转化生长因子-β<sub>1</sub>/Smad信号通路在肾小球系膜细胞的表达及血管紧张素转化酶抑制剂对其影响

doi:10.3877/cma.j.issn.1673-5250.2011.04.005

目的

探讨转化生长因子(transforming growth factor，TGF)-β₁/Smad信号传导通路在肾小球系膜细胞(glomerular measanial cell, GMC)中的作用，观察血管紧张素转化酶抑制剂(angiotensin-converting enzyme inhibitor, ACEI)福辛普利(fosinopril，Fos)在该通路中对磷酸化Smad2/3(phosphorylation-Smad2/3, P-Smad2/3)和Ⅰ型胶原蛋白(collagen Ⅰ，Col Ⅰ)表达的影响，阐明福辛普利的肾保护作用机制。

方法

采用经典方法进行大鼠肾小球系膜细胞复苏、培养和传代后，将其按处理方式分为3组：①TGF-β₁组(TGF-β₁ 5 ng/mL处理)；② Fos组(TGF-β₁ 5 ng/mL＋ Fos 1×10^－4 mol/L处理)；③对照组(空白)。甲基噻唑基四唑(methyl thiazolyl tetrazolium, MTT)比色法分别检测3组肾小球系膜细胞增殖光密度(optical density，OD)值，计算细胞抑制率；间接免疫荧光法检测磷酸化Smad2/3表达情况；酶联免疫吸附测定(enzyme linked immunosorbent assay，ELISA)法测定Ⅰ型胶原蛋白的变化，计算抑制率。

结果

各时间点肾小球系膜细胞增殖荧光强度比较，TGF-β₁组明显高于对照组，Fos组低于TGF-β₁组，差异有显著意义(P<0.05)。磷酸化Smad2/3在对照组仅极微量表达，TGF-β₁组在刺激30 min后，荧光强度显著增强，且集中于细胞核。与TGF-β₁组相比，Fos组磷酸化Smad2/3表达下降，细胞荧光强度较弱，差异有显著意义(P<0.05)。对照组Ⅰ型胶原蛋白仅少量基础性分泌。与对照组相比，TGF-β₁组在各时间点Ⅰ型胶原蛋白分泌量显著上升，差异有显著意义(P<0.05)；与TGF-β₁组比较，Fos组Ⅰ型胶原蛋白在15 min和6 h的表达量比较，差异无显著意义(P>0.05)；在12 h和24 h比较，差异有显著意义(P<0.05)。

结论

人重组转化生长因子-β₁能激活体外培养的大鼠肾小球系膜细胞磷酸化-Smads表达，并激活转化生长因子-β/Smads信号途径，该途径激活能致Ⅰ型胶原分泌增多，并能诱导肾小球系膜细胞增殖，其作用呈一定的时间依赖性关系。福辛普利能部分阻断转化生长因子-β/Smads信号传导通路在大鼠肾小球系膜细胞的激活，抑制Ⅰ型胶原分泌及肾小球系膜细胞增殖，其抑制作用呈一定的时间依赖性关系。

关键词: 肾小球系膜细胞, Ⅰ型胶原蛋白, 转化生长因子-β₁, Smad蛋白, 血管紧张素转化酶抑制剂

Objective

To investigate the role of transforming growth factor (TGF)-β₁/Smad signaling pathway in glomerular mesangial cell (GMC), and the influence of angiotensin-converting enzyme inhibitor(ACEI) fosinopril (Fos) on the expression of phosphorylation-Smad2/3 (P-Smad2/3) and collagen Ⅰ(Col Ⅰ) in this way.

Methods

Glomerular mesangial cells of rat in vitro were cultured, and they were divided into 3 groups: ①TGF-β₁ group (TGF-β₁ 5 ng/mL); ② Fos group (TGF-β₁ 5 ng/mL+ Fos 1×10^-4 mol/L); ③control group (blank). Methyl thiazolyl tetrazolium (MTT) colorimetric method detected cellular proliferation. The change of phosphorylation-Smad2/3 expression was detected by indirect immunofluorescence. The expression of collagen Ⅰ in cell culture supernatant was detected by the enzyme-linked immunosorbent assay (ELISA).

Results

The optical density(OD) value of cellular proliferation was higher than that in TGF-β₁ group when compared with control group(P<0.05), while lower in Fos group when compared with TGF-β₁ group(P<0.05). Phosphorylation-Smad2/3 slightly expressed in control group, while increased obviously in TGF-β₁ group compared with control group(P<0.05). In Fos group, the expression of phosphorylation-Smad2/3 were decreased apparently at 12 h and 24 h than those in TGF-β₁ group(P<0.05).

Conclusion

TGF-β₁ can activate the expression of phosphorylation-Smads and TGF-β₁/Smad signaling pathway in glomerular mesangial cell, which will cause the increased expression of collagen Ⅰ and proliferation of glomerular mesangial cell. Fos can partially block the activation of transforming growth factor-β₁/Smad signaling pathway in glomerular mesangial cell, inhibit the excretion of collagen Ⅰ and proliferation of glomerular mesangial cell.

Key words: glomerular mesangial cell (GMC), collagen Ⅰ(Col Ⅰ), transforming growth factor-β₁(TGF-β₁), Smads, angiotensin-converting enzyme inhibitor (ACEI)

图1　各组肾小球系膜细胞各时间点磷酸化-Smad2/3表达变化 (免疫荧光法，×200)

图2　各时间点ColⅠ蛋白表达量趋势图

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Expression of Transforming Growth Factor-β₁/Smad Signaling Pathway in Rat Glomerular Mesangial Cell and Interventional Effects of Angiotensin-Converting Enzyme Inhibitor

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Expression of Transforming Growth Factor-β1/Smad Signaling Pathway in Rat Glomerular Mesangial Cell and Interventional Effects of Angiotensin-Converting Enzyme Inhibitor

Expression of Transforming Growth Factor-β₁/Smad Signaling Pathway in Rat Glomerular Mesangial Cell and Interventional Effects of Angiotensin-Converting Enzyme Inhibitor