STAT1siRNA转染对高糖培养人肾小球系膜细胞STAT3及转化生长因子β1表达的影响  

Impact of STAT1- siRNA transfection on the expressions of STAT3 and transforming growth factor β1 in human glomerulur mesangial cells induced by high glucose

作　　者：郑晓玉[1] 焦素敏[1] 王力宁[1] 陈莹[1] 杨雪[1] 张瑾[1] 杨爽[1] 高鑫然 范秋灵[1] 

机构地区：[1]中国医科大学附属第一医院肾内科,沈阳110001

出　　处：《中华肾脏病杂志》2013年第1期39-43,共5页Chinese Journal of Nephrology

基　　金：国家973课题（2007CB507405）;国家自然科学基金（81270808）

摘　　要：目的观察信号转导与转录激活子1（STAT1）siRNA转染后高糖培养人肾小球系膜细胞（HMC）的增殖情况及细胞内STAT1、磷酸化（p）STAT1、STAT3、p-STAT3蛋白及下游TGF-β1的表达变化，探讨高糖状态下STAT1和STAT3活性变化及STAT亚型变化对TGF-β1的影响。方法设计并合成针对STAT1基因的3个特异性siRNA序列（STAT1-siRNA），应用Lipofeetamine2000转染试剂将STAT1-siRNA转染入HMC。激光共聚焦显微镜鉴定转染效率，应用Western印迹、实时定量PCR法筛选最有效抑制STATl表达的干扰序列用于后续实验。转染有效干扰序列24h后，用25mmol／L葡萄糖刺激24、48、72h，M1vr法检测各组系膜细胞的增殖情况；Western印迹法检测各组细胞STAT1、p-STATl、STAT3、p-STAT3蛋白的表达；ELISA法检测TGF-β1的表达。结果与对照组相比，高糖可刺激HMC增殖，HMC的P-STAT1、P-STAT3及TGF-β1表达上调（P〈0．05）。转染STAT1-siRNA后高糖刺激HMC，p-STAT3及TGF-β1的表达进一步增高（P〈0．05）。结论高糖可以通过磷酸化方式激活HMC的JAK-STAT信号转导通路。转染STATI-siRNA后，系膜细胞增殖增多，STAT3活性增强。高糖促进HMC分泌TGF--β1，转染STAT1-siRNA后，TGF-β1分泌进一步增加，与肾脏纤维化有关。Objective To observe the cell proliferation and the protein expression of STAT1, phosphorylation of STAT 1 （p- STAT 1）, STAT3, p - STAT3 and transforming growth factor β1 （TGF- β1） in human glomerulur mesangial cells （HMCs） induced by high glucose after STATI-siRNA transfection. Methods Three STAT1- siRNA sequences were designed and synthetized. HMCs in 6-well plate were transiently transfected with STAT1- siRNA using Lipofectamine 2000. After transfection for 48 h or 72 h, STATI mRNA and protein expression were detected by real-time PCR and Western blotting, respectively, to choose the effective sequence in later experiments. After transfection for 24 h and stimulated with 25 mmol/L glucose for 24 h, 48 h, 72 h, cell proliferation was measured by MTT assays, the protein expressions of STAT1, p-STAT1, STAT3 and p-STAT3 were detected by Western blotting, the expression of TGF- β1 was detected by ELISA in each group. Results High glucosecould stimulate HMCs proliferation. The protein expressions of p-STAT1, p-STAT3 and TGF- β1 were increased in the group stimulated by high glucose （P 〈 0.05）. The protein expressions of p-STAT3 and TGF- β1 were further increased in HMCs induced by high glucose after STATI- siRNA transfection （P 〈 0.05）. Conclusions Under high glucose conditions, JAK-STAT signal transduetion pathway of HMCs can be activated, then it is far greater when HMCs are induced by high glucose after STAT1- siRNA transfection. The secretion of TGF- β1 is increased in HMCs under the state of high glucose, and it is further increased after STATI-siRNA transfection, which is related to the kidney fibrosis.

关 键 词：转化生长因子Β1 转染 RNA 小分子干扰 肾小球系膜细胞 高糖 信号转导与转录激活子 

分 类 号：R587.2[医药卫生—内分泌] R692[医药卫生—内科学]