观察来氟米特(leflunomide, LEF)在转化生长因子(transforming growth factor，TGF)-β₁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。

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-β₁组(TGF-β₁ 5 ng/mL)，LEF-1组(LEF 5 μg/mL＋ TGF-β₁ 5 ng/mL)，LEF-2组(LEF 50 μg/mL＋ TGF-β₁ 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。

对照组磷酸化Smad2蛋白少量表达，阳性细胞率为(21.40±1.51)%。TGF-β₁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-β₁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-β₁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-β₁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1 h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。

经转化生长因子-β₁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。

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).

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: TGF-β₁ group (TGF-β₁ 5 ng/mL), LEF-1 group (LEF 5 μg/mL+ TGF-β₁ 5 ng/mL), LEF-2 group (LEF 50 μg/mL+ TGF-β₁ 5 ng/mL), and control group (serum-free RPMI 1640 culture fluid+ 20% fetal bovine serum). The specimens were collected at 15 min, 30 min, 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.

In control group, phosphorylation-Smad2 protein expressed a little, and the cells 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 phosphorylation-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 LEF-1 group and LEF-2 group (P>0.05).

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 .