1,25(OH)_2D_3对甲状旁腺素诱导的肾小管上皮细胞转分化和TGF-β_1的表达的影响

Effects of 1,25(OH)_2D_3 on parathyroid hormone induced transdifferentiation and TGF-β_1 expression in cultured human renal tubular epithelial cells

目的:探讨1,25(OH)2D3对甲状旁腺素(PTH)诱导的肾小管上皮细胞转分化和转化生长因子-β1(TGF-β1)表达的影响。方法:人肾小管上皮细胞(HK-2细胞)培养在含50 mL/L FCS的DMEM/F12培养液中。对照组:加入等体积含50 mL/L FCS的DMEM/F12培养液;PTH刺激组:加入终浓度为10-10 mol/L PTH的含50 mL/L FCS的DMEM/F12培养液;PTH+1,25(OH)2D3干预组:加入10-10 mol/L PTH,同时加入不同浓度(10-10、10-9、10-8、10-7 mol/L)的1,25(OH)2D3。刺激HK-2细胞48 h。半定量RT-PCR法检测细胞中α-平滑肌肌动蛋白(α-SMA)和TGF-β1的基因表达;Western blot法检测细胞中α-SMA和TGF-β1的蛋白表达;免疫细胞化学法检测细胞中α-SMA的表达;ELISA法检测细胞培养上清液中TGF-β1的含量。结果:半定量RT-PCR结果显示,对照组HK-2细胞中几乎无α-SMA的mRNA表达,仅有少量的TGF-β1 mRNA表达;PTH刺激组α-SMA和TGF-β1mRNA表达量与对照组比较明显增加;PTH+1,25(OH)2D3干预组表达量比PTH刺激组显著降低,且随着1,25(OH)2D3浓度的升高呈一定的剂量依赖性(P<0.05)。Western blot结果显示,对照组HK-2细胞中无α-SMA的蛋白表达,仅有少量的TGF-β1蛋白表达;10-10 mol/L的PTH能够明显诱导HK-2细胞中α-SMA的蛋白表达,增加TGF-β1的蛋白表达量;PTH+1,25(OH)2D3干预组,α-SMA和TGF-β1的蛋白表达量比PTH刺激组显著降低(P<0.05)。免疫细胞化学法结果显示,对照组几乎无α-SMA阳性表达的细胞,PTH刺激组可见大量细胞α-SMA表达阳性;PTH+1,25(OH)2D3干预组α-SMA表达阳性的细胞数明显低于PTH刺激组(P<0.05)。ELISA结果显示,对照组细胞上清液中可检测到少量的TGF-β1,PTH刺激组含量显著升高,PTH+1,25(OH)2D3干预组与PTH刺激组比较含量明显降低(P<0.05)。结论:1,25(OH)2D3能够部分拮抗PTH诱导的HK-2细胞转分化和TGF-β1的表达。

AIM： To explore the effects of 1,25（OH）2D3 on parathyroid hormone（PTH） induced transdifferentiation and TGF-β1 expression in cultured human renal tubular epithelial cells.METHODS： 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）2D3;（2） PTH group： 10-10 mol/L PTH;（3） PTH and 1,25（OH）2D3 group： 10-10 mol/L PTH and different concentrations of 1,25（OH）2D3（10-10,10-9,10-8 and 10-7 mol/L）.The gene expressions of α-SMA and TGF-β1 were detected by semi-quantitative RT-PCR.The protein expressions of α-SMA and TGF-β1 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-β1 in the supernatant.RESULTS： The gene expressions of α-SMA and TGF-β1 in PTH group were significantly higher than those in control group（P0.05）.In contrast,they were significantly lower in PTH and 1,25（OH）2D3 group than those in PTH group（P0.05）.Western blot results showed α-SMA could not be detected in normal HK-2 cells,which could be detected in PTH group.TGF-β1 protein expression in PTH group was higher than that in control group.In PTH and 1,25（OH）2D3 group,α-SMA and TGF-β1 protein expressions were significantly lower than those in PTH group（P0.05）.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）2D3 group,the cells of α-SMA positive expressed were significantly less than those in PTH group（P0.05）.ELISA results showed that the level of TGF-β1 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）2D3 group（P0.05）.CONCLUSION： 1,25（OH）2D3 can attenuate PTH-induced transdifferentiation and TGF-β1 expression in cultured human renal tubular epithelial cells.