贵州医科大学学报

2015, v.40;No.175(04) 330-336

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DM大鼠肾组织Id2蛋白表达变化与纤维化病变发生发展关系的研究
Study on the Relationship of Changes of Id2 Protein Expression in Diabetic Rat Kidney Tissues with Occurrence and Development of Fibrosis Lesions

文箐颍;苏博;肖瑛;王圆圆;李霜;刘丽荣;石明隽;郭兵;
WEN Qingying;SU Bo;XIAO Ying;WANG Yuanyuan;LI Shuang;LIU Lirong;SHI Mingjun;GUO Bing;Department of Pathophysiology,Guiyang Medical College;

摘要(Abstract):

目的:动态观察分化抑制因子2(Id2)在糖尿病大鼠血糖控制前后肾小管上皮细胞的表达变化,探讨其在肾小管上皮细胞向间充质细胞转分化过程(EMT)中可能发挥的作用。方法:雄性SD大鼠随机分为正常对照组(N)、糖尿病组(DM),糖尿病胰岛素治疗组(DMT),采用链脲佐菌素(55 mg/kg)尾静脉注射复制Ⅰ型糖尿病大鼠模型,N组和DM组成模后分别于2周(2 w)、8周(8 w)、16周(16 w)、24周(24 w)时各处死6只;成模13周(13 w)起,DMT组大鼠采用胰岛素个体化治疗,以血糖控制在4~7 mmol/L,尿糖阴性为准,分别于16 w和24 w时处死6只大鼠;观察大鼠血糖、24 h尿蛋白并计算肾脏指数,HE、PAS染色光镜下观察肾组织结构的变化,免疫组化观察大鼠肾脏纤维连接蛋白(FN)的蛋白表达情况,Western blotting检测大鼠肾组织分化抑制因子(Id2)、E-钙粘素(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的蛋白变化,RT-PCR检测肾组织Id2 mRNA表达情况。结果:(1)与N组相比,不同时间点DM组血糖、24 h尿蛋白量、肾脏指数均显著升高(P<0.01),并出现肾组织形态学异常改变;而DMT组血糖、24 h尿蛋白量、肾脏指数均低于DM组(P<0.05),肾纤维化病变改善;(2)与N组比较,DM组肾组织Id2蛋白和mRNA的表达从2 w开始减少,16 w、24 w显著减少(P<0.05),并伴有E-cadherin的表达减少(P<0.05)、α-SMA表达增加(P<0.05)和FN蛋白的沉积增多(P<0.05),相关性分析显示Id2蛋白与FN蛋白成负相关(r=-0.931,P<0.05),与E-cadherin蛋白成正相关(r=0.900 0,P<0.05);(3)与DM组相比,DMT组大鼠肾组织Id2蛋白和mRNA表达均明显增加(P<0.05),且E-cadherin的表达增加(P<0.05)、α-SMA的表达及FN在间质沉积减少(P<0.05)。结论:胰岛素在控制血糖能上调肾小管上皮细胞Id2蛋白的表达,恢复Id2对肾纤维化的负调节作用,进而逆转肾小管上皮细胞的EMT及减轻肾间质ECM的沉积,改善DN肾纤维化病变。
Objective: To investigate the expressions of Id2 in the renal tubules of STZ-induced diabetic rats before and after blood glucose control,and explore their roles and relationship in the development of diabetic nephropathy. Methods: SD rats were randomly divided into control( N),diabetes mellitus( DM) and insulin-treated DM groups( DMT). DM model was constructed by injecting streptozotocin( STZ) via tail vein( 55 mg / kg). 48 SD rats were randomly divided into 4 groups: 2 weeks,8 weeks,16 weeks and 24 weeks group. Each group included a control subgroup and a diabetic model subgroup. In addition,for 16 weeks group and 24 weeks group,each group still included an insulintreated subgroup. Since the 13 thweek after modeling,rats in DMT group were given insulin individually. The blood glucose was controlled to the level of 4 ~ 7 mmol / L,and urine glucose remained negative. The blood glucose,24 h urine protein were measured by biochemical method,and the kidney index was measured as well. And the renal fibrosis was examined by HE and PAS staining sections. Immunohistochemistry was employed to assay the expression of FN protein in the rat renal tissue. Western blotting was employed to assay the expression of Id2,E-cadherin and α-SMA protein in the rat renal tissue. In addition,the expression of Id2 mRNA was also examined by RT-PCR. Results:( 1) Compared with group N,the blood glucose,24 h urine protein and the kidney index of group DM increased significantly( P < 0. 01),and renal tissue presented typical morphologic changes at different time points. By comparison,the blood glucose,24 h urine protein and the kidney index of group DMT were significantly lower than those in group DM( P < 0. 05). Lesions of renal fibrosis in DMT rats were obviously relieved.( 2) The expression levels of Id2 protein and mRNA of group DM decreased significantly than that in N group at different time points( P < 0. 05),accompanied by the decreased expression of E-cadherin( P < 0. 05),increased expression of α-SMA( P < 0. 05),and increased deposition of FN protein. Correlation analysis showed Id2 protein was negatively correlated with FN protein( r =-0. 931,P < 0. 05),but positively correlated with E-cadherin( r = 0. 900 0,P < 0. 05).( 3) Compared with DM group,the protein and mRNA expression of Id2 and E-cadherin of DMT group increased significantly( P < 0. 05),while the expression of α-SMA and FN protein decreased. Conclusion: Blood glucose control can up-regulate the expression of Id2 in renal tubular epithelial cells and restore Id2 negative regulation of renal fibrosis,reverse EMT and relieve renal interstitial ECM deposition,and improve DN lesions of renal fibrosis.

关键词(KeyWords): 糖尿病肾病;纤维化;分化抑制因子2;纤维连接蛋白;E-钙黏素;α-平滑肌肌动蛋白;大鼠,Sprague-Dawley
diabetic nephropathy;fibrosis;inhibitors of differentiation;fibronectin;E-cadherin;α-smooth muscle actin;rats,Sprague-Dawley

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81360116);; 贵州省科学技术基金[编号:黔科合J字(2011)2120号]

作者(Author): 文箐颍;苏博;肖瑛;王圆圆;李霜;刘丽荣;石明隽;郭兵;
WEN Qingying;SU Bo;XIAO Ying;WANG Yuanyuan;LI Shuang;LIU Lirong;SHI Mingjun;GUO Bing;Department of Pathophysiology,Guiyang Medical College;

Email:

DOI: 10.19367/j.cnki.1000-2707.2015.04.002

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