抗增殖蛋白抑制转化生长因子-β1诱导的肾脏成纤维细胞增殖和表型改变

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-β1

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。

Objeαive To illuminate the possible role of Prohibitin （PHB） in tubulointerstitial fibrosis. Methods （1）Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis ,26 male and 22 female, aged 7.5 ± 5.5 （2.5-13 years）, and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney disseαed during operation as normal control. Immunohistochemistry was used to deteα the protein expression of PHB and α-smooth muscle aαin （α-SMA）. The correlation between PHB and degree of tubulointerstitial lesion was compared. （2） Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-β1 stimulation were deteαed by Western blotting and RT-PCR analysis. （3）PHB expression plasmid was construαed and transfeαed into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to deteα the PHB and α-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-β1. Results （ 1 ） PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB （ all P 〈 0.01 ）, and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions （ r = - 0. 802,P 〈 0.01 ）. （2） Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-β1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both timedependently and dose-dependently （all P 〈0.01 ）. （3）A recombinant pcDNA3.1 （ - ）/PHB plasmid was successfully construαed. PHB protein expression in the transfeαed NRK-49F cells was 2.54 times higher compared with the non-transfeαed cells. （4） The proportions of the cells in the S and G2/M phases were higher in the NRK-49F cells stimulated by TGF-β1, however, more NRK-49F cells remained in the G0/G1 phase after transfetion of PHB （P 〈0.01 ）. （5）Both α-SMA protein and mRNA were not expressed in the control cells while de novo expression of α-SMA in the NRK-49F cells was increased after the treatment of TGF-β1. Over-expression of PHB did not affeα he basic α-SMA expression but dramatically repressed TGF- β1-initiated α-SMA expression in the NRK-49F cells （P 〈 0.01 ）. Conclusion PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitia） lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-β1.