论文部分内容阅读
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 β subunit (PSMB5) in the directional differentiation of human bone marrow mesenchymal stem cells (h BMSCs) into neuron-like cells. Methods: hBMSCs were infected with lentiviral vector expressing PSMB5 and green fluorescent protein (GFP). The cells were divided into PSMB5 group and GFP control group according to different infectious viruses. The efficiency of infection and protease were verified by RT-PCR and fluorescence spectrophotometry Body activity. The PSMB5 group and GFP control group were preincubated with β-ME for 24 h, then induced with retinoic acid (RA) for 3 days, and then cultured with growth factor (GF) for 3 days. Immunofluorescence The positive rate of Tujl, an early neuron marker in GFP group and PSMB5 group, was detected by staining. At the same time, the induced hBMSCs were injected into the cerebral cortex of mice and the brain slices were collected after 4 weeks. The number of Tuj1 positive cells in GFP group and PSMB5 group was observed by immunofluorescence staining. RESULTS: Overexpression of PSMB5 significantly upregulated proteasome activity. The Tuj1 positive rate in PSMB5 group was 31% ± 8% after in vitro differentiation, which was significantly higher than that in GFP group (22% ± 7%, P <0.01). After 4 weeks of intracerebral injection, the number of Tuj1 positive cells in PSMB5 group was 20.0 ± 8.6, which was significantly higher than that in GFP group (10.7 ± 7.8) (P <0.01). Conclusion: Overexpression of PSMB5 can enhance the proteasome activity of hBMSCs and promote the differentiation of hBMSCs into neuron-like cells.