摘要
目的研究siRNA-SAA干扰对高糖条件培养下的人肾小球系膜细胞(human mesangial cells,HM-Cs)炎症反应及细胞外基质(extra-cellular matrix,ECM)的影响,探讨血清淀粉样蛋白A(serum amyloid A,SAA)在糖尿病肾病(diabetic kidney disease,DKD)发生发展中的作用。方法传代培养的HMCs同步化后分组,脂质体LipofectamineTM2000转染抑制SAA表达的siRNA或无关的siRNA(negative siRNA,neg-siR-NA),用荧光倒置显微镜观察转染效率,利用real-time RT-PCR技术检测SAA及肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)的mRNA表达,ELISA技术检测SAA、纤连蛋白(fibronectin,FN)、TNF-α蛋白的表达情况。结果 SAA-siRNA转染可抑制高糖刺激的HMCs中SAA、FN、TNF-α的表达水平(P<0.05)。结论 SAA是高糖诱导HMCs分泌ECM及炎症反应的重要介质,通过RNA干扰(RNA interference,RANi)技术抑制SAA的表达,可减少高糖刺激下系膜细胞FN、TNF-α的积聚,减少肾小球的ECM和炎症反应,为进一步寻找DKD防治的靶点提供了新的实验依据。
【Objectives】 To observe the effect of SAA-siRNA interference on the inflammatory response and extra-cellular matrix（ECM） in high glucose-induced human mesangial cells（HMCs）,then to explore the role of serum amyloid A（SAA） in the process of diabetic kidney disease（DKD）.【Methods】 Serial subcultivation HMCs were divided into several groups after synchronization.Liposome-Lipofectamine TM 2000 was applied to SAA-siRNA transfection,which could inhibit the expression of SAA,and SAA negative-siRNA to the above cells.The transfection efficiency was observed by fluorescence inverted microscope.The mRNA expression of SAA,tumor necrosis factor-α（TNF-α） was detected by real-time RT-PCR and protein expression of SAA,TNF-α,and fibronectin（FN） was detected by ELISA.【Results】 The expression of SAA,FN,and TNF-α was inhibited by SAA-siRNA transfection in high glucose-induced HMCs（P 0.05）.【Conclusions】 SAA is important medium of inflammation and secretion of ECM induced by high glucose in HMCs.The accumulation of FN and TNF-α of HMCs in high glucose-induced HMCs could be decreased by inhibiting SAA through RNA interference（RANi）,that to mean the ECM and the inflammation of glomerulus could be decreased,which provide a new intervene target for preventing DKD.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2012年第29期31-35,共5页
China Journal of Modern Medicine
基金
辽宁省医学高峰建设工程重点科研项目(No:200938)
辽宁省教育厅资助项目(No:2008800)
