and treatment, and research and development to prevent the occurrence of DN and DN delay the progress of new drugs to provide a theoretical basis and experimental evidence.
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