关键词: 骨髓间充质干细胞, 转化生长因子β1, Snail, 细胞外信号调节激酶1/2, 磷脂酰肌醇3-激酶, 免疫印迹法, 大鼠

Abstract: Objective To examine whether transforming growth factor\|β1(TGF-β1) could induce expression of Snail in bone marrow mesenchy mal stem cells (BMSCs) through activation of extracellular signal\|regulater kinases(ERK) and PI3K. Methods Density gradient centrifugalization combined with adherence method was used to segregate rat BMSCs.The BMSCs were cultivated to the 3rd passage and characterized using flow cytometry technique.After BMSCs were treated with PD98059（0,15,30,50μmol/L）or Wortmannin(0,20,40,80 nmol/L)for 1hour，the levels of p-Akt and p-ERK1/2 with TGF-β1 treatment for 6hours were examined by Western blot to determine the effective concentrations for PD98059 and Wortmanin to inhibit the TGF-β1induced ERK and PI3K activity of cells.Then,we employed these specific inhibitors with respective effective concentrations to incubate BMSCs with TGF-β1 treatment for 6 hours or 24 hours and detect the expression of Snail mRNA and Snail protein by RT-PCR and Western blotting. Results Density gradient centrifugalization combined with adherence method could segregate and purify rat BMSCs effectively.The results of flow cytometry showed that CD29 and CD44 expression were positive while CD34 and CD45 expression was negative for BMSCs.After TGF-β1 treatment，increased levels of p-ERKs and p-Akt were confirmed by Western blotting.TGF-β1induced increased activity of p-Akt and p-ERKs in BMSCs could be inhibited in a concentrationdependent manner by Wortmannin and PD98059.The effective concentrations for PD98059 and Wortmanin to inhibit the TGF-β1induced ERK and PI3K activity of cells were 30μmol/L and 40nmol/L respectively.The significantly decreased levels of the Snail mRNA and Snail prot

Key words: Bone marrow mesenchymal stem cell, TGF-β1, Snail, ERK1/2, PI3K, Western blotting, Rat