饰胶蛋白聚糖与转化生长因子β1对大鼠肾系膜细胞生长及相关信号途径的影响

目的探讨饰胶蛋白聚糖（DCN）与转化生长因子β1（TGF-β1）对大鼠肾系膜细胞（MsC）生长及相关信号途径的影响。方法经脂质体介导将DCN基因转染体外培养的大鼠肾MsC,经筛选和鉴定后,收集阳性细胞克隆的培养上清（DCN上清）。采用流式细胞仪检测经TGF-β1（2μg/L）和（或）DCN上清作用后MsC细胞周期的变化。采用Western印迹法分别检测两者单独或联合作用后,MsC磷酸化丝裂原活化蛋白激酶（p-MAPK）、磷酸化Smad2 （p-Smad2）和p21蛋白表达情况。采用免疫共沉淀方法检测阳性细胞克隆上清中DCN与TGF-β1的结合情况。结果TGF-β1或DCN上清单独作用时均可抑制MsC的增殖,G_2-M期细胞数分别为对照组的81%及86.5%,而两者共同作用时G_2-M期细胞数与对照组差异无统计学意义。经TGF-β1和DCN上清单独作用12 h后,p-ERK1/2表达为对照组的4.4、3.7倍和3.2、3.7倍;p-SAPK/JNK表达为对照组的2.0、1.5倍和1.9、1.5倍;而两者共同作用组p-ERK1/2和p-SAPK/JNK表达与对照组差异无统计学意义。TGF-β1单独作用12 h后,MsC p-Smad2的表达为对照组的2.6倍,而DCN单独作用组及2者联合作用组其表达与对照组差异无统计学意义。DCN单独作用12 h后p21蛋白的表达为对照组的2.6倍,而TGF-β1单独作用及2者联合作用组其表达与对照组差异无统计学意义。阳性细胞克隆上清中DCN可以直接与TGF-β1结合。结论TGF-β1与DCN单独作用均可抑制MsC增殖及激活MAPK信号途径,但2者共同作用时其作用相互抵消。TGF-β1激活Smad信号途径作用可被DCN阻断;DCN上调p21蛋白作用可被TGF-β1阻断。2者通过不同信号分子抑制细胞生长,其作用可能与两者相互结合有关。

Influence of decorin and TGF-β1 on rat mesangial cell growth and relative signal transduction pathway

Objective To explore the effect of decorin and TGF-β1 on rat mesangial cell (MsC) growth and its relative signal pathway. Methods Lipofectin-mediated method was used to transfect DCN vector into MsC. After the screen and identification of transfected MsC, DCN-containing supernatant was collected and it or/and TGF-β1(2 μg/L) was added into the culture medium of normal MsC. Flow cytometer was applied to detect the cell cycle. Western blot analysis was used to examine the expression of phospho-MAPKs, phospho-Smad2 and p21 protein. Inmmunoprecipation was applied to test the combination of DCN and TGF-β1 in supernatant of cultured MsC. Results Compared with normal MsC, the number of G2-M cell stimulated by DCN-containing supernatant or TGF-β1 decreased to 86.5% or 81%（P<0.05），and the expression of phospho-ERK1/2 and phospho-SAPK/JNK was obviously up-regulated(P<0.05), whereas the combination of both DCN and TGF-β1 could not obviously change above expression compared with control group. The protein level of p-Smad2 was increased with stimulation of TGF-β1 and p21 expression was increased with DCN stimulation, but the expression of both p-Smad2 and p21 did not obviously change after combination use of both DCN and TGF-β1 compared with control group. DCN and TGF-β1 in supernatant of cultured MsC could combine with each other. Conclusions TGF-β1 or DCN alone can suppress MsC proliferation and activate MAPKs signal pathway of MsC, but the effect of their combination is antagonistic. DCN can inhibit the activation of TGF-β1/ p-Smad2 signaling pathway. And up-regulation of p21 protein expression caused by DCN can be suppressed by TGF-β1. These all effects may be associated with the combination of DCN and TGF-β1.