Li Xiao-dong, Li Ying, Ding Xin-guo, Gao Shan-lin, Guo Zhi-jun
Department of Nephrology, Tangshan Worker's Hospital, Tangshan, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Feb;28(2):156-9.
To explore the effects of 1, 25(OH)(2);D(3); on parathyroid hormone (PTH) induced transdifferentiation and TGF-β(1); expression in cultured human renal tubular epithelial cells.
HK-2 cells were cultured in DMEM/F12 medium supplemented with 50 mL/L FBS. Cells were divided into three groups. (1) CONTROL GROUP: without PTH or 1, 25(OH)(2);D(3);; (2) PTH group: 10(-10); mol/L PTH; (3) PTH and 1, 25(OH)(2);D(3); group: 10(-10); mol/L PTH and different concentrations of 1, 25(OH)(2);D(3); (10(-10);, 10(-9);, 10(-8); and 10(-7); mol/L). The gene expressions of α-SMA and TGF-β(1); were detected by semi-quantitative RT-PCR. The protein expressions of α-SMA and TGF-β(1); were detected by Western blot. Immunocytochemisty (ICC) was used to measure the expression of α-SMA in HK-2. ELISA was used to assay the level of TGF-β(1); in the supernatant.
The gene expressions of α-SMA and TGF-β(1); in PTH group were significantly higher than those in control group (P<0.05). In contrast, they were significantly lower in PTH and 1, 25(OH)(2);D(3); group than those in PTH group (P<0.05). Western blot results showed α-SMA could not be detected in normal HK-2 cells, which could be detected in PTH group. TGF-β(1); protein expression in PTH group was higher than that in control group. In PTH and 1, 25(OH)(2);D(3); group, α-SMA and TGF-β(1); protein expressions were significantly lower than those in PTH group (P<0.05). ICC results showed that α-SMA was hardly expressed in cells of control group. However, positive expression of α-SMA could be seen in many cells in PTH group. In PTH and 1, 25(OH)(2);D(3); group, the cells of α-SMA positive expressed were significantly less than those in PTH group (P<0.05). ELISA results showed that the level of TGF-β(1); in the supernatant of PTH group was higher than that in control group, which was also higher than that in PTH and 1, 25(OH)(2);D(3); group (P<0.05).
1, 25(OH)(2);D(3); can attenuate PTH-induced transdifferentiation and TGF-β(1); expression in cultured human renal tubular epithelial cells.
探讨1,25(OH)₂D₃对甲状旁腺激素（PTH）诱导的人肾小管上皮细胞转分化及转化生长因子-β₁（TGF-β₁）表达的影响。
HK-2细胞在含50 mL/L胎牛血清的DMEM/F12培养基中培养。细胞分为三组。（1）对照组：不添加PTH或1,25(OH)₂D₃；（2）PTH组：10⁻¹⁰ mol/L PTH；（3）PTH与1,25(OH)₂D₃组：10⁻¹⁰ mol/L PTH及不同浓度的1,25(OH)₂D₃（10⁻¹⁰、10⁻⁹、10⁻⁸和10⁻⁷ mol/L）。采用半定量逆转录聚合酶链反应（RT-PCR）检测α-平滑肌肌动蛋白（α-SMA）和TGF-β₁的基因表达。采用蛋白质印迹法检测α-SMA和TGF-β₁的蛋白表达。采用免疫细胞化学法（ICC）检测HK-2细胞中α-SMA的表达。采用酶联免疫吸附测定法（ELISA）检测上清液中TGF-β₁的水平。
PTH组α-SMA和TGF-β₁的基因表达显著高于对照组（P<0.05）。相反，PTH与1,25(OH)₂D₃组的α-SMA和TGF-β₁基因表达显著低于PTH组（P<0.05）。蛋白质印迹结果显示，正常HK-2细胞中未检测到α-SMA，而PTH组可检测到。PTH组TGF-β₁蛋白表达高于对照组。在PTH与1,25(OH)₂D₃组，α-SMA和TGF-β₁蛋白表达显著低于PTH组（P<0.05）。ICC结果显示，对照组细胞中几乎不表达α-SMA。然而，PTH组许多细胞可见α-SMA阳性表达。在PTH与1,25(OH)₂D₃组，α-SMA阳性表达的细胞明显少于PTH组（P<0.05）。ELISA结果显示，PTH组上清液中TGF-β₁水平高于对照组，也高于PTH与1,25(OH)₂D₃组（P<0.05）。
1,25(OH)₂D₃可减轻PTH诱导的人肾小管上皮细胞转分化及TGF-β₁表达。
