高糖对系膜细胞诱生型一氧化氮合酶表达及细胞外基质合成的影响

Effect of High Glucose on Inducible Nitric Oxide Synthase mRNA Expression and ECM Synthesis in Cultured Mesangial Cells

目的:探讨高糖对体外培养的系膜细胞诱生型一氧化氮合酶(iNOS)mRNA表达的调节作用及NO合成的变化,以及上述变化对细胞外基质(ECM )合成的影响。方法:利用脂多糖(LPS)诱导系膜细胞表达iNOS ,观察5 .6、10、2 5、4 0mmol/L不同糖浓度,在0、4、2 4、4 8h不同时间点培养的系膜细胞iNOSmRNA表达的变化及培养上清中NO、Ⅳ型胶原、纤维连接蛋白含量的变化,以相同渗透压的甘露醇为对照组。细胞增殖测定采用MTT法,iNOS表达采用RT -PCR的方法,NO的检测采用硝酸还原法,Ⅳ型胶原、纤维连接蛋白的测定采用ELISA法。结果:高糖抑制系膜细胞增殖;LPS刺激后系膜细胞可表达iNOS ;高糖浓度时iNOSmRNA表达增加,呈浓度和时间依赖性,相同渗透压的甘露醇无类似作用(P <0 .0 1) ;随着糖浓度的升高和时间延长,实验组上清中NO含量增加,Ⅳ型胶原、纤维连接蛋白含量增加。结论:高糖可上调系膜细胞iNOSmRNA的表达和促进NO、Ⅳ型胶原、纤维连接蛋白的合成。

Objective:To elucidate the effect of high glucose on the mRNA expression of inducible nitric oxide synthase(iNOS) and nitric oxide(NO) synthesis and on extracellular matrix(ECM) synthesis in cultured human mesangial cells(MC).Methods:Cultured human mesangial cells were stimulated with lipopolysaccharide(LPS) to induce iNOS.Normal glucose condition was 5.6 mmol/L and high glucose condition was 10 mmol/L,25 mmol/L,50 mmol/L respectively. Mannitol with same osmolality was used as control.MC proliferation was assessed by means of MTT colorrnetric method. Reverse transcription-polymerase chain reaction(RT-PCR) was used to determine the activity of iNOSmRNA. The aliquot of the medium was used to determine NO by nitrate-reductase assay.Type Ⅳ collagen and fibronectin( FN ) were determined by ELISA.Results:MC proliferation was inhibited by high glucose with the increasing of concentration and duration, reaching peak at 48 h under 50 mmol/L concentration(P<0.05). Mannitol with same osmolality had no significant effects on MC proliferation(P>0.05). The expression of iNOSmRNA induced by LPS was up-regulated by high glucose. There were significant differences in iNOSmRNA expression between mannitol controls and glucose groups(0.145±0.036 vs 0.331± 0.023, P<0.05).NO increased with glucose concentration and duration in experimental group(P<0.05). Both type Ⅳ collagen and FN increased with glucose concentration and duration in experimental group(P<0.05).Conclusion:High glucose up-regulates the expression of iNOSmRNA induced by LPS and increases the concentration of NO, and the expression of Type IV collagen and FN in cultured human mesangial cells.