甲状旁腺素对人肾小管上皮细胞细胞外基质合成与降解的影响

Effects of parathyroid hormone on synthesis and degradation of extracellular matrix in cultured human renal tubular epithelial cells

目的 研究甲状旁腺素（PTH）对人肾小管上皮细胞（HK-2）合成分泌Ⅲ型胶原、纤连蛋白以及对纤溶酶原激活物抑制物1（PAI-1）、基质金属蛋白酶1（MMP-1）、金属蛋白酶1组织抑制剂（TIMP-1）基因表达的影响,以探讨PTH在肾间质纤维化发病机制中的作用.方法 HK-2细胞培养于含5%FBS的DMEM-F12培养液中,加入终浓度为0、10^-12、10^-11、10^-10、10^-9、10^-8 mol/L PTH的培养液刺激HK-2细胞48 h,或10^-8 mol/L PTH刺激HK-2细胞不同时间（0、12、24、48、72 h）.应用半定量RT-PCR法检测细胞中Ⅲ型胶原、纤连蛋白、PAI-1、MMP-1、TIMP-1基因的表达;Western印迹法检测HK-2细胞中Ⅲ型胶原的蛋白表达;ELISA法检测细胞培养上清液中纤连蛋白的含量.结果 PTH呈剂量和时间依赖性促进HK-2细胞中Ⅲ型胶原、纤连蛋白、PAI-1、TIMP-1 mRNA的表达,抑制MMP-1 mRNA的表达,MMP-1/TIMP-1比值降低.PTH呈剂量和时间依赖性增加HK-2细胞中Ⅲ型胶原的蛋白表达及细胞培养上清液中纤连蛋白的含量.结论 PTH通过促进HK-2细胞中PAI-1、TIMP-1的基因表达,抑制MMP-1的基因表达,导致细胞外基质合成增加而降解减少.

Objective To investigate the effects of parathyroid hormone （PTH） on the synthesis and secretion of collagen Ⅲ and fibronectin （FN）, and the expressions of plasminogen activator inhibitor-1 （PAI-1）, matrix metalloproteinase-1 （MMP-1） and tissue inhibitor of metalloproteinase-1 （TIMP-1） mRNA in cultured human renal tubular epithelial cells （HK-2）.Methods HK-2 cells were cultured in DMEM-F12 medium supplemented with 5% FBS. Cells were exposed to different concentrations of PTH （0, 10^-12, 10^-11, 10^-10, 10^-9, 10^-8 mol/L） for 48 h, or 10^-8 mol/L PTH at different time （0, 12, 24, 48, 72 h）. The gene expressions of collagen Ⅲ,FN, PAI-1, MMP-1, and TIMP-1 were detected by semi-quantitative RT-PCR. The protein expression of collagen Ⅲ was detected by Western blotting. The level of FN in the supernatant was assayed by enzyme linked immunosorbent assay （ELISA）. Results PTH increased gene expressions of collagen Ⅲ, FN, PAI-1 and TIMP-1 in a dose- and time-dependent manner, but decreased MMP-1 gene expression. Then the ratio of MMP-1/TIMP-1 was decreased. PTH increased the collagen Ⅲ protein expression in cultured HK-2 cells and the level of FN in the supernatant of cultured HK-2 cells in a dose- and time-dependent manner. Conclusion PTH can up-regulate PAI-1, TIMP-1 gene expressions, and down-regulate MMP-1 gene expression,resulting in elevation of extracellular matrix （ECM） synthesis and reductim of degradation.