PSMB5基因过表达促人骨髓间充质干细胞向神经元样细胞分化

Overexpression of PSMB5 gene promotes human mesenchymal stem cells differentiation into neuron-like cells

目的:明确20S蛋白酶体β白亚单位(PSMB5)在人骨髓间充质干细胞(h BMSCs)向神经元样细胞定向分化过程中的作用。方法:构建PSMB5与绿荧光蛋白(GFP)融合表达慢病毒载体感染h BMSCs,依照感染病毒不同将细胞分为PSMB5组和GFP对照组,并利用RT-PCR和荧光分光光度法验证感染效率和蛋白酶体活性。将PSMB5组和GFP对照组h BMSCs经β-巯基乙醇(β-ME)预诱导24 h,再用视黄酸(RA)诱导3 d,最后用生长因子(GF)持续培养3 d,免疫荧光染色检测GFP组和PSMB5组早期神经元标志物Tujl的阳性率。同时,将诱导后的h BMSCs注入小鼠大脑皮层,4周后收集脑片,免疫荧光染色观察GFP组和PSMB5组Tuj1阳性细胞存活数量。结果:PSMB5基因过表达能够显著上调蛋白酶体活性。经体外诱导分化后PSMB5组Tuj1阳性率达31%±8%,较GFP组22%±7%明显升高(P<0.01)。脑内注射4周后,PSMB5组Tuj1阳性细胞数达20.0±8.6,较GFP组10.7±7.8明显升高(P<0.01)。结论:PSMB5基因过表达能够提高h BMSCs蛋白酶体活性,有助于促使h BMSCs向神经元样细胞分化。

Objective： To clarify the role of 20S proteasome subunit beta 5 （PSMB5） in directed differentiation of hu- man bone marrow mesenchymal stem cells （hBMSCs） into neuron-like ceils. Methods： The human BMSCs were infected by PSMB5 lentiviral vectors with Green Fluorescent Protein （GFP）. Cells were divided into PSMB5 group and GFP con- trol group according to different virus infection. RT-PCR and the proteasome activity assay were used to detect the infec- tion efficiency. The hBMSCs were cultured with growth factors for three consecutive days after pre-induction with 13-mer- captoethanol （13-ME） for 24 h and retinoic acid （RA） for three days. The positive rate of Tujl （ early neuron marker） of both GFP and PSMB5 groups was measured by immunofluorescence staining respectively. The induced hBMSCs were in- jected into the mouse cerebral cortex. After four weeks, brain slices were collected and the number of Tujl-positive cells was observed for two groups by immunofluorescence staining. Results： Overexpression of PSMB5 genes could significantly increase the proteasome activity. After induction culture of hBMSCs, the positive rate of Tujl was within （31 ± 8% ） in the PSMB5 group, and markedly higher than （22 ±7% ）, the rate in the GFP group （P 〈0.01 ）. After 4 weeks of in-tracerebral injection, there were 20.0 ± 8.6 Tujl-positive cells observed in the PSMB5 group, which significantly in- creased compared with that of Tujl -positive cells （ 10.7 ± 7.8 ） in the GFP group（ P 〈 0.01 ）. Conclusion： These results suggest that overexpression of PSMB5 gene could improve proteasome activity of hBMSCs and was helpful promoting differ- entiation of hBMSCs into neuron-like cells.