| ObjectiveOne of the diabetic microvascular diseases, namely diabetic nephropathy(DN), is common in patient who has medical history more than10years.It’s the main death reason in TlDM, however in T2DM,it’s only second to coronary thrombosis and cerebral atherosclerosis.Pathogenesis of DN has not yet been fully elucidated, high blood glucose and fluctuations of blood glucose are the symbol of glucose metabolic disorders in the diabetic patients and also the main cause of nephropathy complications. Normally,at the presence of neurohumoral regulation, blood glucose volatility changes slight.The blood glucose variability,however, is relatively high in diabetic patients. Due to the insulin biological activity or the effect of absolute and relative of the shortage. With the further development of clinical and basic research researchers have gradually recognized that diabetic chronic vascular disease is not only closely related to persistent elevated blood glucose but to intermittent blood glucose. In this study, it is through in vitro simulation experiments with constant high glucose concentration and intermittent blood glucose concentration at the same time acting on rat mesangial cells, we observed the morphological changes of different concentrations at different times through the microscop, identified the proliferation of the cells by MTT method and the expression of TRPC6and TRPC1by RT-PCR.Therefore, we explore and study the fluctuations of blood glucose concentration to promote the pathological mechanisms of injury of diabetic nephropathy,which provide a theoretical basis and experimental evidence of diabetic nephropathy.MethodsWe cultured rat glomerular mesangial cells (GMC)in vitro. GMC in experiment is divided into three groups:(1)Normal control group(NG):The cells were cultured in5.5mmol/L glucose concentration.(2)Constant high glucose group(HG):The cells were cultured in25.0mmol/L glucose concentration.(3)Fluctuations in blood glucose group(FG):At first cells were first cultured for3hours in25.0mmol/L glucose concentration,then cultured for2hours in5.5mmol/L glucose concentration.Repeated three times a day, finally,the cells cultured in5.5mmol/L glucose concentration overnight.Rat glomerular mesangial cells(GMC)were cultured24h,48h,72h respectively,we observed the morphological changes of the cells under the microscope,and identified the proliferation of the cells by MTT method,and measured the expression of TRPC6mRNA and TRPC1mRNA by RT-PCR method.Results1.Morphological changes of the cells were observed. In24h,Cells in normal and stable high blood groups were short strip, short cords; cellular junction were tight;cell nucleus is clear;the cytoplasm outwards pseudopodia of varying lengths.In intermittent blood glucose group,the cellular morphology was flat and branched protoplasmic extension. In48h, except normal control group,morphological changes for the remaining cells in each group showed significant change.In the stable high glucose group, cells were significant increased and hypertrophied,however, In the intermittent blood glucose one, the cellular number were decreased markedly and arranged disorder.Vacuolar degeneration seen in the cytoplasm, part of the cells were apoptotic.The number and morphology of the cells changed significantly. In72h, the cells and the wall-adhering cells were decreased markedly than in48h. It is showed that the most cells occurred the trend of apoptosis and became round in morphology comparing with normal cell.2. Identified the cellular proliferation by MTT method.A significant proliferation was evident in the stable high glucose group in72h. There were statistically differences comparing with normal group(P<0.05).Oppositely, cellular proliferation, fluctuations of blood glucose group in72h, were the most significant inhibitory effect.The differences were obvious(P<0.01) compared with normal groups; This group compared with the other groups, the differences were statistically significant(P<0.05)3. Measured the expression of TRPC6mRNA and TRPClmRNA by RT-PCR.All samples were detected expression of TRPC6mRNA and TRPClmRNA. In normal control group,TRPC6mRNA was strong expression, no difference of the expression at different times. In the other two groups, the expression level of TRPC6were significantly lower than that in the corresponding control groups In24h(P<0.05,P<0.01,respectively), and then reduced gradually with prolongation of time. In72h, the expression level of TRPC6in intermittent blood glucose group was markedly lower than that in the constant one in48h and the differences were obvious(P<0.05). TRPClmRNA,however, the expression level was no difference in different time and concentrations.Conclusions:1. Rat’s glomerular mesangial cells in constant high blood glucose group, the effects of cell proliferation showed the time and concentration dependent,on the contrary,the intermittent blood glucose group inhibited proliferation of the GMC.2. TRPC6mRNA and TRPClmRNA expression on GMC,the expression of TRPC6mRNA on GMC with glucose concentration,however,expression of TRPClmRNA has nothing to do with the blood glucose concentration.3. The intermittent blood glucose group more significantly reduced the expression of transient receptor potential cation channel6(TRPC6) on GMC than the constant high glucose one.The possible reason that under the intermittent high glucose concentration, the rat mesangial cells can not produce adaptive changes,while glucose toxicity enhanced to GMC appear the damage and dysfunction. |
