成年脂肪来源基质细胞分化的星形胶质细胞的超微结构和电生理学。
Ultrastructure and electrophysiology of astrocytes differentiated from adult adipose-derived stromal cells.
机构信息
Department of Neurology, Kailuan General Hospital, Hebei United University, Tangshan, Hebei 063000, China.
出版信息
Chin Med J (Engl). 2011 Sep;124(17):2656-60.
BACKGROUND
Adipose-derived stromal cell (ADSC) differentiation into neural cells in vitro is becoming widely studied. However, there are few reports on astrocytes following differentiation, and particularly on maturation and electrophysiology. In this study, we used various methods to determine ADSC-derived astrocyte maturity.
METHODS
Chemical induction with isobutylmethylxanthine (IBMX) was used to differentiate adult ADSCs into astrocytes followed by hematoxylin-eosin (HE) staining to observe morphology and transmission electron microscopy for cellular ultrastructure assessment. Immunofluorescence was used to detect expression of neural stem cell marker nestin as well as glial markers glial fibrillary acidic protein (GFAP) and S-100. In addition, we measured membrane potentials in bis-(1,3-dibarbituric acid) trimethine oxanol-labeled ADSCs and astrocytes by stimulation with a high potassium solution under an inverted fluorescence microscope. Finally, cell cycle distribution was detected by flow cytometry.
RESULTS
Typical astrocyte morphology was shown by HE staining after 48-hour differentiation. Glial fibril was observed with transmission electron microscopy. GFAP and S-100 were not expressed in the control group, but were expressed within 24-hour differentiation and reached a maximum at day 14 with no change up to day 28. Nestin was weakly expressed in control cells and also reached a maximum at day 14 with the percentage of positive cells constant until day 21 followed by a decrease. Differentiated cell membrane potentials after stimulation with potassium were slightly increased, and then gradually declined over time. There was no significant membrane potential change in the control group. Flow cytometry showed that the percentage of cells in G0/G1 phase was 93% and only 5% in S phase.
CONCLUSION
ADSCs were differentiated into mature astrocytes with typical characteristics including morphology, ultrastructure, marker protein expression, mature potassium channels and mitotic capacity.
背景
脂肪来源的基质细胞（ADSC）在体外向神经细胞分化的研究越来越多。然而，关于分化后的星形胶质细胞，特别是成熟和电生理学方面的报道较少。在本研究中，我们使用了多种方法来确定 ADSC 衍生的星形胶质细胞的成熟度。
方法
用异丁基甲基黄嘌呤（IBMX）化学诱导将成体 ADSC 分化为星形胶质细胞，然后用苏木精-伊红（HE）染色观察形态，用透射电子显微镜观察细胞超微结构。免疫荧光法检测神经干细胞标志物巢蛋白以及神经胶质标志物胶质纤维酸性蛋白（GFAP）和 S-100 的表达。此外，我们通过倒置荧光显微镜在高钾溶液刺激下测量双（1,3-二巴比妥酸）三甲烷醇标记的 ADSC 和星形胶质细胞的膜电位。最后，通过流式细胞术检测细胞周期分布。
结果
48 小时分化后，HE 染色显示典型的星形胶质细胞形态。电镜观察到神经胶质纤维。对照组中不表达 GFAP 和 S-100，但在 24 小时分化时开始表达，第 14 天达到最大值，直到第 28 天没有变化。巢蛋白在对照组细胞中表达较弱，第 14 天也达到最大值，阳性细胞百分比保持不变，直到第 21 天，然后减少。钾刺激后分化细胞的膜电位略有升高，然后随时间逐渐下降。对照组的膜电位没有明显变化。流式细胞术显示 G0/G1 期细胞的百分比为 93%，S 期仅为 5%。
结论
ADSC 分化为具有典型特征的成熟星形胶质细胞，包括形态、超微结构、标记蛋白表达、成熟钾通道和有丝分裂能力。
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