全反式视黄酸对诱导为神经元样细胞的脐带间充质干细胞分化和凋亡的影响

Objective To evaluate the role of different concentration of all-trans retinoic acid (ATRA)on the morphology,proliferation and apoptosis in inducing umbilical cord mesenchymal stem cells (MSC) into neuron-like cells in vitro,and screen the optimal concentration of ATRA.Methods It was an experimental study.The third passage of MSC was placed in 24-well cell culture plates at a density of 1×104/well.After the adherent of cells,the medium was changed to DMEM/F-12 containing different concentration of ATRA(0.25 μmol/L,0.5 μmol/L,1.0 μmol/L,2.0 μmol/L,4.0 μmol/L)for 24 h respectively.The cells cultured without ATRA were taken as the coutrol group.After another 24 h,the morphologic changes of induced cells were observed by inverted microscope and cell proliferation,apoptosis of ATRA was analyzed using the MTT colorimetric assay.We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry.The optimal concentration of ATRA was determined by all the above-mentioned index.According to the nature of the material,analysis of variance (ANOVA) was employed for absorption value and apoptosis rate in different concentration of ATRA for 24 h,t test for further comparision between two groups.T-test were also used between the positive expression of induced neuron-like cells and the control group.Results Compared to the control group,ATRA at the concentration of 0.25 μmoL/L did not inhibit the proliferation of umbilical cord MSC obviously(t=0.72.1.32,P>0.05).Part of MSC wele floating instantly at the moment of adding ATRA of 4.0 μmoL/L and no adherent cells were observed after 24 h'culture.Exposed to ATRA at the concentration of≥1.0 μmol/L for 24 h,the proliferation of MSC were significantly inhibited,showing a dose-dependent manner(t=8.8,18.9,22.1;P<0.01).0.5 μmol/L of ATRA did not affect the proliferation of cells and its moephology remained normal;1.0 μmol/L of ATRA affected very few cells;but 2.0 μmoL/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h.and the cells increased in size and became flattened.Flow cytometry showed that the rate of apoptosis between the control group and≥1.0 μmol/L groups were significantly different(t=9.88,19.95,31.61;P<0.01).Conclusion In the process of inducing umbilical cord MSC into neuron-like cells,0.5 μmol/L ATRA was the optical concentration.≥1.0 μmol/L ATRA can inhibit the cell proliferation,increase the apoptosis of cells significantly and caused obvious damages.

Differentiation and apoptosis effects of all-trans retinoic acid on inducing umbilical cord mesenchymal stem cells into neuron-like cells