Yang Yafeng, Ma Teng, Ge Jun, Quan Xin, Yang Le, Zhu Shu, Huang Liangliang, Liu Zhongyang, Liu Liang, Geng Dan, Huang Jinghui, Luo Zhuojing
Institute of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an, PR China.
Cell Transplant. 2016;25(6):1177-91. doi: 10.3727/096368915X688957. Epub 2015 Sep 2.
Neuron-like cells derived from adipose tissue-derived stem cells (ADSCs) have been considered one of the most promising cells for the treatment of neurodegenerative diseases and neurotrauma in the central nervous system (CNS). Thus far, extensive efforts have been made to facilitate neuronal differentiation of ADSCs, but limited progress has been achieved. In the present study, we tested the possibility of using a combination of electrical stimulation (ES) with Nurr-1 gene transduction to promote neuronal differentiation of ADSCs. The tolerance of ADSCs to ES was first examined by a cell apoptosis assay. The proliferation of cells was characterized using a CCK-8 assay. The morphology of cells was examined by scanning electron microscopy (SEM). The differentiation of ADSCs into neuron-like cells was examined by immunocytochemistry (ICC)-immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme linked immunosorbent assay (ELISA). The gene expression of microtubule-associated protein 2 (MAP-2), β-tubulin, neurofilament 200 (NF-200), octamer binding transcription factor 4 (OCT-4), and glial fibrillary acidic protein (GFAP) after stimulation was examined by qRT-PCR. We found that the optimal intensity of ES for neuronal differentiation of ADSCs was 1 V/cm. In addition, ES combined with Nurr-1 gene transduction increased the neuronal differentiation rate of ADSCs, the length of neurite-like processes, and the secretion of dopamine. Further studies showed that a combination of ES with Nurr-1 gene transduction was capable of promoting the expression of MAP-2, β-tubulin, and NF-200 but decreased the expression of OCT-4 and GFAP. All of these findings indicate that a combination of ES with Nurr-1 gene transduction could facilitate neuronal differentiation of ADSCs, which raises the possibility of its application in the treatment of neurodegenerative diseases and neurotrauma in the CNS.
源自脂肪组织干细胞（ADSCs）的类神经元细胞被认为是治疗中枢神经系统（CNS）神经退行性疾病和神经创伤最有前景的细胞之一。迄今为止，人们为促进ADSCs向神经元分化付出了巨大努力，但进展有限。在本研究中，我们测试了电刺激（ES）与Nurr-1基因转导相结合促进ADSCs向神经元分化的可能性。首先通过细胞凋亡检测来考察ADSCs对ES的耐受性。使用CCK-8检测法来表征细胞增殖情况。通过扫描电子显微镜（SEM）检查细胞形态。通过免疫细胞化学（ICC）-免疫荧光染色、定量实时聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法和酶联免疫吸附测定（ELISA）来检测ADSCs向类神经元细胞的分化情况。通过qRT-PCR检测刺激后微管相关蛋白2（MAP-2）、β-微管蛋白、神经丝200（NF-200）、八聚体结合转录因子4（OCT-4）和胶质纤维酸性蛋白（GFAP）的基因表达。我们发现，促进ADSCs向神经元分化的最佳ES强度为1 V/cm。此外，ES与Nurr-1基因转导相结合提高了ADSCs的神经元分化率、类神经突的长度以及多巴胺的分泌。进一步研究表明，ES与Nurr-1基因转导相结合能够促进MAP-2、β-微管蛋白和NF-200的表达，但降低了OCT-4和GFAP的表达。所有这些发现表明，ES与Nurr-1基因转导相结合能够促进ADSCs向神经元分化，这增加了其在治疗CNS神经退行性疾病和神经创伤中应用的可能性。
