摘要
Objective：Hyperglycemia stimulates secretion of transforming growth factor-βl （TGF-βl） in cultured glomerular mesangial cells, thereby increases production of extracellular matrix （ECM）. We examined the effect of antisense mRNA for Smad2 on high glucose-induced ECM production in rat mesangial cells. Methods..A mammalian expression vector, pES2a, which expresses antisense Smad2 mRNA and green fluorescent protein （EGFP）, was transfected into mesangial cells. Following incubation in high glucose medium, EGFP expression and Smad2 mRNA level were determined by fluorescence microscopy and PCR, respectively. Secreted fibronectin and type IV collagen were assessed by enzyme-linked immunosorbent assay. Results ：Within 48 h of incubation in high glucose medium, Smad2 mRNA level significantly increased by 1.6 fold in association with increases in prodtaction of both fibronectin （from [45.86±2.73] to [84.19±6.81] ng/ml） and type IV collagen （from [16. 28±0. 90] to [55.27±4.75] ng/ml） in nontransfected cells （P〈0.05）. In pES2a-transfected cells, the high glucose-induced increase in Smad2 mRNA was abrogated completely, in parallel with significant suppression of the high glucose-indtmed increase in fibronectinproduction （[54.44±4.99] ng/ml） and type Ⅳ collagen （[20.96±2.47] ng/ml）. An empty vector was without effects. Coneluslon：These findings demonstrate that Smad2 plays a critical role in mediating high glucose-stimulated ECM production in mesangial cells, indicating that inhibition of Smad2 activity by antisense Smad2 mRNA may be an effective means to attenuate glomerular matrix accumulation in diabetic nephropathy.
Objective:Hyperglycemia stimulates secretion of transforming growth factor-β1 (TGF-β1) in cultured glomerular mesangial cells, thereby increases production of extracellular matrix (ECM). We examined the effect of antisense mRNA for Smad2 on high glucose-induced ECM production in rat mesangial cells. Methods:A mammalian expression vector, pES2a, which expresses antisense Smad2 mRNA and green fluorescent protein (EGFP), was transfected into mesangial cells. Following incubation in high glucose medium, EGFP expression and Smad2 mRNA level were determined by fluorescence microscopy and PCR, respectively. Secreted fibronectin and type Ⅳ collagen were assessed by enzyme-linked immunosorbent assay. Results:Within 48 h of incubation in high glucose medium, Smad2 mRNA level significantly increased by 1.6 fold in association with increases in production of both fibronectin (from [45.86±2.73] to [84.19±6.81] ng/ml) and type Ⅳ collagen (from [16.28±0.90] to [55.27±4.75] ng/ml) in non-transfected cells (P<0.05). In pES2a-transfected cells, the high glucose-induced increase in Smad2 mRNA was abrogated completely, in parallel with significant suppression of the high glucose-induced increase in fibronectin production ([54.44±4.99] ng/ml) and type Ⅳ collagen ([20.96±2.47] ng/ml). An empty vector was without effects. Conclusion:These findings demonstrate that Smad2 plays a critical role in mediating high glucose-stimulated ECM production in mesangial cells, indicating that inhibition of Smad2 activity by antisense Smad2 mRNA may be an effective means to attenuate glomerular matrix accumulation in diabetic nephropathy.
作者简介
E-mail： ysqdd@hotmail.com
