目的:探讨人骨髓间充质干细胞(hMSCs)的体外培养生长特性及其在体外定向分化为神经样细胞的条件。方法:采用Ficoll-paque(1.077g/ml)分离液密度梯度分离hMSCs,显微镜下观察其生长特性,测定生长曲线;取传至3～6代的hMSCs,用阿魏酸钠对其进行诱导培养,并以β-巯基乙醇作对照观察诱导培养后细胞的形态变化,分别在6h、1d、3d和7d通过免疫荧光细胞化学染色方法测定诱导后细胞的神经元烯醇化酶(neuron specific enolase,NSE)和胶质纤维酸性蛋白(glia fiber acidprotein,GFAP),进行定量分析。结果:体外培养条件下hMSCs呈长梭形,细胞生长曲线呈S形,细胞倍增时间约为72h。阿魏酸钠诱导培养后6h可见细胞形态明显变化,NSE和GFAP表达阳性。24h后诱导细胞表现为典型的神经细胞样形态。第3天,NSE和GFAP表达最高,分别为67±3.5%和39±1.8%。结论:人骨髓间充质干细胞在体外具有自我更新能力及多分化潜能;阿魏酸钠具有诱导体外培养的人骨髓间充质干细胞向神经细胞分化的作用。
To investigate the mechanisms of bone marrow mesenchymal stem cells (MSCs) transplantation on neurological function following focal cerebral ischemia in rats.Methods Forty Sprague-Dawley rats, subjected to middle cerebral artery occlusion (MCAo) for 2 hours by intraluminal vascular occlusion, were divided into MSC transplantation group (TG) and cerebral ischemia control groups (CG) at random. MSCs were administrated via carotid artery at 24 hours after MCAo in T groups. At 1, 2, 4 and 12 weeks after MCAo, rats were killed in batches to investigate dynamically the regeneration of microvessels and proliferation of neuronal progenitors in ischemic region by immunohistochemistry and in situ hybridization.Results ①Notable microvessel proliferation in the injured cortex was found 1 week after MCAo and peaked at 2 weeks, then decreased at 4 and 12 weeks. The microvessel density (MVD) in the focal cortex of rats treated with MSC was significantly higher than that of C groups at 1,2,4 and 12 weeks after MCAo (P<0.05). Plenty of VEGFmRNA positive signals were found in cortical and perivascular neurons at 1 and 2 weeks after MCAo, and were more intensive in the T groups than those in C groups at 4 and 12 weeks after MCAo. ②At 1 week after MCAo, numerous nestin -positive cells (NPC) in the injured cortex of parietal lobe, caudate putamen, ependyma ,subependymal zone were observed and the NPC count showed the greatest amount of positive cells at the first week after MCAo, decreased at 2 weeks; and at 12 weeks after MCAo, only scanty NPC scattered in the focal cortex of C groups, whereas, in the T groups, the amount of positive cells remained great.The NPC amount of T groups at each time interval was significantly higher than that of C group(P<0.05) . ③At 1 week after MCAo, large amounts BrdU-labeled cells in the ischemic cortex, caudate putamen, ependyma ,subependymal zone were seen and positive-cell count appeared highest at the first week after MCAo, decreased after 2 weeks; and at 4 and 12 weeks , only scanty BrdU po
Human marrow stromal cells (hMSCs) are multipotential stem cells, capable of differentiating into bone, cartilage,fat and muscle. Several recent reports demonstrated that hMSCs have been also differentiated into neural cells.However, only a few reported inducers are applicable for clinical use. This work is to explore the effects of sodium ferulate (SF) on differentiation of hMSCs into neural cells in vitro. We found that hMSCs could be induced to the cells with typical neural morphology when cultured with SF. The cells express neural proteins, such as nestin,neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). About 30% of the hMSC-derived cells expressed nestin when cultured with SF for 3 h, but no expression was detected after 24 h. The percentages of positive cells for NSE or GFAP were about 67% and 39% separately at 6 h, and reached the plateau phage after treatment with SF for 3 days. The data suggest that SF can induce hMSCs to differentiate into neural-like cells in vitro.
