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目的 观察高糖刺激大鼠系膜细胞后微小RNA-148b(miR-148b)的表达变化,及其对靶基因腺苷酸活化的蛋白激酶α1(AMPKα1)的调控作用和对细胞外基质蛋白分泌的影响.方法 大鼠系膜细胞分3组:正常糖组(5.5 mmol/L葡萄糖)、高渗组(5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇)和高糖组(25.0 mmol/L葡萄糖),实时定量PCR检测miR-148b的表达.转染miR-148b抑制物进一步探讨靶向抑制miR-148b的作用.实时定量PCR检测AMPKα1 mRNA的表达;Western印迹法检测AMPKα1、葡萄糖调节蛋白78 (GRP78)、C/EBP同源蛋白(CHOP)、纤连蛋白(FN)、Ⅳ型胶原(ColⅣ)蛋白的表达;免疫荧光法检测ColⅣ的表达.结果 高渗组与正常对照组miR-148b表达差异无统计学意义(P＞0.05);与正常对照组比较,高糖组细胞内miR-148b的表达上调,GRP78、CHOP、FN、ColⅣ表达均显著上调(均P＜0.05),AMPKα1 mRNA及蛋白表达均下调(均P＜0.01).与高糖组比较,转染miR-148b抑制物的高糖组miR-148b及GRP78、CHOP、FN、ColⅣ蛋白表达均显著下调(均P＜0.05),AMPKα1mRNA及蛋白表达均上调(均P＜0.05);转染miR-148b阴性对照的高糖组上述指标与高糖组相比差异均无统计学意义(均P＞ 0.05).结论 高糖可上调体外系膜细胞miR-148b的表达,靶向抑制AMPKα1的表达,从而诱导内质网应激及系膜细胞产生过多细胞外基质蛋白.抑制miR-148b可上调靶基因AMPKα1的表达,从而抑制高糖诱导的系膜细胞内质网应激亢进,减少细胞外基质蛋白的聚积.

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells,and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion.Methods Rat mesangial cells were divided ino 3 groups:normal glucose (NG,5.5 mmol/L glucose) group,hypertonic (MA,5.5 mmol/L glucose+19.5 mmol/L mannitol) group and high-glucose (HG,25.0 mmol/L glucose) group.MiR-148b expression was detected by real time PCR.Then miR-148b inhibitor was transfected to rat mesangial cells.Their protein expressions of AMPKα1,glucose regulated protein 78 (GRP78),C/EBP homologous protein (CHOP),fibronectin (FN) and collagen Ⅳ were detected by Western blotting.The expression of AMPKα1 mRNA was detected by real time PCR.The expression of collagen Ⅳ was also detected by immunofluorescence.Results Compared with NG group,HG group showed up-regulated miR-148bexpression,down-regulated AMPKαl mRNA and protein expressions,and up-regulated CHOP,GRP78,collagen Ⅳ and FN expressions (all P ＜ 0.05).HG-induced mesangial cells with miR-148binhibitor had up-regulated AMPKα1 mRNA and protein expressions,and down-regulated CHOP,GRP78,collagen Ⅳ,FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P ＜ 0.05).Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells,then activate endoplasmic reticulum stress to induce extracellular matrix excretion.MiR-148b inhibitor up-regulates AMPKα1 expression,inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.