血清淀粉样蛋白A-siRNA沉默对波动性高血糖培养下肾小球系膜细胞分泌细胞外基质及炎症因子的影响

The effect of serum amyloid A-siRNA silence on fluctuated high glucose-induced secretion of extracellular matrix and inflammation factors in human mesangial cells

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＋SAA-siRNA（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 observe the effect of serum amyloid A-siRNA silence on the inflammatory response and extro-cellular matrix （ECM） in fluctuated high glucose-induced human mesangial cells （HMCs）, to explore the relationship between fluctuated high glucose and chronic kidney disease （CKD）in diabetes,and to evaluate the role of SAA in the process of CKD. Methods Synchronized HMCs were divided into 6 groups. The transfection efficiency was observed with fluorescence microscope. The mRNA expression of SAA and tumor necrosis factor-α （TNF-α） were detected by real-time RT-PCR, the intro- cellular SAA protein was detected by Western Blot, and the protein expression of SAA, TNF-α and fibronectin （FN） were detected by ELISA. Results The expression of SAA,FN and TNF-α in the high glucose （HG） group was higher than in the normal glucose group （P〈0.05）. The expression of the above factors in the fluctuated glucose （FG） group was higher than in the HG group （P〈0. 05）. The expression of the above factors in the FG＋SAA-siRNA group was obviously lower than in the FG group （P〈0.05）. Conclusion Fluctuated high glucose evokes more expression of SAA,FN and TNF-α compared with stable high glucose. Through the silencing of SAA expression by SAA-siRNA, the secretion of FN and TNF-α in fluctuated high glucose-induced HMCs could be decreased and the accumulation of ECM and inflammationof HMCs could be decreased, which providing a new experimental evidence for preventing CKD.