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目的研究SAA-siRNA沉默对波动性高糖培养下人肾小球系膜细胞(HMCs)炎症反应及细胞外基质(ECM)的影响,探讨波动性高糖与糖尿病慢性肾脏疾病(CKD)的关系及血清淀粉样蛋白A(SAA)在CKD发生发展中的作用。方法传代培养5~9代的HMCs,同步化后分6组进行实验。荧光倒置显微镜观察siRNA转染效率;real-time RT-PCR技术检测SAA及TNF-α的mRNA表达;Western Blot印迹检测细胞内SAA蛋白的表达;ELISA法检测细胞上清液中SAA、纤连蛋白(FN)及肿瘤坏死因子α(TNF-α)的表达情况。结果分组培养48 h后,高血糖(HG)组SAA、FN及TNF-α的表达较对照(NC)组升高(P<0.05),波动性高血糖(FG)组较HG组表达升高(P<0.05);波动性高血糖+SAAsiRNA(FG+SAA-siRNA)组SAA、FN及TNF-α表达较FG组降低(P<0.05)。结论波动性高血糖可引起HMCs更高水平SAA、FN及TNF-α的表达;通过SAA-siRNA沉默SAA表达,可减少波动性高血糖刺激下HMCs的FN、TNF-α分泌,减少肾小球ECM积聚和炎症反应,为CKD防治提供新的实验依据。
Objective To investigate the effect of SAA-siRNA silencing on inflammatory reaction and extracellular matrix (ECM) in human mesangial cells (HMCs) under fluctuating high glucose conditions and to explore the relationship between fluctuating high glucose and chronic kidney disease (CKD) in diabetes And serum amyloid A (SAA) in the occurrence and development of CKD role. Methods The passage 5-9 passages of HMCs were subcultured and divided into 6 groups for experiment. The expression of SAA and TNF-α mRNA was detected by real-time RT-PCR, the expression of SAA protein was detected by Western Blot and the expression of SAA and fibronectin (FN) and tumor necrosis factor alpha (TNF-alpha) expression. Results After 48 h of culture, the expression of SAA, FN and TNF-α in hyperglycemia (HG) group was significantly higher than that in NC group (P <0.05), but higher in HG group than in HG group (P <0.05). The expression of SAA, FN and TNF-α in the group of fluctuating hyperglycemia plus SAAsiRNA (FG + SAA-siRNA) was lower than that in the FG group (P <0.05). Conclusions Vasopathic hyperglycemia can induce higher levels of SAA, FN and TNF-α in HMCs. SAA-siRNA silencing SAA expression can reduce the secretion of FN and TNF-α in HMCs induced by fluctuating hyperglycemia and decrease glomerular ECM accumulation and inflammatory response, provide a new experimental basis for the prevention and treatment of CKD.