The Effects Of Antisense Smad2 Oligodeoxynudeotides On Fibronection And Lamimin Secretion Of Human Renal Mesangial Cells With High Glucose

PrefaceDiabetic nephropathy(DN)is one of the most serious chronic complicationses of diabetes mellitus(DM),and it is the leading cause that results in end-stage kidney failure.The pathogenetic mechanism of diabetic nephropathy is still unclear,a great deal of researches have expressed various factors participating in its development, among which transforming growth factor-β1(TGF-β1)/Smad signaling pathway plays a prominent role in it.TGF-βsignalling is an important cellular regulator of growth and differentiation.The principal intracellular mediators of TGF-βsignalling are smad proteins,which upon TGF-βstimulation can accumulate in the nucleus and regulate the transcription of target genes.Smad 2 is one of the Smad proteins,which is a substrate of TGF-βreceptor.Thus,transient association and phosphorylation of Smad 2 by the TGF-βreceptor is necessary for nuclear accumulation and initiation of signaling.The main expression of DN is glomerulosclerosis and tubulointerstitial fibrosis.TGF-β1 systems is activated by high glucose in DM,and Smad2 proteins can increase the secretion of extracellular matrix(ECM)through transfer TGF-βsignaling,which can make glomerular basement membrane thicker and result in DN.Fibronection(FN)and lamimin(LN)is the important composition of ECM,in this study antisense Smad2 oligodeoxynudeotides(ODN)were transferred transiently into human renal mesangial cells through lipofectamine 2000,which can restrain the activation of Smad2 gene directly.The objective of this research is to observe the effects of antisense Smad2 ODN on FN and LN secretion,to explore the action of Smad2 in DN and find a new way to retard the progress of glomerulosclerosis.Object and method1,MaterialCell:Human renal mesangial cell(4200,America ScienCell Research Laboratory)Reagent:Cell complete medium(4201,America ScienCell Research Laboratory), Lipofectamine 2000(America Invitrogen Life Technologies),sense and antisense Smad2 oligodeoxynudeotides(Shanghai sangon bio-engineering technical service limited company),FN,LN ELISA reagent box(Shanghai senxiong company).Instrument:The refrigerator,the centrifuge,the thermostate,the microscope,the microscope of fluorescence,the cell-culture laboratory and so on.2,Experimental techniqueThe human renal mesangial cells were maintained according to the directions and used at passages 5 to 8 for the experiment.To observed the transfer efficiency,the ODNs were mixed with Lipofectamine 2000 at the different ratio and transferred into human renal mesangial cells.The following experiment choosed the group of high transfer efficiency.The human renal mesangial cells at passages 5 to 8 were divided into 6 groups:①high glucose group:glucose 30mmol/L,②antisense Smad2 ODN group:glucose 30mmol/L,antisense Smad2 ODN 0.8μg,Lipofectamine 2000 2.0μl,③sense Smad2 ODN group:glucose 30mmol/L,sense Smad2 ODN 0.8μg,Lipofectamine 2000 2.0μl,④Lipofectamine 2000 group:glucose 30mmol/ L,Lipofectamine 2000 2.0μl,⑤normal glucose group:glucose 5.0mmol/L,⑥mannitol group:mannitol 25mmol/L, glucose 5.0mmol/L.The concentrations of FN and LN in different time(0,18,24,48,72h)were examined by ELISA. ResultThe different ratio of ODNs and Lipofectamine 2000 result in different transfer efficiency.The group of high transfer efficiency was ODN:Lipofectamine 2000=0.8μg:2.0μl=1:2.5.High glucose group significantly increased concentrations of FN and LN in the culture medium treated with normal glucose group,antisense significantly decreased concentrations of FN and LN in the culture medium treated with high glucose group,the secretion of FN and LN of sense group have no decreases,the secretion of FN and LN of mannitol group was similar with that of normal glucose group,concentrations of FN and LN of Lipofectamine 2000 group was similar with that of high glucose group.ConclusionHuman renal mesangial cells with high glucose can increase the secretion of FN and LN,Smad2 antisense ODN can restrain the secretion of FN and LN,Lipofectamine 2000 can't restrain the secretion of FN and LN,the factor which can increase the secretion of FN and LN isn't high osmotic pressure but high glucose-stimulated.