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目的探讨尿毒症患者血清对人肾小管上皮细胞株(HK-2)转化生长因子-β1(TGF-β1)基因表达和蛋白分泌的影响。方法收集广东医学院附属医院2002-022003-03在无菌条件下的40份尿毒症病人血清和20名正常人血清,应用超滤离心装置将尿毒症患者血清分离成分子质量>10000u,5000~10000u,<5000u3个组分。应用酶联免疫吸附试验(ELISA)检测HK-2细胞培养上清液TGF-β1蛋白分泌量;逆转录聚合酶链反应(RT-PCR)方法检测TGF-β1mRNA表达。结果不同浓度尿毒血清组TGF-β1基因表达和蛋白分泌量均较正常对照组增高,以10%尿毒血清组最高;分子质量>10000u的组分TGF-β1基因表达和蛋白分泌量最高,分子质量5000~10000u的组分TGF-β1基因表达轻度增高;分子质量<5000u的组分TGF-β1基因表达和蛋白分泌与正常对照组相比差异无显著性。结论尿毒症毒素通过影响HK-2细胞TGF-β1基因表达和蛋白分泌从而在人肾小管-间质纤维化中起重要作用,而以分子质量大于10000u的尿毒症毒素起主要作用。
Objective To investigate the effect of serum of uremic patients on the gene expression and protein secretion of transforming growth factor-β1 (TGF-β1) in human renal tubular epithelial cell line (HK-2). Methods The serum of 40 patients with uremia and 20 normal human serum under aseptic conditions were collected. The serum of uremic patients was separated by ultrafiltration centrifugation into molecular masses> 10000u, 5000 ~ 10000u, <5000u3 components. The secretion of TGF-β1 in HK-2 cell culture supernatant was detected by enzyme linked immunosorbent assay (ELISA). The expression of TGF-β1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) Results The levels of TGF-β1 gene and protein secretion in uremic serum at different concentrations were higher than those in normal control group, and were highest in 10% uremic serum group. TGF-β1 gene expression and protein secretion were the highest at molecular weight> 10000u, and molecular weight The expression of TGF-β1 gene of 5000 ~ 10000u was slightly increased. The expression of TGF-β1 gene and protein secretion of molecular weight <5000u had no significant difference compared with the normal control group. Conclusions Uremic toxins play an important role in human tubulointerstitial fibrosis by affecting TGF-β1 gene expression and protein secretion in HK-2 cells, whereas uremic toxins with a molecular mass greater than 10000u play a major role.