Jin Wei, Xing Yi-qiao, Yang An-huai, Yang Yan-ning, Ai Ming
Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, China.
Zhonghua Yan Ke Za Zhi. 2010 Jun;46(6):542-9.
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.
It was an experimental study. The third passage of MSC was placed in 24-well cell culture plates at a density of 1 × 10(4)/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 control 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 comparison between two groups. T-test were also used between the positive expression of induced neuron-like cells and the control group.
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 were 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 morphology 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).
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.
评估不同浓度全反式维甲酸（ATRA）在体外诱导脐带间充质干细胞（MSC）向神经元样细胞分化过程中对细胞形态、增殖及凋亡的作用，并筛选出ATRA的最佳浓度。
本研究为实验性研究。将第3代MSC以1×10⁴/孔的密度接种于24孔细胞培养板。细胞贴壁后，分别更换为含不同浓度ATRA（0.25 μmol/L、0.5 μmol/L、1.0 μmol/L、2.0 μmol/L、4.0 μmol/L）的DMEM/F-12培养基培养24 h。未加ATRA培养的细胞作为对照组。再过24 h后，用倒置显微镜观察诱导细胞的形态变化，采用MTT比色法分析ATRA对细胞增殖及凋亡的影响。另取一组对照组和ATRA组，用Annexin V-FITC/PI联合流式细胞术检测诱导细胞的凋亡及阳性染色率。通过上述各项指标确定ATRA的最佳浓度。根据资料性质，对不同浓度ATRA作用24 h后的吸光度值及凋亡率采用方差分析，两组间进一步比较采用t检验。诱导神经元样细胞阳性表达与对照组间比较也采用t检验。
与对照组相比，0.25 μmol/L的ATRA对脐带MSC增殖无明显抑制作用（t = 0.72，1.32，P > 0.05）。加入4.0 μmol/L ATRA时部分MSC即刻漂浮，培养24 h后未见贴壁细胞。≥1.0 μmol/L的ATRA作用24 h，MSC增殖受到明显抑制，呈剂量依赖性（t = 8.8，18.9，22.1；P < 0.01）。0.5 μmol/L的ATRA不影响细胞增殖，细胞形态正常；1.0 μmol/L的ATRA仅影响极少量细胞；但2.0 μmol/L的ATRA培养24 h较1 h对细胞增殖的抑制作用明显增强，细胞体积增大、变扁平。流式细胞术显示对照组与≥1.0 μmol/L组凋亡率差异有统计学意义（t = 9.88，19.95，31.61；P < 0.01）。
在脐带MSC向神经元样细胞诱导过程中，0.5 μmol/L ATRA为最佳浓度。≥1.0 μmol/L ATRA可明显抑制细胞增殖，增加细胞凋亡，并造成明显损伤。
