The Mechanism Of The Preventive Effect Of Extracelluar Matrix On Oxidative Stress-induced Senescence In Stem Cells

[Objective]:Umbilical cord derived mesenchymal stem cells(UC-MSCs)can be obtained by a convenient and harmless way.It can expand in vitro for a longer time.At the same time,using UC-MSCs would not cause ethical controversy.Therefore UC-MSCs can be wider used in the area of regenerative therapy.In vivo,stem cells grow in extracellular matrix(ECM).However,once losing the microenvironment in vivo,stem cells are more prone to premature aging under the influence of various adverse factors in vitro.It greatly reduces the practical application value of stem cells.Decelluarized extracelluar matrix(DECM)mimics the microenvironment of stem cells in vitro.The aim of this study is to investigate whether DECM can regulate the senescence of UC-MSCs caused by hydrogen peroxide(H2O2)via nuclear factor erythroid-2-related factor(NRF-2).[Methods]:Firstly,we determined that 200 ?M is the proper concentration of H2O2 to induce the premature senescence of UC-MSCs.We cultured UC-MSCs on tissue culture polystyrene(TCPS)flasks and DECM respectively,and used H2O2 to induce celluar senescence.The capacity of proliferation was evaluated by the the Cell Counting Kit-8.The SA-?-gal staining kit was used to mark senescence cell.Flow cytometry test cell cycle distribution.The ability of osteogenic differentiation was analyzed by Alizarin Red S staining and RT-qPCR.The expression of aging and osteogenesis related genes was detected by RT-qPCR.We silienced the expression of NRF-2 by small interfering RNA(siRNA)to determine if DECM rejuvenate UC-MSCs via NRF-2.[Results]:Compared with cells cultured on TCPS flasks,the cells cultured on DECM showed stronger proliferation ability regardless of the pretreatment of H2O2.The cell cycle distribution results showed that UC-MSCs cultured on DECM flasks exhibited a significantly decreased proportion in the G0/G1 phase.The cells cultured on DECM flasks showed an incrased positive rate of SA-?-Gal staining,higher expression of P53,P21 and low expression of NRF-2.The results of Alizarin Red S staining and RT-qPCR analysis of osteogenesis related genes showd that the capacity of osteogenic differentiation of UC-MSCs cultured on DECM flasks is superior to those on TCPS flasks.After the silencing the expression of NRF-2 of UC-MSCs cultured on DECM flasks by siRNA,the expression levels of P53 and P21 and the positive rate of SA-?-Gal staining were significantly increased,confirming the key role of NRF-2 in the anti-aging process of DECM.[Conclusion]:DECM provides a suitable microenvironment for UC-MSCs in vitro that is beneficial for their proliferation and osteogenic differentiation and DECM can effectively reserve the senescence of cells via NRF-2.