【作者】 刘晔；

【导师】 傅余芹；

【作者基本信息】 山东大学 ， 内科学， 2014， 硕士

【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(P<0.05),而β-catenin的水平明显增高(P<0.05),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(P<0.05),显著提高β-catenin的水平(P<0.05),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(P<0.01),相反β-catenin的表达量则明显降低(P<0.01); HG+NaHS组与NG组相比较,ZO-2、Nephrin的表达量仍有明显下降(P<0.01),相反β-catenin的表达量仍有明显升高(P<0.01)。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。更多还原

【Abstract】 ObjectiveTo observe the expressions of cystathionine-y-lyase(CSE), zona occludens-2(ZO-2), Nephrin and β-catenin in high glucose-induced mouse podocytes injury and the changes of ZO-2, Nephrin and β-catenin after treated by hydrogen sulfide, to investigate the mechanisms of high glucose-induced podocytes injury, effects of hydrogen sulfide on Diabetic Nephropathy and its most possible protective mechanisms underlying.MethodsSubculture of mouse podocytes(MPCs) in vitro were randomly divided into4groups:(1)high glucose(HG group):after treated by non-serum media for24h, each group was respectively cultured in high glucose(30mmol/L)1640media for0,12,24,48h;(2)normal glucose(NG) group;(3) normal glucose+DL-propargylglycine (PPG) group:each group was cultured for24h by serum starvation and then different concentrations of PPG intervened for48h;(4) high glucose+NaHS (HG+NaHS):each group were cultured by serum starvation for24h, the cells were disposed to1640media with30mmol/L glucose and100μmol/L NaHS for48h. Mannitol were used to adjust osmotic pressure to30mmol/L in all the groups to avoid the interference of different osmotic pressure.After treating for a certain time, the cells were collected for further detection. The expression of ZO-2, Nephrin, β-catenin and cystathionine-y-lyase(CSE) were determined by western blot.Results1. Compared with HG Oh group, the expressions of Nephrin, ZO-2and CSE were significantly reduced in the cells treated with high glucose for12,24,48h (P<0.05), while the levels of P-catenin were elevated obviously (P<0.05), four proteins changed in a time-dependent manner.2. Compared with NG group, the levels of ZO-2, Nephrin were decreased significantly in normal glucose groups cultured with different concentrations of PPG (P<0.05). In contrast, the levels of β-catenin elevated significantly (P<0.05). Three proteins presented greater variation with different concentration of PPG.3. Compared with HG group, high glucose-induced podocytes treated by NaHS induced a more significant expressions of ZO-2and Nephrin (P<0.01), whereas there was a severe reduction of β-catenin in HG+NaHS group (P<0.01). Relative to NG group, the expressions of ZO-2and Nephrin in HG+NaHS group were decreased obviously (P<0.01), the level of β-catenin in HG+NaHS group was higher than that in NG group (P<0.01)ConclusionsOur results suggested high glucose induced mouse podocytes injury by reducing ZO-2expression, excessive activation of Wnt/β-catenin pathway and decreasing the level of Nephrin in a time-dependent manner. On the other side, downregulation of CSE contributes to hyperglycemia induced podocyte injury also in a time-dependent way. That suggested the reduction of endogenous H2S triggered by the CSE decline probably is one of mechanisms in high glucose induced podocytes injury. Exogenous H2S protects against hyperglycemia induced podocyte injury possibly through upregualtion of ZO-2and inhibition of Wnt/β-catenin pathway, and then reducing the loss of Nephrin.更多还原