来氟米特对转化生长因子β_1诱导的体培养的大鼠肾小球系膜细胞中Smad2表达的影响

Influence of Leflunomide on the Expression of Smad2 of Rat Glomerular Mesangial Cell Induced by Transforming Growth Factor β_1

目的观察来氟米特(leflunomide,LEF)在转化生长因子(transforming growth factor,TGF)-β_1刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)表达Smad2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,经鉴定造模成功后第7代用于实验。按照对模型大鼠体外培养的肾小球系膜细胞的处理方式不同,将其分为TGF-β_1组(TGF-β_1 5 ng/mL),LEF-1组(LEF 5μg/mL+TGF-β_1 5 ng/mL),LEF-2组(LEF 50μg/mL+TGF-β_1 5 ng/mL)和对照组(无血清RPMI 1640培养液+20%胎牛血清)。分别于15 min,30 min,1 h,2 h和6 h收集标本,用间接免疫荧光法检测各组磷酸化Smad2(phosphorylation-Smad2,p-Smad2)蛋白表达变化;采用荧光半定量RT-PCR法检测各组Smad2mRNA表达变化。结果对照组磷酸化Smad2蛋白少量表达,阳性细胞率为(21.40±1.51)%。TGF-β_1组阳性细胞率为(70.00±3.23)%,30 min时表达开始升高,1 h及2 h时表达明显增高,6 h表达开始下降。各时间点LEF-1组和LEF-2组磷酸化Smad2蛋白表达下降,阳性细胞率分别为(23.60±2.80)%和(25.81±1.29)%,细胞荧光强度呈减弱趋势。与TGF-β_1组相比,LEF-1组和LEF-2组磷酸化Smad2蛋白表达下降,差异有显著意义(P<0.05)。但LEF-1组和LEF-2组与对照组比较,差异无显著意义(P>0.05)。肾小球系膜细胞中Smad2mRNA相对表达量,TGF-β_1组显著高于对照组,2 h时达高峰。LEF-1组和LEF-2组Smad2mRNA显著低于TGF-β_1组,差异有显著意义(P<0.01);但LEF-1组和LEF-2组间比较,差异无显著意义(P>0.05)。LEF-1组和LEF-2组Smad2mRNA相对表达量1h时较低,之后逐渐升高,说明随时间延长,来氟米特对体外培养的肾小球系膜细胞干预作用逐渐减弱。结论经转化生长因子-β_1刺激后,体外培养的大鼠肾小球系膜细胞磷酸化Smad2蛋白和Smad2mRNA相对表达量分别增加,来氟米特可降低Smad2蛋白和Smad2mRNA表达水平,为来氟米特的肾脏保护作用提供理论依据。

Objective To observe influence of leflunomide （LEF） on the expression of Smad2 of rat glomerular mesangial cell （GMC） induced by transforming growth factor β1（TGF-β1）. Methods After establishing the cultured glomerular mesangial cells of rat in vitro, the seventh generation was used in this experiment after identification. This experiment included 4 groups： TGFq31 group （TGF-β1 5 ng/mL）, LEF-1 group （LEF 5 gg/mL＋ TGF β1 5 ng/mL）, LEF2 group （LEF 50 μg/mL＋ TGF-β1 5 ng/mL）, and control group （serum free RPMI 1640 culture fluid＋ 20G fetal bovine serum）. The specimens were collected at 15 min, 30 rain, 1 h, 2 h, and 6 h. Expression changes of phosphorylation-Smad2 protein in 4 groups were detected by indirect immunofluorescence. Changes of Smad2mRNA expression were analyzed by fluorescence semiquantitative PCR. Results In control group, phosphorylation Smad2 protein expressed a little, and the ceils staining positive was （21.40± 1.51）%. In TGF-1 group, cells staining positive was （70.00±3.23）%. It started to increase at 30 min and reach the peak at 1 h and 2 h, and then began to decrease at 6 h. The expression of phospborylation-Smad2 in LEF-1 group and LEF-2 group decreased at different time points, the cells staining positive were （23.60±2.80）% and （25.81±1.29）%, respectively. Compared with TGF-β1 group, expression of phosphorylation-Smad2 in LEF-1 group and LEF-2 group decreased （P〈0.05）. But there had no significant difference between control and LEF-1 group or LEF-2 group （P〉0.05）. Expression of Smad2mRNA in mesangial cell in TGF-β1 group was higher than those of other three groups （P〈0. 01）, but no significant difference was found between LEF1 group and LEF2 group （P〉0. 05）. Conclusion After the stimulation of transforming growth factor β1, mesangial cell phosphorylation-Smad2 protein and expression of mRNA increase. Leflunomide my decrease the expression level of Smad2 protein and mRNA expression level to provide the theoretical evidence for the renal protection