摘要
目的建立新生大鼠前扣带皮层（ACC）神经元的体外培养方法,并进行纯度和生长状态检测。方法从新生鼠ACC分离出神经元,采用低浓度胰酶长时间消化、阿糖胞苷处理、差速培养等方法进行体外原代培养。在培养的第7天,应用兔抗大鼠微管相关蛋白2（MAP2）对培养的神经元进行纯度鉴定,利用微量滴定（MTT）法测定培养第712天ACC神经元的生长状态。结果从新生大鼠ACC分离的神经元,培养至第5天,可见多数细胞长出2个以上突起且呈多极形态并交织成网;第7天神经元有多种形式的接触,互相迁移靠拢,部分神经元聚集成团。MAP2细胞免疫荧光染色结果表明,ACC神经元阳性率大于80%,MTT结果显示,培养第79天的ACC神经元可保持较好的生长状态,之后细胞活性降低。结论新生大鼠ACC神经元可进行体外原代培养,纯度和活力检测表明,培养第79天的神经元可作为ACC神经元相关研究的体外细胞模型。
Objective To explore the optimal condition of primary culture for anterior cingulate cortex（ACC） neurons in neonatal rats and identify their purity and viability.Methods Neurons were isolated from the ACC of neonatal rats,and then digested with a low concentration of tripsin for a long time.When the cells were cultured till day 4,Ara-c was added to purify them.The cultured ACC neurons were identified by the microtubule-associated protein2（MAP2） immunocytochemical method.Viability of ACC neurons in the cell cycle（about 14 d） was investigated by the measurement of optical density（OD） values at 570 nm using the MTT method.Results ACC neurons from neonatal rats were successfully cultured under the present experimental condition.On day 5 of culture,most cells appeared multi-polar and had more than two ecptoms and their interlacing looked like a piece of a net.On day 7,many kinds of connections presented among these cultured cells and even aggregation of cells appeared.The cultured neurons were stained by immunocytochemistry（ICC） and the green immunoreactive product was seen in neurons under a fluorescence microscope,indicating that the cultured neurons were positive.The MTT experiment shows that viability of ACC neurons cultured from day 7 to day 9 was the best.Conclusion ACC neurons in neonatal rats can be cultured in vitro,and primary cultured ones may serve as a cell model in vitro for research into ACC,indicated by the result of purity and viability in ACC neurons.
出处
《山东大学学报（医学版）》
CAS
北大核心
2011年第5期54-57,共4页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助项目(30872433)
关键词
扣带回
神经元
免疫组织化学
原代培养
活力鉴定
Gyrus cinguli； Neurons； Immunohistochemistry； Primary culture； Viability assessment；
作者简介
李芸（1986-），女，硕士研究生，主要从事痛情绪的基础研究。E-mail：liyun—wish2009@126．com
张孟元（1962-），男，博士，教授，主要从事心外科手术的麻醉。E—mail：zhangmy717@163．com
王公明（1971-），男，博士，副教授，主要从事痛情绪的基础研究和临床麻醉。E-mail：tagmwangl971@163．com
