转化生长因子-β1诱导下肾小球系膜细胞Smad2的表达与Ⅳ型胶原分泌的相关性研究及来氟米特的干预作用  

Study of Correlation Between the Expression of Smad2and Collagen Type ⅣSecretion of Rat Glommerular Mesangial Cell Stimulated by Transforming Growth Factor-β1and the Effect of Interventions by Leflunomide

作　　者：胡雯辉[1] 于力[1] 温跃强[1] 张瑶[1] 郝志宏[1] 陈蓉燕[1] 

机构地区：[1]广州医学院附属广州市第一人民医院儿科,广州510180

出　　处：《中华妇幼临床医学杂志（电子版）》2013年第4期372-377,共6页Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition)

基　　金：广东省自然科学基金项目(5000424);广州市科技计划项目(2012A011028)~~

摘　　要：目的探讨转化生长因子(TGF)-β1诱导肾小球系膜细胞(GMC)中Smad2的表达与Ⅳ型胶原(ColⅣ)表达的相关性及来氟米特(LEF)的干预作用。方法建立体外培养大鼠GMC模型,经鉴定后第7代用于实验。按照对大鼠GMC模型处理方式将其分为:TGF-β1组(培养液+TGF-β1 5ng/mL),LEF-1组(培养液+LEF 5μg/mL+TGF-β1 5ng/mL),LEF-2组(培养液+LEF 50μg/mL+TGF-β1 5ng/mL)与对照组(仅加入培养液)。分别于15min,30min,1h,2h和6h收集标本,采用酶联免疫吸附测定(ELISA)法测定细胞培养上清液ColⅣ表达。采用间接免疫荧光法检测各组磷酸化(P)-Smad2蛋白表达;采用荧光半定量RT-PCR法检测各组Smad2mRNA表达。结果在各时点,①ColⅣ表达量比较:TGF-β1组显著高于对照组(P<0.05),LEF-1与LEF-2组均显著低于TGF-β1组(P<0.05)。②P-Smad2表达量比较:在对照组仅少量表达,TGF-β1组表达明显升高,与对照组比较,差异有显著意义(P<0.05);LEF-1与LEF-2组的P-Smad2表达较TGF-β1组显著下降,差异有显著意义(P<0.05)。③Smad2mRNA表达量比较:在TGF-β1组表达显著高于对照组(P<0.05),而在LEF-1组与LEF-2组显著低于TGF-β1组(P<0.01);但LEF-1组与LEF-2组比较,差异无统计学意义(P>0.05)。④GMC上清液中,ColⅣ与Smad2mRNA表达量呈正相关(r=0.707,P<0.05)。结论经TGF-β1刺激后,体外培养大鼠GMC中P-Smad2表达及ColⅣ表达量均增加,因此LEF可降低Smad2表达与ColⅣ表达水平,减轻细胞外基质(ECM)沉积。本研究结果为LEF的肾保护作用提供了实验依据。Objective To observe the correlation between the expression levels of Smad2 and collagen type IV （ColIV）secretion of rat glomerular mesangial cell（GMC） stimulated by TGF-β1, whether leflunomide （LEF） influences the expression levels of Smad2 and Coliv secretion of rat GMC stimulated. Methods After establishing the cultured GMC of rat in vitro ,the seventh passages of cells were used in the experiment after identification. The experiment incluced 4 groups： TGF-β1 group（cell cultured medium and 5 /zg/mL TGF-β1 were added）, LEF-1 group（low dose of LEF treated group, besides normal cell cultured medium and 5 ng/mL TGF-β1,5 μg/mL LEF were added） ,LEF-2 group（high dose of LEF treated group,besides normal cell cultured medium and 5 ng/mL TGF-β1,50μg/mL LEF were added）, cotrol group （nomal cell cultured medium was added）. Each group was collected at 15 min, 30 min, 1 h, 2 h and 6 h. CollV secretion was detected by the enzyme-linked immunosorbent-assay （ELISA）. The expressions levels of P-Smad2 protein were analyzed by indirect immunofluorescence, real time （RT）-PCR were used to detect the mRNA expression of Smad2. Results ①At each time, CoIIV secretion level was singnificantly higher in the protein expression in TGF-β1 group than that in control group （P〈0.05）, but singnificantly lower than that in LEF-1 group and LEF-2 group （P〈0.05）. ②In control group, phosphorylation（P）-Smad2 protein expressed a little, while stimulation of cells staining positive in TGF-β1 group was singnificantly higher than those in control group（P〈0.05）. Compared with the stimulation,the expressions of P-Smad2 in LEF-1 group and LEF-2 group were significantly decreased （P〈0.05）. ③Expression of Smad2 mRNA in TGF-β1 group was higher than those other three groups （P〈0. 01） ,but no significant difference in the expressions were found between those in LEF-1 group and LEF-2 group （P 〉 0. 05）. ④ The content of Col IV secretion was positively correlated with th

关 键 词：系膜细胞 转化生长因子-Β1 SMAD2 Ⅳ型胶原 来氟米特 

分 类 号：R692.6[医药卫生—泌尿科学]