论文部分内容阅读
目的研究抗增殖蛋白(prohibitin,PHB)在肾间质纤维化发生中的作用。方法 (1)检测48例原发性肾小球肾炎患儿肾组织中 PHB 蛋白表达,并比较其与肾小管间质损伤程度的相关性。(2)观察 PHB 在大鼠肾脏成纤维细胞(NRK-49F)中亚细胞定位,以 Western 印迹和 RT-PCR 测定NRK-49F 受到转化生长因子β1(TGF-β1)刺激后 PHB 表达的变化。(3)构建 PHB 表达质粒并转染,观察 PHB 对 NRK-49F 细胞周期以及表达α-平滑肌肌动蛋白(α-SMA)蛋白质和 mRNA 的影响。结果(1)PHB 蛋白主要表达于肾间质细胞和肾小管上皮细胞的胞质,随肾小管间质损伤程度加重而逐渐减弱(组间比较,均 P<0.01),PHB 表达量与肾小管间质损伤程度显著负相关(r=-0.802,P<0.01)。(2)激光共焦显微镜下见 PHB 主要分布于 NRK-49F 的细胞质,细胞核亦有较弱表达。TGF-β1刺激后 PHB 蛋白和 mRNA 表达均下调,呈现时间和剂量依赖关系(组间比较,P<0.01)。(3)成功构建 PHB 真核表达质粒,转染48h 细胞中 PHB 蛋白量升高约2.54倍(与未转染组比较,P<0.01)。(4)转染 PHB 基因明显抑制 TGF-β1所诱导的细胞增殖,使更多的细胞处于 G_0/G_1期(与FGF-β1组比较,P<0.01),而对未受刺激的细胞无影响(P>0.05)。(5)转染 PHB 基因明显抑制TGF-β1所诱导的α-SMA 蛋白质和 mRNA 表达(与 TGF-β1组比较,P<0.01),而对α-SMA 基础表达无影响(与 FGF-β1组比较,P>0.05)。结论 PHB 在肾组织中的表达水平可以反映肾小管间质损伤程度,外源性 PHB 显著抑制 TGF-β1诱导的成纤维细胞增殖和表型改变。
Objective To study the role of prohibitin (PHB) in the pathogenesis of renal interstitial fibrosis. Methods (1) To detect the expression of PHB in renal tissue of 48 children with primary glomerulonephritis and to compare their correlation with the degree of tubulointerstitial injury. (2) The subcellular localization of PHB in rat renal fibroblasts (NRK-49F) was observed. The changes of PHB expression of NRK-49F stimulated by TGF-β1 were detected by Western blotting and RT-PCR. (3) PHB expression plasmid was constructed and transfected to observe the effect of PHB on the cell cycle of NRK-49F cells and the expression of α-smooth muscle actin (α-SMA) protein and mRNA. Results (1) PHB protein was mainly expressed in the cytoplasm of renal interstitial cells and renal tubular epithelial cells, and gradually weakened with the degree of tubulointerstitial injury (all P <0.01) The degree of interstitial injury was significantly negatively correlated (r = -0.802, P <0.01). (2) Under the laser confocal microscope, PHB mainly distributed in the cytoplasm of NRK-49F, and the nucleus also showed weaker expression. After TGF-β1 stimulation, PHB protein and mRNA expression were down-regulated, showing a time-and dose-dependent relationship (between groups, P <0.01). (3) The PHB eukaryotic expression plasmid was successfully constructed, and the PHB protein content increased about 2.54 times (P <0.01 compared with untransfected cells) 48 h after transfection. (4) Transfection of PHB gene significantly inhibited the proliferation of cells induced by TGF-β1, and more cells in G_0 / G_1 phase (P <0.01 compared with FGF-β1 group), but had no effect on unstimulated cells (P> 0.05). (5) Transfection of PHB gene significantly inhibited α-SMA protein and mRNA expression induced by TGF-β1 (compared with TGF-β1 group, P <0.01), but had no effect on the basic expression of α-SMA Compare, P> 0.05). Conclusion The expression level of PHB in renal tissue can reflect the degree of tubulointerstitial injury. Exogenous PHB can significantly inhibit the proliferation and phenotype of fibroblast induced by TGF-β1.