Hao Hsiao-Nan, Parker Graham C, Zhao Jane, Barami Kaveh, Lyman William D
Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI, USA.
Alcohol Clin Exp Res. 2003 Aug;27(8):1310-7. doi: 10.1097/01.ALC.0000080671.56559.EF.
Exposure to ethanol (EtOH) can be deleterious to the developing central nervous system. The mechanisms by which EtOH exposure induces neural pathology in utero remain unclear. However, EtOH-induced increases in protein kinase C (PKC) have been associated with apoptosis in human primary cell cultures. Although the toxic effects of EtOH on differentiated neural cells have been studied in laboratory animal models, the susceptibility of the human neural stem cells (NSCs) that predominate in the central nervous system during embryonic development has not been addressed.
For this study, fetal human brain cells, which satisfied the criteria for NSCs by being CD133-positive, nestin-positive, and differentiated glial fibrillary acidic protein-positive human astrocytes, were studied. The cytotoxic potential of EtOH in NSC and astrocyte cultures was studied by using morphological and biochemical methods. In addition, membrane and cytosolic fraction PKC activity for each cell type was assessed.
NSC showed a dose-dependent increase in EtOH-induced toxicity as estimated by terminal transferase-mediated dUTP nick end labeling (TUNEL) stain and viability assays. TUNEL staining indicating DNA degradation consistent with programmed (apoptotic) cell death was detectable in 90% of NSC 16 hr after 2 hr exposure to 10 mM EtOH. NSC also showed a concentration-dependent increase in membrane, but not cytosol, PKC activity over the same EtOH dose range. By contrast, astrocytes showed no cytotoxic effects at any concentrations of EtOH used (0-10 mM). PKC activity of both the membrane and cytosolic fragments from astrocytes also was unaffected by this range of doses.
This study demonstrates the susceptibility of human NSCs, compared with astrocytes, to EtOH and indicates that alterations in PKC signal transduction in NSC may play a role in EtOH-induced neuropathological processes.
接触乙醇（EtOH）可能对发育中的中枢神经系统有害。EtOH暴露在子宫内诱发神经病理学的机制仍不清楚。然而，EtOH诱导的蛋白激酶C（PKC）增加与人类原代细胞培养中的细胞凋亡有关。尽管在实验动物模型中已经研究了EtOH对分化神经细胞的毒性作用，但在胚胎发育期间中枢神经系统中占主导地位的人类神经干细胞（NSC）的易感性尚未得到研究。
在本研究中，对符合NSC标准的胎儿人脑细胞进行了研究，这些细胞为CD133阳性、巢蛋白阳性且分化为胶质纤维酸性蛋白阳性的人类星形胶质细胞。通过形态学和生化方法研究了EtOH在NSC和星形胶质细胞培养物中的细胞毒性潜力。此外，还评估了每种细胞类型的膜和胞质部分的PKC活性。
通过末端转移酶介导的dUTP缺口末端标记（TUNEL）染色和活力测定估计，NSC显示出EtOH诱导的毒性呈剂量依赖性增加。在暴露于10 mM EtOH 2小时后16小时，90%的NSC中可检测到TUNEL染色，表明DNA降解与程序性（凋亡）细胞死亡一致。在相同的EtOH剂量范围内，NSC的膜PKC活性也呈浓度依赖性增加，但胞质PKC活性未增加。相比之下，星形胶质细胞在任何使用的EtOH浓度（0-10 mM）下均未显示细胞毒性作用。星形胶质细胞膜和胞质片段的PKC活性也不受该剂量范围的影响。
本研究表明，与星形胶质细胞相比，人类NSC对EtOH敏感，并表明NSC中PKC信号转导的改变可能在EtOH诱导的神经病理过程中起作用。
