摘要
目的:探讨天然脑活素(NC)诱导骨髓间充质干细胞(MSCs)向神经元样细胞分化的作用。方法:取6-8周龄SD大鼠,无菌获取胫骨和股骨骨髓,全骨髓贴壁筛选法分离培养并纯化大鼠MSCs,应用天然脑活素诱导MSCs向神经元分化。倒置相差显微镜追踪比较观察分化过程中细胞形态的变化,免疫细胞化学和逆转录-聚合酶链反应(RT-PCR)方法检测神经元特异性标志物:巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达情况。免疫细胞化学检测细胞微管相关蛋白-2(MAP-2)的表达。结果:经全骨髓贴壁筛选法获得了纯度较高(90%以上)的大鼠MSCs,天然脑活素含药血清诱导分化后的细胞呈现双极、多极和锥形的典型神经元细胞的形态,分别从mRNA和蛋白水平上证实诱导分化后的细胞表达神经元标记物NSE和nestin,不表达神经胶质细胞标记物GFAP。诱导分化后细胞MAP-2的表达明显。结论:天然脑活素可诱导MSCs向神经元样细胞的定向分化。
AIM： Natural cerebrolysin （NC） is a Chinese traditional medicine, formulated by tall gastrodis tuber, ginseng and ginkgo leaf at 2： 1：2 quality ratio. This study is to investigate whether NC can induce rat mesenchymal stem cells （MSCs） to differentiate into neuron-like cells in vitro. METHODS： The SD rats about 6 -8 week old Were used for experiment. MSCs were separated from shin bone and/or thigh bone marrow of rats by holo - marrow and wall sticking methods. MSCs passages were cultured and depurated in vitro. The purified MSCs were induced to differentiate ori- entedly into neuron - like cells by incubating the cells with NC. The morphology of cells was observed under inverted phase contrast microscope. The expressions of neuron - specific enolase （ NSE）, microtubule - associated protein 2 （ MAP - 2）, nestin and glial fibrillary acidic protein （GFAP） were detected by immunocytochemistry and reverse transcription -poly- merase chain reaction （ RT - PCR）. RESULTS： MSCs were separated and cultured successfully by holo - marrow and wall sticking methods. The high purity （above 90% ） of MSCs was acquired by passaged proliferation. Most of the MSCs incubated with NC differentiated into neuron - like cells, revealing cytodendrite, showing typical nerve cell＇s morphous with two polar, muhipolar and taper. After incubated with NC, the cells expressed NSE and nestin, and not expressed GFAP at mRNA and protein level. By immunochemical staining, we observed that MSCs treated by NC were MAP -2 expression positive obviously. CONCLUSION ： Rat MSCs could be induced to differentiate orientedly into neuron - like cells by natural cerebrolysin.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第8期1548-1553,共6页
Chinese Journal of Pathophysiology
基金
广东省科学计划重点资助项目(No.2006B35630007)
深圳市科技局重大资助项目(No.JH200506030643A)
关键词
天然脑活素
骨髓间充质干细胞
神经元样细胞
Natural cerebrolysin
Bone marrow mesenchymal stem cells
Neuron- like cells
