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目的观察人肾脏系膜细胞在血管紧张素Ⅱ(AngⅡ)刺激下mRNA稳定因子人类抗原R(HuR)蛋白对细胞周期蛋白D1(cyclinD1)蛋白的作用。方法体外培养人肾脏系膜细胞,根据AngⅡ对其的刺激时间即0、3、6、9、24 h分别记为0A、3A、6A、9A、24A组。应用流式细胞仪检测0A组与24A组细胞周期;免疫细胞化学方法观察各组HuR蛋白的亚细胞定位变化;Western blot方法检测各组全细胞与胞质内HuR蛋白及全细胞cyclinD1表达水平;RT-PCR方法检测各组cyclinD1的RNA水平。干扰RNA方法观察抑制HuR蛋白表达后AngⅡ刺激下对全细胞cyclinD1表达的保护效应。结果与0A组比较,24A组细胞增殖趋势明显;HuR蛋白亚细胞定位从胞核转移到胞质,其中3A组变化最显著;全细胞HuR蛋白表达水平不变,胞质内HuR蛋白表达增强,其中3A组最显著(P<0.05);cyclinD1蛋白表达增强,其中24A组最显著(P<0.05),而RNA水平保持不变(P>0.05)。与转染前相比,干扰RNA转染后3A组胞质HuR蛋白表达明显减弱(P<0.05);24A组cyclinD1蛋白表达明显减弱(P<0.05),而RNA水平不变(P>0.05)。结论①AngⅡ可刺激人肾小球系膜细胞增殖;②AngⅡ刺激下人肾脏系膜细胞核内HuR蛋白向胞质内转移;③HuR蛋白参与AngⅡ刺激下cyclinD1蛋白表达过程。
Objective To observe the effect of HuR protein, an mRNA stabilizing factor, on the expression of cyclin D1 protein in human renal mesangial cells stimulated by angiotensin Ⅱ (Ang Ⅱ). Methods Human mesangial cells were cultured in vitro. The stimulating time of AngⅡ was recorded as 0A, 3A, 6A, 9A, 24A respectively at 0, 3, 6, 9 and 24 hours. The cell cycle of group 0A and group 24A was detected by flow cytometry. The subcellular localization of HuR protein in each group was observed by immunocytochemistry. The expression of HuR protein and the expression of cyclinD1 in whole cell and cytoplasm were detected by Western blot. The mRNA level of cyclinD1 in each group was detected by RT-PCR. The effect of Ang Ⅱ on the expression of cyclinD1 in whole cell after HuR protein expression was observed by interfering RNA method. Results Compared with 0A group, the cell proliferation tendency was obvious in 24A group. The subcellular localization of HuR protein was transferred from the nucleus to the cytoplasm. The 3A group had the most significant change. The expression level of HuR protein in the whole cell was unchanged and the expression of HuR protein in the cytoplasm was enhanced. (P <0.05). The expression of cyclinD1 protein was enhanced in group 3A, and the level of cyclinD1 was the highest in group 3A (P <0.05), while the level of RNA remained unchanged (P> 0.05). Compared with those before transfection, the expression of HuR protein in 3A group was significantly decreased (P <0.05), while the expression of cyclinD1 in 24A group was significantly decreased (P <0.05) . Conclusions ① AngⅡ stimulates the proliferation of human glomerular mesangial cells; ② HuR protein translocates to the cytoplasm in human renal mesangial cells stimulated by AngⅡ; ③ HuR protein is involved in the expression of cyclinD1 in AngⅡ.