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目的:研究肾损伤分子1(KIM-1)与高糖环境下人近端肾小管上皮细胞(HK-2)自噬的关系,探讨KIM-1参与糖尿病肾病进展的可能机制。方法:体外培养HK-2分五组:(1)对照组(D-葡萄糖5.6 mmol/L);(2)高渗组(D-葡萄糖5.6 mmol/L+D-甘露醇24.4 mmol/L);(3)高糖组(D-葡萄糖30 mmol/L);(4)高糖+KIM-1siRNA组;(5)高糖+siRNA对照组。分别于培养8h、16h、24h进行测定。Western印迹法检测细胞KIM-1、自噬标志蛋白微管相关蛋白l轻链3Ⅱ型(LC3Ⅱ)蛋白的表达;实时定量PCR法(real-time PCR)法检测细胞KIM-1、LC3ⅡmRNA的表达。透射电镜下观察细胞内自噬体的形成。结果:与对照组相比,高糖组细胞KIM-1、LC3Ⅱ蛋白及mRNA表达呈时间依赖性增加(P<0.05),细胞内自噬体形成数量呈时间依赖性增多(P<0.05)。与高糖组相比,KIM-1 siRNA转染组细胞KIM-1、LC3Ⅱ蛋白及mRNA表达显著减少(P<0.05),自噬体形成数量减少(P<0.05)。结论:下调KIM-1的表达能显著抑制高糖条件下肾小管上皮细胞LC3Ⅱ的表达和自噬体的形成,提示KIM-1可能通过调控高糖环境下肾小管上皮细胞自噬参与糖尿病肾病的进展。
Objective: To investigate the relationship between KIM-1 and autophagy in proximal human proximal tubular epithelial cells (HK-2) under high glucose conditions and to explore the possible mechanism of KIM-1 involved in the progression of diabetic nephropathy. METHODS: HK-2 cells were cultured in vitro in five groups: (1) control group (5.6 mmol / L D-glucose); (2) hypertonic group (5.6 mmol / L D-glucose + 24.4 mmol / L D-mannitol) ; (3) high glucose group (D-glucose 30 mmol / L); (4) high glucose + KIM-1siRNA group; Respectively cultured 8h, 16h, 24h determination. Western blotting was used to detect the expression of KIM-1 and LC3Ⅱprotein, and the expression of LC3Ⅱ and KIM-1 mRNA was detected by real-time PCR. The formation of autophagosomes was observed under transmission electron microscope. Results: Compared with the control group, the protein and mRNA expressions of KIM-1 and LC3Ⅱ in high glucose group increased in a time-dependent manner (P <0.05) and the number of autophagosomes increased in a time-dependent manner (P <0.05). Compared with high glucose group, the expression of KIM-1 and LC3Ⅱ protein and mRNA in KIM-1 siRNA transfected group decreased significantly (P <0.05) and the number of autophagosome decreased (P <0.05). CONCLUSION: Down-regulation of KIM-1 expression significantly inhibits the expression of LC3II and autophagosome formation in renal tubular epithelial cells under high glucose conditions, suggesting that KIM-1 may be involved in the pathogenesis of diabetic nephropathy by regulating autophagy of renal tubular epithelial cells under high glucose conditions progress.