饰胶蛋白聚糖对大鼠肾系膜细胞生长的抑制作用及其信号转导途径的研究

Study on the inhibitory effect of decorin on rat mesangial cell growth and its signal transduction pathway

目的探讨饰胶蛋白聚糖(DCN)对大鼠系膜细胞(MsC)生长的抑制作用及其信号转导分子MAPKs和p21表达的影响。方法经脂质体介导将DCN基因转染体外培养的大鼠MsC,筛选和鉴定后,收集阳性细胞克隆的培养上清(DCN上清),将其加入正常MsC的培养液中,采用流式细胞仪检测细胞周期。用Western印迹法分别检测MAPKs,包括细胞外调节激酶(ERK)1/2、应激活化蛋白激酶(SAPK),氨基末端激酶(JNK)和p38和p21蛋白表达;用免疫荧光法观察p21在细胞中的表达。结果DCN上清明显抑制正常MsC的增殖,G2-M期细胞数明显减少,仅为对照组的35%(P<0.05);磷酸化ERK1/2及SAPK/JNK表达增强,分别为对照组的2.2倍、1.4倍及1.7倍、1.8倍;磷酸化p38无明显变化。DCN抗体呈浓度依赖性抑制磷酸化ERK1/2、SAPK/JNK的表达上调。DCN上清还可使细胞p21的表达明显增强,而DCN抗体也同样呈浓度依赖性地抑制其上调表达的作用,ERK1/2及SAPK/JNK通路抑制剂U0126和circumin均可抑制其上调表达的作用,分别为对照组的64%和61%;而p38通路抑制剂SB203580则对其无影响。结论DCN对肾MsC的生长有抑制作用,其机制可能经ERK1/2、SAPK/JNK和p21蛋白介导。

Objective To explore the inhibitory effect of decorin（DCN） on rat mesangial cell （MsC） growth and on the expression of mitogen-activated protein kinases （MAPKs） and p21 protein. 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 added into the culture medium of normal MsC, then flow cytometer was used to detect the cell cycle. Western blot analysis was used to explore the changes of MAPK and p21 protein. Immunofluorescence was adapted to detect the expression of p21 on cultured MsC. Results Compared with normal MsC, the number of G2-M cells treated with DCN-containing supernatant decreased to 35% （P 〈 0.05 ）, while the expression of phospho-ERK1/2 and phospho-SAPK/JNK on MsC was enhanced significantly （P 〈 0.05）. DCN antibodies suppressed the increasing of phospho-ERKl/2 and phospho-SAPK/JNK in a dose-dependent manner. Western blot analysis and immunofluorescence revealed that DCN- containing supernatant up-regulated the expression of both total and nuclear p21 protein on MsC. This enhanced expression was attenuated by DCN antibody . Inhibitors U0126 and eireumin of ERK and SAPK/JNK pathway could inhibit total and nuclear p21 up-regulation caused by D（：N-eontaining supernatant （P 〈 0.05）, while the inhibitor SB203580 of p38 pathway had no similar effect on MsC. Condusion DCN-mediated transduction possibly by signaling molecule ERK1/2 and SAPK/JNK and p21 protein can suppress the cultured mesangial cell growth.