采用海马原代神经元和离体脑片建立乙醇性痴呆体外模型的比较  

Setting up alcohol-associated dementia models in vitro with primary-cultured hippocampal neuron and brain slice

作　　者：刘勇[1] 曾玉娥[2] 杨海玉[3] 

机构地区：[1]江西省人民医院病理科,南昌330006 [2]南昌大学医学院,南昌330006 [3]江西省人民医院临床医学研究所,南昌330006

出　　处：《中华神经医学杂志》2014年第1期41-44,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81060111）

摘　　要：目的建立和比较不同的乙醇性痴呆（AAD）体外研究模型，为进一步探讨其发病机制提供方法学参考。方法取胎鼠海马进行原代神经元培养及鉴定，给予不同浓度的乙醇作用24h．采用四甲基偶氮唑蓝（MTT）比色法检测细胞存活率以及Hoechst33342染色观察细胞凋亡状况。另外，取新生大鼠海马切取脑片进行离体培养，采用HE染色观察不同时间点乙醇对海马脑片的损伤作用。结果原代培养的海马神经元高表达神经元特异性烯醇化酶（NSE），低表达神经胶质纤维酸性蛋白（GFAP）。乙醇（50～100mol／L）作用24h可明显抑制海马神经元存活并呈浓度依赖关系；乙醇（50mol／L，24h）可诱导海马神经元显著凋亡。海马脑片HE染色证实短时乙醇作用（50mol／L,30min）即可导致细胞明显损伤，而长时乙醇作用（50mol／L，24h）可导致海马形态结构严重破坏。结论在AAD发病机制研究中，原代海马神经元适用于建立慢性乙醇诱导损伤模型，而离体海马脑片适用于急性乙醇毒性研究。Objective To set up the different alcohol-associated dementia （AAD） models in vitro and provide methods for researching the mechanism of AAD. Methods Hippocampal neurons got from fetal rats were primary cultured for 6 days and identified. Then, the cells were treated with different doses of ethanol （25-100 mol/L） for 24 h. The cell viability was analyzed with MTT assay. The staining with Hoechst33342 was used to observe the cell apoptosis. In addition, hippocampi of newborn rats 7-10 days after birth were taken out and cut to 300 μm thickness of slices; the morphological changes of the brain slices were observed with HE staining at different time points after ethanol administration. Results Primary-cultured hippocampal neurons highly expressed neuron-specific enolase （NSE） and lowly expressed glial fibrillary acidic protein （GFAP）. And the cell viability was significantly decreased by ethanol administration （50-100 mol/L, 24 h） in a dose-dependent manner. Increased apoptosis cells were detected when cells were treated with 50 mol/L concentration of ethanol for 24 h. For hippocampal slices, acute ethanol administration （50 mol/L, 30 min） induced significant cell apoptosis and chronic ethanol administration （50 mol/L, 24 h） resulted in the serious damage of hippocampal morphology. Conclusions The models that primary-cultured hippocampal neuron apoptosis induced by chronic ethanol administration is suitable for researching the mechanism of AAD. Hippocampal slices are more sensitive for ethanol toxic effects and may be used for the research of acute alcohol toxicity.

关 键 词：乙醇性痴呆 体外研究 海马神经元 原代培养 离体脑片 

分 类 号：R749.16[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]