高糖对小鼠足突细胞的损伤及硫化氢干预的影响

发布时间：2018-04-18 21:24

本文选题：糖尿病肾病 + 硫化氢　；参考：《山东大学》2014年硕士论文

【摘要】：目的观察高糖诱导的小鼠足突细胞损伤中胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)、紧密连接蛋白2(ZO-2)、裂孔蛋白(Nephrin)及β-连环蛋白(β-catenin)的表达变化及硫化氢干预后ZO-2、Nephrin和β-catenin蛋白水平的变化,探讨高糖诱导的足突细胞损伤机制、硫化氢对糖尿病肾脏疾病的作用及其保护机制。方法将体外传代培养的小鼠足突细胞(MPCs)系分为(1)高糖组(HG组)：各组细胞脱血清饥饿24h后,用含3Ommol/L葡萄糖的1640培养基分别刺激0、12、24、48h；(2)正常糖组(NG组)；(3)正常糖+DL-炔丙基甘氨酸(DL-propargylglycine, PPG)组(NG+PPG组)：各组细胞脱血清饥饿24h后,用含不同浓度PPG的1640培养基干预48h；(4)高糖+NaHS组(HG+NaHS组)：各组细胞脱血清饥饿24h后,用含3Ommol/L葡萄糖和100μmol/L NaHS的1640培养基处理48h。以上各组细胞均用不同浓度的甘露醇将渗透压调至3Ommol/L,避免渗透压的差异对本实验结果的影响。处理后,用Western blot检测各组细胞CSE、ZO-2、Nephrin及β-catenin蛋白水平表达差异。结果 1.与高糖Oh组相比,高糖处理12h、24h、48h均显著降低CSE、Nephrin、ZO-2的表达(P0.05),而β-catenin的水平明显增高(P0.05),4种蛋白变化均表现出时间依赖性； 2.与正常糖组相比,正常糖培养加不同浓度的PPG可显著抑制ZO-2、Nephrin的表达(P0.05),显著提高β-catenin的水平(P0.05),3种蛋白变化呈浓度依赖性； 3.与高糖组相比,高糖诱导的足突细胞加NaHS培养后,ZO-2、Nephrin的表达量有明显增多(P0.01),相反β-catenin的表达量则明显降低(P0.01); HG+NaHS组与NG组相比较,ZO-2、Nephrin的表达量仍有明显下降(P0.01),相反β-catenin的表达量仍有明显升高(P0.01)。结论本研究结果提示高糖对足突细胞的损伤可能是通过降低ZO-2表达、过度激活Wnt/β-catenin通路、减少Nephrin的表达量来实现的,ZO-2、β-catenin、Nephrin三种蛋白的变化表现出时间依赖性,同时高糖诱导的足突细胞CSE表达降低,也具有时间依赖性,提示CSE降低引起的内源性硫化氢释放减少可能是足突细胞损伤的重要机制之一。而外源性硫化氢对高糖诱导的足突细胞损伤具有一定保护作用,其机制可能与增加ZO-2表达、抑制Wnt/β-catenin通路,进而减少Nephrin的丢失有关。
[Abstract]:PurposeTo observe the changes of cystathionine- 纬 -lyase (CSEN), tight junction protein 2ZO-2, rifting protein Nephrinin (Nephrinin) and 尾 -catenin (尾 -catenin) and the changes of ZO-2Nephrin and 尾 -catenin protein levels in mouse podocyte cells induced by high glucose, and the changes of ZO-2Nephrin and 尾 -catenin protein levels after hydrogen sulfide intervention.To investigate the mechanism of high glucose induced injury of podocyte, the effect of hydrogen sulfide on diabetic kidney disease and its protective mechanism.MethodMouse podocyte cells (MPCs1) were divided into high glucose group (HG group) and high glucose group (HG group). The cells in each group were deprived of serum for 24 hours after starvation.The 1640 medium containing 3Ommol/L glucose was used to stimulate the 1640 medium containing 3Ommol/L glucose for 48 h respectively) the normal glucose group was treated with DL-propargyl glycine (DL-propargyl glycine) and the normal glucose group (DL-propargyl glycine): after 24 hours of starvation, the cells in each group were starved for 24 hours after deserting.1640 medium containing different concentrations of PPG was used to intervene for 48 h. HG NaHS group in high glucose NaHS group was treated with 1640 medium containing 3Ommol/L glucose and 100 渭 mol/L NaHS for 48 h.The osmotic pressure was adjusted to 3Ommol / L with different concentrations of mannitol to avoid the effect of the difference of osmotic pressure on the results of the experiment.After treatment, Western blot was used to detect the expression of ZO-2Nephrin and 尾 -catenin protein.Result1.Compared with the high glucose group, the protein expression of Nephrininine ZO-2 in CSE was significantly decreased at 24 h or 48 h after treatment with high glucose, while the level of 尾 -catenin was significantly higher than that in the control group, and the changes of 尾 -catenin were all time-dependent.2.Compared with the normal glucose group, normal glucose culture and different concentrations of PPG could significantly inhibit the expression of ZO-2Nephrin and increase the level of 尾 -catenin in a concentration-dependent manner.3.Compared with the high sugar group,On the contrary, the expression of 尾 -catenin in HG NaHS group was significantly lower than that in NG group, but the expression of 尾 -catenin in HG NaHS group was still significantly lower than that in NG group, but the expression of 尾 -catenin was still significantly increased in HG NaHS group compared with NG group.ConclusionThe results suggest that the changes of ZO-2 and 尾 -catenin in podocyte may be time-dependent by decreasing the expression of ZO-2, over-activating the Wnt/ 尾 -catenin pathway and reducing the expression of Nephrin.At the same time, the decrease of CSE expression in podocyte induced by high glucose is also time-dependent, which suggests that the decrease of endogenous hydrogen sulfide release induced by the decrease of CSE may be one of the important mechanisms of podocyte injury.The exogenous hydrogen sulfide may protect podocyte from high glucose induced podocyte injury by increasing ZO-2 expression, inhibiting Wnt/ 尾 -catenin pathway and reducing the loss of Nephrin.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

【参考文献】