摘要
目的探讨转化生长因子-β1(TGF-β1)是否能诱导小鼠足细胞株凋亡以及诱导凋亡的途径。方法(1)体外培养小鼠足细胞株;(2)应用不同浓度(10^(-1)~10^+ng/L)TGF-β1诱导小鼠足细胞凋亡;(3)10~3ng/L TGF-β_1诱导不同时间(12、24、48h)后观察足细胞凋亡情况;(4)采用流式细胞仪检测Annexin V、Caspase 3;(5)实时定量PCR及Western印迹法检测p38丝裂原活化蛋白激酶(p38MAPK)、Smad2、Smad3、Smad7mRNA及蛋白质的表达情况。结果(1)小鼠足细胞在不同浓度(10^(-1)~10~4ng/L)TGF-β_1刺激下,细胞凋亡率明显高于正常对照组(均P<0.05);(2)随着TGF-β_1浓度增加、凋亡时间增加,足细胞凋亡率增高(P<0.05);p38MAPK、Smad2、Smad3、Smad7 mRNA表达增加;TGF-β_110~3ng/L为最佳诱导小鼠足细胞凋亡浓度,24h为最佳诱导凋亡时间;(3)与正常对照组比较,10~3ng/LTGF-β_1诱导后Caspase 3阳性细胞比率显著增加(P<0.01),p38MAPK、Smad2、Smad3、Smad7蛋白质表达亦显著增加(P<0.01)。结论 TGF-β_1能诱导小鼠足细胞凋亡,呈浓度及时间依赖性。TGF-β_1通过Smad通路、p38MAPK通路以及线粒体通路诱导小鼠足细胞凋亡。
Objective To investigate the effect of TGF-β1 on apoptosis of mouse podocytes and its signal pathway. Methods Cultured mouse podocytes were treated with different concentrations of TGF-β1（0,1x10-1N104 ng/L） for 24 h, or treated with 103 ng/L TGF-β1 for different time duration （12, 24, 48h）. Annexin V and Caspase 3 were measured by flow cytometry. The mRNAs and proteins of P38MAPK, Smad2, Smad3 and Smad7 were detected by real time PCR and Western blotting. Results Compared to controls（0ng/L TGF- 15 1） the ratio of apoptotic podocytes and mRNA expressions of p38MAPK, Smad2, Smad3 and Smad7 were increased in TGF-β1 （10-1-104ng/L） treatment groups in a concentration and time-dependent manner （P〈0.05）. Treatment with TGF--β1 （103rig/L） for 24 h induced maximal apoptosis in podocytes. The ratio of Caspase3-positive podocytes was increased after treated with TGF--β1（103ng/L, 24h） compared to controls（P〈0.01）. Conclusion TGF-β1induces the apop- tosis of mouse podocytes time-dependently and dose-dependently, in which Smad, p38MAPK signal pathway and mitochondri- on might be involved.
出处
《浙江医学》
CAS
2013年第19期1718-1721,共4页
Zhejiang Medical Journal
基金
浙江省中医药管理局立项资助课题(2006C108)
