Inhibition of Notch signaling pathway promotes the differentiation of bone marrow mesenchymal stem cells into islet / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effect of Notch signaling during bone marrow mesenchymal stem cells (BMSC) differentiating into islet in vitro.Methods The specific inhibitor of γ-secretase DAPT was used to inhibit the Notch signaling pathway.Mter induction,DTZ staining,indirect immunofluorescence staining,RT-PCR and Westenn blotting were used to detect the expression of insulin,glucagon,Pdx-1 and Ngn3.Results (1) Identification of BMSCsIndirect immunofluorescence staining showed that BMSCs could express CD59 and CD90,which both were makrers of mesenchymal stem cells.Besides,BMSCs could express nerve culluar markers such as NSE,GFAP,suggesting multi-directional differentiation.(2) The result of MTT showed DAPT could inhibit the cell proliferation in a time-dependent manner and a dose-dependent mannar.Besides,DAPT could inhibit the expression of target gene of Notch signal pathway in a time-dependent manner and a dosedependent mannar.After treated by 1,5,20 μmol DAPT,the expression of Hes1 had reached to 92.06％,71.40％ and 46.89％ of controls respectively,suggesting efficiency of inhibition on Notch reached 7.94％,28.6％ and 53.11％ respectively (all P＜ 0.05).(3) Indirect immunofluorescence staining showed the expression of pancreas-specific markers such as insulin and glucagon were much higher in DAPT treated BMSCs than that in controls,which was confirmed by RT-PCR and Western blotting analyses.The proportion of insulin-producing cells differentiated from DAPT treated BMSCs was (74.03 ± 3.96)％,which was higher than that from controls[(36.49 ± 3.24)％,P＜ 0.05].(4)Furthermore,RT-PCR and Western blotting analysis showed that the expressions of Pdx-1 and Ngn3 were earlier than that of insulin and glucagon,and the expressions of Pdx-1 and Ngn3 were higher in DAPT treated BMSCs than that in controls.Conclusions Notch signaling pathway plays a role in the differentiation of BMSCs into islet in vitro.Pharmacological interference with Notch signaling pathway may provide a novel method to obtain islet for therapeutic use.