中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2014年
1期
41-44
,共4页
乙醇性痴呆%体外研究%海马神经元%原代培养%离体脑片
乙醇性癡呆%體外研究%海馬神經元%原代培養%離體腦片
을순성치태%체외연구%해마신경원%원대배양%리체뇌편
Alcohol-associated dementia%In vitro study%Hippocampal neuron%Primary culture%Brain slice
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24 h,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(50 mol/L,24 h)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(50 mol/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.
目的 建立和比較不同的乙醇性癡呆(AAD)體外研究模型,為進一步探討其髮病機製提供方法學參攷. 方法 取胎鼠海馬進行原代神經元培養及鑒定,給予不同濃度的乙醇作用24 h,採用四甲基偶氮唑藍(MTT)比色法檢測細胞存活率以及Hoechst33342染色觀察細胞凋亡狀況.另外,取新生大鼠海馬切取腦片進行離體培養,採用HE染色觀察不同時間點乙醇對海馬腦片的損傷作用. 結果 原代培養的海馬神經元高錶達神經元特異性烯醇化酶(NSE),低錶達神經膠質纖維痠性蛋白(GFAP).乙醇(50～100mol/L)作用24h可明顯抑製海馬神經元存活併呈濃度依賴關繫;乙醇(50 mol/L,24 h)可誘導海馬神經元顯著凋亡.海馬腦片HE染色證實短時乙醇作用(50mol/L,30min)即可導緻細胞明顯損傷,而長時乙醇作用(50 mol/L,24h)可導緻海馬形態結構嚴重破壞. 結論 在AAD髮病機製研究中,原代海馬神經元適用于建立慢性乙醇誘導損傷模型,而離體海馬腦片適用于急性乙醇毒性研究.
목적 건립화비교불동적을순성치태(AAD)체외연구모형,위진일보탐토기발병궤제제공방법학삼고. 방법 취태서해마진행원대신경원배양급감정,급여불동농도적을순작용24 h,채용사갑기우담서람(MTT)비색법검측세포존활솔이급Hoechst33342염색관찰세포조망상황.령외,취신생대서해마절취뇌편진행리체배양,채용HE염색관찰불동시간점을순대해마뇌편적손상작용. 결과 원대배양적해마신경원고표체신경원특이성희순화매(NSE),저표체신경효질섬유산성단백(GFAP).을순(50～100mol/L)작용24h가명현억제해마신경원존활병정농도의뢰관계;을순(50 mol/L,24 h)가유도해마신경원현저조망.해마뇌편HE염색증실단시을순작용(50mol/L,30min)즉가도치세포명현손상,이장시을순작용(50 mol/L,24h)가도치해마형태결구엄중파배. 결론 재AAD발병궤제연구중,원대해마신경원괄용우건립만성을순유도손상모형,이리체해마뇌편괄용우급성을순독성연구.
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 newbom 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.
