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

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B1（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NRK49F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G0／G1期（与TGF-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TGF-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。

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1

OBJECTIVE: 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 dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-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-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. 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-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfetion of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-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 tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.