26S蛋白酶体参与人骨髓间充质干细胞向神经元样细胞分化

26S proteasome regulates differentiation of human bone marrow stromal cells into neural-like cells

目的:研究人骨髓间充质干细胞(hBMSCs)向神经元样细胞逐步分化过程中26S蛋白酶体的作用。方法:贴壁分离获得的hBMSCs先经β-巯基乙醇(β-ME)预诱导24h,随后用视黄酸(RA)诱导3d,最后再用生长因子(GF,10μg/LbFGF、20μg/LNGF)持续培养3d。免疫荧光染色观察不同诱导阶段神经前体细胞标志物nestin、早期神经元标志物Tuj1、成熟神经元标志物NF的表达。免疫荧光染色和RT-PCR分析不同诱导阶段26S蛋白酶体的表达变化。应用含26S蛋白酶体抑制剂的培养液(5μmol/LMG132、10μg/LbFGF、20μg/LNGF)作用于经β-ME/RA诱导后的细胞,NF免疫荧光染色观察蛋白酶体抑制剂对hBMSCs向神经元样细胞分化的影响。结果:未诱导的hBMSCs几乎不表达nestin、Tuj1和NF;经β-ME/RA诱导后神经前体细胞标志物nestin和早期神经元标志物Tuj1表达率增高(34.41%±1.27%,27.79%±1.27%);经β-ME/RA/GF诱导后成熟神经元标志物NF表达率迅速升高(56.72%±2.40%)。免疫荧光染色和RT-PCR结果证实,未诱导hBMSCs26S蛋白酶体表达水平较低;经β-ME/RA诱导后个别细胞26S蛋白酶体染色增强,26S蛋白酶体mRNA表达水平增至1.33倍;经β-ME/RA/GF诱导后26S蛋白酶体深染细胞增多,26S蛋白酶体mRNA表达水平增至1.77倍。应用26S蛋白酶体抑制剂MG132后,NF阳性细胞表达率显著降低(37.59%±1.52%)。结论:hBMSCs在β-ME/RA/GF诱导下可逐步向神经元样细胞分化,26S蛋白酶体在诱导过程中表达水平逐步增高,26S蛋白酶体抑制剂能够抑制hBMSCs向成熟神经元分化,提示26S蛋白酶体可能参与诱导hBMSCs向神经元样细胞的成熟分化。

AIM： To investigate the process of human bone marrow stromal cells （hBMSCs） differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS： Purified hBMSCs were treated with β-mercaptoethanol （β-ME） for 1 day and retinoic acid （RA） for 3 days, followed by growth factor （10 μg/L bFGF or 20 μg/L NGF） for another 3 days. Immunofluorescence was performed to detect the expression of nestin （a neural precursor cells marker）, Tuj1 （a premature neuronal marker）, and neurofilament （NF, a mature neuronal marker） at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS： Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells （34.41%±1.27%） and Tuj1-positive cells （27.79%±1.27%） were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA （1.33）, as well as by β-ME/RA/GF （1.77）, was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells （37.59%±1.52%）. CONCLUSION： After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.