来氟米特干预肾小球系膜细胞Smad2的表达及Ⅳ型胶原分泌的体外研究  

Interventions of leflunomide on Smad2 expression and collagen type IV secretion of Rat Glommerular Mesangial Cell in vitro

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,510180

出　　处：《广州医药》2013年第3期1-6,共6页Guangzhou Medical Journal

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：目的观察来氟米特(leflunomide,LEF)在转化生长因子β1(TGF-β1)刺激大鼠肾小球系膜细胞(GMC)表达Smad2及Ⅳ型胶原(ColⅣ)过程中发挥的作用。方法建立体外培养大鼠肾小球系膜细胞模型,经鉴定后第7代用于实验。按照对大鼠肾小球系膜细胞模型处理方式不同,实验分4组,对照组(只加培养液),TGF-β1组(TGF-β1 5 ng/ml),LEF-1组(LEF 5 ng/ml+TGF-β1 5 ng/ml)和LEF-2组(LEF 50 ng/ml+TGF-β1 5 ng/ml)。分别于15min、30min、1h、2h和6h收集标本,用酶联免疫吸附测定(ELISA)方法测定细胞培养上清液的ColⅣ胶原蛋白表达量的变化。用间接免疫荧光法检测各组磷酸化Smad2(P-Smad2)蛋白表达的变化;采用荧光半定量RT-PCR法检测各组Smad2 mRNA表达的变化。结果在各时间点,TGF-β1刺激组ColⅣ分泌量高于对照组(P<0.05),LEF各组Ⅳ胶原分泌低于TGF-β1(P<0.05)。对照组P-Smad2蛋白有少量的表达;TGF-β1组表达升高,有统计学意义(P<0.05),与TGF-β1组相比,两LEF组P-Smad2表达下降,有统计学意义(P<0.05)。肾小球系膜细胞中,TGF-β1组Smad2 mRNA表达高于对照组。LEF-1组和LEF-2组Smad2 mRNA表达水平低于TGF-β1组(P<0.01)。但两LEF组间比较无统计学意义(P>0.05)。LEF干预作用有时间性,随时间延长,其LEF干预作用逐渐减弱。结论经TGF-β1刺激后,体外培养的大鼠肾小球系膜细胞磷酸化-Smad2表达及Ⅳ胶原分泌增加,来氟米特可降低Smad2表达和Ⅳ胶原分泌水平,减轻细胞外基质(ECM)的沉积,为来氟米特的肾脏保护作用提供实验依据。Objective To observe whether leflunomide influences the expression of smad2 and collagen type Ⅳsecre- tion of rat glommerular mesangial cell stimulated by TGF-β1. Methods After establishing the cultured glomerularmesangial ceUs of rat in vitro, the geventh passages of cells were used in the experiment after identification. The experiment included 4 groups ： control group （ Ctrl group）, TGF-β1 group （TGF group）, leflunomide treated group （ LEF 1 group, LEF 2group） . Each of cell group were collected at 15minutes, 30minute, 1 hour, 2hour and 6hour. The collagen Ⅳ secretion was detected by the enzyme-linked immunosorbent-assay （ELISA） . The change expressions of P-Smad2 protein were analyzed by indirect immu- nofluorescence, real time PCR were used to detect the mRNA expression of Smad2. Results At each time, the collagen Ⅳ secretion was significantly higher in the protein expression of TGF group than in control group （P 〈 0. 05 ）, but significantly low- er in all LEF group （P 〈 0. 05） . In control group, phosphorylation-Smad2 protein expressed a little, in TGF-i31 group, stim- ulation of cells staining positive was significantly higher in control group （P 〈 0. 05） . Compared with the stimulation, the ex- pression of phosphorylation-Smad2 in leflunomide groups were significantly decreased （P 〈 0.05） . Expression of Smad2 mRNA in TGF-β1 group was higher than those of other three groups （P 〈 0. 01 ）, but no significant difference in the expression was found between LEF-1 and LEF-2 （ P 〉 0. 05 ） . LEF possesses timeliness to Smad2 in inhibition, which effect decreases gradu- ally with time passing. Conclusion TGF-β1 can increase the expression of Smad2 and collagen Ⅳ secretion , leflunomide may inhibit the expression levels of Smad2 and collagen Ⅳ secretion induced by TGF-β1, thereby reducing the ECM deposition and offering the theoretical evidence for the renal protection.

关 键 词：系膜细胞 TGF-β1 SMAD2 Ⅳ胶原 来氟米特 

分 类 号：R726.9[医药卫生—儿科]