인간 중간엽 기질세포에서 H2O2에 의한 산화성 손상에 대한 Necrostatin의 보호 효과

Abstract

Stem cell based therapy using bone marrow derived mesenchymal stromal cells (BM-MSCs) is a feasible, safe and potential alternative therapeutic strategy for intractable neurological disease. During traditional cell culture process, it is inevitable for culturing cells to be injured by physical and/or chemical stresses. One of these is that cell culture imposes a state of oxidative stress on cells. Necrostatin, a small tryptophan- based molecule, has been reported to inhibits receptor-interacting protein (RIP)-1 kinase and programmed necrosis. We studied whether Necrostatin protected cells against oxidative stress during BM-MSCs culture process by measuring viability, intracellular ROS of cells and analysis of Immunocytochemistry and cell surface antigen by FACS. Pre-treatment of 1 μM to 10 μM of Necrostatin in H2O2 - induced oxidative stress cells were significantly increased cellular proliferation and viability depending on concentration of Necrostatin and 10 μM of Necrostatin were showed the highest cell proliferation rate and viability among 1- 1000 μM of Necrostatin compared with control group. The cellular morphology with pre-treated Necrostatin showed typical spindle like shape but in only H2O2 treated cells bizarre morphologic changes were shown. Also In pre-treatment of Necrostatin at 10 μM gene expressions of Oct4, FGF1, PGF, IL4 IGF1, IL10, TNFαIP6 and TGF-β1 were increased by real time PCR analysis. In immunocytochemistry study, expressions of Oct4, Nanog, Sox2, CD44 and CD54 were increased compared to control group. As results of FACS analysis Necrostatin treated cells showed more than 95% in antigen expression of CD73, CD49C, CD105, CD44, CD29 and CD105 and less than 5% of CD34, CD45 and CD106. These results were suggested the Necrostatin might protect cells against oxidative stess during BM-MSCs culture.