Sun Qiaoyu, Ou Ya, Wang Shujuan, Yuan Xiaodong, Zhang Wenjiang, Deng Hongliang, Zhang Lili
Department of Neurology, Affiliated Kailuan General Hospital of Hebei United University, No. 57 East Xinhua Road, Tangshan, 063000, Hebei Province, People's Republic of China.
J Mol Neurosci. 2014 Aug;53(4):608-16. doi: 10.1007/s12031-014-0227-5. Epub 2014 Jan 14.
Autophagy plays different roles in the growth and development process of different cells. The role of autophagy in the differentiation process of adult adipose-derived stromal cells (ADSCs) into astrocytes is unclear. We researched the role of autophagy in the induction process by adding autophagy agonist rapamycin, which was not added in the control group. Immunocytochemistry showed that the expression of glial fibrillary acidic protein (GFAP) was increased gradually with the extending reaction time and had reached the peak on the 14th day. Typical autophagy ultrastructure, including autophagic bodies and self-macrophage lysosomal, was shown under transmission electron microscopy (TEM) when cells were induced for 14 days. By methyl thiazolyl tetrazolium (MTT) assay, we found that the number of living cells was reduced gradually, and early apoptosis rate was increased by flow cytometry. We observed that the differentiation ratio, the number of living cells, and the positive expression rates of GFAP in the rapamycin group were higher than those in the control group when ADSCs were induced for 48 h and 7 days (P < 0.01); however, the rates of early apoptosis were lower than those in the control group. The positive rate of microtubule-associated protein light chain 3 (LC3) in the rapamycin group had been up to 88.87% on the 7th day (P < 0.01), but not obvious with extending time. After 14 days of induction, the optical density (OD) value of surviving cells was declined, and early apoptosis rate was increased gradually. The results showed that adding autophagy agonist to the inducers may enhance intensity of autophagy, shorten the induction time, and improve the efficiency of differentiation.
自噬在不同细胞的生长和发育过程中发挥着不同的作用。自噬在成年脂肪来源的基质细胞（ADSCs）向星形胶质细胞分化过程中的作用尚不清楚。我们通过添加自噬激动剂雷帕霉素来研究自噬在诱导过程中的作用，对照组未添加该激动剂。免疫细胞化学显示，胶质纤维酸性蛋白（GFAP）的表达随着反应时间的延长而逐渐增加，并在第14天达到峰值。当细胞诱导14天时，透射电子显微镜（TEM）下显示出典型的自噬超微结构，包括自噬体和自噬溶酶体。通过甲基噻唑基四氮唑（MTT）法，我们发现活细胞数量逐渐减少，流式细胞术检测显示早期凋亡率增加。我们观察到，当ADSCs诱导48小时和7天时，雷帕霉素组的分化率、活细胞数量和GFAP阳性表达率均高于对照组（P<0.01）；然而，早期凋亡率低于对照组。雷帕霉素组微管相关蛋白轻链3（LC3）的阳性率在第7天时高达88.87%（P<0.01），但随时间延长不明显。诱导14天后，存活细胞的光密度（OD）值下降，早期凋亡率逐渐增加。结果表明，在诱导剂中添加自噬激动剂可能会增强自噬强度，缩短诱导时间，并提高分化效率。
