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目的:探讨维生素D3抑制肾小管上皮细胞纤维化的作用和可能的机制,为临床有效防治肾小管上皮细胞纤维化提供理论基础。方法:低氧处理后Western blot检测肾小管上皮细胞系HKC细胞中E-cadherin,α-SMA的表达。不同浓度维生素D3作用于HKC细胞,real time PCR及Western blot检测的HKC细胞中Runx3的mRNA表达水平和蛋白表达水平。构建RUNX3基因的小干扰RNA载体并将其转染HKC肾小管上皮细胞,经G418筛选获得稳定表达的细胞系。Western blot检测维生素D3对转染后的低氧处理的HKC细胞EMT的影响。结果:低氧处理后的HKC细胞上皮标志物E-cadherin表达显著下降,而间皮标志物α-SMA的表达显著增加,提示HKC细胞发生EMT。不同浓度的维生素D3作用HKC中Runx3的mRNA水平和蛋白水平表达逐渐增加。成功构建了RUNX3的小干扰RNA载体并将其转染HKC细胞;筛选到稳定的敲除RUNX3的肾小管上皮细胞系。结果显示,与对照组相比,维生素D3不能抑制敲除RUNX3的HKC细胞的EMT的发生。结论:维生素D3通过翻译水平正性调控RUNX3的表达进而抑制肾小管上皮细胞的EMT,从而抑制肾脏纤维化的发生。
OBJECTIVE: To investigate the effect of vitamin D3 on renal tubular epithelial cell fibrosis and its possible mechanism, providing a theoretical basis for effective prevention and treatment of renal tubular epithelial cell fibrosis. Methods: The expressions of E-cadherin and α-SMA in renal tubular epithelial cell line HKC were detected by Western blot after hypoxic treatment. HKC cells were treated with different concentrations of vitamin D3. Runx3 mRNA and protein levels in HKC cells were detected by real time PCR and Western blot. The small interfering RNA vector of RUNX3 gene was constructed and transfected into HKC renal tubular epithelial cells, and stable cell line was obtained by G418 screening. Western blot was used to detect the effect of vitamin D3 on the EMT of transfected hypoxic-treated HKC cells. Results: The expression of E-cadherin in HKC cells was significantly decreased after hypoxia treatment, while the expression of mesothelial marker α-SMA was significantly increased, suggesting that EMT occurred in HKC cells. Different concentrations of vitamin D3 role in HKC Runx3 mRNA and protein levels gradually increased. The small interfering RNA vector of RUNX3 was successfully constructed and transfected into HKC cells. The stable renal tubular epithelial cell line that knocked out RUNX3 was screened out. The results showed that vitamin D3 can not inhibit the EMT of HKC cells knocking out RUNX3 compared with the control group. Conclusion: Vitamin D3 can inhibit renal tubular epithelial cells EMT by regulating the expression of RUNX3 at the level of translation, thereby inhibiting the occurrence of renal fibrosis.